Identification and Characterization Using Cytochrome p450

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Insect Science (2011) 18, 484494, DOI 10.1111/j.1744-7917.2010.01380.x

ORIGINAL ARTICLE

Identification and characterization of a cytochrome P450CYP6CX1 putatively associated with insecticide resistancein Bemisia tabaci

Hua-Mei Zhuang1, Kuan-Fu Wang1, Lin Zheng1, Zu-Jian Wu1, Tadashi Miyata2 and Gang Wu1

1Key Laboratory of Biopesticide and Chemical Biology, Ministry of Education, Fujian Agriculture and Forestry University, Fuzhou, Fujian,

China, 2Laboratory of Applied Entomology, Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya, Japan

Abstract The novel full length of cytochrome P450 gene has been isolated in insecticide-

resistant (named CYP6CX1v1) and -susceptible (named CYP6CX1v2) Bemisia tabaci,

which was identified as B biotype, in Shangjie, Fujian, China (Sj). CYP6CX1 (1 940 bp

contained a 1 557 bp open reading frame) included conserved domains common to CYP6

members, such as heme-binding motif PFGEGPRFCIA, putative meander-binding se-

quence ETLR and PERF in helix-K, oxygen-binding motif AGLDPV and conserved

sequence PEKFNP near the carboxyl end. There were four different replacements of

amino acid residues between R and S B. tabaci (Thr300 Ala, Thr354Pro, Arg486His and

Ile503Thr), among which the substitution Ile503Thr was located in the substrate recogni-

tion sites region. The mRNA transcription level of CYP6CX1v1 was 2.38-fold as high as

that ofCYP6CX1v2. The results indicated that the CYP6CX1 from the B biotype B. tabaci

in Sj was one of the CYP6members, and enhancedCYP6CX1 expression and substitute of

amino acid residues might be involved in the resistance mechanisms in fieldB. tabaci.

Key words Bemisia tabaci, biotype, cytochrome P450s monooxygenases, insecticide

resistance

Introduction

Bemisia tabaci (Gennadius) is a very important cos-

mopolitan insect pest of cruciferous crops and causes

serious losses due to the direct (phloem feeding) or in-

direct damage (transmitting plant viruses and excreting

honeydew) (Gocmen & Devran, 2002). It is considered

to be a highly cryptic species complex, and more than 24

biotypes ofB. tabaci have been identified by various tech-

niques (Roditakis et al., 2005). Different B. tabaci vary

in species of host plant, spread ability, adaptability to dif-

ferent habitats, transmitting plant viruses and resistance

Correspondence: Gang Wu, Department of Plant Protection,

Fujian Agriculture and Forestry University, Fuzhou 350002, Fu-

jian, China. Tel & fax: +86 591 87646115; email: newugan@

163.com

to insecticides, depending on different biotypes. Among

the 24 biotypes, B-biotype of B. tabaci, due to its wide

spread ability, high fecundity, polyphagous nature, fast

adaptability to insecticides and being a vector of many

geminiviruses, is one of the most damaging biotypes in

numerous crops world-wide (Karunkeret al., 2008). Fur-

ther, B-, A-, Q-, Cv- and No-B-biotype have been found in

China to-date, based on the sequence comparison of mito-

chondrial cytochrome oxidase I (mt-COI) gene sequences

as markers, and B biotype was the most widespread and

damaging biotype (Liu et al., 2007; Qiu et al., 2009).

Because of the indiscriminate application of insecti-

cides, B. tabaci had developed significant resistance to

numerous insecticide classes (Prabhaker et al., 2008;

Fernandez et al., 2009). Resistance to approximately

35 active ingredients had been reported for B tabaci

in at least 20 countries world-wide (Roditakis et al.,

2005). Among the detoxification systems, it is known

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A novel P450 gene isolated from Bemisia tabaci 485

that P450s (encoded by CYP genes) plays a dominant

role in the metabolism of a wide variety of both endoge-

nous and xenobiotic substances, thus contributing numer-

ous functions, including growth, development, nutrition

and xenobiotic detoxification in insects (Karunker et al.,

2008; Bautista etal., 2007). Only monooxygenase activitycorrelates with imidacloprid, thiamethoxam, acetamiprid

and neonicotinoid resistance, among metabolizing en-

zymes such as esterases, glutathione S-transferases and

cytochrome P450-dependent monooxygenases (Rauch &

Nauen, 2003). P450 and carboxylesterase (CarE) are in-

volved in a highly resistant B. tabaci strain, while glu-

tathione S-transferase (GST) is not different between re-

sistant and susceptible B. tabaci (Roditakis et al., 2006).

