喜盐芽孢杆菌D8基因文库的构建及甘氨酸甜菜碱转运蛋白betH基因的筛选

喜盐芽孢杆菌(Halobacillus)D8是一株产生芽孢的革兰氏阳性中度嗜盐菌,能够耐受2 5 %NaCl。以其总DNASau3AI部分酶切的片段为供体、pUC18为载体,构建了该菌株的基因文库,共获得约90 0 0个重组质粒。通过菌落原位杂交、菌落PCR检测及DNA序列测定,从该文库中筛选到含有完整的甘氨酸甜菜碱次级转运系统基因的重组质粒,将此基因命名为betH基因。序列分析发现,betH基因的大小为15 15bp ,编码由5 0 5个氨基酸组成的BetH蛋白,分子量为5 6 1kD。经蛋白疏水性分析,推测为含有12个跨膜区的跨膜蛋白,与Oceanobacillusiheyensis甘氨酸甜菜碱转运蛋白、枯草芽孢杆菌(Bacillussubtilis)OpuD、楚氏喜盐芽孢杆菌(Halobacillustrueperi)BetH、单核细胞增生利斯特氏菌(Listeriamonocytogenes)BetL、嗜盐海球菌(Marinococcushalophilus)BetM和耐盐芽孢杆菌(Bacillushalodurans)甘氨酸甜菜碱转运蛋白的氨基酸同源性分别为6 4 %、5 1%、4 9%、4 8%、4 3%和4 4 %。

Construction of the genomic library of Halobacillus sp. D8 and isolation of the glycine betaine transporter betH gene

Halobacillus sp. D8 is a sporing-forming , gram-positive moderately halophilic bacterium which could tolerate up to 25%(W/V) NaCl. A genomic library ofHalobacillus sp. D8 was constructed using pUC18 as vector, and 9000 recombinant plasmids were obtained. By dot blot hybridization, colony PCR and DNA sequencing, the entire glycine betaine transporterbetH gene was isolated from the constructed library. Inspection of the sequenced 4.3 kb DNA region revealed the presence of three ORFs. The putative ORF ofbetH is 1515bp long, encoding a 505-residue protein (BetH) with a calculated molecular mass of 56.1kD. Hydrophobicity plot analysis of BetH indicated a transmembrane protein containing 12 transmembrane regions. Homology searches for BetH of strain D8 in the GenBank using the BLAST program revealed significant sequence identities to other glycine betaine transporters: the putative glycine betaine transporter ofO. iheyensis(64% identity), OpuD ofB. subtilis(51% identity), BetH ofH. trueperi(49% identity), BetL ofL. monocytogenes (48% identity), BetM of M. halophilus(43% identity) and the putative glycine betaine transporter ofB. halodurans(44% identity).