青枯菌M5菌株特异基因组消减文库的构建

Construction of a Subtracted Gene Library of Ralstonia solanacearum M5

为筛选青枯菌M5菌株特有的序列,利用SSH技术对青枯菌生理小种5号菌株M5(tester)和生理小种1号菌株GMI1000(driver)基因组进行了比较。分别提取二者基因组DNA,RsaⅠ酶切,将酶切后M5菌株DNA分为两份,分别与接头1和接头2R连接,然后进行两轮杂交和两轮PCR扩增,并检测连接效率与消减效率。将获得的消减PCR产物,即差异基因/差异DNA片段,与pGEM-T载体连接,转化E.coli DH5α感受态细胞,建立消减文库。随机挑取96个白色克隆,经菌液PCR法测定,80个为预期阳性,含有预期的插入片段。首次构建了青枯菌M5菌株特异基因的抑制消减文库,为进一步筛选、克隆青枯菌M5菌株特异核苷酸片段奠定了基础。

To screen the specific sequences of Ralstonia solanacearum race 5 strain M5, SSH technique was used to compare the genomic differences between M5 （tester） and GMI1000 （driver, race 1）. The genomic DNA of M5 and GMI1000 were isolated and digested with Rsa I to give a range of blunt-ended fragments respectively. The digested tester DNA was then subdivided into two portions, which were ligated with adaptor 1 and adaptor 2R. Following twice hybridizations and two times PCR, the efficiency of ligation and subtraction detected. The subtracted fragments were cloned into pGEM-T vector and transformed into E.coli DH5a competent cells to construct a subtracted DNA library. From 96 clones picked randomly, 80 clones containing a 100-500bp insert were identified to be positive by PCR analysis. It＇s the first construction of subtracted gene library for screening the race 5 specific genomic DNA.