Molecular Strategies for Molecular Strategies for detection of insertion of detection of insertion of genes in transgenic plants genes in transgenic plants

Dr Anil KumarAssociate Professor & In chargeDept. Of Molecular Biology &Genetic Engineering, CBSHGBPUAT Pantnagar UttranchalPin-263245

Genes and Genes and ProteinsProteins

Gene(a piece of DNA)

traitprotein

translationtranslation

mRNA

transcriptiontranscription

Any agriculturally important trait can be introduced by incorporation of a useful gene

Transgenic Technology : Art and Transgenic Technology : Art and science of creating designer cropsscience of creating designer crops

• Identification of useful gene (s)• Creation of a suitable gene construct• Transfer of this construct in to plant cells/tissues in

vitro ( maintained as organized explants such as immature embryos, stem sections, cotyledons etc. Using tissue culture techniques, through a process called transformation.

• Selection of transformed cell lines or seedling using a suitable marker system and regeneration of fertile plants from the transformed cells.

• Analysis of transformed plants for several aspects including stable integration, expression and genetic behavior of transgene(s)

DESIGNER CROPS OF SUPERIOR TRAITS• Agronomic Traits

– Biotic Stress• Insect Resistance• Disease Resistance

– Viral, Bacterial, Fungal, Nematode• Weed, herbicide tolerance, parasitic weeds

– Abiotic Stress• Drought, Cold, Heat, Salinity, Poor soils

• Quality Traits– Yield- Nitrogen Assimilation, Starch Biosynthesis, O2 Assimilation – Processing– Shelf-life– Reproduction: sex barriers, male sterility ,seedless ness– Nutrients (Nutraceuticals)

• Macro: Protein, Carbohydrates, Fats, Fiber• Micro: Vitamins, Minerals, Antioxidatnts, Isoflavonoids,

Phytoestrogens, Condensed tannins• Anti-nutrients: Phytase, Allergen and Toxin removal

– Taste– Fiber, quality, strength, natural colors– Architecture– Ornamentals: color, shelf-life, morphology, fragrance

• Novel Crop Products– Oils – Proteins: nutraceuticals, therapeutics, vaccines– Polymers

• Renewable Resources, Biofuels, feedstocks for synthetics

How are Transgenic crops made ?

Locating Genes for Plant TraitsIdentifying & locating genes for agriculturally important traits: Most limiting step in transgenic process

Little is Known about specific genesto enhance yield potentialimprove stress tolerancemodify chemical properties of harvested productsotherwise affect plant characters

Identifying a single gene for a trait is not sufficientMust understand how the gene is regulatedWhat other effects it might have on the plantHow it interacts with other genes active in the same biochemical pathway

Designing Genes for Insertion

Once a gene has been isolated & cloned : it must be suitablymodified for effective insertion into a plant

A promoter sequence must be added for the gene to be correctly expressed

Sometimes, the cloned gene is modified to achieve greater expression in a plant

The termination sequence signals to the cellular machinery that the end of the gene sequence has been reached

A selectable marker gene is added to the gene "construct" in order to identify plant cells or tissues that have successfully integrated the transgene.

Simplified representation of a constructed transgene, containingnecessary components for successful integration and expression

Promoters used for Transgene expression

It is the ON/OFF switch : controls when & where in the plant the gene will be expressed

Most promoters used are CONSTITUTIVE : cause gene expression throughout the life cycle of plant in most tissues.

CaMV 35S Promoter : Most commonly used & gives high degree of expression in plants

Other promoters : More Specific : respond to cues in the plant’s internal & external environment

Promoter from cab gene (encoding chlorophyll a/b binding proteins) : Light – inducible

Promoters from Arabidopsis : Specifically & rapidly induced by natural plant stress/wounding related semiochemical cis-jasmone

How are Transgenic How are Transgenic Plants Produced?Plants Produced?

