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Genetics
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Mohd bin Mahmud @ Mansor, FIAT
Transgenic plants
Introduction• 25,000 field safety and production trials
• 60 genetically engineered plants and animals
• 45 countries
• 6500 in the USA alone (18,000 sites)
• 74% of the soybeans
• 71% of the cotton
• 32% of the corn
• Has been practiced for hundreds of years
• Slow and uncertain
• Sexual cross between two lines, back-cross with offspring and one of the parents until a plant with desired characteristics is obtained.
• Limited to only plants that can sexually hybridized
Introduction
Introduction• Recombinant DNA technology
• Offers unlimited possibility
• Greater specificity
• Genes from incompatible plants, animals, bacteria or insects
• Sexual compatibility becomes irrelevant
• Process is faster since the gene can be directly selected
• We will together discuss how foreign genes can be introduced into plant
• Using Ti-plasmid system (Agrobacterium tumefaciens)
• "Gene gun"
• Also some major products of genetic engineering in plants
• Production of insect-tolerant and herbicide-tolerant crops
Introduction
Ti-plasmid System• Ti-plasmid as vector
• Agrobacterium tumefaciens is a plant pathogenic soil bacterium
• Causes crown gall disease
• The key is 200kb circular DNA plasmid - Ti plasmid
• During infection, T-DNA is transferred and inserted randomly into the genome of the host plant
• The T-DNA contains genes for the synthesis of:
1. Food for the bacterium (amino acids - opines)
2. Plant hormones (auxin and cytokinin)
3. Vir genes
• Both are expressed at high level
• Overexpression of hormones leads to?
Ti-plasmid System
• Virulence genes:
Located at 35kb region on the Ti-plasmid
Needed for the production of trans-acting proteins that essential for plant cell transformation
25 genes in 7 operons
Switched on by chemicals (acetosyringone) produced by wounded plant cells
Ti-plasmid System
• The signals perceived by virA protein, transduced to virG protein
• Activating upstream region of other vir genes and activates the expression of the other vir genes
• The activation of vir genes will excised T-DNA from the plasmid
Ti-plasmid System
Ti-plasmid System
• Flanked by two borders
• Sequence called RB and LB
• Involve in excision of the T-DNA sequence
• Starts at RB follows by a nick at LB as a single strand
• Coated by virE proteins (coded by virE gene in vir-regions
• Transported into plant cells via membrane channel formed by virB protein
Ti-plasmid System
Ti-plasmid System
Ti-plasmid System
Ti-plasmid System
Ti-plasmid System
Ti-plasmid System
The binary vector system• Developed from the concept of Ti-plasmid system
• Essential functions for transfer are supplied separately.
• Namely?
A. ?
B. ?
• Neomycin phototransferase II
resistance to kanamycin
• Contains polylinker
Facilitate insertion of
the DNA
Contains series of unique
restriction sites
The binary vector system
The binary vector system
The Gene Gun• Using standard bacterial vectors
• 1 micrometer in diameter
• Tungsten or gold
• Coated with DNA (gene of interest + selectable marker)
• Fired with a gun with velocities over 400 mps directly into plant tissue of interest
• Then transferred to a nurse cell culture plate
• Major advantages:
Possibility of adding a transgene to organelles that have their own DNA genome
Need special markers:
1. Spectinomycin
2. Streptomycin
The Gene Gun
The Gene Gun
Insect resistance: Bt-crops• Bacillus thuringiensis - apore forming soil bacterium
• Crystals consists of one or more delta-endotoxins or cry proteins of 130kDal
• Upon digestion by larvae, proteases in the insect's gut cleave the protein into an active N-terminal 65-79 kDal
• The toxin acts by binding to receptors on the cell membrane of the midgut epithelial cells
• Inserts itself into the membrane, forms pores that kill the epithelial cells
• Eventually kills the insect by colloid osmotic lysis
• We have the information, so now what can we do? Discuss.
Insect resistance: Bt-crops