枯草芽孢杆菌渗透压调节基因proB的克隆和表达

用PCR扩增的方法从耐盐的枯草杆菌中克隆出一个13kb长的DNA片段，经功能检测，证明正向插入片段与大肠杆菌的脯氨酸营养缺陷特性(proB-)能够营养互补。含有该重组质粒的大肠杆菌DH5α在基本培养基上的耐盐能力从2%提高至4%。通过引物步行法测定了该插入片段的核苷酸序列。利用DNAsis软件进行序列分析发现，该片段第122～1235bp核苷酸编码一个由370个氨基酸组成的蛋白质分子，其上游存在非典型的-10区，典型的-35区和核糖体结合位点，起始密码子处有最佳翻译起始效率的侧翼核苷酸序列。将其与Genebank中的已知基因的序列和编码的氨基酸序列进行同源性比较，结果表明该片段与枯草杆菌168的核苷酸序列、氨基酸序列的同源性分别为81%和90%。证明该基因确实是一个proB基因。通过与三十个不同种属微芽生物proB基因的氨基酸序列比较，发现该蛋白存在有可能与形成酶的活性中心和三维结构有密切关系的几个绝对保守的区域。

Cloning and Expression of a Osmoregulatory Gene pro B from Halotolerant Bacillus subtilis

A 1.3 kb fragment is cloned from halotolerated Bacillus subtilis 93151 with PCR amplification method, and its positively-directionally inserted fragment can complemented with proB-E. coli by function test. Halotolerated ability of E. coli DH5 alpha having this recombination plasmid rises from 2% to 4% in minimal medium. The nucleotide sequence of this fragment is obtained by primer walking method. Nucleotide sequence of this fragment 167-1269 bp translates a protein which has 370 amino acid by sequence analysis through DNAsis program. There are non-typical-10 sequences, typical-35 sequences and a Ribosome binding site of this fragment in its upstream sequence, and there is flanking sequence, which has best efficiency of beginning translating. Homologues comparision, of nucleotide and amino acid sequences of this fragment and those of gene in gene bank shows that homogenous of Nucleotide sequences and amino acid sequences of this fragement and Bacillus subtilis 168 are respectively 81%, 90%, which prove that this gene is certainly a pro B gene. This protein translated by this fragment has several absoluter conservative domain which have been correlating closely with forming active center of enzyme and tri-dimension structure of active center, compared amino acid sequences of this fragment and pro B genes of thirty kinds of different microorganism.