Screening The Salt-tolerance Gene From Halophile Colonized In Ancient Brine Well By Metagenomic Library

The salt-tolerance breeding of bacteria is an important part and a heat spots insalt-tolerance organism engineering for a long time. With the developing of biotechnology, itis an important way that foreign DNA was transferred into the organism and expressedagainst the salt environmental stress by the recombinant DNA and transgenic technology. Soscreening the salt-tolerance genes has played a key role in the salt-tolerance breeding.The halobionts are important objects for screening the salt-tolerance gene. Brine Well, asa special man-made saline environments, has laying considerable halophiles. With thelong-term against with the salt stress, halophile has evolved an efficient mechanism to senseand transduct the salt signal and can specific expression some genes to resistance the saltstress.The salt-tolerance protein plays a critical role in extrusion of Na+to keep the cytoplasmisoosmotic with the environment and to avoid intoxication of living cells, and, its activity ofcell is essential for organism to survive saline conditions. So, in this study, we constructedmetagenomic library of halophiles from Brine Well to screening salt-tolerance genes to enrichthe genetic material of the salt-tolerance bacteria breeding.In this study, chromosomal DNA was prepared from cells harvested from aerobic growthat30°C、150r/min for14³0days in brine with1%casamino acids added and partiallydigested with Mbo I. The DNA fragments with1–10kb were separated by agaroseelectrophoresis and ligated into pUC18, which had been digested with BamHI anddephosphorylated with bacterial alkaline phosphatase, using T4DNA ligase. The recombinantplasmids were transformed into competent cells of E.coli KNabc, the antiporter-deficientstrain, to construct metagenomic library for screening the salt-tolerance genes conferringantiporter activity of the stains on the Amp-LBK plates with0.2mol/L NaCl by functionalcomplementation. Finally,98salt-tolerance clones, which mostly contain1-2kb foreign DNAfragment, has been obtained.The positive E. coli clones were sequenced and analytical resultshowed that these genes screened encoded proteins as flowing: AdoMet_MTasessuperfamily、 AAT-I superfamily、 PMSR superfamily、 OM-channels superfamily andArsB/NhaD permease superfamily, respectively.And, the gene belongs to ArsB/NhaDpermease superfamily is an ArsB permease gene, named K-ArsB （Accession: JN168102）. Thesalt-tolerance clones include K-ArsB named E. coil K-ArsB, and the protein encoded byK-ArsB named K-ArsB.With the bioinformatics study of the screened salt-tolerance gene K-ArsB, we found itcontain one open reading frame （ORF,993bp）, a start codon （ATG）, a start codon （TAG）, aShine-Dalgarno （AGGAGG）,-10region （GTTAAT） and-35region （TTGAT）. The ORF waspredicted to encode a protein consisted of330amino acid residue with a predicted molecular mass of34999.81Da and a pI of7.02. A comparison of topological model by PSIPREDrevealed that the ORF product were fold by α-helix and a little no-regular coil into itssecondary structure. The protein encoded by K-ArsB is an integral membrane protein, havebeen shown to translocate sodium, arsenate, antimonite, sulfate and organic anions acrossbiological membranes in all three kingdoms of life. A homology search revealed that K-ArsBhad the highest homology with the ArsB from Klebsiella sp. MS92-3（95%, accession:ZP₀8303599） and Escherichia hermannii NBRC105704（94%, accession: ZP₀9808970）and a lower similarity to the ArsB from Escherichia sp. TW09308（89%, ZP₀9461528） andSerratia proteamaculans568（87%, accession: YP₀01478619） and Yersinia frederikseniiATCC33641（87%, accession: ZP04631221）. Hydropathy analysis of the deduced proteinrevealed,9transmembrane segments were found by DAS program.The sodium dodecyl sulfate polyacrylamide gel electropheresis（SDS-PAGE） showed thatK-ArsB encode an specific protein with a molecular mass of34999.81Da. The Na+-resistantand alkali tolerance research showed that K-ArsB can significant enhance the E. coil KNabc’sability of Na+-resistant and alkali tolerance. So, the salt-tolerance gene K-ArsB screened bymetagenomic library is a satisfactory gene. This study was significant in not only helping usunderstand the mechanism of maintain the intracellular environment homeostasis forhalophiles in the Dagong Ancient Brine Well, but also significance to exploit and find newsalt-tolerance gene in it.