枯草芽孢杆菌proA基因突变对提高大肠杆菌转化子渗透压耐受能力的影响  

Influence of the Mutated proA Gene from Bacillus subtilis on Improving Osmotic Tolerance of Escherichia coli Transformant

作　　者：魏红波[1] 曹军卫[1] 陈明清[1] 刘瑞杰[1] 

机构地区：[1]武汉大学生命科学院,湖北武汉430072

出　　处：《武汉大学学报（理学版）》2006年第2期207-212,共6页Journal of Wuhan University:Natural Science Edition

基　　金：武汉大学科技创新基金(204270023)

摘　　要：将枯草芽孢杆菌93151野生型菌株的proB基因和耐盐突变株93151-14的proA基因,进行体内重组,筛选出含有野生型proB和突变型proA杂合基因的转化子.此杂合基因能够与脯氨酸营养缺陷型大肠杆菌JM83功能互补.分别测定了大肠杆菌JM83三种转化子(分别含有野生型proBA基因,双突变proBA基因和杂合proBA基因)的耐盐能力,发现含有杂合proBA基因转化子的耐盐能力(0.45 mol/L)尽管比含有双突变proBA基因的转化子(0.5 mol/L)要低一些,但比含有野生型proBA基因的转化子(0.3 mol/L)要高得多.测定了3种转化子在不同盐浓度下生长时的胞内自由脯氨酸含量,发现其含量均随着盐浓度的上升而提高,表明其耐渗透压胁迫能力的提高与胞内自由脯氨酸的积累密切相关.但在相同盐浓度下,含有杂合proBA基因转化子的胞内自由脯氨酸含量要低于含有双突变proBA基因的转化子的胞内自由脯氨酸含量,但明显高于含有野生型proBA基因的转化子的胞内自由脯氨酸含量.说明尽管proB基因的突变在提高细胞耐高渗胁迫的能力中,有重要作用但proA基因的突变也发挥了不可忽视的作用.ProB gene from the wild type strain of Bacillus subtilis 93151 and proA gene from the salt tolerant mutant of Bacillus subtilis 93151-14 were amplified using PCR method respectively. PCR products were ligated into the both sides of the linear plasmid pBE2 digested by using pst Ⅰ and Sac Ⅰ. The ligation mixtures were transformed into proline auxotrophic strain Escherichia coli JM83 for in vivo recombination. Tbe transformant harboring the recombination vector containing the wild-type proB gene and the mutated proA hybrid gene were selected by functional complementation of E. coli JM83. The salt-tolerance of three transformants （containing the hybrid proBA gene, the wild type proBA gene and the mutated proBA gene, respectivly） was examined. The salt-tolerance of transformant containing the hybrid proBA gene （0.45 mol/L） is higher than thai of transformant containing the wild type proBA gene （0.3 mol/L）, although the salt-tolerance of it is lower than that of transformant containing the mutated proBA gene （0.5 mol/L）. Intracellular free proline levels of three E. coli JM83 transformants increased in response to the elevated salt concentration. It is suggested that the accumulation of intracellular free proline play a particularly important role in improving the osmotolerance stress of E. colitransformant. In the same salt concentration, intracellular free proline level of transformant containing the hybrid proBA gene was lower than that of transformant carrying the mutated of proBA gene, but it is higher than that of transformant containing wild-type proBA genes obviously. The contribution of the mutation of proA gene could not be neglected in the improvement of the osmotic stress tolerance,although the mutation of proB gene was a key factor in improving osmotic stress tolerance. osmotic stress tolerance;

关 键 词：枯草芽孢杆菌耐盐突变株 proA基因突变 渗透压胁迫 脯氨酸合成 

分 类 号：Q754[生物学—分子生物学]