Cloning, Bioinformatics and Activity Analysis of the MnhB Gene from the Moderately Halophilic Bacterium Halobacillus Y5

Abstract

Original Research Article Objective: The mnhB gene of Halobacillus Y5 was cloned to clarify its basic biological information. Methods: The genome of Halobacillus Y5 was extracted and the gene fragments were obtained by PCR, then 400~900 bp fragments were recovered and linked to vector pUC18. The constructed recombinant plasmids were transformed to competent E.coli KNabc by functional complementation, and the strain containing the gene of Na + /H + antiporters was screened for bioinformatics analysis. The positive monoclonal strains were screened by specific medium. Results: mnhB gene has two CDS regions, respectively 305 bp and 128 bp, encoding 96 amino acids. The predicted relative molecular weight is 11048.16 Dt, the isoelectric point is 4.66, the fat coefficient is 130.40, and the instability coefficient is 29.15. It is a stable protein with a half-life of 30 h. Comparison of similarity between mnhB from Halobacillus Y5 and 5 sequences, the homology of wp_101843976.1, wp_082234107.1, wp_079529991.1, wp_128522668.1, wp_08502981.1 were 93%, 69%, 78%, 69%, 67%, respectively. Through phylogenetic tree analysis, the Na + /H + antiporters from Halobacillus Y5 is in a separate branch, which may be a new member of Na + /H + antiporter. Na + (Li + , K)/H + antiport activity was detected from everted membrane vesicles prepared from E. coli /pUCmnhB but not those of /pUC18. Conclusion: A new mnhB gene was successfully cloned, which laid a theoretical foundation for the development and utilization of Halobacillus Y5.