MULTIGENE ENGINEERING IN

PLANTSLenina.nk

Multigene engineering in plants

Majority of agronomic traits are quantitative and are controlled polygenetically.Multigene transfer enables – 1) manipulate entire metabolic pathways 2) express multimeric proteins 3) study complex genetic control circuits Methods for multigene engineering Nuclear genome transformation chloroplast genome transformation

Methods for nuclear gene transformation

A. Crossing Transgenic Lines

Homozygous transgenic parents are used.

Eg:Reconstruction of bacterial enzymatic pathway of organic mercury detoxification.

S.P. Bizily et al ,.( 2000)

B.Sequential Transformation

Is the repetitive insertion of transgenes into a plant.

E.g. The restoration of fertility in transgenic male sterile tobacco by anti sense RNA system.

Hird et al .,( 2000).

C. Co transformation

Linked Genes Unlinked Genes

Agrobacterium mediated

Direct transfer

Assisted direct transfer

Artificial plant chromosomes

Operon system

Agrobacterium - standard binary vectors

Direct transfer- conventional vectors

Naqvi et al ., (2010)

Chloroplast Transformation

Size-120-160kb ~150 genesHigh copy number.

Basic set of genes t RNA r RNA Some ribosomal proteins Polypeptide components of photosystem. 8 large sub units of RUBISCO 4 sub units of chloroplast specific RNA polymerase.

Site Specific Integration By Homologous Recombination

No Positon Effect

No Vector Sequence

No Gene Scilencing

Bock ( 2007 )

Genetically engineering crop plants for disease

resistance via the chloroplast genome is

desirable to achieve high levels of expression

and to prevent pollen-mediated escape of

transgenes.

Maternal inheritance and gene containment

Daniell et al, .(2001)

lack of toxicity of transgenic pollen to non-target insects

Compartmentalization help to protect from proteolytic enzymes.

Purification is also easy

No pleiotropic effects

Daniell et al.,(2003)

High Expression & Normal Growth

Integration Of Transgenes In Chloroplast Genome

Hetero Plasmic Chloroplast - A mix of wild type & transformed

chloroplast genome.

1) Inter plastidic 2) Intraplastidic

• Homoplastomy- All chloroplasts are wild type or transformed.

Integration Of Transgenes In Chloroplast Genome

Application Of Multigene Engineering Through Nuclear Genome

In Metabolic Engineering

Tailoring a metabolic pathway

Nutritional value enhancement

For dissecting metabolic pathways

Modification of endogenous pathways

To synthesize a novel compound or macromolecule

Metabolic engineering of carotenoid pathway

Carotenoids are secondary metabolites .

They are also required by animals as metabolic precursors and antioxidants

Some of them are precursors of pro-vitamin A

To combat vit A deficiency.

The transgenic concept introduce , under endosperm –specific

regulation all genes necessary to establish the biochemical pathway for beta carotene , the most effective precursor to provit A.

Golden rice/provit rice

3 genes engineered in SGR-1

IPP

Geranylgeranyl diphosphate

Phytoene

Lycopene

-carotene(vitamin A precursor)

Phytoene synthase

Phytoene desaturase

Lycopene-beta-cyclase

ξ-carotene desaturase

Daffodil gene-psy

Single bacterial gene;-crtlperforms both functions

Daffodil gene-lcy

Vitamin APathway

is completeand functional

Potrykus et al,.(2000)

Golden Rice 1 produced 1.6ug/g of total carotenoids

Golden Rice 2 produces 37ug/g of total carotenoids (31ug/g of beta-carotene)

Carotenoid content in golden rice

Development of super nutritious maize

4 genes for the vitamins β-carotene, ascorbate, and folate transferred to maize genome

β-carotene:Psy1 : corn (Zea mays) phytoene synthasecrtI: Pantoea ananatis gene encoding carotene

desaturase

ascorbatedhar: rice dehydroascorbate reductase

folate GCH1: E. coli folE gene encoding GTP cyclohydrolase

Capell et al.,(2009)

In canola, 7 transgenes were introduced using an Agrobacterium mediated linked cotransformation strategy.

idi ,crtE,crtB, crtI, crt Y, crt Z, crt W genes from 3 sp:

Carotenoid content was 657 µg/g of seed

In potato, 3 transgenes introduced . crtI, crt B, crt Y

Quality Improvement In Oilseeds

Reconstruction of PUFA pathwayso Poly unsaturated fatty acid

FA with multiple double bond

Aliphatic chain with >20C

• Play major role in human health.

• 2 classes-w6 & w3

LC-PUFA biosynthesis pathway

Sayanova,o et al.,(2009)

Recent Development In PUFA Pathway Engineering

Constraints in pathway engineering of PUFA

Napier.A(2007)

Approaches to overcome the bottlenecks in LC-PUFA yields

Identification of improved desaturases

Identification of specific acyl-exchange mechanisms

Controlling the flux of LC-PUFAs into TAGs

Napier et al,(.2007)

Over expression of bacterial operon impart resistance to lepidopteran pests.

Bt cry2Aa2 operon is transferred to tobacco plants resulted in

the accumulation of Cry2Aa2 protein at 46.1% of tsp

Expression of an antimicrobial peptide, MSI-99 as a dicistron in

transgenic chloroplasts. Daniell et al, .(2001)

Impart resistance to biotic and abiotic stresses

The integration of a yeast trehalose-6 phosphate synthase (TPS) gene

as a dicistron in transgenic chloroplasts was shown to confer drought

tolerance Lee et al ,.(2003)

PHB production through multi gene engineering

Hyper expression of foreign protein

Over expression of the B cry2Aa2 operon in chloroplasts leads to formation of insecticidal crystals.

Miller et al., Nat Biot. 2001. 19:71-4

Operon expression & crystal formation

Bt cry2Aa2 operon(4kb) is used .

Vector used is pLD CtV2

Foreign gene integration determined by PCR screening.

Southern blot analysis of To & T1 generation

cry2Aa2 protein expression &quantification

INSECT BIOASSAY

Foreign protein accumulated at 45.3% of the total soluble protein.

Insects that are normally difficult to control (10-day old cotton bollworm, beet armyworm) were killed 100% after consuming transgenic leaves

DISCUSSION

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