1、農(nóng)桿菌介導(dǎo)轉(zhuǎn)化法 農(nóng)桿菌是普遍存在于土壤中的一種革蘭氏陰性細(xì)菌，它能在自然條件下趨化性地感染大多數(shù)雙子葉植物的受傷部位，并誘導(dǎo)產(chǎn)生冠癭瘤或發(fā)狀根。,1,專業(yè)精制,農(nóng)桿菌 感染柳樹 產(chǎn)生 冠癭瘤,2,專業(yè)精制,原理： 根癌農(nóng)桿菌和發(fā)根農(nóng)桿菌細(xì)胞中分別含有Ti質(zhì)粒和Ri質(zhì)粒，其上有一段T-DNA，農(nóng)桿菌通過侵染植物傷口進(jìn)入細(xì)胞后，可將T-DNA插入到植物基因組中。,3,專業(yè)精制,因此，農(nóng)桿菌是一種天然的植物遺傳轉(zhuǎn)化體系。人們將目的基因插入到經(jīng)過改造的T-DNA區(qū)，借助農(nóng)桿菌的感染實(shí)現(xiàn)外源基因向植物細(xì)胞的轉(zhuǎn)移與整合，然后通過細(xì)胞和組織培養(yǎng)技術(shù)，再生出轉(zhuǎn)基因植株。 農(nóng)桿菌介導(dǎo)法起初只被用于雙子葉植物中

2、，近年來，農(nóng)桿菌介導(dǎo)轉(zhuǎn)化在一些單子葉植物（尤其是水稻）中也得到了廣泛應(yīng)用。,4,專業(yè)精制,5,專業(yè)精制,6,專業(yè)精制,Story 冠癭瘤病：雙子葉植物經(jīng)常發(fā)生，因腫瘤著生地面在近地面的根莖交界處，形似帽狀而得名。 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 Vi

3、rE2:ssb protein,nls,enhances the efficincy of t-strand transport into the nucleus VirC: Virc1 and Virc2 bind to overdrive,55,專業(yè)精制,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

4、its mobilization to the plant cell. Because its transfer 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,unpubli

5、shed results)in trans to the ti plasmid vir region.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).,56,專業(yè)精制,6)T-DNA integration integration sites: random? hot spots(actively transcribed)? homology between plant junctions and T-DNA sequences? Model:illegitimate recombination,57,專業(yè)精制,Table1 Summary of vir G