Vanda Mimi Palmer Thesis
-
Upload
mohd-hairul-ab-rahim -
Category
Documents
-
view
320 -
download
8
description
Transcript of Vanda Mimi Palmer Thesis
1
Abstract of thesis presented to the Senate of Universiti Putra Malaysia in fulfillment of
the requirement for the degree of Master of Science
CHEMICAL COMPOSITION OF FLORAL VOLATILES AND EXPRESSION
OF SCENT-RELATED GENES IN VANDA MIMI PALMER
By
MOHD HAIRUL AB. RAHIM
November 2010
Chairman: Parameswari a/p Namasivayam, PhD
Faculty: Biotechnology and Biomolecular Sciences
Vanda Mimi Palmer is an orchid hybrid of Vanda Tan Chay Yan and Vanda tessellata.
The flower of this orchid produces a sweet fragrance during daylight hours at the open-
flower stage. Lately, a lot of effort has been channeled into understanding the fragrance
pathway in scented flowers but none in Vandaceous orchids. This study aims to
investigate on the molecular and biochemical aspects of the fragrance in Vanda Mimi
Palmer. Scent emission analysis of this orchid was carried out at different developmental
stages and at different time points in a 24-hour cycle. Gas chromatography mass
spectrometry (GC-MS) analysis has shown that the scent of Vanda Mimi Palmer is
dominated by metabolites from the terpenoid, benzenoid and phenylpropanoid pathways.
Identified volatile compounds derived from terpenoid pathway are linalool, ocimene and
nerolidol. Meanwhile, methylbenzoate, phenylethanol, benzyl acetate and phenylethyl
acetate are the metabolites identified from the benzenoid and phenylpropanoid pathways.
Scent emission of Vanda Mimi Palmer is also developmentally and temporally regulated.
2
On the molecular biology aspect, fragrance-related cDNA transcripts have been identified
by a differential screening of the Vanda Mimi Palmer‟s floral cDNA library. Reverse-
Northern analysis was carried out by hybridizing the putative positive clones with two
cDNA probes representing mRNA transcripts of bud and fully-open flower during the
daylight hour separately. The clones that showed up-regulated expression in fully-open
flowers were selected for sequencing. From the sequencing results, putative 4-(cytidine
5′-diphospho)-2-C-methyl-D-erythritol kinase (VMPCMEK), putative cytochrome P450
(VMPCyP450), and an unknown protein (VMPA28) were selected for molecular
characterization. The three transcripts with a putative phenylacetaldehyde synthase
(VMPPAAS), a previously isolated transcript from Expressed Sequence-Tags (ESTs),
were subjected to full-length cDNA isolation and expression analyses by real-time RT-
PCR. Expression analyses of these transcripts were investigated in different tissues, at
different developmental stages, and time points in a 24-hour cycle using real-time RT-
PCR. The transcripts are highly expressed in floral tissues compared to vegetative tissues
as well as developmentally and temporally regulated. In conclusion, from the
biochemical and molecular work on the fragrance, there are two putative biochemical
pathways which might be involved in fragrance biosynthesis in Vanda Mimi Palmer that
are the terpenoid, and also the benzenoid and phenylpropanoid pathways.
3
Abstrak tesis yang dikemukakan kepada Senat Universiti Putra Malaysia sebagai
memenuhi keperluan untuk ijazah Master Sains
KOMPOSISI KIMIA HARUMAN DAN EKSPRESI GEN-GEN BERKAITAN
WANGIAN DALAM VANDA MIMI PALMER
Oleh
MOHD HAIRUL AB. RAHIM
November 2010
Pengerusi: Parameswari a/p Namasivayam, PhD
Fakulti: Bioteknologi dan Sains Biomolekul
Vanda Mimi Palmer ialah orkid kacukan di antara Vanda Tan Chay Yan dan Vanda
tesellata. Bunga orkid ini yang telah berkembang sepenuhnya mengeluarkan bau yang
harum pada waktu siang. Kebelakangan ini, perhatian diberikan terhadap tapak jalan
biokimia penghasilan wangian bagi bunga wangi selain daripada orkid Vanda. Kajian ini
bertujuan untuk mengkaji wangian Vanda Mimi Palmer yang merangkumi aspek-aspek
biokimia dan biologi molekul. Analisis bagi wangian yang dihasilkan oleh orkid ini
dijalankan pada setiap peringkat perkembangan bunga dan juga masa yang berbeza dalam
kitaran 24 jam sehari. Analisis dijalankan menggunakan alat kromatografi gas-
spectrometrik jisim (GC-MS). Analisis GC-MS tersebut menunjukkan wangian Vanda
Mimi Palmer didominasi oleh metabolit daripada tapak jalan terpenoid, dan juga,
benzenoid dan phenylpropanoid. Pengeluaran wangian Vanda Mimi Palmer juga didapati
dikawalatur mengikut peringkat perkembangan bunga dan juga peredaran masa. Dalam
aspek biologi molekul, transkrip cDNA berkaitan penghasilan wangian telah dikenalpasti
melalui penyaringan pembezaan perpustakaan cDNA bunga (floral cDNA library) Vanda
4
Mimi Palmer. Analysis „reverse-Northern‟ dijalankan dengan menghibridkan secara
berasingan klon-klon positif bersama dua „cDNA probe‟ yang berbeza mewakili transkrip
mRNA bagi peringkat kudup dan juga bunga kembang penuh. Klon-klon yang
menunjukkan ekspresi yang lebih tinggi bagi peringkat bunga kembang penuh
berbanding kudup dipilih untuk jujukan. Daripada keputusan analisis jujukan, 4-(cytidine
5′-diphospho)-2-C-methyl-D-erythritol kinase putatif (VMPCMEK), cytochrome P450
protein putatif (VMPCyP450), dan transkrip protein yang belum dikenalpasti (VMPA28)
dipilih untuk pencirian biologi molekul. Ketiga-tiga transkrip tersebut bersama transkrip
phenylacetaldehyde synthase putatif (VMPPAAS) yang dipencilkan daripada “Expressed
Sequence-Tags” (ESTs) bunga Vanda Mimi Palmer dipilih untuk pencirian termasuk
pemencilan cDNA lengkap dan juga analisis ekspresi menggunakan tindakbalas rantaian
polimerase masa nyata (RT-PCR). Analisis ekspresi tersebut dijalankan bagi tisu yang
berbeza, peringkat perkembangan bunga yang berbeza dan masa yang berbeza dalam
kitaran 24 jam sehari menggunakan RT-PCR. Transkrip tersebut menunjukkan ekspresi
yang tinggi pada tisu bunga berbanding tisu vegetatif, dan dikawalatur oleh peringkat
perkembangan bunga dan juga peredaran masa. Kesimpulannya, daripada hasil kajian
biokimia dan biologi molekul, dua tapak jalan biokimia telah dikenalpasti
berkemungkinan terlibat bagi penghasilan wangian dalam Vanda Mimi Palmer iaitu tapak
jalan terpenoid dan juga tapak jalan benzenoid dan phenylpropanoid.
5
ACKNOWLEDGEMENTS
I would like to express my utmost gratitude to my supervisor, Dr. Parameswari a/p
Namasivayam, for her patience, encouragement, time as well as precious advice and
guidance, leading me throughout this research project. My sincere appreciation is also
extended to my co-supervisors, Assoc. Prof. Dr. Janna Ong Abdullah and Prof. Dr.
Gwendoline Ee Cheng Lian for their guidance, support and technical advice.
Special thanks to Malaysia Toray Science Foundation (MTSF) for giving me a research
grant for the screening and isolation of putative fragrance-related cDNAs. Another
special thanks to Universiti Putra Malaysia for supporting my work on biochemical and
molecular characterization of the fragrance of Vanda Mimi Palmer through Research
University Grant Scheme (RUGS) and also for providing me my stipend for the last two
years through Graduate Research Fellowship (GRF). My deepest appreciation also goes
to Chemistry Department, Faculty of Science UPM for giving me permission to use GC-
MS for my biochemical analysis of the scent of Vanda Mimi Palmer and also to En.
Zainal Abidin Kasim for his help in my biochemical analysis with GC-MS.
I would like to thank all my lab mates in Molecular Biology Laboratory, Biotech 3, UPM
as well as all of the laboratory staff in the laboratory, for their technical guidance,
support, care, forgiveness, valuable ideas and experience. My deepest appreciation is
extended to family for their endless support, care and love, accompanying me through all
the happiness and sadness in my study.
6
CHAPTER 1
INTRODUCTION
Floral scent or floral fragrance is an important constituent for perfume and food
industries. The extracts from flowers including jasmine and rose have been used
extensively in fragrance and flavour industries. Besides that, there is always a high
demand for scented flowers from aromatherapy industries especially in Malaysia and
Thailand. In horticultural and agricultural industries, floral scent is very important for
pollination of crops. Floral scent studies have been well established in some scented
flowers including Clarkia breweri, Antirrhinum majus, Rosa hybrida and Petunia
hybrida in both biochemical and molecular aspects covering three fragrance biosynthetic
pathways that are terpenoid, lipoxygenase-catalyzed fatty acid derivatives, and also
benzenoid and phenylpropanoid pathways (Pichersky and Dudareva, 2007).
Orchids with fragrance have higher demand in the orchid industry and fetching higher
prices compared to non-fragrance orchids (Eric Kok, Manager, Malaysian Orchids Sdn.
Bhd., pers. comm. on 20th
May 2008). In orchid industry, extensive work has been
focused on hybridizing scented orchid with non-scented orchid in order to produce flower
with attractive colour appearances. Most of the progenies produced have diluted scent or
no scent at all. In orchids, floral scent identification started in the early 1990s (Kaiser,
1993) but the knowledge on fragrance biosynthetic pathways is still far from being
understood. More recently, a few fragrance-related cDNAs were identified from
expressed sequence-tags (ESTs) of Phalaenopsis bellina (Hsiao et al., 2006) and the only
7
cDNA that has been well characterized is geranyldiphosphate synthase that is involved in
the biosynthesis of geranyl diphosphate, a precursor for monoterpenes biosynthesis
(Hsiao et al., 2008).
Besides Phalaenopsis bellina there are a lot of fragrance orchids which are still not well
studied for their fragrance characteristics including Vanda Mimi Palmer. Vanda Mimi
Palmer is a well-known commercial orchid hybrid especially in Malaysia and Thailand.
This orchid produces a sweet smelling fragrance during day time in fully-open flower
stage (Janna et al., 2005). This orchid had won several awards for its sweet smelling
fragrance including the Champion Award for Fragrant Orchid organized by the Royal
Horticultural Society of Thailand in 1993 and the Best Orchid Fragrance in the 17th
World Orchid Conference in 2002 (Nair and Arditti, 2002). Thus, Vanda Mimi Palmer
with its fragrance emission characteristic was chosen for this study in order to understand
its fragrance biosynthetic pathways.
The knowledge on the sequences of fragrance-related cDNAs isolated from Vanda Mimi
Palmer can be used for transformation into non-scented orchids and other non-scented
flowers in order to increase the commercial value of the orchids and other ornamental
flowers. Besides that, understanding on the fragrance biosynthetic pathways of Vanda
Mimi Palmer will assist in the cloning of fragrance-related cDNAs into bacterial and
yeast expression vector for production of fragrance compounds in bulk. The knowledge
of the proportion of each compound in the fragrance of Vanda Mimi Palmer will facilitate
8
the production of custom-made perfume of the same smell as Vanda Mimi Palmer either
biologically or chemically synthesized.
The specific objectives for this study were:
1) to determine the constituents of the scent of Vanda Mimi Palmer in comparison to
its parents,
2) to isolate and characterize selected putative fragrance-related transcripts of Vanda
Mimi Palmer, and
3) to analyze the expression profile of the selected putative fragrance-related cDNAs
of Vanda Mimi Palmer
9
CHAPTER 2
LITERATURE REVIEW
2.1 Orchid – An Introduction
Orchids are classified under the Orchidaceae, one of the largest families of flowering
plants with an estimated population of 20,000 to 35,000 species (Dressler, 1993;
Mabberly, 1997). More than 800 orchid genera have been identified from the entire world
including Aranda, Aranthera, Cattleya, Dendrobium, Oncidium, Phalaenopsis,
Paphiopedilum and Vanda. In Malaysia, there are more than 120 genera and 2000 species
that have been discovered (Hamdan, 2008). An orchid flower consists of three sepals and
three petals. The petals and sepals are usually nearly alike where petals are located in the
first whorl while sepals in the second whorl of the flower. One of the petals is often
highly modified to form the lip or labellum, and is complicated in shape (Seidenfaden
and Wood, 1992).
The habitats of orchids vary such as mountainous forests, highlands, tropical mountain
forests and also lowlands (Fadelah et al., 2001). In nature, there are epiphytic orchids
which grow on branches and trunks of trees, terrestrial orchids which grow on soil and
lithophyte orchids which grow on rocks (Hamdan, 2008). The epiphytic orchids use
branches and trunks of trees only for support purpose without taking anything from the
trees. Living up on the trees helps the orchids to get away from competition with other
plants on the forest floor and escape from mineral contaminants on soil (Rittershausen
10
and Rittershausen, 2008). The source of nutrients for epiphyte and lithophyte are from
organic substances of dead leaves, mosses and insects meanwhile for terrestrial orchids,
the nutrients for growth are directly from the soil (Hamdan, 2008).
To date, more than 100,000 orchid hybrids have been established in the world either by
crossing between the same genera (interspecific hybrid) or by crossing with different
genera (intergeneric hybrid) (Hands, 2006). The first orchid hybrid in the world is
Calanthe Dominiyi produced in 1856, a cross of Calanthe masuca and Chalanthe furcata
(Sheela, 2008). The list of new orchid hybrids is now controlled by The Royal
Horticultural Society, England (RHS). Lately, a few hundreds orchid hybrids with
commercial values are established annually for orchid industry (Hamdan, 2008). In
Malaysia, the Vandaceous hybrids like Dendrobiums and Oncidiums are the most popular
cut-flower cultivated since they are easily grown and cultivated in Malaysia‟s climate
(Fadelah et al., 2001).
In the floriculture industry, the demand on orchid species and orchid hybrids is very high
from all over the world. The demand on orchids is high due to their aesthetic values
(exotic and limited sources) including colour, scent appearance and morphology. In
orchid industry, the price of scented orchids is often higher compared to scentless orchids
(Eric Kok, Manager, Malaysian Orchids Sdn. Bhd., pers. comm. on 20th
May 2008).
11
2.2 Vanda Mimi Palmer and Its Parents
Vanda Mimi Palmer is a cross between Vanda tessellata and Vanda Tan Chay Yan
(Motes, 1997) (Figure 1). The special characteristic of Vanda Mimi Palmer compared to
other orchid hybrids is the fragrance characteristic. Vanda Mimi Palmer has won a few
international awards for its strong sweet fragrance such as the Champion Award for
Fragrant Orchid organized by the Royal Horticultural Society of Thailand in 1993 and the
Best Orchid Fragrance in the 17th
World Orchid Conference in 2002 (Nair and Arditti,
2002).
Vanda Mimi Palmer could have inherited its fragrance and colour characteristics from
Vanda tessellata, an epiphytic orchid from Sri Lanka, India and Burma (Kaiser, 1993;
Motes, 1997). Vanda tessellata has inflorescences of 25cm to 30cm long and grey-green-
brown flowers. The lip is white at the side and violet purple in the middle. The shape of
Vanda Mimi Palmer‟s flower closely resembles Vanda Tan Chay Yan‟s which is a hybrid
of Dutch Vanda Josephine and Vanda dearei (Yeoh, 1978). Vanda Tan Chay Yan has
round and flat petals and sepals. This hybrid has won many awards such as First Class
certificate (the highest award of the Royal Horticultural Society) in 1954, the Trophy for
The Best Vanda at the Second World Conference in Hawaii and numerous Singapore and
Malayan Prizes. In 1960s, this hybrid lost its popularity as commercial cut orchid due to
its disability to flower more than twice a year (Yeoh, 1978).
12
Vanda tessellata Vanda Tan Chay Yan
Vanda Mimi Palmer
Figure 1: Vanda Mimi Palmer and Its Parents. Vanda Mimi Palmer is a hybrid of
Vanda tessellata (adapted from Hamdan, 2008) and Vanda Tan Chay Yan.
13
In Malaysia, the demand for Vanda Mimi Palmer is high due to the fragrance emitted by
its flower rather than its beautiful colour and structure (Eric Kok, Manager, Malaysian
Orchids Sdn. Bhd., pers. comm. on 20th May 2008). Floral extracts from Vanda tessellata
(one of the parents of Vanda Mimi Palmer) have been used in some local traditional
practices for medicinal purposes in India, such as treatment for inflammatory conditions
and instilled into the ear as remedy for otitis (Chopra et al., 1956). Besides that, the
extract from the leaves in the form of paste is applied to the human body for cooling
down a fever (Chopra et al., 1956; Basu et al., 1971). Root extract from Vanda tessellata
had also been used for rheumatism treatment, fever, dyspepsia, bronchitis and also
nervous problem (Kirtikar and Basu, 1975). A scientific study on the extracts of Vanda
tessellata has shown inflammatory property against acute inflammation induced by
carrageenan, serotonin and formaldehyde (Suresh Kumar et al., 2000). Besides that,
alcohol extract from Vanda tessellata has shown an enhancement of male sexual activity
in normal mice (Suresh Kumar et al., 2000). Thus, Vanda Mimi Palmer might have some
medicinal properties as Vanda tessellata since half of the gene pool of Vanda Mimi
Palmer was derived from Vanda tessellata.
2.3 The Biological Importance of Floral Scent
In general, floral buds do not have scent, and the fragrance characteristic of a flower
appears during anthesis as the petals open (Schade et al., 2001). Floral scent emission
patterns vary among species. Some flowering plants such as Citrus medica and
Odontoglossum constrictum emit scent primarily during day time meanwhile some other
14
plants such as Petunia hybrida and Clarkia breweri emit their scent at the highest level
during night time (Altenburger and Matile, 1988). Floral scent emission patterns are
different among species due to the control of cicardian clock, photoperiod and also
adaptation to specific pollinators‟ active time (Verdonk et al., 2003).
Floral scent is one of the factors that attract pollinators to help in pollination. The
pollinator varies among plant species including birds, insects and bats. Some flowering
plants need very specific pollinators for their pollination (Dobson, 1994) as they are
attracted to specific odors or scent emitted by flowering plants. For example, beetles are
attracted to flowers that have musty, spicy and fruity odors (Kaiser, 1993; Frowine, 2005)
while bees and flies are attracted and help in pollination of sweet scented flowers that can
be detected by human nose (Reinhard et al., 2004).
Besides pollination purpose, some plants emit volatiles such as monoterpenes,
sesquiterpenes and hormones such as salicylic acid, jasmonic acid and ethylene from
their vegetative tissues to defend themselves against pathogenic microorganisms and
insects‟ attack (Arimura et al., 2005; Wei et al., 2007). Volatiles produced by some green
leaves have been reported to reduce bioactivity and performance of herbivores and
sometimes have antifungal activity (Kaori et al., 2006).
15
2.4 The Economic Importance of Floral Scent
Floral scent or flower fragrance is very important in perfumery, cosmetic, agricultural
and cut flower industries. Flower fragrance produced by flowering plants such as
jasmine, roses, and lavender are pleasant to human sensory system and have potential
application as perfume ingredients (Rees, 1991). The high demand on floral fragrances
for perfumery and food industries has caused researchers to focus on fragrance-related
biochemical compounds and their biosyntheses (Knudsen et al., 1993). The knowledge on
natural floral fragrances and their specific components are used in perfume production to
produce synthetic perfumes and mimic the natural floral fragrance (Verdonk et al., 2003).
An example of the highly commercialized floral fragrances in perfumery industry are
rose (Rosa hybrida) (Zuker et al., 1998; Guterman et al., 2002) and jasmine (Jasminum
grandiflorum) (Kaiser, 1993).
In agricultural industry, pollination of crops is very important for fruit development. The
highest yield can be obtained whenever the highest pollination occurs in field with the
help of pollinators. Domesticated crops from other parts of the world might not be
suitable for local pollinators due to drastic changes of morphology and biochemistry of
the plants (Pichersky and Dudareva, 2007). Commercialization of the plants might also
be prevented by the lack of natural pollinators. Domestication of natural pollinators of the
plant into other territory is also not usually successful due to the lack of ability of the
pollinators to adapt to the new environment (Buchmann and Nabhan, 1996). Scent
16
engineering of local and new introduced plant species into new territory might enhance
pollination by local pollinators (Pichersky and Dudareva, 2007).
In cut-flower industry which is known as a multi-billion dollar industry, extensive work
on breeding of cultivated flowers to improve their vase life, shipping characteristics, and
visual aesthetic values such as shape and colour has contributed to the lost of their
original scent (Vainstein et al., 2001). Genetic engineering approach by transformation of
selected genes for the selected traits might restore the original scent in the plants. Besides
that, the production of scent in scentless flowering plants or modification of floral scent
can also be done by genetic engineering to increase the commercial values of the flower
in cut-flower industry (Pichersky and Dudareva, 2007).
2.5 Floral Scent and Its Volatile Compounds
The scent of scented flowers varies between species due to the combination of the
compositional and the level of each compound (Knudsen et al., 1993; Dudareva et al.,
2000). The entire floral organs are involved in floral scent emission but petal is the main
source of floral scent in most flowers (Pichersky et al., 1994). Floral scents are stored in
special oil glands such as trichome before released to the air as volatiles (Effmert et al.,
2006). Analysis on volatile compounds in floral scent by headspace with gas
chromatography-mass spectrometry (GC-MS) method has led to the discovery of more
than 1700 chemical structures (Knudsen and Gershenzon, 2006). Floral scent is a
complex mixture of low molecular mass molecules such as monoterpenes,
17
sesquiterpenes, benzenoids, phenylpropanoids and fatty acid derivatives (Knudsen et al.,
1993). Besides that, there are also other compounds in floral scents such as nitrogen and
sulfur containing compounds including indole, a compound from amino acid metabolism
(Knudsen and Gershenzon, 2006).
In floral scent studies, more than 500 terpenoid compounds have been identified
including monoterpene (C10), sesquiterpene (C15), diterpenes (C20) and irregular terpenes
(Knudsen and Gershenzon, 2006). Examples of monoterpenoid compounds identified in
floral scent studies are linalool, ocimene, mycrene, nerol, citranellol and geraniol.
Linalool compound has been identified in floral scent of snapdragon (Antirrhinum majus)
(Nagegowda et. al, 2008), Clarkia breweri (Raguso and Pichersky, 1995), Arabidopsis
thaliana (Chen et al., 2003) and a lot of orchid species such as Dendobium beckleri,
Dendobium brymerianum, and Phalaenopsis violacea (Kaiser, 1993). Other
monoterpenoid compounds such as mycrene and ocimene were detected in the scent of
Anthirrinum majus (Dudareva et al., 2003), Nicotiana suaveolens (tobacco) (Raguso et
al., 2003), Arabidopsis thaliana (Chen et al., 2003) and also in some orchids such as
Platanthera chlorantha, Polystachya cultriformis and Zygopetalum crinitum (Kaiser,
1993).
Besides monoterpenoid compounds, sesquiterpenoids such as germacrene D, farnesene,
caryophyllene, copaene and nerolidol were also detected in floral scent of many plant
species. Germacrene D has been detected in the scent of Rosa hybrida (Hendel-
Rahmanim et al., 2007), Petunia hybrida (Verdonk et. al, 2003), and some orchids such
18
as Aerangis confusa, Aerangis biloba, and Dendrochilum cobbianum (Kaiser, 1993).
Caryophyllene, another volatile sesquiterpene compound, was also detected in many
scented flowers including Arabidopsis thaliana (Chen et al., 2003), carnation (Dianthus
caryophyllus) (Schade et al., 2001), and some orchids such as, Cattleya lawrenceana,
Cattleya percivaliana, Dendrobium trigonopus and Dendrochilum cobbianum (Kaiser,
1993). Besides germacrene D and caryophyllene, nerolidol is another compound of
sesquiterpene found in scented flowers including Antirrhinum majus (Nagegowda et. al,
2008), Nicotiana suaveolens (tobacco) (Raguso et al., 2003) and also in some orchids
such as Diaphananthe pulchella, Epidendrum ciliare, Masdevallia estradea and
Zygopetalum crinitum (Kaiser, 1993).
The other class of volatile compounds that is highly distributed among scented flowers is
benzenoids and phenylpropanoids. So far, more than 300 volatile compounds of this class
have been identified in floral scent of plant species which include methylbenzoate,
methylsalicylate, phenylacetaldehyde, phenylethyl acetate, benzyl acetate, phenylethanol,
eugenol and isoeugenol (Knudsen and Gershenzon, 2006). Methylbenzoate has been
detected in some scented flowers such as Petunia hybrida (Verdonk et. al, 2003),
Antirrhinum majus (Nagegowda et. al, 2008), Stephanotis floribunda (Pott et al., 2002)
and also in some orchids such as Dendrobium trigonopus, Encyclia baculus and Laelia
perinii (Kaiser, 1993). Benzyl benzoate has been identified to be emitted by floral organs
of some scented flowers including Petunia hybrida (Verdonk et. al, 2003), Nicotiana
suaveolens (Raguso et al., 2003), Stephanotis floribunda (Pott et al., 2002) and also in
some orchids such as Dendrobium moniliforme, Dendobium monophyllum, Dendrobium
19
williamsonii, and Dendrochilum cobbianum (Kaiser, 1993). Eugenol and isoeugenol are
compounds of benzenoid and phenylpropanoid classes which also contribute to the floral
scent of scented flowers including Petunia hybrida (Verdonk et. al, 2003), Clarkia
breweri (Raguso and Pichersky, 1995), Stephanotis floribunda (Pott et al., 2002) and also
some orchids such as Angraecum bosseri, Himantoglossum hircinum, Lycaste aromatica,
Phalaenopsis violancea, and Platanthera bifolia (Kaiser, 1993).
Other important class of floral scent compounds is fatty acid derivatives which are
derived from lipoxygenase pathway such as hexanol, hexanal, nonanal, pentadecane,
decanal and dodecanal (Knudsen and Gershenzon, 2006). Volatile of fatty acid
derivatives are normally detected in vegetative tissues and play an important role in plant
defense. Traces of fatty acid derived compounds have been detected in floral scent of
some scented flowers (Knudsen and Gershenzon, 2006). In the floral scent of Petunia
hybrida, some of the fatty acid derivatives that were detected included decanal,
dodecanal, 3-hexenal and 2-hexenal (Verdonk et al., 2003). Besides that, some orchids
were also reported to emit fatty acid derivatives such as octanal, 2-heptanol, nonanal,
decanal, methyl decanoate and ethyl decanoate (Kaiser, 1993; Hsiao et al., 2006).
2.6 The Fragrance Biosynthetic Pathway and Molecular Biology of Floral Scent
To date, many volatile compounds have been identified but only few enzymes and genes
involved in the fragrance biosynthetic pathways have been characterized. Therefore, the
mechanisms of fragrance formation are still not fully understood (Dudareva et al., 2000).
20
Most of the volatile compound analyses have been done by using gas chromatography-
mass spectrometry (Guterman, 2002). For many years, research on floral scent was
focused on its chemical rather than biological aspects due to the complexity of the studies
involved in volatile emission (Vainstein et al., 2001). There are three major biosynthetic
pathways involved in floral scent production which are terpenoid, benzenoid/
phenylpropanoid and lipoxygenase pathways (Croteau and Karp, 1991). Common
modifications such as hydroxylation, acetylation and methylation have been described
even though the pathways leading to the final products have not been fully characterized
(Guterman et al., 2002).
Terpenoid compounds are synthesized via the terpenoid pathway (Figure 2) which is
localized in both the plastid and cytosol (McCaskill and Croteau, 1995; Lichtenthaler,
1999; Rohmer, 1999). There are two initial pathways in the terpenoid pathway;
Methylerythritol Phosphate (MEP) Pathway in the plastid (Lichtenthaler, 1999; Rohmer,
1999) and Mevalonic Acid (MVA) Pathway in the cytosol (McCaskill and Croteau,
1995). Both pathways play an important role in the production of dimethylallyl
diphosphate (DMAPP) and its isomer isopentenyl diphosphate (IPP) (McCaskill and
Croteau, 1995; Lichtenthaler, 1999; Rohmer, 1999). Condensation of one DMAPP
molecule with one IPP molecule generates the production of geranyl diphosphate (GPP)
which is the main precursor for monoterpenes such as linalool, ocimene, and mycrene
(Ogura and Koyama, 1998; Poulter and Rilling, 1981) meanwhile condensation of a
DMAPP with two IPP molecules generates the production of farnesyl diphosphate (FPP)
which is the main precursor for sesquiterpenes such as caryophyllene, germacrene D and
21
Figure 2: Terpenoid Biosynthetic Pathway in Plastid and Cytosol. This diagram was
adopted from Nagegowda et al., 2008.
Abbreviations: DMAPP, dimethylallyl diphosphate; GA-3P, glyceraldehydes-3-
phosphate; DXP, 1-deoxy-D-xylulose-5-phosphate; DXR, DXP reductoisomerase; MEP,
2-C-methyl-D-erythritol-4-phosphate; DXS, DXP synthase; FPP, farnesyl diphosphate;
FPPS, farnesyl diphosphate synthase; GPP, geranyl diphosphate; GPPS, geranyl
diphosphate synthase; GGPP, geranylgeranyl diphosphate; GGPPS, geranylgeranyl
diphosphate synthase; HMG-CoA, 3-hydroxy-3-methylglutaryl-CoA; HMGR, 3-
hydroxy-3-methylglutaryl-CoA reductase; IPP, isopentenyl diphosphate; MVA,
mevalonic acid.
22
nerolidol (McGarvey and Croteau, 1995). At present, several fragrance-related genes,
cDNAs and enzymes responsible for the production of terpenoid have been isolated and
characterized from several plants such as linalool synthase from Clarkia breweri
(Pichersky et. al, 1995), Arabidopsis thaliana (Chen et. al, 2003), Antirrhinum majus
(Nagegowda et. al, 2008), ocimene synthase from Antirrhinum majus (Dudareva et. al,
2003), mycrene synthase from Antirrhinum majus (Dudareva et. al, 2003) and
germacrene D synthase from Rosa hybrida (Guterman et al., 2002).
Benzenoid and phenylpropanoid pathway (Figure 3) is another fragrance biosynthetic
pathway identified in the plant system besides the terpenoid pathway. Benzenoids and
phenylpropanoids are derived from phenylalanine as the main precursor (Gang et al.,
2001). The pathway starts with the deamination of L-phenylalanine to trans-cinnamic
acid catalyzed by L-phenylalanine ammonia lyase (PAL), followed by shortening of the
C2 unit of the side chain of cinnamic acid for formation of benzaldehyde compound via a
few proposed pathways such as CoA-dependent ß-oxidative, CoA-independent-non-ß-
oxidative pathway or a combination of these two pathways (Boatright et al., 2004). The
benzenoid pathway is proceeded with the oxidation of benzaldehyde to benzoic acid
which is the main precursor for the formation of volatile benzenoids and
phenylpropanoids such as benzylbenzoate, benzylacetate, methybenzoate, and
phenylethyl acetate (Boatright et al., 2004). There are other phenylpropanoids emitted in
scented plants such as eugenol, isoeugenol, and methyleugenol which are synthesized via
other routes without going through benzoic acid as an intermediate. Coniferyl alcohol and
coniferyl acetate play a role in this other routes as intermediates with cinnamic acid as the
23
Figure 3: Benzenoid and Phenylpropanoid Biosynthetic Pathway. This chart was
adopted from Pichersky and Dudareva, 2007. It represents a compilation of reactions and
enzymes in several scented plants including Clarkia breweri and Petunia hybrida.
Abbreviations: BEAT, acetyl-coenzyme A:benzylalcohol acetyltransferase; BPBT,
benzoyl-CoA:benzylalcohol/2-phenylethanol benzoyltransferase; BSMT, benzoic
acid/salicylic acid carboxylmethyltransferase; BZL, benzoate:CoA ligase; CFAT,
coniferyl alcohol acyltransferase; EGS, eugenol synthase; IEMT, S-adenosyl-L-
methionine:(iso)eugenol O-methyltransferase; IGS, isoeugenol synthase; PAAS,
phenylacetaldehyde synthase; PAL, phenylalanine ammonia-lyase; SAMT, salicylic acid
carboxyl methyltransferase.
24
main precursor for the production of these compounds (Boatright et al., 2004: Pichersky
and Dudareva, 2007).
There are other volatile phenylpropanoid compounds derived from phenylalanine without
going through cinnamic acid such as phenylacetaldehyde and phenylethanol (Boatright
et. al, 2004). The pathway starts with the transamination of phenylalanine to
phenylpyruvate followed by decarboxylation to phenylacetaldehyde (Vuralhan et al.,
2003; Boatright et al., 2004). Reduction of phenylacetaldehyde produces phenylethanol
while oxidation of phenylacetaldehyde will lead to the production of an ester
phenylacetate (Erlich 1907; Vuralhan et al., 2003). Phenylacetaldehyde synthase (PAAS)
plays an important role in the decarboxylation of phenylalanine to phenylacetaldehyde
(Boatright et al., 2004; Kaminaga et al., 2006). PAAS has been reported to be a cytosolic
homotetradimeric enzyme that belongs to group II pyridoxal 5‟-phosphate-dependent
amino acid decarboxylase (Sandmeier et al., 1994). In floral scent studies, two PAAS
have been identified from Petunia hybrida (PhPAAS) and Rosa hybrida (RhPAAS) that
share 64% identity (Kaminaga et al., 2006). PhPAAS and RhPAAS were reported to
share ~50-60% identity with other plant decarboxylases such as tyrosine decarboxylases,
tryptophan decarboxylases and aromatic amino acid decarboxylases (Kaminaga et al.,
2006).
The third pathway related to fragrance biosynthesis is lipoxygenase pathway. The
products of this pathway are derived from C18 fatty acids (linoleic and linoleic acids)
which are cleaved into C6 and C12 components by hydroperoxide lyase (Feussner and
25
Wasternack, 1998). Hydroperoxide lyase produces either 3-cis hexenal or hexanal which
are the common constituents of volatiles in green leaf or flower depending on the C18
substrate (Knudsen et al., 1993). This 3-cis hexenal or hexanal can be further converted
to alcohols (3-cis-hexenol or hexanol) or 3-hexenyl acetate (D‟Auria et al., 2002).
2.7 Floral Scent Studies on Orchids
Floral scent studies on orchids was initiated in early 1980s. However, early work was
focused on detection of volatile compounds emitted by orchid flowers using gas
chromatography-mass spectrometry (GC-MS) method (Kaiser, 1993). Identification of
floral scent compounds in orchids has led to extensive biochemical and molecular studies
of other scented plants such as Clarkia breweri and Petunia hybrida. However, the floral
scent biosynthetic pathways of orchids are still far from understood (Hsiao et al., 2006).
Identification of volatile compounds has been carried out on the scent of more than 180
orchid species and hybrids including Cattleya araguaiensis, Cymbidium formosanum,
Dendrobium carniferum, Dendrobium superbum, Oncidium curcutum, Phalaenopsis
violacea, Vanda tessellata (Kaiser, 1993) and Phalaenopsis bellina (Hsiao et al., 2006)
by GC-MS. Based on the GC-MS analysis on the orchids‟ scent, monoterpenoids and
sesquiterpenoids are the highly distributed compounds among orchid species. Some of
those include linalool, mycrene, ocimene, germacrene D and nerolidol. Benzenoids and
phenylpropanoid compounds that are also found in scented orchids include
methylbenzoate, benzyl benzoate, benzyl acetate, phenylethyl acetate, eugenol and
26
isoeugenol. Besides that, there were traces of other compounds detected in scented
orchids such as fatty acid derivatives, indole and formanilide (Kaiser, 1993; Hsiao et al.,
2006).
In Vanda tessellata (one of the parents of Vanda Mimi Palmer), more than 20 volatile
compounds have been identified in its scent including linalool, mycrene, ocimene, methyl
benzoate, methyl isobutyrate, cinnamic aldehyde, cinnamic alcohol, methyl cinnamate,
benzyl acetate, phenylethyl acetate and indole. Methylbenzoate was the highest volatile
compound emitted by Vanda tesellata representing 61.5% of the total scent, followed by
linalool (23%), cinnamic aldehyde (5.1%) and methyl cinnamate (4.6%). Other minor
compounds identified in the scent of Vanda tessellata were methyl isobutyrate, methyl 2-
methylbutyrate, α-pinene, mycrene, ocimene, benzyl acetate, methyl salicylate, 3-
phenylpropanoid, cinnamic aldehyde, α-ionone, cinnamic alcohol and indole (Kaiser,
1993).
On the molecular biology aspect, only recently a group of researchers from Taiwan have
reported expressed sequence-tags (ESTs) on a scented orchid species Phalaenopsis
bellina (Hsiao et al., 2006). Isolation and identification of putative fragrance-related
cDNAs was achieved by comparing floral ESTs sequences of Phalaenopsis bellina with
ESTs of a non-scented orchid, Phalaenopsis equestris. From their work, the
monoterpenes biosynthetic pathway of linalool, mycrene and geraniol was elucidated
using the bioinformatics approach, Pathway and Literature (PAL) finder program. From
the ESTs of Phalaenopsis bellina, several fragrance-related cDNAs that encode for
27
geranyl diphosphate synthase, epimerase, lipoxygenase, diacylglycerol kinase, O-
methytransferase, and cytochrome P450 monooxygenase were isolated. The only
fragrance-related cDNA which has been well characterized in orchids is geranyl
diphosphate synthase (PbGDPS) from Phalaenopsis bellina (Hsiao et al., 2008). Real-
time RT-PCR analysis has shown that the expression of PbGDPS gene increased
gradually once the bud open and reached the highest peak on the fifth-day after bud-
opening. After that, the expression decreased gradually until the end of the flower‟s life.
The same expression pattern was reported on the emission of monoterpene compounds
such as linalool and geraniol. Protein characterization has shown the PbGDPS enzyme is
a homodimeric enzyme which can catalyze the formation of both geranyl diphosphate
(GDP) and farnesyl diphosphate (FPP) which are the main precursors for the production
of monoterpenoids and sesquiterpenoids, respectively.
2.8 Floral Scent Studies in Vanda Mimi Palmer
Preliminary work on scent analysis of Vanda Mimi Palmer was carried out by nose
detection during day time from 8.00am to 5.00pm and at different floral developmental
stages (Janna et al., 2005). The study shows that emission was at the highest level
between 12-2pm when the flower was fully-open. Subsequent molecular biology work on
Vanda Mimi Palmer was focused on the construction of a floral cDNA library of Vanda
Mimi Palmer representing almost all mRNA transcripts of fully-open flower at different
time points in a 24-hour cycle and at different flower developmental stages such as early
bud (green), mature bud (red), half-open flower and fully-open flower (Chan, 2009; Chan
28
et al., 2009).Two fragrance-related cDNAs were isolated from a preliminary sequencing
of 100 clones from the floral cDNA library. The transcripts are 1-deoxy-D-xylulose 5-
phosphate reductoisomerase (accession number: EU145744) (Chan et al., 2009) and
lipoxygenase (Chan, 2009). Besides that, a suppression subtraction hybridization (SHH)
was also carried out to isolate more fragrance-related cDNAs by hybridizing the
substracted open flower cDNA library with two different cDNA probes of open flower
and bud stage during day time separately. From the SSH work, another two fragrance-
related cDNAs were successfully isolated which are sesquiterpene synthase (accession
number: EU145743) and alcohol acyltransferase (accession number: EU145742) (Chan,
2009).
Molecular characterization has been carried out on the 1-deoxy-D-xylulose 5-phosphate
reductoisomerase, by subjecting it to a full-length cDNA isolation and gene expression
analysis by real-time RT-PCR. The expression studies were carried out in different
tissues, at different floral developmental stages and at different time points in a 24-hour
cycle. Besides that, other fragrance-related cDNAs such as sesquiterpene synthase and
alcohol acyltransferase were also characterized in the same manner (Chan, 2009).
Characterization of the fragrance-related transcripts showed upregulated expression in
floral tissues especially in the petal and sepal compared to vegetative tissues such as leaf,
shoot and root. Expression analyses of the fragrance-related transcripts at different
developmental stages and different time points in a 24-hour cycle has shown that
fragrance biosynthesis in Vanda Mimi Palmer is developmentally and rhythmically
regulated (Chan, 2009; Chan et al., 2009).
29
CHAPTER 3
MATERIALS AND METHODS
3.1 Plant Material
Orchid plants (Vanda Mimi Palmer and Vanda Tan Chay Yan) used for this study were
purchased from the United Malaysian Orchids Sdn. Bhd., a nursery located in Rawang,
Selangor. The purchased plants were maintained in the nursery by Mr. Eric Kok. Both
Vanda Mimi Palmer and Vanda Tan Chay Yan used in this study were grown separately
in pots with charcoal under tropical climate (12 hours in light followed by 12 hours in
dark), temperature between 25-300C and exposed to 70-80% of sunlight. Samples for
volatile analysis such as flowers and buds were directly captured from the orchid plants
without detaching the flowers. For essential oil extraction, the flowers were directly
processed after being detached from the mother plants without freezing in liquid nitrogen.
Meanwhile, samples for RNA work such as flowers, buds, leaves, shoots and roots were
detached from the mother plant, frozen in liquid nitrogen and stored in -800C before use.
All of the plants used for volatile analyses, essential oil extraction as well as RNA
extraction were brought to Universiti Putra Malaysia and placed outside of laboratory
building with almost similar condition to the nursery at Rawang (not directly exposed to
the sun) to ensure growth as well as scent biosynthesis and emission are similar to when
the plants are grown in the nursery.
30
3.2 Analysis of the Scent of Vanda Mimi Palmer by GC-MS
Determination of the constituents of the scent of Vanda Mimi Palmer was carried out by
gas chromatography-mass spectrometry (GC-MS) to identify the volatile compounds
emitted by Vanda Mimi Palmer at different developmental stages and also at different
time points in a 24-hour cycle. Emission analysis of the floral scent of Vanda Mimi
Palmer was carried out by GC-MS at three different floral developmental stages; bud,
half-open flower and fully-open flower. Temporal emission analysis was carried out by
GC-MS for every two hours interval (12am, 2am, 4am, 6am, 8am, 12pm, 2pm, 4pm,
6pm, 8pm and 10pm) in a 24-hour cycle to determine the emission pattern of each single
compound. Besides that, GC-MS analysis on open flower of Vanda Tan Chay Yan was
carried out in order to compare with the volatiles emitted by Vanda Mimi Palmer. All of
the volatile emission analyses were carried out in three replicates using flowers from
different mother plants. Average of the three replicates of the volatile analysis in a 24-
hour cycle was used to plot graphs with the error bar showing the standard error for the
three replicates.
Volatiles emitted by single flower were captured by Solid Phase Micro-Extraction
(SPME) (Supelco, USA). The SPME with silica fiber that was coated with 100µm
polydimethylsiloxane (PDMS) was used to absorb the volatiles emitted by the flowers of
Vanda Mimi Palmer and Vanda Tan Chay Yan. A single flower was put into a modified
funnel without detaching the flower from the flower stalk (Figure 4). The back of the
funnel was covered with aluminum foil. The SPME holder was pressed to allow the silica
31
Figure 4: Solid Phase Micro Extraction (SPME) Used to Capture Volatile
Compounds Emitted by the Flower of Vanda Mimi Palmer. The flower was trapped
and captured by the SPME for 15 minutes.
Funnel
Silica fiber of SPME
SPME holder
Flower
Aluminium foil
Retort stand
32
fiber in the SPME emerge from the SPME syringe and captured volatiles produced by the
flower for 15 minutes. After that, The SPME fiber was thermally desorbed for 1 minute
at 2500C in an injector port of Shimadzu GC-MS (Shimadzu, Japan) with the port in
splitless injector mode. Volatile compounds were separated by using a capillary HP-5
column (50m x 0.32mm, film thickness 1.05µm) with helium (21 kPa) as a carrier gas.
The GC oven was programmed 450C for 1 minute followed by an increase of 10
0C per
minute to 2800C. The temperature 280
0C was extended for 10 minutes. Mass spectra of
the eluted compounds were recorded for the m/z value of 30-300. The spectrum given by
each compound was compared to the National Institute of Standards and Technology
(NIST) spectral library 2002 (Scientific Instrument Services, USA).
3.3 Extraction and Analysis of Essential Oil of Vanda Mimi Palmer
Essential oil was extracted from Vanda Mimi Palmer by soaking 100 grams of open
flower of Vanda Mimi Palmer in 800ml hexane for 48 hours. After soaking, the debris
was removed from hexane with essential oil by filtering with 1MM Whatman filter paper
(GE healthcare, USA). The essential oil was recovered by evaporating in a 250 ml round
bottom flask with a neck size of 24/29 using a rotary evaporator machine with water bath
set at 450C. This step was repeated until all of the hexane was evaporated from the
essential oil. The weight of the round bottom flask was recorded before and after the
evaporation process by the evaporator machine. After that, the essential oil in paste form
was dissolved in 5ml acetone and then adjusted to 10 part per million (ppm). A volume of
1.5µl of the 10ppm essential oil was analyzed by GC-MS using the same protocol as
33
described in section 3.2. The same approach was applied to the flowers of Vanda Tan
Chay Yan.
