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【目的】探索大肠埃希氏菌Escherichia coli FtsZ突变体FtsZ~(E75A)、FtsZ~(R78G)和FtsZ~(D82A)对FtsZ自身组装和FtsZ-MreB相互作用的影响。【方法】利用常规分子克隆和定点突变技术,构建FtsZ及其突变体表达载体,亲和纯化得到相应的目标蛋白;通过同源重组构建QN6(ftsZ::yfp-cat)、QN7(ftsZ~(E75A)::yfp-cat)、QN8(ftsZ~(R78G)::yfp-cat)和QN9(ftsZ~(D82A)::yfp-cat)菌株;利用活细胞成像技术观察FtsZ及其突变体的胞内定位模式;免疫沉淀和细菌双杂交实验检测FtsZ/FtsZ*-FtsZ*或FtsZ/FtsZ*-MreB间的相互作用;光扫描检测定点突变对FtsZ组装特性的影响。【结果】FtsZ~(E75A)、FtsZ~(R78G)和FtsZ~(D82A)突变体的功能活性降低、各突变体在E.coli内不能正确的定位和形成功能性Z环;FtsZ/FtsZ*-FtsZ*单体间的相互作用减弱或消失,FtsZ*-MreB相互作用破坏;FtsZ突变体体外聚合效率降低。【结论】FtsZ E75、R78和D82是影响FtsZ正确组装和功能及FtsZ-MreB相互作用的重要氨基酸。
【Objective】 To investigate the effects of Escherichia coli FtsZ mutant FtsZ ~ (E75A), FtsZ ~ (R78G) and FtsZ ~ (D82A) on FtsZ self-assembly and FtsZ-MreB interaction. 【Method】 FtsZ and its mutant expression vector were constructed by conventional molecular cloning and site-directed mutagenesis. The target protein was obtained by affinity purification. QN6 (ftsZ :: yfp-cat), QN7 (ftsZ ~ (FtsZ ~ (R82G) :: yfp-cat) and QN9 (ftsZ ~ (D82A) :: yfp-cat) were used to observe the expression of FtsZ and its mutants by live cell imaging The interaction between FtsZ / FtsZ * -FtsZ * or FtsZ / FtsZ * -MreB was detected by immunoprecipitation and bacterial two-hybrid assay. The effect of site-directed mutagenesis on FtsZ assembly was detected by optical scanning. 【Results】 The functional activities of FtsZ ~ (E75A), FtsZ ~ (R78G) and FtsZ ~ (D82A) mutants were decreased. Each mutant could not correctly locate and form a functional Z loop in E. coli. FtsZ / FtsZ * -FtsZ * interactions weaken or disappear, FtsZ * -MreB interaction is destroyed; FtsZ mutant in vitro polymerization efficiency decreases. 【Conclusion】 FtsZ E75, R78 and D82 are important amino acids that affect the correct assembly and function of FtsZ and the interaction of FtsZ-MreB.