The metabolisms of P450s are critical in resistance to in-

secticides in the field populations of B. tabaci (Karunker

et al., 2008, 2009; Kang et al., 2006). The total num-

ber of P450 genes, a superfamily, in insects registered

in the GenBank, is over 1 000. The P450 genes of in-

sects belong to CYP4, CYP6, CYP9, CYP12, CYP15,

CYP18, CYP28, CYP49 and CYP308 families. Among

these families, CYP4, CYP6, CYP9, CYP12, CYP18 and

CYP28 are found only in insect species (Berge et al.,

1998; Amenya et al., 2008; Guo et al., 2009). The to-

tal 256 full-length sequences of P450 genes in insects

are registered. Among them, 20% are CYP6 members

and 45% CYP4 members (Guo et al., 2009). CYP4 and

CYP6 members are thought to be involved in the resis-

tance to insecticides in insect species because of signif-

icant over-expression. A complementary DNA (cDNA)

microarray from B. tabaci was used to monitor changesin gene expression in a resistant B. tabaci population.

One hundred and eleven expressed sequence tags (ESTs)

were identified that are differentially up-regulated in the

resistant strain after pyriproxyfen treatment. Many of the

up-regulated ESTs belong to families usually associated

with resistance and xenobiotic detoxification, and some

ESTs belong to P450 families (Ghanim & Kontsedalov,

2007). Eleven distinct P450 cDNA sequences from B-

and Q-biotype B. tabaci were cloned and were classified

as members of the CYP4 or CYP6 families. In addition,

one full-length P450 gene CYP6CM1 was obtained, and

constitutive over-expression, structural model and func-tional characterization of CYP6CM1, which was asso-

ciated with imidacloprid resistance, have been reported

(Karunker et al., 2008, 2009). However, there are few

studies regarding P450 genes in B. tabaci.

To study the potentially involved metabolic resistance

by P450 gene, we investigated resistance levels and iso-

lated a novel CYP6 P450 gene putatively associated with

insecticide resistance in the resistant field, and susceptible

insectarium, populations ofB. tabaci.

Materials and methods

Sources of insects

A field population of thesubnymph ofB. tabaci wascol-

lected from commercial crucifer (Brassica oleracea var.italica L.) vegetable fields in Shangjie, Minhou, Fujian,

China (Sj), and introduced into an insectarium under field

conditions in September 2006.B. tabaci in the insectarium

was fed on cauliflower. The insectarium was constructed

with a stainless-steel net and a glass roof at the Fu-

jian Agriculture and Forestry University (FAFU), Fuzhou,

China. The insectarium excluded external B. tabaci and

was free from insecticides. The insectarium population of

B. tabaci was collected from the insectarium in September

2009, and tentatively used as a related susceptible popula-

tion (named as insectarium B. tabaci) in this study. Mean-

while, a field population of the subnymph of B. tabaci

was collected from Sj in September 2009, and was used

as the insecticide-resistant population (named as field B.

tabaci). Insecticides were not applied in the fields for at

least 1 week before the subnymphs of the field B. tabaci

were collected.The subnymphsofB. tabaci collectedfrom

the field and the insectarium were then put into large vials

in an environment chamber at 25 1C for a photoperiod

of 16 : 8 h L : D, and provided with 15% honey solu-

tion. The newly emerged adults of B. tabaci were used

for experiments. The history of the insecticide applica-

tion in Sj was the same as that described by Kang et al.

(2006). To control B. tabaci, fenvalerate and chlopyrifos

had been used in Sj for more than 30 years, and avermectinmore than 10 years. The experiments to study the optimal

reaction conditions for cloning the CYP6 gene and mes-

senger RNA (mRNA) transcription was conducted from

September 2008 by using the B. tabaci which were col-

lected from the insectarium in FAFU and the fields in

Sj. However, the B. tabaci collected from the insectarium

and the field in Sj in September and October 2009 were

used as insecticide-susceptible and -resistant populations

of B. tabaci, respectively, to compare the differences in

insecticide susceptibility andCYP6 gene between the in-

sectarium and field populations ofB. tabaci.

