1株中度嗜盐菌质粒基因组文库的构建及2个膜蛋白基因的克隆和序列分析

Construction of Plasmid Genomic Library of One Moderately Halophilic Bacteria & Cloning,Sequence Analysis of Two Membrane Protein Genes

中度嗜盐菌是生活在高盐环境中的微生物,以1株从蜢子虾酱中分离的中度嗜盐菌盐脱氮枝芽胞杆菌(Virgibacillus halodenitrificans)为研究对象,通过酶切,筛选DNA片段然后连接pUC19载体并转化大肠埃希菌,构建了该菌株的质粒基因组文库,推测可获得5×103个阳性克隆,覆盖约15 Mb的遗传信息。随机选取部分克隆进行测序,在测序结果中筛选到了2个具有完整ORF的膜蛋白基因分别为编码Na+H+逆向转运蛋白的NhaC基因,编码一离子通道蛋白的MscS基因,GenBank登陆号分别为JX849200、JX849202。其中NhaC基因ORF全长为1 446 bp,编码481个氨基酸,包括10个跨膜结构域;MscS基因的全长ORF为822 bp,编码273个氨基酸的蛋白质,包括3个跨膜区,与其他菌种的序列比对表明第3个跨膜区保守性最强。

Moderately halophilic bacteria are microorganism living in high salt environment. A moderately halophilic bacteria Virgibacillus halodenitrificans was separated from a shrimp paste as research object. Plasmid genomic library of the strain was constructed. DNA fragments were selected after genomic DNA digestion by restriction enzyme, and then were linked with pUC19 vector and transformed into E. coli to construct the plasmid genomic library of the strain. It was speculated that about 5 × 10^3 positive clones were obtained, covering about 15 Mb of genetic information. Part of the clones was selected to carry out sequencing. From the sequencing results two membrane protein genes possessing complete ORF respectively encoding NhaC gene of Na＋ H＋ reverse transporter protein, MscS genes of encoding an ion channel protein were screened. The GenBank accession numbers of the two genes were JX849200, JX849202 respectively. Among them the NhaC gene was 1 446 bp length ORF, encoded 481 amino acids, including 10 transmembrahe domains. MscS gene had 822 bp length ORF, encoded a protein of 273 amino acids, including 3 transmembrane domains. The sequence comparison analysis with other strain seeds indicated that the third transmembrane domain＇ s conservativeness was the strongest.