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Ti Plasmid & Agrobacterium Mediated gene transfer
- 1. Ti Plasmid & Agrobacterium Mediated gene transfer Prof. Niketan Deshmukh Rai University Ahmedabad
- 2. Agrobacterium Gram Negative Soil Borne Rod Shaped Motile Nature’s own Genetic Engineer Crown Gall
- 3. Introduction WHAT ARE PLASMIDS ? PLASMIDS are double stranded, closed circular DNA molecules, which exist in the cell as extra chromosomal units. Transmitted from one bacterium to another (even of another species) via horizontal gene transfer. Engineered plasmids are widely used as vectors in DNA cloning
- 4. Ti Plasmid Ti plasmid is a tumor-inducing plasmid or tumor induction plasmid. Found in Agrobacterium tumefaciens bacteria .
- 5. 1. T-DNA Region :This region has the genes for the Biosynthesis of Auxin (aux), Cytokinin (cyt), and Opine (ocs) T-DNA Border : A set of 24 bp sequence present on either side of T-DNA 2. Virulence Region : The genes responsible for the transfer of T- DNA into the host plant are located outside the T-DNA ant the region referred to as vir or virulence region 3. Opine Catabolism region : Uptake and Metabolism of Opine Organization of Ti-Plasmid A B C D E G
- 6. Mechanism of T-DNA transfer and integration
- 7. 1. Signal Induction : Wounded Plant cells release certain phenolic compounds which are recognized as signals by agrobacterium. 2. Attachment : The Agrobacterium attaches to palnt cells through polysaccharides, particularly via cellulose fibers 3. Production of Virulance Proteins : Wound Phenolic compounds Signal Vir A Plant CellBacteria Vir A Vir G Vir B,C,D,ESignal
- 8. 4. Production of T-DNA Strand : The right and left border of T-DNA are recognized by Vir D1/D2 proteins and these proteins involved in the production of ss-DNA. 5. Transfer of T-DNA out of Agrobacterium : Vir B form transport apparatus and ss-T- DNA in association with vir D2 exported from the bacterial cell. 6. Transfer of T-DNA into plant cells and its Integration : In plant cell ss T-DNA get covered with vir E2 for its protection. Vir D2 and E2 interact with variety of plants proteins which influences T-DNA transport and its integration into plant genome. (illegitimate Recombination) Transcription Translation Crown GallIntegration
- 9. virA - Transports acetosyringone into bacterium, activates virG post- translationally (by phosphorylation) virG - Promotes transcription of other vir genes virD2- Endonuclease/integrase that cuts T-DNA at the borders but only on one strand. virE2 - Can form channels in membranes virE1 - Chaperone for virE2 virD2 & virE2 also have NLSs, gets T-DNA to the nucleus of plant cell virB - Operon of 11 proteins, gets T-DNA through bacterial membranes Function of Vir genes
- 10. B B A G D 2 D 2 B C D E D1/D2 D1/D2 E2 SS-T DNA Wounded Plant cell E2 Ti 1 2 3 4 5 6 Transcription Translation Production of auxin, cytokinin and opine Autostimulation of Cell division Bacteria
- 11. Thanks God ! It’s Over at last ……. Thank you Prof. Niketan Deshmukh