Insecticides

Chlorpyrifos (technical grade, 95% pure) from Jinbo

Pesticide Co., Ltd., Zhibo, Shangdong, China; fenvaler-

ate (technical grade, 96% pure) from Sumitomo Chemi-

cal Co., Ltd., Osaka, Japan; avermectin (technical grade,

95.7% pure) from North China Pharmaceutical Group

Corporation Aino Co., Ltd., Hebei, China, were used.

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486 H. M. Zhuang et al.

Table 1 Oligonucleotide primers used in this study.

ProductPrimers Sequences (5 to 3)

size (bp)

C1-J-2195 5-TTGATTTTTTGGTCATCCAGAAGT-3 844

L2-N-3014 5-TCCAATGCA CTAATCTGCCATATTA-3

AP 5-GGCCACGCGTCGACTAGTACTTTTTTTTTTTTTTTT-3

AUAP 5-GGCCACGCGTCGACTAGT-3

GDP-F 5-CGGA(A/G)AC(A/G/C/T)(A/C/T)(C/T)(A/G/C/T)(A/C)G(A/G/C/T)AA(A/G)TA(T/C)CC-3 250

GDP-R 5-CGGG(A/G/C/T)CC(A/G/C/T)(G/T)

(A/G/C/T)CC(A/G)AA(A/G/C/T)GG-3

GSP1 5-GCCGCTGGAATCATAAGACC-3 519

GSP2 5-TACTTTCCCGACCCAGAG-3 436

UPM 5-CTAATACGACTCACTATAGGGCAAGCAGTGGTATCAACGCAGAGT-3

NUP 5-AAGCAGTGGTATCAACGCAGAGT-3

GSP3 5-GGTATGTAGGACCCAGGCAC-3

GSP4 5-TTTCATTGACGACCTGCTCC-3 1211

CYP6CX1v2-F 5

-CGGGTATCCTAAAAAAATGG-3

1559CYP6CX1v2-R 5-TGGTTATCAGTCCGAGGGCTT-3

qF 5-CGAACTGGCGTATCACC-3 248

qR 5-CCGCTGGAATCATAAGACC-3

-actin -F 5-GCTGCCTCCACCTCATT-3 129

-actin-R 5-ACCGCAAGATTCCATACCC-3

Bioassays

The bioassay for adult B. tabaci was conducted by the

dry film method (Kang et al., 2006). Briefly, 2 mL ace-

tone solution of insecticide were poured into a glass vial(1.2 cm diameter, 10 cm length), and capped with a rub-

ber plug. The solution in the vial was swirled for 10 s.

Then, the excess solution was poured off, and the vial

was placed on a wire rack upside down. Control vials

were treated with acetone only in the same manner. Adult

insects were introduced into the vial and left in con-

tact with the insecticide in an environment chamber at

25 1C at a photoperiod of 16 : 8 h L : D, and pro-

vided with 15% honey. Insect mortality was recorded

12 h after their introduction. Each lethal concentration

at 50% (LC50) was calculated with five concentration lev-

els and corresponding mortalities. No mortality was ob-

served in the control during the bioassays. Adults, which

did not respond to pencil tip prodding, were judged to be

dead.

Biotype identification

Single adults of B. tabaci was used for DNA extrac-

tion according to the method of Luo et al. (2002). Mi-

tochondrial cytochrome oxidase I (mt COI) gene se-

quences were selected as a marker gene to identify the

biotype of B. tabaci of Sj in our study. The univer-

sal primers, C1-J-2195 and L2-N-3014 (Table 1), were

used for the amplification of mt-COI gene. Polymerasechain reaction (PCR) was done at a 25 L volume, and

the DNA was first denatured for 5 min at 94C; fol-

lowed by 35 cycles at 94C for 30 s, 50C for 30 s

and 72C for 60 s; and a final extension for 7 min at

72C. PCR products were electrophoresed as above, and

the sequencing was carried out for five adult individu-

als ofB. tabaci. The fragment was named Origin-FJ. The

pure sample (Origin-FJ) was sequenced by Guangzhou

Office, Shanghai Invitrogen Biotechnology Co., Ltd,

China (Shanghai Invitrogen), and analyzed by Clustalw2

(http://www.ebi.ac.uk/Tools/clustalw2/). The nucleotide

sequences of mt-COI of B. tabaci were compared with

those of Texas-B-biotype, Argentina-B-biotype, India-B-

biotype and Israel-B-biotype. The sequencing result of

mt-COI of Origin-FJ was 844 bp. Intercepting 720 bp of

Origin-FJ corresponding to the sequence of United States

Texas-B, homology to other four sequences of B-biotypes

of B. tabaci, were analyzed by the Clustalw2 (Fig. 1).

