ti plasmid

Agrobacterium

1. Ti Plasmid By – AKSHAY PAREEK M.Sc. Biotech II sem Department of Biotechnology

2. CONTENT INTRODUCTION Ti Plasmid Agrobacterium tumefaciens Ti Plasmid stucture Vir (virulent) Gene Structure of T-DNA Infection Process Ti Plasmid-Derived Vector Systems Agrobacterium mediated transformation Process of Transformation

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. Agrobacterium tumefaciens A. tumefaciens is a gram-negative soil bacterium which naturally transforms plant cells, resulting in crown gall (cancer) tumors. Infects plants through breaks or wounds . Tumor formation is the result of integration of T-DNA (Transfer DNA) in plant genome.

6. Ti Plasmid stucture

7. Vir (virulent) Gene Transfer the T-DNA to plant cell Acetosyringone (AS) (a flavonoid ) released by wounded plant cells activates vir genes. virA,B,C,D,E,F,G (7 complementation groups, but some have multiple ORFs), span about 30 kb of Ti plasmid.

8. Function of vir genes virA - transports acetosyringone into bacterium, activates virG post- translationally (by phosphorylation ) virG - promotes transcription of other vir genes virD 2 - 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

9. Opines Derivatives of amino acids synthesized by T-DNA Ti plasmids can be classified according to the opines produced : Nopaline plasmids Octopine plasmids Agropine plasmids

10. Nopaline plasmids : carry gene for synthesizing nopaline in the plant and for utilization (catabolism) in the bacteria. Octopine plasmids : carry genes to synthesize octopine in the plant and catabolism in the bacteria. Agropine plasmids : carry genes for agropine synthesis and catabolism.

11. Structure of T-DNA LB SHOOTY LOCUS ROOTY LOCUS ONC REGION tms2 tms1 tmr ocs GGCAGGATATATTCAATTGTAA GGCAGGATATATACCGTTGTAAT octopine synthase

12. Infection Process

13. Ti Plasmid-Derived Vector Systems Using Ti plasmid as a vector it is possible to insert a desired DNA sequence (gene) into the T DNA region of Ti plasmid. There are several limitations to use Ti plasmids directly as cloning vectors :- LARGE SIZE. TUMOR INDUCTION PROPERTY. ABSENCE OF UNIQUE RESTRICTION SITES.

14. Agrobacterium plasmids are disarmed by deleting naturally occurring T-DNA encoded oncogenes and replacing them with foreign genes of interest . The right and left border sequences of T-DNA which is required for T-DNA integration. A multiple cloning site. An origin of replication A selectable marker gene

15. Agrobacterium mediated transformation The important requirements for Agrobacterium - mediated gene transfer in higher plants are as follows:- The plant explants must produce acetosyringone for activation of Vir genes . The induced Agrobacterium should have access to cells that are competent for transformation. Explants include cotyledon, leaf, thin tissue layer, peduncle, hypocotyls, stem, microspores

16. Explants (cotyledon, hypocotyls, microspore) Co-cultivation to allow infection Transformed and non-transformed tissue Transformed tissue/callus Transformed shoots Rooted shoots Adult plants Process of Transformation Agrobacterium Antibiotics to kill bacteria ( carbenicillin ) Selective media to kill non transformed tissues (addition of Kanamycin )

17. Acknowledgement Dr. Sandeep Kumar (HOD) Department of Biotechnology NIET, Nims University Jaipur Dr. Sandeep Tripathi (Assistant Professor) Department of Biotechnology NIET, Nims University, Jaipur All Faculty members All seniors, Batch mates & Juniors

18. THANK YOU