3.4 Isolation of Total RNA
The RNA extraction procedure was based on Yu and Goh method (2000) with minor
modifications (Chan et al., 2009). One gram samples of Vanda Mimi Palmer from
different tissues/organs (petal, sepal, leaf, root and shoot), at different floral
developmental stages (young bud (green), mature bud (red), half-open flower, fully-open
flower, and 14-day old flower) and at different time points in a 24 hour cycle (12am,
2am, 4am, 6am, 8am, 12pm, 2pm, 4pm, 6pm, 8pm and 10pm) were ground saperately
into fine powder in liquid nitrogen. The ground samples were mixed thoroughly with
20ml of pre-warmed (650C) extraction buffer [100mM Tris-Cl (pH 7.5) containing 20mM
EDTA (pH 8), 2M NaCl, 2% (w/v) hexadecyl (cetyl) trimetyl ammonium bromide
(CTAB), 1% (w/v) polyvinylpyrrilidone (PVP), and 2% (v/v) β-mercaptoethanol]. The
mixture was then incubated at 650C for 15 minutes followed by centrifugation at 12,857 x
g, 40C for 15 minutes to pellet the cellular debris. After the centrifugation, recovered
supernatant was mixed thoroughly with an equal volume of chloroform:iso-amylalcohol
(24:1). The mixture was shaken vigorously followed by centrifugation at 12,857 x g, 40C
for 15 minutes. After the centrifugation, chloroform:iso-amylalcohol (24:1) extraction
was carried out twice to recover the top aqueous phase. Lithium chloride (LiCl) was then
added to the recovered top aqueous phase to a final concentration of 2M followed by
incubation at 40C overnight. Recovered RNA was then pelleted by centrifugation at
34
12,857 x g, 40C for 30 minutes. The RNA pellet was rinsed in cold 80% (v/v) ethanol and
air-dried before dissolving in 100µl diethyl pyrocarbonate (DEPC)-treated water. The
RNA samples were then stored at -200C until further use but no longer than two months.
The quantity and quality of the total RNA were determined by spectrophotometric
readings at 230nm, 260nm, and 280nm using the Biophotometer (Eppendorf, Germany)
and nanospectrophotometer (Implen, Germany). The integrity of RNA was checked by
formaldehyde denaturing agarose gel electrophoresis (see section 3.4.1).
3.4.1 Formaldehyde Denaturing Agarose Gel Electrophoresis
Formaldehyde denaturing agarose gel of 1.2% (w/v) was prepared by melting 0.4 gram
agarose powder in 30ml of 1X F buffer [20mM MOPS buffer (pH 7) containing 1mM
EDTA and 5mM NaOAc]. The melted agarose was allowed to cool to 500C and
formaldehyde was added to a final concentration of 6% (v/v). The formaldehyde agarose
gel was then poured into a gel cast and allowed to solidify. While waiting for the gel to
be solidified, 1µg of total RNA was added into sample buffer [1X F buffer containing
50% (v/v) formamide and 6% formaldehyde (v/v)] followed by addition of a final
concentration of 0.24X loading dye [0.25%(w/v) bromophenol blue, 0.25% xylene
cyanol, 30% (v/v) glycerol] and 1µg of ethidium bromide. The sample was then
incubated at 650C for 10 minutes and placed on ice immediately before loading into the
gel. Gel electrophoresis was carried out in a formaldehyde running buffer [1X F buffer
containing 6% (v/v) formaldehyde] at 45 Volts for 1 hour. The agarose gel was destained
35
for 30 minutes in autoclaved DEPC-treated water and viewed using a gel documentation
system (Bio-Rad, USA).
3.5 Isolation of PolyA+
mRNA
The PolyATract® mRNA Isolation Systems III (Promega, USA) was used to isolate the
polyA+ mRNA from total RNA. One miligram of total RNA was made up to a final
volume of 500µl by addition of nuclease-free water provided in the kit. The RNA sample
was then incubated at 650C for 10 minutes. Annealing reaction was then carried out by
adding 150pmol biotinylated oligo(dT) probe and Sodium Saline Citrate (SSC) buffer,
pH 7 to a final concentration of 0.5X (diluted from 20X SSC provided in the kit) to the
RNA solution for the oligomers to anneal with polyA+ mRNA. The mixture was then
mixed gently and allowed to completely cool at room temperature.
While waiting for the RNA sample to completely cool, streptavidin-paramagnetic
particles (SA-PMPs) (provided in the kit) were washed by gently flicking the bottom of
the SA-PMPs‟ tube until the particles were completely dispersed. The SA-PMPs were
then captured by placing the tube in a specific magnetic stand (provided with the kit) for
about 30 seconds until the SA-PMPs were completely accumulated on the wall of the
tube. Recovered supernatant was carefully removed and the SA-PMPs particles were
washed three times with 300µl of 0.5X SSC buffer (diluted from 20X SSC provided in
the kit), each time being captured by magnetic stand as described above. The washed SA-
PMPs particles were finally resuspended in 100µl of 0.5X SSC buffer.
36
The total RNA from the annealing reaction was then added to the washed SA-PMPs. The
mixture was then incubated at room temperature for 10 minutes and mixed gently for
every 2 minutes. The SA-PMPs particles were then captured again as described above
and the recovered supernatant was carefully removed without disturbing the SA-PMPs
particles. The SA-PMPs particles were washed four times with 300µl of 0.1X SSC buffer
(diluted from 20X SSC provided in the kit). Each washing was carried out by gently
flicking the bottom of the tube until the SA-PMPs particles were completely dispersed.
The final supernatant was removed as much as possible without disturbing the SA-PMPs
particles.
These steps were followed by elution of polyA+
mRNA from the recovered SA-PMPs
particles. The SA-PMPs particles were resuspended by gently flicking in 100µl of RNAse
free water. The SA-PMPs particles were then magnetically captured and the eluted
mRNA was transferred into a sterile RNAse free microcentrifuge tube. The elution step
was repeated by resuspending the SA-PMPs pellet in 200µl of RNAse free water. The
recovered polyA+
mRNA was precipitated by addition of a final concentration of 0.3M
sodium acetate (NaOac) pH 5.2 and 80% (v/v) isopropanol. The precipitation was carried
out at -200C overnight. After the overnight incubation, the mixture was centrifuged at
12,857g at 40C for 20 minutes. Recovered mRNA pellet was washed with 75% (v/v)
ethanol and air-dried. The mRNA pellet was then dissolved in 20µl RNAse-free water
and stored in -200C for subsequent use. The quantity and purity of the eluted polyA
+
mRNA was determined by spectrophotometric readings using a Biophotometer
(Eppendorf, Germany).
37
3.6 Double-stranded cDNA Synthesis
Universal Riboclone®
cDNA synthesis system (Promega, USA) was used for double-
stranded cDNA synthesis from mRNA isolated in section 3.5. There are two major parts
involved in the cDNA synthesis which are first-strand cDNA synthesis and second-strand
cDNA synthesis. First-strand cDNA synthesis was carried out by addition of 1µg of oligo
(dT) into 2µg of mRNA sample. Nuclease-free water (provided in the kit) was added up
to a total volume of 15µl. The mixture was then incubated at 700C for 10 minutes and
placed on ice immediately followed by addition of 40 units of RNAsin® and 5µl of 5X
first-strand buffer (provided in the kit) into the mixture. The mixture was heated at 420C
for 5 minutes followed by addition of 30 units of AMV reverse transcriptase and sodium
pyrophosphate to a final concentration of 2mM. The mixture was then incubated at 420C
for 60 minutes. The first-strand cDNA synthesized was then used as template for the
synthesis of second-strand cDNA.
Second-strand cDNA synthesis was carried out by adding 40µl of 2.5X second-strand
buffer (provided in the kit), 5µg of acetylated Bovine Serum Albumin (BSA), 25 units of
DNA polymerase I, and 0.8 units of RNAse H into the synthesized first strand-cDNA.
Nuclease-free water was then added up to a total volume of 100µl followed by incubation
at 140C for two hours. The double-stranded cDNA synthesized was then heated at 70
0C
for 10 minutes to stop the cDNA synthesis. The double-stranded cDNA was then placed
on ice and 0.2 unit of T4 DNA polymerase was added into the mixture followed by
incubation at 370C for 10 minutes. After the incubation, EDTA with a final concentration
38
of 20mM was added to stop the reaction. Phenol-chloroform extraction was then carried
out by addition of equal volume of phenol:chloroform:isoamyl-alcohol (25:24:1) into the
synthesized double-stranded cDNA. The mixture was then vortexed briefly, followed by
a centrifugation at 15,871 x g at room temperature for 1 minute. After the centrifugation,
recovered aqueous phase was precipitated by adding sodium acetate to a final
concentration of 0.3M (pH5.2) and two volumes of cold absolute ethanol (pre-chilled at
40C). The precipitation was carried out at -80
0C for 30 minutes. Centrifugation at 15,871
x g for 15 minutes was then carried out at 40C to pellet the pure double-stranded cDNA.
After centrifugation, the supernatant was discarded and the recovered double-stranded
cDNA in pellet form was rinsed with 70% (v/v) ethanol. The pellet was then air dried,
dissolved in 20µl of Tris-EDTA (TE) buffer pH 8 and then stored at -200C until further
use.
3.6.1 Quantification of Double-stranded cDNA
The quantity of the double-stranded cDNA synthesized was determined by ethidium
bromide plate assay. A volume of 30ml of 0.8% (w/v) agarose gel was prepared in 1X
Tris-acetate-EDTA (TAE) buffer, pH 8. The agarose was melted and allowed to cool to
~500C. Ethidium bromide to a final concentration of 0.1µg/ml was added into the molten
agarose. The agarose mixture was mixed well by swirling and then poured into a 100 mm
Petri dish. A few columns and lanes were plotted at the back of the Petri dish before the
agarose solution containing ethidium bromide was poured into it. The agarose solution
was allowed to solidify.
39
Standard DNA (Lambda DNA) (Fermentas, Canada) used in the ethidium bromide assay
were diluted in nuclease-free water. The concentrations of standard used for the ethidium
bromide plate assay were 200ng/µl, 150ng/µl, 100ng/µl, 75ng/µl, 50ng/µl, 25ng/µl,
10ng/µl and 5ng/µl. The standards and the synthesized double-stranded cDNA in section
3.6 were spotted onto the solidified ethidium bromide plate. Both standards and cDNA
spotted on the plate were allowed to be absorbed by the agarose placed in a fume hood
for about 20 minutes. The ethidium bromide assay was viewed using a gel documentation
system (Bio-Rad, USA). The concentration of the cDNA sample was determined by
comparing the signal intensity produced by the cDNA with the standard.
3.7 cDNA Library Screening
A floral cDNA library of Vanda Mimi Palmer previously constructed by Miss Chan Wai
Sun (Department of Microbiology, Faculty of Biotechnology and Biomolecular Sciences,
Universiti Putra Malaysia) was used for this study. The cDNA library was constructed by
using the Zap-cDNA® Gigapack
® III Gold Cloning Kit (Stratagene, USA). The cDNA
library represents all mRNAs expressed in Vanda Mimi Palmer‟s flower at all different
flowering stages in a 24-hour cycle. The titer of the cDNA library provided was 7.8 x 109
pfu/ml. In this study, the floral cDNA library was hybridized with fully-open flower
cDNA probe of Vanda Mimi Palmer containing pool of mRNA transcripts expressed by
fully-open flower during daylight hours (8am, 10am, 12pm, 2pm, 4pm and 6pm). The
clones that gave positive signals were selected as putative fragrance-related cDNA
candidates for further characterization.
40
3.7.1 Probe Labeling
The double-stranded cDNAs synthesized in section 3.6 was used as templates for probe
preparation. The double-stranded cDNA was labelled with biotin by using the NEBlot®
Phototape® Kit (New England BioLabs Inc, USA) according to the manufacturer‟s
instruction. The protocol of probe labeling used was based on random priming labeling
method. The probe labeling was carried out with 1µg double-stranded cDNA as template
and nuclease-free water was added to a total volume of 34µl. A control reaction was
prepared by using 1µg of unbiotinylated lambda DNA (Hind III digested lambda DNA)
(provided in the kit) as the template. The same condition was applied for the control
reaction. Incubation at 970C for 5 minutes was then carried out to break the hydrogen
bond between the double-stranded cDNA. The denatured double-stranded cDNA was
then placed on ice for 5 minutes followed by the addition of 10µl of 5X labelling buffer
containing biotinylated random octamers (provided in the kit), 5 units of klenow
fragment (3‟-5‟exo-) and dNTPs mix a with final concentration of 5mM of dTTP, 5mM
dGTP, 5mM dCTP, and 5mM of the mixture of dATP and biotynilated-dATP (Biotin-14-
dATP) (provided in the kit) and the mixture was incubated at 370C for 20 hours. The
reaction was then terminated by the addition of EDTA (pH 8) to a final concentration of
20mM. Purification was then carried out by adding two volumes of cold absolute ethanol
and 10M lithium chloride (LiCl) to a final concentration of 0.14M and a precipitation
step was then carried by incubation at -800C for 30 minutes. The mixture was centrifuged
at 15,871 x g, at 40C for 15 minutes to pellet the synthesized probe. The pellet was then
41
washed with 70% (v/v) ethanol. After air dried, the pelleted probe was dissolved in 20µl
of TE buffer and stored at -200C for further use.
3.7.1.1 Detection of Labeling Efficiency
The probe synthesized was subjected to a serial dilution of 1/5, 1/52, 1/5
3, 1/5
4, and 1/5
5
in nuclease-free water in order to check the labeling efficiency of the probe. One
microliter of each diluted probe was spotted onto a Hybond-N+ nylon membrane
(Amersham Bioscience, UK) and air dried for 10 minutes. The membrane was then
soaked in 0.4N NaOH for 2 minutes followed by three times agitation in 2X SSC buffer
for 2 minutes. The membrane was then air dried before continuing with SDS detection
method. The SDS detection method was initiated by agitating the membrane in a washing
buffer [2.5mM sodium phosphate buffer, pH 7.2 containing 0.5% (w/v) sodium dodecyl
sulphate (SDS) and 12.5mM NaCl] for 5 minutes. The membrane was then agitated in
10ml blocking solution [25mM sodium phosphate buffer, pH 7.2 containing 5% (w/v)
SDS and 125mM NaCl] for 15 minutes at room temperature. The blocking solution was
then added with streptavidin-alkaline phosphatase conjugate (Promega, USA) at a
dilution factor of 1/10,000 and agitated for 10 minutes. The blocking solution was then
discarded and the membrane was washed twice with washing buffer. The first wash was
carried out for 5 minutes while the second wash for 1 hour. The washing buffer was then
discarded and the membrane was equilibrated in 10ml of detection buffer [20mM Tris-Cl
pH 9.5 containing 20mM NaCl and 2mM MgCl2] for 5 minutes. Then, the membrane was
transferred into a transparent plastic bag and was spread evenly with 200µl of ready to
42
use CDP-star® (Roche, USA), a chemiluminescent substrate. The CDP-star
® was allowed
to equilibrate for 2 minutes and the excess CDP-star and bubbles were removed by
rolling a glass rod on the plastic bag. The plastic bag was then sealed, placed in an X-ray
cassette followed by an exposure to an X-ray film (Kodak, USA) for 30 minutes. After
the exposure, the film was developed in a developer solution (Kodak, USA). The X-ray
film was rinsed in distilled water, followed by fixation in a fixer solution (Kodak, USA)
in a dark room to visualize the signals on the X-ray film.
3.7.2 Primary Screening
In this study, 500,000 pfu of the floral cDNA library was plated onto 10 NZY (Appendix
A) agar plates (150 mm petri dishes) with XL1-Blue MRF cells, a recombinant
Escherichia coli host strain provided in the Zap-cDNA® Gigapack
® III Gold Cloning Kit
(Stratagene, USA). Plaques were allowed to grow to hairpin size for 8 hours at 370C. The
plaques were then transferred onto a 147 mm diameter plaque lift nylon membrane
(Amersham Biosciences, UK) and hybridized with the fully-open flower cDNA probe.
The clones that give positive signals on X-ray film were cored out and used for secondary
screening.
3.7.2.1 Preparation of Bacterial Culture for Infection.
A loop of the XL1-Blue MRF cells from a glycerol stock was streaked onto LB agar
(Appendix A) containing 12.5µg/ml tetracycline. The plate was incubated overnight at
43
370C. After overnight incubation, a colony of the XL1-Blue MRF was picked and
inoculated into 5ml of LB broth (Appendix A) with supplements [10mM MgSO4, 0.2%
(w/v) maltose]. The culture was incubated at 370C overnight in an incubator shaker,
shaking at 200rpm. The overnight bacterial culture was then subcultured into 50ml LB
broth with supplements and incubated at 370C in the incubator shaker, shaking at 200rpm
until the OD600 reached ~0.5. The bacterial culture was then transferred into two tubes
equally and centrifuged at 12,857 x g at room temperature for 2 minutes to pellet the
bacterial cells. After the centrifugation, the bacterial pellet was resuspended in half of the
original volume with 10mM MgSO4 and the OD600 was then adjusted to 0.5 for infection
purpose.
3.7.2.2 Preparation of Filter for Primary Screening
A serial dilution of the floral cDNA library of Vanda Mimi Palmer was carried out in SM
buffer [50mM Tris-Cl, pH 7.5 containing 100mM NaCl, 8mM MgSO4.7H2O and 0.002%
(w/v) gelatin] from the original stock to produce a titer of 780pfu/µl for plating purpose.
The diluted cDNA library in a volume of 64.1µl (~50,000 pfu) was mixed with 600 µl of
XL1-Blue MRF cells (OD600 = 0.5) and incubated at 370C for 15 minutes to allow phage
particles in the cDNA library to attach to the XL1-Blue MRF cells. NZY top agarose (see
Appendix A) at ~370C in a volume of 6.5ml was mixed immediately with the mixture in
a 15ml sterile centrifuge tube and then poured onto a NZY plate. The plate was swirled
quickly to spread the NZY top agar evenly on the NZY plate before it solidified. The
44
plate was then incubated at 370C in an incubator for 8 hours to allow the formation of
plaques. The plate was then stored at 40C at least for 2 hours prior to plaque lift.
3.7.2.3 Transferring the Plaques onto Membrane (Plaque Lift)
Commercial plaque lift Hybond-N+, nylon membrane (Amersham Biosciences, UK) with
a diameter of 147 mm was used to transfer the plaques formed on NZY agar plates. The
membranes were marked by cutting at three sites asymmetrically. A cut membrane was
initially placed onto the plate containing plaques for 2 minutes and the cut sites were
marked at the bottom of the plate. A second plaque lift was repeated for the same plate by
placing another membrane for 4 minutes, as a duplicate. The membranes were then air
dried for 15 minutes. Each of the membrane (plaque side up) was placed onto a 3MM
Whatman paper (GE healthcare, USA) pre-wetted with a denaturing solution [1.5M
NaCl, 0.5M NaOH] for 2 minutes followed by a neutralization solution [0.5M Tris-Cl
buffer, pH 8 containing 1.5M NaCl] for 5 minutes. The membranes were then rinsed in a
rinsing solution [0.2M Tris-Cl buffer, pH 7.5 containing 2X SSC) for 25 seconds in the
same condition. The membranes were then air-dried for 30 minutes and then baked at
800C for 2 hours. The NZY agar plates were kept at 4
0C up to two weeks for further use.
3.7.2.4 Pre-hybridization and Hybridization of Membrane
Hybridization was carried out based on the instruction manual of Phototape® -Star
Detection Kit (New England Biolabs Inc., USA) with modifications. Firstly, baked nylon
45
membranes were pre-wetted in a 2X SSC buffer for 2 minutes. The membranes were then
transferred into 15ml of pre-warmed (650C) pre-hybridization buffer and pre-hybridized
in a HB-1000 Hybridization machine (Techne, UK) for 30 minutes at 550C. While pre-
hybridizing, a mixture of 1µg of biotin-labeled open-flower cDNA probe, 80µg of
sheared salmon sperm DNA and 10X SSC buffer was denatured in boiling water for 10
minutes. After denaturing, the probe mixture was added into the pre-hybridization
solution in a hybridization bottle and hybridization was carried out at 550C for 16 hours.
After 16 hours of hybridization, the membranes were washed twice in low stringency
condition with 2X washing solution [2X SSC, 0.1% SDS] by agitation at room
temperature for 5 minutes. The 2X washing solution was discarded, followed by high
stringency wash with 0.5X washing solution [0.5X SSC, 0.1% (w/v) SDS] at 550C for 15
minutes. The high stringency wash was repeated for 30 minutes with fresh 0.5X washing
solution in the same manner. After the high stringency wash, the membranes were
equilibrated in washing buffer [2.5mM sodium phosphate buffer, pH 7.2 containing 0.5%
(w/v) SDS and 12.5mM NaCl] for 5 minutes. Blocking step was then carried out by
agitating the membranes in blocking solution [25mM sodium phosphate, pH 7.2
containing 5% (w/v) SDS and 125mM NaCl] for 15 minutes at room temperature. The
membranes were then agitated in fresh blocking solution with streptavidin-alkaline
phosphatase conjugate (Promega, USA) at a dilution factor of 1/10,000 for 15 minutes at
room temperature. The membranes were then washed twice with washing buffer [2.5mM
sodium phosphate, pH 7.2 containing 0.5% (w/v) SDS and 12.5mM NaCl]. The first
wash was carried out for 5 minutes while the second wash for 60 minutes. The
46
membranes were then equilibrated in detection buffer [20mM Tris-Cl pH 9.5 containing
20mM NaCl and 2mM MgCl2] for 10 minutes. The membranes were then transferred into
a transparent plastic bag and 500µl of ready to use CDP-star® (Roche, USA), a
chemiluminescent substrate was added and spread evenly on the membrane. The CDP-
star® was allowed to equilibrate for 2 minutes and the excess CDP-star and bubbles were
removed by rolling a glass rode on the plastic bag. The plastic bag was then sealed,
placed in an X-ray cassette followed by an exposure to an X-ray film (Kodak, USA)
overnight. After the exposure, the film was developed as described in section 3.7.1.1.
3.7.2.5 Coring of Positive Clones
A light box was used to align back the plates with the positive signals on X-ray film.
Each of the positive plaques was cored out into a fresh 1.5ml microcentrifuge tube
containing 500µl SM buffer and 20µl chloroform. The tubes were then vortexed briefly
and incubated at room temperature for 30 minutes. After that, the tubes were stored at
40C for further use.
3.7.2.6 Secondary Screening
A set of serial dilution (10-1
, 10-2
, 10-3
, 10-4
, and 10-5
) was prepared for the cored out
phages from primary screening in SM buffer. A volume of 20µl of each phage dilution
was mixed with 350µl of XL1-Blue MRF cells (OD600=0.5) in a fresh 1.5ml sterile
microcentrifuge tube separately. The mixtures were then incubated at 370C for 15
47
minutes to allow the phage particles to attach onto the bacterial cells. Subsequently, the
mixtures were resuspended with 3.5ml of ~370C NZY agarose top agar and immediately
poured onto NZY agar plates. The plates were swirled evenly before the agarose
solidified, sealed with parafilm and incubated at 370C for 16 hours. The plates were then
kept at 40C for at least 2 hours before transferring the plaques onto membrane. The
dilution of 10-3
was chosen to be applied for all of the clones cored out in primary
screening because this dilution gave 50 to 100 plaques on each NZY agar plate (100 mm
petri dish).
Plaque lifting was carried out in the same manner as described in the primary screening
in section 3.6.2.3 with 87mm diameter immobilon membranes (Millipore, USA). The
subsequent steps such as pre-hybridization, hybridization, washing and detection were
carried out in the same condition as mentioned in section 3.7.2.4. Positive plaque from
each plate that represents each positive clone was cored out and resuspended with 500µl
SM buffer and 20µl chloroform in a sterile 1.5ml microcentrifuge tube. The tube was
then vortexed briefly and incubated at room temperature for at least 30 minutes before
storing at 40C for further use.
3.8 Single Clone In Vivo Excision
Phagemids of the putative positive clones were subjected to in vivo excision. Initially,
SOLR cells and XL1-Blue MRF cells were cultured in 5ml LB broth (see Appendix A)
with supplement [10mM MgSO4, 0.2% (w/v) maltose] separately. The culture was
48
incubated at 300C with shaking at 200 rpm for 16 hours. The 5ml overnight culture was
sub-cultured into 50ml of LB with supplement. The culture was incubated at 300C with
shaking at 200rpm until the OD600 reached 0.5. The tube containing the culture was then
centrifuged at 1000g for 10 minutes to pellet the bacterial cells. The pellet was then
resuspended in 25ml of 10mM MgSO4. The OD600 of the resuspended bacterial cells was
then adjusted to 1.0.
In vivo excision was initiated by addition of 200µl of XL1-Blue MRF cells (OD600 =1.0),
250µl of phage (stock from secondary screening in SM buffer with chloform) and 1µl of
helper phage into 1.5ml microcentrifuge tube followed by incubation at 370C for 15
minutes to allow attachment of phage to XL1-Blue MRF cells. The mixture was then
added into 3ml of LB broth with supplements in a 15ml centrifuge tube followed by
incubation at 370C with shaking at 200rpm for 16 hours. After the incubation period, the
cultures were heated at 700C for 20 minutes followed by a centrifugation at 1000 x g for
15 minutes. After the centrifugation, recovered filamentous phages in supernatants were
transferred into fresh microcentrifuge tubes and stored at 4 0C for further use.
Plating of the excised phagemid was carried out by mixing 100µl of filamentous phages
of each clone with 400µl of SOLR cells (OD600 = 1.0) separately. The mixture was then
incubated at 370C for 15 minutes followed by plating of 200µl of the mixture on LB plate
containing 100µg/ml of ampicillin separately. The plate was incubated at 370C for 16
hours. After the incubation period, a single colony grown on the plate was re-streaked on
a fresh LB plate containing 100µg/ml of ampicillin and incubated at 370C for 16 hours.
49
The single colony was used for colony PCR reaction, plasmid mini preparation and also
for glycerol stock preparation. The same procedure was applied for all of the clones cored
out in secondary screening.
3.9 Reverse-Northern Analysis
Reverse-Northern analysis was carried out in this study to select putative positive clones
that show up-regulated expression in fully-open flower stage compared to bud stage of
Vanda Mimi Palmer as candidates for fragrance-related cDNAs. Reverse-Northern
applied Southern Blotting method whereby PCR product of cDNA inserts were used in
the experiment instead of genomic DNA.
Firstly, colony PCR amplification of cDNA insert from each clone was performed. Single
colony of each clone grown on LB-ampicillin (100µg/ml) agar plates were picked out
using a tooth pick and swirled into PCR mix [1X PCR buffer (Invitrogen, USA), 1.5mM
MgCl2 (Invitrogen, USA), 0.2µM T3 universal primer (5‟-ATTAACCCTCACTAAAG-
3‟), 0.2µM T7 universal primer (5‟-AATACGACTCACTATAG-3‟), 0.2mM dNTPs
(Bioron, Germany), 1 unit Taq DNA polymerase (Invitrogen, USA)]. The PCR
conditions were as follow: 1 cycle of pre-denaturation at 950C for 5 minutes, followed by
35 cycles of denaturation at 950C for 15 seconds, annealing at 55
0C for 45 seconds and
extension at 720C for 1 minute, followed by final extension at 72
0C for 5 minutes. The
PCR amplification was carried out by using MJ Cycler and Master Cyler (Bio-Rad,
USA). The PCR products were then loaded in duplicate on 1.5% (w/v) agarose gel and
50
electrophoresis was carried out at 80 Volts for 40 minutes. The gels were stained in
1µg/ml ethidium bromide solution for 1 minute followed by de-staining in distilled water
for 30 minutes. The gels were then viewed using a gel documentation system (Biorad,
USA). After viewing using the gel documentation system, the gels were used for
Southern Blotting (Sambrook and Russell, 2001).
In Southern Blotting, the gels were denatured in 0.4 N NaOH (denaturing solution) for 15
minutes and blotted onto a Hybond-N+ nylon membranes (Amersham Bioscience, UK)
through gravitational transfer overnight in 0.4 N NaOH (Sambrook and Russell, 2001).
After the overnight transfer, the wells position on agarose gels were marked on the
membranes by using a pencil. The membranes were then air dried and baked at 800C for
2 hours. The baked membranes were then hybridized with two different probes
separately: open flower cDNA probe and bud cDNA probe. The open flower and bud
cDNA probes were synthesized separately using NEBlot® Phototape Kit (New England
BioLabs Inc, USA) as described in section 3.7.1. The hybridization, washing and
detection steps were same as mentioned in section 3.7.2.4. The clones that showed
stronger signals with open flower probe compared to bud probe of Vanda Mimi Palmer
were selected for partial sequencing and further characterization.
3.10 Elimination of Cymbidium mosaic Virus Coat Protein cDNA Transcripts
In preliminary sequencing of 100 clones of the floral cDNA library by Miss Chan Wai
Sun, half of the clones in the floral cDNA library were reported to be contaminated by
51
Cymbidium mosaic virus coat protein cDNA transcript. The clones that represent the
Cymbidium mosaic virus coat protein cDNA transcript (see Appendix B) were eliminated
from the screened clones (section 3.9) by hybridizing the PCR-amplified inserts of all the
in vivo excised clones with Cymbidium mosaic coat protein cDNA probe. The
membranes used in reverse-Northern analysis in section 3.9 were stripped off by using
hot-SDS method as described in the manual of Hybond+
nylon membrane (Amersham
Bioscience, UK). Boiled 0.1% SDS (w/v) was poured onto each membrane in a tray and
was agitated for 10 minutes. The stripped membranes were re-hybridized with
Cymbidium mosaic virus coat protein cDNA probe as described in section 3.7.1.
The template used for probe synthesis was PCR-amplified from plasmid containing
Cymbidium mosaic virus coat protein cDNA transcript. The amplification was carried out
using gene specific primers (CMV-F 5‟-AGAGCCCACTCCAACTCCA-3‟ and CMV-
R 5‟-GCAGGCAGAGCATAG AGACTG-3‟) that amplified a partial cDNA sequence
(650bp) of the Cymbidium mosaic virus coat protein. The PCR reaction was carried out in
a final volume of 20µl containing PCR mix [1X PCR buffer (Invitrogen, USA)
containing 1.5mM MgCl2 (Invitrogen, USA), 0.2µM CMV-F primer, 0.2µM CMV-R
primer, 0.2mM dNTPs (Bioron, Germany) and 1 unit Taq DNA polymerase (Invitrogen,
USA)] and 100ng plasmid as initial template. The PCR conditions were as follow: 1
cycle of pre-denaturation at 940C for 3 minutes, followed by 35 cycles of denaturation at
940C for 30 seconds, annealing at 53
0C for 45 seconds and extension at 72
0C for 45
seconds, and a final extension at 720C for 5 minutes.
52
3.11 Plasmid Mini Preparation
The clones that showed up-regulated expression in fully-open flower stage compared to
bud stage of Vanda Mimi Palmer were subjected to plasmid mini preparation as
described in Sambrook et al., 1989 with minor modifications. Initially, single colonies of
in vivo excised clones were inoculated in 3ml LB broth containing 100µg/ml ampicillin.
The cultures were then incubated in an incubator shaker at 370C, shaking at 200rpm for
16 hours. After the incubation period, the cultures were centrifuged at 15,294 x g for 1
minute at room temperature. The supernatants were then discarded and the recovered
pellets were resuspended in 200µl of Solution I [25mM Tris-Cl buffer pH 8 containing
10mM EDTA] followed by incubation on ice for 10 minutes. After that, 400µl of
Solution II [0.2N NaOH, 0.5% (w/v) SDS] was added into each tube and mixed gently by
inverting the tubes for several times followed by 10 minutes incubation on ice. After that,
300µl of Solution III [3M sodium acetate pH 4.8] was added into the tubes and mixed
completely. The tubes were then incubated on ice for 15 minutes followed by
centrifugation at 15,294 x g for 15 minutes at 40C. The supernatant was transferred into
fresh microcentrifuge tube and 50µg RNAse A was added into the tube. Removal of
RNA from plasmid DNA by RNAse A was carried out by incubation at 370C for 30
minutes.
After the incubation with RNAse A, 400µl of phenol: chloroform: isoamylalcohol
(25:24:1) was added into the tube. The mixture was then vortexed completely and
centrifuged at 15,294 x g at room temperature for 10 minutes. After that, top aqueous
53
phase was transferred into new fresh microcentrifuge tube followed by precipitation.
Plasmid DNA was precipitated by adding 0.1 volume of 3M NaOAc, pH 5.2 and 0.6
volume of isopropanol and this was followed by incubation at -200C for 1 hour. The
plasmid DNA was pelleted by centrifugation at 15,294 x g at 40C for 20 minutes. The
supernatant was discarded and the pellet was rinsed with 70% (v/v) ethanol. The pellet
was air dried and dissolved in 20µl Tris-EDTA (TE) buffer, pH 8.
3.12 DNA Sequencing and Sequence Analysis
All of the clones that showed up-regulated expression in open flower stage compared to
bud stage of Vanda Mimi Palmer after the removal of Cymbidium mosaic virus coat
protein transcripts were sequenced using T3 primer. The sequencing reaction was carried
out by Medigene Sdn. Bhd. and First Base Sdn. Bhd., Kuala Lumpur, Malaysia. Vector
and adaptor sequences were eliminated before proceeding with sequence analysis. All the
sequences were subjected to contigs and singletons analyses using the CAP3 software
(http://pbil.univ-lyon1.fr/cap3.php) (Huang and Madan, 1999). The tentative unique
genes were compared against the GeneBank non-redundant database at the National
Centre for Biotechnology Information (NCBI) (http://www.ncbi.nlm.nih.gov) using
BLASTX and BLASTN with a cut off value less than 1e-05
and score more than 80. The
Expressed Sequence-tags (ESTs) were then classified into functional groups based on
their putative functionality (Zhao et al., 2006; Lindqvist et al., 2006).
54
3.13 Verification of Fragrance-related cDNA by RT-PCR Analysis
Total RNA samples for the verification of fragrance-related cDNA was extracted from
flowers and buds collected at 12.00 noon separately using the same protocol as described
in section 3.4. The total RNA samples were used as template for first strand cDNA
synthesis. PolyA tail mRNAs in the total RNA were reverse-transcribed into first strand
cDNA by using Quantitect reverse transcription kit (Qiagen, Germany) according to the
manufacturer‟s instruction. Initially, 1µg of total RNA template and 2µl of 7X of gDNA
wipe buffer (provided in the kit) were transferred into a clean PCR tube followed by
addition of RNAse-free water to a total volume of 14µl. The mixture was then incubated
at 420C for 2 minutes and chilled on ice quickly. The remaining components of the kit
were then added into the mixture which include reverse transcriptase enzyme (1µl),
Quantiscript RT buffer (4µl), and RT primer mix (1µl). The mixture was then incubated
at 420C for 30 minutes followed by incubation at 95
0C for 3 minutes to inactivate the
reverse transcriptase enzyme. The first strand cDNA for each sample was aliquoted into a
few microcentrifuge tubes and kept at -200C for storage.
Forward and reverse primers (Table 1) of each selected transcript were designed using
Primer 3 software with advance setting including the size of primers between 18-22bp,
melting temperature (Tm) of the primers were between 50-600C and the product size
ranged between 150-250bp. The primers were synthesized by First Base Sdn. Bhd.,
Malaysia. Gradient RT-PCR was carried out for each primer set for each clone of interest
containing cDNA insert to get the best annealing temperature. The gradient RT-PCR
55
Table 1: Characteristics of Primers Used in Verification. Primers for putative
fragrance-related transcripts and endogenous control that were used in RT-PCR.
Target/ Amplicon
Length (bp)
Primers
Primer Sequences
Optimal
Annealling
Temperature
VMPCyP450
(201bp)
Forward
Reverse
5‟ GCTGTTTTCATGTCTGGAAGC 3‟
5‟ TCCTGTTTGTGACGGCTCTT 3‟
530C
VMPEST (182 bp)
Forward
Reverse
5‟ GCAACGCTCTCATGGTTTAT 3‟
5‟ AAAAGCCTCGAAAAATCTGA 3‟
570C
VMPCMEK
(202bp)
Forward
Reverse
5‟ GTACACGGAAACGATCACTG 3‟
5‟ AACATGCAAGCCAAACATT 3‟
590C
VMP36
(227bp)
Forward
Reverse
5‟- CTCCCGCATTGACCATAAAT-3‟
5‟-GGAACCACACCCAAACTCTC-3‟
550C
VMP48
(200bp)
Forward
Reverse
5‟-TTGGATGTCGTGAAGGCAAT-3‟
5‟- CAACACAAGAAGATAGCACAGCA-3‟
530C
VMP59
(221bp)
Forward
Reverse
5‟-CGAGGAAGACGAAGAGGAAG-3‟
5‟-CGAAAAATAGAACAGAGCCATAG-3‟
530C
VMP83
(202bp)
Forward
Reverse
5‟-GCTGGTTAGGGTGAAGCAT-3‟
5‟-AAAAACATAGACAAATGGAGACC-3‟
530C
VMP90
(243bp)
Forward
Reverse
5‟-GGAAAGGAAGAAAAGCAGCA-3‟
5‟-CGACACCAAGAAACATCTCC-3‟
590C
VMP96
(231bp)
Forward
Reverse
5‟-TCGCCTTCTCTCATCTCTGAA-3‟
5‟- CAAGCCCACGCATAAAAGTA-3‟
590C
VMPA28
(202bp)
Forward
Reverse
5‟-GTACACGGAAACGATCACTG-3‟
5‟-AACATGCAAGCCAAACATT-3‟
590C
56
Target/ Amplicon
Length (bp)
Primers
Primer Sequences
Optimal
Annealling
Temperature
VMPA46
(182bp)
Forward
Reverse
5‟-GGATGTTCTACGGGTGGAC-3‟
5‟-AGAGAGGAGCACAGCTTTATTT-3‟
590C
VMPA54
(180bp)
Forward
Reverse
5‟-AAAAGCAGCGGTTTATGAAG-3‟
5‟-CCAAACGAAAACTCAGGAAT-3‟
530C
Elongation factor
(507bp)
-endogenous control
Forward
Reverse
5‟ CCGACCGCAAGGAGAGTTAT 3‟
5‟ AAGCCACGGAACAAAAACAG 3‟
550C
57
conditions were as follow: 1 cycle of pre-denaturation at 940C for 3 minutes, followed by
35 cycles of denaturation at 940C for 30 seconds, annealing at 50-60
0C for 45 seconds
and extension at 720C for 45 seconds, and a final extension at 72
0C for 5 minutes. The
best annealing temperature for each primer set for each sequence of interest was chosen
based on the highest amount (most intense band) of PCR-amplified products viewed
using a gel documentation system (Biorad, USA) with minimum or without primer
dimers.
After determining the optimum annealing temperature, RT-PCR was carried out using the
same PCR protocol as described above with specific annealing temperature (see Table 1
for the annealing temperature used) for each sequence of interest. The RT-PCR products
were analyzed by agarose gel electrophoresis separately and viewed using a gel
documentation system (Biorad, USA). After viewing, the agarose gels with RT-PCR
product for each clone were transferred onto nylon membranes separately by Southern
blot method as described in section 3.9. The membranes were baked at 800C for 2 hours.
The membranes were then hybridized with specific probe labeled with biotin representing
each cDNA of gene of interest. The membranes were hybridized for 16 hours with
specific probe separately followed by washing and detection steps as described in section
3.7.2.4. Putative elongation factor, a housekeeping gene cDNA transcript was obtained
from Miss Chan Wai Sun and used as an endogenous control for the RT-PCR reaction.
58
The probes used for the hybridization were synthesized using NEBlot®
phototape kit as
described in section 3.7.1. The cDNA inserts of each clone were PCR-amplified using T3
(5‟-ATTAACCCTCACTAAAG-3‟) and T7 (5‟-AATACGACTCACTATAG-3‟)
universal primers prior to the probe synthesis. The PCR-amplified cDNA inserts of each
clone were purified using SpinPrep PCR clean-up kit (Novagen, Germany) according to
the manufacturer‟s instruction. A volume of 100µl of PCR product was mixed completely
with 400µl of SpinPrep bind buffer A (provided in the kit). The mixture was transferred
into a SpinPrep PCR filter with a receiver tube and centrifuged at 15,294 x g for 1
minute. The flow through was discarded and 400µl of the SpinPrep bind buffer A was
transferred into the filter and centrifuged at 15,294 x g for 1 minute. The PCR-amplified
insert at the surface of the filter‟s membrane was washed with 500µl of SpinPrep wash
buffer B (provided in the kit). The wash step was carried out by a centrifugation at 15,294
x g for 1 minute. The flow through was discarded and the filter was centrifuged again for
2 minutes to remove excess of wash buffer. After that, 30µl of pre-warmed (700C)
SpinPrep elution buffer C (provided in the kit) was transferred into the filter and allowed
to stand for 3 minutes at room temperature. The filter was transferred into a fresh 1.5ml
microcentrifuge tube and centrifuged at 15,294 x g for 1 minute. The same procedure was
applied to all the clones selected for the verification of putative fragrance-related
transcripts.
3.14 Full-length cDNA Isolation of Fragrance-related Transcripts
Full-length cDNA isolation was carried out for three selected putative fragrance-related
cDNAs from the verified transcripts in section 3.13 [putative 4-(cytidine 5′-diphospho)-2-
59
C-methyl-d-erythritol kinase (VMPCMEK), putative cytochrome p450 protein transcript
(VMPCyP450) and an unknown protein transcript (VMPA28)] and a putative fragrance-
related cDNA transcript (putative phenylacetaldehyde synthase (VMPPAAS) isolated by
Miss Chan Wai Sun from the floral cDNA library of Vanda Mimi Palmer.
The cDNA templates for full-length cDNA isolation were synthesized using the SMART
RACE cDNA Amplification Kit (Clonetech, USA) according to the manufacturer‟s
instruction. The templates used for full-length cDNAs isolation were the 5‟- and 3‟-
RACE-Ready cDNAs synthesized from total RNA of Vanda Mimi Palmer at open flower
stage collected during day time at 12.00 noon. PCR amplification of 5‟- region was
carried out by using 5‟- RACE-Ready cDNA while 3‟- RACE-Ready cDNA was used as
template for PCR amplification of 3‟- region. For 5‟-RACE-Ready cDNA, 1µg of total
RNA sample was mixed with 1.7 μM of 5‟-CDS primer A (5'–(T)25V N–3' ) (N = A, C,
G, or T; V = A, G, or C) and 1.7 μM of SMART II A oligo (5'–
AAGCAGTGGTATCAACGCAGAGTACGCGGG–3'). Meanwhile for 3‟-RACE-Ready
cDNAs preparation, 1µg of total RNA sample was mixed with 1.7 μM of 3‟-CDS primer
A (5'–AAGCAGTGGTATCAACGCAGAGTAC(T)30V N–3' ) (N = A, C, G, or T; V =
A, G, or C). The tubes for 5‟-RACE-cDNA and 3‟-RACE cDNA were incubated at 700C
for 2 minutes followed by chilling on ice before addition of 1X first-strand buffer
(provided in the kit), 2mM of DTT (provided in the kit), 1mM of dNTP mix (provided in
the kit) and 1μl of MMLV reverse transcriptase (provided in the kit) for each tube. The
tubes were incubated at 420C for 1.5 hours to synthesize first-stand cDNA. After the
incubation period, 100µl of tricine-EDTA buffer (provided in the kit) was added into
60
each of the 5‟-RACE and 3‟-RACE first-strand reaction products. The 5‟- and 3‟-RACE-
Ready cDNAs were then heated at 720C for 7 minutes to inactivate the reverse-
transcriptase enzyme. The 5‟- and 3‟-RACE-Ready cDNAs were then stored at -200C.
PCR amplification of 5‟- and 3‟-regions of the selected putative fragrance-related cDNA
transcripts was carried out by using the Advantage 2 Polymerase Mix (Clonetech, USA)
with gene specific primer for each transcript (Table 2) and universal primer mix (UPM)
provided in the SMART RACE cDNA amplification kit (Clonetech, USA) according to
the manufacturer‟s instruction with minor modifications. The 5‟ cDNA fragments of
VMPCMEK, VMPPAAS, VMPCyP450 and VMPA28 were PCR-amplified in 50µl PCR
reaction separately with 1X Advantage 2 PCR buffer (provided in the kit), 2.5μl of 5‟-
RACE-Ready cDNA, 1X Universal primer A mix (UPM) (see Table 2), 0.2 μM of gene
specific primer (listed in Table 2), 0.2mM of dNTP mix, and 1X Advantage 2
Polymerase Mix. The gene specific primers were designed manually at the conserved
region of the partial sequence of the cDNA transcript by considering the high melting
temperature (Tm) in the range of 65-720C with high GC content especially at the 3‟ end
of the primer, the size of the primers designed ranged between 25-30bp and synthesized
by Bioneer, Korea. The PCR amplification of 5‟- cDNA fragments were carried out by
pre- denaturation at 940C for 1 minute followed by 35 cycles of denaturaton at 94
0C for
30 seconds, annealing at 680C for 30 seconds and extension at 72
0C for 3 minutes. Final
extension was carried out at 720C for 5 minutes. The 3‟ cDNA fragment of VMPCyP450
and VMPA28 cDNAs were amplified by using the 3‟-RACE-Ready cDNA as the
template in the same manner as the 5‟- RACE PCR.
61
Table 2: Primers Sequences for the Isolation of Full-length Transcripts. Primers of
fragrance-related transcripts were designed and used to isolate the 5‟-, 3‟-ends and ORF.
All of the primers except for UPM primers were synthesized by Bioneer, Korea.
Meanwhile UPM primers were provided in the Advantage2 polymerase mix (Clonetech,
USA).
