农杆菌介导法.ppt

1、农杆菌介导转化法 农杆菌是普遍存在于土壤中的一种革兰氏阴性细菌，它能在自然条件下趋化性地感染大多数双子叶植物的受伤部位，并诱导产生冠瘿瘤或发状根。,农杆菌 感染柳树 产生 冠瘿瘤,原理： 根癌农杆菌和发根农杆菌细胞中分别含有Ti质粒和Ri质粒，其上有一段T-DNA，农杆菌通过侵染植物伤口进入细胞后，可将T-DNA插入到植物基因组中。,因此，农杆菌是一种天然的植物遗传转化体系。人们将目的基因插入到经过改造的T-DNA区，借助农杆菌的感染实现外源基因向植物细胞的转移与整合，然后通过细胞和组织培养技术，再生出转基因植株。 农杆菌介导法起初只被用于双子叶植物中，近年来，农杆菌介导转化在一些单子叶植物（

2、尤其是水稻）中也得到了广泛应用。,Story 冠瘿瘤病：双子叶植物经常发生，因肿瘤着生地面在近地面的根茎交界处，形似帽状而得名。 1907年， Smith pilot protein? nuclear localization,nuclear localization signal(nls)?(vird2) overdrive(24bp DNA sequence):an enhancer,stimulates T-DNA transfer,enhances t-dna copy number SS DNA VirE2:ssb protein,nls,enhances the efficincy

3、 of t-strand transport into the nucleus VirC: Virc1 and Virc2 bind to overdrive,two conserved domains of 13 and 5 to 7 bp as follows: TGGATATAT TG N PuG TGTAAAT NC CT TC Only these 25bp direct repeats at the ends of the T-DNA are required in cis for its mobilization to the plant cell. Because its tr

4、ansfer is unaffected by (a)deletions of the internal portion of native T-DNAs or (b)placement of cloned fragments carrying only T-DNA border repeats,either on a separate plasmid (19,36)or on the Agrobacterium chromosome (37;s.satchel,d.corbin,unpublished results)in trans to the ti plasmid vir region

5、.these findings form fhe basis of the design of modified and simplified t-dna molecules fhat are useful as vectors to transform plant cells with cloned dna fragments of interest(57,72,113).furthermore,deletion of the first 6 bp or the last 10bp of the 25bp sequences blocks t-dna transfer(101).,6)T-DNA integration integration sites: random? hot spots(actively transcribed)? homology between plant junctions and T-DNA sequences? Model:illegitimate recombination,Table1 Summary of v