Professional Documents
Culture Documents
Sathiyaraj Srinivasan
사티야라지 스리니바산
Dept. of Oriental Medicinal
materials and Processing
Kyung Hee University,
Republic of Korea – 449 701
Gene Transfer Technique
To transfer a gene from one DNA molecule to another DNA molecule.
Transient transfection
Stable transfection
Agrobacterium tumefaciens
Mechanism of infection of Bacterium in plants
Ti Plasmid :
The tumor inducing plasmid (pTi) contains a portion that is
transferred to the plant cell is Transfer DNA (T-DNA)
Vir D1 Topiosomerase; Helps Vir D2 to recognise and cleave within the 25bp
border sequence
Vir D2 Covalently attaches to the 5I end of the T-strand, thus forming the T-DNA
Complex. Also guides the T-DNA complex through the nuclear pores
Vir C Binds to the 'overdrive' region to promote high efficiency T-strand
Synthesis
Vir E2 Binds to T-strand protecting it from nuclease attack, and intercalates with
lipids to form channels in the plant membranes through which the
T-complex passes
Vir E1 Acts as a chaperone which stabilises Vir E2 in the Agrobacterium
Vir B & Vir Assemble into a secretion system which spans the inner and outer
bacterial membranes. Required for Export of the T-complex and Vir E2
D4
into the plant cell
Important genes encoded by
Ti plasmid
1. Cytokinins
(plant hormone for cell plant division and tumorous growth)
• Virulence region (vir) carried on pTi, but not in the transferred region
(T-DNA). Genes code for proteins that prepare the T-DNA and the
bacterium for transfer.
3. T-DNA encodes genes for opine synthesis and for tumor production.
chvA pscA
chvB
T-DNA
tra bacterial conjugation
pTi
vir genes
opine catabolism
transfer to the plant
oriV inc pTi’s are in the same inc group.
Ti Plasmid
T-DNA
region
DNA between
L and R borders is
transferred to plant
as ssDNA;
Tumor-
producing
genes
T-DNA encoded
Opine catabolism genes can be
substituted by
Virulence region target genes
ORI
The basis of Agrobacterium-mediated genetic
engineering
T-DNA of A. tumefaciens is excised and integrates into
the plant genome as part of the natural infection
process.
Any foreign DNA inserted into the T-DNA will also be
integrated.
Ti-plasmid based vectors
Disarmed Ti plasmid
Disarmed Ti plasmid
capable for infection
with gene of interest Form
(no vir genes) co-integrated plasmid Intermediate vector
after homologous with T-region
recombination on T-DNA and gene of interest
Helper vector (transferred by conjugation)
for infection Helper vector
(with vir genes) for transfer of
intermediate plasmid into A.tum
Co-integrated vectors
(hybrid ti-plasmids)
In a modern labs
rarely used
DISADVANTAGES:
1) Long homologies required between the Ti plasmid
and the E. coli plasmids (pBR322 based Intermediate vectors)
making them difficult to engineer and use
Phenolics AGROBACTERIUM
detected by the
VirA/VirG two T-DNA VirD2 attaches to
component exposed 5I end
sensor system.
Bacterial
Plasmid
Formation of T-Pilus
VirA VirG
PLANT CELL
Phenolics
Produced by VIP1 associates with
Wounded the complex to target
Plant cell it to the nucleus
VIP2 associates the
complex to
Gene of interest
4. Plant cells are selected on kanamycine
5. Presence of transgene confirmed by PCR
6. Whole plant could be grown from transformed cells !!!
Transgenic Plants
tomato, potato and flax in past
Since the first widespread release in 1996, land devoted to transgenic crops
has increased by 10 - 50% yearly
90% of Canadian canola is GMO - mostly transgenic
20+ countries suspected of growing transgenic crops without official
approval
Global area of transgenic crops
(ISAA Brief. Global Review of Commercialised Transgenic crops: 1998 & 2001)
60
Millions of hectares
Acreage of transgenic 50
crops has gone from 40
nothing in 1995 to 30
around 135 million
20
acres in 2001.
10
0
1995 1997 1999 2001
Approved Transgenic plants
Soybean Carnations
Corn Potato
Cotton Flax
OilSeed rape Papaya
Sugarbeet Chicory
Squash Rice
Tomato Melon
Tobacco
Types of GM crops (1998)
40
35
Soybean and corn are the 30
major GM crops 25
area
20
* Large acreage
15
* Grown in the USA 10
* Can be regenerated 5
Acreage of potatoes is small 0
Soybean
Cotton
Corn
Oil Seed
(<0.1 million hectares)
Types of genetic modification
Millions of hectares
25
20
>99% of all 15
transgenic crops 10
are either herbicide 5
or insect resistant 0
resistance
Herbicide
Others
Insect
<1% have other
traits