Transcript Ti质粒

DNA重排研究进展专题
Ti-质粒与植物基因组的相互作用
蒋舜媛 董霞 任昶
2002-10-08
What is Ti-plasmid?
Ti-plasmid, ——（short for tumor-inducing
plasmid）is a large (150~200 kb) doublestranded circular DNA plasmid in a soil
bacterium called Agrobacterium tumefaciens,
which causes what is known as produces
uncontrolled growths (tumors, or galls),
normally at the base (crown) of the plant. Tiplasmid is the key to this tumor production.
Figure 1. In the process of causing crown gall
disease, the bacterium Agrobacterium tumefaciens
inserts a part of its Ti plasmid—a region called
T-DNA—into a chromosome of the host plant.
Figure 2. Simplified representation of the major regions of the Ti plasmid of
A. tumefaciens. The T-DNA, when inserted into the chromosomal DNA of
the host plant, directs the synthesis of nopaline, which is then utilized by the
bacterium for its own purposes. T-DNA also directs the plant cell to divide in
an uncontrolled manner, producing a tumor.
Structure of Ti-plasmid
T-DNA区
毒性区 (Vir-region, vir)
质粒复制起点 (Origin of replication, ori)
质粒结合转移位点
(Transfer function site, tra)
冠瘿碱分解位点 (ocs or nos)
Two types of Ti-plasmid
OCS plasmid (octopine )
 pTiAch5,
pTiA6NC, pTiB653, pTiAg162
NOS plasmid (nopaline )
 pTiT37,
pTiT38
土壤农杆菌-植物DNA转移体系步骤
植物敏感细胞和土壤农杆菌相互作用
土壤农杆菌的毒性区基因被激活
T-DNA的切割和T复合物生成
T复合物由土壤农杆菌经植物细胞膜
进入植物细胞
T-DNA整合到植物染色体上
Figure 1. In the process of causing crown gall
disease, the bacterium Agrobacterium tumefaciens
inserts a part of its Ti plasmid—a region called
T-DNA—into a chromosome of the host plant.
Interaction of Agrobacterium Ti-plasmid
DNA and plant cells
植物敏感细胞和土壤农杆菌相互作用
植物受伤产生大量对土壤农杆菌感染敏
感的细胞

受伤细胞产生一些低分子量的酚类物质可明
显刺激土壤农杆菌Ti质粒上毒性区表达

乙酰丁香酮（AS），α-羟基乙酰丁香酮
Ti质粒毒性区基因激活及
T-DNA复合物生成
Insert picture
Integration of T-DNA into the
chromosome of host plant
T-DNA整合到植物染色体上
首先在植物靶DNA上出现一个缺刻(nick)
随着DNA解链，宿主细胞的5’—3’外切酶活性使缺刻
扩大成裂口(gap)
T-DNA侵入裂口，末端与靶DNA单链上的少数核苷酸
配对形成异源二倍体；
T-DNA悬挂在外侧的末端被切割除去，T-DNA与靶
DNA末端相连；
靶DNA上链出现缺刻，以整合后的T-DNA下链为模板
合成T-DNA的第二条链，完成整合过程。
References via NCBI
The interaction of Agrobacterium Ti-plasmid DNA and plant cells (1980)
Transgene integration in aspen: structures of integration sites and mechanism
of T-DNA integration. (2002)
How does the T-DNA of Agrobacterium tumefaciens find its way into the
plant cell nucleus? (1993)
T-DNA integration: a mode of illegitimate recombination in plants. (1991)
Transfer and Integration of T-DNA without Cell Injury in the Host Plant.
(1997)
Integration of Agrobacterium T-DNA into a tobacco chromosome: possible
involvement of DNA homology between T-DNA and plant DNA. (1990)
Illegitimate recombination in plants: a model for T-DNA integration. (1991)
DNA rearrangement associated with the integration of T-DNA in tobacco: an
example for multiple duplications of DNA around the integration target.
(1995)
The DNA sequences of T-DNA junctions suggest that complex T-DNA loci
are formed by a recombination process resembling T-DNA integration. (1999)
Two different but related mechanisms are used in plants for the repair of
genomic double-strand breaks by homologous recombination. (1996)
Production of transgenic
plants with recombinant Tiplasmid
Formation of Cointegrate Ti-plasmid
Generation of a transgenetic plant
via the growth of a cell transformed
by T-DNA
Figure 3. (a) To
produce transgenic
plants, an
intermediate vector of
manageable size is
used to clone the
segment of interest. In
the method shown
here, the intermediate
vector is then
recombined with an
attenuated
(“disarmed”) Ti
plasmid to generate a
cointegrate structure
bearing the insert of
interest and a
selectable plant
kanamycin-resistance
marker between the TDNA borders, which
is all the T-DNA that
is necessary to
promote insertion. (b)
The generation of a
transgenic plant
through the growth of
a cell transformed by
T-DNA.
Formation of Cointegrate Ti-plasmid
Attenuate the natural Ti-plasmid.

have the entire right-hand region of its T-DNA,
including tumor genes and nopaline-synthesis
genes deleted, rendering it incapable of tumor
formation.
Prepare a intermediate vector.
Incorporate the intermediate vector.
Fig 3.(a) To produce transgenic plants, an intermediate vector of
manageable size is used to clone the segment of interest. In the method
shown here, the intermediate vector is then recombined with an attenuated
(“disarmed”) Ti plasmid to generate a cointegrate structure bearing the
insert of interest and a selectable plant kanamycin-resistance marker
between the T-DNA borders, which is all the T-DNA that is necessary to
promote insertion.
Generation of a transgenetic plant via the
growth of a cell transformed by T-DNA
Bacterial infection
transformed plant cell
Cell culturation  callus
Inducing the differentiation of shoots
and roots, and transferring to soil
 transgenetic plant
Fig.3 (b) The generation of a transgenic plant
through the growth of a cell transformed by T-DNA.
infectio
n
Figure 4. T-DNA and any DNA contained within it are
inserted into a plant chromosome in the transgenic plant and
then transmitted in a Mendelian pattern of inheritance.
Thanks!
The End