The results indicated that there was a very small differ-

ence among Origin-FJ and the other four B-biotypes ofB.

tabaci. The internal consistency was higher than 99.7%.

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Fig. 1 The comparison of mitochondrial cytochrome oxidase I sequence among Origin-FJ, Texas-B, Argentina-B, India-B and Israel-B.

Identical residues were designated by dashes. The different nucleic acids are indicated by a dark background. The consistency of the

sequence of Origin-FJ (the same pair of comparison sites divided by the total sites) was 99.7% (with Texas-B, GenBank AF164675),

100% (with Argentina-B, GenBank AF340216), 99.7% (with India-B, GenBank AF321927) and 99.7% (with Israel-B, GenBank

AF418671), respectively.

Therefore, Origin-FJ in this study was identified as B-

biotype.

RNA extraction and cDNA synthesis

RNA for subsequent P450 gene amplification and

cloning was prepared from both insectarium and field

populations ofB. tabaci; 150 to 200 adults of the field

B. tabaci adults were homogenized in 1 mL Trizol

Reagent (Invitrogen, Shanghai, China). Each RNA sam-

ple was treated with RNase-free Dnase (TaKaRa, Shang-

hai, China). Reverse transcription was then performed on

3 g of each RNA sample in a 20-L reaction using Su-

perscript III Reverse Transcriptase Kit (Invitrogen) and

Adapter primer (AP, Table 1), following the suppliers

instructions.

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PCR amplification of P450 cDNA fragments, cloning

and sequence analysis

For theamplification of P450s gene, degenerate primers

GDP-F and GDP-R (Table 1) were used. These primers

were designed based on sequences surrounding heme-binding regions of CYP6 families. Thermocycling con-

ditions consisted of an initial denaturation step at 94C

for 4 min, followed by 35 cycles (94C for 30 s, 45C

for 30 s, 72C for 30 s) and a final extension at 72C for

7 min. PCR products of the expected size were purified

from 1% (w/v) agarose/TAE gel using TaKaRa Agarose

Gel DNA Purification Kit and cloned using the pMDTM

18-T Vector (TaKaRa). Plasmids were sent to Shanghai

Invitrogen, and sequenced by automated DNA sequencer

ABI model 3700. The fragment 1 with 250 bp was ob-

tained. Based on fragment 1, we designed GSP1 (Table 1)

as reverse primer, together with Abridge Universal Am-

plification primers (AUAP, Table 1) as forward primer, to

amplify a fragment by PCR, consisting of denaturation at

94C for 5 min followed by 35 cycles of 94C for 30 s,

50C for 30 s and 72C for 2 min and final extension

at 72C for 10 min. The fragment 2 with 519 bp was

obtained.

Rapid amplification of cDNA ends (RACE)

3- and 5- RACE reactions were performed to com-

plete the cDNA sequence ofCYP6CX1 gene of the field

B. tabaci. 3-RACE was performed by using GSP2 andAUAP (Table 1), and PCRconditions were 94Cfor5min,

followed by 35 cycles of 94Cfor30s,50Cfor30s,72C

for 1 min and a final extension step of 72C for 10 min.

The fragment 3 was obtained. For 5-RACE amplifica-

tion, the first-strand cDNA was synthesized from RNA

using the SMARTTM race cDNA amplification kit (Clon-

tech, Takara, Japan). 5-RACE used Universal Primer A

Mix (UPM) and GSP3 (Table 1), and rounds of PCR (35

cycles) consisted of denaturation at 94C for 5 min fol-

lowed by 35 cycles of 94C for 30 s, 61C for 30 s and

72C for 2 min and a final extension at 72C for 10 min.