•• AgrobacteriumAgrobacteriumtumefacienstumefaciens

•• Gene Gun / Gene Gun / BiolisticsBiolistics•• ElectroporationElectroporation

Commonly Used Methods:Commonly Used Methods:

Protoplast Immatureembryo (IE)

Embryo derivedcalli (EDC)

Electroporation/PEG mediatedtransformation

Biolistictransformation

Agrobacteriummediated

transformationSelection in presence ofSelectable marker gene

Plant regeneration

Molecular characterizationevent selection/gene expression/function of transgene

Development of transgenic plants

Transformation System

Transgenic Plants- From Lab to the field

Transgenic lines obtained throughTransformation and regeneration

Laboratory analysis to confirmStable integration of transgene(s)Number of copies of the transgene(s)Expression of transgene(s)

Monitoring and analysis of transgenics in aContainment (Glasshouse) Facility for

Stability of transgene expressionAgronomically desirable expression of transgenic traitGenetic behavior of transgene(s)Biosafety evaluation and risk assessment

Small plot (<500m2) field experimentsTo evaluate agronomic performanceAnd to further analyze biosafety

Large scale field testing at multiple sites

Commercilization of transgenic varietyAfter risk assessment and evaluation ofNet benefit offered by the transgenic

Monitoring of transgenic variety during commercialization

PRODUCTS GENE INTROGR-ESSED

GENETICALLY ALTERED TRAITS

WHEAT

BRASSICA

BRASSICA

TOMATOCARROT

PAT

OSMOTIN

ANNEXIN

Pr M EPr M EGUS

HERBICIDE RESISTANCE (BASTA)

TOLERANCE TO ALTERNARIA BLIGHT AND SALINITY

MODULATION OF HYPERSENSITIVE RESPONSE AGAINST ALTERNARIA BLIGHT AND POWDERY MILDEW

PRODUCTION OF EDIBLE VACCINE AGAINST JAPANESE ENCEPHALITIS VIRUS

TRANSGENIC CROPS DEVELOPED SO FAR AT PANTNAGAR

Core Characterization•Gene(s)

– Source(s)– Molecular characterization– Insert / copy number / gene

integrity

•Protein(s)– History of safe use and

consumption– Function / specificity /

mode-of-action– Levels– Toxicology / allergenicity

testing

•Food/Feed Composition

– Proximate analysis– Key nutrients– Key anti-nutrients– Animal performance

assessment

•Environmental– Host organism– Safety to non-target

organisms– Soil degradation, toxicity– Outcrossing, weediness

METHODS TO CHECK GENE INTEGRATIONGene that facilitates the detection of genetically modified plants

during development

Use of selectable marker genes--Antibiotic resistance markers-Herbicide resistance markers-Non-antibiotic based markers

These genes coming up

Use of a reporter gene-gene whose expression is easy to observe. Used to “report” gene expression, regulation and localization

•Luciferase (luc) gene•Green fluorescent protein (GFP•GUS (uidA) gene

Antibiotic/Herbicideresistance Marker

Substrate for selection

npt II (Neomycin Phosphotransferase)

G418, Kanamycin, Neomycin

hpt (Hygromycin Phosphotransferase)

Hygromycin

dhfr (Dihydrofolate reductase) Methotrexate, Trimethoprim

ble Bleomycin

gat (Gentamycin Acetyltransferase) Gentamycin

bar & pat (Phosphinothricinacetyltransferase)

Phosphenothricin(Bialaphos, Basta)

epsps (5-enolpyruvylshikhimate-3-phosphate synthase)

Glyphosate

Risks & Concerns of using Antibiotic/HerbicideResistance Genes

Horizontal Gene TransferTransfer of these genes from the transgenic plants to the microbes

Make the bacteria in the guts of animals and humans resistant to antibiotics

Make antibiotic medicines less effective

Transfer to soil microbes : A field study shows that transgenic DNA persists for 2 years after the GM crop has been harvested

Other genes used as Selectable Marker Genes

man A gene from E coliencodes mannose phosphate transferase and confer

upon transformed cells ability to use mannose as a sole carbon source

Ipt (isopentenyl transferase) gene from Agrobacteriumlocated on T-DNA and induces cytokinin synthesis. Plants

selected on the basis of their ability to produce shoots from callus on medium lacking cytokinins.

