海南根瘤菌I66 nodD基因DNA片段的克隆  

Cloning of the DNA Fragment Containing nodD Gene of Rhizobium hamanense I66

作　　者：温尚昆[1] 李小红[1] 杨苏声[1] 

机构地区：[1]中国农业大学生物学院微生物学系,北京100094

出　　处：《农业生物技术学报》2002年第1期50-52,共3页Journal of Agricultural Biotechnology

摘　　要：从海南根瘤菌（Rhizobium hainanense）I66提取总DNA,用Xba I完全酶切,然后用苜宿中华根瘤菌（Sinorhizobiummeliloti）042B的nodD DNA片段作探针进行Southern杂交,发现2条含有nodD基因DNA片段的阳性条带,分别为4.0和6.0kb。电泳回收含有4.0～6.0kb的DNA片段,用pUC18作载体连接,并转化E.coliDH5α,构建含有nodD的部分基因文库。提取该文库的质粒与nodD探针做点杂交,筛选到nodD的阳性克隆,称为pUOD75。再将pUOD75的nodD片段克隆到pBBR-MCS-5,构建成pBOD75。用两亲本杂交,把pBOD75转入豌豆根瘤菌蚕豆生物型（Rhizobium　eguminosarum bv.viciae）LPR5045,用类黄酮化合物毛地黄黄酮和染料木黄酮分别诱导其转化子。结果发现被毛地黄黄酮诱导的转化子显现蓝色。海南根瘤菌I66 nodD基因在毛地黄黄酮诱导下得到表达,说明其表达产物可能具有结瘤调节功能。Total DNA of Rhizo bium.hainanense I66 was digested with Xba I enzyme ,and the digested DNA was carried out by Southern hybridization with Sinorhizobium meliloti nod D probes.The result showed that there were two positive fragments containing nodD genes.The partial library was constructed by inserting the 4.0 kb and 6.0 kb DNA fragments of the total DNA digested with Xba I into pUC18. By Southern dot blot analysis of the plasmids in the partial library,two positive clones containing nodD were obtained.and one of them was named pUOD75.The pBOD75 was constructed by inserting nodD fragment to pBBR-MCSS-5 and transferred into Rhizobium leguminosarum bv.viciae LPR5045,blue colour was shown on the plate by induction of luteolin.The results indicated that the pBOD75 had the function of nodD gene.

关 键 词：海南根瘤菌 nodD基因 DNA片段 基因克隆 

分 类 号：S154.381[农业科学—土壤学] Q939.114[农业科学—农业基础科学]