[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.