plant genetic engineering


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Plant Genetic Engineering:

Plant Genetic Engineering Abhisek Dwivedy School of Biological Sciences B601- Genetic Engineering NISER

Ti Plasmid:

Ti Plasmid

Ti Plasmid:

Ti Plasmid Agrobacterium tumeifaciens 200kb, T-DNA integration into genome, genes for synthesis of auxin , ctyokinin and opine Genes for auxin & cytokinin are removed and the gene of interest is inserted between the two borders Strategies of insertion Co integration vectors - homologous recombination using a small vector plasmid Binary vectors -disarmed T DNA cloned into a small plasmid, manipulated and transformed back to A . tumifaciens . Selection marker commonly used are Kanamycin resistance gene or herbicide resistance gene Infection through wounds using leaf disks Grown on plates containing Ampicillin and Kanamycin Since wounding in monocots causes lignifications, preferably used in dicots (can be used in dicots adding acteosyringone

Ti plasmid:

Ti plasmid promoters for opines synthesis are exploited for high level of transgene expression promoter for CaMV 35S RNA is also employed Positional effects: difference in expression due to difference in location of transgene in the genomes (low expression when present near telomeres) Transgene silencing: presence of loxP sites can enhance or silence the expression of a gene

Direct Nuclear Transformation:

Direct Nuclear Transformation Protoplast Transformation : cell wall removed via cellulase / pectinase treatment Foreign DNA inserted via chemical/ electrporetic or liposomal method Cell well grows back after 10 days and cells also start dividing Selection by Kanamycin resistance Biolistics : DNA is coated over small metal particles (gold etc) and fired onto the cell forcing the DNA to enter nucleus and fit into the genome Such process is complex and my results in multiple copy insertion at random locations

Viral Vectors:

Viral Vectors CaMV : 8000bp ds circular DNA, maximum insert size less than 1kb High error prone methods due to RNA intermediates, and a narrow host range (cauliflower, turnip, cabbage) Geminivirus : ss DNA genome, replication via a ds DNA intermediate Can infect both monocots and dicots Less error prone as compared to CaMV and show more stable transient gene expression Infect crop plants Show high level of gene rearrangement thus sometimes are undesirable TMV and Potato X virus also used

Chloroplast Transformation:

Chloroplast Transformation

Chloroplast Transformation:

Chloroplast Transformation Chloroplast genome is arranged into operons , so multiple genes can be simultaneously expressed and since one chloroplast has more than one genome expression is usually high Homologus recombination of more than 400bp homologus sequence flanked at both ends Chloroplast specific promoters Antibiotic resistance gene aadA gives resistance against Spectinomysin ( aminoglycoside ) Kanamycin resistance also used Betained Aldehyde Dehydrogenase - BADH that converts betaine aldehyde to glycine betaine is also used for selection Double membrane of chloroplast is a barrier Can be used in high production of human therapeutic genes

Commercial Exploitation:

Commercial Exploitation Delayed Ripening : Antisense mRNA sequence expressed using 35sRNA CaMV promoter, resulted in low production of enzyme polygalacturonase thus reduced degradation of ploygalaturonic acid. Insecticide resistance : using B. thuringiensis cry1Ac toxin, a 130kDa protein that is insoluble at low condition so harmless to human, but in the mid-gut of Lepidopterans where pH is >9, the dissolve and cause septicemia leading to death of the insect. Herbicide resistance : Glyphosate is an inhibitor of aromatic amino acid synthesis and is used as herbicide. It targets the enzyme EPSPSase , thus resistance is conferred either by over expressing the protein or by using a mutant variety of enzyme resistant to herbicide. Viral resistance : resistance is conferred by applying super-infection, wherein the coat protein of TMV is expressed in the plant which prevents other viruses to infect. Fungal Resistance : expression of chitinase gene, chitinase degrades the chitin present in fungus by hydrolysis thus confers resistance.

Terminator Technology:

Terminator Technology

Terminator Technology:

Terminator Technology In the transgenic plant RIP not synthesized due to presence of blockade flanked by loxP sites Due to absence of Tetracyline TET-R bind to Cre promoter the preventing Cre to act at lox P On addition of tetracycline, TET-R is inhibited, thus Cre is expressed and it excises the loxP flanked blockade But the seeds now produced lack the blockade, so RIP is expressed leading to sterility

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