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

LIANG Hua-zhong et al(Key Laboratory of Food Biotechnology of Sichuan,College of Bioengineering,Xihua University,Chengdu 610039)

Na+/H+ antiporter gene;Metagenomic library;Halophiles;Dagong Ancient Brine Well

[Objective] This study aimed to screen a 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 protein encoded by Na+/H+ antiporter gene.[Method] Metagenomic DNA libraries of halophiles from the Dagong Ancient Brine Well were screened for genes with Na+/H+ antiporter activity on the 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 in not only helping us understand the necessity of the existence of Na+/H+ antiporter in the Dagong Ancient Brine Well to maintain the intracellular environment homeostasis for halophiles,but also enriching our knowledge about the different mechanisms of Na+/H+ antiporter in halophiles in such an extreme environment.