Betaine aldehyde dehydrogenase gene (BADH) from SpinachUsed with chloroplast genome.Converts toxic betaine

aldehyde to non-toxic glycine betaine, which also serves as an osmoprotectant and confer drought/salt tolerance

GUS (uidA) gene

X-Gluc Blue precipitate5-bromo-4-chloro-3-inolyl-β-D-glucuronic acid

β-glucuronidase

gus expression in Roots

Green fluorescent protein (GFP)

Protein identified form luminescent jellyfish Aequorea victoria.GFP has now been produced in a number of heterologouscell types and there appears to be little requirement for specific additionalfactors for post-translational modification of the protein, which may beautocatalytic or require ubiquitous factors. Many structural variants nowavailable commercially (e.g. red fluorescent protein)

A. thaliana C24 wild type (left)35S-mgfp4-ER transformed

(right)Aequorea victoria

Luciferase (luc) gene

The glow is widely used as an assay for luc expression, which acts as a "reporter" for the activity of any regulatory elements that control its expression. Luciferase is particularly useful as a reporter- low-light cameras can detect bioluminescence, in real time and with high sensitivity, in living cells and organisms.

Crystal structure of luciferasePlants (Arabidopsis)

Significance of Molecular Significance of Molecular characterizationcharacterization

• Need for rigorous molecular characterization of each transgenic plant submitted for review for predicting the safety of a novel food.

• Molecular characterization of transgenic plants provide attention to regulators about the information in terms of food, feed or environmental safety consideration.

Three Aspects to ConsiderThree Aspects to Consider1. The transformation system– Agrobacterium-mediated– Protoplast system– Microparticle bombardment

2. Molecular characterization of the inserted DNA– Insert number– Insert composition

3. Genetic stability of the introduced trait– Segregation analysis– Integron stability

WHY IT IS NEEDED?

Introgression of Introgression of TransgeneTransgene to to Product DevelopmentProduct Development

Utility of Molecular CharacterizationUtility of Molecular Characterization

• May be able to address issues related to positional effects, pleiotropic effects, and gene silencing

• Provides information on the composition and integrity of the inserted DNA

• Ensures that the developer has appropriately characterized the genetic modification

Transcription

Splicing

Translation

NUCLEUS

CYTOSOL

USE OF PCR TO CHECK GENE INTEGRATIONUSE OF PCR TO CHECK GENE INTEGRATION

Different stages of hardening of annexin transformed Brassica plants.(a) Rooted PCR positive shoots transferred in plastic pots covered with

polybags kept for hardening at transgenic glass house.(b) A PCR positive plant growing in the pot.(c) A hardened PCR positive plant growing in plantation pot after 2 months.(d) Hardened PCR positive plants growing at transgenic glass house.

Annexin PCR of T1 progeny of T0 Brassica plantLane M = 100 bp ladderLanes 1-10 = T1 progeny plants of T0 plant

1.5 kb1.0 kb

M 1 2 3 4 5 M 6 7 8 9 10

THIS EXPERIMENT HAS REVEALED THAT A TRANSFORMANT PLANT HAS MOSAICS OF GENE WHEN GROWN IN VITRO FROM CALLUS. • THE GENE MOSAICS COULD OCCUR AT BOTH ORGAN AND TISSUE OR CELLULAR LEVEL.

INHERITANCE OF ANNEXIN TRANSGENE IN TINHERITANCE OF ANNEXIN TRANSGENE IN T11 PLANTSPLANTS

Edible vaccine against Japanese Encephalitis virusEdible vaccine against Japanese Encephalitis virus

Callusing Shoot initiation

Rooting in solid medium

Rooting in liquid medium

Different stages of regeneration in tomato

Presence of Transgene in tomato by PCR amplification

Transformed plant kept for hardening

Transgene silencing

Reduced/abolished expression of foreign gene

Loss of expression : Not due to loss of transgene but due to their inactivation

Concept of gene Space : Genomes are made of isochores (long stretches of DNA with high compositional homogeneity)

If a GC rich transgene is integrated into a GC isochoreor an AT rich transgene is integrated into an AT isochore : It is Transcribed

If a GC rich transgene is integrated into the AT rich gene space or vice versa : It is Inactivated, as there is no compositional homogeneity with the neighboringsequences

Position Dependent & Sequence Dependent Gene silencing

Transgene integrates into a genomic region containing heterochromatin.

