水稻白叶枯病菌 c-di-GMP 受体 Clpxoo 关键结合功能位点的确定

摘要

The purpose of this study is to identify the critical residues in Clpxoo of a c-di-GMP signal receptor in Xanthomonas oryzae pv. oryzae(Xoo). By the gene mutation of amino acid site of Clpxoo protein,the construction of expressing vector,induced protein expressed, and analysis of Ni-NTA Resin affinity chromatography,the prokaryotic expression of Clpxoo and point mutants was conducted and the protein was purified.. The binding affinities of c-di-GMP with native and variant Clpxoo were measured by isothermal titration calorimetry(ITC) experiments. Under optimized conditions for protein expression and purification,the proteins of Clpxoo point mutants and point mutants of Clpxoo that does not bind with c-di-GMP were acquired successfully while using gene site-directed mutagenesis and bridging PCR. Results showed that site 70 of aspartic acids and site 99 of glutamic acid were found to be the key residues for their binding to c-di-GMP.%旨在确定水稻白叶枯病菌（Xanthomonasoryzaepv.oryzae，Xoo）c-di-GMP 信号受体 Clpxoo 的关键功能位点。通过对 Clpxoo 蛋白氨基酸位点基因突变，表达载体构建、蛋白诱导表达及其 Ni-NTAResin 亲和层析，进行了 Clpxoo及其点突变蛋白的原核表达和产物纯化；通过等温滴定量热法（ITC），检测了 Clpxoo及其点突变蛋白与 c-di-GMP 的结合作用。利用基因定点突变和桥接 PCR 方法，在优化的诱导表达和纯化条件下，成功地获得了 Clpxoo 点突变蛋白和与 c-di-GMP 不发生结合的 Clpxoo 点突变体。结果表明，Clpxoo 蛋白第70位天冬氨酸和第99位谷氨酸是与 c-di-GMP 结合的关键功能位点。