Target/
Amplicon
Length (bp)
Primers
Primer Sequences
Optimal
Annealling
Temperature
VMPCMEK
5‟ region
VMPCMEK 5‟ GSP
UPM (long primer)
UPM (short primer)
5‟-GACCCTGTGGACGAAGTTGGCTCTG-3‟
5‟-CTAATACGACTCACTATAGGGCAAGCAGT
GGTATCAACGCAGAGT-3‟
5‟-CTAATACGACTCACTATAGGGC-3‟
680C
VMPCMEK
ORF
VMPCMEK ORF
forward
VMPCMEK ORF
reverse
5‟- CGC TTC TCA GCT TTT CTC CTA ACA ATG
GCC T -3‟
5‟- TTT TCC TGT TTG TGA CGG CTC TTC TCT
GCT CA -3‟
680C
VMPPAAS
5‟ region
VMPPAAS 5‟ GSP
UPM (long primer)
UPM (short primer)
5‟- AGCGTGAATCTTCTTGGTACAACCACCTC
-3‟
5‟-CTAATACGACTCACTATAGGGCAAGCAGT
GGTATCAACGCAGAGT-3‟
5‟-CTAATACGACTCACTATAGGGC-3‟
680C
VMPPAAS
ORF
VMPPAAS ORF
forward
VMPPAAS ORF
reverse
5‟- GAACTCCAGAAAATGGGCAGCCTTCCCA
C-3‟
5‟- CTCAGAGTGTTTTGAGTTTCCAACCCAGC
T-3‟
680C
VMPCyP450
5‟ region
VMPCYP450 5‟ GSP
UPM (long primer)
UPM (short primer)
5‟-CAGTAGTGTCCCTCCCTGCAATAACA-3‟
5‟- CTAATACGACTCACTATAGGGCAAGCAGT
GGTATCAACGCAGAGT-3‟
5‟-CTAATACGACTCACTATAGGGC-3‟
680C
VMPCyP450
3‟ region
VMPCYP450 5‟ GSP
UPM (long primer)
UPM (short primer)
5‟-TGAGAGCTAAACAAAACGGGCATCA 3‟
5‟- CTAATACGACTCACTATAGGGCAAGCAGT
GGTATCAACGCAGAGT-3‟
5‟-CTAATACGACTCACTATAGGGC-3‟
680C
62
Target/
Amplicon
Length (bp)
Primers
Primer Sequences
Optimal
Annealling
Temperature
VMPCyP450
ORF
VMPCyP450 ORF
forward
VMPCyP450 ORF
reverse
5‟-GCTGCCACTAATGTCTTCTTCCTCAAGCTC
C-3‟
5‟- GTCTGCACTTCACTTATGGACAACAAACA
GAC-3‟
680C
VMPA28
5‟ region
VMPA28 5‟ GSP
UPM (long primer)
UPM (short primer)
5‟-AAATATCCCGCAACCTGTCCCACCT-3‟
5‟-CTAATACGACTCACTATAGGGCAAGCAGT
GGTATCAACGCAGAGT-3‟
5‟-CTAATACGACTCACTATAGGGC-3‟
680C
VMPA28
3‟ region
VMPA28 3‟ GSP
UPM (long primer)
UPM (short primer)
5‟-TGCCGAGCATTTGATGGACGAAAGT-3‟
5‟-CTAATACGACTCACTATAGGGCAAGCAGT GGTATCAACGCAGAGT-3‟
5‟-CTAATACGACTCACTATAGGGC-3‟
680C
VMPA28
ORF
VMPA28 ORF
forward
VMPA28 ORF
reverse
5‟-TGTGAGATTAGTTCAATTCTTAGGCACC
CCAG-3‟
5‟-GCTTGTAGACAGCAACATGCAAGCCAAA
CATT-3‟
680C
63
3.14.1 Purification of RACE-PCR Products
The RACE-PCR products were purified by using GeneAll Exspin Combo GP (GeneAll
Biotechnology, Korea) according to the manufacturer‟s instruction. The RACE-PCR
products were electrophoresed on 1.0% (w/v) agarose gel and in 1X TAE buffer, pH 8
(see Appendix A) at 80 Volts for 30 minutes. The gel was viewed using a gel
documentation system (Bio-Rad, USA). The band that showed expected size of PCR
product was excised using ethanol-cleaned razor blade. The excised gel slice was
weighed and transferred into a new fresh tube and 300µl of GB buffer (provided in the
kit) was added for each 100mg of the agarose gel slice. The mixture was incubated at
500C for 10 minutes and vortexed for every 2-3 minutes. The mixture in a volume of
700µl was then transferred into SV column (provided in the kit) and centrifuged at
15,294 x g for 1 minute. The flow through was discarded and the step was repeated until
finished all the mixture. After that, 500µl of fresh GB buffer was applied to the SV
column and centrifuged at 15,294 x g for 30 seconds. A volume of 700µl of washing
buffer (Buffer NW) (provided in the kit) was applied into the SV column and centrifuged
at 15,294 x g for 30 seconds. The flow through was discarded and the SV column was
centrifuged at 15,294 x g for 2 minutes to remove the excess washing buffer. The SV
column was then transferred into a fresh new 1.5ml microcentrifuge tube and 30µl of
elution buffer (Buffer EB) (Provided in the kit) was transferred into the SV column and
allowed to stand for 1 minute. A final centrifugation was carried out at 15,294 x g for 1
minute to recover the purified RACE-PCR product. The same procedure was applied for
purification of all RACE-PCR products in this study.
64
3.14.2 Preparation of Competent Cells
Competent cells were prepared by calcium chloride treatment using Escherichia coli
DH5α strain (Sambrook et al., 1989). A single colony of DH5α from an LB (see
Appendix A) agar plate was inoculated into 5ml of LB broth (see Appendix A). The LB
broth culture was incubated at 370C overnight in an incubator shaker with shaking at
200rpm. A volume of 2ml of the overnight culture was transferred into 20ml fresh LB
broth in a 50ml centrifuge tube and incubated in the incubator shaker at 370C with
shaking at 200rpm for 2 hours. After the incubation period, the tube was centrifuged at
1000 x g for 5 minutes at 40C. The pellet was resuspended in 10ml of 75mM cold
calcium chloride (CaCl2) followed by incubation on ice for 20 minutes. After the
incubation period, the bacterial cells were centrifuged at 1000 x g for 5 minutes at 40C.
The pellet was resuspended in 2ml of 75mM cold CaCl2, followed by addition of 15%
(v/v) glycerol. A volume of 100µl of competent cells with 15% (v/v) glycerol was
aliquoted into 1.5ml microcentrifuge tubes. The stocks were kept at -800C for future use.
3.14.3 Cloning and Transformation of RACE-PCR Product
The purified RACE-PCR product in section 3.14.1 was cloned into the yTA cloning
vector (Yeastern, Taiwan) according to the manufacturer‟s instruction. An amount of
150ng of the purified RACE-PCR product was transferred into a 0.2ml PCR tube
followed by addition of ligation components provided in the kit; 1µl of ligation buffer A,
1µl of ligation buffer B, 50ng of yT&A cloning vector, and 1unit of yT4 DNA ligase.
65
Nuclease-free water was then added up to a total volume of 10µl. The ligation reaction
was carried out at 40C overnight.
The ligation product was transformed into Escherichia coli DH5α competent cells
prepared in section 3.14.2 by heat shock transformation method (Sambrook et al., 1989).
A volume of 2.5µl of the ligation product was transferred into the 100µl of competent
cells followed by incubation on ice for 20 minutes. After the incubation period, a heat-
shock treatment was carried out by incubating the tube at 420C for 90 seconds followed
by incubation on ice for 1 minute. A volume of 1ml of LB broth (see Appendix A) was
added into the tube. The tube was then incubated at 370C with shaking at 250rpm for 45
minutes. After that, the tube was centifugated at 15,294 x g for 1 minute and 800µl of
supernatant was discarded. The pellet was then resuspended in the remaining supernatant.
A volume of 100µl of the resuspended DH5α cells was spreaded onto LB plate
containing 2.5mg of ampicillin (see Appendix A) with 0.8mg of X-gal and 1.4mg of
IPTG for blue and white screening. The plate was then incubated at 370C incubator oven
for 16 hours.
After the 16 hours incubation period, the transformants (white colonies) were re-streaked
onto a fresh LB plate containing 2.5mg of ampicillin. The plate was then incubated at
370C incubator oven for 16 hours. A single colony of each transformant was subjected for
colony PCR reaction with M13 forward (5' GTAAAACGACGGCCAGT 3') and M13
reverse (5' GCGGATAACAATTTCACACAGG 3') universal primers to select for a
transformant with the exact insert size. The colony PCR was carried out as described in
66
section 3.8 except for the universal primers used were M13 forward and reverse instead
of T3 and T7. The clone with the expected size of PCR product was subjected for
plasmid mini preparation (see section 3.11) and sent for sequencing with both M13
forward and reverse primers. The same procedure was applied for all 5‟- and 3‟- RACE-
PCR products.
3.14.4 Isolation of Open Reading Frame (ORF) of the Putative Fragrance-related
Transcripts
The open reading frame (ORF) of VMPCMEK, VMPPAAS, VMPCyP450 and VMPA28
were isolated by PCR-amplification using the gene specific primers (see Table 2). The
amplifications were carried out in 50µl reaction containing 0.2µM forward primer (see
Table 2), 0.2µM reverse primer (see Table 2), 1mM of dNTP mix (Clonetech, USA),
2.5μl of 5‟-RACE-Ready cDNA and 1X Advantage 2 Polymerase Mix (Clonetech,
USA). The gene specific primers were designed manually at the conserved region of the
partial sequence of the cDNA transcript by considering the high melting temperature
(Tm) in the range of 65-720C with high GC content especially at the 3‟end of the primers.
The size of the primers ranged between 30-34bp and synthesized by Bioneer, Korea. The
PCR cycling parameters used were as follow; pre-denaturation at 940C for 1 minute
followed by 35 cycles of denaturation at 940C for 30 seconds, annealing at 68
0C for 30
seconds and extension at 720C for 3 minutes. Final extension was carried out at 72
0C for
5 minutes. The PCR products were electrophoresed on 1.0% (w/v) agarose gel in 1X
TAE buffer (see Appendix A) at 80 Volts for 30 minutes. The gel was viewed using a gel
67
documentation system (Bio-Rad, USA). The band that showed the expected size of PCR
product was excised using ethanol-cleaned razor blade. The PCR products were purified
by using GeneAll Exspin Combo GP (GeneAll Biotechnology, Korea) according to the
manufacturer‟s instruction (see section 3.14.1). The purified PCR products were cloned
into yT&A cloning vector (see section 3.14.3) and transformed into Escherichia coli
DH5α competent cells (see section 3.14.3). A transformed colony was grown overnight
for plasmid mini preparation (see section 3.11) and sent for sequencing. The sequencing
was carried out by Macrogen Inc, Korea.
3.14.5 Sequence Analysis of the Cloned ORF
The ORF sequences of the putative fragrance-related cDNAs were analyzed by using
BLASTX program (NCBI GeneBank) (http://www.ncbi.nlm.nih.gov) to search for the
homologous sequences. Bioedit software version 7.0.1 (Hall, 1999) was used to translate
the cDNA sequences of the putative fragrance-related transcripts into amino acid
sequences. The amino acid sequences were then analyzed by BLASTP program (NCBI
GeneBank) (http://www.ncbi.nlm.nih.gov). Clustal W multiple alignment program
applying BLOSUM62 matrix in the Bioedit software was used to align the amino acid
sequences of the putative fragrance-related transcripts with homologous sequences from
other plants based on default setting. Expasy program was used to compute the
theoretical pI and predict molecular weight (MW) of the proteins (Expasy)
(http://br.expasy.org/tools/). Phylogenetic trees were constructed using the Mega version
4 software (Tamura et al., 2007) with Neighbour-Joining method to determine the genetic
68
relationship between the deduced proteins of putative fragrance-related transcripts with
closely related proteins available in NCBI GenBank database. Unrooted trees were
produced with 1000 sample replication. Motifs search and signal peptide prediction was
carried out by Localizome software (http://localodom.kobic. re.kr/LocaloDom/index.htm)
(Lee et al., 2006), and Expasy tool (http://br.expasy. org/tools/).
3.15 Expression Studies of Fragrance-related Transcript by Real-time RT-PCR
Expression analysis of the putative fragrance-related transcripts (VMPPAAS,
VMPCMEK, VMPCyP450 and VMPA28) was carried out by real-time RT-PCR in
different tissues, at different developmental stages and different time points in a 24-hour
cycle. For expression studies in different tissues, total RNA was isolated from both floral
(bud, open-flower, petal, sepal and lip) and vegetative tissues (leaf, shoot, root and stalk).
For expression studies at different flower developmental stages, total RNA was isolated
from young bud (green), mature bud (red), half-open flower, fully-open flower and 14-
day old fully-open flower. All the samples for expression analysis on different tissues and
developmental stages were collected at 12.00pm in the afternoon since the scent emission
of Vanda Mimi Palmer was detected at high level at this time. For expression studies at
different time points in a 24-hour cycle, fully open flowers were collected for every 2
hours starting at 8.00 am until 6.00 am on the next day. The procedure used for total
RNA extraction from all the above mentioned samples was as described in section 3.4.
69
For expression analysis, first-strand cDNA was synthesized from 1µg of total RNA using
Quantitect reverse transcription kit (Qiagen, Germany) as described in section 3.13. Real-
time RT-PCR reaction was carried out by using Brilliant® SYBR
® Green QPCR master
mix (Stratagene, USA) with 1µl of 10X diluted cDNA template with gene specific
primers (150nM forward primer and 150nM reverse primer). The primers used for the
real-time expression analysis were designed at the 3‟ end. The primers were designed
using Primer 3 software with advanced setting including size of primers in the range of
20-25bp, melting temperature (Tm) between 55-650C and the product size between 150-
250bp. The primers were synthesized by Sigma-Aldrich, USA. The primers‟ sequence
used for expression studies of gene of interest VMPPAAS, VMPCMEK, VMPCyP450,
and VMPA28 are listed in Table 3. Four replicates were prepared for each sample and
they are known as technical replicates. Besides that, 4 replicates of negative control
known as non-template control (NTC) were included for each real-time RT-PCR reaction
using the same mastermix with the gene of interest without any template.
3.15.1 Optimization for Real-time RT-PCR
Gradient real-time RT-PCR was carried out to select the best annealing temperature for
each putative fragrance-related transcript without any primer dimers. Gradient real-time
RT-PCR was carried out by using iQ5 cycler (Biorad, USA) utilizing the following
program: 950C for 10 min; 40 cycle of 95
0C for 30 sec, 55-65
0C for 30 sec (10
0C
temperature range) and 720C for 30 sec; 81 cycles for melting curve analysis; 10 sec for
each 0.50C (55-95
0C). Melt curve analyses were carried out for genes of interest and
70
Table 3: Primers Sequences for the Real-time RT PCR. Primers were designed at 3‟
end of the gene of interest and were synthesized by Sigma-Aldrich, USA. The primers
were used to analyze the expression of each putative fragrance-related transcript in
different tissues, at different developmental stages and different time points in a 24-hour
cycle.
Target/ Amplicon
Length (bp)
Primers
Primer Sequences
Annealing
Temperature
VMPCMEK
(201bp)
Forward
Reverse
5‟-GCTGTTTTCATGTCTGGAAGC-3‟
5‟-TCCTGTTTGTGACGGCTCTT-3‟
590C
VMPPAAS Forward
Reverse
5‟-ACGAAATGTTGGGAAATGAATA-3‟
5‟-ACTTGCCTTCTCTTGAACCA-3‟
590C
VMPCyP450
(182 bp)
Forward
Reverse
5‟-GCAACGCTCTCATGGTTTAT-3‟
5‟-AAAAGCCTCGAAAAATCTGA-3‟
570C
VMPA28
(202bp)
Forward
Reverse
5‟-GTACACGGAAACGATCACTG-3‟
5‟-AACATGCAAGCCAAACATT-3‟
590C
Endogenous
control /
Amplicon length
(bp)
Actin
(236bp)
Forward
Reverse
5‟-CAGTGTTTGGATTGGAGGTTC-3‟
5‟- CCAGCAGCAGTCAGGAAAA-3
590C
Alpha tubulin (227bp)
Forward
Reverse
5‟- CTCCCGCATTGACCATAAAT-3‟
5‟-GGAACCACACCCAAACTCTC-3‟
560C
Cyclophilin
(200bp)
Forward
Reverse
5‟-TTGGATGTCGTGAAGGCAAT-3‟
5‟-
CAACACAAGAAGATAGCACAGCA-3‟
590C
71
endogenous controls to check for sequence specificity amplification (see Appendix H).
The gradient real-time RT-PCR reaction was also carried out for each reference genes
(endogenous controls) using the same protocol as described above for the putative
fragrance-related transcript. The endogenous controls selected for the expression studies
were actin (Accession no: AF246716), alpha tubulin (Accession no: GW687608)
and cyclophilin (Accession no: GU942927). The optimal annealing temperature for
transcripts of interest and housekeeping genes are listed in Table 3.
PCR Amplification Efficiency Test
PCR amplification efficiency test was performed on all putative fragrance-related
transcripts and reference genes as decribed in the Guide to Performing Relative
Quantification of Gene Expression Using Real-time Quantive PCR (Applied Biosystems,
2004). First strand cDNA of fully-open flower of Vanda Mimi Palmer was used for PCR
amplification efficiency test. The cDNA was diluted in a series of five fold (50, 5
-1, 5
-2,
5-3
, and 5-4
). The PCR amplification was carried out utilizing the following program:
950C for 10 min; 40 cycle of 95
0C for 30 sec, annealing for 30 sec (see Table 3) and
extension at 720C for 30 sec; 81 cycles for melting curve analysis; 10 sec for each 0.5
0C
(55-950C). A standard curve was plotted against the log input of template cDNA amount
and the CT value of each dilution. The PCR efficiency was estimated by the slope of
standard curve (Equation A, in Appendix C), calculated using Equation B (Appendix C).
The accepted PCR efficiency is ranged between 90-110% and slope in the range of 3.1-
72
3.6 as decribed in the Guide to Performing Relative Quantification of Gene Expression
Using Real-time Quantive PCR (Applied Biosystems, 2004).
3.15.3 Expression Analysis of Putative Fragrance-related Transcripts
The relative quantity (Q) of cDNA transcript of each gene was calculated by the software
using Equation C (see Appendix C). Normalization expression was carried out by
normalizing the relative cDNA quantity of each gene of interest with cDNA of reference
genes (endogenous control) using Equation D (see Appendix C) where the relative
quantity of all reference genes was used for normalization factor (see Equation E in
Appendix C). Finally, the normalized expression level of the target gene was rescaled
with the calibrator using Equation F (see Appendix C).
For expression studies at different tissues and developmental stages, bud was used as
calibrator while for relative expression studies at different time points, the relative
quantity of the transcripts at 12.00am was used as calibrator. The calibrator for each
expression analysis was selected as a reference tissue for comparative amount of
transcript in each tissue studied whereby the expression of the calibrator is equal to 1.
Up-regulated expression refers to the relative amount of the transcript in the tissue that is
more than 1 compared to the calibrator while down-regulated expression refers to the
relative amount of the transcript in the studied tissue that less than 1 in comparison to the
calibrator.
73
Expression of each putative fragrance-related transcript was analyzed using iQ5 software
(Biorad, USA) by applying ∆∆CT comparative method. The ∆∆CT Comparative method
was used to estimate the relative expression level of the fragrance-related transcripts. The
normalized expression results were plotted in graphs for the relative quantity of the
transcripts in different tissues, at different developmental stages and different time points
in a 24-hour cycle compared to the calibrator.
74
CHAPTER 4
RESULTS
4.1 Biochemical Analysis on Flowers of Vanda Mimi Palmer
4.1.1 GC-MS Analysis of the Scent of Vanda Mimi Palmer
Floral scent compounds of Vanda Mimi Palmer were captured by solid phase micro-
extraction (SPME) and were determined by Gas chromatography-mass spectrometry
(GC-MS). GC-MS analysis (Figure 5 and Table 4) shows that the scent of Vanda Mimi
Palmer was dominated by terpenoids (linalool and ocimene) as well as benzenoids
(methylbenzoate and benzyl acetate) and a phenylpropanoid (phenylethyl acetate). A few
other compounds were also detected at low level such as linalool oxide, phenylethanol,
nerolidol, indole and formanilide. Determination of the compounds was based on
similarity of the mass spectra and the spectrum fragments of the compounds with the
compounds in the NIST mass spectral library 2002 with the cut off value of Similarity
Index (SI) equal to or more than 90%. Floral scent analyses in a 24-hour cycle in Figures
6 and 7 show that the emission of major terpenoids compounds (ocimene, linalool) as
well as benzenoids (methylbenzoate and benzyl acetate) and the phenylpropanoid
(phenylethyl acetate) started as early as 6.00am, and increased gradually until it reached
the highest peak at 2.00 noon. After that, the emission of the compounds decreased
gradually until 6.00pm. None of the volatile compounds was detected from the flowers
by GC-MS at night (from 8.00pm until 4.00am). During the highest peak of the scent
75
Figure 5: Gas Chromatogram of Volatile Compounds Emitted by Fully-open Flower of Vanda Mimi Palmer. The compounds
detected were: (1) ocimene, (2) linalool oxide, (3) linalool, (4) methylbenzoate, (5) phenylethanol, (6) benzyl acetate, (7) formanilide,
(8) phenylethyl acetate, (9) indole, and (10) nerolidol.
76
Table 4: Volatile Compounds Emitted by Vanda Mimi Palmer with Their Relative
Retention Time and m/z Fragments
Peak Relative
retention time
(min)
Main spectrum fragments
(m/z)
Compound
name
Monoterpene
1
2
3
8.636
9.147
9.592
36,41,53,67,79,93,105,121
41,43,59,81,93,112
41,43,69,71, 93,107,121,136
Ocimene
Linalool oxide
Linalool
Sesquiterpene
10
16.492
41,43,69,71,93,107,123,136,162
Nerolidol
Benzenoid
4
5
9.702
10.030
51,77,105,136
39,51,65,78,91,105,122
Methylbenzoate
Benzyl acetate
Phenylpropanoid
6
8
10.783
11.926
39,43,65,79,91,108,150
39,43,65,78,91,104
Phenylethanol
Phenylethyl
acetate
Indole
9
13.084
39,50,63,74,90,117
Indole
Formanilide
7
12.260
39,52,65,76,93,161
Formanilide
77
Emission of Terpenoid Compounds in a 24-hour cycle
0
250000
500000
750000
1000000
1250000
1500000
1750000
2000000
2250000
2500000
12am 2am 4am 6am 8am 10am 12pm 2pm 4pm 6pm 8pm 10pm
Time
Ab
un
da
nc
e
ocimene
linalool
nerolidol
linalool oxide
Figure 6: Emission of Terpenoids by Fully-open Flower of Vanda Mimi Palmer in a
24-hour Cycle: Ocimene, Linalool, Nerolidol and Linalool Oxide. The volatile
compounds of fully-open flower of Vanda Mimi Palmer were captured by solid phase
micro-extraction (SPME) before injection into GC-MS injector port. The experiment was
carried out for 12 hours in light, followed by 12 hours in dark. The error bars represent
the standard deviation of the relative amount of the volatile compounds detected in three
replicates of scent analysis.
78
Emission of Benzenoid/Phenylpropanoid Compounds in a 24-hour cycle
0
250000
500000
750000
1000000
1250000
1500000
1750000
2000000
2250000
2500000
2750000
3000000
3250000
3500000
3750000
4000000
12am 2am 4am 6am 8am 10am 12pm 2pm 4pm 6pm 8pm 10pm
Time
Ab
un
da
nc
e
methylbenzoate
benzyl acetate
phenylethyl acetate
phenylethanol
Figure 7: Emission of Benzenoids and Phenylpropanoids of Vanda Mimi Palmer in
a 24-hour Cycle: Methylbenzoate, Benzyl Acetate, Phenylethyl Acetate and
Phenylethanol (Phenylethyl Alcohol). The volatile compounds of fully-open flower of
Vanda Mimi Palmer were captured by solid phase micro-extraction (SPME) before
injection into GC-MS injector port. The experiment was carried out for 12 hours in light,
followed by 12 hours in dark. The error bars represent the standard deviation of the
relative amount of the volatile compounds detected in three replicates of scent analysis.
79
emission of Vanda Mimi Palmer (at 2.00pm), the scent of Vanda Mimi Palmer was
represented by 34.8% of terpenoids, 62.6% of benzenoids and phenylpropanoids, and
2.6% traces of other compounds including indole and formanilide (Figure 8A). The major
terpenoids detected in the scent of Vanda Mimi Palmer which were ocimene and linalool,
representing 34% of the total scent (ocimene 16.3% and linalool 17.7%) (Figure 8B).
Another terpenoids including linalool oxide (monoterpene) and nerolidol (seisquiterpene)
were detected at trace levels during the highest peak of scent emission which represent
0.3% and 0.5%, respectively, of the total scent (see Figure 8B). While for benzenoids and
phenylpropanoids, phenylethyl acetate was detected at the highest level representing
26.4% of the total scent of Vanda Mimi Palmer, followed by benzyl acetate (23.7%),
methylbenzoate (11.1%) and phenylethyl alcohol (phenylethanol) (1.4%) (see Figure 8B).
Scent analyses of Vanda Mimi Palmer at three developmental stages (bud, half-open
flower, and fully-open flower) in Figure 9 show no detectable volatile compounds
emitted at bud stage. In the half-open flower stage, emission of terpenoids incuding
linalool and ocimene was detected at a very high level compared to other compounds. In
the fully-open flower stage, scent emission of Vanda Mimi Palmer is at the highest level.
In contrast to half-open flower stage, the benzenoid and phenylpropanoid compounds
including benzyl acetate and phenylethyl acetate were detected at the highest level from
fully-open flower stage.
80
benzenoids and
phenylpropanoids
62.6%
other compounds
2.6%
terpenoids
34.8%
ocimene
16.3%
linalool oxide
0.3%
linalool
17.7%
methylbenzoate
11.1%
phenylethyl alcohol
1.4%
formanilide
0.4%
benzyl acetate
23.7%
phenylethyl acetate
26.4%
indole
2.2%
nerolidol
0.5%
Figure 8: Percentage of (A) Terpenoid, Benzenoid and Phenylpropanoid, and other
Compounds and (B) Each Compound in the Scent of Fully-open Flower of Vanda
Mimi Palmer. The pie charts show the percentage of each individual compounds and
class of compounds detected in the scent of Vanda Mimi Palmer during the highest peak
of scent emission at 2.00pm.
(A)
(B)
81
Figure 9: Comparison of Volatiles Emitted by Vanda Mimi Palmer at Three
Different Flower Developmental Stages: (A) bud (B) half-open flower and (C) fully-
open flower. The compounds are; (1) ocimene, (2) linalool oxide, (3) linalool, (4)
methylbenzoate, (5) phenylethanol, (6) benzyl acetate, (7) formanilide, (8) phenyletyl
acetate, (9) indole and (10) nerolidol.
82
The volatile compounds emitted by Vanda Mimi Palmer were compared with compounds
emitted by both parents, Vanda Tan Chay Yan (non-scented orchid) and Vanda tessellata
(scented orchid) in order to understand the fragrance characteristic of Vanda Mimi
Palmer. Emission of some volatile compounds including ocimene, decane and copaene in
Vanda Tan Chay Yan (Figure 10) were determined using GC-MS. The same GC-MS
analysis was not carried out for Vanda tessellata due to inavailability of the sample
source. Thus, a previously reported result by Kaiser (1993) on the scent of Vanda
tessellata was used for comparison. In his study, fully-open flower of Vanda tessellata
was trapped into a vessel for three hours (11.00 am-2.00 pm) and pumped into 10mg of
pure activated charcoal and then dissolved in 10-50µl of carbon disulphate followed by
10-30µl of ethanol solution before injecting into GC-MS (GC- Carlo Erba FTV 4160
chromatograph, MS- Varian MAT Model 212/CH-5 mass spectrometer with Finnigan
INCOS data system) (Kaiser, 1993). A comparison of the volatile compounds emitted by
Vanda Mimi Palmer and both parents is shown in Table 5. From the comparison,
ocimene was the only compound found to be emitted by Vanda Mimi Palmer and both of
its parents. Some of the compounds that were detected in the scent of Vanda Mimi
Palmer were also present in one of its parents (Vanda tessellata, a scented orchid) which
included benzyl acetate, methylbenzoate and linalool (Kaiser, 1993). Meanwhile, other
compounds emitted by Vanda Mimi Palmer included nerolidol, phenylethanol and
phenylethyl acetate were not detected in both parents.
83
Figure 10: Comparison of Volatiles Emitted by Fully-open Flower of (A) Vanda Mimi Palmer and (B) Vanda Tan Chay Yan. The compounds are: (1) ocimene, (2) linalool oxide, (3) linalool, (4) methylbenzoate, (5) phenylethanol, (6) benzyl acetate, (7)
formanilide, (8) phenyletyl acetate, (9) indole and (10) nerolidol (11) decane and (12) copaene.
84
Table 5: Comparison of Terpenoids, Benzenoids and Phenylpropanoids Emitted by
Vanda Mimi Palmer, Vanda tessellata and Vanda Tan Chay Yan
Compound Vanda Mimi
Palmer
Vanda Tan Chay
Yan
Vanda tessellata
(Kaiser, 1993)
Monoterpene
α-pinene
Linalool
Linalool oxide
Mycrene
Ocimene
Sesquiterpene
Copaene
Nerolidol
-
√
√
-
√
-
√
-
-
-
-
√
√
-
√
√
-
√
√
-
-
Benzenoid
Benzaldehyde
Benzyl acetate
Benzyl alcohol
Cinnamyl alcohol
Methyl benzoate
Methyl cinnamate
Methyl salicylate
Phenylpropanoid
Phenylethanol
Phenylethyl acetate
-
√
-
-
√
-
-
√
√
-
-
-
-
-
-
-
-
-
√
√
√
√
√
√
√
-
-
Note: The “ √ ” indicates the presence of the compound in the volatiles of the flowers
while the absence of the compound is indicated by “ - ”.
85
4.1.2 Analysis of Essential Oil of Vanda Mimi Palmer and Vanda Tan Chay Yan
Essential oil extraction was carried out on fully-open flower of Vanda Mimi Palmer and
Vanda Tan Chay Yan separately in order to understand the scent production and storage
in Vanda Mimi Palmer. The essential oil was extracted by soaking the flowers for 48
hours in hexane and followed by GC-MS analysis. The GC-MS detected 63 compounds
in the essential oil of Vanda Mimi Palmer and 63 compounds in the essential oil of Vanda
Tan Chay Yan (Appendix D, subsection (c) and (d)). Comparison between the
compounds detected in the essential oil of both flowers is presented in Table 6. Some of
the volatile compounds detected in the scent of Vanda Mimi Palmer (Figure 4 in section
4.1.1) which included linalool, methylbenzoate, phenylethanol, benzyl acetate and
phenylethyl acetate were also detected in the essential oil of Vanda Mimi Palmer (see
Table 6). Besides that, other intermediate compounds such as benzyl alcohol and benzyl
benzoate were also detected in the essential oil of Vanda Mimi Palmer. In addition,
another two sesquiterpenes which were germacrene and copaene were detected in the
essential oil of Vanda Mimi Palmer. Based on the comparison, phenylethanol compound
which was detected in the scent of Vanda Mimi Palmer., was also detected in the
essential oil of both Vanda Mimi Palmer and Vanda Tan Chay Yan. Interestingly, the
presence of phenylethanol compound in the essential oil of Vanda Mimi Palmer was
detected at high level representing 11.18% of the total essential oil (see Table 6). The
essential oil analysis shows that the terpenoids represent 2.09% of while benzenoids and
phenylpropanoids represent 26.51% of the total essential oil (Figure 11).
86
Table 6: Comparison of Terpenoids, Benzenoids and Phenylpropanoids Detected in
Essential Oils of Vanda Mimi Palmer and Vanda Tan Chay Yan
Compound
Vanda Mimi Palmer
Vanda Tan Chay Yan
Monoterpene
1-hydroxylinalool
Linalool
Sesquiterpene
Copaene
germacrene D
-
√ (1.79 %)
√ (0.17 %)
√ (0.13 %)
√ (0.38 %)
-
-
-
Benzenoid
Benzylacetaldehyde
Benzyl acetate
Benzyl alcohol
Benzyl benzoate
Methylbenzoate
Naphthalene
Phenylpropanoid
Phenylethanol
Phenylethyl acetate
Vanillin
√ (1.55 %)
√ (2.02 %)
√ (4.98 %)
√ (0.20 %)
√ (3.37 %)
√ (0.15 %)
√ (11.18 %)
√ (0.85 %)
√ (2.21 %)
√ (3.57 %)
-
-
-
-
-
√ (2.00 %)
-
-
Note: The “ √ ” indicates the presence of the compound in the essential oil of the flowers
while the absence of the compound is indicated by “ - ”. The full list and percentage of
volatiles, semi-volatiles and non-volatiles in the essential oil of both Vanda Mimi Palmer
and Vanda Tan Chay Yan can be referred to Appendix D, in subsection (c) and (d).
87
terpenoids
2.09%
benzenoids and
phenylpropanoids
26.51%
other compounds
71.40%
Figure 11: Composition of Terpenoids, Benzenoids and Phenylpropanoids, and
Other Compounds in the Essential Oil of Vanda Mimi Palmer.
88
4.2 Molecular Studies of Fragrance-related cDNA Transcripts of Vanda Mimi
Palmer
4.2.1 Isolation of Putative Fragrance-related cDNAs
4.2.1.1 Floral cDNA Library Screening
Isolation of fragrance-related cDNAs from Vanda Mimi Palmer was carried out by
screening the floral cDNA library representing all mRNA transcripts expressed in a 24-
hour cycle (12 hours in light and 12 hours in dark) and at different developmental stages
including bud, half-open flower, and fully-open flower. In a primary screening, 500,000
phages (500,000 pfu) were screened by hybridizing the floral cDNA library with fully-
open flower cDNA probe of Vanda Mimi Palmer representing all mRNA transcripts
expressed in fully-open flower between 8.00am to 6.00pm, a period when strong scent
emission of Vanda Mimi Palmer was detected (refer Figures 6 and 7). From the primary
screening (Figure 12a), 800 plaques showed positive signals on X-ray film by
autoradiography detection method. The plaques with positive signals represent mRNA
transcripts that are expressed between 8.00am to 6.00pm in fully-open flower of Vanda
Mimi Palmer. Secondary screening was applied to all the putative positive plaques
(Figure 12b) and the plaques with the strongest signal on X-ray film were cored out and
selected for in vivo excision.
89
Figure 12: Autoradiograph Containing Positive Signals (dark spot) of Putative
Positive Plaques from (a) Primary Screening; (b) Secondary Screening. From the
secondary screening, individual plaques with the strongest signal were cored out for in
vivo excision.
90
4.2.1.2 Reverse-Northern Analysis
All the clones were in vivo excised to phagemid form and used for reverse-Northern
analysis. The reverse-Northern analysis was carried out by hybridizing PCR-amplified
inserts (Figure 13a) with two different cDNA probes synthesized from mRNA transcripts
of fully-open flower (scent emitting stage) and bud (non-scent emitting stage) of Vanda
Mimi Palmer separately in order to isolate the clones that putatively represent fragrance-
related transcripts. From a total of 800 putative positive clones, 246 clones showed up-
regulated expression in fully-open flower stage during day time compared to the bud
stage (Figures 13b and 13c). A previous study on floral cDNA library of Vanda Mimi
Palmer has shown that half of the library was contaminated by transcripts encoding
Cymbidium mosaic virus coat protein (Chan, 2009). Thus, clones containing Cymbidium
mosaic virus coat protein transcripts (Figure 13d) were removed by hybridizing the PCR-
amplified cDNA inserts of the clones with probes representing the Cymbidium mosaic
virus coat protein transcripts. Upon eliminating transcripts related to Cymbidium mosaic
virus, only 62 clones were selected for plasmid isolation and sequenced as candidates for
fragrance-related cDNAs.
4.2.1.3 Sequencing and Analysis
Single pass sequencing result of the 62 clones were assembled into 5 contigs and 52
singletons using the Cap3 sequence assembly program (Appendix E). All of the 62
sequences (100%) were readable sequences with more than 285bp. The tentative unique
91
Figure 13: Hybridization of (a) PCR Amplified Inserts with Three Different Probes;
(b) Fully-open Flower cDNA Probe, (c) Bud Stage cDNA Probe, and (d) Cymbidium
mosaic Virus cDNA Probe. White-circles show selected clones that have up-regulated
expression in fully-open flower of Vanda Mimi Palmer during day time compared to bud
stage after removal of clones containing Cymbidium mosaic virus coat protein cDNA.
Lane 1-25 represents PCR product of putative positive clones isolated from the screening
of floral cDNA library.
92
gene sequences (TUG) were aligned against the GeneBank nucleotide and protein
database (BLASTX) to search for sequence similarity with the cut off value less than 1e-
05 and score more than 80. From the BLASTX search, fifty-seven tentative unique gene
sequences (TUG) were similar to the sequences in NCBI database (Appendix E). The
other five sequences had e-value more than 1e-05
and were classified as sequences with
no-significant hit due to no similarity sequence in the NCBI database. All the ESTs were
classified into eleven groups based on their putative functionality (Zhao et al., 2006). The
groups are metabolism (19%), unknown (16%), protein synthesis (11%), cellular
transport (10%), cell cycle and DNA processing (10%), protein with binding function
(8%), energy (8%), cell rescue, defense and virulence (6%), development (6%),
biogenesis of cellular component (3%) and transcription (3%) (see Figure 14 and
Appendix E). The unknown group represents ESTs with unknown function including the
sequences that hit to hypothetical proteins and also with non-significant hit sequences.
4.2.1.4 Verification of Putative Fragrance-related cDNAs with Up-regulated
Expression in Fully-open Flower Compared to Bud of Vanda Mimi Palmer
Based on BLASTX and literature search results, it was postulated that some cDNA
transcripts classified in metabolism and unknown group might be potential putative
fragrance-related cDNAs. Thus, a verification was carried out for thirteen selected clones
(potential putative fragrance-related cDNA clones) by RT-PCR using two different
93
metabolism
19%
unknown
16%
protein synthesis
11%cellular transport
10%
cell cycle and DNA
processing
10%
protein with binding
function
8%
energy
8%
Cell rescue, defence
and virulence
6%
Development
6%
biogenesis of cellular
component
3%
transcription
3%
Figure 14: Classification of the Clones with Up-regulated Expression in Fully-open
Flower Compared to Bud of Vanda Mimi Palmer. The 62 clones with up-regulated
expression in fully-open flower compared to bud of Vanda Mimi Palmer were classified
into eleven groups based on their putative functionalities.
94
tissues which are fully-open flower and bud to choose for the cDNAs clones that have
up- regulated expression in the fully-open flower. Three cDNA clones from the
metabolism group including putative esterase (VMPEST), putative 4-(cytidine 5′-
diphospho)-2-C-methyl-d-erythritol kinase (VMPCMEK) and putative cytochrome p450
protein (VMPCyP450) were selected for the verification because the selected transcripts
have been reported to be involved in fragrance biosynthesis in other plants. Besides that,
another 10 putative fragrance-related transcripts from unknown group including
hypothetical protein and no significant hit protein transcripts were selected for
verification of their expression levels in the two different tissues. The cDNAs from this
group could be novel transcripts involved in the fragrance biosynthetic pathway.
From the verification results, 5 clones showed up-regulated expression in fully-open
flower compared to bud, 3 clones showed down-regulated expression in fully-open
flower compared to bud and 6 clones showed equal expression in both fully-open flower
and bud stages (see Table 7). Three putative fragrance-related cDNA clones which
showed up-regulated expression in fully-open flower compared to bud of Vanda Mimi
Palmer were selected for full-length cDNA isolation and expression analysis by real-time
RT-PCR. The selected cDNA clones were putative 4-(cytidine 5′-diphospho)-2-C-
methyl-d-erythritol kinase (hereafter referred as VMPCMEK), putative cytochrome p450
protein (hereafter referred as VMPCyP450) and an unknown protein cDNA transcript
(hereafter referred as VMPA28).
95
Table 7: Verification of Putative Fragrance-related cDNAs with Up-regulated
Expression in Fully-open Flower Compared to Bud of Vanda Mimi Palmer by RT-
PCR; (a) cDNA transcripts of the selected clones were PCR-amplified using first-strand
cDNA of the selected tissues. (b) The PCR products were hybridized with specific probe
representing the cDNA sequence of each transcript (reverse Northern). Each lane
representing different tissues selected for the verification; 1) bud of Vanda Mimi Palmer
2) fully-open flower of Vanda Mimi Palmer. “No significant hit” and “hypothetical
protein” transcript sequences were named as unknown protein.
Clone Name PCR and reverse Northern Housekeeping gene
(elongation factor)
51 Putative cytochrome
P450 (VMPCyP450)
(up-regulated in fully-
open flower stage)
1 2
1 2
31 Putative esterase
(VMPEST)
(down-regulated in fully-
open flower stage)
1 2
1 2
71 Putative 4-(cytidine 5'-
diphospho)-2-C-methyl-
D-erythritol kinase
(VMPCMEK)
(up-regulated in fully-
open flower stage)
1 2
1 2
33 Hypothetical protein
(up-regulated in fully-
open flower stage)
1 2
1 2
A36 Unknown protein
(equal expression)
1 2
1 2
(a)
(b)
(a)
(b)
(a)
(b)
(a)
(b)
(a)
(b)
(a)
(b)
(a) (b)
(a)
(b)
(a)
(b)
(a)
(b)
96
Clone Name PCR and reverse Northern Housekeeping gene
(elongation factor)
48 Hypothetical protein
(up-regulated in fully-open
flower stage)
1 2
1 2
59 Unknown protein
(down-regulated in fully-
open flower)
1 2
1 2
83 Hypothetical protein
(equal expression)
1 2
1 2
90 Hypothetical protein
( down-regulated in fully-
open flower stage )
1 2
1 2
96 Unknown protein
(down-regulated in fully-
open flower)
1 2
1 2
A28 Unknown protein
(up-regulated in fully-open
flower)
1 2
1 2
A46 Unknown protein
(equal expression)
1 2
1 2
A54 Hypothetical protein
(equal expression)
1 2
1 2
(a)
(b)
(a)
(b)
(a)
(b)
(a)
(b)
(a)
(b)
(a)
(b)
(a)
(b)
(a)
(b)
(a)
(b)
(a)
(b)
(a)
(b)
(a)
(b)
(a)
(b)
(a)
(b)
(a)
(b)
(a)
(b)
97
4.2.2 Cloning and Characterization of Selected Fragrance-related Transcripts
Molecular characterization was carried out on three putative fragrance-related transcripts
that showed up-regulated expression in fully-open flowers of Vanda Mimi Palmer. The
selected transcripts were putative 4-(cytidine 5'-diphospho)-2-C-methyl-D-erythritol
kinase (VMPCMEK), putative cytochrome P450 protein (VMPCyP450) and an unknown
protein (VMPA28). In addition, a putative phenylacetaldehyde synthase (VMPPAAS)
previously identified from the floral ESTs of Vanda Mimi Palmer by Miss Chan Wai Sun
was also selected for molecular characterization. Putative fragrance-related cDNA
transcripts were subjected to full-length cDNA isolation and gene expression analysis.
Gene expression analysis was carried out in different tissues, at different developmental
stages, and different time points in a 24-hour cycle.
4.2.2.1 Sequence and Expression Analyses of Putative Phenylacetaldehyde Synthase
(VMPPAAS)
The full-length sequence of VMPPAAS cDNA is 1709 bp consisting of 1524bp open
reading frame (ORF) flanked by a 92bp 5‟ untranslated region (UTR) and a 93bp 3‟UTR
including a poly-A tail (Figure 15). The ORF encodes a protein of 508 amino acid
residues (Figure 16). The predicted molecular weight of this protein is 56.1 kD with an
isoelectric point (pI) of 7.1.