The repressive chromatin structure & DNA methylation can spread into the transgenic locus from the flanking genomic DNA

Homology Dependent Gene Silencing

Caused by Multiple copies of transgene (Repeat induced gene silencing)

Affect not only the stability of transgene but also alter the activity of endogenous gene (Cosuppression)

Transcriptional Gene Silencing

No mRNA is produced from silenced gene

Affected loci : nucleation points for heterochromatin formation & DNA methylation

Post – Transcriptional Gene Silencing

Transcription is required for silencing to take place

Induce degradation of mRNA : very little accumulate in cytoplasm

Transgene ExpressionTransgene Expression

• So expression of transgenes has been attributed to:– copy number - the number of transgene

copies integrated into the host genome– “positional effects” - the position of the T-DNA

integration site in the host genome may affect the level of expression

– variable arrangements of transgene sequences in the host genome e.g. multiple copies in direct or inverted repeats

Detection of Insert NumberDetection of Insert Number

• Refers to the number of sites where the transgenic element is incorporated into the host genome.

• This is deduced by digesting genomic DNA with a restriction enzyme that does not cut within the transgenic element followed by Southern blot analysis with a probe specific to one or more of the introduced genes.

• More than one band = more than one insertion site.

• This should be repeated with at least one other restriction enzyme to confirm the number of inserts.

gggccat

ttaaggattcc-32p

aattcc32p-taagg

cccggta

32pt

Insert NumberInsert Number

Gene A Gene B

BamHI

Inserted Element

Genomic DNA BamHI digest

32P-labelled cDNA probe

Southern blot

Should yield a single band if one insert

3.5 kb

11 49 50 52 53 54 56 57 59 62 64 90 94 95 96 97 98 99 100102 NT PC

3.5 kb

109 135 136 146 147 150 151 152 154 155 156 157 NT PC

Southern analysis showing the integration of crtI gene cassette in selected PCR+ primary transgenics of BR29 (Golden indica rice) developed by Agro-transformation.Enzyme = Eco RI, Probe = PCR generated 1.03 kb crtI fragment, Positive control Eco RI released 3.5 kb fragment of pCaCar; lines showing single insertion are marked by red rectangles (Datta et al., unpublished data).

Fig. 1A

Fig. 1B

Example: Insert NumberExample: Insert Number

Detection Of Detection Of TransgeneTransgene Copy Copy NumberNumber

• This is not the same as the number of insertion sites

• Digestion with one or more (i.e. can be single or double digests) restriction enzymes that either do not cut within the transgenic element, or cut only once but not within the sequence complementary to the hybridization probe

• This should yield one band per inserted element. • Usually this is done with more than one

restriction enzyme, or combination of enzymes.

Transgene Copy NumberTransgene Copy Number

Gene A Gene B

EcoRI

Inserted Element

32P-labelled cDNA probe

GenomicDNA

EcoRI digest Southern blotA

A

B

BXhoI

XhoI digest Southern blot

A single band from each digestion indicates a single copy

Transgene RearrangementsTransgene Rearrangements

• Rearrangements of transgenic sequences may be observed in Southern blot analyses as hybridizing fragments of a different size than the full-length DNA insert.

• Larger fragments are indicative of concatenation (head to head or head to tail). Concatemers may be deduced by digesting genomic DNA with a restriction enzyme that cuts at a single site within the transgenic element; multiple copies of the DNA insert will then be resolved by Southern blot analysis.

• Larger than full length fragments of transgenic DNA may also be caused by interspersion of inserted DNA with host DNA (oats, rice).

