ti plasmid
TRANSCRIPT
Ti Plasmid
By –AKSHAY PAREEKM.Sc. Biotech II semDepartment of Biotechnology
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
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 .
Ti Plasmid
• Ti plasmid is a tumor-inducing plasmid or
tumor induction plasmid.
• Found in Agrobacterium tumefaciens
bacteria .
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.
Ti Plasmid stucture
Vir (virulent) Gene
1. Transfer the T-DNA to plant cell
2. Acetosyringone (AS) (a flavonoid) released
by wounded plant cells activates vir genes.
3. virA,B,C,D,E,F,G (7 complementation
groups, but some have multiple ORFs),
span about 30 kb of Ti plasmid.
Function of vir genes• 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
Opines
• Derivatives of amino acids synthesized by T-DNA
• Ti plasmids can be classified according to the
opines produced :
1. Nopaline plasmids
2. Octopine plasmids
3. Agropine plasmids
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.
Structure of T-DNA
LB
SHOOTY LOCUS ROOTY LOCUS
ONC REGION
tms2 tms1 tmr
ocs
GGCAGGATATATTCAATTGTAA GGCAGGATATATACCGTTGTAAT
octopine synthase
Infection Process
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.
• 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
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
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)
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
THANK YOU