海南根瘤菌I66结瘤基因nodD的克隆及其功能检测-微生物学专业论文.docxVIP

摘要摘 摘要 摘 要 海南根瘤菌(Rhizobium hainanense)166分离自我国海南省。(是我国特 有的根瘤菌资源。]“ f提取166的总DNA， 经酶切验证其质量适合于构建部分基因文库。根据不 同根瘤菌的nod D基因的上下游保守序列设计引物，)，以166的总DNA为模板， 再以该引物进行PCR扩增，得到约700bp的片段／用地高辛标记法将其标记为探 针。 再将其与含s meJiloti的nod D基因的质粒进行Southern杂交呈阳性，)7。 证明所得片段为166 nod D基因的一部分。 用Sac I完全酶切Z66的总DNA后，f电泳，将凝胶从上到下分成5部分， 分别回收DNA，以其为模板，以上述引物进行PCR扩增，把呈阳性的片段连接 到pUCl8载体上，构建部分基因文库。提取转化子的质粒，以其为模板，以上 ^／ 述相同的引物进行PCR扩增，初步筛选阳性克隆，再以点杂交验证，j得到6个 阳性克隆，选取其一命名为pUCTD，／用SacI酶切质粒pUCTD，得到2．2kb的插 ＼ 入片段，由于插入的DNA片段的大小合适，故无需亚克隆，可直接将质粒送去 、，／ 测序。)测序结果显示为，166 nod口基因与s meliloti nod D基因高度同源。 ， 回收SacI酶切后2．2kb大小的插入片段，将其连接到具有广泛寄主的载体 pBBRlMCS．5上，筛选转化子，经酶切验证，得到pUCTD．1。三亲本杂交把 质粒pUCTD一1转入豌豆根瘤菌蚕豆生物型LPR5045中进行类黄酮诱导试验，结 果，所得的接合子能在含有毛地黄黄酮的FY平板上呈现蓝色，说明166的nodD 基因能接受苜蓿分泌的毛地黄黄酮的诱导，激活其他结瘤基因的转录，其NodD 蛋白具有结瘤调节的功能。 关键词：海南根瘤菌166，结瘤基因，PCR扩增筛选，功能检测 y √ × ∥ I !旦奎些查堂堡主堂丝堡兰————Abstract !旦奎些查堂堡主堂丝堡兰———— Abstract Rhizobium hainansense 166 was isolated from root nodules of Desmodium sinuatum from Hainan Province． The total DNA of 166 was extracted by the routine way and detected it by the enzyme digesting to make sure its quality be suitable for the constructing 166 nod D gene library．Conserved sequences were obtained as seven rhizobium nod D gene were contrasted using the software DNAMAN；And oIigonucleotide primers for PCR were designed from these sequences．Basing the 166 total DNA．PCR was performed and its product was hybridized with plasmid pYSDl亿ontainingmeliloti nO扣)by SouthelTl dot blot．The positive dot proved that the PCR reaction product containing 700bp DNA fragement was part of 166 nod D gene． Sacl was selected to digest the the 166 total DNA。Afler electrophoresis on a 1％agarose gel．the gel was cleaved into five parts from the upper to the bottom under the UV Extracted them from the gel and screened tflem by PCR．The positive bands containing 1．3—2．3 kb Sac／fragments were ligated with plasmid pUC l 8 and the partial 166 nod D gene library was constructed．Then the positive colonies containing nodD were found by the PCR