logging in or signing up ti plasmid presentation-2 yashloksingh11 Download Post to : URL : Related Presentations : Share Add to Flag Embed Email Send to Blogs and Networks Add to Channel Uploaded from authorPOINT lite Insert YouTube videos in PowerPont slides with aS Desktop Copy embed code: Embed: Flash iPad Dynamic Copy Does not support media & animations Automatically changes to Flash or non-Flash embed WordPress Embed Customize Embed URL: Copy Thumbnail: Copy The presentation is successfully added In Your Favorites. Views: 304 Category: Entertainment License: All Rights Reserved Like it (0) Dislike it (0) Added: October 09, 2011 This Presentation is Public Favorites: 0 Presentation Description part-2 Comments Posting comment... Premium member Presentation Transcript Agrobacterium - mediated Gene Transfer: Agrobacterium - mediated Gene Transfer Most common method of engineering dicots, but also used for monocots Pioneered by J. Schell (Max-Planck Inst., Cologne) Agrobacteria soil bacteria, gram-negative, related to Rhizobia species: tumefaciens - causes crown galls on many dicots rubi - causes small galls on a few dicots rhizogenes - hairy root disease radiobacter - avirulentSlide 2: Crown galls caused by A. tumefaciens on nightshade. More about Galls: http:// waynesword.palomar.edu/pljuly99.htm http://kaweahoaks.com/html/galls_ofthe_voaks.htmlAgrobacterium tumefaciens: Agrobacterium tumefaciens the species of choice for engineering dicot plants; monocots are generally resistant (but you can get around this) some dicots more resistant than others (a genetic basis for this) complex bacterium – genome has been sequenced; 4 chromosomes; ~ 5500 genesInfection and tumorigenesis: Infection and tumorigenesis Infection occurs at wound sites Involves recognition and chemotaxis of the bacterium toward wounded cells galls are “real tumors”, can be removed and will grow indefinitely without hormones genetic information must be transferred to plant cellsTumor characteristics : Tumor characteristics Synthesize a unique amino acid, called “opine” octopine and nopaline - derived from arginine agropine - derived from glutamate Opine depends on the strain of A. tumefaciens Opines are catabolized by the bacteria, which can use only the specific opine that it causes the plant to produce. Has obvious advantages for the bacteria, what about the plant?Elucidation of the TIP (tumor-inducing principle): Elucidation of the TIP (tumor-inducing principle) It was recognized early that virulent strains could be cured of virulence, and that cured strains could regain virulence when exposed to virulent strains; suggested an extra- chromosomal element. Large plasmids were found in A. tumefaciens and their presence correlated with virulence: called tumor-inducing or Ti plasmids.Ti Plasmid : Ti Plasmid Large ( 200-kb) Conjugative ~10% of plasmid transferred to plant cell after infection Transferred DNA (called T-DNA ) integrates semi-randomly into nuclear DNA Ti plasmid also encodes: enzymes involved in opine metabolism proteins involved in mobilizing T-DNA ( Vir genes)Slide 8: auxA auxB cyt ocs LB RB LB, RB – left and right borders (direct repeat) auxA + auxB – enzymes that produce auxin cyt – enzyme that produces cytokinin Ocs – octopine synthase, produces octopine T-DNA These genes have typical eukaryotic expression signals!Slide 9: auxA auxB Tryptophan indoleacetamide indoleacetic acid (auxin) cyt AMP + isopentenylpyrophosphate isopentyl-AMP (a cytokinin) Increased levels of these hormones stimulate cell division. Explains uncontrolled growth of tumor.Vir (virulent) genes: Vir (virulent) genes On the Ti plasmid 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.Vir gene functions (cont.): Vir gene functions (cont.) virA - transports AS into bacterium, activates virG post-translationally (by phosphoryl.) virG - promotes transcription of other vir genes virD 2 - endonuclease/integrase that cuts T- DNA at the borders but only on one strand; attaches to the 5' end of the SS virE2 - binds SS of T-DNA & can form channels in artificial 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 membranesSlide 12: VirE2 may get DNA-protein complex across host PMSlide 13: Monocots don't produce AS in response to wounding. Important : Put any DNA between the LB and RB of T-DNA it will be transferred to plant cell! Engineering plants with Agrobacterium: Two problems had to be overcome: (1) Ti plasmids large, difficult to manipulate (2) couldn't regenerate plants from tumors Binary vector system: Binary vector system Strategy: 1. Move T-DNA onto a separate, small plasmid. 2. Remove aux and cyt genes. 3. Insert selectable marker ( kanamycin resistance) gene in T-DNA. 4. Vir genes are retained on a separate plasmid. 5. Put foreign gene between T-DNA borders. 6. Co-transform Agrobacterium with both plasmids. 7. Infect plant with the transformed bacteria.Slide 15: Binary vector systemSlide 16: Making a transgenic plant by leaf disc transformation with Agrobacterium.Slide 17: Thank you You do not have the permission to view this presentation. In order to view it, please contact the author of the presentation.