98
3 GCG GGA TCG CGG CAA GGC AGA AGG CAA CCA AAA AAC AAA AAA AAA 47
48 AAA AAC AAG CTT GTT GCC TTT CCC ATT CCC TAG GAA CTC CAG AAA 92
>>>>>>>>>>>>>>>
93 ATG GGC AGC CTT CCC ACC GAA CCA TTC CTG CCA CTA GAC CCA GAC 137
1 M G S L P T E P F L P L D P D 15
>>>>>>>>>>>>>>>>>>>>>>>>>>>>>
VMPPAAS ORF Forward primer
138 CGC TTC ACC AAA GAA TCC AAA GCC GTC GTC GAC TTC ATC GCC GAC 182
16 R F T K E S K A V V D F I A D 30
183 TAC TAC CGC CAA ATA GAA CTC TTC CCT GTT CGC AGC CAA GTA AAG 227
31 Y Y R Q I E L F P V R S Q V K 45
228 CCA GGC TAT CTC CAT GAC CGC ATT CCA AAC ACT CCC CCC ATC CTC 272
46 P G Y L H D R I P N T P P I L 60
273 TCC GAA CCC ATC ACC ACA ATC CTC CAC GAC ATT AAA ACA GAC ATC 317
61 S E P I T T I L H D I K T D I 75
318 TTT CCC GGA CTA ACC CAC TGG CAA AGC CCC AAT TTT TAC GGC TAC 362
76 F P G L T H W Q S P N F Y G Y 90
363 TAC CAA GCC AAT GCC AGC ACC CCC GGT TTC GCC GGA GAG ATG CTC 407
91 Y Q A N A S T P G F A G E M L 105
408 TGT TCC GGC CTC AAC GTC GTC GGC TTC AGC TGG ATC GCT TCC CCT 452
106 C S G L N V V G F S W I A S P 120
453 GCC GCC ACT GAA CTA GAA ACC ATC ATC ATG GAC TGG ATG GCC AAG 497
121 A A T E L E T I I M D W M A K 135
498 ATG CTC AAA CTT CCA TCA ACC TTC CTT TCC GGA CAC CTC GGC GGC 542
136 M L K L P S T F L S G H L G G 150
543 GGC GGT GGC GTA ATC CAC GGC AGC ACG TGC GAA GCG GTG CTC TGC 587
151 G G G V I H G S T C E A V L C 165
588 ACC CTC GCC GCT GCT AGA GAT AAC GCT TTG AGC AAG AGC GAC GGC 632
166 T L A A A R D N A L S K S D G 180
633 GAA GGG ATC ACG AAG CTG ACG GTA TAT GTC TCT GAT CAG ACA CAT 677
181 E G I T K L T V Y V S D Q T H 195
678 TTT ACG GTT CAG AAG GCG GCG AAG TTG GTT GGA ATC CCG ACG CGG 722
196 F T V Q K A A K L V G I P T R 210
723 AAC TTA CGG GTG ATA TCG ACT TCG AGG GAG ACA GGG TAT GCC TTG 767
211 N L R V I S T S R E T G Y A L 225
768 ACG GCG GAG ATT GTG AGG GCG GCG ATG GAT GCT GAT GTG GCG GCA 812
226 T A E I V R A A M D A D V A A 240
813 GGG ATG GTG CCG CTG TAT TTG TGT GGC ACG GTG GGG ACG ACG GCT 857
241 G M V P L Y L C G T V G T T A 255
858 GTG GGG GCG GTG GAC CCG ATA AGG GAG ATC GGG GAG GTT GCG AGG 902
256 V G A V D P I R E I G E V A R 270
903 GAG TTC GGG GTG TGG TTC CAC GTG GAC GCG GCG TAT GCG GGG AGC 947
99
271 E F G V W F H V D A A Y A G S 285
948 GCT GGG ATT TGC CCT GAG TTC CGG CGG TTC TTT GAT GGA GTG GAG 992
286 A G I C P E F R R F F D G V E 300
993 ACG GCT GAT TCC TTT AGT TTG AAT CCG CAT AAA TGG CTG CTC GCA 1037
301 T A D S F S L N P H K W L L A 315
1038 AAC ATG GAC TGT TGT TGC CTT TGG GTA AGA TGT GCA ACG AAG CTC 1082
316 N M D C C C L W V R C A T K L 330
1083 GTA GAC TCG TTA TCG ACC AAG CCG GAG ATA TTG ACA AAC AGT GCT 1127
331 V D S L S T K P E I L T N S A 345
1128 AGC GAA GAT GGC AAA GTG ATT GAC TAC AAA GAT TGG CAG GTC GCA 1172
346 S E D G K V I D Y K D W Q V A 360
1173 CTG AGT CGT AGG TTT CGT GCA ATG AAG CTA TGG ATA GTC ATC AGA 1217
361 L S R R F R A M K L W I V I R 375
1218 CGA TTT GGA GTT GCT AAC CTG ATG GAG CAC ATC AGG AGC GAT GTG 1262
376 R F G V A N L M E H I R S D V 390
1263 GAG ATG GCC AAG CAT TTC GAG AGA CTT GTC GCC GAG GAT GAG AGG 1307
391 E M A K H F E R L V A E D E R 405
1308 TTT GAG GTG GTT GTA CCA AGA AGA TTC ACG CTC GTT TGT TTT AAA 1352
406 F E V V V P R R F T L V C F K 420
1353 TTG AGG TAT GTG GGA GAA GAT ATT GAT GAA GAG GAG GGG ACG AAA 1397
421 L R Y V G E D I D E E E G T K 435
>>>>>>>
1398 TGT TGG GAG ATG AAT AAG AAG TTG CTC GAT TCG GTG AAC GAA AGT 1442
436 C W E M N K K L L D S V N E S 450
>>>>>>>>>>>>>>>>>>>>>
VMPPAAS RT-PCR Forward Primer
1443 GGA CGA GCA TTC ATG ACC CAT GCG GTT GTT TGC GGG CAG TTT GTG 1487
451 G R A F M T H A V V C G Q F V 465
1488 CTG CGG TTT GCA CTT GGC GCC ACG TTG ACA GAG ATA CGA CAT GTG 1532
466 L R F A L G A T L T E I R H V 480
1533 GAG GAG ACA TGG AGG TTG GTT CAA GAG AAG GCA AGT GAG TTG TTG 1577
481 E E T W R L V Q E K A S E L L 495
<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<
VMPPAAS RT-PCR Reverse Primer
1578 ATG ATT ACG AAT GAG CTG GGT TGG AAA CTC AAA ACA CTC TGA GAT 1622
496 M I T N E L G W K L K T L * 508
<<< <<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<
VMPPAAS ORF reverse primer
1623 AGC TCA CTT AAA ATA AAA AGA GAA ATA ATA GTT TCA AAT GAA ATA 1667
1668 AAA TTA TAA AAA ATT TAA AAA TTT AAA GAA AAA AAA AAA AAA 1709
Figure 15: The Nucleotide and Deduced Amino Acid Sequence of VMPPAAS
(Putative Phenylacetaldehyde Synthase). The open reading frame (ORF) of
VMPPAAS starts at nucleotide 93. The asterisk (*) indicates stop codon. The putative
polyadenylation signal is bold and underline. The location of gene specific primers used
in real-time PCR analysis and ORF isolation are shown by arrow heads (>>>> and <<<<).
100
Figure 16: PCR Product of the Open Reading Frame (ORF) of Putative
Phenylacetaldehyde Synthase of Vanda Mimi Palmer (VMPPAAS). The ORF
represents 508 amino acids. The PCR product was electrophoresed on 1% (w/v) agarose
gel. Lane M: 100bp marker (Vivantis, Malaysia); lane 1 and 2: gap; lane 3: PCR product
of ORF of VMPPAAS.
1500bp
1000bp
500bp
M 1 2 3
~1550bp 2000bp
3000bp
101
BLASTX and BLASTP analysis showed that VMPPAAS has the closest similarity to
putative tyrosine/dopa decarboxylase (AAK50420.1) and pyridoxal-dependent
decarboxylase conserved domain containing protein (ABB47543.1) of Oryza sativa from
japonica-cultivar group, corresponding to 57% identity. The VMPPAAS also shows
similarity to tyrosine decarboxylase of Aristolochia contorta (ABJ16446.1) and
tyrosine/dopa decarboxylase of Papaver somniferum (AAC61844.1) corresponding to
55% and 54% identities, respectively. Besides that, VMPPAAS also shows similarity to
fragrance-related amino acid sequences of well studied scented flowers including
aromatic L-amino acid decarboxylase of Rosa hybrida (BAF64844.1) (56% identity),
phenylacetaldehyde synthase of Rosa hybrida (ABB04522.1) (56% identity) and
phenylacetaldehyde synthase of Petunia hybrida (ABB72475.1) (54% identity).
Localizome analysis using Localizome software (Lee et al., 2006) revealed a domain for
pyridoxal-dependent decarboxylase in the amino acid sequence of VMPPAAS. In
addition, the same software also predicted no signal peptide present in the amino acid
sequence of VMPPAAS and in all of the closely related protein sequences. Prediction of
motifs present in the deduced protein sequence of VMPPAAS and the closely related
proteins by the Expasy tool showed a conserved motif for a pyridoxal phosphate
attachment site (see Figure 17). Besides that, there are two N-myristoylation sites
(GVxxGS and GAxxTE), three casein kinase II phosphorylation sites (TxxE, SxxE, and
TxxE), two N-glycosylation sites (NAST and NESG), and a site for cAMP- and c-GMP-
dependent protein kinase phosphorylation (RRxT) in the VMPPAAS amino acid
sequence (see Figure 17). There is another motif (VHVDAAY) shared by VMPPAAS
102
Figure 17: Alignment of VMPPAAS (Putative Phenylacetaldehyde Synthase) with
Other Closely Related Protein Sequences from the GeneBank. The VMPPAAS amino
acids sequence was aligned with putative tyrosine/dopa decarboxylase (AAK50420.1) of
Oryza sativa, tyrosine/dopa decarboxylase of Papaver somniferum (AAC61844.1),
aromatic L-amino acid decarboxylase of Rosa damascena (BAF64843.1),
phenylacetaldehyde synthase of Rosa hybrida (ABB04522.1) and phenylacetaldehyde
synthase of Petunia hybrida (ABB72475.1). The pyridoxal phosphate attachment site and
HVDAAY motif are highlighted in box. Four stars (****) indicate the conserved motif
for N-myristoylation sites (GVxxGS and GAxxTE) meanwhile two stars (**) indicate the
conserved motif for casein kinase II phosphorylation sites (TxxE, SxxE and TxxE). The
conserved motif for N-glycosylation sites (NAST and NESG) are marked with three stars
(***).
VHVDAAY motif
Pyridoxal phosphate attachment site
NAST
***
TxxE
***
GVxxGS
****
SxxE
**
TxxE
**
NESG
***
103
and other decarboxylases protein either from plants or bacteria (Connil et al., 2002) (see
Figure 17). A phylogenetic tree (Figure 18) was constructed using MEGA version 4.0
(Tamura et al., 2007) to estimate the genetic relatedness between amino acid sequence of
VMPPAAS with other plant decarboxylases. In the phylogenetic tree, VMPPAAS is
clustered together with protein sequences from aromatic amino acid of Ricinnus
communis, and supported by the bootstap value of 93 for the branch.
Relative expression study of VMPPAAS transcript in different tissues (Figure 19) of
Vanda Mimi Palmer showed up-regulated expression in floral tissues including petal,
sepal and lip but down-regulation in vegetative tissues including leaf, shoot, root and
stalk. Among the floral tissues, petal has shown the highest expression of VMPPAAS
transcript which is more than 20,000-fold expression compared to bud tissue as the
calibrator. In the sepal, the expression of VMPPAAS transcript was also detected at high
level (nearly to 15,000-fold expression) compared to the calibrator. Meanwhile in the lip,
the VMPPAAS transcript was detected at 5000-fold higher expression compared to the
calibrator.
Relative expression analysis at five different flower developmental stages shows that the
VMPPAAS transcript was differentially expressed during the flower life cycle (Figure
20). In bud stages including young and mature buds, the expression of VMPPAAS
transcript was down-regulated compared to half-open and fully-open flower stages. At
14-day fully-open flower, the expression of VMPPAAS transcript decreased drastically at
very low level.
104
VMPPAAS (Vanda Mimi Palmer)
OsTyDC(Oryza sativa-japonica cultivar...
RcAADC (Ricinus communis)
PsTyDC(Papaver somniferum)
RsAADC (Rosa hybrida)
RhPAAS (Rosa hybrida)
RdAADC (Rosa x damascena)
PhPAAS (Petunia hybrida)
CaTDC(Camptotheca acuminata)
AtTyDC (Arabidopsis thaliana)
ZmTyDC1 (Zea mays)
OsTDC (Oryza sativa Japonica Group)
ZmTyDC2 (Zea mays)
Figure 18: Phylogenetic Tree of VMPPAAS with Homologous Proteins. The
phylogenetic tree is constructed by MEGA software version 4.0 using amino acid
sequence of VMPPAAS with the homologous proteins available in GeneBank database
(NCBI) including tyrosine decarboxylase of Oryza sativa (AAK50420.1), aromatic
amino acid decarboxylase of Ricinus communis (EEF36965.1), tyrosine/DOPA
decarboxylases Papaver somniferum (AAC61844.1), aromatic L-amino acid
decarboxylase of Rosa hybrida (BAF64844.1), phenylacetaldehyde synthase of Rosa
hybrida (ABB04522.1), aromatic L-amino acid decarboxylase of Rosa damascene
(BAF64843.1), phenylacetaldehyde synthase of Petunia hybrida (ABB72475.1),
tryptophan decarboxylases of Camptotheca acuminata (AAB39708.1), tyrosine
decarboxylase of Arabidopsis thaliana (AAL69507.1), tyrosine/DOPA decarboxylase 1
of Zea mays (ACG29316.1), tryptophan decarboxylase of Oryza sativa (BAD35168.1)
and tyrosine/dopa decarbxylase 2 of Zea mays (ACG46884.1).
105
0
5000
10000
15000
20000
25000
bud fully-
open
flower
petal sepal lip leaf root shoot stalk
Tissues
Rel
ativ
e
Exp
ress
ion
of
VM
PP
AA
S
(fo
ld c
han
ge)
Figure 19: Relative Expression Study of VMPPAAS Transcript in Different Tissues
of Vanda Mimi Palmer. The floral tissues are bud, fully-open flower, petal, sepal and lip
meanwhile the vegetative tissues are leaf, root, shoot and stalk. The quantitative
expression level of VMPPAAS in each tissue was calculated relative to the calibrator
which was the bud collected at 12.00 noon. Standard error between three replicates of
relative gene expression in each tissue is indicated by error bar.
0
15000
30000
45000
60000
75000
90000
105000
young bud
(green)
mature bud
(red)
half-open
flower
fully-open
flower
14-day old
flower
Developmental Stages
Rela
tive
Exp
ress
ion
of V
MPP
AAS
(fol
d ch
ange
)
Figure 20: Relative Expression Analysis on VMPPAAS Transcript at Different
Developmental Stages During Flower Development. This study was carried out on
five flower developmental stages of Vanda Mimi Palmer including young bud, mature
bud, half-open flower, fully-open flower and 14-day old fully-open flower. Quantitative
measurement of VMPPAAS expression in each flower developmental stage was
expressed relative to the calibrator which was the young bud collected at 12.00 noon.
Standard error between three replicates of relative gene expression at each developmental
stage is indicated by error bar.
106
Relative expression study of VMPPAAS transcript at different time points in a 24-hour
cycle (12 hours in light, followed by 12 hours in dark) (Figure 21) shows a differential
expression. From the analysis, the expression of VMPPAAS was higher at night (7.00pm
to 7.00 am) compared to day time (7.00am to 7.00pm). From 8.00am until 6.00pm (in
light) the expression level was lower compared to night. However, after 6.00pm the
amount of VMPPAAS transcript increased more than three fold with the highest peak at
10.00pm.
4.2.2.2 Sequence and Expression Analyses of Putative 4-(cytidine 5'-diphospho)-2-C-
methyl-D-erythritol Kinase (VMPCMEK)
The full-length cDNA sequence of VMPCMEK is 1446bp, comprising 1200bp ORF,
51bp of 5‟ UTR and 195bp of 3‟UTR with a polyadenylation tail (poly-A tail) (Figure
22). The ORF encodes a protein of 400 amino acid residues (Figure 23) with a predicted
molecular weight of 44.1 kD and an isoelectric point (pI) of 8.4.
BLASTX and BLASTP analysis (NCBI) shows the deduced amino acid sequence of
VMPCMEK exhibited similarity to 4-(cytidine 5'-diphospho)-2-C-methyl-D-erythritol
kinase from other plants (66-73% identity). It has the highest similarity to 4-(cytidine 5'-
diphospho)-2-C-methyl-D-erythritol kinase of Nicotiana benthamiana (ABO87658.1),
107
0.00
0.50
1.00
1.50
2.00
2.50
3.00
3.50
4.00
12am 2am 4am 6am 8am 10am 12pm 2pm 4pm 6pm 8pm 10pm
Time
Rela
tive e
xp
ressio
n o
f V
MP
PA
AS
(fo
ld c
han
ge)
Figure 21: Relative Expression Analysis of VMPPAAS at Different Time Points in a
24-hour Cycle. Quantitative expression of VMPPAAS at each time point was expressed
relative to the calibrator which was the fully-open flower collected at 12.00 noon.
Standard error between three replicates of relative gene expression at each time point is
indicated by error bar.
108
1 GGC TGC TGC AAA AAG CAT AAC TCC CTA CGC TTC TCA GCT TTT CTC 45
>>>>>>>>>>>>>>>>>>>>>>>
46 CTA ACA ATG GCC TCT TTC TCT AAT CAT CTG ATT TCA TCG TTC TCA 90
M A S F S N H L I S S F S 13
>>>>>>>>>>>>>>>>>
VMPCMEK ORF Forward Primer
91 TGC TCG AGA AGA AGT GCT TCA CTG CCC CGA AGA GGG AAT ACC CCC 135
14 C S R R S A S L P R R G N T P 28
136 TCG ATC TTT CGA CAT GGT CAT TAC TCT TTC AAA TCT TGG TCT GAG 180
29 S I F R H G H Y S F K S W S E 43
181 GTC AGC GGG AAC AAG TAT GGT AGA ATC CTC ATT TGT GCG GCA GAA 225
44 V S G N K Y G R I L I C A A E 58
226 ACT GGG AGG AGG CAA GTG GAG ATT GTT TAT GAT CCG GAG GAG AGG 270
59 T G R R Q V E I V Y D P E E R 73
271 TTT AGT GGA CTG GAG GGT GAA GTA GAT GAT AAC AAC AAG CTT TCT 315
74 F S G L E G E V D D N N K L S 88
316 AGG TTG ACC CTA TTC TCG CCG TGT AAG ATT AAT GTT TTC TTG AGG 360
89 R L T L F S P C K I N V F L R 103
361 ATA ACT GGA AAG AGG AAT GAT GGG TTT CAT GAT TTG GCC TCT TTG 405
104 I T G K R N D G F H D L A S L 118
406 TTT CAT GTA ATC AGT TTA GGA GAT ACG ATT AAA TTC TCC TTG TCA 450
119 F H V I S L G D T I K F S L S 133
451 CCA TTA AAG AGA AAG GAT CGC CTG TCA ACT AAT GTG CCG GGA GTT 495
134 P L K R K D R L S T N V P G V 148
496 CCA GTT GAT GAT AGA AAT TTG ATA ATC AGA GCT CTC AAT CTT TAC 540
149 P V D D R N L I I R A L N L Y 163
541 AGG AAG AAG ACA GGC ACA AAC AAT TTC TTC CAG ATT GAG CTT GAC 585
164 R K K T G T N N F F Q I E L D 178
586 AAA AAA GTT CCT ACT GGT GCT GGG CTT GGT GGT GGA AGT AGT AAC 630
179 K K V P T G A G L G G G S S N 193
631 GCA GCA ACT GCT TTA TGG GCT GCC AAC CAG TTC AGT CGT TCT CTT 675
194 A A T A L W A A N Q F S R S L 208
676 GTT ACT GAA AAA GAG CTT CAG GAT TGG TCA GGT GAA ATT GGT TCA 720
209 V T E K E L Q D W S G E I G S 223
721 GAT ATT CCT TTT TTT TTC TCT AAT GGG GCT GCA TAT TGT ACC GGT 765
224 D I P F F F S N G A A Y C T G 238
766 AGG GGA GAG GTT GTT AAA GAA CTT CCT TTT GCA TTG CCC AAG GAC 810
239 R G E V V K E L P F A L P K D 253
811 CTG CCA ATG GTT CTT ATA AAG CCC CAA GAA GCA TGT CCA ACC GCC 855
254 L P M V L I K P Q E A C P T A 268
856 GAA GTG TAC AAG CGA CTT CAT CTT GGT AAA ACT AGT TCA GTT GAC 900
269 E V Y K R L H L G K T S S V D 283
901 CCG TTG ACT CTG CTA GAA AAG ATA TCT CTA AAT GGA ATA TCT CAA 945
109
284 P L T L L E K I S L N G I S Q 298
946 GAT GTC TGC ATA AAT GAT CTT GAA CCC CCT GCA TTT GAT GTT TTG 990
299 D V C I N D L E P P A F D V L 313
991 CCA TCC TTG AAG AAG TTG AAG CAA CGT GTG CTA GCT GCA GGG CGT 1035
314 P S L K K L K Q R V L A A G R 328
1036 GGC CAG TAT AGT GCT GTT TTC ATG TCT GGA AGC GGA AGC ACC ATT 1080
329 G Q Y S A V F M S G S G S T I 343
>>>>>>>>>>>>>>>>>>>>>>>>>>>
VMPCMEK RT-PCR Forward Primer
1081 GTG GGA ATT GGT TCA CCA GAC CCA CCT CAA CTT GTT TAT GAT GAG 1125
344 V G I G S P D P P Q L V Y D E 358
1126 GAT GAA TAC AAT GAT GTT TTC ATA ACA GAG GCT TCC TTT CTC ACT 1170
359 D E Y N D V F I T E A S F L T 373
1171 CGG CAA CAG AAT CAG TGG TAC GCA GAG CCA ACT TCG TCC ACA GGG 1215
374 R Q Q N Q W Y A E P T S S T G 388
1216 TCT TTG AGC AGA GAA GAG CCG TCA CAA ACA GGA AAA TAA TTA CGA 1260
389 S L S R E E P S Q T G K * 400
<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<
VMPCMEK ORF Reverse Primer
<<<<<<<<<<<<<<<<<<<<<<<<<<
VMPCMEK RT-PCR Reverse Primer
1261 TAA TTT TTT TAC ATT CTA GAC CTT CTA ATT TTA ATT TTT CTC ACA 1305
1306 TAA AAT CAT ATT GTA TTA CTG TAC TTA TTG TTC ATG CAA GAA AGA 1350
1351 TCG ATC AAG CTA TCT TTC ATG AAT GAG CAA AAT ATG CAA TTT TAA 1395
1396 AAG GCA CAT TTA CAT GCT TAA AAA AAA AAA AAA AAA AAA AAA AAA 1440
1441 AAA AAA 1446
Figure 22: The Nucleotide and Deduced Amino Acid Sequences of VMPCMEK
(putative 4-(cytidine 5'-diphospho)-2-C-methyl-D-erythritol kinase). The open
reading frame (ORF) of VMPCMEK starts at nucleotide 52. The asterisk (*) indicates
stop codon. The putative polyadenylation signal in the nucleotide sequence is bold and
underline. The location of gene specific primers used in real-time PCR analysis and ORF
isolation are shown by arrow heads (>>>> and <<<<).
110
Figure 23: PCR Product of the Open Reading Frame (ORF) of Putative 4-(cytidine
5'-diphospho)-2-C-methyl-D-erythritol Kinase of Vanda Mimi Palmer
(VMPCMEK). The PCR product was electrophoresed on 1% (w/v) agarose gel. Lane M:
100bp marker (Vivantis); Lane 1: PCR product of ORF of VMPCMEK.
1200bp
1000bp
500bp
M 1
~1200bp
111
corresponding to 73% identity. The closest similarity was followed by 4-(cytidine 5'-
diphospho)-2-C-methyl-D-erythritol kinase of Solanum lycopersicum (AAF87717.1) and
Catharanthus roseus (ABI35992.1), corresponding to 72% identity. Besides that, the
VMPCMEK sequence also showed similarity to two 4-(cytidine 5'-diphospho)-2-C-
methyl-D-erythritol kinase proteins of Ginkgo biloba (AAZ80385.1 and AAZ80384.1),
corresponding to 70% and 69% identity, respectively.
VMPCMEK and all of the closely related proteins were predicted to have no signal
peptide. Prediction of motifs by Expasy tool revealed three conserved N-myristoylation
sites (GAxxCT, GQxxAV and GSxxTI) shared by VMPCMEK and other closely related
proteins (Figure 24). The software also predicted the presence of another conserved site
in VMPCMEK and the closely related protein sequences which is cAMP- and cGMP-
dependent protein kinase phosphorylation site (RKxT). Besides that, a conserved motif
for ATP-binding site for the functional activity of 4-(cytidine 5'-diphospho)-2-C-methyl-
D- erythritol kinase (Kim et al., 2008) was also detected in the sequence of VMPCMEK
and its closely related proteins (Figure 24).
A phylogenetic tree (Figure 25) was constructed using MEGA software version 4.0
(Tamura et al., 2007) to estimate the genetic relatedness between amino acid sequence of
VMPCMEK with its closely related proteins. From the phylogenetic tree, VMPCMEK
amino acid sequence is not clustered together with 4-(cytidine 5'-diphospho)-2-C-
methyl-D- erythritol kinase proteins from Solanum lycopersicum, Nicotiana
benthamiana, and Cantharanthus roseus.
112
Figure 24: Alignment of VMPCMEK with Other Closely Related Protein Sequences
Downloaded from the GeneBank Database. The VMPCMEK is aligned with
HbCMEK (4-(cytidine 5'-diphospho)-2-C-methyl-D-erythritol kinase) of Hevea
brasiliensis (BAF98293.1), SlCMEK (4-(cytidine 5'-diphospho)-2-C-methyl-D-erythritol
kinase) of Solanum lycopersicum (AAF87717.1), CrCMEK (4-(cytidine 5'-diphospho)-2-
C-methyl-D-erythritol kinase) of Catharanthus roseus (ABI35992.1), ZmCMEK (4-
(cytidine 5'-diphospho)-2-C-methyl-D-erythritol kinase) of Zea mays (ACG34338.1) and
NbCMEK (4-(cytidine 5'-diphospho)-2-C-methyl-D-erythritol kinase) of Nicotiana
benthamiana (ABO87658.1). Residues 189 through 204 (highlited in box) are the
conserved ATP-binding site for the functional activity of 4-(cytidine 5'-diphospho)-2-C-
methyl-D-erythritol kinase (Kim et al., 2008). There are three putative conserved motifs
for N-myristoylation site shared by VMPCMEK with other closely related plant proteins
(GAxxCT, GQxxAV and GSxxTI). The motifs are marked with four stars (****).
Besides that, there is another conserved motifs for cAMP- and cGMP-dependent protein
kinase phosphorylation site (RKxT) marked with two stars (**).
RKxT
**
GAxxCT ****
GQxxAV GSxxTI
**** ****
113
VMPCMEK (Vanda Mimi Palmer)
HbCMEK (Hevea brasiliensis)
SrCMEK (Stevia rebaudiana)
SlCMEK (Solanum lycopersicum)
NbCMEK (Nicotiana benthamiana)
CrCMEK (Catharanthus roseus)
AtCMEK (Arabidopsis thaliana)
ZmCMEK (Zea mays)
GbCMEK1 (Ginkgo biloba 1)
GbCMEK2 (Ginkgo biloba 2)
Figure 25: A Phylogenetic Tree of VMPCMEK with Homologous Proteins. The
phylogenetic tree is constructed by MEGA software version 4.0 using amino acid
sequence of VMPCMEK with the homologous proteins available in GeneBank database
(NCBI) including other 4-(cytidine 5'-diphospho)-2-C-methyl-D-erythritol kinase from
Hevea brasiliensis (BAF98293.1), Stevia rebaudiana (ABB88838.3), Solanum
lycopersicum (AAF87717.1), Nicotiana benthamiana (ABO87658.1), Catharanthus
roseus (ABI35992.1), Arabidopsis thaliana (AAG01340.1) Zea mays (ACG34338.1) and
two proteins from Ginkgo biloba (AAZ80384.1) and (AAZ80385.1).
114
Relative expression analysis of VMPCMEK in different tissues (Figure 26) of Vanda
Mimi Palmer showed up-regulated expressions in floral tissues in contrast to vegetative
tissues. Among the floral tissues, petal and sepal showed the highest expression of
VMPCMEK transcript which was approximately two folds higher than the bud
(calibrator). In the lip, the expression of VMPCMEK was detected lower than the bud.
Relative expression analysis of VMPCMEK at different floral developmental stages
showed that VMPCMEK was differentially expressed (Figure 27). The expression level
of VMPCMEK increased gradually from young bud to half-open flower (the highest
level), followed by a decrease in fully-open flower stage. Relative expression study of
VMPCMEK at different time points in a 24-hour cycle (12 hours in light and followed by
12 hours in dark) (Figure 28) showed a differential expression pattern. From the analysis,
expression of VMPCMEK increased gradually from 12.00am to 8.00am, followed by a
gradual decrease until 2.00pm (the lowest peak). After 2.00pm, the expression increased
again gradually until 10.00pm.
4.2.2.3 Sequence and Expression Analyses of Putative Cytochrome P450 Protein
(VMPCyP450)
VMPCyP450 has a full-length cDNA transcript of 1785bp comprising 1614bp of ORF,
18bp of 5‟-UTR and 153bp of 3‟-UTR with a poly-A tail (Figure 29). The VMPCyP450
sequence encodes for 538 amino acid residues (Figure 30) with a predicted molecular
weight of 62.1 kD and an isoelectric point (pI) of 8. A homologous sequence
115
0.00
0.50
1.00
1.50
2.00
2.50
bud fully-
open
flower
petal sepal lip leaf root shoot stalk
Tissues
Rela
tive
Exp
ressio
n o
f V
MP
CM
EK
(fo
ld c
han
ge)
Figure 26: Relative Expression Study of VMPCMEK Transcript in Different
Tissues Including Floral and Vegetative Tissues. The floral tissues are bud, fully-open
flower, petal, sepal and lip meanwhile the vegetative tissues used in the expression study
are leaf, root, shoot and stalk. The quantitative expression level of VMPCMEK in each
tissue was calculated relative to the calibrator which was the bud collected at 12.00 noon.
Standard error between three replicates of relative gene expression in each tissue is
indicated by error bar.
0.00
0.50
1.00
1.50
2.00
2.50
3.00
young bud
(green)
mature bud
(red)
half-open
flower
fully-open
flower
14-day old
flower
Developmental Stages
Rel
ativ
e
Exp
ress
ion
of
VM
PC
ME
K
(fo
ld c
han
ge)
Figure 27: Relative Expression Analysis of VMPCMEK Transcript at Different
Developmental Stages During Flower Development. This study was carried out on
five different flower developmental stages of Vanda Mimi Palmer including young bud,
mature bud, half-open flower, fully-open flower and 14-day old fully-open flower.
Quantitative measurement of VMPCMEK expression in each flower developmental stage
was expressed relative to the calibrator which was the young bud collected at 12.00 noon.
Standard error between three replicates of relative gene expression at each developmental
stage is indicated by error bar.
116
0.00
0.50
1.00
1.50
2.00
2.50
3.00
12am 2am 4am 6am 8am 10am 12pm 2pm 4pm 6pm 8pm 10pm
Time
Rela
tive e
xp
ressio
n o
f V
MP
CM
EK
(fo
ld e
xp
ressio
n)
Figure 28: Relative Expression Analysis of VMPCMEK at Different Time Points in
a 24-hour Cycle. Quantitative expression of VMPCMEK at each time point was
expressed relative to the calibrator which was the fully-open flower collected at 12.00
noon. Standard error between three replicates of relative gene expression at each time
point is indicated by error bar.
117
1 ATA CTG CTG CTG CCA CTA ATG TCT TCT TCC TCA AGC TCC TCA CTT 45
M S S S S S S S L 9
>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>
VMPCyP450 ORF Forward Primer
46 CTG CCA TAC AAA CTC ATT GCT TTC AGC GCA ATC TTC CTC ATC AGT 90
10 L P Y K L I A F S A I F L I S 24
91 TGG ATC TTC CTT CAT AGA TGG GCA CAG AGA AAC CGC AGA GGT CCG 135
25 W I F L H R W A Q R N R R G P 39
136 AAG ACA TGG CCG CTC ATC GGA GCC GCC ATT GAA CTG CTC AAC AAC 180
40 K T W P L I G A A I E L L N N 54
181 TAT GAA CGC ATG CAT GAT TGG ATT ACA GAT TAC TTG TCT GAA TGG 225
55 Y E R M H D W I T D Y L S E W 69
226 AGG ACT GTT ACT GTC CCC TTG CCC TTC ACT TCA TAC ACT TAT ACT 270
70 R T V T V P L P F T S Y T Y T 84
271 GCA GAC CCT GCA AAT GTG GAG CAT ATT CTG AAG ACC AAC TTC AAC 315
85 A D P A N V E H I L K T N F N 99
316 AAC TAT CCC AAG GGA GAG CTA TTT AGA TCA TAT ATG GAG GTA TTG 360
100 N Y P K G E L F R S Y M E V L 114
361 CTG GGA GAT GGG ATA TTT AAC GCA GAT GGA GAG CTG TGG AGG AAG 405
115 L G D G I F N A D G E L W R K 129
406 CAG AGG AAG ACT GCA AGC TTT GAG TTT GCT TCA AAG AAC TTG AGG 450
130 Q R K T A S F E F A S K N L R 144
451 GAA CTG AGC ACC GTT GTG TTT AGA GAG TAT GCT TTG AAA CTA TCT 495
145 E L S T V V F R E Y A L K L S 159
496 GAC ATA TTA TGC CAA GCC TCT TGC AAA GAT CAT CAT CAA GCT GTA 540
160 D I L C Q A S C K D H H Q A V 174
541 GAT ATT CAG GAT TTA TTC ATG AGG ATG ACA ATG GAC TCC ATA TGC 585
175 D I Q D L F M R M T M D S I C 189
586 AAG CTT GGT TTT GGA GTG GAG ATA GGG ACA CTA TCT CCC CAA CTC 630
190 K L G F G V E I G T L S P Q L 204
631 CCT GAT AAC AGC TTT GCT CGA GCT TTC GAC ACC GCG AAC GCG ACC 675
205 P D N S F A R A F D T A N A T 219
676 GTC ACG CGT CGA TTC TTC GAT CCC TTG TGG AGG TTG AAG AGG TTT 720
220 V T R R F F D P L W R L K R F 234
721 CTT TGT GTG GGA TCA GAG GCT GCC CTC AAC CAA AAT ATC AGA ATT 765
235 L C V G S E A A L N Q N I R I 249
766 GTT AAT GAC TTC ACC TCT AAT GTT ATA CGT ACA AGA AAG GCT GAG 810
250 V N D F T S N V I R T R K A E 264
811 ATC ATG AGA GCT AAA CAA AAC GGG CAT CAT GAT GAG ACA AAG CAA 855
265 I M R A K Q N G H H D E T K Q 279
856 GAC ATA CTA TCA AGG TTC ATC GAG CTC GCC AAC ACC GAC AAA GAG 900
280 D I L S R F I E L A N T D K E 294
901 AGT GAT TTC AGC ACG GAA AAA GGT TTA AGA GAT GTG GTG CTA AAC 945
295 S D F S T E K G L R D V V L N 309
118
946 TTT GTT ATT GCA GGG AGG GAC ACT ACT GCT GCA ACG CTC TCA TGG 990
310 F V I A G R D T T A A T L S W 324
>>>>>>>>>>>>>>>>>>
VMPCyP450 RT-PCR Forward Primer
991 TTT ATA TAC ATA TTA GTC ACA CAA CCT CAG GTG GCA CAG AAA CTC 1035
325 F I Y I L V T Q P Q V A Q K L 339
>>>>>>
1036 TAT ATA GAG ATG AAA GAG TTT GAG GAG ATC AGA GCT GAA GAA GAA 1080
340 Y I E M K E F E E I R A E E E 354
1081 AAT ATA AAT TTG GAT TTA TGT AAT TTG GAA GAT ATG GAT TCA TTC 1125
355 N I N L D L C N L E D M D S F 369
1126 AGA AAC AGA TTA TCA GAT TTT TCG AGG CTT TTG GAT TAT GAT TCA 1170
370 R N R L S D F S R L L D Y D S 384
<<<<<<<<<<<<<<<<<<<<<<<<<<
VMPCyP450 RT-PCR Reverse Primer
1171 TTA GCA AGG CTG CAA TAT CTG CAT GCA TGC ATT ACA GAG ACC CTG 1215
385 L A R L Q Y L H A C I T E T L 399
1216 AGG CTG TTT CCT CCT GTT CCT CAG GAC GCG AAA GGC ATT TTG AAG 1260
400 R L F P P V P Q D A K G I L K 414
1261 GAT GAT GTT CTC CCT GAC GGA ACA AAA CTG AGA GCC GGG GAA ATG 1305
415 D D V L P D G T K L R A G E M 429
1306 GTG CTA TAC GTC CCC TAT TCA ATG GGA AGA ATG GAG TAC ATT TGG 1350
430 V L Y V P Y S M G R M E Y I W 444
1351 GGC ATC GAC GCA TCA GAA TTT CGC CCC GAA AGA TGG CTA AAT AAC 1395
445 G I D A S E F R P E R W L N N 459
1396 GAC AAT AAT TCC GTC CAA AAT AAC GTC TCT CCA TTC AAG TTC ACG 1440
460 D N N S V Q N N V S P F K F T 474
1441 GCG TTT CAG GCT GGT CCC AGA ATG TGC TTG GGG AAG GAC TCC GCT 1485
475 A F Q A G P R M C L G K D S A 489
1486 TAT CTG CAG ATG AAG ATG ACA GCA GCG TTA CTC TGC AGG TTC TTT 1530
490 Y L Q M K M T A A L L C R F F 504
1531 CAA TTC AGA CTT GCT CCT CAT CAT CCT CCT GTT AAG TAT AGG ATG 1575
505 Q F R L A P H H P P V K Y R M 519
1576 ATG ATA GTA CTT TCC ATG GCG CAT GGC CTG CAT GTG CTC GTT TGT 1620
520 M I V L S M A H G L H V L V C 534
1621 AGA AGA GGA TCA TGA TTT TTG ATG CAT GGA TCT ATG TTT TAT TAT 1665
535 R R G S * 538
1666 TAA GTT ATT GCG TCT GTT TGT TGT CCA TAA GTG AAG TGC AGA CAA 1710
<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<
VMPCyP450 ORF Reverse Primer
1711 CAT CAT TAT TAT ATG TCA TGC CCC CTA AAT GTT TAC TTC CGA AAA 1755
1756 AAA AAA AAA AAA AAA AAA AAA AAA AAA AAA 1785
Figure 29: The Nucleotide and Deduced Amino Acid Sequence of VMPCyP450
(Putative Cytochrome P450 Protein). The open reading frame (ORF) of VMPCyP450
starts at nucleotide 19. The asterisk (*) indicates stop codon. The putative
polyadenylation signal is bold and underline. The location of gene specific primers used
in real-time PCR analysis and ORF isolation are shown by arrow heads (>>>> and <<<<).
119
Figure 30: PCR Product of Open Reading Frame (ORF) of VMPCyP450 of Vanda
Mimi Palmer. The ORF represents 538 amino acids. The PCR product was
electrophoresed on 1% (w/v) agarose gel. Lane M: 100bp marker (Vivantis, Malaysia);
Lane 1: ORF of clone VMPCyP450.
1500bp
500bp
M 1
~1600bp
120
analysis by BLASTX and BLASTP (NCBI) shows that VMPCyP450 exhibited similarity
to sequences that encode cytochrome P450 proteins from other plants (39-67% identity).
From the BLASTX and BLASTP analyses, VMPCyP450 has the closest similarity to
putative cytochrome P450 protein of Oryza sativa (ABF 94184.1), and cytochrome P450
protein of Populus trichocarpa (EEE89622.1), corresponding to 66% identity. Besides
that, VMPCyP450 also showed similarity to cytochrome P450 monooxygenase of
Petunia hybrida (AAZ39646.1) (45% identity) and cytochrome P450 monooxygenase
from other plants.
Localizome analysis predicts that VMPCyP450 protein does not have any signal peptide.
Most of other plant cytochrome P450 proteins were predicted by the Localizome software
to have signal peptide except cytochrome P450 protein of Arabidopsis thaliana,
cytochrome P450 protein of Populus trichocarpa and cytochrome P450 monooxygenase
of Petunia hybrida. VMPCyP450 and all the closely related proteins were predicted to
have a transmembrane domain by the Localizome software. The deduced VMPCyP450
protein was predicted to have 11 amino acids at the N-terminal (non-cytosolic), 21 amino
acids located in the membrane and 506 amino acids in cytosol. Expasy programme
prediction shows that three N -myristoylation sites (GxxxAD, GVxxGT and GsxxAL)
were shared by VMPCyP450 and its closely related proteins (Figure 31). Besides that,
there was another conserved site for tyrosine kinase phosphorylation (see Figure 31). A
phylogenetic tree (Figure 32) was constructed using the MEGA version 4.0 (Tamura et
al., 2007) to estimate the genetic relationship of VMPCyP450 with other cytochrome
121
Figure 31: Alignment of VMPCyP450 with Other Closely Related Protein Sequences
Downloaded from GeneBank Database NCBI. The VMPCyP450 was aligned with
cytochrome P450 of Ricinus communis (EEF39957.1) putative cytochrome P450 of
Arabidopsis thaliana (AAG60111.1), putative cytochrome P450 protein of Oryza sativa
from Japonica Cultivar (AAL84318.1), cytochrome P450 of Zea mays (ACG35470.1)
and cytochrome P450 monooxygenase of Petunia hybrida (AAZ39646.1). There are
three N-myristoylation sites (GxxxAD, GVxxGT and GsxxAL) shared by VMPCyP450
with other closely related proteins which are marked with four stars (****). There is
another putative conserved region for tyrosine kinase phosphorylation site (RxxExxxY)
marked with five delta symbol (∆∆∆∆∆
).
GIxxAD
****
GSxxAL
****
RxxExxxY ∆∆∆∆∆
GVxxGT
****
122
VMPCyP450 (Vanda Mimi Palmer)
PhP450 (Petunia x hybrida)
MtP450 (Medicago truncatula)
GmP450 (Glycine max)
RcP450 (Ricinus communis)
PtP450 (Populus trichocarpa)
AtP450 (Arabidopsis thaliana)
CaP450 (Capsicum annuum)
OsP450 (Oryza sativa-japonica cultiva...
ZmP450 (Zea mays)
Figure 32: A Phylogenetic Tree of VMPCyP450 and Homologous Proteins. The
phylogenetic tree is constructed by MEGA software version 4.0 using amino acid
sequence of VMPCyP450 with the homologous proteins available in GeneBank database
(NCBI) including cytochrome P450 of Ricinus communis (EEF39957.1), cytochrome
P450 of Populus trichocarpa (EEE89622.1) cytochrome P450 of Arabidopsis thaliana
(AAG60111.1), cytochrome P450 of Capsicum annuum (ACD10924.1), cytochrome
P450 protein of Oryza sativa from Japonica Cultivar (AAL84318.1), cytochrome P450 of
Zea mays (ACG35470.1), cytochrome P450 monooxygenase of Petunia hybrida
(AAZ39646.1), cytochrome P450 monooxygenase of Medicago tranculata
(ABC59094.1) and cytochrome P450 monooxygenase of Glycine max (Soy
bean)(ABC68403.1).
123
P450 proteins. The phylogenetic tree shows that VMPCyP450 amino acid sequence was
clustered together with cytochrome P450 monooxygenase of Medicago truncatula,
Glycine max and Petunia hybrida and supported by the bootstrap value of 99 for the
branch.
Relative expression analysis in different tissues (Figure 33) of Vanda Mimi Palmer
including floral and vegetative tissues by real-time RT-PCR shows that the VMPCyP450
had up-regulated expression in floral tissues especially in the lip. The other floral tissues
including petal and sepal showed lower expressions of VMPCyP450 compared to the lip.
For vegetative tissues such as leaf, root, shoot, and stalk, the expression of VMPCyP450
was detected at a very low level. Expression analysis of VMPCyP450 transcript at
different flower developmental stages (Figure 34) shows a developmentally regulated
pattern. Meanwhile, the expression analysis of VMPCyP450 at different time points in a
24-hour cycle (Figure 35) also shows a differential expression pattern.
4.2.2.4 Sequence and Expression Analyses of Unknown protein (VMPA28)
The full-length sequence of VMPA28 cDNA transcript is 972bp containing 591bp ORF
flanked with 148bp 5‟UTR and 233bp 3‟UTR including a poly-A tail (Figure 36). The
ORF encodes a protein of 197 amino acid residues (Figure 37). Expasy tool (Prolite) has
predicted 22.32kD as the molecular mass of VMPA28 protein with an isoelectric point of
9.06. The Expasy tool also predicts the presence of a N-glycosylation site (NTSN), two
124
0.00
1.00
2.00
3.00
4.00
5.00
6.00
bud fully-
open
flower
petal sepal lip leaf root shoot stalk
Tissues
Rel
ativ
e
Exp
ress
ion
of
VM
PC
yP45
0
(fo
ld c
han
ge)
Figure 33: Relative Expression Analysis of VMPCyP450 in Different Tissues
Including Floral and Vegetative Tissues. The floral tissues are bud, fully-open flower,
petal, sepal and lip meanwhile the vegetative tissues used in the expression study are leaf,
root, shoot and stalk. The quantitative expression level of VMPCyP450 in each tissue
was calculated relative to the calibrator which was the bud collected at 12.00 noon.
Standard error between three replicates of relative gene expression in each tissue is
indicated by error bar.
0.00
0.50
1.00
1.50
2.00
2.50
3.00
3.50
4.00
young bud
(green)
mature bud
(red)
half-open
flower
fully-open
flower
14-day old
flower
Developmental Stages
Rel
ativ
e
Exp
ress
ion
of
VM
PC
yP45
0
(fo
ld c
han
ge)
Figure 34: Relative Expression Study of VMPCyP450 at Different Flowering
Developmental Stages. This study was carried out on five flower developmental stages
of Vanda Mimi Palmer including young bud, mature bud, half-open flower, fully-open
flower and 14-day old fully-open flower. Quantitative measurement of VMPCyP450
expression in each flower developmental stage was expressed relative to the calibrator
which was the young bud collected at 12.00 noon. Standard error between three replicates
of relative gene expression at each developmental stage is indicated by error bar.
125
0.00
1.00
2.00
3.00
4.00
5.00
6.00
7.00
12am 2am 4am 6am 8am 10am 12pm 2pm 4pm 6pm 8pm 10pm
Time
Rela
tive E
xp
ressio
n o
f V
MP
CyP
450
(fo
ld e
xp
ressio
n)
Figure 35: Relative Expression Study of VMPCyP450 at Different Time Points in a
24-hour Cycle. Quantitative expression of VMPCyP450 at each time point was
expressed relative to the calibrator which was the fully-open flower collected at 12.00
noon. Standard error between three replicates of relative gene expression at each time
point is indicated by error bar.
