Ti plasmid

Shirin Shahana .N
BScBiotechnology
SIAS

1. T i P L A S M I D S h i r i n S h a h a n a . N 3 y r - B s c . B i o t e c h n o l o g y S I A S

2. P L A S M I D S • P l a s m i d s are double stranded , closed circular DNA molecules , which exist in the cell as e x t r a chromosomal units . • T h e s e a r e t ransmitted from one bacterium to another ( even of anothe r species ) via horizontal gene transfer . • Engineered plasmids are widely used as vectors in DNA cloning . E g : T i p l a s m i d

3. T i P L A S M I D • T i plasmid is a tumor - inducing plasmid or tumor induction plasmid . • I t i s f o und in Agrobacterium tumefaciens bacteria . • I t is a gram - negative soil bacterium which naturally transforms plant cells , resulting in crown gall ( c a n c e r ) tumors . • T h e y i nfects plants through breaks or wounds . • T umor formation is the result of integration of T - DNA ( Transfer DNA ) in plant genome .

4. T i P L A S M I D & V I R U L E N C E G E N E S • T h e 3 i m o p r t a n t r e g i o n s o f T i p l a s m i d : - 1 ) T - D N A r e g i o n • T h i s r e g i o n has the genes for the biosynthesis . of anxin ( anxcytokinin ( W ) and opine ( cs ) , and is flanked by left and right borders . • T - DNA borders : - A set of 24 kb sequences present on either side ( right & left ) of T - DNA are also transferred to the plant cells . • I t is clearly established that the night border is more entical for ' T - DNA transfer .

5. 2 ) V i r u l e n c e r e g i o n •The genes responsible for the transfer of T - DNA into host plant are located outside T - DNA and the region is reffered to as vir or virulence region . •Acetosyringone( a flavonoid ) released by wounded plant cells activates vir genes . • At least nine vir - gene operons have been identified . • These include wir A , vir G wir B1 , vir CI . vir DI , D2 vir D4 and vir El.E2 . 3 ) O p i n e C a t a b o l i s m r e g i o n •This region codes for proteins involved in the uptake and metabolism of opines . • D e rivatives of amino acids synthesized by T - DNA .

6. Ti plasmids can be classified according to the opines produced : - 1. Nopaline plas mid s : - C arry gene for synthesizing nopaline in the plant and for utilization ( catabolism ) in the bacteria . 2. Octopine plasmids : - C arry genes to synthesize octopine in the plant and catabolism in the bacteria . 3. Agropine plasmids : - C arry genes for agropine synthesis and catabolism .

7. T i p l a s m i d - D e r i v e d V e c t o r S y s t e m s Using Ti plasmid as a vector it is possible to insert a desired DNA sequence ( gene ) into the TDNA region of Ti plasmid . There are several limitations to use Ti plasmids directly as cloning vectors d u e t o : - - 1 ) L a r g e s i z e . 2 ) T u m o u r i n d u c t i o n p r o p e r t y . 3 ) A b s e n c e o f u n i q u e r e s t r i c t i o n s i t e s .

8. • 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 is required for T - DNA integration . 1 ) M ultiple cloning site . 2 ) O rigin of replication . 3 ) S electable marker gene .

9. A g r o b a c t e r i u m m e d i a t e d t r a n s f o r m a t i o n The important requirements for Agrobacterium mediated gene transfer in higher plants are as follows : - 1 ) The plant explants must produce acetosyringone for activation of Vir genes . 2 ) The induced Agrobacterium should have access to cells that are competent for transformation . 3 ) Explants include cotyledon , leaf , thin tissue layer , peduncle , hypocotyls , stem , microspores .

10. T H A N K Y O U . . . ! !