巴西固氮螺菌ntrBC基因的克隆与核苷酸序列分析

以EMBL3为载体,构建了巴西固氮螺菌(Azospirillum brasilense)Yu62的基因文库.以巴西固氮螺菌Yu62中PCR扩增出的450bp DNA 片断作为探针,对该基因文库进行筛选,得到了10个阳性克隆(EA1—EA10),其中含有两种不同类型的克隆,分别以EA4和EA9为代表.对EA4的杂交分析发现目的基因位于2.9kb EcoRI片段上.DNA序列分析结果表明该片段含有完整的ntrC编码区,其编码产物由480个氨基酸组成.分子量为53469;ntrC上游是完整的ntrB编码区,其编码产物由400个氨基酸组成,分子量为43487.对相应的NtrC和NtrB氨基酸序列进行同源性分析,说明巴西固氮螺菌与根瘤菌的亲缘关系较与其它自生固氮菌的更为接近.

CLONING AND SEQUENCING OF ntrBC GENES FROM AZOSPIRILLUM BRASILENSE

A gene library of Azospirillum brasilense Yu62 was constructed inEMBL3. The library was screened with PCR amplified fragment as a special probe.Ten positive plaques (EA1-EA10) were selected. Detection results showed they contained two different types of clones, representing as EA4 and EA9 respectively.Southern hybridization of EA4 displayed that target gene was located in a 2.9kbEcoRI fragment. Sequence of this fragment had allowed the position and identification of ntrC gene, which encoding a protein of 53469, consisted of 480 aminoacids. In the upstream of ntrC, a complete ntrB coding region was also found,which encoding a protein of 43487, consisted of 400 amino acids. Homologousanalysis of the deduced amino acid sequences of ntrC and ntrB from different bacteria demonstrated that A. brasilense was closer to Rhizobia than to other free-livingdiazotrophs.