转座子标签法对阴沟肠杆菌B8拮抗水稻白叶枯病菌相关基因片段的克隆与检测  

Cloning and detection of the antagonistic related genes of enterobacter cloacae B8 to Xanthomonas oryzae pv.oryzae with transposon tagging method.

作　　者：黄海宁[1] 余旭平[1] 陈卫良[1] 龚鸿飞[1] 李德葆[1] 

机构地区：[1]浙江大学生物技术研究所,浙江杭州310029

出　　处：《浙江大学学报（农业与生命科学版）》2006年第3期265-269,共5页Journal of Zhejiang University:Agriculture and Life Sciences

基　　金：国家863"基金资助项目(104-04-01-01)

摘　　要：阴沟肠杆菌B8是从水稻叶面分离获得的具有拮抗水稻白叶枯病菌等多种病原细菌的广谱拮抗菌.为研究其拮抗机理,我们应用转座子标签法对阴沟肠杆菌B8中与抗水稻白叶枯病菌拮抗活性相关的DNA片段进行了克隆.通过自杀性质粒pZJ25介导,将Tn5转座到B8的染色体上,从而筛选到2株拮抗活性丧失的菌株B8B和B8F;用Tn5片段为探针,分别对B8、B8B、B8F进行Southern杂交,结果在B8中无特异条带,而在B8B和B8F中各有一条约20 kb和9 kb的EcoRⅠ特异条带;分别提取二株突变菌株的基因组DNA,BamHⅠ酶切,克隆于pBS的BamHⅠ位点上,利用Tn5上的Kan抗性,分别筛选到质粒pTLB和pTLF,获得包含Tn5部分序列的B和F二个基因片段,大小约为6 kb和7 kb;应用pTLB全质粒和pTLF中约0.8 kb的BamHⅠ+HpaⅠ外源F片段为探针,对B8、B8B、B8F进行Southern杂交,证实我们克隆到Tn5转座位点周围的二个片段,且他们分别位于B8菌株染色体DNA的二个不同位置.Enterobacter cloacae B8, which was firstly isolated from the surface of rice leaves, is an antagonistic bacterium against Xanthomonas oryzae pv. oryzae and many other bacteria pathogens. For the understanding of the antagonistic mechanism of E. cloacae B8 against X. oryzae pv. oryzae, transposon tagging technique was adopted to clone antagonistic related genes of E. cloacae B8. Mediated with suicidal plasmid pZJ25, Tn5 transposed to the chromosome of B8, and resulted in the isolation of two strains named BSB and B8F, which lost the antagonistic character. Southern blot of EcoRⅠ cut DNA of BS, BSB, BSF with Tn5 fragment as probe showed that there was a band about 20 kb or 9 kb in size in BSB and BSF respectively, but no band in BS. Genomic DNA of two mutated strains were isolated respectively, cut with BamHⅠ , ligated to BarnHⅠ cut vector pBS. Using the Kan resistance of Tn5 fragment, two plasmids named pTLB and pTLF were isolated, and two DNA fragments of B and F with Tn5 about 6 kb and 7 kb were obtained. Southern blot of B8, B8B, BSF DNA with probe of total plasmid pTLB or 0.8 kb BamHⅠ＋HpaⅠ foreign DNA fragment of pTLF （the F fragment） showed that two DNA fragments around the transposition sites were cloned, which were in two different positions in B8 geonome.

关 键 词：阴沟肠杆菌B8 水稻白叶枯病 拮抗相关基因 克隆 

分 类 号：Q785[生物学—分子生物学]