The PCR product was used for re-amplification usingNested Universal Primer (NUP) and GSP4 (Table 1), and

rounds of PCR (35 cycles) consisting of denaturation at

94C for 5 min followed by 35 cycles of 94C for 30 s,

62C for 30 s and 72C for 2 min with a final extension at

72C for 10 min. The fragment 4 was obtained. The initial

CYP6CX1 cDNA fragment (fragments 1 and 2) and cDNA

ends obtained by 3-RACEand 5-RACE (fragments 3 and

4) were edited and assembled for full-length cDNA of the

field B. tabaci (named CYP6CX1v1). The clones of the

initial CYP6CX1 cDNA fragment andthe 3-and5-RACE

(fragments 3 and 4) in the insectarium population of B.

tabaci were carried out in the same way as in the field

B. tabaci. The primers for cloning the internal fragments

in the insectarium B. tabaci were designed based on the

sequence of CYP6CX1v1. The primers used in 3

- and5-RACE in the insectarium B. tabaci were the same as

those in the fieldB. tabaci. The full length of CYP6CX1

in the insectarium B. tabaci (named CYP6CX1v2) was

edited and assembled. The full-length nucleotide se-

quence of the open reading frame (ORF) in the insec-

tarium B. tabaci was confirmed by PCR amplification

using primers CYP6CX1v2-F (at positions 4968) and

CYP6CX1v2-R (reverse complementary to nucleotides

at 1 6231 643) (Table 1). The RT-PCR products were

purified directly from bands excised from agarose gels

and cloned into pMDTM 18-T Vector (TaKaRa). Positive

clones were sent to Shanghai Invitrogen to be sequenced.

Software including DNAMAN (http://www.lynnon.com/)

and ClustalW2 were used to analyze the gene

sequences.

Real-time quantitative PCR (qPCR) in adult B. tabaci

Quantitative PCR was conducted using an MiniOpticon

System for real-time PCR Detection (Bio-Rad, Hercules,

CA, USA) with the SYBR Premix Ex Taq (TaKaRa) kit.

Reaction mixtures (final volume 20 L) contained 2

SYBR Premix Ex Taq, 200 nmol/L of each primer, 2 L

cDNA and 7.2 L Rnase-free water. PCR conditionswere 95C for 10 s, followed by 40 cycles of 95C for

6 s and 55C for 25 s. Fluorescence was measured after

each cycle. Relative mRNA expression of CYP6CX1v1

and CYP6CX1v2 was measured in reference to the -

actin gene. Based on the sequence of CYP6CX1v1 and

CYP6CX1v2, the same region of nuclotide acid sequence

was selectedto design the primers qF and qR, for qPCR. In

addition, the primers, -actin-F and-actin-R, were used

for amplifying the-actin gene (Table 1). Standard curves

of target gene and reference gene were made, using trip-

licate serial dilutions with six different cDNA concentra-

tions covering a 3 125-fold concentration range. The ho-mogeneity of the PCR products was confirmed by melting

curve analysis. The mean normalized expression value of

P450 gene was calculated by comparing the threshold cy-

cle (Ct)of the gene tothat of-actin gene according to the

equations of standard curves of target gene and reference

gene (-actin gene), respectively (Larionov et al., 2005).

In brief, the equation of standard curve of -actin gene

wasy=0.295x+ 6.78 (r2 = 0.999), and the equation of

standard curveofCYP6CX1 gene wasy=0.322x+8.89

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Table 2 Comparisons on susceptibility to insecticides in the field and insectarium populations of Bemisia tabaci.

Insectarium population Field population

Insecticides LC50 (95% CI)SlopeSE

LC50 (95% CI)SlopeSE

Resistance

(mg/L) (12 h) (mg/L) (12 h)ratio

Fenvarelate 88.0 (75.4103) 2.78 0.22 3 940 (3 4774 464) 3.17 0.21 44.8

Chlorpyrifos 43.9 (24.778.9) 4.18 0.96 1 019 (8991 155) 2.84 0.18 23.2

Avermectin 0.16 (0.140.18) 2.23 0.14 5.76 (3.718.93) 5.08 1.09 36.0

LC50, lethal concentration at 50%.

(r2 = 0.995); X = Ct value. The relative expression was

CYP6CX1 = 100.322Ct1+8.89/100.295Ct2+6.78, where

Ct1= threshold cycle of target gene and Ct2 = threshold

cycle of reference gene. Each reaction was performed in

triplicate to minimize variation within experiments, and

the mean of at least three independent biological replicateswas calculated.

Statistical analysis

The bioassay data were analyzed for x2, LC50 values,

and their 95% confidence intervals (95% CI) by probit

analysis using a data processing system (Tang & Feng,

1997). t-tests were also calculated using the data process-

ing system (Tang & Feng, 1997).