126
2 AAA AAC GTC GTT GTG TCT CGG GGT CGT TGG GGA GAA TTT CTT AAT 46
47 AAC AGT CGG AAA AAA GGT TCC CTA ATG ATA AGC GGG ACA GTT AGC 91
92 GCA ACT AAA TTA ATG TGA GAT TAG TTC AAT TCT TAG GCA CCC CAG 136
>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>
VMPA28 ORF forward Primer
137 ATT TTA CAT TTT ATG TTT TCC GGT TCG TTT TTT TTG GGA AAT TTT 181
M F S G S F F L G N F 11
182 TTA GTG AAT AAC AAT TTC ACC CAG GAA AAC AGT TAT GAC CAT GAT 226
12 L V N N N F T Q E N S Y D H D 26
227 TAC GCC AAG CTT GCA TGC CTG CAG GTT GAC TTT AGA GGG GAT CCA 271
27 Y A K L A C L Q V D F R G D P 41
272 AAT TTT AAA TAT CCC GCA ACC TGT CCC ACC TTT TCT CCC CTT TTC 316
42 N F K Y P A T C P T F S P L F 56
317 GTC TCT TTT TCC CTG TTT CTC TTT CTC ATC CGA TTG ATG GAT CTT 361
57 V S F S L F L F L I R L M D L 71
362 AGG GCG GCG ATA GTC GCC GCC GCC GGC GAG CGT TGG ACG GAG GAG 406
72 R A A I V A A A G E R W T E E 86
407 CGG CAC TCC CGC TTC CTC AAC TCG ATC GAA AGT ACT TTC GTC CAT 451
87 R H S R F L N S I E S T F V H 101
452 CAA ATG CTC GGC ATC CAT CCC GAC GGC GAT AAC CTC CGC CGA TGC 496
102 Q M L G I H P D G D N L R R C 116
497 GCG GCG AGG CTC GAC CGT CGT GTT CCC GAT TGC ATC GCC GGT AAA 541
117 A A R L D R R V P D C I A G K 131
542 GAG TCT GCG AAG AGT TCT CAG ATG CGA TCG CCG GAT AGG AGG CCT 586
132 E S A K S S Q M R S P D R R P 146
587 GCT GCC ATT ACT GCG GGC GCC AAC ACC TCT AAT TGT ACA CGG AAA 631
147 A A I T A G A N T S N C T R K 161
>>>>>>>>>>>>>>
632 CGA TCA CTG CGG CGA TAT GAT GCG TCG CTA GAC CAG GTG GTG CCG 676
162 R S L R R Y D A S L D Q V V P 176
>>>>>>>>>>>
VMPA28 RT-PCR Forward Primer
677 GAG TTC AAA AAT AAG AAC GTC GGC GAG GAT GCA TCC AAG CGA AAG 721
177 E F K N K N V G E D A S K R K 191
722 TTT GAA GAC GCA GCA CAT TAA CAA TGA GAA CAA TAT TGG AGT CTA 766
192 F E D A A H * 197
767 AAG CTG CAG CTG TCT TCA TCT CTT GCT GTC TAC AAG CAA TGT TTG 811
<<<<<<<<<<<<<<<<<<<<<<<<<<< VMPA28 ORF Reverse Primer
812 ACT TGC ATG TTG AAA GGA ATG TAG AAA TGT GTT CTT ATA TAT TAT 856
<<<<<<<<<<<<<< VMPA28 RT-PCR Reverse Primer
<<<<<<<<<<<<<<
857 GTT TTA GAG GAA ACG TAT TTA TGA TAA TTT TTG CTT GTT AAA GTT 901
902 GGT TTT TGC CAT TTC AAC ATG GTT TCA TTC TGT GAA CAT TTT AGA 946
947 TCA AAA AAA AAA AAA AAA AAA AAA AAA 973
Figure 36: The Nucleotide and Deduced Amino Acid Sequences of VMPA28. The
open reading frame (ORF) of VMPA28 starts at nucleotide 149. The asterisk (*) indicates
stop codon. The putative adenylation signal is bold and underline in the nucleotide
sequence. The location of gene specific primers used in real-time PCR analysis and ORF
isolation are shown by arrow heads (>>>> and <<<<).
127
Figure 37: PCR Product of Open Reading Frame (ORF) of an Unknown Protein of
Vanda Mimi Palmer (VMPA28). The PCR product was electrophoresed on 1% (w/v)
agarose gel. Lane M: 100bp marker (Vivantis); Lane 2: ORF of clone VMPA28.
1000bp
500bp
M 1 2
~ 700bp
128
protein kinase C phosphorylation sites (SxK, SxR) and a N-myristoylation site
(GAxxSN). BLASTX and BLASTP analyses (NCBI) show the deduced amino acid of
VMPPA28 had no significant similarity to any known sequence in the GeneBank
database. Localizome analysis shows there was no signal peptide in the deduced protein
sequence of VMPA28.
Expression analysis of VMPA28 was carried out in different tissues, at different flower
developmental stages and also at different time points in a 24-hour cycle by real-time RT-
PCR. Analysis of VMPA28 transcript in different tissues (Figure 38) shows a slight up-
regulated expression in floral tissues compared to vegetative tissues. For analysis of
VMPA28 transcript at different flower developmental stages (Figure 39), the expression
of VMPA28 was developmentally-regulated where the transcripts level increased
gradually from the bud to the fully-open flower stage, followed by a gradual decrease
until the end of flower life-time. The transcript was also found to be differentially
expressed at different time points in a 24-hour cycle (Figure 40).
129
0.00
0.40
0.80
1.20
1.60
bud fully-
open
flower
petal sepal lip leaf root shoot stalk
Tissues
Rela
tive
Exp
ressio
n o
f V
MP
A28
(fo
ld c
han
ge)
Figure 38: Relative Expression Study of VMPA28 Transcript in Different Tissues
Including Floral and Vegetative Tissues. The floral tissues are bud, fully-open flower,
petal, sepal and lip meanwhile the vegetative tissues used in the expression study are leaf,
root, shoot and stalk. The quantitative expression level of VMPA28 in each tissue was
calculated relative to the calibrator which was the bud collected at 12.00 noon. Standard
error between three replicates of relative gene expression in each tissue is indicated by
error bar.
0.00
0.50
1.00
1.50
2.00
2.50
3.00
3.50
4.00
young bud
(green)
mature bud
(red)
half-open
flower
fully-open
flower
14-day old
flower
Developmental Stages
Rel
ativ
e E
xpre
ssio
n o
f V
MP
A28
(fo
ld c
han
ge)
Figure 39: Relative Expression Study of VMPA28 at Different Flower
Developmental Stages. The study was carried out in five flower developmental stages
of Vanda Mimi Palmer including young bud, mature bud, half-open flower, fully-open
flower and 14-day old fully-open flower. Quantitative measurement of VMPA28
expression in each flower developmental stage was expressed relative to the calibrator
which was the young bud collected at 12.00 noon. Standard error between three replicates
of relative gene expression at each developmental stage is indicated by error bar.
130
0.00
0.50
1.00
1.50
2.00
2.50
3.00
3.50
4.00
4.50
5.00
12am 2am 4am 6am 8am 10am 12pm 2pm 4pm 6pm 8pm 10pm
Time
Rela
tive E
xp
ressio
n o
f V
MP
A28
(fo
ld c
han
ge)
Figure 40: Relative Expression Study of VMPA28 at Different Time Points in a 24-
hour Cycle. Quantitative expression of VMPPAAS at each time point was expressed
relative to the calibrator which was the fully-open flower collected at 12.00 noon.
Standard error between three replicates of relative gene expression at each time point is
indicated by error bar.
131
CHAPTER 5
DISCUSSIONS
5.1 Biochemical Analysis of the Scent of Vanda Mimi Palmer
Biochemical analysis of the scent of Vanda Mimi Palmer by gas chromatography-mass
spectrometry (GC-MS) showed a fluctuating pattern of volatile emission by fully-open
flower of Vanda Mimi Palmer in a 24-hour cycle with the highest level detected during
the daytime (in light) but none at night (in dark). This emission pattern was observed in
most roses and snapdragon (Anthirrhinum majus) whereby the highest level of scent
emission was reported to occur during daytime (Helsper et al., 1998; Picone et al., 2004;
Dudareva et al., 2003). Meanwhile, for some other plants such as in Petunia hybrida and
Stephanotis floribunda, the highest level of scent emission occurs at night (Verdonk et
al., 2003; Pott et al., 2002). The pattern of scent emission of Vanda Mimi Palmer in a 24-
hour cycle might be controlled by some factors such as photoperiod and circardian clock
like in other scented plants (Lu et. al, 2002). Besides that, flowers of Vanda Mimi Palmer
also showed a developmentally regulated pattern of scent emission whereby no volatile
compound was detected in the bud stage. Emission of volatile compounds was detected
from the half-open flower stage and increased to the maximum level in the fully-open
flower stage. The same pattern of scent emission was previously reported in other scented
flowers including Clarkia breweri and Anthirrhinum majus (Pichersky et al., 1994;
Nagegowda et al., 2008).
132
Based on the GC-MS analysis of the scent of Vanda Mimi Palmer, volatile compounds
emitted by the flowers are derived from the terpenoid, benzenoid and phenylpropanoid
groups. Thus, there are possibly two main pathways involved in the fragrance
biosynthesis of Vanda Mimi Palmer which are terpenoid as well as benzenoid and
phenylpropanoid pathways. Hsiao et al. (2006) reported that the volatile compounds in
Phalaenopsis bellina (a scented orchid from Taiwan) are derived from the three
pathways: terpenoid, lipoxygenase, as well as benzenoid and phenylpropanoid pathways
(Hsiao et al., 2006). These three fragrance biosynthetic pathways were also reported in
other well studied scented flowers like Rosa hybrida (Guterman et al., 2002), Clarkia
breweri (Pichersky et al., 1995), Petunia hybrida (Boatright et al., 2004) and
Anthirrhinum majus (Nagegowda et al., 2008).
The GC-MS analysis of the scent of Vanda Mimi Palmer showed that there are four
volatile compounds (linalool, ocimene, linalool oxide and nerolidol) which might have
been derived from the terpenoid pathway. Linalool, ocimene and linalool oxide are
classified as monoterpenes (Croteau and Karp, 1991; Knudsen and Gershenzon, 2006)
while nerolidol is classified as a sesquiterpene (Knudsen and Gershenzon, 2006;
Nagegowda et al., 2008). Monoterpenes and sesquiterpenes are the common compounds
in the scent of scented orchids including Phalaenopsis bellina (Hsiao et al., 2006),
Dendrobium beckleri and Phalaenopsis violacea (Kaiser, 1993). The terpenoid
compounds emitted by Vanda Mimi Palmer‟s flower were also detected in the scent of
other scented flowers including Anthirrhinum majus (Linalool, ocimene and nerolidol)
(Dudareva et al., 2003; Nagegowda et al., 2008), and Clarkia breweri (Linalool)
133
(Pichersky et al., 1994). Thus, the terpenoid pathway in Vanda Mimi Palmer involved in
the biosynthesis of linalool, ocimene and nerolidol compounds might be closely related to
the terpenoid pathway in other well studied scented flowers. The terpenoid pathway for
the floral scent biosynthesis in those well studied scented flowers could be used for the
study of the terpenoid pathway in Vanda Mimi Palmer.
Besides terpenoids, there were four other compounds in the scent of Vanda Mimi Palmer
which might be derived from the benzenoid and phenylpropanoid pathway such as
methylbenzoate, benzyl acetate, phenylethanol and phenylethyl acetate. The same
benzenoid and phenylpropanoid compounds were reported to be present in the scent of
other scented orchids like Dendrobium trigonopus, Dendrochilum cobbianum and Vanda
tessellata (Kaiser, 1993), and in other scented flowers like Rosa hybrida (Shalit et al.,
2003), Petunia hybrida (Verdonk et al., 2003), Clarkia breweri (Raguso and Pichersky,
1995; Dudareva et al., 1998) and Anthirhinum majus (Dudareva et al., 2000). In
Phalaenopsis bellina, some other benzenoid and phenylpropanoid compounds such as 3-
phenyl-2-propen-1-ol, 3-methylphenyl butanoic ester, and 2-methylphenyl butanoic ester
were detected in its floral scent (Hsiao et al., 2006). Benzyl acetate which was detected in
the scent of Vanda Mimi Palmer, was also reported to be present in the scent of Rosa
hybrida (Shalit et al., 2003) and Clarkia breweri (Raguso and Pichersky, 1995; Dudareva
et al., 1998). Another detected compound, methylbenzoate was also reported in many
scented flowers like Petunia hybrida (Verdonk et al., 2003) and Anthirhinum majus
(Dudareva et al., 2000). Besides that, phenylethanol, a compound which is also present in
the scent of Vanda Mimi Palmer, was identified in the scent of Rosa hybrida (Shalit et
134
al., 2003), Petunia hybrida (Verdonk et al., 2003), Clarkia breweri (Raguso and
Pichersky, 1995; Dudareva et al., 1998), and Rosa hybrida (Shalit et al., 2003). Thus, the
benzenoid and phenylpropanoid pathways in Vanda Mimi Palmer which could possibly
be involved in the biosynthesis of methylbenzoate, benzyl acetate, phenylethyl acetate
and phenylethanol compounds might be closely related to the benzenoid and
phenylpropanoid pathways in well studied scented flowers especially like Petunia
hybrida, Clarkia breweri and Rosa hybrida.
Solid phase Micro-extraction (SPME) method used in this study is one of the advanced
methods utilised to capture volatile compounds emitted by scented flowers. In this study,
a modified funnel was used to collect and accumulate the volatiles emitted by Vanda
Mimi Palmer flowers in a special trap before being captured by the SPME. It is possible
that there might be some other compounds in the scent of Vanda Mimi Palmer emitted in
traces amount that could not be detected in this study using the above method (capturing
was just for 15 minutes). Trace compounds could be detected and identified using a
combination of modern headspace attached directly to the GC-MS where the volatiles
emitted by the scented flowers are accumulated in a special chamber for 2-3 hours and
then concentrated with a special pump prior injection into the GC-MS port.
Unfortunately, this approach was not employed in this study due to inavailability of the
system in Universiti Putra Malaysia. There is another method used in the floral scent
studies in rose by Hendel-Rahmanim et al. (2007) and Farhi et al. (2010) on Rosa hybrida
whereby a few grams of the rose petals were soaked in hexane for a few hours. The
debris was removed and the supernatant containing hexane with the extracted compounds
135
was filtered and then concentrated with nitrogen before being injected into the GC-MS
injector port. In this study, the above mentioned method was applied to extract the
essential oil from Vanda Mimi Palmer using hexane and a large amount of flowers
(100grams). Most of the compounds detected at high levels in the scent of Vanda Mimi
Palmer were also detected in the essential oil of Vanda Mimi Palmer. However, there
were some compounds including ocimene and nerolidol which were not found in the
essential oil. One possible reason for non-detectability of ocimene is because the
compounds might not be stored in the cell long enough prior to its release into the air. As
for nerolidol, this compound could be synthesized at very low amount compared to other
compounds such as linalool, methylbenzoate, benzyl acetate and phenylethyl acetate in
the floral tissues of Vanda Mimi Palmer. Besides that, a lot of other compounds which
are not emitted by fully-open flower of Vanda Mimi Palmer were also detected in the
essential oil (see Appendix D, subsection (c) and (d)). This could be due to the nature of
hexane itself as a powerful solvent to extract out non-polar compounds from the plant
samples.
In Vanda Mimi Palmer, the emitted scent is dominated by ocimene and linalool
compounds in the morning (8.00am-12.00 noon). At this time, other compounds were
detected at very low levels especially the compounds derived from the benzenoid and
phenylpropanoid pathways. However, the emission levels of benzenoid and
phenylpropanoid compounds especially benzyl acetate and phenylethyl acetate increased
drastically after 12.00 noon, higher than the levels of the terpenoid compounds (linalool
and ocimene). Thus, the percentage of each compound of the scent of Vanda Mimi
136
Palmer is different at different time points during daytime. This might be the special
characteristic of the scent of Vanda Mimi Palmer to attract different pollinators at
different time points during daytime. Unfortunately no comparison can be made with
other well studied scented flowers such as Petunia hybrida, Clarkia breweri, Antirrhinum
majus and Rosa hybrida as their percentage of each compound in the scent at different
time points and their functions to attract specific pollinators at different time points were
not investigated.
Comparison of the volatiles emitted by Vanda Mimi Palmer (scented orchid) and its
parents, Vanda Tan Chay Yan (non-scented orchid) and Vanda tessellata (scented orchid)
in Table 6 (section 4.1.1) shows ocimene was the only compound detected in the scent of
Vanda Mimi Palmer and both of its parents. Ocimene is a monoterpene derived from the
terpenoid pathway (Dudareva et al., 2003). An ocimene synthase from Vanda Mimi
Palmer is postulated to be involved in the final step of terpenoid biosynthetic pathway.
This enzyme could be involved in catalyzing the formation of ocimene from geranyl
diphosphate, a precursor for monoterpenoid biosynthesis. The transcript of this enzyme
has yet to be identified in Vanda Mimi Palmer. For another monoterpene compound
which is linalool, a linalool synthase transcript has been identified in the floral ESTs of
Vanda Mimi Palmer (Teh Siow Ling, Master student, Faculty of Biotechnology and
Biomolecular Sciences, Universiti Putra Malaysia, pers. comm. on 18th September 2008).
This enzyme might be involved in catalyzing the formation of linalool from geranyl
diphosphate. The linalool synthase gene might be derived from Vanda tessellata since
137
linalool was also detected in the scent of Vanda tessellata previously by Kaiser (Kaiser,
1993) using GC-MS.
Besides that, the formation of nerolidol (a sesquiterpene) was postulated to be catalyzed
by sesquiterpene synthase (Chan, 2009). The transcript of sesquiterpene synthase was
identified from the floral ESTs of Vanda Mimi Palmer. This sesquiterpene gene in Vanda
Mimi Palmer could be contributed by the gene pool from Vanda Tan Chay Yan since
coupane, a sesquiterpene was identified to be emitted by fully-open flower of Vanda Tan
Chay Yan (see Figure 10, in section 4.1.1). In contrast, no sesquiterpene was detected in
the scent of Vanda tessellata (another parent of Vanda Mimi Palmer) as reported by
Kaiser (Kaiser, 1993). The combination of GC and MS used by Kaiser in his work
(Kaiser, 1993) at that time was sensitive enough to detect the presence of sesquiterpenes
since some sequiterpenes were detected in other scented orchids including Aerangis
confuse (germacrane D and nerolidol), Dendrobium virgineum (nerolidol), Laelia anceps
(nerolidol), Oncidium longipes (nerolidol), and Polystachya fallax (caryophyllene and α-
farnesene) (Kaiser, 1993). In Vanda tessellata, sesquiterpene synthase might not be
expressed if it is present as a recessive allele of the gene. While in Vanda Tan Chay Yan,
seisquiterpene synthase gene might be represented by a dominant allele. The hybrid of
these orchids could bring heterozygous dominant characteristic of the sesquiterpene
synthase. Interestingly, there were another two sesquiterpenes identified in the essential
oil of of Vanda Mimi Palmer (germacrene and copaene) (see Table 6) besides nerolidol
that was identified in the scent of Vanda Mimi Palmer.
138
Volatiles comparison between Vanda Mimi Palmer and its parents (see Table 4, in
section 4.1.1) shows that two benzenoids (benzyl acetate and methylbenzoate) were
detected in the scent of both Vanda Mimi Palmer and Vanda tessellata (Kaiser, 1993).
These compounds could be derived from the benzenoid pathway. A number of
benzenoids identified in the scent of Vanda tessellata (Kaiser, 1993) were not detected in
the scent of Vanda Mimi Palmer such as benzaldehyde, benzyl alcohol, cinnamyl alcohol,
methyl cinnamate and methyl salicylate. Similarly, some compounds were only detected
in the scent of Vanda Mimi Palmer including phenylethanol and phenylethyl acetate.
Based on the differences, it was postulated that some modifications might have occurred
in the benzenoid and phenylpropanoid pathways of Vanda Mimi Palmer compared to its
parent Vanda tessellata which showed emission of benzenoids and phenylpropanoids
(Kaiser, 1993). Benzaldehyde, benzyl alcohol and cinnamyl alcohol which were detected
in the scent of Vanda tessellata as final products might be intermediates or precursors for
methylbenzoate, benzylacetate and phenylethyl acetate biosynthesis in Vanda Mimi
Palmer. In addition, phenylethanol compound was identified in the essential oil of both
Vanda Mimi Palmer (scented orchid) and Vanda Tan Chay Yan (non-scented orchid).
The phenylethanol might be used directly or acts as an intermediate for other
phenylpropanoids involved fragrance and non-fragrance metabolism. In addition, the
benzenoid and phenylpropanoid pathway has been involved in the biosynthesis of other
non-fragrance compounds including flavanoids and high molecular weight
phenylpropanoids (Lacombe et al., 1997; Shirley, 2001; Boerjan et al., 2003; Takashi et
al., 2007; Derikvand et al., 2008). Interestingly, in Vanda Mimi Palmer‟s essential oil, the
phenylethanol compound was detected at high level, 11.18% of the total essential oil. In
139
Vanda Mimi Palmer, the phenylethanol emission was only detected at trace level during
the highest peak, while the other phenylpropanoid which is phenylethyl acetate was
detected to be emitted during day time at very high level (see Figure 7). This suggested
that phenylethanol might be the main precursor for phenylethyl acetate in Vanda Mimi
Palmer.
Most of the compounds in the scent of Vanda Mimi Palmer were also detected in its
essential oil including linalool, methylbenzoate, benzyl acetate, phenylethanol and
phenylethyl acetate. The compounds might be stored in special oil glands or trichomes
for few hours before they are released into the air as volatiles since a lot of oil glands or
trichomes were detected on the surface of the petal and sepal of fully-open flowers of
Vanda Mimi Palmer (Janna Ong Abdullah, unpublished data). Besides that, two
intermediates of the scent‟s compound such as benzyl alcohol and benzyl benzoate were
detected in the essential oil of Vanda Mimi Palmer (see Table 6, in section 4.1.1). The
intermediate compounds might be the precursors for the production of benzyl acetate and
phenylethyl acetate which were detected in the scent of Vanda Mimi Palmer. In addition,
none of the fragrance compounds in the scent of Vanda Mimi Palmer was detected in the
essential oil of Vanda Tan Chay Yan except for phenylethanol. This phenylethanol might
be involved in non-fragrance activities since the compound was not detected in the
volatiles of fully-open flowers of Vanda Tan Chay Yan. In the essential oils of Vanda
Mimi Palmer and Vanda Tan Chay Yan, a lot of non-fragrant compounds were identified
(see Appendix D, subsection (c) and (d)). This might be due to the nature of the hexane
extraction method itself which could extract out most of the non-polar compounds from
140
the sample. Alternatively, pure essential oil could be isolated by a technique called
hydro-distillation. However, the hydro-distillation method has its limitation since it
requires a large amount of flowers.
Based on biochemical analysis on volatiles of Vanda Mimi Palmer and comparison with
its parents‟ scent compounds, it seems that the fragrant characteristic of Vanda Mimi
Palmer might be contributed by a pool of genes from both parents. In other scented
flower especially roses, the scent produced by Rosa chinensis (Chinese rose), an ancestor
of modern roses (Rougetel, 1988) is different from the scent of modern rose cultivars
(Wu et al., 2004). A lot of compounds in the scent of the Chinese rose are not found in
modern roses cultivars and some of them do not have any fragrance because breeding
was only focused on the beautiful colour and shape of the flower instead of the fragrance
itself (Yomogida, 1992; Zuker et al., 1998; Wu et al., 2004). For example, 1,3,5-
trimethoxybenzene compound (TMB) synthesized from ploroglucinol, was identified in
the scent of Rosa chinensis but was not detected in the scent most of modern rose
varieties but a related compound which is 3,5-dihyroxytoulene synthesized from orcinol
was detected in many modern roses. Biochemical modifications could have occured in
the fragrance biosynthetic pathway of roses by interaction of genes and enzymes derived
from parents of rose hybrids (Flament et al., 1993; Lavid et al., 2002; Wu et al., 2004).
141
5.2 Molecular Studies of Vanda Mimi Palmer
5.2.1 Isolation of Putative Fragrance-related cDNAs
Thirteen putative fragrance-related cDNA clones were isolated from the floral cDNA
library of Vanda Mimi Palmer and preliminarily characterized by reverse-Northern
analysis with two different probes representing mRNAs of fully-open flower and bud
stage separately. The aim was to select cDNAs with up-regulated expression in fully-
open flower stage (high fragrance emission) compared to bud stage (no fragrance
emission) of Vanda Mimi Palmer which might be potential fragrance-related cDNAs.
From the verification of the putative fragrance-related cDNAs with up-regulated
expression in fully-open flower compared to bud of Vanda Mimi Palmer in section
4.2.1.4, VMPCMEK (putative 4-(cytidine 5′-diphospho)-2-C-methyl-d-erythritol kinase)
was selected for further characterization because it showed higher expression in fully-
open flower of Vanda Mimi Palmer compared to the bud. This could be due to the
involvement of VMPCMEK transcripts in the early steps of the terpenoid pathway (see
Figure 41) for the biosynthesis of monoterpenes and sesquiterpenes such as linalool,
ocimene and nerolidol as reported in section 4.1.1.
Besides VMPCMEK, VMPCyP450 was also selected for further characterization because
of its high expression in the fully-open flower of Vanda Mimi Palmer compared to the
bud. In the bud, this VMPCyP450 might be involved in other metabolism especially for
142
PLASTID CYTOSOL
Figure 41: Elucidation of Terpenoid Pathway in Vanda Mimi Palmer. The pathway is
elucidated based on the terpenoid pathway of Anthirrhinum Majus (Nagegowda et al.,
2008). VMPCMEK and VMPCyP450 which was identified and isolated in this study are
shown in circles. Other putative fragrance related cDNAs that were isolated from floral
ESTs of Vanda Mimi Palmer are shown in boxes (Janna Ong Abdullah, Associate
Professor, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra
Malaysia, pers. comm. on 26th October 2009).
Pyruvate + glyceraldehyde 3-phosphate
1-Deoxy-D- 5-phosphate (DXP)
2-C-Methyl-D-erythritol 4-phosphate (MEP)
4-(Cytidine 5‟-diphospho)-2-C-
Methyl-D-erythritol (CDP-ME)
4-(Cytidine 5‟-diphospho)-2-C-
Methyl-D-erythritol 2-phosphate
(CDP-ME)
2-C-Methyl-D-erythritol 2,4-cyclodiphosphate
(cMEPP)
1-Hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate
(HMBPP)
3S-Hydroxy-3-methylglutaryl-CoA
(HMG-CoA)
3R-Mevalonic acid (MVA)
Mevalonic acid 5-phosphate
Mevalonate diphosphate
Dimethylallyl
diphosphate
(DMAPP)
Isopentenyl
diphosphate (IPP)
Dimethylallyl
diphosphate
(DMAPP)
Isopentenyl
diphosphate
(IPP)
Geranyl diphosphate (GPP)
Farnesyl diphosphate (FPP)
Linalool Ocimene
Linalool oxide Nerolidol
VMPCMEK
VMPCyP450
VMPLIS
VMPHMGR
VMPDXS
VMPDXR
VMPHDS
VMPFPPS
VMPSQS
VMPAACT
2 Acetyl-CoA Acetoacetyl-CoA
143
the biosynthesis of non-fragrance compounds since cytochrome P450 has been reported
to be involved in metabolism in the lipoxygenase pathway and also phenolic metabolism
(Ehlting et al., 2006). Another transcript selected for further characterization is an
unknown protein designated as VMPA28. The verification result shows the expression of
VMPA28 transcript was slightly higher in the fully-open flower compared to the bud,
suggesting this transcript might be involved in other metabolisms in the plant system
which has yet to be investigated.
5.2.2 Molecular Characterization of Putative Fragrance-related Transcripts
5.2.2.1 Sequence and Expression Analysis of Putative Phenylacetaldehyde Synthase
(VMPPAAS)
The BLASTX and BLASTP analyses (NCBI) show the deduced amino acid sequence of
VMPPAAS is 49-63% similar to other plant decarboxylases such as phenylacetaldehyde
synthases, tryptophan decarboxylases, tyrosine decarboxylases and aromatic amino acid
decarboxylases. GC-MS analysis of the scent of Vanda Mimi Palmer shows the presence
of phenylethyl acetate, which could be synthesized via the benzenoid and
phenylpropanoid pathways. Phenylacetaldehyde synthase from Petunia hybrida catalyzed
the decarboxylation of phenylalanine to phenylacetaldehyde (Kaminaga et al., 2006),
which might be the potential precursor for the production of phenylpropanoids including
phenylethanol and phenylethyl acetate in Vanda Mimi Palmer. Previously in floral scent
studies, two phenylacetaldehyde synthases have been identified from Petunia hybrida
(PhPAAS) and Rosa hybrida (RhPAAS). The PhPAAS and RhPAAS share ~50-60%
144
identity with other plant decarboxylases including tyrosine decarboxylases, tryptophan
decarboxylases and aromatic amino acid decarboxylases (Kaminaga et al., 2006). Motif
prediction by Expasy tool showed a pyridoxal phosphate attachment site in the deduced
amino acid sequence of VMPPAAS and other plant decarboxylases (see figure 17).
Besides that, a conserved VHVDAAY motif has been identified in the deduced amino
acid sequence of VMPPAAS as reported in other decarboxylase proteins from plants and
bacteria by Connil et al., 2002 (see figure 17). Thus, VMPPAAS could be classified as
aromatic amino acid decarboxylase which might be involved in decarboxylation of
aromatic amino acids in Vanda Mimi Palmer especially for phenylalanine. The
involvement of VMPPAAS in decarboxylation of phenylalanine to phenylacetaldehyde
can only be confirmed by enzymatic assay (see Figure 42). Phylogenetic analysis of
VMPPAAS with other plant decarboxylases shows VMPPAAS is not clustered together
in the same group as PhPAAS and RhPAAS since Rosa hybrida and Petunia hybrida
come from different family and genera compared to Vanda Mimi Palmer. Localization
analysis by Localizome software shows no transit peptide in VMPPAAS amino acid
sequence, suggesting this protein might be localized in cytosol.
Relative expression analysis in different tissues shows that the putative
phenylacetaldehye synthase (VMPPAAS) was up-regulated in floral tissues compared to
vegetative tissues. Among the floral tissues of Vanda Mimi Palmer, petal showed the
highest expression of VMPPAAS and followed by sepal. In other scented flowers, petals
have been identified as the main source of scent emission and biosynthesis. Most of the
145
Figure 42: Elucidation of Benzenoid and Phenylpropanoid Pathways of Vanda Mimi
Palmer. This elucidation is based on benzenoid and phenylpropanoid metabolism of
Petunia hybrida (Boatright et al., 2004; Pichersky and Dudareva, 2007).
146
well studied fragrance-related transcripts have shown higher expression levels in petal
compared with the other floral parts including in Petunia hybrida (Verdonk et al., 2003),
Clarkia (Onagraceae) (Pichersky et al., 1994), and Rosa hybrida (Shalit et al., 2003). In
sepal, the high level of VMPPAAS expression (slightly lower than petal) might be due to
the similar structure and function of sepal and petal in orchid. This is supported by earlier
histological work on Vanda Mimi Palmer that showed the presence of many oil glands
(trichomes) in petals and sepals (Janna Ong Abdullah, unpublished data) suggesting
potential sites for fragrance biosynthesis and accumulation.
Relative expression analysis of putative VMPPAAS at different developmental stages
shows a developmentally regulated pattern. This expression study complements the result
of GC-MS analysis on the scent of Vanda Mimi Palmer at different developmental stages
(in section 4.1.1) whereby the emission of phenylethyl acetate in Vanda Mimi Palmer is
developmentally regulated. In bud stages including young and mature buds, the
expression of VMPPAAS showed a down-regulated expression compared to half-open
and fully-open flower stages. In addition, from the GC-MS analysis, no volatile
compound was detected in the bud stage. In other scented flowers including Clarkia
breweri, Anthirrhinum majus, Rosa hybrida and Petunia hybrida (Pichersky et al., 1994;
Nagegowda et al., 2008), no emission of fragrance-related compounds were detected at
the bud stage. Thus, during early stages of flower development, the floral tissues of
Vanda Mimi Palmer might not be ready for fragrance biosynthesis. In half-open flower
stage, the expression of VMPPAAS increased drastically and reached the highest level at
fully-open flower stage. At 14-day old fully-open flower, the VMPAAS expression level
147
decreased drastically compared to the early fully-open flower stage. The developmentally
regulated pattern of fragrance biosynthesis in Vanda Mimi Palmer is also similar to other
fragrance-related transcripts of S-adenosyl-L-methionine: benzoic acid carboxyl methyl
transferase (BAMT) from Anthirrinum majus (Dudareva et al., 2003), geranyl
diphosphate synthase from Phalaenopsis bellina (Hsiao et al., 2008), and linalool
synthase from Antirrhinum majus (Nagegowda et al., 2008).
Relative expression analysis of VMPPAAS in the fully-open flower of Vanda Mimi
Palmer in a 24-hour cycle shows a differential expression whereby the expression is up-
regulated at night time and down-regulated during day time. The expression pattern of the
VMPPAAS in a 24-hour cycle is opposite compared to the emission of phenylethyl
acetate in a 24-hour cycle whereby the emission of phenylethylacetate compounds from
the fully-open flower was detected at high levels during day time and not detected at
night (see section 4.1.1). This might be due to VMPPAAS being not directly involved in
catalyzing the formation of the end product (phenylethyl acetate) but involved in the
formation of the main precursor for the end product. In addition, the precursor might be
stored in cells for few hours before being used for subsequent reaction and released as
volatile (phenylethyl acetate) during day time. The gene that encodes the rate-limiting
enzyme for the formation of phenylethyl acetate (not identified yet) might show high
expression during day time because phenylethyl acetate emission was detected high
during day time (see section 4.1.1). In other scented flowers such as Petunia hybrida and
Stephanotis floribunda, the expression of fragrance-related genes and emission of
volatiles were very high level during night time and very low level during the day (Pott et
148
al., 2002; Boatright et al., 2004). Meanwhile, in Anthirrhinum majus, both fragrance-
related genes expression (mycrene synthase and ocimene synthase) and volatiles emission
were detected at high level during the night (Dudareva et al., 2003). In other scented
flowers including Petunia hybrida (Boatright et al., 2004), Anthirrhinum majus
(Dudareva et al., 2003; Nagegowda et al., 2008) and Stephanotis floribunda (Pott et al.,
2002), the volatiles emission pattern and expression profile of fragrance-related genes
which are involved in the formation of end product are usually similar.
5.2.2.2 Sequence and Expression Analysis of Putative 4-(cytidine 5'-diphospho)-2-C-
methyl-D-erythritol Kinase (VMPCMEK)
The deduced amino acid sequence of VMPCMEK shows 66-72% identity to 4-(cytidine
5'-diphospho)-2-C-methyl-D-erythritol kinase from other plants including Hevae
brasiliensis, Solanum lycopersicum, Catharanthus roseus and Ginkgo biloba. Besides
that, VMPCMEK shared the conserved ATP-binding site for functional activity of 4-
(cytidine 5'-diphospho)-2-C-methyl-D-erythritol kinase (Kim et al., 2008). In addition,
there are few other putative conserved motifs shared by VMPCMEK and other plant 4-
(cytidine 5'-diphospho)-2-C-methyl-D-erythritol kinase as predicted by the Expasy tool.
The 4-(cytidine 5'-diphospho)-2-C-methyl-D-erythritol kinase has been reported to be
involved in phosphorylation of 4-(cytidine 5′-diphospho)-2-C-methyl-D-erythritol into its
phosphorylated form, 4-(cytidine 5′-diphospho)-2-C-methyl-D-erythritol 2-phosphate in
the terpenoid pathway (Kim et al., 2008) (see Figure 41 in section 5.2.1). The subsequent
reactions in the terpenoid pathway might produce the main precursors including
149
dimethylallyl diphosphate (DMAPP) and Isopentenyl diphosphate (IPP) (see Figure 41 in
section 5.2.1) for biosynthesis of monoterpenes and sesquiterpenes (Ogura and Koyama,
1998; Poulter and Rilling, 1981; McGarvey and Creteau, 1995). The Localizome
software predicted the presence of signal peptide in VMPCMEK amino acid sequence.
Thus, VMPCMEK might be localized in plastid since it is predicted to be involved in the
early step of terpenoid pathway, the methyl erythritol phosphate (MEP) pathway which
has been reported to occur in plastid (Lichtenthaler, 1999; Rohmer, 1999).
Relative expression analysis of putative 4-(cytidine 5'-diphospho)-2-C-methyl-D-
erythritol kinase (VMPCMEK) in different tissues showed up-regulated expression in the
tested floral tissues compared to vegetative tissues. VMPCMEK is predicted to be
involved in the earlier path of terpenoid pathway which might contribute to the
biosynthesis of volatile terpenoids including linalool, ocimene and nerolidol, as detected
in the floral scent of Vanda Mimi Palmer by GC-MS analysis (see section 4.1.1). In the
bud, no fragrance compounds had been detected by GC-MS but the VMPCMEK
expression level was relatively high in bud compared to vegetative tissues. This might be
due to the involvement of terpenoid pathway for synthesis of other non-volatile
terpenoids including carotenoids, gibberelin and sterol which might be used either in
primary or secondary metabolisms (Bremly, 1997).
Analysis of VMPCMEK expression at different flower developmental stages shows a
developmentally regulated pattern in accordance to volatile emission pattern (in section
4.1.1), suggesting the terpenoids emission is developmentally regulated. Terpenoid
150
pathway was also reported to be important for gibberellin biosynthesis whereby
gibberellins were reported to play an important role in plant growth and development
(Bremley, 1997). VMPCMEK might be involved in the early step of the terpenoid
pathway which contributes for bud development. Thus, during bud stage of Vanda Mimi
Palmer, VMPCMEK might play an important role for flower development while at open
flower stage (once the flower open until end of the flower life-time), VMPCMEK might
contribute for fragrance biosynthesis.
VMPCMEK shows a differential expression in a 24-hour cycle but the expression pattern
is totally different compared to VMPPAAS. This could be due to VMPCMEK and
VMPPAAS might be involved in different pathways in fragrance biosynthesis of Vanda
Mimi Palmer. Besides that, the expression of VMPCMEK and emission pattern of
terpenoids including linalool and ocimene (see Figure 6 in section 4.1.1) are also
different. At 2pm, the expression of VMPCMEK is at the lowest level while the emission
level of linalool and ocimene compound are detected at the highest level. The expression
of VMPCMEK increased gradually from 12.00am until 8.00am, and then decreased
gradually until the lowest peak at 2.00pm while the emission of terpenoids including
linalool and ocimene (see Figure 6 in section 4.1.1) were detected at very low levels as
early as 6.00am and increased gradually until the highest peak at 2.00pm. This might be
due to the fragrance compounds or their intermediates might not be directly emitted after
being synthesized and the compounds might be stored in trichomes for a few hours before
being released into the air as scent. The precursors of the final compounds might be
accumulated in cytosol or plastid before being used for the final product biosynthesis.
151
Besides that, the volatiles emission might not be controlled at gene expression level, but
influenced by environment factors like heat and light. This is supported by the fact that
the emission of volatiles from the flower of Vanda Mimi Palmer were detected at very
high level in the early afternoon, a period when highest temperature usually occur in
Malaysia.
5.2.2.3 Sequence and Expression Analysis of Putative Cytochrome P450 Protein
(VMPCyP450)
BLASTX and BLASTP analysis (NCBI) shows the deduced amino acid of VMPCyP450
exhibited 39-67% similarity to other plant cytochrome p450 proteins including p450
protein from Oryza sativa, Populus trichocarpa, Ricinus communis, Capsicum annum
and also cytochrome P450 monooxygenase of Petunia hybrida, a well studied scented
flower. Cytochrome p450 proteins have been reported to be involved in modification of
monoterpene compounds into another compound such as oxidation of linalool to linalool
oxide (Hsiao et al., 2006; Dudareva et. al., 2004; Dudareva and Pichersky, 2000). Thus,
VMPCyP450 is postulated to be involved in fragrance biosynthetic pathway of Vanda
Mimi Palmer since GC-MS analysis of the scent of Vanda Mimi Palmer detected traces
of linalool oxide. Besides that, cytochrome P450s have been reported to be involved in
other fragrance biosynthetic pathways including benzenoid, phenylpropanoid and
lipoxygenase pathways (Ehlting et al., 2006). Thus, in Vanda Mimi Palmer, VMPCyP450
might be involved in fragrance biosynthesis eventhough the exact function of the protein
in fragrance biosynthetic pathway of Vanda Mimi Palmer is still far from understood.
Phylogenetic analysis of VMPCyP450 with closely related proteins from other plants
152
shows VMPCyP450 is clustered together with cytochrome P450 monooxygenase of
Petunia hybrida (a well studied scented flower). Thus, VMPCyP450 might be involved in
fragrance biosynthesis like Petunia hybrida eventhough the cytochrome P450
monooxygenase is not well studied in Petunia hybrida. Localization analysis by
Localizome shows no signal peptide in the VMPCyP450 amino acid sequence. The same
analysis shows the presence of a transmembrane domain in the VMPCyP450 amino acid
sequence. Thus, VMPCyP450 could possibly be located in the plastid membrane like
cyctochrome C which is involved in photosynthesis. So, VMPCyP450 might be involved
indirectly in the terpenoid pathway of Vanda Mimi Palmer since monoterpene
biosynthesis in plant mostly occurs in plastid (Lichtenthaler, 1999; Rohmer, 1999).
Relative expression analysis of putative cytochrome P450 protein (VMPCyP450) in
different tissues showed up-regulated expression in floral tissues compared to vegetative
tissues. Among the floral tissues, the lip shows the highest expression, more than two
times higher than petal and sepal. This contrasting result compared to other putative
fragrance-related transcripts (VMPPAAS and VMPCMEK) in Vanda Mimi Palmer could
be due to the involvement of VMPCyP450 in other metabolisms besides fragrance
biosynthesis. As reported by Seidenfaden and Wood (1992), lip of orchid, which is a
modified petal, has a complicated structure. Transformation of a putative P450 gene into
Phalaenopsis flowers (orchidecae genera) showed a possibility for colour modification of
flowers compared to wild type where the anthocyanins level showed an increased (Su and
Shu, 2003). Thus, in Vanda Mimi Palmer, VMPCyP450 might also be involved in the
bright colour formation especially in the lip. In petal and sepal, VMPCyP450 might be
153
involved indirectly in fragrance biosynthesis as cytochrome P450 proteins were reported
to be involved indirectly in fragrance biosynthesis including terpenoid, benzenoid and
phenylpropanoid, as well as lipoxygenase pathways as in other plants (Ehlting et al.,
2006). In Vanda Mimi Palmer, VMPCyP450 might be involved in terpenoids as well as
benzenoid and phenylpropanoid pathways because volatile fragrance compounds from
both classes were detected in the scent of Vanda Mimi Palmer (see section 4.1.1).
Real-time PCR result on VMPCyP450 expression at different developmental stages
shows a developmentally regulated pattern like other putative fragrance-related
transcripts (VMPPAAS and VMPCMEK). This result is expected since the fragrance
compounds emission detected by GC-MS (see section 4.1.1) is developmentally regulated
eventhough the specific location of VMPCyP450 in the fragrance biosynthetic pathway is
still not well understood. Expression analysis of VMPCyP450 at different time points in a
24-hour cycle shows a differential expression throughout the day. The pattern is totally
different compared to other putative fragrance-related transcripts (VMPPAAS and
VMPCMEK). This could be because VMPCyP450 is not only involved in fragrance
biosynthesis but also other metabolic functions in Vanda Mimi Palmer.
5.2.2.4 Sequence and Expression Analysis of Unknown Protein (VMPA28)
Analysis of the deduced amino acid sequence of an unknown protein transcript
(VMPA28) of Vanda Mimi Palmer shows no significant hit to any known proteins in the
Genebank database. Thus, sequence analysis with closely related proteins including
clustal W alignment and phylogenetic analysis cannot be carried out. Localization
154
analysis by Localizome software shows VMPA28 does not have any signal peptide in its
amino acid sequence. Thus, VMPA28 might be localized in the cytosol. Motif search by
Expasy tool shows the presence of some sites including a N-glycosylation site (NTSN),
two protein kinase C phosphorylation sites (SxK, SxR) and a N-myristoylation site
(GAxxSN). The function of the VMPA28 can only be confirmed by functional study of
this protein.
Real-time PCR analysis on the expression of the VMPA28 transcript in different tissues
showed a slightly higher expression in floral tissues compared to vegetative tissues. This
could be due to the involvement of VMPA28 in both fragrance and non-fragrance
metabolisms eventhough the function of VMPA28 protein is still far from understood.
Expression analysis of VMPA28 transcript at different flowering developmental stages
shows a developmentally regulated pattern like other putative fragrance related
transcripts (VMPPAAS, VMPCMEK and VMPCyP450). The result is in accordance to
the GC-MS result on the scent of Vanda Mimi Palmer whereby the scent emission is
developmentally regulated. Meanwhile, expression analysis of VMPA28 transcript in a
24-hour cycle showed a differential expression which is totally different compared to
other putative fragrance-related transcripts selected (VMPPAAS, VMPCMEK and
VMPCyP450) for molecular characterization. Thus, the involvement of VMPA28 in
fragrance metabolism in Vanda Mimi Palmer could be different to these other putative
fragrance-related transcripts selected for molecular characterization.
