2. Ti- 质粒与植物基因组的相互作用 蒋舜媛 董霞 任昶 2002-10-08 DNA 重排研究进展专题

3. What is Ti-plasmid? Ti-plasmid, —— （ short for tumor-inducing plasmid ） is a large (150~200 kb) double- stranded 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. Ti- plasmid is the key to this tumor production.

4. 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.

5. 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.

6. 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)

7. Two types of Ti-plasmid OCS plasmid (octopine ) pTiAch5, pTiA6NC, pTiB653, pTiAg162 NOS plasmid (nopaline ) pTiT37, pTiT38

8. 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

9. 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.

10. 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

11. Ti 质粒毒性区基因激活及 T-DNA 复合物生成 Insert picture

12. 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。

13. 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)

14. Production of transgenic plants with recombinant Ti- plasmid Formation of Cointegrate Ti-plasmid Generation of a transgenetic plant via the growth of a cell transformed by T-DNA

15. 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 T-DNA 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.

16. 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.

17. 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.

18. 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

19. Fig.3 (b) The generation of a transgenic plant through the growth of a cell transformed by T-DNA. infectio n

20. 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.

21. Via NCBI, from Modern Genetic Analysis 11. Applications of Recombination DNA Technology Recombinant DNA Technology in Eukaryotes Molecular Cell Biology 8. Genetic Analysis in Cell Biology 8.5 Gene Replacement and Transgenic Animals

22. The End Thanks!