硝酸菌norB基因的克隆及序列分析

Cloning and Sequencing of NorB Gene of Nitrobacterium

目的扩增、克隆硝酸菌的norB基因。方法通过PCR扩增硝酸菌的norB基因,然后克隆至T载体中,并转化进E.coliJM109中。通过兰白斑挑选阳性转化子。对质粒测序,并与Genbank上的序列进行同源性分析。结果同源性搜索发现所有扩增到的基因无论在核酸序列水平还是在蛋白序列水平都与Genbank上的汉堡硝化杆菌norB基因同源,且同源性高达96%～98%。结论扩增获得的基因经过基因序列分析证实为硝酸菌norB基因。

Objective To amplify and sequence the distinctive norB gene of Nitrobacterium. Methods The norB gene was amplified by PCR and was cloned into T-vector and then transformed into E.coli JM109. After white-and-blue selection, positive colonies were sequenced with T7 sequencing primers by Takara Company. Spliced with Vector NTI Suite software, the homologous analysis of sequences were conducted in Genbank through Internet. Results The results showed that all of norB genes obtained from the nitrite-oxidizing bacteria were homologous with the norB gene of Nitrobacterium hamburgensis in Genbank. The homology ranged from 96% to 98%, and there were no frameshift mutations, which guaranteed the correct ORF. Conclusion The obtained genes are norB genes indeed.