• Smaller than full-length fragments are evidence of deletions and truncations

Gene IntegrityGene Integrity

• The goal is to determine if the gene(s) of interest are intact, or whether there have been truncations/deletions

• Digest genomic DNA with restriction enzymes to isolate the gene of interest

• Hybridize with a gene-specific probe.• Resolved band should be the same size as that

isolated from the plasmid.• Alternatively, PCR with 5’- and 3’-terminal

specific primers in order to amplify a fragment of the same size as the inserted gene

Gene Integrity: MON 810Gene Integrity: MON 810•Southern blot analysis of plasmid DNA (lane 1), control genomic DNA (lane 2), and transgenic maize DNA (lane 3) digested with NcoIand EcoRI and hybridized with a 32P-labelled probe specific to the cry1Ab gene.

nptII

ori-pUC

cryIA(b)

BamHI 5336

MON810 Example: Plasmid MON810 Example: Plasmid BackboneBackbone

Southern blot analysis of plasmid DNA (lane 1), control genomic DNA (lane 2), and transgenic maize DNA (lane 3) digested with NcoI and EcoRIand hybridized with 32P-labelled probes specific for the nptII region (lanes 1-3) or the ori-pUC region (lanes 4-6).

nptII

ori-pUC

cryIA(b)

BamHI 5336

Genetic StabilityGenetic Stability• For each novel trait, the pattern and stability of inheritance

must be demonstrated as well as the level of expression of the trait.

• Serological techniques are generally used to measure trait expression (gain of function) ( either qualitatively [e.g., Western immunoblotting, enzyme linked immunosorbent assay (ELISA), etc.] or quantitatively (e.g., ELISA, radioimmunoassay, etc.).

• If the new trait is one that does not result in the expression of a new or modified protein (e.g., transgenic plants containing inserted antisense sequences i.e loss of function) then its inheritance will have to be determined by examining the DNA insert directly or by measuring RNA transcript production.

Transformation Vector Map (Partial), Transformation Vector Map (Partial), Southern and Enzyme AssaySouthern and Enzyme Assay

Fig 2 represents partial map of transformation vector containing a bacterial transgene (hph) encodes hygromycinphosphotransferaseunder the control of CaMV35S constitutive promoter.

Fig 3 represents a Southern blot analysis showing integration of transgene (expected size) in To and T1 offspring plants.

Fig 4 represents enzyme assay for HPH of the same To and T1 plants shown in the Southern blot confirming inheritance and functioning of the transgene.

Hazard Identification RequiresHazard Identification Requires

• Knowledge of which genes are expressed• Characteristics, concentration and

localization of expressed products• Consequences of expression – effects on

metabolite pools• And, in cases where the modification results

in the production of antisense mRNA, the consequences of altering the expression of an endogenous gene must be evaluated

Characterize Expressed ProteinCharacterize Expressed Protein• Identity

– Immunological cross-reactivity (Western blotting, ELISA)

– Physiochemical properties (size, stability to heat and proteolytic digestion)

• Functionality– Enzymatic activity in the case of introduced

enzymes [e.g. phosphinothricin acetyltransferase(PAT)]

– Biological activity• Where appropriate, similarity to products from

traditional sources

WESTERN BLOTTING TO CHECK GENE EXPRESSION WESTERN BLOTTING TO CHECK GENE EXPRESSION

Nitrocellulose membrane

Gel-ve electrode

+ve electrode

Control Transgenic

Control transgenic

DOT BLOTTING TO CHECK GENE EXPRESSION INDOT BLOTTING TO CHECK GENE EXPRESSION INTRANSGENIC PLANTS TRANSGENIC PLANTS

Patterns of ExpressionPatterns of Expression• How, when, and where• Based on the promoter used (constitutive,

tissue-specific, inducible, etc), is the novel protein expressed in the expected tissues and under the expected conditions?

• Presence or absence and amounts of expressed protein should be determined for a range of plant tissues (e.g. roots, leaves, seeds, pollen)

• And, from a food safety perspective, levels of expression of the protein(s) in the edible portions of the plant are critical

Microarrays for detecting gene and protein expression

TransgenicNormal

Microarrays for detecting gene and protein expression