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1、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,韦仟蕾捂桐建藉重慌检炬后茬凝揪涧造绰宜诗么豫异里晦皱帕盛香边酬臼Ti质粒与植物基因组的相互作用Ti质粒与植物基因组的相互作用,What is Ti-plasmid?,Ti-plasmid, （short for tumor-inducing plasmid）is a large (150200 kb) double-stranded circular DNA plasmid in a soil bacterium called Agrobacterium tumefaciens, which c
2、auses 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.,蛛条镀脓裕五影银嗣臼羞喳盟森瞎酥杯帮抱筹厚簿柒医适聂延屑予裤吻恨Ti质粒与植物基因组的相互作用Ti质粒与植物基因组的相互作用,Figure 1. In the process of causing crown gall disease, the bacterium Agr
3、obacterium tumefaciens inserts a part of its Ti plasmida region called T-DNAinto a chromosome of the host plant.,岗寄匀赏隆隙却胚裕螺守缺景遥冕闪谈慈缝珊叼蜘因囊蹭取撮弛爱扮期族Ti质粒与植物基因组的相互作用Ti质粒与植物基因组的相互作用,Figure 2. Simplified representation of the major regions of the Ti plasmid of A. tumefaciens. The T-DNA, when inserted into
4、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.,摸质硝堆札抛翱索孽附舷疫谩搁诛宏东凉挥枉阶监涩挑院察耿听到珠伺僻Ti质粒与植物基因组的相互作用Ti质粒与植物基因组的相互作用,Structure o
5、f 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),渝议柄乖甭亨罕剥峙殖茶敞栋琳姥瘤巨卫鲸爵詹泼柔赌兔颇壁楷砷闰泥狄Ti质粒与植物基因组的相互作用Ti质粒与植物基因组的相互作用,Two types of Ti-plasmid,OCS plasmid (octopine ) pTiAch5, pTiA6NC, pTiB653, pTiAg162 NOS plasmid (no
6、paline ) pTiT37, pTiT38,匠髓叮盂阂氟秦漓抢侣戊托笺蘸伺拆侨置矮递鹏染今探矩瞪嘱釉卤撂驰阵Ti质粒与植物基因组的相互作用Ti质粒与植物基因组的相互作用,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,铝颈趣咸讽畜啡短拜冤很舆娜燥枢器真选靡丝羞巩矗兢褒律恬娠零食碟弦Ti质粒与植物基因组的相互作用Ti质粒与植物基因组的相互作用,Figure 1. In the process of causing crown gall
7、 disease, the bacterium Agrobacterium tumefaciens inserts a part of its Ti plasmida region called T-DNAinto a chromosome of the host plant.,雕芭译脯吼乳喂待晓仪颖锦奠吻眩弛豌迸肖串镰埋貉盾厉冯卡生莫骇彪环Ti质粒与植物基因组的相互作用Ti质粒与植物基因组的相互作用,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
8、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），-羟基乙酰丁香酮,辊帐前滞重回胯葡豺厌腑俯储脐蚁佛忆哟厨吗褒矛皮菱蛋岛但极孙颊炔韧Ti质粒与植物基因组的相互作用Ti质粒与植物基因组的相互作用,Ti质粒毒性区基因激活及 T-DNA复合物生成,Insert picture,渴提颐垃傈硕施浓球赦诀惕狸墓弱鬼尽同肩验巍耶俩沦挤租坯赐帖花旬男Ti质粒与植物基因组的相互作用Ti质粒与植物基因组的相互作用,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
9、DNA上出现一个缺刻(nick) 随着DNA解链，宿主细胞的53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。,婉肄酒物斌樟仓骄谨彰孕核庸懒络遮妹乐轨滚庐锑族喇甥西樊堑乔为灼净Ti质粒与植物基因组的相互作用Ti质粒与植物基因组的相互作用,References via NCBI,The interaction of Agrobacterium Ti-plasmid DNA
10、 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
11、) 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 rea
12、rrangement 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 differ
13、ent but related mechanisms are used in plants for the repair of genomic double-strand breaks by homologous recombination. (1996),站裔惰骋摔蚤匈卢压滔住懒铣组碑豫稳瓦藻最佣汞轨例哲哄腆圾有喳虾糜Ti质粒与植物基因组的相互作用Ti质粒与植物基因组的相互作用,Production of transgenic plants with recombinant Ti-plasmid,Formation of Cointegrate Ti-plasmid Generation o
14、f a transgenetic plant via the growth of a cell transformed by T-DNA,粤价类审抱后蜂挛爪肖咋隅狠速孪靖堡梁吭支歪禁窖征棘姨耀邱抡鹃祝烙Ti质粒与植物基因组的相互作用Ti质粒与植物基因组的相互作用,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 intermedia
15、te 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 o
16、f a transgenic plant through the growth of a cell transformed by T-DNA.,丸丽尤升都执繁懊伎裔纤漂克待躯郭胺蒸屉滋扫声毙宵回旅朋换父氖惠来Ti质粒与植物基因组的相互作用Ti质粒与植物基因组的相互作用,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 ge
17、nes deleted, rendering it incapable of tumor formation. Prepare a intermediate vector. Incorporate the intermediate vector.,首遵嫂缠弹酱偶喘鹿俗掉侄晕惕滁五短练椒豫努操题箍姓公青侮如庄拷询Ti质粒与植物基因组的相互作用Ti质粒与植物基因组的相互作用,Fig 3.(a) To produce transgenic plants, an intermediate vector of manageable size is used to clone the segment of
18、 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 n
19、ecessary to promote insertion.,玻符脯恃侥扁雄大石塔抿喇者怂涸服嗜英硷桶扎碑沿仑递袭涨坛趣罢链龟Ti质粒与植物基因组的相互作用Ti质粒与植物基因组的相互作用,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 tran
20、sferring to soil transgenetic plant,抠抱因系拒薄忻拇古忠洼迪诉蓉规史击斧时馒微嘻设止闺轧娇犁掠境钩舟Ti质粒与植物基因组的相互作用Ti质粒与植物基因组的相互作用,Fig.3 (b) The generation of a transgenic plant through the growth of a cell transformed by T-DNA.,infection,伐鼻桐祁烙苍押剂俯架韦惫提阑谐啼谈株匹薛雏颓桨玖袜搅于隘伴甩撕按Ti质粒与植物基因组的相互作用Ti质粒与植物基因组的相互作用,Figure 4. T-DNA and any DNA co
21、ntained within it are inserted into a plant chromosome in the transgenic plant and then transmitted in a Mendelian pattern of inheritance.,新沿家习构澎架赵姬飘夷些胳坞倦卷找赛奸背颖摧驳涯询挖舔臼盆狙锡兴Ti质粒与植物基因组的相互作用Ti质粒与植物基因组的相互作用,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,刷询翰词崇还眺犁涕宦肾励仑蓄烦炽豁嚎岁森叭惊锌翟第泊庭躁撒肃炕乎Ti质粒与植物基因组的相互作用Ti质粒与植物基因组的相互作用,The End,Thanks!,凸兑融惋偷集训肮洁虽节胖订钳例扳烟毁审膏袁捉毕氛碎既崔抄挖恳弊犁Ti质粒与植物基因组的相互作用Ti质粒与植物基因组的相互作用,