Results

Determination of resistance levels to insecticides

in B. tabaci

Significant resistances to fenvalerate, chlorpyrifos and

avermectin were found in the field population ofB. tabaci,

as compared to those in the insectarium population of B.

tabaci. The resistance ratios were 44.8 for fenvalerate,

23.2 for chlopyrifos and 36 for avermectin, respectively.

The resistances to fenvalerate and avermectin were high

(Table 2).

Cloning and sequencing analyses ofCYP6CX1

Based on sequences of the fragment 1 (250 bp), frag-

ment 2 (519 bp), fragment 3 (436 bp) and fragment

4 (1 211 bp), a novel complete sequence P450 cDNA

(CYP6CX1v1) with 1 940 bp in the field B. tabaci

was obtained by overlaying the cloned sequences. The

CYP6CX1v1 contained a 1 557 bp ORF encoding 518

amino acid residues. The predicted isoelectric point of

the cDNA-deduced protein was 8.56 and the molecu-

lar weight (MW) was 58 784 Da, within the range (46

60 kDa) of other reported cytochrome P450s (Nelson

et al., 1993). The deduced amino acid sequence con-

tains important conserved domains common to CYP6

members, such as the oxygen- binding motif AGXXPX(i.e., AGLDPV at position 322327), the heme-binding

decapeptide PFXXGXXXCXA (i.e., PFGEGPRFCIA at

position 454464) and the putative meander-binding se-

quences EXXR and PXRF (ETLR at 380383 and PXRF

at 436439) in helix-K. In addition, a conserved domain

(FPDP, at 428431) common to another CYP6 member

(Y/FPD/EP) and a PXXFXP near the carboxyl end (i.e.,

PEKFNP at position 431436) were found (Figs. 2 and 3)

(Nelson et al., 1993; Karunker et al., 2008). A BLAST

(www.ncbi.ie) search indicated that CYP6CX1 amino acid

sequence was identical to CYP6 families that shared the

highest homology. A phylogenetic tree of B. tabaci (Bt)

P450s deduced amino acid sequences and selected P450sfrom another eight insect species is shown in Fig. 4. The

protein encoded by CYP6CX1 had the highest amino

acid identity with CYP6CM1vB from B. tabaci (Gen-

Bank EU642555, 43%) andCYP6CM1vQ from B. tabaci

(GenBank EU344879, 43%). The identity of CYP6CX1

to the CYP6 subfamilies from other insect species, in-

cluding Tribolium castaneum (Ta), Anopheles funestus

(Af), Culex quinquefasciatus (Cq), Nilaparvata lugens

(Nl), Blattella germanica (Bg), Anopheles gambiae (Ag),

Anopheles minimus (Am) andLygus lineolaris (Ll), were

from 32% to 34%. Therefore, CYP6CX1v1 was a typical

cytochrome P450 gene. The phylogenetic analysis placedCYP6XC1v1 and CYP6XC1v2 in the CYP6 family and

CYP6Csubfamily.

Comparison ofCYP6CX1 between R and S B. tabaci

To analyze the differences between CYP6CX1v1 from

the fieldB. tabaci andCYP6CX1v2 from the insectarium

B. tabaci, alignment ofCYP6CX1v1 andCYP6CX1v2 nu-

cleotide sequences (Fig. 2) and their putative CYP amino

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Fig. 3 Alignment of deduced amino acid sequences ofCYP6CX1v1 andCYP6CX1v2. Identical residues are designated by dashes. The

four amino acid substitutions at positions 300, 354, 486 and 503 are indicated by a dark background. Conserved domains common to

CYP6 members are boxed, such as the heme-binding decapeptide PFXXGXXXCXA (position 454464), the oxygen-binding domain

AGXXPX (position 322327), a conserved amino acid sequence PXXFXP (position 431436), a putative meander-binding sequence

EXXR (position 380383) and PXRF (position 436439) in helix-K.

Discussion

Polymerase chain reaction-based cloning strategies with

degenerate primers make it possible to rapidly amplify

members of CYP superfamilies in many insects. In this

study, we used a pair of degenerate primers (Table 1)

based on sequences surrounding heme-binding regions of

CYP6 to amplify a short P450s fragment, then accord-

ing to the short fragment, we designed four gene-specific

primers used for another fragment and 3- and 5-RACE

reactions. Just as described in the Results section, six con-

served domains, which were the conservative sequences

of CYP6 family (Nelson et al., 1993; Karunker et al.,2008), existed in CYP6CX1. Because there were no differ-

ences in the base sequences corresponding to those of the

primers used for 3- and 5-RACE between CYP6CX1v1

and CYP6CX1v2, the 3- and 5-RACE in both S and R

B. tabaci could be carried out successfully by using the

same primers.

