自贡大公古盐井中嗜盐菌的Na~+/H~+离子逆转运蛋白基因筛选(英文)

目的]筛选四川自贡大公古盐井中嗜盐微生物的Na+/H+离子逆转运蛋白基因,并对其结构和编码蛋白进行分析。方法]构建自贡大公古盐井中嗜盐微生物的Na+/H+离子逆转运蛋白宏基因组文库,通过与Na+/H+离子逆转运蛋白基因缺陷宿主菌株E.coliKNabc的功能互补筛选Na+/H+离子逆转运蛋白基因。并通过生物信息学方法对该基因的起始密码子、终止密码子、ORF、-35区、-10区和SD序列以及编码蛋白的分子量、等电点、疏水区域、跨膜区域、系统进化和耐盐特性进行分析。结果]筛选到1个新的Na+/H+离子逆转运蛋白基因m-nha,该基因能够赋予E.coliKN-abc在盐和碱性条件下良好生长的能力。结论]由于m-nha编码蛋白在氨基酸序列和结构上不同于以往报道的Na+/H+离子逆转运蛋白,故鉴定该基因为1个新的Na+/H+离子逆转运蛋白基因。该研究结果对于理解古盐井中嗜盐微生物的嗜盐机制,开发利用古盐井中的基因资源及寻找新的耐盐基因具有重要的意义。

Screening an Na~+/H~+ Antiporter Gene from the Halophiles Colonizing in the Dagong Ancient Brine Well of Zigong City,China

Objective] This study aimed to screen an Na+/H+ antiporter gene from the halophiles colonizing in the Dagong Ancient Brine Well in Zigong City,China,and then analyze the gene structure and properties of the protein encoded by this gene.Method] Metagenomic DNA libraries of halophiles from the Dagong Ancient Brine Well were used for screening genes with Na+/H+ antiporter activity in antiporter-deficient E.coli KNabc strain by functional complementation.Then the start codon,stop codon,ORF,-35 region,-10 region and SD sequence of Na+/H+ antiporter gene,as well as the molecular weight,isoelectric point,hydrophobic region,transmembrane domain,phyletic evolution and salt resistance of protein encoded by the gene were investigated.Result] A new Na+/H+ antiporter gene m-nha was obtained,which rendered the antiporter-negative mutant E.coli KNabc cells with both the resistance to Na+ and the ability to grow under alkaline conditions.Conclusion] The structure and amino acid sequence of M-Nha was different from the previously reported Na+/H+ antiporters,and the m-nha gene disclosed from the Dagong Ancient Brine Well was identified as a novel Na+/H+ antiporter gene.This study was significant not only in helping us understand the salt tolerance of halophiles in ancient brine wells and develop and utilize the genes resource,but also in exploring new salt-tolerant genes.