155
CHAPTER 6
CONCLUSION
The objectives of this study have been successfully achieved by biochemical analysis of
the scent of Vanda Mimi Palmer as well as isolation and molecular characterization of
putative fragrance-related transcripts that might be involved in the fragrance biosynthetic
pathways of this orchid hybrid. Two fragrance biosynthetic pathways have been
elucidated to play a role in biosynthesis of the fragrance which are terpenoid and also
benzenoid and phenylpropanoid pathways. In the terpenoid pathway, a putative
fragrance-related transcript, putative 4-(cytidine 5′-diphospho)-2-C-methyl-d-erythritol
kinase (VMPCMEK) which was identified by differential screening of floral cDNA was
postulated to be involved in the early step of this pathway. The VMPCMEK transcript
might play an important role in the biosynthesis of monoterpenes and sesquiterpene
including linalool, ocimene, and nerolidol. Meanwhile, a putative phenylacetaldehyde
synthase (VMPPAAS) isolated from ESTs of Vanda Mimi Palmer might be involved in
the early path of benzenoid and phenylpropanoid pathway, possibly involved in the
biosynthesis of phenylethanol and phenylethyl acetate compounds which were detected in
the floral scent of this orchid. Besides that, there were two putative fragrance-related
genes encoding putative cytochrome P450 protein (VMPCyP450) and an unknown
protein (VMPA28) were also isolated and characterized at molecular level. These genes
might contribute to the fragrance of Vanda Mimi Palmer indirectly.
156
Combination of GC-MS analysis of the scent of Vanda Mimi Palmer and expression
analysis of putative fragrance-related transcripts at different developmental stages have
shown that the floral scent biosynthesis and emission in Vanda Mimi Palmer is
developmentally and rhythmically regulated. The knowledge from this work is important
for preliminary understanding of fragrance characteristics and its biosynthetic pathway in
Vanda Mimi Palmer. In order to gain a deeper insight on the fragrance biosynthesis in
Vanda Mimi Palmer, future work should focus on isolation of full-length cDNA of rate-
limiting enzymes that are responsible for the biosynthesis of fragrance compounds. The
fragrance-related cDNA should be cloned into bacterial system and confirm their
involvement in fragrance biosynthetic pathway by enzymatic assays. Besides that, future
work should also focus on transformation of the fragrance-related cDNA into other plant
system including non-fragrance orchids and other flowers to increase their commercial
values.
157
BIBLIOGRAPHY
Altenburger, R. and Matile, P. (1988). Rhythms of fragrance emission in flowers. Planta
174: 242-247.
Arimura, G., Kost, C. and Boland, W. (2005). Herbivor-induced, indirect plant defences.
Biochimica et Biophysica Acta 1734: 91-111.
Basu, K., Das gupta, B., Bhattacharya, S.K., Lal, R. and Das, P.K. (1971). Anti-
inflammatory principles of Vanda roxburghii. Current Science. 40: 86-87.
Beaulieu, J.C. and Grimm, C.C. (2001). Identification of volatile compounds in
cantaloupe at various developmental stage using solid phase micro-extraction.
Journal of Agricultural and Food Chemistry 49: 1334-1352.
Boatright, J., Negre, F., Chen, X., Kish, C.M, Wood, B., Peel, G., Orlova, I., Gang, D.,
Rhodes, D. and Dudareva, N. (2004). Understanding in vivo benzenoid metabolism
in petunia petal tissue. Plant Physiology 135: 1993-2011.
Boerjan, W., Ralph, J. and Baucher, M. (2003). Lignin biosynthesis. Annual Review of
Plant Biology 54: 519-546.
Bremly, P.M. (1997). Isoprenoid metabolism. In P.M. Dey and J.B. Harborn. Plant
Biochemistry (pp. 417-437). London : Academic Press Ltd.
Buchmann, S. and Nabhan, G. (1996). The Forgotten Pollinators. Washington: Island
Press.
Chan, W.S., Abdullah, J.O., Namasivayam, P., Mahmood, M. (2009). Molecular
characterization of a new 1-deoxy-D-xylulose 5-phosphate reductoisomerase
(DXR) transcript from Vanda Mimi Palmer. Scientia Horticulturae 121: 378-382.
Chan, W.S. (2009). Identification and Characterization of Selected Full-length
Fragrance-related Transcripts from Orchid (Vanda Mimi Palmer), Master Thesis,
Universiti Putra Malaysia.
Chen, F., D‟Auria, J.C., Tholl, D., Ross, J.R., Gershenzon, J., Noel, J.P., and Pichersky,
E. (2003). An Arabidopsis thaliana gene for methylsalicylate biosynthesis,
identified by biochemical genomic approach, has a role in defense. Plant Journal
36: 577-588.
Chin, S.T., Nazimah, S.A.H., Quek, S.Y., Che Man, Y. B., Abdul Rahman, R. and Mat
Hashim, D. (2007). Analysis of volatile compounds from Malaysian durians (Durio
158
zibethinus) using headspace SPME coupled to fast GC-MS. Journal of Food
Composition and Analysis 20: 31-44.
Chopra, R.N., Nayar, S.L. and Chopra, I.C. (1956). Glossary of Indian Medicinal Plants.
New Delhi: New Delhi C. S. I. R.
Connil, N., Breton, Y. L., Dousset, X., Auffray, Y., Rince, A. and Pre vost, H. (2002).
Identification of the Enterococcus faecalis Tyrosine Decarboxylase Operon
Involved in Tyramine Production. Applied and Environmental Microbiology 68:
3537-3544.
Croteau, R., and Karp, F. (1991) Origin of natural odorants. In P.M. Muller and
D.Lamparsky. Perfume: Art, Science and Technology (pp. 101-126). New York:
Elsevier Applied Sciences.
D‟Auria, J.C., Chen, F. and Pichersky, E. (2002). Characterization of an acyltransferase
capable for synthesizing benzyl benzoate and other volatile esters in flowers and
damaged leaves of Clarkia breweri. Plant Physiology 130: 466-476.
Derikvand, M.M., Sierra, J.B., Ruel, K., Pollet, B., Do, C.T., Thevenin, J., Buffard, D.,
Jouanin, L. and Lapierre, C. (2008). Redirection of the phenylpropanoid pathway to
feruloyl malate in Arabidopsis mutants deficient for cinnamoyl-CoA reductase 1.
Planta 227: 943-956.
Dobson, H.E.M. (1994). Floral volatiles in insect biology. In E.A. Bernays. Insect Plant
Interactions Volume.5 (pp. 47-81). Bota Raton: CRC Press.
Dressler, R.L. (1993). Phylogeny and classification of the orchid family. Oregon:
Dioscorides Press.
Dudareva, N., D‟Auria, J.C., Nam, K.H., Raguso, R.A. and Pichersky, E. (1998). Acetyl-
CoA: benzylalcohol acetyltransferase – an enzyme involved in floral scent
production in Clarkia breweri. Plant Journal 14: 297-304.
Dudareva, N., Murfitt, L. M., Mann, C.J, Gorestein, N., Kolosova, N., Kish, C.M.,
Bonham, C. and Wood, K. (2000). Developmental regulation of methyl benzoate
biosynthesis and emission in snapdragon flowers. Plant Cell 12: 949-961.
Dudareva, N. and Pichersky, E. (2000) Biochemical and Molecular Genetic Aspects of
Floral Scents. Plant Physiology 122: 627-633.
Dudareva, N., Martin, D., Kish, C.M., Kolosova, N., Gorestein, N., Faldt, J., Miller, B.
and Bohlman, J. (2003). (E)-ß-ocimene and myrcene synthase genes of floral scent
biosynthesis in snapdragon: Functional and expression of three terpene synthase
genes of a new terpene synthase subfamily. Plant Cell 15: 1227-1241.
159
Dudareva, N., Pichersky, E. and Gershenzon, J. (2004) Biochemistry of Plant Volatiles.
Plant Physiology 135: 1893-1902.
Effmert, U., Buss, D., Rohrberk, D. and Piechulla, B. (2006). Localization of the
synthesis and emission of scent compounds within the flower. In N. Dudareva and
E. Pichersky. Biology of Floral Scent (pp. 105-124). Florida: CRC Press.
Ehlting, J., Hamberger, B., Million-Rousseau, R. and Werck-Reichhart, D. (2006).
Cytochrome P450 in phenolic metabolism. Phytochemistry Review 5: 239-270.
Erlich, F. (1907). Uber die Bedingungen der Fuselolbildung and uber ihren
Zusammenhang mit dem Eiweissaufbau der Hefe. Berichte der Deutschen
Chemischen Gesellschaft . 40: 1027-1047.
Fadelah, A.A., Zaharah, H., Rozaily, Z., Nuraini, I., Tan, S.L. and Hamidah, S. (2001).
Orchids The Living Jewels of Malaysia. Kuala Lumpur: Malaysia Agricultural
Research and Development Institute.
Faegri, K. and Van der Pijl, L. (1979). The Principles of Pollination Ecology. London:
Pergamon Press.
Farhi, M., Lavie, O., Masci, T., Hendel-Rahmanim, K., Weiss, D., Abeliovich, H. and
Vainstein, A. (2010). Identification of rose phenylacetaldehyde synthase by
functional complementation in yeast. Plant Molecular Biology 72: 235-245.
Feussner, I. and Wasternack, C. (1998) Lipoxygenase catalyzed oxygenation of lipids.
Lipid-Fett 100: 146-152.
Flament, I., Debonneville, C. and Furrer, A. 1993. Volatile constituents of roses:
characterization of cultivars based on the headspace analysis of living flower
emissions. In R. Teranishi, R.G. Buttery, and H. Sugisawa. Bioactive Volatile
Compounds from Plants (pp. 269-281). Washington: American Chemical Society.
Frowine, S.A. (2005). Fragrant Orchids. Oregon: Timber Press Inc.
Gang, D.R., Wang, J., Dudareva, N., Kyoung, H.N., Simon, J.E., Lewinsohn, E. and
Pichersky, E., (2001). An investigation of the storage and biosynthesis of
phenylpropenes in sweet basil. Plant Physiology 125: 539-555.
Guterman, I., Monshe, S., Naama, M., Dan, P., Mery, D.Y., Gil, S., Einat, B., Olga, D.,
Mariana, O., Michal, E., Jihong, W., Zach, A., Pichersky, E., Efraim, L., Dani, Z.,
Vainstein, A. and David, W. (2002). Rose Scent: Genomic approach to discovering
novel floral fragrance-related genes, The Plant Cell 14: 2325-2338.
Hall, T.A. (1999). BioEdit: a user-friendly biological sequence alignment editor and
analysis program for Windows 95/98/NT. Nucleic Acids Symposium 41: 95-98.
160
Hamdan, O. (2008). Variasi Dunia Orkid : Panduan Penanaman dan Penjagaan. Shah
Alam: Alaf 21 Sdn. Bhd.
Hands, G. (2006) Pocket Guide to Orchids. England: D&S Books Ltd.
Hendel-Rahmanim, K.H., Masci, T., Veinstein, A. and Weiss, D. (2007). Diurnal
regulation of scent emission in rose flowers. Planta 226: 1491-1499.
Helsper, J.P.F.G., Davies, J.A., Bouwmeester, H.J., Krol, A.F., and Van Kampen, M.H.
(1998). Circadian rhythmicity in emission of volatile compounds by flowers of
Rosa hybrida L. cv. Honesty. Planta 207: 88-95.
Hites, R.A. (1997). Gas Chromatography Mass Spectrometry. In S.A. Settle. Handbook
of Instrumental Techniques for Analytical Chemistry (pp. 609-626). New Jersey:
Prentice Hall PTR.
Hsiao, Y.Y., Tsai, W.C., Huang, T.H., Wang, H.C, Wu, T.S, Leu, Y.L., Chen, W. H. and
Chen, H.H. (2006). Comparison of transcripts in Phalaenopsis bellina and
Phalaenopsis equestris (Orchidaceae) flowers to deduce monoterpene biosynthesis
pathway. BMC Plant Biology 6: 14.
Hsiao, Y.Y., Jeng, M.F., Tsai, W.C., Chuang, Y.C., Li, C.Y., Kuoh, C.S., Chen, W.H.
and Chen, H.H. (2008). A novel homodimeric geranyl diphosphate synthase from
the orchid Phalaenopsis bellina lacking a DD (X)2-4D motif. The Plant Journal 55:
719-733.
Huang, X. and Madan, A. (1999). CAP3: A DNA sequence assembly program. Genome
Research 9: 868-877.
Jaillais, B., Bertrand, V. and Auger, J. (1999). Cryo-trapping/SPME/GC analysis of
cheese aroma. Talanta 48: 747-753.
Janna O.A, Tan, L.P., Maziah, M., Puad, A. and Azlan, J.G. (2005). Dull to behold, good
to smell……Vanda Mimi Palmer. Biotech Communication 1: 21-25.
Kaiser, R. (1993). The scent of orchids: Olfactory and chemical investigations.
Amsterdam: Elsevier Science Publishers B.V.
Kaminaga, Y., Schnepp, J., Peel, G., Kish, C.M., Gili, B.N., Weiss, D., Orlova, I., Lavie,
O., Rhodes, D., Wood, K., Porterfield, D.M., Cooper, A.J.L., Schloss, J.V.,
Pichersky, E., Veinstein, A. and Dudareva, N. (2006). Plant phenylacetaldehyde
synthase is a bifunctional homotetradimeric enzyme that catalyzes phenylalanine
decarboxylation and oxidation. Journal of Biological Chemistry 281: 23357-23366.
Kaori, S., Kishimoto, K., Ozawa, R., Kugimiya, S., Urahimo, S., Arimura, G., Horiuchi,
J., Nishioka, T., Matsui, K. and Takabayashi, J. (2006). Changing green leaf
volatile biosynthesis in plants: An approach for improving plant resistance against
161
both herbivores and pathogens. Proceedings of The National Academy of Sciences
103: 16672-16676.
Kim, S.M., Kim, Y. B., Kuzuyama, T., Kim, S.U. (2008) Two copies of 4-(cytidine 5‟-
diphospho-2-C-methyl-D-erythritol kinase (CMK) gene in Ginkgo biloba:
molecular cloning and functional characterization. Planta 228: 941-950.
Kirtikar, P.K. and Basu, B.D. (1975) Indian Medicinal Plants, Second Edition (Bishen, S.
and Mahendran, P.S, First edition 1935). Dehra Dun: New Connaught Place
Knudsen, J.T., Tollesten, L. and Bergstrom, G.L. (1993). Floral scents-A checklist of
volatile compounds isolated by headspace techniques. Phytochemistry 33: 253-280.
Knudsen, J.T. and Gershenzon, J. (2006). The chemical diversity of floral scent. In N.
Dudareva and E. Pichersky. In N. Dudareva and E. Pichersky. Biology of Floral
Scent (pp. 105-124). Florida: CRC Press.
Lacombe, E., Hawkins, S., Van Doorsselaere, J., Piquemal, J., Goffner, D.,
Poeydomenge, O, Boudet, A.M. and Grima-Pettenati, J. (1997). Cinnamoyl CoA
reductase, the first committed enzyme of the lignin branch biosynthetic pathway:
cloning, expression and phylogenetic relationship. Plant Journal: 429-441.
Lavid, N., Wang, J., Shalit, M., Guterman, I., Bar, E., Beuerle, T., Menda, N., Shafir, S.,
Zamir, D., Adam, Z., Vainstein, A., Weiss, D., Pichersky, E. and Lewinsohn, E.
(2002). O-methyltransferase involved in biosynthesis of voloatile phenolic
derivatives in rose petals. Plant Physiology 129: 1899-1907.
Lee, S., Lee, B., Jang, I., Kim, S. and Bhak, J. (2006). Localizome: a server for
identifying transmembrane topologies and TM of eukaryotic proteins utilizing
domain information. Nucleic Acids Research 34: 99-103.
Lichtenthaler, H.K. (1999). The 1-deoxy-D-xylulose-5-phosphate pathway of isoprenoid
biosynthesis in plants. Annual Review of Plant Physiology and Plant Molecular
Biology 50: 47-66.
Liu, T.T. and Yang, T.S. (2002). Optimization of solid-phase microextraction analysis for
studying change of headspace flavour compounds of banana during ripening.
Journal of Agricultural and Food Chemistry 50: 653-657.
Lindqvist, C., Scheen, A.C., Yoo, M.J., Grey, P., Oppenheimer, D.G., Leebens-Mack,
J.H., Soltis, D.E., Soltis, P.S. and Albert, V.A (2006). An expressed sequence tag
(EST) library from developing fruits of an Hawaiian endemic mint (Stebogyne
rugosa, Lamiaceae): characterization and microsatellite markers. BMC Plant
Biology 6: 16.
162
Lu, S., Xu, R., Jia, J.W., Phang, J., Matsuda, S.P.T. and Chen, X.Y. (2002). Cloning and
Functional Characterization of a _-Pinene Synthase from Artemisia annua That
Shows a Circardian Pattern of Expression. Plant Physiology 130: 477-486.
Mabberly, D.J. (1997). The Plant (Second edition). Cambridge: Cambridge University
Press.
McCaskill D and Croteau R. (1995). Monoterpene and sesquiterpene biosynthesis in
glandular trichomes of peppermint (Mentha × piperita) rely exclusively on plastid-
derived isopentenyl diphosphate. Planta 197:49–56.
McGarvey, D. J. and Croteau, R. (1995). Terpenoid metabolism. Plant Cell 7: 1015-
1026.
Motes, M.R. (1997). Vandas: Their Botany, History, and Culture. London: Timber Press.
Nair, H. and Arditti, J. (2002). Award and trophies. Proceedings of the 17th
World Orchid
Conference “Sustaining Orchids for the Future”. Sabah: Natural History
Publications (Borneo) Sdn. Bhd.
Nagegowda, D.A., Gutensohn, M., Wilkerson and C.G., Dudareva N. (2008). Two nearly
identical terpene synthases catalyze the formation of nerolidol and linalool in
snapdragon flowers. The Plant Journal 55: 224-239.
Ogura K. and Koyama, T. (1998). Enzymatic aspects of isoprenoid chain elongation.
Chemical Review 98: 1263-1276.
Pichersky, E. and Dudareva, N. (2007). Scent engineering: toward the goal of controlling
how flowers smell. Trends in Biotechnology 25: 105-110.
Pichersky, E., Lewinsohn, E. and Croteau, R. (1995). Purification and characterization of
S-linalool synthase, and enzyme involved in the production of floral scent in
Clarkia breweri. Archieves of Biochemistry and Biophysics 306 : 803-807.
Pichersky, E., Raguso, R.A., Lewinsohn, E. and Croteau, R. (1994). Floral scent
production in Clarkia (Onagraceae) I. Localization and developmental modulation
of monoterpenes emission and linalool synthase activity. Plant Physiology 126:
1533-1540.
Picone, J.M., Clery, R.A., Watanabe, N., MacTavish H.S. and Turntbull, C.G.N. (2004).
Rhythmic emission of floral volatiles from Rosa damascena semperflorens cv.
„Quatre Saisons‟. Planta 219: 468-478.
163
Pott, M.B., Pichersky, E. and Piechulla, B. (2002). Evening-specific oscillation of scent
emission, SAMT enzyme activity, and mRNA in flowers of Stephanotis floribunda.
Journal of Plant Physiology 159: 925-934.
Poulter , C.D. and Rilling, H.C. (1981). Prenyl transferase and isomerase. In J.W. Porter
and S.L. Spurgeon. Biosynthesis of Isoprenoid Compounds Vol. 1 (pp. 162-209).
New Jersey: John Willey and Sons Inc.
Raguso R.A and Pichersky E. (1995). Floral volatiles from Clarkia breweri and Clarkia
concinna (Onagraceae): recent evolution of floral scent and moth pollination. Plant
System Evolution 194: 55-67.
Raguso, R.A., Levin, R.A., Foose, S.E., Holmberg, M.W. and McDade, L.A. (2003).
Fragrance chemistry, nocturnal rhythms and pollination “syndromes” in Nicotiana.
Phytochemistry 63: 265-284.
Rees, J.D. (1991). Reverse enzyme synthesis in microemulsion-based organogels.
Biochemica et Biophysica Acta 1073: 493-501.
Rohmer, M. (1999). The discovery of a mevalonate-independent pathway for isoprenoid
biosynthesis in bacteria, algae and higher plants. Natural Product Report 16: 565-
574.
Reinhard, J., Srivivasan, M.V. and Zhang, S. (2004). Scent-triggered navigation in
honeybees. Nature 427: 411.
Rittershausen, B. and Rittershausen, S. (2008). Orchid Basics. London: Octopus
Publishing Group Ltd.
Rougetel, H.L. (1988) Chinese ancestors and their descendents. In A Heritage of Roses.
London: Unwin Hyman.
Sandmeier, E., Hale, T. I. and Christen, P. (1994). Multiple evolutionary origin of
pyridoxal-5'-phosphate-dependent amino acid decarboxylases. Europe Journal of
Biochemistry 221: 997-1002.
Sambrook, J. and Russel, D.W. (2001). Molecular Cloning: A Laboratory Manual. New
York: Cold Spring Harbor Laboratory Press.
Sambrook, J., Fritsch, E.F., Maniatis, T. (1989). Molecular Cloning: A Laboratory
Manual. New York: Cold Spring Harbor Laboratory Press.
Sananayake, U.M., Wills, R.B.H. and Lee, T.H. (1977). Biosynthesis of eugenol and
cinnamic aldehyde in Cinnamomum zeylanicum. Phytochemistry 16: 2032-2033.
164
Schade F., Legge R.L. and Thompson J.E. (2001). Fragrance volatiles of developing and
senescing carnation flowers. Phytochemistry 56: 703-710.
Seidenfaden, G. and Wood, J.J. (1992). The orchids of Peninsular Malaysia and
Singapore: A revision of R.E. Holttum: Orchids of Malaya. Fredensborg: Olsen &
Olsen.
Shalit, M., Guterman, I., Volpin, H., Bar, E., Tamari, T., Menda, N., Zach, A, Zamir, D.,
Vainstein, A., Weiss, D., Pichersky, E. and Lewinsohn, E. (2003). Volatile ester
formation in Roses; Identification of an acetyl-coenzyme A; Geraniol/citronellol
acetyltransferase in developing rose petals. Plant Physiology 131: 1868-1876.
Sheela, V.L. (2008). Flowers for Trade: Vol. 10 Horticulture Sciences Series, New Delhi:
New India Publishing
Shirley, B.W. (2001). Flavanoid biosynthesis. A colourful model for genetics,
biochemistry, cell biology and biotechnology. Plant Physiology 126: 485-493.
Su, V. and Shu, B.D. (2003). Cloning and expression of a putative cytochrome P450 gene
that influences the colour of Phalaenopsis flowers. Biotechnology Letters 25: 1933-
1939.
Suresh Kumar, P.K., Subramoniam, A. and Pushpangadan, P. (2000). Aphrodisiac
activity of Vanda tessellata (Roxb.) Hook. Ex Don extract in male mice. Indian
Journal of Pharmacology 32: 300-304.
Takashi, R., Githiri, S.M., Hatayama, K., Dubauzet, E.G., Shimada, N., Aoki, T., Ayabe,
S., Iwashina, T., Toda, K. and Matsumura, H. (2007). A single base deletion in
soybean flavanol synthase gene is associated with magenta flower color. Plant
Molecular Biology 63: 125-135.
Tamura, K., Dudley, J., Nei, M. and Kumar, S. (2007) MEGA4: Molecular Evolutionary
Genetics Analysis (MEGA) software version 4.0. Molecular Biology and Evolution
24:1596-1599.
Tholl, D. and Rose, U.S.R. (2006). Detection and Identification of floral scent
compounds. In N. Dudareva and E. Pichersky. Biology of Floral Scent (pp. 3-25).
Florida: CRC Press.
Tumlinson, H. J., Paré, P.W. (1997). De Novo Biosynthesis of Volatiles Induced by
Insect Herbivory in Cotton Plants. Plant Physiology 114: 1161-1167.
Vainstein, A., Lewinsohn, E., Pichersky, E. and Weiss, D. (2001) Floral fragrance. New
Inroads into an Old Community. Plant Physiology 127: 1383-1389.
165
Verdonk, J.C., de Vos, C.H.R., Verhoeven, H.A., Haring, M.A., Van Tunen, A.J. and
Schuurink, R.C. (2003). Regulation of floral scent production in petunia revealed by
targeted metabolomics. Phytochemistry 62: 997-1008.
Vuralhan, Z., Morais, M.A., Tai, S.L., Piper, M.D.W. and Pronk, J.T. (2003).
Identification and characterization of phenylpyruvate decarboxylase genes in
Saccharomyces cerevisiae. Applied Environmental Microbiology 69: 4534-4541.
Wei, J., Wang, L., Zhu, J., Zhang, S., Nandi, O.I and Kang, L. (2007) Plants Attract
Parasitic Wasps to Defend Themselves against Insect Pests by Releasing Hexenol.
Plos One 9: 852
Wu, S., Watanabe, N., Mita, S., Dohra, H., Ueda, Y., Shibuya, M. and Ebizuka, Y.
(2004). The key role of phloroglucinol O-methyltransferase in biosynthesis of Rosa
chinensis. Plant Physiology 135: 95-102.
Yeoh, B.C. (1978) After the war. In Orchids: A Publication Commemorating The Golden
Anniversary of The Orchid Society of South East Asia, ed. Teoh, E.S. pp. 18-23.
Singapore: Times Periodicals.
Yomogida, K. (1992). Scent of modern roses. Koryo 175: 65-89.
Yu, H. and Goh, C.J. (2000). Identification and characterization of three orchid MADS-
Box genes of the AP1/AGL9 subfamily during floral transition. Plant Physiology
123: 1325-1336.
Zhao, Y.H., Wang, G.Y., Zhang, J.P., Yang, J.B., Peng, S., Gao, L.M., Li, C.Y., Hu, J.Y.,
Li, D.Z. and Gao L.Z. (2006). Expressed Sequence Tags (ESTs) and Phylogenetic
Analysis of floral genes from a Paleoherb Species, Asarum caudigerum. Annals of
Botany 98: 157-163.
Zuker, A., Tzfira, T. and Vainstein, A. (1998). Genetic engineering for cut-flower
improvement. Biotechnology Advance 16: 33-79.
166
APPENDIX A
GENERAL SOLUTIONS
TAE buffer LB broth (1 L)
242 g Tris base 10 g tryptone
57.1 ml glacial acetic acid 10 g NaCl
100 ml 0.5 M EDTA pH 8 5 g yeast extract
Adjust to pH 7.0
(For LB agar, 20g of bacteriological agar
must be added)
SM buffer TE buffer
5.8 g NaCl 10 mM Tris-HCl pH 8
2 g MgSO4.7H2O 1 mM EDTA
50 ml 1 M Tris-HCl pH 7.5
5 ml 2% (w/v) gelatin
NZY agar (1 L) Pre-hybridization buffer (100 ml)
5 g NaCl 10 ml 50 X Denhardt‟s solution
2 g MgSO4.7H2O 25 ml 20 X SSC
5 g yeast extract 2.115 ml 1 M NaH2PO4
10 g NZ amine 2.885 ml 1 M Na2HPO4
15 g agar 0.5 g SDS
Adjust to pH 7.5
NZY top agar (1 L) 20 X SSC buffer
5 g NaCl 3 M NaCl
2 g MgSO4.7H2O 0.3 M sodium citrate
5 g yeast extract
10 g NZ amine
7 g agar
Adjust to pH 7.5
167
APPENDIX B
Cymbidium mosaic Virus Coat Protein Transcript Sequence
1 AATAAAGTAGCTATAGGCCCCTGGCGAGGGTTAAGTTACCACAATAATTTGAAATAATCA
61 TGGGAGAGCCCACTCCAACTCCAGCTGCCACTTACTCCGCTGCCGACCCCACTTCTGCAC
>>>>>>>>>>>>>>>>>>>>
Forward Primer (CMV-F) 5’-AGAGCCCACTCCAACTCCAG-3’
121 CCAAGTTGGCCGACCTGGCTGCCATTAAGTACTCACCTGTCACCTCCTCCATCGCCACCC
181 CCGAAGAAATCAAGGCCATAACCCAATTGTGGGTCAACAACCTTGGCCTCCCCGCCGACA
241 CAGTGGGTACCGCGGCCATTGACCTGGCCCGCGCCTACGCTGACGTCGGGGCGTCCAAGA
301 GTGCTACCCTGCTCGGTTTCTGCCCTACGAAACCTGATGTCCGTCGTGCCGCTCTTGCCG
361 CGCAGATCTTCGTGGCCAACGTCACCCCCCGCCAGTTTTGCGCTTACTACGCAAAAGTGG
421 TGTGGAATCTGATGCTGGCCACTAACGATCCGCCCGCCAACTGGGCCAAGGCTGGTTTCC
481 AGGAGGATACCCGGTTTGCTGCCTTTGACTTCTTCGACGCCGTCGATTCCACTGCCGCGC
541 TGGAGCCTGCCGAATGGCAGCGCCGCCCTACTGACCGTGAACGTGCTGCGCACTCGATCG
601 GGAAGTACGGCGCCCTTGCCCGTCAGCGTATCCAAAACGGCAACCTCATCACCAACATTG
661 CCGAGGTCACCAAGGGCCATCTTGGCTCCACCAACAGTCTCTATGCTCTGCCTGCACCCC
<<<<<<<<<<<<<<<<<<<<<
Reverse primer (CMV-R) – 5’-GCAGGCAGAGCATAGAGACTG-3’
721 CTACTGAATAACGCCAAACTTAATAAGGCGTGTGGTTTTCTAAAGTTTGTTTCCACTACT
781 GGCGTAATATATTTAGCCAGATAAATAAAAAAAAAAAAAAAAA
168
APPENDIX C
Equations Used in Expression Analysis of the Putative Fragrance-related
Transcripts
Equation A (Standard curve)
y = mx + c
where y = CT values, m = slope of the standard curve, x = log input of template amount,
c = y-intercept of the standard curve line.
Equation B (PCR amplification efficiency)
E = (10 -1/slope
– 1) x 100
where E = PCR amplification efficiency, slope = m as mentioned in Equation A
Equation C (Relative quantity)
Q = E(lowest CT – sample CT)
where Q = Relative sample quantity, E = PCR efficiency
Equation D (Normalization expression)
Normalized expression level of target n = Qtarget n/ NFtarget n
where Qtarget n = relative sample quantity of target n, NFtarget n = normalization factor of target n
Equation E (Normalization Factor)
NF = (Q sample (ref 1) * Q sample (ref 2) * …………. * Q sample (ref n))1/n
Where NF = normalization factor, Q sample (ref 1) = relative quantity of reference gene 1 (endogenous control),
Q sample (ref 2) = relative quantity of reference gene 2 (endogenous control), n = number of endogenous
control genes used
Equation F (Scaled Normalized Expression)
Rescaled normalized expression target = normalized expression leveltarget n
normalized expression levelcalibrator
169
APPENDIX D
GC-MS Analysis on Vanda Mimi Palmer and Vanda Tan Chay Yan
a) Data for GC-MS analysis of the scent of Vanda Mimi Palmer
170
171
172
173
174
175
176
177
178
179
180
181
b) Data for GC-MS analysis of the volatiles emitted by Vanda Tan Chay Yan
182
183
184
185
c) Data for GC-MS analysis of the essential oil of Vanda Mimi Palmer
186
187
d) Data for GC-MS analysis of the essential oil of Vanda Tan Chay Yan
188
189
APPENDIX E
Contigs and Singletons
No Clone name cDNA transcript Contig/
singletone 1 1 putative zinc finger (C2H2 type) family protein Contig 1
2 29 putative zinc finger (C2H2 type) family protein Contig 1
3 17 putative ATP binding / alanine-tRNA ligase Contig 2
4 47 putative ATP binding / alanine-tRNA ligase Contig 2
5 11 putative phospholipid transfer protein Contig 3
6 40 putative phospholipid transfer protein Contig 3
7 A44 putative phospholipid transfer protein Contig 3
8 57 putative S18.A ribosomal protein Contig 4
9 60 putative S18.A ribosomal protein Contig 4
10 72 putative enoyl-ACP-reductase Contig 5
11 82 putative enoyl-ACP-reductase Contig 5
12 2 putative elongation factor Singleton 1
13 3
putative 2,3-biphosphoglycerate independent
phosphatase
Singleton 2
14 4 putative DNA ligase Singleton 3
15 5 putative cycloartenol synthase Singleton 4
16 10 putative coated vesicle membrane like protein Singleton 5
17 24 putative vacuolar sorting protein Singleton 6
18 25
putative zinc finger (C3HC4-type RING finger) family
Singleton 7
19 27 putative protein kinase Singleton 8
20 30 putative ATP-dependent protease Singleton 9
21 31 putative esterase/ lipase/ thioesterase family protein Singleton 10
22 33 hypothetical protein Singleton 11
23 34 putative LITAF-domain-containing protein Singleton 12
24 35 putative kinesin Singleton 13
25 36 no significant hit protein Singleton 14
26 38 putative pleiotropic drug resistance like protein Singleton 15
27 42 putative flowering time control protein isoform Singleton 16
28 44 putative glycosyl hydrolase family protein Singleton 17
29 45 putative Leucine aminopeptidase Singleton 18
30 48 hypothetical protein Singleton 19
31 51 putative cytochrome P450 monooxygenase Singleton 20
32 52 Putative photosystem II core complex protein Singleton 21
33 59 no significant hit protein Singleton 22
34 61 putative abscisic stress ripening protein Singleton 24
190
No Clone name cDNA transcript Contig/
singletone 35 62 putative alpha tubulin Singleton 25
36 63 putative syntaxin-like protein Singleton 26
37 66
putative KH domain-containing protein / zinc finger protein-like
Singleton 27
38 68 putative RNA binding protein Singleton 28
39 69 putative GRF protein Singleton 29
40 71
putative 4-diphosphocytidyl-2-C-methyl-D-
erythritol kinase
Singleton 30
41
73
putative dihydrolipoamide S-acetyltransferase
precursor
Singleton 31
42 74
putative Ubiquinol-cytochrome c reductase complex ubiquinone-binding protein
Singleton 32
43 75 putative Aci-reductonedioxygenase Singleton 33
44 76 putative thioredoxin-dependent peroxidase Singleton 34
45 77 putative mitochondrial dicarboxylate carrier protein Singleton 35
46 78 putative 6-phosphogluconolactonase family protein Singleton 36
47 81 putative calmodulin-binding protein Singleton 37
48 83 hypothetical protein Singleton 38
49 85 putative jasmonate ZIM-domain protein Singleton 39
50 86 putative translation elongation factor 1 alpha Singleton 40
51 90 hypothetical protein Singleton 41
52 91 putative MADS box AP3-like protein Singleton 42
53 94 putative 40S ribosomal protein Singleton 43
54 95 putative 60S ribosomal protein Singleton 44
55 96 no significant hit protein Singleton 45
56 A28 no significant hit protein Singleton 46
57 A38 putative GRF-interacting factor Singleton 47
58 A40 putative chaperon/ heat shock protein Singleton 48
59 A46 no significant hit protein Singleton 49
60 A54 hypothetical protein Singleton 50
61 A58 putative pectate-lyase like protein Singleton 51
62 A66 beta-ketoacyl-CoA synthase Singleton 52
191
APPENDIX F
BLASTX Results
Sequencing results of 62 clones up-regulated expressed in fully-open flower of Vanda
Mimi Palmer after elimination of Cymbidium mosaic Virus transcripts
No
Clone name
cDNA transcript
Classification
Score
E.value
1 1
putative zinc finger
(C2H2 type) family protein
protein with binding
function
164
5.00e-39
2 2
putative elongation
factor
protein synthesis
211
3.00e-53
3 3
putative 2,3-
biphosphoglycerate
independent phosphatase
Metabolism
291
3.00e-77
4 4
putative DNA ligase
cell cycle and DNA
processing
432
1.00e-119
5
5
putative cycloartenol
synthase
biogenesis of cellular
component
291
2.00e-77
6 10
putative coated vesicle
membrane like protein
cellular transport
325
2.00e-87
7
11
putative phospholipid
transfer protein
cellular transport
180
5.00e-44
8 17
putative ATP binding /
alanine-tRNA ligase
Transcription
289
2.00e-76
9 24
putative vascoular
sorting protein
cellular transport
448
2.00e-124
10 25
putative zinc finger
(C3HC4-type RING finger) family
protein with binding
function
258
3.00e-67
11 27
putative protein kinase
cell cycle and DNA
processing
161
2.00e-38
12 29
putative zinc finger
(C2H2 type) family
protein
protein with binding
function
135
1.00e-30
192
No
Clone name
cDNA transcript
Classification
Score
E.value
13 30
putative ATP-dependent
protease
Energy
396
1.00e-108
14 VMPEST
putative esterase/ lipase/
thioesterase family
protein
Metabolism
333
1.00e-89
15 VMPA33
hypothetical protein
Unknown
106
1.00e-32
16 34
putative LITAF-domain-
containing protein
Metabolism
191
3.00e-47
17 35
putative kinesin
cell cycle and DNA
processing
141
3.00e-32
18 VMPA36
no significant hit protein
Unknown
19 38
putative pleiotropic drug
resistance like protein
Cell rescue, defence and
virulence
128
3.00e-28
20 40
putative phospholipid
transfer protein
cellular transport
180
6.00e-44
21 42
putative flowering time
control protein isoform
Development
102
5.00e-20
22 44
putative glycosyl
hydrolase family protein
Metabolism
241
1.00e-62
23 45
putative Leucine
aminopeptidase
protein synthesis
234
4.00e-60
24 47
putative ATP binding /
alanine-tRNA ligase
Transcription
289
2.00e-76
25 VMPA48
hypothetical protein
Unknown
121
3.00e-26
26 VMPCyP450
putative cytochrome
P450 monooxygenase
Metabolism
154
3.00e-36
27 52
Putative photosystem II
core complex protein
Energy
111
3.00e-23
28 57
putative S18.A ribosomal
protein
protein synthesis
254
2.00e-66
193
No
Clone name
cDNA transcript
Classification
Score
E.value
29
VMP59
no-significant hit protein
unknown
30 60
putative S18.A ribosomal
protein
protein synthesis
254
2.00e-66
31 61
putative abscisic stress
ripening protein
Cell rescue, defence and
virulence
221
1.00e-56
32 62
putative alpha tubulin
cell cycle and DNA
processing
90.1
4.00e-17
33 63
putative syntaxin-like
protein
cell cycle and DNA
processing
184
5.00e-45
34 66
putative KH domain-
containing protein / zinc
finger protein-like
protein with binding
function
115
1.00e-24
35 68
putative RNA binding
protein
protein with binding
function
214
8.00e-54
36 69
putative GRF protein
Development
445
2.00e-123
37 VMPCMEK
putative 4-
diphosphocytidyl-2-C-
methyl-D-erythritol
kinase
Metabolism
169
7.00e-41
38 72
putative enoyl-ACP-reductase
Metabolism
154
6.00e-38
39 73
putative dihydrolipoamide
S-acetyltransferase
precursor
Metabolism
204
1.00e-51
40 74
putative Ubiquinol-
cytochrome c reductase
complex ubiquinone-
binding protein
Energy
123
4.00e-27
41 75
putative Aci-reductonedioxygenase
Metabolism
294
5.00e-78
194
No
Clone name
cDNA transcript
Classification
Score
E.value
42 76
putative thioredoxin-
dependent peroxidase
Energy
155
1.00e-36
43
77
putative mitochondrial
dicarboxylate carrier
protein
cellular transport
138
1.00e-31
44 78
putative 6-
phosphogluconolactonas
e family protein
Metabolism
102
1.00e-20
45 81
putative calmodulin-binding protein
protein with binding function
94.4
3.00e-18
46 82
putative enoyl-[acyl-
carrier protein] reductase
Metabolism
417
4.00e-115
47 VMP83
hypothetical protein
Unknown
255
2.00e-66
48 85
putative jasmonate ZIM-
domain protein
Cell rescue, defence and
virulence
105
3.00e-21
49 86
putative translation
elongation factor 1 alpha
protein synthesis
259
8.00e-68
50 VMP90
hypothetical protein
Unknown
82.4
2.00e-14
51 91
putative MADS box
AP3-like protein
Development
183
3.00e-45
52 94
putative 40S ribosomal
protein
protein synthesis
241
2.00e-62
53 95
putative 60S ribosomal
protein
protein synthesis
176
1.00e-42
54 VMP96
no significant hit
protein
Unknown
195
Note: The “no significant hit” proteins are the sequences which not homologous to any
sequence in NCBI database for the cut off e-value of 1e-05
and score more than 80. The
clones selected for verification by RT-PCR are highlighted and the abbreviations is only
apply for the selected clones for the verification.
