作　　者： ; ; ; ;

机构地区： 中国农业大学生物学院农业微生物资源及其利用农业部重点实验室

出　　处： 《农业生物技术学报》 2003年第6期628-632,共5页

摘　　要： 从海南根瘤菌(Rhizobium hainanense)166提取总 DNA,用 EcoR Ⅰ酶进行完全酶切和电泳,然后用苜蓿中华根瘤菌(Sinorhizobium meliloti)042B 的 nodABC DNA 片段作探针进行 Southern 杂交,发现1条含有 nodABC 基因 DNA 片段的阳性条带,大小为2.1～2.5kb。通过电泳回收该 DNA 片段,用 pUC18作载体连接,并转化大肠杆菌(Escherichia coli)DH5α,从而构建了含有 nodABC 的部分基因文库。提取该文库的质粒与 nodABC 探针做菌落杂交,筛选到 nodABC 的阳性克隆,称为pUER12。将含有 nodABC 的 DNA 片段与 EcoR Ⅰ酶切的 pBBR-MCS-5连接,转化大肠杆菌 DH5α,获得转化子 pBER12。用三亲本杂交方法,把 pBER12转入苜蓿中华根瘤菌 AK1657,将其接合子进行结瘤实验,验证了 pBER12具有 nodABC 基因的功能。然后将含有166 nodABC 片段的 DNA与 EcoR Ⅰ酶切的 pGEM-7Z(+)连接、测序和进行同源性比较,发现166 nodABC 编码的蛋白与苜蓿中华根瘤菌具有高度的同源性。 Total DNA of Rhizobium hainanense I66 was digested with Eco R Ⅰ,and the digested DNA was carried out by Southern hybridization with Sinorhizobium meliloti nodABC as probe.The result showed that there was a positive DNA fragment containing nodABC genes.The partial library was constructed by inserting the 2.1～2.5 kb DNA fragments of the total DNA digested with Eco R Ⅰ into pUC18 plasmid.One positive clone was obtained by Southern blot of the colonies in the partial library,and it was named pUER12.The pUER12 was digested by Eco R Ⅰ,and the DNA fragment containing nodABC was ligated with pBBR-MCS-5, then transformed to Escherichia coli DH5 α and obtained pBER12.The pBERI2 containing I66 nodABC gene was transferred into S.meliloti pAK1657 by triparental mating.The results indicated that the pBER12 had the function of nodABC genes.I66 nodABC were sequenced and their nucleotide acid and amino acid were compared with other rhizobia nodABC genes,and showed that the proteins encoded by I66 nod ABC were highly homologous to Sinorhizobium meliloti.

关 键 词： 海南根瘤菌 基因 克隆 测序 功能

领　　域： [生物学]