革兰氏阳性中度嗜盐菌甘氨酸甜菜碱转运蛋白基因的克隆和功能分析微生物学专业论文.docxVIP

AbstractHalobaeillus Abstract Halobaeillus trueperi was isolated from hypersaline sediments of the Great Salt Lake in U．S．A， which could endure salt concentration from O．5％to 25％NaCI(w／v)．and grow optimally in media containing 5％一10％NaCI． Halobaeillus trueperi accumulates glycine betaine under condition of high osmolarity．A partial fragment of the glycine betaine transporter 6efH gene was obtained from the genome of／／．trueperi with degenerate primers．Through Southern blot hybridization and inverse PCR，a 5．1 kb EcoRl fragment containing the beth gene was sequenced．The betH gene was predicted to encode a 55．2 kDa protein (504 amino acid residues)with 12 transmembranes regions．BetH showed 56％identity to the OpuD of Bacillus subtilis and belonged to BCCT family．Its putative promoter region was highly homologous to aB-dependent promoter of丑subtilis．A 2．6 kh fragment including the betH gene was subeloned into pUCl8 and transformed into the丑eoli MKHl3。colonies appeared on the plate ofthe selective M9 medium．The accumulation of glycine betaine in E coli MKHl3 was examined using”C nuclear magnetic resonance spectroscopy． Depending on the conserved amino acids of glyeine betaine ABC transporter,a pair of degenerate primers were designed．With H trueperi genemic DNA and degenerate primers．a 560 bp PCR fragment was obtained and labeled as a probe．After／-／．trueperi genomic DNA was digested with different endonucleases，Southern blot result showed a 2．6 kb positive fragment digested by EeoRl and IPCR was carried out to obtain the flanking sequence．BLAST result showed the fragment containing the opuAA and its 5’upsteam sequence was obtained．Then，a piece of degenerate primer was designed according to the conserved amino acids ofglycine betaine ABC transporter system glyeine betaine-binding protein as a reversed primer．Combined with the opuAA-up。a 2．3 kb fragment was obtalned through PCR。 BLAST result showed a fragment which contained the partial opuAA，the whole opuAB and partial opuA