Download Lecture 3 Ti plasmid derived vector system The simplest way to

Lecture 3
Ti plasmid derived vector system
The simplest way to exploit Ti plasmid to genetically transform plants is just
inserting the desired DNA sequence into the T-DNA region and then use the Ti plasmid
and A.tumefaciens to deliver and insert this gene into the genome of the susceptible plant
cell. Though Ti plasmids are effective natural vectors they had certain limitations.

The phytohormone produced by transformed cells growing in culture prevents
their regeneration into mature plants. Hence auxins and cytokinin genes must be
removed from the Ti –plasmid derived cloning vector.

The opine synthesis gene must be removed as it may divert plant resources into
opine production in transgenic plant.

Generally, Ti- plasmids are large in size (200-800 kb).For effective cloning, large
segments of DNA that are not essential for cloning has to be removed.

As Ti plasmid does not replicate in E.coli,Ti-plasmid based vectors require an
ori that can be used in E.coli
To overcome these constraints, Ti plasmid based vectors were organized with the
following components:

A selectable marker gene that confers resistance to transformed plant cells. As
these marker genes are prokaryotic origin, it is necessary to put them under the
eukaryotic control (plant) of post transcriptional regulation signals, including
promoter and a termination- poly adenylation sequence, to ensure that it is
efficiently expressed in transformed plant cells.

An origin of replication that allows the plasmid to replicate in E.coli.

The right border sequence of the T-DNA which is necessary for T-DNA
integration into plant cell DNA.

A polylinker (MCS) to facilitate the insertion of cloned gene into the region
between T-DNA border sequences.
As these cloning vectors so organized lacked vir genes, they cannot effect the transfer
and integration of T-DNA region into host plant cell. So two Ti-plasmid derived
vector systems were developed. They include:
1. Binary vector system
2. Co-integrate vector system
1. Binary vector system
The binary vector system contains either E.coli or A.tumifaciens origins of DNA
replication, i.e.an E.coli - A.tumifaciens shuttle vector or a single broad host range ori. In
either case no vir genes are present on the binary cloning vector. All the cloning steps are
carried out in E.coli before the vector is introduced into A.tumifaciens. The A.tumifaciens
strain carries a modified (disarmed) Ti plasmid that contains a complete set of vir genes
but lack portions of T-DNA region, so that this T-DNA cannot be transferred. With this
system, the defective Ti plasmid synthesizes the vir gene products that mobilize the TDNA region of the binary cloning vector plasmid. By providing the proteins encoded by
the vir genes, the defective Ti plasmid acts as helper plasmid ,enabling the T-DNA from
binary cloning vector to be inserted into the plant chromosomal DNA. Since transfer of
T-DNA is initiated from the right border, the selectable marker which will eventually be
used to detect the presence of the T-DNA inserted into the plant chromosomal DNA is
placed next to the left border. If selectable marker is present adjacent to the right border,
only small portion of T-DNA will be transferred resulting in a plant with only selectable
marker and no gene of interest. Few binary vectors are developed with selectable markers
one adjacent to the right and the other adjacent to the left border.
Ti plasmid vector systems
are often working as binary vectors
T DNA region removed
Gene of interest
Plant selectable marker
Bacterial selectable
marker
Disarmed
Ti
plasmid
ori for E.coli
Virulence
region
ori for A. tumefaciens
HELPER
plasmid
ori for A. tum
DISADVANTAGE: Depending on the orientation,
plasmids with two different origins of replication may be unstable in E. coli
ADVANTAGE: small vectors are used, which increases transfer efficiency
from E. coli to Agrobacterium.
No intermolecular recombination is needed
2. Co-integrate vector system
In the co integrate vector system the cloning vector has a plant selectable marker
gene, the target gene, the right border, an E.coli origin of the DNA replication, and a
bacterial selectable marker gene. The co-integrate vector recombines with the modified
( disarmed) Ti plasmid that lacks both the tumor producing genes and the right border of
the T-DNA within A.tumifaciens, and the entire cloning vector becomes integrated into
the disarmed Ti plasmid to form a recombinant Ti plasmid. The co-integrate cloning
vector and the disarmed helper Ti plasmid both carry homologous DNA sequence that
provide shared site for in vivo homologous recombination, normally these sequence lie
inside the T-DNA region. Following recombination, the cloning vector becomes part of
the disarmed Ti plasmid, which provides the vir genes necessary for the transfer of TDNA to host plant cell. The only way that this cloning vector can be maintained in
A.tumifaciens is a part of the co-integrated structure. In this co-integrated configuration
genetically engineered T-DNA region can be transferred to plant cells.