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