No
Clone name
cDNA transcript
Classification
Score
E.value
55 VMPA28
no significant hit
protein
Unknown
56 A38
putative GRF-interacting factor
Development
117
5.00e-25
57 A40
putative chaperon/ heat
shock protein
Cell rescue, defence and
virulence
161
2.00e-38
58 A44
putative phospholipid
transfer protein
cellular transport
180
4.00e-44
59 VMPA46
no significant hit
protein
Unknown
60 VMPA54
hypothetical protein
Unknown
127
3.00e-28
61 A58
putative pectate-lyase
like protein
biogenesis of cellular
component
201
2.00e-50
62 A66
beta-ketoacyl-CoA
synthase
Metabolism
191
3.00e-47
196
APPENDIX G
Expressed Sequence-Tags (ESTs) of Vanda Mimi Palmer
>1-1-T3 hypothetical protein
TTGGAGATTCTCGCTTCGCCGCACCCACAGCCGCGGCAGCATCTGCGGCGGTGCAGACCCGGATACGGTGAAATAAAA
GGTAGAATTTTGGTCAGAGGGCGGTAAGGAGAGGGAGCGGAAGGGGAGGAAGTGGAAGATGACGGGAAAAGCGAAG
CCGAAGAAGCACACGGCGAAGGAAATAGCAGCGAAGGTTGATGCAGCGACGACGAATCGCGGCGGGGGTAAGGCTGG
GCTCGCGGACCGATCGGGACTGGATAAGGGAGGGCATGCTAAATTCGAGTGCCCTCACTGCAGGATAACCGCGCCTGA
TATGAAGTCGATGCAGATCCATCATGAAGCCCGACACCCTAAGATTCCTTTCGAAGAATCGAAGCTCACCGATCTTCAT
GCCTCCAACGTTGGCGATTCGTCCAAGCCCCGCCCAGGGGTTCGGGGAAGCTTCAAAAAATAGCTTCAGCTTTGCTATT
CCGAATAGCACTGTGAGCTATTCAATAACCTTTGCTTTCCTTCTGTCATATTTAGCTTCAACTTTTACGATGCCCTAATGT
ATTTTTATGATATTTGCTCGACTGCATCTTGTCATCGCTGCTACAAGTAAGAACATGTGAGATTCCTTGTGTCATGGTTA
TCGGAGGACTTGAATTTGAACTATTTGTAGTAGTTTTCTTAAACGGTTGCTTGCTTTAAGGTAATGCGCTTCCCATAGCC
GATTA
>2-2-T3 elongation factor
CACCCTGGTCAGATTGGCAACGGCTACGCTCCCGTCCTGGACTGCCACACCTCTCACATTGCCGTCAAGTTTGCCGAAA
TCCTGACCAAGATCGACAGGCGGTCTGGCAAGGAGCTCGAGAAGGAGCCCAAGTTCCTGAAGAATGGCGATGCCGGTT
TCGTGAAGATGATTCCCACGAAGCCGATGGTGGTCGAGACCTTCTCAGAGTATCCTCCGCTGGGCCGTTTCGCCGTGAG
GGACATGAGGCAAACAGTGGCTGTTGGAGTCATCAAGAGCGTCGAGAAGAAGGATCCCAGTGGAGCCAAGGTTACCA
AATCTGCTGCCAAGAAGAAATAAAGCTGAATGGATAAACTCTTTGAGTCTTGTTTATGTTAAGTTATCTTTGTGTTGGTC
TAATCCGGTGCAGCTTTCCAGTATCAGATTCCAGATCCGGTGCTTGATATGCTGTGGCGGTGAGGTTTTGGTGTCTTTTA
AGCTTTGTTTTATGCGACTGTTTTAATTTGTTTGAGTTGAGAAGACTGCGTTTTCATTTTAACAGCTCTTTTGATTATGTT
TCTTGCACTGCTTGTTAAATTTATATTTATACATTTTTTAGGGTCTATATTATAAATTTATTTTTGTCTT
>3-3-T3 2,3 phosphoglycerate independent phosphatase
CTTTAACGTTCAACCTAAAATGAAGGCACTTGAAATTGCCGAGAAGGCAAGGGATGCTATTCTCAGTGGCAATTTTGAC
CAGGTACGAGTTAATATACCAAACGGTGACATGGTAGGCCACACTGGTGATATTGGGGCTACTACTGTGGCTTGTGAG
GCTGCAGACGTAGCTGTAAAGATGATTCTTGATGCCATTGAGCAAGTGGGTGGCATCTATGTTGTCACTGCAGATCATG
GCAATGCTGAGGACATGGTGAAAAGGGGTAAATCTGGGCAGCCACTTTTTGACAAAGAAGGGAAAGTTCAAATTCTCA
CCTCTCACACCCTTCAACCGGTTCCTATCGCCATCGGCGGCCCCGGCTTGAGTCCAGGTGTTCGTTTCCGGAGTGATGTG
CCTGATGGTGGCCTTGCCAATGTTGCTGCTACTGTGCTGAACTTACATGGTTTTGAAGCTCCCAGTGACTATGAAACCAC
CCTAATTGAAGTAGTTGAGAACTAAATATACTGTGTAATTTTCTATTTATAATATGCTAATGTGATTTTTATATTAGTTA
AGCTCATTGAAAATCTTAAAATAAGTTTACTCCATTGAACTGGAGAATGTTGTCAGGTTTATGTTGTCACAGTTGAGGG
GGCTGTGGGCAAAGTTTTGGGTTGTTTCACTATAGTTTATATTTGTGTGGAATCTCTCCTCACAATGACTTTCTTGCTTTG
AATATTTACGCCACTTTCGAGGGATGTTGTTTTTGCTTAAAGTTTCATATGTTGTATTATTGGATAGTTGGTTATGGCAA
AGACGATGATTGCTT
>4-4-T3 DNA ligase
TTTTGATATTCTCTACATTAATGGAAGGCCTCTCCTCCATGAGCAACTCAAAGTTCGCAAGGAGGAACTTTACAAATGT
TTTGTGGAGATACCCGGAGAATTTAGTTTTGCTACTGCAATAACATCTAATGATCTTGAAGAAATACAGAAGTTCTTAG
AAAACGCTGTCAGCTCCAGTTGTGAAGGGTTGATTATCAAAACTTTGGACAAAGATGCTACATATGCGCCTTCAAAACG
GTCAAACAACTGGTTAAAATTGAAAAAGGATTATATGGACAGTGTAGGAGACACTTTAGATCTGGTGCCCATTGCTGCC
TTCCATGGCCGAGGAAAACGCACAGGTGTTTATGGTTCTTTTCTTCTTGCTTGCTATGATGAACAAAATGATGAATACC
AAAGCATATGTAACATTGGTACTGGATTTTCTGAATTGCAGCTTGAAGAGAGGTCCAAAAGTCTTGGAAGTAAAGTTAT
TCCAAAACCAAAACCATACTACAGAGTAGCTGATTCAATGAATCCTGATGTCTGGTTCGAACCTGCAGAGGTTTGGGTA
GTAAAGGCAGCGGATTTGAGCATTAGTCCTGTTCATCGTGCTGCTTCTGGCATTGTGGATCCAAATAAGGGTATTTCTCT
GCGATTTCCTCGTCTACAACATGTCCGAGATGACAAAAACCCAGAACAAGCCACAACGTCTGAGCAGGTTGCTGAAAT
GTATCGTGCACAAAAGATCAATCATATGAACAATCAAGAGGAAGAGGATGACGAATGACGTGGTTTGCTGCTTGCAAT
CAAGTCTATCATTTCTTTGAAGTTGGCAGTGTAGTTTTCCTGGTAGAGTCTAATGCATTTACTTGATAAACTTCTTATTCT
ACTTTCTTGTGCTAGTACTTTGCCTATTAAATGTAAACNTGAATCTATGCTATGACGTTTCGTTTC
>5-5-T3 cycloartenol synthase/ squelene cyclase
ATTTCATTGAGAAGATACAGAAACCAGATGGTTCATGGTATGGTTCTTGGGCTGTTTGCTTCACTTATGGAATATGGTTT
GGAGTGAAAGGACTAATTGCAGCTGGAAAATCATATCAAAATAGTCCTTCTATCCAAAAAGCATGTAATTTCCTGTTAT
CTAAACAACTAGCTTCTGGTGGTTGGGGGGAGAGTTATCTGTCTTGTCAACATAAGGTGTACACAAATCTTGAAGGAGG
TCGCACTCATGCAGTAAACACAGCATGGGCCATGTTGGCTCTAATAGATGCTGGACAGGCTGAAAGAGATCCAAAGCC
ATTGAATCGAGCAGCAAAAACCTTGATTAACATGCAGCTGGAGAGCGGCGAATTCCCTCAACAGGAAATTATGGGAGT
197
TTTCAACAGAAACTGCATGATCAGCTATTCAGCATATCGCAACATCTTCCCAATCTGGGCGCTCGGAGAATACCGCACT
CGGGTTTTGAAGGCAAGCAATTAACATAACTCCATGTCTGAGATAAACAGCTCCTCGAACCTCATGCTTTTGTCTCTTGC
AAAAGCATTCTTGCTTGGAAAACAATAATGTGATATTTTAATGGAGGAAAATATATTAAAGATTCCATTCT
>6-10-T3coated vesicle membrane like protein
CTCAGCGGTGGGCCTTTTTTCATGTTTGCAATCGGCTGTTGGGATTAGGTTTGTGATAGACAGGGAAGAGTGCTTCTCCC
ATGAAGTTCCATATGAAGGAGACACTGTTCATGTTTCGTTTGTTGTGATTAAGTCTGAGACACCCTGGCATTACGGAAA
TGAGGGCGTGGATCTCGTGGTGAAAGATCCATCTGGCAATCAAATTCATGATTTTCGTGATAAGATAAGCGATAAATTT
GAATTTATAGTTCGCAAGAAAGGGCTTCATCGCTTTTGCTTCACCAACAAATCTCCATATCATGAAACAATAGATTTTG
ATGTTCAAGTCGCCCACTTCACATACTTTGAAGAACATGCGAAGGATGAGCATTTATCTCCTCTCCTTGAACAAATCAA
CAAGTTAGAAGACGCTCTTTACAATATTCAATTTGAGCAGCATTGGCTGGAGGCTCAGACCGAACGTCAAGCAATTGTA
AATGAAGGGATGAGCAGAAGGGCAATACACAAGGCACTTTTTGAATCTGCAGCACTGATTGGAGTCATTGTGCTACAA
GTGTATCTCCTCCGTCGCCTTTTTGAGCGAAAGCTTGGAACCTCTAGGGTTTAGCCAACTCAGGAGCCTGCAACTTCCAT
CACATGTTTCTTCTTTCTTCCAAACCTCTGACTTCATTCGTAACAAAATGAGCTTGTTATACAGGGAATCTTCATTCAAC
ACTGGCTTGTCTTTTTGGCTTGTTCCAGTGCTTCCCTGATCTAATAACCAGGTAATTATCAAATTATTCAGTTTGTTGCTT
AGTAAATGAGAATATATCTGTTTGTGGCAGC
>7-11-T3 phospholipid transfer protein
GTTATCACTCATTGTCAATGGCTCGCTCCACCGCTTTTGCAGTCGTATGCATAGTGTCCTTCCTCCTTGTTTCCGGCGTTT
TCCGCGAGGCGAGCGGGGCCATCAGTTGCGGTCTGGTGGCCTCATCCGTGTCATCGTGTATCAACTATATTCGAGGCGT
TGGCTCGCTCTCACCAGCATGTTGCAGTGGGGTGAAGAGACTCAACTCGCTGGCGCGCACAACCCCTGACCGTCAGGC
GGCATGTTCTTGCCTCAAGAGTTTCGCCAACCGTATCCCTAATCTGATCCCTTCCCGTGCTGCTGGACTTCCTAGCAGCT
GCGGAGTCAGCGTTCCATATCCCATCAGTACCTCCACCGACTGCTCCAAGGTGCACTGAGCTTCTTGGACGGGCTACAA
ATTACTGTTTTCGTATGATCTACAGAATAAAGTGGTCTCTGGGTCTAGTGAGGGTTGTTAATATCTTGTTGTTTGTGCGT
TTCTGTCTTATTTTAATTTATGTTCCTGTATTGTTATAAGGCTACACCCTTGGATGTGGTGAGTTTAATATTCCTGCT
>8-17-T3 ATP binding/ tRNA ligase
CGGCCGCTCTAGAACTAGTGGATCCCCCGGGCTGCAGGAATTCGGCACGAGGGCGACATCCTCCGGCACCTCGTTTGC
AATGGTACAAGTACCAGCTCGCAGGTCCTGAACGACGAGGCTGACGACGACTATGGCGAAGCAGAGTCCAACGAAGC
TTGCCTACTTCGAGGGCATGTACGCGCTTCAGTCCACTGCCACCTTCCTCTCATTCGAGCAGGTAGATGGCCGATTGGC
GATGATCCTTGATTCAACCATCTTCCACCCTCAGGGTGGTGGGCAGCCAGCTGACAAAGGGGTCATTTGCGGTTCAGGC
TTCAGGTTCGTGGTGGAGGATGTCCGGTCCAACGATGGGGTGGTCTTTCATTTTGGATACATTGATGACTCTCAAGCTA
ACTGTGAATCCAACCTCAAAGAGAGACAAGAAGTTACCTTACAAGTTGATGCACAACGACGTGATCTCAATTCCAGGA
TTCATTCTGCTGGCCATTTATTGGATATTTGTATGCACAATGTAGGCTTATCTCACCTAAAGCCTGGAAAGGGTTATCAT
TTCCCTGACGGGCCATTTGTTGAATATATTGGAGTAATCTCCCCAGATCAACTGCAGATCAAACAAAAAGAGCTTGAAG
AAGAAGCAAATTCATTAATCAGTATTGGTGGAAAGGTTTCTGCTTCTGTTTTACCTTATGATGAAGCTGCCAGGTGGTG
CAATGGTGATCTCCCTAGTTACATTTTGAAGGGCAGCACCCCAAGAATTGTGAGGCTAGGTAACAATGCCGGATGCCCC
TGTGGAGGGACTCACGTTGCTGATATTTCAGACATAAAAAGCTTTAAGATTACCCAAATTCGATCAAAGAAAAGAATC
ACAAAGATCAGCTACAGCATCAACCCATGAGCTCTGTATTTCTCTGCAACATTGTTGATCTATTGCGATGATTAGTGAG
CTCCTATCATACCTGAATGTATCACCTAATTTTATTATGTTTTTGTATGATTAATCACAGTTGTTATTATTGGTCTATAGA
AATGTGAACTTTGCCT
>9-24-T3 Vascoular sorting protein
GGAACATTCTGCCTAGGCTGTACCTTCTTTGCACGGTCAGATCTATTTATATAAAATCAAAAGAGGCTCCTGCAAAGGA
TATCCTTAAGGATATTGTGGAAATGTGCCGGGGTGTACAACACCCTGTCCGGGGTCTCTTTCTGAGGAATTACCTCTGTC
AGATCAGTAGGGATAAGTTGCTTGATATTGGTTCAGACTATGAAGGGGATGGCGGCAGTGTTATCGATGCTGTTGAATT
TGTGCTTCAGAACTTTACAGAGATGAATAAACTGTGGGTCCGAATGCATTATCAGGGACCAGTTTGGGAAAAGGAGAA
ACGTGTAAAAGAGAGGAGTGAGCTTCGTGATCTTGTGGGAAAAAACCTCCATGTTCTTAGCCAAATAGATGGTGTTGAT
CTTGGTATGTACAAAGAAAATGTGCTTCCTAGGGTATTAGAACAGGTGGTCAATTGCAAAGATGAACTAGCCCAACATT
ATTTGATGGATTGTATAATCCAAGTGTTTCCAGATGAATATCACTTGCAAACTCTTGAATCATTATTAGGCGCATGTCCA
CAACTTCAGCCCACTGTTGATGTTATGGCAGTCCTATCTCAGCTCATGGATAGATTATCCAATTATGCAGCCTCTAGTAC
GGAGGTTTTACCAGAATTTTTGCAAGTTGAAGCTTTTGCCAAATTAAGCAGTTACATTGGAAAGGTCATTGATTCGCAG
CCTGAAATGCCAATTTTTGGTGCCATCGGCTTATACGTCTCACTTCTTACATTTACTCTTCATGTCCATCCAGATCGTCTT
GATTATGTGGATCAAGTTCTGGGAGAGTGTGTTAGAAAATATCTGGAAAATCGAGATTGAGGATAGCAAAGCATTAAA
CAAATAGTTGCTCTTTAAGTGCTCCAATGGAAAAGTACAATGACATAGAATATTGTTTTAAAGCTGCTANTATCCAGTG
TCATGAGCACCTTGNTATGCNNAAGAAATCATGCAGTGNNTATCAGAGCATATGAGATAACCGCATATC
198
>10-25-T3 zinc finger (c3Hc4-type ring finger) family
CTCCCTTATGTTGACCACCAAAAGGCAAAGACTATAAAGAATGATTTTAATTTGCATAAAGACATGATTCGGTTGGAAG
TGGATGTCCAGAACCCGGAGGAGTATCTGGTTTCCTTTGTCTATGATGCTGTCGTGGACGGGAGGTAATCCGCTTGCGT
CACCATATACTACTTTGCTAAAGAAGGAGCCAACTGTAGTTTTTCCTCAACCGAAGAAGACATCTATAAACCCAGGAAT
TATCTTTTTGAGAAGGGAACGGCCAAAAATTTCTGTCAACCATCTGGGCATGGCATTGATTTAGGCTTCTTTGAGTTAG
AGAGTCTTTCAAAGCCAGTAAATGGAGAAGTCTTCCCACTAGTTATTTATGCCAAGGCATGCGGGCCAACTGATGGAG
ATGGCCACTCTTCTCAATCTTCTGCCACTCATGTGCAGATTACATTAGCCGTCATTGAGAAGAATAGTAAAGGAGAGTT
TGAAGCTAAAGTTATAAAGCAAATCCTATGGATTGCCGGCGAACGCTACGAGCTGCAAGAAATCTTTGGCCTGGAAAA
TTCTTCAACTGAACAGATTGATGATGGTGATGATGATATTGGTAAGGAGTGCGTAATCTGCTTGACAGAACCTAGGAGC
ATTGCAGTCTTTCCCTGCAGGCATTTGTGTATGTGTAGTGATTGTGCAAAATCTTTGAGGGTCAAATCAAATAAGTGTCC
CATTTGTCGTCAACCTGTTGAGAAACTAATGGAAATCAATGTAAATTTGAGGAGCTCTGATAGGTAAGATCGGCATCAG
TCTTCATGAAATTTGACAGAGGT
>11-27-T3 putative protein kinase
ATATTTGTTTGGCTGATTTTGCTAAGAGATGAGTTACGGCAGAGATGATCAAAATGATGGAATAGAGAAAAGGAAGTT
TATGAATTCTGAACCAAAAACAGCACCAGATTCTGTCCATCCTGGCAGGCCGTCCTTTGACTGCAACTATACTATTCCA
GTTGAGAACTTGGAGATTGATAAAAAGCTTTTGATTGATCCGAAGAATCTTTATATAGGATCCAAGATTGGTGAAGGTG
CTCATGGAAAAGTTTATGAGGGAAAATTTAGGGAACAAGTTTTAGCACTCAAAATTATCAATAGTGGCAACACGAATG
AGGAGAAAATGACAATTCAGGCTCGTTTCATTCGAGAAGTCAATATGATGTCTAGAGTGAAACATGAGAATCTTGTTA
AGTTCATTGGAGCTTGCAAGCACCCTATCATGGTTATTGCTACAGAGTTGCTACCTGGGATGTCCTT
>12-29-T3zinc finger (C2H2 type) family protein
GGAAGAAGACGGGAAAAGCGAAGCCGAAGAAGCACACGGCGAAGGAAATAGCAGCGAAGGTTGATGCAGCGACGAC
GAATCGCGGCGGGGGTAAGGCTGGGCTCGCGGACCGATCGGGACTGGATAAGGGAGGGCATGCTAAATTCGAGTGCC
CTCACTGCAGGATAACCGCGCCTGATATGAAGTCGATGCAGATCCATCATGAAGCCCGACACCCTAAGATTCCTTTCGA
AGAATCGAAGCTCACCGATCTTCATGCCTCCAACGTTGGCGATTCGTCCAAGCCCCGCCCAGGGGTTCGGGGAAGCTTC
AAAAAATAGCTTCAGCTTTGCTATTCCGAATAGCACTGTGAGCTATTCAATAACCTTTGCTTTCCTTCTGTCATATTTAG
CTTCAACTTTTACGATGCCCTAATGTATTTTTATGATATTTGCTCGACTGCATCTTGTCATCGCTGCTACAAGTAAGAAC
ATGTGAGATTCCTTGTGTCATGGTTATCGGAGGACTTGAATTTGAACTATTTGTAGTAGTTTTCTTAAACGGTTGCTTGC
TTTAAGGT
>13-30-T3 ATP dependent protease
GGATTTGGTCGATTAGGGATGATTTGCTTGTGCCGTCTTCTCCCTACTTCCCTGTGGAGGCTCAGGGCGGACAAGGACC
ACCGCCAATGGTGCAGGAGCGGTTTCAGAGCGTTATTAGCCAGCTATTCCAGCATAGAATTATACGCTGTGGTGGTGCG
GTCGATGATGACATGGCAAATATAATTGTAGCGCAGCTTCTTTATCTCGATGCAGTGGATCCTACGAAGGACATCGTCA
TGTATGTGAACTCTCCTGGAGGATCAGTGACAGCTGGTATGGCTATATTTGATACGATGAGGCATATTCGTCCTGATGT
TTCCACTGTATGTGTTGGATTAGCAGCCAGTATGGGTGCTTTCATACTAAGTTCTGGCACCAAGGGAAAACGCTACAGC
CTACCAAACTCCAGAATCATGATTCATCAGCCCCTTGGCGGCGCCCAAGGAGGACAAACTGATATAGACATTCAGGCA
AATGAGATGCTTCATCACAAGGCCAATTTGAATGGATATCTAGCTTACCACACGGGCCAGAGTTGGGAGAAAATAAAC
CAGGATACTGATCGAGACTTCTTCATGAGCGCCAAAGAGGCAAAAGATTATGGCTTAATTGATGGAGTAATAATGAAT
CCACTCAAAGCTCTTCAGCCTCTCTCAGCTTGAGATGATGAAGAAGAAGAATCAGATGAGTTCTACGACCATGATCAGA
GAAATCAGAGCTTAAGGATTGCAGTAATCTGCTCTGCTGCGATACTAAAACTTCCGACCGTTGAGAGCACTCATTGCCT
GATATAGTCAGTATTATTCGTCTTATTGTGCCTATCTAGAGTTAGAGTTAGCTCACAATGTTGCTTGAATATTTATCAAT
TAAATGAATATCAGAAAATTGCGGTTTT
>14-31-T3 esterase/lipase/thioesterase family protein
GTAAGGCGTTTCCGCGAAGAGGTCAGTTCACAGTTGGAATTCTGCGTCTCTATCGACGATGTCGTCTCCTGGAGTGTCC
GAGCAAAGGGTTGAAATTTTAAACAACTATGGAGAGAAACTTGTTGGTGTGCTGCATTTAGCAGGTTCCAAGAATCTG
GTGATCTTATGCCATGGATTTCGCTCCACAAAGGATGAGAAACTGTTACTTAACCTCATTGCTGCACTAACGAAAGAAG
GTGTGAGCGCCTTTCGCTTTGATTTTGCTGGAAATGGGGAAAGTGAAGGTGAATTTCAATATGGAAACTATCGCAAGGA
AGCTGACGACTTACGAGCTGTGGTGCTATATTTCTTAGAGCAAAATTTTAAAATTTGTGCTATTACTGGGCATAGTAAA
GGAGGAAATGTGGTGCTTCTTTATGCATCTACGCATAATGATGTCCCTCTCATCATTAATCTTTCTGGCCGTTTTGCATT
GGAAAGAGGAATTGAAGGACGCCTGGGGAAAGAATTCATGCAAAGAATAAAGAAAGATGGCTTTATTGATGTCAAGG
ATAAAACAGGAAAATTTGAATACCGGGTGACAGAAGAAAGCTTGATGGATCGTCTAACCACAGACATGCGTGCAGCAT
GCCATGCCATTGATAAGAAATGCAGGGTTTTGACAATCCATGGTTCAGCAGATGAAATTGTACCTTCAGAAGATGCCTT
TGAGTTCGACAAAGTCATACCCAACCACAAGCTTCATATCATTGAAGGTGCTGACCATTGCTATACTGCATGCCAAGCA
GAGCTGGCTTCTCTTGTTCTAGACTTCATAAAATCTGATCAGGTCGTCGATGCTACCACAGCACAAGTGATGTAAGAGT
TTTTCAAGCTCATTGTTGTTACTTTATCTTTCAATTTGTCACGCCATGGTCATTACTGTCTAGCTTGATGTTTCATTTTCTA
TATGTAATG
199
>15-33-T3 Hypothetical protein
ATGAAACCCTAATTTTTGAGCTTTTGGATTTCAAAAGCGAGATTGAAGACAATGCCAGTGCTGTTTGGTTTCTACGTGA
CATTGCTCGTGAACAGGATGCAGAAGATAGCATGGTCCAAGAACACTCAGGAACAATAGAAGTTGCTGGCTTACAGTA
TAGAGATGCTCCTGTAATATATCAAACAGCAGTTGGTCAAATGACCATTTCCAAGGAAAGCCAAGGAATGGAGGGACA
CAAAAATGGGGGGGGGTTTAAGGGGAAATATTTCGCGTCCCAGGATTTTGCGGTGAGGGGGGCGCGTGATCACTGCAT
ATGAGCAAATTTTGGACAATTCCTTAAGTGAGAGTAGAAGCATTGAGGAAGCTGATCCAACAGTTTCTGGCAGCCTATC
TGCTGCAGAAGTCTTTAAACTATCAGCTGCAAGCTTTAAGGTACATGACTGGAATCTCTTTGGTGCTGGGGCTTGACTA
ATTATTTAATATGATTTTAAGAATTTTTGGTTATGAAAAACATTTTCATATTATTTTAATATGATTTTAAGATTTTATTTG
ACTTGTATTTTTATTTTTTAAAACTGGTGCTTAATCTGTAATTCTGTTCAACTATAATTATTTTTACAGGCGATGAAGCAA
CTGATGATGGGAATTT
>16-34-T3 LITAF domain containing protein
GTGAAACGGAAGAACTGATGGCAGCGAAGGGTTCAGGCGACGAACCTGCTCTTGGGGTGCCCTACCACTATGCATCCG
GCGGTGCCGGGGTTTCCGGATATGCCCAAGGTCCGAATCCACAGCCTGCATTCTACGTGGCGCAGCACCCGCATCAAG
CTGGGTTGATCCCGCCGAATGCGGTTTTCGGAGATCCAAAGGGCATCCCGCTCCAGCAGACGATGTTCCGGGATACCCC
CGCACCTTTCGAGTGCGTTTACTGTGGATCATCCAGTCTTACCACCATCAGATCCAAACCAAGTCTAGCTGCTCTTGTTG
GTTGCATGATGCCATTTATGCTGGGAGTATGTTTTCTTTGCCCTTCCATGGACTGCCTCTGGCACAAATATCACTATTGC
CCAAATTGTAAAGAGAAGGTTGCCTACTTTGAAAAATCAGACCCTTGTATTGTGGTGGATCCAACTAATTGGACCGAGC
CCAGCTATGCCATCCCCAGGTTGGTTTGAACTCTCTTTGAAGTTTTTGCGGATATATATATATATATATATATATATATA
TATATATATATACGGTTTTTGCTTTTGGATTTGTAAATAATGAAGTCCTCTTTGT
>17-35-T3 putative kinesin
AGGAAGAGNCCTTAATAGCAGCTCATAGAAAAGAAATTGAAAATACAATGGAGATAGTTCGGGAGGAAATGAATTTA
TTGGCGGAGGTGGACCGACCAGGAAGCCAAATTGACACGTATGTATCGCAGCTCAGCTTCCTGCTGTCGCGGAAGGCT
GCCGGGCTGGTCGGCCTACAAGCTCGCCTGGCCAGATTTCAGCACCGGCTGAAAGAGCACGAGATTCTCAGTCGCAAG
AAGGCTTTGAGGTAATAAATGATCTGCGTGCTTTTCTTTCTTTTTAATGTATCTTCTACCAGCAGAATGTCCCCTTCCCTC
TCTCTGCTTTTTCTTGCTGATTGTTATTGCCACAGATTTCTCATATTCTCATTCTTGAAGTTATACAATAGCGGCAATGCC
GTGTGATTTACCTTTATGTGTCGTGTTTGGCCTTCTTTTATTGTTGGGAGTGAGGGTGGGTTGGTCTTTCGGAATGAAAG
AAGAAGAATGTATGGAAGCCATTGTTGATAATAGTATAGTATTTATTT
>18-36-T3 hypothetical protein
ANNAGCCNTGTGCTTTTATTTCTGGGGCCTGCTTTTGTCTATCTTGTGCCTGAGGACAATGTCTGGGAGGTAGTCTTACA
GGTTCTGGTGGCCTTAGTGTGTGTCGTTGGCGGATCCGCCTCCTTCGCTGCTTCAAATTTCTTGTCAAACTTGCAGAGAT
CCAATTGAGTTTTGGTAGTCTTGTGCTTCTGACGTGGTCATTTTGGTGCAAATGTTAGCTTGTTACTTGCTCGGATTCCTT
CTGAACTCTATGTATTAGGCAATAACAAGCAAGTCAGCTTCTGGGGAGAACTATGCATGGACAGTAGGTATTTGCTAAA
TTCTTTTTAAGATTCAGGTAATATATATATATATATATATATATATATATATATATATATATAGATATGTATGTATGTATT
TATGTATGTATGTATGGAACTGTACCTTCGGAGTATCATACAATAATTAAGCTCTAAAAATTGAATGCAATAATTTTCA
GCT
>19-38-T3 pleiotropic drug resistance like protein
GTNNAAATTCCACGGCCGAGGATGCCTATATGGTGGAGATGGTATTTCTGGGCATGCCCAATTGCGTGGACACTTTATG
GATTAGTCGCTTCGCAGTTTGGTGATATAGAAGACGAGCTCGAATCTGGTGAGATTGTGTCTCAATTTGTAAAAAGTTA
CCTTGGATTCAGACATGACTTCTTGGGCGTGGTTGCGGCTGTAATCGTTGGGATTCCAGCACTCTTTGCCTTCATGTTTG
GATTTTCAATTAAAGCTCTAAACTTCCAAAGAAGATGAAGGAATTGATGAAGTCCTAAAAGATGCCCTGGTGCTAGGA
AGTTTAACATATTCATTTTGTTAATATTCTTCCACAAGAAGAATGAAAAGTTCTCAGAGGGGGTAATAATGAAGTGAAG
GATATAATCTTTCCTGACCTATATATGAACAAGAAAATATGCTCATGAATATGTTCCTCGATGTACTTAATTTGGGCATC
AGTTCAAATGTAGAGAAGTTAGGATGGATGTTGAGAGGTCTGATTTATCTTTGTCAACTATTCAAATGTAATAGTGCAA
ATATAAAACTTTACTTGGTGGATATATAATGGGGAGATATATTAACACT
>20-40-T3 phospholipid transfer protein
GTAGAGNGTTATCACTCATTGTCAATGGCTCGCTCCACCGCTTTTGCAGTCGTATGCATAGTGTCCTTCCTCCTTGTTTCC
GGCGTTTTCCGCGAGGCGAGCGGGGCCATCAGTTGCGGTCTGGTGGCCTCATCCGTGTCATCGTGTATCAACTATATTC
GAGGCGTTGGCTCGCTCTCACCAGCATGTTGCAGTGGGGTGAAGAGACTCAACTCGCTGGCGCGCACAACCCCTGACC
GTCAGGCGGCATGTTCTTGCCTCAAGAGTTTCGCCAACCGTATCCCTAATCTGATCCCTTCCCGTGCTGCTGGACTTCCT
AGCAGCTGCGGAGTCAGCGTTCCATATCCCATCAGTACCTCCACCGACTGCTCCAAGGTGCACTGAGCTTCTTGGACGG
GCTACAAATTACTGTTTTCGTATGATCTACAGAATAAAGTGGTCTCTGGGTCTAGTGAGGGTTGTTAATATCTTGTTGTT
TGTGCGTTTCTGTCTTATTTTAATTTATGTTCCTGTATTGTTATAAGGCTACACCCTTGGATGTGGTGAGTTTAATATTCC
TGCTAT
200
>21-42-T3 flowering time control protein isoform
TATAATCTCCTTTGCTAGACCTGCAAACAGCAGAATACGATGGAGAGGCATCGCTTCGGTGACGGTGGAAAGTACTCG
AGAATGCCGTCACGGTGGCCATCAGATACCCAACCAAACCAGCACCAACGTTACCCTAGAGGCGGTGGAATAGCCGGC
GGCGGCGAAGGCTTTGGTGGCCGATACCACCCTTACCGTGGCCAGCCTGATTATTCTTCCGCCGGCGGTCAGGGTTTCC
GCGATGGTCATGGGGACTTGGGTAGTCATCAGATGCCGATGGGTGGTCAGAAGCGAGGGTTTGCGGGCAGAGGAGGTT
CTCCAGACTTTGGTGAGGGAAACAAGTTTGCCAAACTGTTCATTGGGTCGGTTCCAAAGACTGCAACTGAAGAAGATAT
TCGTCCTTTATTTGAGGAACATGGTGATGTTGTTGAAGTGGTTTTAATTAAGGATTGGCGAACAGGTCACCAACAAGGT
AGATTCAGATGTTTTCTCATGTTCGTGATACATTGAGGTTTTCTTTAACAGGTCCTTTGGGGTTTGGGGAGCAATTTATT
TATAAGTCCTCTCAGATAATGTTTTTATCTGATTAGAGTGGTTGTATTCTGTTGCAACCAGCCCATCATTATGAGGTCAG
GGTGGTATTGAGTTTCTCAAAGAAAAAGGGAAACTTGACATTGTCGTTTCTATATTTACAGCAGCCAAAATGTTGATGA
ACTGTTTACACGTGTTGATGCTTATTGATTAAAAGCTTGGTATGCAATTAAGGCATTTTTGAACTGCTTATAGCAGTGGA
AGGGAATGATGTCTTTTGAGCCTTTCTTCTCTAAAAGATCTATGGCAGTTGTTCTTATAAGCTTAGTATTTATGTGCAAC
GGTGATGAGAACTATACACATATTTTTACTTAATAAAGCTTTGCTCTTCTTGCATTTTATGCAAATACT
>22-44-T3 glycosyl hydrolase family protein 43
GAAGCACCGGCCGTGTTCAAACATAAAGGAACATACTATATGATAACTTCAGGGTGCACAGGGTGGGCGCCGAACAGG
GCAATGGCGCATGCGGCGGAGGCGATGATGGGGTTATGGGAGACGATTGGTAACCCTTGTGTCGGCGGGAACAAAATA
TTCGAACTGACCACTTTCTTTTCACAGAGCACTTTCATAGTTCCGATTTCGGGGCTGCCGGGGGTGTTCATTTTCATGGC
AGATCGGTGGAATCCGTCGGAGCTGAGAGATTCCAGGTATGTTTGGCTGCCTCTGACAGTAGGTGGAGTGGTTGATGA
ACCATTAGAATACAATTTTGGGTTTCCAGTTTGGTCTAAGGTCTCCATTTTCTGGCATAGGAGATGGAGACTTCCGAATT
GGTGAACTGCTTAACATTGAGTGTACAATATTTTGTAGATTGTTTTACATATGGTGTATAGTGTAATAAATGTTTTTGTA
GATTTT
>23-45-T3 Leucine aminopeptidase
GTAAANNNGGTGTCGAAAAGATTGTTGATTTGGCAACGCTAACTGGTGCTTGCATTGTTGCCCTTGGACCCAGCATTGC
AGGAATCTTCACTCCCAACGATGAACTAGCAAAAGAGATCACTGCTGCTTCCGTGTTAACCGGTGAGAAATTTTGGAGG
TTGCCTTTGGAGGAGAGTTATTGGGAGTCAATGAAGTCGAGTGTTGCTGATATGGCTAATAGTGGAGGCCGTCATGGCG
GCGCTATCTTAGCTGCACTTTTCCTCAAACAGTTTGTGGATGAAAAGGTCCAGTGGGTGCATATAGATGTAGCTGGTCC
AGTTTGGAATGATAAGAAGCGCGTAGCCACAGGGTTTGGTGTATCAACTTTGGTTGAATGGGTTCTCAAGAATTCTTCT
TAAATCAAATACTGAGATGCAGCAAATGGAATAGAACAAACTTTTTTCAGGTTCAGCTCATGAAAGCATTAGGAAACA
TTCTCAAATTTACTTCAAATAAAAGAACTTTGATCTGATTTTCTGTTGTCTGATATTTTATTAGTCTGCTATGTATTAGTG
TTGTCTGTTGTCATAATTTTTATGATTTGTAGCTCTGAGTGGCAGTGTAAACTAGCTGAATTTTGAATGAATTTGAAGTG
TTGTGAGGCTTTTGGCTTGTTAAATAAACAGTCAAAACTTTGTTGTGTTTGACTTGTTTATTGATGCAGTTTGCAAACAT
GAAAATTTATTTCAAAGCTTTATAAAAATTTTAAAGTC
>24-47-T3 ATP binding / alanine-tRNA ligase
GCGANNCCTCCGGCACCTCGTTTGCAATGGTACAAGTACCAGCTCGCAGGTCCTGAACGACGAGGCTGACGACGACTA
TGGCGAAGCAGAGTCCAACGAAGCTTGCCTACTTCGAGGGCATGTACGCGCTTCAGTCCACTGCCACCTTCCTCTCATT
CGAGCAGGTAGATGGCCGATTGGCGATGATCCTTGATTCAACCATCTTCCACCCTCAGGGTGGTGGGCAGCCAGCTGAC
AAAGGGGTCATTTGCGGTTCAGGCTTCAGGTTCGTGGTGGAGGATGTCCGGTCCAACGATGGGGTGGTCTTTCATTTTG
GATACATTGATGACTCTCAAGCTAACTGTGAATCCAACCTCAAAGAGAGACAAGAAGTTACCTTACAAGTTGATGCAC
AACGACGTGATCTCAATTCCAGGATTCATTCTGCTGGCCATTTATTGGATATTTGTATGCACAATGTAGGCTTATCTCAC
CTAAAGCCTGGAAAGGGTTATCATTTCCCTGACGGGCCATTTGTTGAATATATTGGAGTAATCTCCCCAGATCAACTGC
AGATCAAACAAAAAGAGCTTGAAGAAGAAGCAAATTCATTAATCAGTATTGGTGGAAAGGTTTCTGCTTCTGTTTTACC
TTATGATGAAGCTGCCAGGTGGTGCAATGGTGATCTCCCTAGTTACATTTTGAAGGGCAGCACCCCAAGAATTGTGAGG
CTAGGTAACAATGCCGGATGCCCCTGTGGAGGGACTCACGTTGCTGATATTTCAGACATAAAAAGCTTTAAGATTACCC
AAATTCGATCAAAGAAAAGAATCACAAAGATCAGCTACAGCATCAACCCATGAGCTCTGTATTTCTCTGCAACATTGTT
GATCTATTGCGATGATTAGTGAGCTCCTATCATACCTGAATGTATCACCTAATTTTATTATGTTTTTGTAATGATTAATC
ACAGTTGTTATTATGGGTCTATAGGAAAATGTGAAACTTTGCCT
>25-48-T3 hypothetical protein
NNNAGATTGAGGTGTAGTGGAATTTGCTCGGATGAACACCCTATCAGAACTGAGCTTGTGAGTTTTGCTTCCCTCTTTG
CCCCATTGCGGCCATCAGTGAAGATAAATCCTCAAGCAGCTACCAGATTCATTGAGCATTCTTTGCCTGATTTGGCACC
AGACCAGAGGAAGAGTCTCCATAATATCAGTACAGGCAAGGGTGACCGGACTCCTTTCATGACAAACAGAGCGAAGA
AGAAAACGAAATACCAATCTTTTGAGCAGCAGTCTGCGCGTGCTGCTGCGCAGGAGTTTCTTGAGAAGGCAGCAAGGG
AGCTCTTTGGCTCAAACGATTCGGACGTGAAGGGTCCATTGCAGAACCTTAAATCAGATGATGAAGATGATGAATAAA
TGCTTTGATTTATTTTTTATTTTTTTATTTATTTCTTTTACTTCAGATAAATTGTTCTCTTTTCTTCGTGAAGTATTTGGTAA
ATTATGTTTGATAAAAAACACTTTTTTTTTCTAAGAACCTTAATTTTATGGTAGT
201
>26-51-T3 cytochrome P450-like protein
CTGAGATCATGAGAGCTAAACAAAACGGGCATCATGATGAGACAAAGCAAGACATACTATCAAGGTTCATCGAGCTCG
CCAACACCGACAAAGAGAGTGATTTCAGCACGGAAAAAGGTTTAAGAGATGTGGTGCTAAACTTTGTTATTGCAGGGA
GGGACACTACTGCTGCAACGCTCTCATGGTTTATATACATATTAGTCACACAACCTCAGGTGGCACAGAAACTCTATAT
AGAGATGAAAGAGTTTGAGGAGATCAGAGCTGAAGAAGAAAATATAAATTTGGATTTATGTAATTTGGAAGATATGGA
TTCATTCAGAAACAGATTATCAGATTTTTCGAGGCTTTTGGATTATGATTCATTAGCAAGGCTGCAATATCTGCATGCAT
GCATTACAGAGACCCTGAGGCTGTTTCCTCCTGTTCCTCAGGTTGGGTTCAAAAAAATAAAAGGGAAGATTTACATAGA
TTTACATATATTTATGTTAAATATATAAATATTT
>27-52-T3 putative photosystem II core complex proteins
CCAACCACCCTCTCTTCCATCCCTGACCTCCTCTGCCATCGCCGCTGCGGTATTCTCCTCCCTAAGCTCAGCGGACGCTG
CCCTTGCAGTGCAACAGATCGCCGACATTGCCGATGGTGACAGCCGGGGCATTGCTCTCCTAATTCCCATAGTTCCGGC
CATCGGTTGGGTTCTTTACAACATTCTCCAGCCGGCGTTGAACCAGATCAACAGGATGAGGAGTGTGAAAGGGCTTGCC
ATTGGGTTGGGGCTCGGACTGGGCCTGAACTCAGCAACCAGTGCATCAGCTGGAGAAGTGGCGGTGATTGCTGAGGCG
GCTTCTAGGGATAATAGGGGCCTACTGTTGCTAGTTCCAGTGGGTGTGGCGATTGCTTGGGTGCTGTTCAATATACTTCA
GCCAGCGTTAAACCAGATCAATAGGATGAGGGAAAGGTAAGGTACGAACGAAGTAGAGTTTGAGCCTTGCCGTTTGTG
ATTGAGTTCTTGGATGATTATAGCTTCCTTTGTATTCATGGTCATTATATTTTATCGAGGCACATTGTAGTATGCCATGAT
TATTAGATATAATTCTTTTGCTGC
>28-57-T3 S18.A ribosomal protein
AAACCCTAGCATTCGGAAAATTTTTGAAGCAGAGAAAAAATGTCTCTAATTGCGAACGAGGATTTCCAGCACATTCTTC
GTGTGCTGAACACGAACGTGGATGGGAAGAAGAAGATCATGTTTGCTCTCACCTCCATCAAGGGTATTGGTCGCCGTTT
TGCTAACATCGTCTGCAAGAAGGCCGATGTCGACATGAACAAGAGAGCTGGTGAACTCTCTGCTGCTGAACTTGAGAA
TCTCATGACTGTAGTTGCAAATCCACGCCAATTCAAAATCCCAGATTGGTTTTTAAACAGAAAGAAGGATTACAAGGAT
GGTCGGTACTCACAAGTGGTTTCAAATGCATTGGACATGAAATTGAGAGATGATCTCGAAAGATTGAAGAAGATCAGA
AATCATCGGGGCCTGCGTCATTATTGGGGGCTTCGTGTTCGTGGGCAGCACACAAAGACTACTGGGAGGAGAGGAAAG
ACTGTCGGCGTGTCGAAGAAGCGTTGATCCTCCACCTCATTCCTGCTTTGCTTCTCTACTGCATTGTTATGTTGTCTCTCC
ATTGGAGGTTGAAACTGAAATTTTGGGCGACTT
>29-59-T3 unnamed protein product
NNNNAGNGCTCGAGAACGATGCTTCCACCTCCACCCAATAGCATGAGAACGATGCCTCCACCTCCGCCGAAATTCCAG
TCGGATCATAACTTGAGAATAATGCCTCCACCTCCGAAGTTTCGATCCGTTCTTAATGGTAATTCAGAAATGAGAGCAG
CAGCTGTTCACAGAGATTCAGAGAAAGCTAGTTTAGAACGGGTTCCTGATACCCTATTGAAGCTGGTTGAGTATGGCGA
GGAAGACGAAGAGGAAGATGACATGGTTGGCTCAGCCGAAGAATCCTGTCGAAGTGATATAATGCAAAGCACGAGCT
CAAAACCTTTTTGGGCTGTATAATAAGTAGTCTTTTGAAATTTTAGTTGGCATGTCAACATGTTTAATCTCCTTCCATTG
GCTCTCAGGCCTTCTCTGATTGATGATGAGCTTTGAAGCTATGGCTCTGTTCTATTTTTCGATTTGGATGATAAATTTAGT
GAGAAAATTTGATTTAT
>30-60-T3 S18.A ribosomal protein
CGGCGGCGGGGAGTGGAAACCCTAGCATTCGGAAAATTTTTGAAGCAGAGAAAAAATGTCTCTAATTGCGAACGAGGA
TTTCCAGCACATTCTTCGTGTGCTGAACACGAACGTGGATGGGAAGAAGAAGATCATGTTTGCTCTCACCTCCATCAAG
GGTATTGGTCGCCGTTTTGCTAACATCGTCTGCAAGAAGGCCGATGTCGACATGAACAAGAGAGCTGGTGAACTCTCTG
CTGCTGAACTTGAGAATCTCATGACTGTAGTTGCAAATCCACGCCAATTCAAAATCCCAGATTGGTTTTTAAACAGAAA
GAAGGATTACAAGGATGGTCGGTACTCACAAGTGGTTTCAAATGCATTGGACATGAAATTGAGAGATGATCTCGAAAG
ATTGAAGAAGATCAGAAATCATCGGGGCCTGCGTCATTATTGGGGGCTTCGTGTTCGTGGGCAGCACACAAAGACTAC
TGGGAGGAGAGGAAAGACTGTCGGCGTGTCGAAGAAGCGTTGATCCTCCACCTCATTCCTGCTTTGCTTCTCTACTGCA
TTGTTATGTTGTCTCTCCATTGGAGGTTGAAACTGAAATTTTGGGCGACTT
>31-61-T3 abscisic stress ripening protein
GCTCGGTGCCATAGCCGCTGGTGCTTTTGCACTGCATGAGAAGCACAAGGCAGAGAAAGACCCTGAGCACGCCCATAA
GCACAAGATAGAAGAGGAAATTGCAGCAGCAGCTGCAGTTGGTGCCGGTGGTTATGCCTTCCATGAGCATCACGAGAA
GAAAGAAGCCAAGGAAGAGGAGAAAAAGCACCATCACCACCACTTTTAAAGCTTTCAACTATATCAAGACATCCATTA
CTATGTGTTTGTAATTTATATATATATATATATATATATATATATTTTTTGGG
>32-62-T3 alpha tubulin
CTCCCGCATTGACCATAAATTTGATCTCATGTATGCGAAGCGTGCTTTTGTGCATTGGTATGTTGGAGAAGGAATGGAA
GAAGGTGAGTTTTCCGAGGCTCGGGAGGATCTTGCAGCCCTCGAGAAAGACTACGAGGAAGTCGGTGCTGAGGGTGCT
GAAGATGAAGGGGAAGATCCGGATGACTATTGAGTTAGTGGGGATTCATTGAGAGTTTGGGTGTGGTTCCAGTCTTGTT
GATTTGTTTTGTTGTACTCGTGCTAGATATGCTTTCATATTGGCATATATTTCAAACCTTTGTGGTGGTGTTCTTCCATGC
GTGATTTTCCTTTTTGGATTTTTAAAAGTTTACTGGGTGTTAAATGGAATTGCTTAGATT
202
>33-63-T3 syntaxin-like protein
GTANCGGCCTGATGAAGAGGTAGAACTTTGTCTTTTTTTAATTTCTGAATGATTTGAATGTTTTTAAGATCCATAGTTTC
TACCATAATTGTTGGCTCTGCAGACTATCGACCAGCTGATAGAGACAGGAAATAGCGAGCAAATTTTCAAGAAGGCAA
TACAAGAGCAAGGACGTGGCCAGATAGTGGACACCGTTGCAGAGATCCAGGAGCGTCATGATGCCATTAAAGATTTAG
AAAGGAAGCTTCTTGAGCTGCAGCAGGTATTCTTCGACATGGCGATATTGGTCGAAGCACAAGGTGACTTGCTCGATAA
CATTGAATCCCAGGTTTCAAGTGCGGTTGACCACGTTCAATCTGGAACAACAGCTCTGCAGACGGCGAAGAGACTGCA
AAAGAATTCTCGTAAGTGGATGTGCATAGCCATTATAATTCTTCTAATTATTGTCATAGTGATCGTTGTAGCTGTCATTA
AGCCTTGGAGCAAATAGTTTCCTTACAACAGACAGCTGAAGGAACTTCAGGATCGATCGATGGATCAATCAATCAATC
AGGTGATCAACTGTTTTGTATGTCATATATTTAATATTATTATTTATTTTCGCCAACTCTTTCTGCATCTGTTGATATTTTT
GTCGTATACGCTGTCGAGAGCAGCAATTTTTTTTTAATGAATTGGATACTGTTAGCGAAAAATAAATTTGTTATTATTAT
TATTATTTTTAT
>34-66-T3 KH domain-containing protein / zinc finger protein-like
AGNGANCTGCCGTTTAACTGGAGCTAGACTGTTCATACGTGAGCATGAGAGTGATCCAAATCTTAGGAACATTGAGCT
GGAAGGAACATTTGATCAGATCAAGCAGGCGACTGGGTTGGTCAGGGAACTGATTGTCAACATCAGTACTAGTGCTGC
TCCTGTGCCCGTGAAGGCAGCTGGTGGTCTTGGGGCAGGAGGTGGTGGTGGTAGTGGTGGTCCTGGGAGCAATTTTAA
GACAAAGATGTGTGACAATTTCACCAAGGGAAGCTGCACCTTTGGAGAGAGGTGCCATTTTGCTCATGGGGCGGCTGA
GCTGCGCAAGGCAACTGCTTGAAATGTTTTAACCACAACTTTCTAAGGTCATTTGTAGCAAGCGCCGGTTCGTTCGTTTT
GTAGGTGGACTCGGAACTGTTTATGAACTAGTTGATTTCTTT
>35-68-T3 putative RNA binding protein
GTTAGGATTGCAGATTCGAGCGAGCAGATTCGGGCATTGACGGAAATGAATGGTGTTTATTGTTCTTCTAGACCTATGC
GGGTTGGTCCAGCAGCTAGTAAGAGGTCTGCCGATGCACAACAGCATGACTCTGCAAAAGCTTCATTCCAAAGCTCAC
