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MULTIGENE ENGINEERING IN
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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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