枯草芽孢杆菌耐盐突变株proA基因克隆及proBA基因渗透压调节功能研究

利用TaKaRaLAPCRTM试剂盒扩增枯草芽孢杆菌 931 5 1耐盐突变株proA基因的未知下游序列。根据测序结果 ,设计引物 ,克隆出发菌株和突变株全长proBA基因。将出发菌株和突变株的proBA基因分别转化大肠杆菌JM83(proBA- ) ,均能够与其功能互补。SDS PAGE分析其表达产物 ,有两条分子量分别约为 4 0kD和 4 5kD的新蛋白带出现。测定 4种转化子 (分别含有出发菌株和突变株proB基因的大肠杆菌 1 1 2 5 2转化子及proBA基因的大肠杆菌JM83转化子 )的耐盐能力。发现含有突变株proB或proBA基因转化子的耐盐能力 ,均比相应的含有出发菌株proB或proBA基因的转化子高。另外含有出发菌株和突变株的proBA基因转化子的耐盐能力 ,也均比相应的仅含proB基因的转化子高 ,表明枯草芽孢杆菌的ProA比大肠杆菌的ProA更为有效。测定所有JM83转化子胞内自由脯氨酸 ,发现其含量随盐浓度的上升而提高 ,其中含突变菌株proBA基因的转化子提高更为显著

proA Gene Cloning and Function Analysis of proBA Gene in Osmoregulation from A Salt-tolerant Mutant of Bacillus subtilis

The unknown downstream sequence of the proA gene from a salt tolerant mutant of Bacillus subtilis 93151 was cloned using TaKaRa LA PCR TM in vitro cloning kit. Based on the sequencing result, primers were designed to amplify the complete proBA genes from 93151 and its mutant. proBA gene expression could complement the proline auxotrophy E.coli JM83 Analysis of proBA gene products using SDS PAGE showed that two independent novel proteins with molecular weights of 40kD (ProB) and 45kD (ProA) appeared. The salt tolerance of the four transformants (1 1252 containing proB genes and JM83 carrying proBA genes from 93151 and its mutant) were examined. This showed that transformants containing the proB or proBA gene from the mutant were more resistant to osmotic stress than those from 93151, indicating that the mutation in proB gene assuredly improved their resistance to osmotic stress. Besides, JM83 transformants harbouring proBA genes were also more salt tolerant than 1 1252 transformants with just proB genes, suggesting that ProA of B. subtilis is more effective than that of E.coli. Intracellular free proline levels of JM83 transformants increased in response to the elevated salt concentrations, and the proline contents of JM83 transformant carrying the mutated proBA gene enhanced more remarkably than that of JM83 transformant carrying the proBA gene from 93151 It suggests that the improvement in osmotolerance of JM83 transformant carrying the mutated proBA gene relates closely with the accumulation of intracellular free proline.