AGGGAAACCAATCAGAAAGTGACCCAAACAACACTACAATTTTTGTCGGTGGCCTGGATCCTAATGCTACGGAGGATA
TGCTGCGGCAGGTTTTTAGCCCATATGGGGAACTGGCTCATGTGAAGATACCTGTTGGAAAGCGTTGTGGCTTTGTCCA
GTTTGTTAGGAGGGCTTGTTCAGAGGAGGCTCTACTGATGCTTCAGGGAACTCAGCTTGGCGGTCAAAAGATGCGGCTT
TCATGGGGTCGTAGTCCTTCAAGCAAGCAGTCTCAGCAGCAGGAAACTAATCAGTGGAATGGAAACTACTATGGTTAT
GGCCAAAGCTATGAAGGATATGGGTATGCTCAAGCTCCTCAGGATCCGAACATGTATTCATATGGAACTTATCCAGGAT
ACGCAAATTATCAGCAACCACAGTGAGCTTTACTCGGATTGTGGCCTTTAACTTGCAAATGGTAGCAGTTTGCAAGCGG
TATCTTCAACTTCACCAGAAGACGGTCTATAGTTATGTCTGCAGGGTATTTTAACATGGATATAATTGGTTGTGCTTTCT
TCATCTTCAGAGGCTGCGTTAGTTATGCTTTTTAAGCTTCTCTTAGCTATGATTCCTGAGTGTGTTGTCTTAACGCTTGTT
ACCTTTTCTCCTGAGTTTCAATGGTTAAAGAGTATCAATTTAACTGAGTACAAGTTATGATTTATGGATTATTAATGAAT
CAATCTGGATCAATTCTAGCTTTGCTGA
>36-69-T3 GF14 protein
AAAAGAGANCGCGATCGTTGAGACGAAGATCTAGAAAAGGTTACTGGATCATTATAAAGCAGAAGTAAAGATGTCGTC
TTCTGAGTCTTCTCGTGAGGATAATGTTTATATGGCAAAGCTTGCAGAGCAAGCTGAGCGATATGAGGAAATGGTTGAG
TTCATGGAGAAGGTGGCCAAGACGGGAAGTGTGAATGAGTTGTCTGTGGAGGAGCGCAACCTCCTCTCTGTGGCCTAT
AAAAATGTTATCGGAGCTAGACGGGCCTCGTGGAGGATAATATCCTCCATTGAGCAGAAAGAGGAAGGCCGTGGCAAT
GAGGATCGTGTGACCATTATCAAGGAATATCGTGGAAAAATTGAGACCGAGCTCAGCAAGATCTGTGATGGAATCTTG
AAGCTGCTTGATTCTCATCTGATTCCCTCTGCTTCTGCTGCTGAATCAAAGGTTTTCTACCTAAAGATGAAGGGTGATTA
CCACAGGTATCTTGCTGAGTTTAAAACTGGAGCAGAGAGGAAGGAGGCTGCTGAGAGCACACTACTGGCGTACAAATC
TGCTCAGGATATTGCATTGGCGGAACTGGCCCCTACTCATCCAATAAGACTGGGGTTGGCGCTGAATTTCTCAGTTTTCT
ATTATGAAATCCTTAACTCTCCAGATCGTGCCTGCAATCTTGCAAAACAGGCCTTTGATGAGGCCATCTCTGAATTGGA
CACCCTTGGCGAAGAGTCCTACAAGGATAGCACATTGATCATGCAACTTCTCCGAGACAACTTGACTCTATGGACTTCT
GACATAACGGAGGATGCTGGGGATGAGATCAAAGAATCCTCAAAACATGAATCATGAGGAGCTGGACCTCATTTTCAT
TTCATTTTAAGTAGATATTATTGCTTTGAAAGACTTTGTTGTGTGCGTGAACTTCTTACTTTTAACTAATGAAACAACAG
CCCTGTATATGGCGACTGGATGTGAAGATGGCGTTTTTTAATGGTTATATTAAGCTTGTGGTGTGCATCATTATGCTATT
TAAGGGCTTTCGAATTACTTTTTAGTAGATGTCAGCTTGCTACTGTTT
>37-71-T3 isopentenyl monophosphate kinase
GTNAAGCGACTTCATCTTGGTATAACTAGTTCAGTTGACCCGTTGACTCTGCTAGAAAAGATCTCTCTAAATGGAATAT
CTCAAGATGTCTGCATAAATGATCTTGAACCCCCTGCATTTGATGTTTTGCCATCCTTGAAGAAGTTGAAGCAACGTGT
GCTAGCTGCAGGGCGTGGCCAGTATAGTGCTGTTTTCATGTCTGGAAGCGGAAGCACCATTGTGGGAATTGGTTCACCA
GACCCACCTCAACTTGTTTATGATGAGGATGAATACAATGATGTTTTCATATCAGAGGCTTCCTTTCTCACTCGGCAAGA
GAATCAGTGGTACGCAGAGCCAACTTCGTCCACAGGGTCTTTGAGCAGAGAAGAGCCGTCACAAACAGGAAAATAATT
ACGATAATTTTTTTACATTCTAGACCTTCTAATTTTAATTTTTCTCACATAAAATCATATTGTATTACTGTACTTATTGTT
CATGCAAGAAAGATCGATCAAGCTATCTTTCATGAATGAGCAAAATATGCAATTTTAAAAGGCACATTTACATGCTT
203
>38-72-T3 enoyl-[acyl-carrier-protein] reductase (NADH)/ oxidoreductase
AAGTGATGAGTCCCGACGGCTGTCGGGGGGGCTCTTTTCCAGATTGCTATCCGANCAAAGAGAGTGATGCAGTGTTTGA
TAGTCCTGAGGATGTCCCTGATGAAATTAAGACGAACAAGCGTTATTCAGGTTCTTTATACTGGACTGTGAAGGAAGTT
GCTGAATGCGTAAAGGATGACTTTGGAAGTATAAATATTCTTGTGCATTCCCTTGCTAACCGGCCAGAGGTGACCTAGC
CTCTATTGGAGACTTCTGAACAGGGGTATCTTGCAACTTTATCTGTTTTCTTCTACTCCTTTAAATCTCTACTCGAGGGG
GGCCGTCCTATAATAAATCCATGGGTAGCTAACATATCTCTAACTTACATTGCTTCTGAGAGAATCTTTCCAGTATATGT
TGTAGCATGATTTCGGCAAAAGCTGCACTTGAGAGTGATACTCAATTACTACCTTTTAGAATCA
>39-73-T3 dihydrolipoamide S-acetyltransferase precursor
AAAAGACTCAGTTAATGACATTGTCATAAAAGCTGTAGCACTAGCGTTGAGGAATGTTCCTGAAGCAAATGCTTATTGG
AGTGATGAGAAGGGTGAGGCCATCATATCCAGCTCCATTGACATATCAATTGCGGTGGCTACAGAGAAGGGCTTAATG
ACACCAATTGTAAGGAACGCAGACGAGAAGACATTATCAACTATATCCTCAGAGGTTAAAGAATTGGCTGAGAAGGCA
CGTAATGGAAAACTTAAACCTGAGCAGTTTCAAGGTGGGACTTTTAGCATATCAAATCTGGGAATGTTCCCTGTAGACC
ACTTTTGCGCGATTATAAATCCTCCACAGGCATGCATTCTTGCTGTTGGTCGAGGTTATCAAGTGGTTGAGCCTGTCAGT
GGCAGCGGTGGAATCGAGAAGCCTGGAATGGTGACAAAGATGAGCTT
>40-74-T3 Ubiquinol-cytochrome c reductase complex ubiquinone-binding protein QP-C
GAGGAAAGCAGAGCGAGAGGGAGAGATGGGGAAGACGCCGGTGAGGATGAAGGCAGTGGTTTATTCCCTATCGCCAT
TCCAGCAAAAGGTTATGCCGGGATTGTGGAAAGATCTTCCGACCAAGATTCACCACAAAATCTCTGATAATTGGCTAAG
CACTGTTCTTCTCCTAGGCCCGCTCATCGGAACCTACTCGTATGTTCAGCATTACAAGGAGAAGGAGAAGCTCGCGCAC
AGGTATTGAATCTGAAGTCTTGCAGAAGATGCTGAATATGGATTGCAGTATCTGAAGATTTTTTCCTGCATAAATACTG
AGACTTTTTTTGAAGTAAATTAGATTAATAACTGCATTTTGGGCACTGGTGGGAGTATTTGTTTAATTATTTGAGTAATA
ATTATCGACATAATTGAATGTGATAGATCTTCCCAGCGTTGGTGTTTTATTTTTTGGGTCATTTACATGCATACAATACA
ATTCTT
>41-75-T3 1,2-dihydroxy-3-keto-5-methylthiopentene dioxygenase 2 (Aci-reductonedioxygenase 2)
NTGAGCACCGCACGCCCAAGCAGTTTGTTTCTATTGACAAACTTGCAGAACTTGGTGTTCTCAGCTGGCGACTGAATGC
TAATGACTATGAAAATGATGAAGCGCTAAAAAAGATTCGTAAGGCGAGGGGCTACTCTTATATGGACATAGTTGAAGT
TTGCCCAGAGAAGCTGCCAAATTATGAAGCCAAGATCAAGAGCTTCTTTGAGGAACATTTGCACACCGATGAAGAAAT
TCGTTATTGTCTTGAAGGAAGCGGTTATTTTGATGTGAGGGATGAAAATGACCAATGGATTCGTGTAGCTGTGAAGAAA
GGGGCAATGATTGTATTGCCTGCTGGAATTTACCATCGATTTACTCTAGACACAAATGACTACATAAAGGCTTTACGAC
TTTTTGTCGGTGAGCCTGTGTGGACTCCACACAACCGACCCAATGATCATCTCCCTGCCAGAAAGGAGTACCTTGATTT
GTTGGGGAAGAAGTCAGCTGCTGGTGGCCATCAAGCCATTGAGGCTCACTGAGGTCATTGGATAAGTACATCTCTTAAC
ACCAGTCTTAGCTGCCGACTGCGATTCCTGCTTTAATCTTATATAATCTCATTTCATGAGTGGTTTTTCACGTGTGGGTTT
GATGTGATGCCAGATGTTTCTGTTGTCTTATAGAATAAAATCGGTTGCTTTATGCCATGCCAAAAGCGTGAACATGTGG
GATTTGTTTATCTTCTGTTCCTGCTGCATGCTGGCTACTTGTATGACGCTTTATGTTAAATAAGTTTGATGAGTTTGCC
>42-76-T3 thioredoxin-dependent peroxidase
GCGTGGACGAGATCCTTCTAATTAGCGTCAATGANCCATTTGTGTTGAAGGAGTGGGCGAAGAGTTACACGGACAACA
AGCACGTGAAGTTCCTCGCCGATGGCTCAGCTAACTACACGAAGGCTCTTGGCCTGGAGCTGGATCTGAGCGAGAAGG
GGCTCGGGCTGCGTTCTCGCAGGTATGCGATCCTGGTGGACGACCTCAAGGTTAAGGTGGCTAACATTGAGGAGGGTG
GAGATTTCACCATCTCTGGTGCTGATGAGATCCTCAAGTCTCTGTGATACGCCTTCTGCTGTTTGTTGCCTCTTTTTGAGA
CGCGCTCGTTTCACGTTTCTCTTTGAATAAAAGCGCTTCTTAGCTCTTCCTGTTTTGTTTTGTGTGAAAGAGAACACAGTT
CTATAAAGTTCAAGTGTTTATTTGGTTTACTCTTTAAGGTTTACCTGCAAGAAGTTTGTATTGTTGTTTGAAGTTGGTAA
AGATCTTAATTTTATGCC
>43-77-T3 putative mitochondrial dicarboxylate carrier protein
GANTCCGCCGCCGGATTAGTGGCAGCGGTGGCGTCGAATCCGGTGGATGTGGTGAAGACGAGGGTGATGAATATGAAG
GTTGAGAAGGGGGCACCGCCGCCGTATGCCGGAGCGCTGGACTGTGCGTTGAAGACGGTGAGGGCGGAGGGACCTAT
GGCGCTTTATAAAGGGTTTATTCCTACTGTTTCTCGTCAGGGGCCCTTCACCGTCGTTCTCTTCGTCACGCTGGAGCAGG
TGCGCGCGCTTCTGAAAGATTTCTAGGTTCATCTCTATGTGAGATTTGTTGGATTGATTTGTTATGATGCCGAAAATGTA
TTGCCGCTCTGGGGGCTATGAGCAGCAGAGCGGCTAGTGTTTGTTCTTCAGAAGAATCAATTAAAATCTGAGTTTTTCT
ATTATT
>44-78-T3 6-phosphogluconolactonase family protein NAANANACCCGAGTCACCGCCAGAAAGGATAACCTTTTCGATTCCAGTGATCAACTCAGCATCAAACGTCGCCATTTTG
GCTACGGGTGATGACAAGGCCATGGCTCTGCAGTTCGTTGTCGATCACAGCTCTAGTTTTGATGCCTTTTCGCTGCCCGC
AAGGTTGGTGAACCCGACCGAAGGGAAGCTGTTGTGGTTCGTGGACAAAGCGGCAGCCTCGTTCATCGACTCTGCCGA
TGAAAGCGAGCATTTTGAAATTTAGCAGCTATGTATGTGTTCTGTATGTAGATGAGTACAGTGGGTTTGATTAGTTACTT
CTTTTGTTTAGGCCCTCTTTGAGATATCTGAAGGAAGCTGTGGTTAAGATGTGAGGGTAAAGCAGTAGATGAGGAAGTG
GATTAATAAGCATAGTGCAACATTTTGGATGGAGGGCAGTAATCTGTGAGAGACATTGCTTTTATTTTAATTAGTGAAA
GAAAATTCTCAAGAGTTCTGAATTTTC
204
>45-81-T3 calmodulin-binding protein
TTGAAGAGGAAGACGGCGCTGCCGTACAAGCAGAGTTTTTTAAACTGGCTCAGATTCGCTCCGGCGTTGCGTAGTTTTA
GTTCTTCGGGGGCCGGCGATAATCATTCACCCTATCGTGGGAAAACCATGGCAGGGTCGAAGTCAATGCCTCGCTTCAG
AGAAGGGAATTAATTGAGCAAAATTGAATGAATTTGGTCAAGAAAGTTAGTTAGATTTTTTTTTCTTTTTTTTTTATTTG
CTATGATAATTGATGAGTTTTAAAGTAGTTAAATTCATTTTAAGATGAGCATAATATAAATTTTAAATGATTTATCATGA
TTATGCTTATCTTAAAATAAATATAATAATTTTAAACCTATCTTCATCAAAACATTCTGACTATTTTTTCTTCATCTAATC
AGCTATGCTTGTTGTACATAAAGGTGAGTGTGTTGTGTCTAAGTGTGTAAATTTCTACTTTGAAATGAGAAAGGATGTC
T
>46-82-T3 enoyl-[acyl-carrier protein] reductase
AGATTCTTGTTGGAACATGGGTGCCTGCATTGAACATATTTGAGACTAGCTTAAGGCGTGGAAAGTTTGATGAGTCCCG
ACGGCTGTCAAATGGCTCTCTTTTCGAGATTGCTAAGGTTTATCCCCTTGATGCAGTGTTTGATAGTCCTGAGGATGTCC
CTGATGAAATTAAGACAAACAAGCGTTATTCAGGTTCTTCAAATTGGACTGTGAAGGAAGTTGCTGAATGCGTAAAGG
ATGATTTTGGAAGTATAGATATTCTTGTGCATTCCCTTGCTAACGGGCCGGAGGTGACCAAGCCTCTATTGGAGACTTC
CAGAAAGGGGTATCTTGCAGCTTTATCTGCTTCCAGCTACTCCTTTATATCTCTACTCCAGCATTTCCTTCCTATAATAA
ATCCAGGGGGAGCTACGATATCTCTAACTTACATTGCTTCTGAGAGAATCATTCCAGGATATGGTGGAGGCATGAGTTC
GGCAAAAGCTGCACTTGAGAGTGATACTAAAGTACTAGCATTTGAAGCAGGAAGGAAAAAGCAAGTTAGAGTAAATG
CAATCTCTGCTGGTCCGCTGGGAAGCCGTGCTGCAAAAGCCATTGGTTTCATTGAGAAGATGATAGAGTATTCACATGC
TAATGCGCCATTACAGAAAGAACTGCTAGCAGATGAAGTTGGTAATGCTGCGGCTTTCTTGGTGTCCCCATTAGCTTCT
GCTATCACTGGCTCCGTAGTTTATGTCGACAATGGATTAAATACAATGGGTTTGGCAATTGACAGCCCATCTCTCTCAGT
CGAGTGATAAAGCAGAAAATCTGCATAATGATTGGTTTTTTAATAACAAGTATCCCCTTCTGTTTCAATCTCCTGCGGCA
TTAATGAATAATAATTGAGTTGGTTTCTTTCCTAGTTTCATTCGAGGTCCAGTGGTTTTATACATGTAGCTATGTGATGA
TATGATGATTCCTTTCAGTGNGTACAGTGAAAATACATTTTGATG
>47-83-T3 hypothetical protein OsI_021037
GNAANAATTCAGGAAGAAGGTCCTTATGATGATCTTGATGAGGAAAAGGGCCTCTCTCGTCGTTGCCAGCTTGTGCTCG
CATTCCTTGCATTTTTTTTACTGTTTACAACATTTTGCCTTATTATCTGGGGAGCGAGCAGACAGTACAAAGCAGATGTG
GTGGTGAAGAGCTTGTCAATGGATGATTTTTATTCGGGAGAGGGTTCGGATAGCACTGGTGTTCCGACCAAGTTGATCA
CGGTGAACTGTTCTCTGAAGATCAATGTGTATAATCCAGCATTCACATTTGGTATTCATGTCACTTCTAGTCCCATTAAT
CTCAAGTACTTGGATATTGTCATTGCTACTGGTCAAATAGAGAACTACTACCAGCCCAGAAAAAGCCATAGAACCATGT
CTTTAATACTAAGGGGAGACAAGGTTCCTCTATATGGAGCTGGTGCTGCCTTACCTCTATCCAATAGTGGTGGTTCTGTT
CCATTAACTTTGGAATTTGATTTGGTTACCCGAGCTAATGTGGTGGGAAAGCTGGTTAGGGTGAAGCATCAGAAGCATG
CCTCATGCCAAATTGTAGTTGATTCTAGCAAGAACAAGGCCATAAAGTTGTCAAAAAATGCTTGTACCTATGACTAAGT
CCCTTTATCTTCATGTTTTACTAGCAGATAATCTGCTCATGATGAGGTGAAAGAAGGAAGAGTAGTTGCATGGTCTCCA
TTTGTCTATGTTTTTTTTATGTTCATTTTTGTATATTGTTGCAATAAGACAATTTGAAGATGAGTTTTGACTTGAATTTCC
TGTACTGTAGAACTTATACTTTGTTGGTAAGTGGAAGAAGGCATTTCTCTTGT
>48-104013_85_T3 jasmonate ZIM-domain protein 1
AAGAAATCGATCGATCGAGGCGGAGGATTCAACTAGTCTTTGGGATTTTCACACTCAACGATAGAGGGTATATGGCAA
AGAGGCAGGAGAAGTCGAACTTCTCCATCACCTGCGGCCTCTTGAGCCAGTACATCAAGGAAAAGGGCAGCCTTGCTG
ATCTAGGGCTTCTCGATTCTGCTCGATTAGGCAAACATGAGGCTTATCGGCCGCTGACAACCAGGAGCTTGCTCTCAGG
AGTAGGTTTCTCCATCAATGACCCTAAAGACACCAAATCCATGGAGCTTTTTCACAAGAGTATTGGTTTCCTTCCGGCC
>49-86-T3 translation elongation factor 1 alpha
GNNAAGACCTGAAACGTGGTTTTGTGGCTTCTAGTTCAAAGGATGATCCTGCTAGGGAAGCCGCCAATTTCACTTCTCA
GGTCATCATCATGAACCACCCTGGCCAGATTGGCAATGGTTATGCTCCGGTCCTTGACTGCCACACTTCCCACATTGCC
GTCAAATTTGCAGAGATCTTGACCAAGATAGACAGGCGGTCCGGCAAGGAGCTCGAGAAGGAGCCAAAATTTCTCAAG
AATGGAGATGCAGGTTTCGTGAAGATGATTCCCACCAAGCCTATGGTGGTTGAGACGTTCTCCGAGTATCCGCCGCTCG
GAAGGTTTGCTGTGAGGGACATGAGGCAGACGGTGGCGGTCGGGGTCATCAAGAGCGTGGAGAAGAAGGATCCGAGT
GGAGCGAAGGTGACCAAATCGGCTGCCAAGAAGAAATGAATTGTGCGTTGTTGTTTGAATAAGGAGGAGCGGGAATCC
ATCGAGTTGGTTTCTGGTGTTTGGATGCAGAACTGGGTGCTTGACAGACGGTGGCACTGCTCGCTTCAGTTATCTTTTTA
GTTGTTGTCTGTGTTGTTTGTTTTTCTTGTGTTGAAGGCTGTTGTACTGCTTTTCTATGGTTGTATTATTTATCGCTGCTTA
TTATGATTGTACCGTTGTTGTTGTATGAGTTTGAATTTGATGTTATTTGAGTTTTTGTGTTCT
>50-90-T3 hypothetical protein
NGCGATCAGGTTCCAAGACTTGATTTTTATTTTCCTATGGAAGTTCACAAAGATAGCAATGAACGTTTATCCATACCCA
CATTTGGCGAGTGGGATGGGAAGGTGGGTCTACCGGACTACTCGGTTGATTTTACTAAGATTCGAGAAAATCGGCGGC
AGAACAAGAGTCGGGTGAGCTTGGGGAATGAGGATGAGCTCCTACATCGTACTGGTGTAAATAGTGACCAGAATGTTG
ACGCTCTCAAGAAGACACTCCCACTTCATCAGAAGCATGTTAACTCCACAGTGGAAAGGAAGAAAAGCAGCAGATATT
TCAACTGTTGTCGTTGTTTGGGGGCCTGAAACTGTGGCTTCATCCACACATTATGTTGCACGTGGAGGTGAGGGCCTTTG
AAGCTCTTCAGCCATCCTTTTCAAATTTCTTTCACCTCCGTTTTGTCGCTCAATCAAATGTTATGGTGATTGCAAACTACT
205
TGGATTAGGATTTTTGTTATGAAGAAATGCAGGATTGGAGATGTTTCTTGGTGTCGTTCTATAAAGATGTAATGTTTGGA
TTGCTTGTTAGATTCACATATTTTTATTATTATTCGATCGCATTGTTGTAGATAGATTTTATATTTTATGTACATGAAATT
TTATCTTATTTATGATTAGTAATTTCTTATTCTAATCC
>51-91-T3 MADS box AP3-like protein 17
NCNCCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCCCTTGAGCAAACTTTGGAAGAGTCTCTGAGAATTGT
TAGGCATAGAAAGTATCATGTGATCGCCACACAAACTGACACTTACAAGAAAAAGCTTAAAAGCACAAGGGAAACAT
ACCGCGCTCTAATACATGAACTGGATATGAAAGAGGAGAATCCGAACTACGGTTTTAATGTTGAGAACCACAGCAGAA
TTTATGAAAACTCAATTCCAATGGTGAATGACTGCCCTCAGATGTTTTCCTTTAGGGTTCATCCAAATCAGCCTAATCTG
CTCGGTTTGGGTTATGAATCACACGATCTTAGCCTTGCATGATCAGCAGTACTATTATAAAAGTTTATGATTTTATTGCA
TTTTTATTATATGTTTGAAACTTTAGAATTATGAGATGGGGGATCTAGTC
>52-94-T3 40S ribosomal protein S15a
TTTGAANGATGCTCTCAAGAGCATGTACAATGCTGAGAAGCGTGGGAAAAGACAAGTTAAAATCAGACCATCTTCCAA
AGTTATCATCAAGTTCCTCCTCGTCATGCAGAAGCATGGCTATATCGGTGAGTTCGAGTATGTCGATGATCATAGAGCG
GGCAAGATTGTGGTGGAATTGAATGGGCGTTTGAACAAGTGTGGTGTCATTAGCCCAAGGTTTGATGTTGGTGTTAAGG
ATATTGAGCCATGGACTGCAAGATTGCTCCCTTCGCGTCAGTTTGGGTATATTGTGCTGACAACATCAGCAGGCATCAT
GGATCATGAGGAAGCTAGGAGGAAGAATGTAGGAGGCAAGGTTCTTGGATTCTTTTACTAGTTAAAAAATGTTTTGAA
TGAATTTTGAAGATTGTTGTCTTTTAAGTGCATTTATTTGATGTTAGCCTGCATTTCTCCTTAACTTTGAATGGTGTAAGG
CTACTTGTTATCTTTGGATCTTGAGGCATATTTCCCATTTGTGTTTGCGTATTTTTTTGCTAAGGTTACCGTTGAAAATAT
TGCAGAAAATTTTATTTT
>53-104016_95_T3 60S ribosomal protein L14 (RPL14A)
CCCTTCATAGCTTCCAGCAGATCGCAGCAGCAGTCATGCCTTTCCAGAGGTACGTGGAGATCGGGAGGGTGGCACTCGT
CAACTATGGGAAGGAGTATGGGAGACTTGTTGTTATCGTCGATGTCATCGATCAGAATCGAGCCTTAGTTGATGCTCCT
GACATGGTTCGAGGACAAATGAACTTCAAGAGGCTGTCGCTGACTGATATCAAGATTGACATCCCCCGAATTCCCAAG
AAGAGCAAACTGATCGAGGCCATGCAAGCAGCTGATGTGAAGAATAGATGGGAGAAGAGCTCTTGGGGGAGGAAGCT
GATTGTGCAAAAGAAGAGAGCTGAACTGAATGATTTTGATAGATTTAAGGTCATGTTGGCTAAAATTAAGAGAGGAGA
TTCCATCCGACGTGAGCTTTCAAAGCTAAAGAAGAGTGCTTCAAAGTGAGGCTTTTGAAGAATTAATGGCCGATTCTGT
GTCCATTATTGCAGAATTTAAGTTTGAGAGTCAACCGTTAGAACAAAATGTAGTTTTGGTCGCTGTTATGAACTTAAAA
GTTTGATGTTTTGATATTTGGATTTTTTTAAAAAAATTTGTAACTGTTTGCTTTATTATTTGAATGAATTTAAGAATTGGA
TTATTATTAATTATTCCACAGTAGAAGGAGTAATTTGTGAAACTGATTTCTGTTAATGTTTGATTGCAGATTCTACAAAC
GCAATCTTCAAAATGTATTTTTGATTGTTCAATATAGATTTTAGAAATGTCTCAATTTGTTT
>54-96-T3 hypothetical protein OsJ_006705
CTTGATTTTGCTCAGAGATCTATGCCTGTTATTGGATATAGCGCTCATGCAGGTAACAGCGGGCCAATGGCTTCTGAGT
CAGTGCCTGGTTAAAATGTCCATTTCTAAGTCGCCGAATAATGCACCTCCATAATCAATCATGAAAGGACCCCCTGGGC
TAGTTTATGTATGAATGCAAAGCCTTTATCACCATCATTGTCTGCACCATCATCGTCAACATGGGAAGGAGGTTAGCTTT
TGTTGAAGTTTAGGCGAAATTGCTCATGCCTTAACGAGCACATATCAGTTTTCGCCTTCTCTCATCTCTGAATTTGGTCT
CTTCTAACATGTAAACTTTAGAAACAGTGGGGAGTAGCCGGGAGGTTTCTTCTTCCGGGGAGCTTTATTTTGTTCGCTCT
GCATGGGAACTCCAGTAAATTTATTAGTGTCCTGGACTATACAGGTTTGATCCTGGAGATCGATCGTTTCTGCAGTTTTA
AAACTTGAAACTATCAAAAAGGTACTTTTATGCGTGGGCTTGGTGGTGTAATTATTCTGTTAGAATCTATATTTAGAGG
CTTC
>55-28 hypothetical protein
AAATATCCCGCAACCTGTCCCACCTTTTCTCCCCTTTTCGTCTCTTTTTCCCTGTTTCTCTTTCTCATCCGATTGATGGATC
TTAGGGCGGCGATAGTCGCCGCCGCCGGCGAGCGTTGGACGGAGGAGCGGCACTCCCGCTTCCTCAACTCGATCGAAA
GTACTTTCGTCCATCAAATGCTCGGCATCCATCCCGACGGCGATAACCTCCGCCGATGCGCGGCGAGGCTCGACCGTCG
TGTTCCCGATTGCATCGCCGGTAAAGAGTCTGCGAAGAGTTCTCAGATGCGATCGCCGGATAGGAGGCCTGCTGCCATT
ACTGCGGGCGCCAACACCTCTAATTGTACACGGAAACGATCACTGCGGCGATATGATGCGTCGCTAGACCAGGTGGTG
CCGGAGTTCAAAAATAAGAACGTCGGCGAGGATGCATCC
>56-38 GRF interacting factor
CTCCTCACTTGCCCGCCGCCATTCTTGTCGATTACCCACTGTTCCCTCAAAGATCCCCTTTATTCGCTTAACCCACCACC
AAAGCTGAATTTCAGAGTCCAGGACTCAAAAAGCCGAAATCTTTCCGTGTAGAAAATACGACAAGCCAACACAGGAAT
GCAGCAGCCCTCGCATCCGATGTCCCAAATTTCTCCGGGCAACATTACCACAGAGCAGATTCAGAAGTATTTGGATGAA
AACAAGCAGCTGATTTTGGCAATTTTGGATAATCAGAACTTGGGCAAACTTGCTGAATGTGCTCAGTACCAAGCCCAGC
TCCAAAAGAATCTGCTTTATCTCGCCGCCATAGCTGATGCACAGCCTCCAACTCCTTCAGTCCGTCCTCAGGTTTTCTCT
206
GCACAATTTTCAAGCTTATCCTCATATATCTGGTTGTGCATAGAAGTAGAAATTTGTTAATGCTGACAGTTTGAGGAGC
GAGCTCATGGAACGAAGACTATTCAAAATTTGGGTTTGTTTATGGCTCGGTGACTATCATGCTTACTTGAAACAACTTT
GGTTTACCTCGGTATAGATCAAATAATTTTGCTTGAGATGAAATCTAAATAAAGGGTTGGTTTGTTCTATATATAATGA
AGTCTTTTACCTAGCACACTATTAGATATGGTTGTGAAGAGCTTCTTATAATAAAAAATTTTGGGAAAGC
>57-40 chaperon heatshock protein
GCAAGCTGAAGATGGATGCCAGAGTTTTCAGATTGGAGAACCCCCTCTTCTACGCACTGCAAAATTTGGTGGACCTGCC
GGAAGAAATGGAGAAAGCCTTCAACGCTCCGACGAAGAAATATGTACGCGACGCGCGGGCGATTGCTTCCACGCCGGC
AGACGTGATTGAGAAGCCGACGGCCTACGAGTTCGTCCTAGATATGCCGGGAGTGAAATCTGGCGATATAAAGGTCCA
GGTAGAGGAGGGTAACGTGCTCGCCATTACAGGGGAGCGGCGCCGGGAGGACGAAGACAAGGAAGGCGGCGTAAGTT
ACCTTCGATTGGAACGACGGGTAGGGCAATTTCTGCGCAAGTTCTCTCTCCCGGATGACTCCAATCTCGAAGCGATAAC
CGCTGTCAGCCGTGATGGTGTTCTGACAGTTACCGTGCAGAAGCTGCCGCCGCCCGAGCCTAAGAAACCCAAGACAAT
CGAGGTCAAGATTGCGTGAATTGATCTGTTCGTTTTGAATTTTTAGGGTTTGCTTTACTGTCATTATGAAGTATGTTGAG
GTTGTGTCTAAGGATTGGATCCTTGTGATCTGCTTTGTTTCCAATCGCTTTTTGATATATCTATAATACTATCAGTAAAG
ATTTTGTGGCTTTGGG
>58-44 phospholipid transfer protein
CTCGCTCCACCGCTTGTGTTGCAGTCGTATGCATAGTGTCCTTCCTCCTTGTTTCCGGCGTTTTCCGCGAGGCGAGCGGG
ACCGTCACTTGCGGCCAGGTGGCCTCAAACGTGGCACAGTGCATCGGCTATATTCGAGGCGCTGGCTCGCTCACACCAG
CATGTTGCAGTGGGGTGAGGAAACTCAACGGGCTGGCGAGCACCACCCCTGACCGTCAGACGACATGTTCTTGCCTCA
AGACTCTCGCCGGCAGTATCAAGGATCTGAACCGTGGCCTTGCTGCTGGACTTCCTGGAAAGTGCGGCGTCAACGTTCC
ATATCCCATCAGCACCTCCACCGACTGCTCCAAGGTGCGCTGAGCTCCAAGGACGGGCTACTCATCGAGCACTGCTTTG
GTTCGATGATCTGCAGAATAAAGTGGAGACTGGATCTATTGAAGCTATTTTATGTATTGTTGTTTGTGGGTTCTGTCTTT
CGATTTTAGTTTATTGTTTTGCAAGAGGGTGCACCCTTGGTTGTGGTGTGCTGGATATTGTTGCTGTAATATTATCACTTT
TTCGACTAATATGAGTTGCAGCATATTATAATT
>59-46 hypothetical protein
GGAGAAGCCTAAGGAGCCGGAGAAGGAGAAGCCTAAACAGCCTGAGCCGCCTCAAGGGGGCCAGCCGGTCTGGCCGC
CCGGTCCACACATGCCATGCTGCTGCTGGAGACCATGCTATGAGCAGTACTACGGCGGCTGTCGGTGCTGCTCCTGTGG
GATGTTCTACGGGTGGACGGGGCCGCCATTGCCACCGGCTATGGGCACTCCGTCAACATCCACGTGTCAGTTCTTCTGC
GAGGAGGATCCTTCTACATGTACCATTATGTAATAATATAGTCCCTTTCGTTCAAATTATGAGCCATTAATAGAGATGA
TAAATAAAGCTGTGCTCCTCTCTATAGTGCATTGAGTTATAATAACTCAAGAGATATATGTCAGTTTTATAATATTTTGT
CTTCTTTATTTGAATATGTAATTTTGTTTTCATCATGGTGAAAGATATATGTCAACTTTGCAATATTTTGTTTTCTTT
>60-54 hypothetical protein
CTGAGAAGCAGTCCTCGTACACGTACTGGGTGAGGGAGACGAAGGACGACGCGGCGCCGCTGCAGGTGCCTCGGAAG
CTCACCCCGGAGGACGTTTCTCAGCAATCCCAGCCTAACTTGTTGGGATCCGTATGGAATCAGGCCGGAACAtGGGAGG
AAAAAAATCTAAATTCATGGGCAAATAGTAGAATTAAGGAGCTTCTCAGTTCATTGTCCTTGGAGTTTTCTAATGGAAA
AGCAGCGGTTTATGAAGTTACCAAATGTTCAGGGGATGCATTTTTGATCACGGTGCGGAAC
>61-58 pectate lyase protein
GCGATTATGATGATGGTTTGATTGATATCaCaCgAGAGAGCACTGACaTCaCTATCTCgAGATGCCACTTTGCAATGCATG
ATAAAACAATGCTTATTGGGGCTGATAGCAGCCATATTACTGATAGATGTATCCGGGTGACAATACATCACTGTTTTTT
TGATGGAACAAGACAGAGACACCCTCGTGTTAGATTTGGGAGAGTTCACCTCTACAATAATTATACAAGAAATTGGGG
TATATATGCAGTATGCGCTAGTGTGGAAGCaCAGGTTCTCTCTCAGTGCAATATATATGAAGCCGGAGAG
>62-66 hypothetical protein
CCCACCCCTTCCCTATCTTCCATGATAATCAACCACTACAAGCTTAGGGGAAACATCATCAGCTACAACCTTGGTGGTA
TGGGATGTAGTGCTGGACTCATCTCCATAGATCTTGCAAATCGTCTTCTTCAAGTTCATCCCAACTCCTATGCTTTGGTT
ATCAGCATGGAGAACATTACTCTCAATTGGTACTTTGGAAATACCCGTTCCATGCTCGTTTCGAATTGCTTGTTTCGAAT
GGGCGGCGCAGCTATTCTCCTATCCAACAGACGGTCAGATCGCCGTCGATCCAAGTATCAGTTAGTCCATACGGTTCGC
ACGCATAAGGGCGCCGACGAAAAATGTTTTGCTTGTGTGACTCAACAAGA
207
APPENDIX H
Real-time RT-PCR Data
1) PCR Efficiency for Putative Fragrance-related cDNAs and Housekeeping Genes
(D)
(C)
(B)
(A)
208
Notes: PCR efficiency of putative fragrance-related cDNA and housekeeping gene
cDNA transcripts for real-time RT-PCR analysis. (A) putative actin (B) putative
cyclophylin (C) putative alpha tubulin (D) VMPPAAS (E) VMPCMEK (F) VMPCyP50
and VMPA28
(F)
(E)
(G)
209
2) Expression Analyses of Putative Fragrance-related cDNA Transcripts and
Reference Genes Transcripts
a) Expression Analyses in Different Tissues
VMPPAAS
Tissues Calibrator Relative Quantity Standard Error
bud x 1 0.238
fully-open flower 8333 168.76
petal 20256 888.56
sepal 15879 487.98
lip 4439 567.47
leaf 0.5 0.007
root 0.3 0.0065
shoot 0.3 0.0054
stalk 0.4 0.076
VMPCMEK
Tissues Calibrator Relative Quantity Standard Error
bud x 1 0.16757
fully-open flower 1.63 0.20777
petal 1.763 0.2645
sepal 1.81 0.1436
lip 0.609 0.02515
leaf 0.009 0.00095
root 0.015 0.0042
shoot 0.012 0.00155
stalk 0.012 0.00121
VMPCyP450
Tissues Calibrator Relative Quantity Standard Error
bud x 1 0.17897
fully-open flower 3.094 0.27044
petal 1.12 0.17177
sepal 1.912 0.18797
lip 5.497 0.30796
leaf 0.001 0.00008
root 0.003 0.00047
shoot 0.001 0.00005
stalk 0.002 0.00016
210
VMPA28
Tissues Calibrator Relative Quantity Standard Error
bud x 1 0.10546
fully-open flower 1.17 0.09434
petal 1.266 0.10744
sepal 1.352 0.04426
lip 1.054 0.06094
leaf 0.586 0.06464
root 0.62 0.04724
shoot 0.482 0.04496
stalk 0.644 0.06778
b) Expression Analyses at Different Flower Developmental Stages
VMPPAAS
Stages Calibrator Relative Quantity Standard Error
young bud (green) x 1 0.045
mature bud (red) 8.711 0.578
half-open flower 49277.86 4691.799
fully-open flower 83509.328 6075.784
14-days old fully-open flower 7617.29 1163.816
VMPCMEK
Stages Calibrator Relative Quantity Standard Error
young bud (green) x 1 0.10003
mature bud (red) 1.508 0.21186
half-open flower 2.273 0.22069
fully-open flower 1.822 0.23132
14-days old fully-open flower 0.123 0.10549
VMPCyP450
Stages Calibrator Relative Quantity Standard Error
young bud (green) x 1 0.10579
mature bud (red) 2.734 0.27015
half-open flower 3.344 0.49873
fully-open flower 3.041 0.22863
14-days old fully-open flower 2.094 0.29847
VMPA28
Stages Calibrator Relative Quantity Standard Error
young bud (green) x 1 0.25742
mature bud (red) 1.98 0.24924
half-open flower 2.5 0.34808
fully-open flower 3.5 0.26529
14-days old fully-open flower 2.8 0.28955
211
c) Expression Analyses at Different Time Points in a 24-hour Cycle
VMPPAAS
Time Calibrator Relative Quantity Standard Error
12am x 1 0.09914
2am 0.601 0.01281
4am 0.474 0.03276
6am 0.328 0.13436
8am 0.023 0.05484
10am 0.012 0.00981
12pm 0.011 0.0071
2pm 0.001 0.00021
4pm 0.001 0.00021
6pm 0.154 0.04827
8pm 1.404 0.01737
10pm 3.361 0.29116
VMPCMEK
Time Calibrator Relative Quantity Standard Error
12am x 1 0.18511
2am 1.87 0.22595
4am 1.954 0.27654
6am 2.437 0.30942
8am 2.613 0.34981
10am 2.179 0.33652
12pm 1.424 0.25509
2pm 0.857 0.09788
4pm 1.13 0.12365
6pm 1.15 0.17864
8pm 1.342 0.18711
10pm 1.778 0.25471
VMPCyP450
Time Calibrator Relative Quantity Standard Error
12am x 1 0.18057
2am 0.158 0.02676
4am 0.347 0.06629
6am 0.654 0.06927
8am 0.813 0.10608
10am 0.819 0.09146
12pm 0.445 0.07637
2pm 1.56 0.16879
4pm 2.378 0.11831
6pm 3.808 0.23655
8pm 4.586 0.33069
10pm 6.367 0.10896
212
VMPA28
Time Calibrator Relative Quantity Standard Error
12am x 1 0.2131
2am 1.562 0.17269
4am 2.013 0.2717
6am 2.196 0.27402
8am 2.981 0.39576
10am 1.376 0.02264
12pm 0.635 0.18099
2pm 1.879 0.2764
4pm 4.188 0.2486
6pm 2.18 0.2475
8pm 0 0
10pm 0 0
213
3) Melting Curve analysis
(a) VMPPAAS
(b) VMPCMEK
(c) VMPCyP450
(d) VMPA28
214
(e) Actin (Reference gene)
(f) Cyclophilin (Reference gene)
(g) Tubulin (Reference gene)
215
BIODATA OF THE AUTHOR
Mohd Hairul Ab. Rahim was born on 18 September 1985 in Telok Mas, Melaka. He went
to SK Telok Mas primary school from 1992-1997. He continued his secondary education
in SMK (A) Sultan Muhammad, Melaka from 1998-2000, and then SM(A)P Kajang from
2001-2002 to obtain his Sijil Pelajaran Malaysia (SPM). His pre-university education was
completed in Malacca Matriculation College. In July 2004, he entered Universiti Putra
Malaysia to begin his undergraduate study and completed his Bachelor of Science degree
in Biotechnology with a CGPA of 3.372. He continued his Master degree in the field of
Plant Biotechnology at Universiti Putra Malaysia in 2007.
He was active in extracurricular activities throughout his undergraduate studies. He was a
member of the Students‟ Representative Council of Universiti Putra Malaysia for
2005/2006 session and also an Exco of BioMix Society, an undergaduate society of
Faculty of Biotechnology and Biomolecular Sciences UPM for 2004/2005 session.
Besides that, he was involved in many students‟ programs including „Sambutan Hari As-
syura Universiti Putra Malaysia 2006 organized by the Students‟ Representative Council
of Universiti Putra Malaysia (The Director of the programme), commitee members of
„Faculty Night 2005 and 2006‟ organized by BioMIX Society and a facilitator for
„Minggu Perkasa Putra 2005‟ (orientation week for incoming new students of Universiti
Putra Malaysia). During his undergraduate studies, he was selected to present a poster
presentation at 18th Intervarsity Biochemistry Seminar for his final year project. He
received the Public Service Department (JPA) scholarship for his undergraduate studies.
216
His interest on molecular biology and plant biotechnology started in 2006 during his
practical training at Molecular Biology Laboratory, Makmal Biaklon, Felda Agricultural
Services under the supervision of Dr. Sharifah Shahrul Rabiah Syed Alwee. During his
postgraduate studies, he had presented in some conferences and seminars such as Asia
Pacific Conference on Plant Tissue Culture and Agrobiotechnology (APaCPA) 2007
(poster presentation), 5th Malaysian International conference on essential oils, fragrance
and flavour Materials (MICEOFF5) (oral presentation), 18th
Scientific Meeting of the
Malaysian Society for Molecular Biology and Biotechnology (MSMBB) (Poster
presentation) and 2009 Plant Biotechnology Postgraduate Symposium (Oral
presentation). He won the first prize for best oral presenter for the Molecular Biology,
Agriculture and Physiology category in the 2009 Plant Biotechnology Postgraduate
Symposium. Besides that, he was also a recipient for a research grant from Malaysia
Toray Science Foundation (MTSF) 2008 for the screening of fragrance-related cDNAs
from Vanda Mimi Palmer. During his postgraduate studies, he was financially supported
by the Graduate Research Fellowship (GRF), Universiti Putra Malaysia.
217
LIST OF PUBLICATIONS
Terpenoid, Benzenoid and Phenylpropanoid Compounds in the Floral Scent of Vanda
Mimi Palmer. Mohd-Hairul, A.R., Namasivayam, P., Cheng Lian G.E. and Abdullah,
J.O. (2010). Journal of Plant Biology 53: 358-366.
Putative Phenylacetaldehyde Synthase Transcript of Vanda Mimi Palmer: Sequence and
Expression Analysis. Mohd-Hairul, A.R., Chan, W.S., Namasivayam, P., Cheng Lian
G.E. and Abdullah, J.O. (2010) International Journal of Botany. Published online on
September 2010. http://scialert.net/abstract/?doi=ijb.0000.20116.20116#
Screening, Isolation and Molecular Studies of Putative Fragrance-related Transcripts of
Vanda Mimi Palmer. Mohd-Hairul, A.R., Namasivayam, P., Abdullah, J.O. and Cheng
Lian G.E. (2010). Status: Submitted