In accordance with the P450 nomenclature system

(Nebert et al., 1991), the subsequent Arabic numeral after

CYP indicated the different gene families of CYP super-

families. The subsequent capital letter after the Arabic

numeral indicated different gene subfamilies. The amino

acid sequence with identity higher than 40% belongs to

the same gene family, such as CYP6. CYP6CX1v1 and

CYP6CX1v2 obtained in this study showed higher than

40% identity to the CYP6 genes of other insect species,

and belonged to CYP6. In our previous results, a fragment

(1 002 bp from the field B. tabaci) were submitted and

named as CYP6CX1v1 by GenBank Submissions Staff

(GenBank GQ292715). CYP6CX1v1 had 49% identity to

CYP6CM1 (GenBank EU642555) fromB. tabaci reported

by Karunkeret al. (2008). Based on the fragment (Gen-Bank GQ292715), the full-length CYP6 genes were iso-

lated successfully in both field and insectarium B. tabaci

in this study, and were then named as CYP6CX1v1 and

CYP6CX1v2, respectively. CYP6CX1v1 andCYP6CX1v2

showed the highest identity to CYP6CM1 (43%), and

could be identified as a new CYP6family member.

CYP6A2 containing three point mutations were consti-

tutively over-expressed and involved in DDT metabolism

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Fig. 4 Phylogenetic tree of P450 deduced amino acid sequences of Bemisia tabaci (Bt) and selected P450 from other insect species.

Tribolium castaneum (Ta), Anopheles funestus (Af), Culex quinquefasciatus (Cq), Nilaparvata lugens (Nl), Blattella germanica (Bg),

Anopheles gambiae (Ag),Anopheles minimus (Am),Lygus lineolaris (Ll). The identify ofCYP6CX1 to the otherCYP6subfamilies from

other insect species and the GenBank accession number are shown in parenthesis after the CYP name: 6BK14-Ta (33%, EFA05731),

6BK17-Ta (33%, ABX64450), 6BK4-Ta (33%, NP_001123875), 6BK12-Ta (32%, EFA12529), 6BQ5-Ta (33%, EFA02819), 6BQ7-Ta

(32%, EFA02821), 6BQ13-Ta (32%, EEZ99338), 6P9-Af (32%, ABC87786), 6P13-Af (33%, ABO77953), 6P3-Ag (33%, AF487534),

6P7-Am (34%,AAR88141), 6BB1v2-Cq(32%, XP_001847401), 6a8-Cq (34%,XP_001870174),6X1v2-Ll (33%,AAL15174), 6X1v3-

Ll (33%, AAM94461), 6X1v1-Ll (33%, AAL15173), 6cm1-Bt (43%, ACS92724), 6CM1vB-Bt (43%, ACD84797), 6CM1vQ-Bt (43%,

ACA51846), 6AX1-Nl (34%, CAH65681), 6J1-Bg (33%, AF281325).

0.0

0.5

1.0

1.5

2.0

2.5

3.0

Relativefoldchange

Insectarium Fie ld

Fig. 5 Messenger RNA (mRNA) expression ofCYP6CX1 gene

in the insectarium (white) and field (black) adults of Bemisa

tabaci. Relative quantities indicate that the levels of transcripts

are normalized to the internal standard (-actin gene). The

R mRNA expression in B. tabaci was expressed relative to

that in insectarium B. tabaci, set at 1. Each bar represents the

mean SD of three independent experiments.

of D. melanogaster. The location of the mutations in a

model of the 3D structure of the CYP6A2 protein sug-

gested that some of them may be important for enzyme

activity of this molecule. This has been verified by het-

erologous expression (Berge et al., 1998). Two amino

acid residue mutation of CYP gene, coupled with up-

regulation of mRNA expression (2.1-fold higher mRNA

expression in pyrethroid-resistant strains), was possibly

related to resistance development in the tarnished plant

bug (Zhu & Snodgrass, 2003). There were six putative

substrate recognition sites (SRS16) in CYP6CM1 in B.

tabaci (Karunkeret al., 2009). A total of four amino acid

residue replacements were found in CYP6CX1v1. The

amino acid replacements ofCYP might be involved in in-

secticide resistance or result from polymorphism amongthe individuals. Because the substitution Ile503Thr was

located in the SRS6 region (DRETFTLNP), the muta-

tion might be important in the function of CYP6CX1v1.

