The Bioinformatics Analysis And The Expression Study Of ButA Gene From Tetragenococcus Halophilus CICC10469

Tetragenococcus halophilus CICC10469 was found in the soy sauce brewing process,it is able to produce amino-acids and other substances under 12-18 % of the salt concentrations.This paper mainly focused on the structure prediction and expression level of the salt tolerance-rated gene butA.In the first part,several bioinformation softwares were used to analyze the protein ButA systematically and comprehensively.According to the preliminary inference by PSI-BLAST,the protein ButA may be a permease of the BCCT family.The protein also displayed the SWTLf YWa WW motif in the location of amino acids numbered 353 to 367.The phylogenetic tree showed that was provide credible basis to study ButA.The Prot Param analyzed the physical and chemical properties of ButA.Prot Scale analyzed ButA has several hydrophobic peptides.The TMHMM analyzed ButA has 12 transmembrane regions.COILS and PSIPRED predicted ButA has random coils.The bioinformatics were used to study how ButA transprot GB,and provided basis theory to explore the salt tolerant mechanisms.In the second part,we used the real-time fluorescence relative quantitative(RT-q PCR)technology.The RT-q PCR analyzed the expression of butA gene under the different salt concentrations.Results showed that Na~+ regulate the butA expression.Under hypertonic environment(> 1 M),butA expression increased by the Na~+,while in low permeability environment(< 1 M),butA expression increased firstly,and then decreased with the increase of Na~+.The T.halophilus membrane proteins were isolated by Triton X-114,and tested by SDS-PAGE.By the iodine crystal precipitation to measured the contents of intracellular glycine betaine(GB)from different salt tolerate T.halophilus.Results showed that in both hypertonicity and hypotonicity,the intracellular GB was increased with Na~+.At the last part,the pET32a(+)– butA vector was transformed into E.coli BL21,and the IPTG used to induce expression.And then,the HIS label used to purified protein.The AKTA Explorer 100/10 protein purification system determined a 68 mM imidazole concentration elution effect was the best.Then,the His Trap HP gravity column with 68 mM imidazole Elution Buffer was used to get a vast purified protein.By the iodine crystal precipitation to measured the contents of intracellular GB.Results showed that whether the presence or absence of high permeability stress,ButA protein heterologous expression were raised the ability of E.coli BL21 transport GB.