The other three amino acid replacements might re-

sult from polymorphism. Nevertheless, the functions

of the four amino acid replacements should be ver-

ified by heterologous expression and metabolisms

in vitro.

C 2010 The Authors



10/11


Besides amino acid replacement, P450-mediated resis-

tance to insects might be associated with another two

mechanisms, P450 over-expression or the combined ef-

fects of aminoacid replacement and P450 over-expression

(Hemingway et al., 2004). The major mechanism in all

samples investigated appeared to be enhanced detoxifica-tion by cytochrome P450 monooxygenases, based on the

mRNA transcription of 11 distinct P450 cDNA sequences

from B- and Q-biotype B. tabaci (Karunkeret al., 2008).

In this study, significantly higher expression ofCYP6CX1

might be involved in the resistance to fenvalerate, chlor-

pyrifos and avermectin in the field B. tabaci collected

from Sj. However, the evidence to correlate the relation-

ship between the transcription level of mRNA and re-

sistance mechanisms was weak because only 2.38-fold

difference in CYP6CX1 transcription level between the

insectarium and the fieldB. tabaci was found. However,

it is known that P450-mediated oxidative degradation is

the major mechanism of insecticide resistance in the field

population ofB. tabaci in Sj, based on the synergistic ef-

fects on the susceptibility of insecticides, including chlor-

pyrifos, fenvalerate, avermectin and imidaclorpid, by us-

ing enzyme inhibitors (Kang et al., 2006). Although the

B. tabaci strains in this study were different from those

used in Kang et al. (2006), the two field populations ofB.

tabaci used in this study and by Kang et al. (2006) were

collected from Sj, and the collection times for the two

insect populations were close (i.e., 2008 and 2006, respec-

tively). Fenvalerate and chlopyrifos has been used in Sj for

more than 30 years to control B. tabaci, and avermectin

for more than 10 years. Because P450 gene was verifiedto be involved in the resistance to fenvalerate, chlopy-

rifos and avermectin in the Sj population of B. tabaci

(Kang et al., 2006), and the insecticides used to control B.

tabaci were similar in Sj during 2006 to 2009 according

to our field investigation, the resistance mechanism to the

three insecticides might be similar in the field B. tabaci

used in this study and in Kang et al. (2006). Neverthe-

less, it was known that cytochrome P450s played a crucial

role in insecticide resistance of insects through metabolic

detoxification, andCYP6family P450 was widely known

to be involved in metabolic resistance to pyrethrins and

organophosphates in some other insect species (Nikouet al., 2003; Bautista et al., 2007; Amenya et al., 2008;

Zhou et al., 2010). It has been speculated that CYP6CX1

might be involved in resistances to the three insecticides

in the fieldB. tabaci in Sj.

In this study, a novel P450 gene putatively associated

with insecticide resistance was identified from B. tabaci.

Because the fieldB. tabaci showed significant resistance

to three different classes of insecticides (i.e., fenvarerate,

avermectin and chlorpyrifos), besides the P450 gene, such

as CYP6CX1v1, other detoxification enzymes and spe-

cific insensitive targets might be involved in the resistance

in B. tabaci depending on the different insecticides. The

direct relationships between the mutation or expression

and the function ofCYP6CX1 in insecticide resistance in

B. tabaci should be conducted in our further research.The susceptible and the resistant populations in this

study were collected from Sj in 2006 and 2009, respec-

tively and therefore, did not share a common genetic

background, although the insectarium and the field pop-

ulations of B. tabaci originated from Sj. Strains with the

same genetic background are needed for studying resis-

tance mechanisms. In addition, insectarium B. tabaci was

not really a susceptible strain (susceptible homozygote),

but a related susceptible strain with some susceptible and

resistant heterozygote; back-cross experiments should be

conducted in the future to obtain a susceptible and resis-

tant homozygote with the same genetic background.

Acknowledgments

We thank C. W. Li, graduate student at FAFU, for his

help in collecting the insects from the field and rearing

them. This work was financially supported bythe National

Natural Science Foundation of China (30771413), the

National Natural Science Foundation of Fujian Province

(009J01071) and the Key Program of Education Ministry,

China (207055, JA06010).

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Accepted June 20, 2010

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Identification and Characterization Using Cytochrome p450

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