c-di-GMP抑制转录调控因子Clpxoo与葡聚糖酶基因启动子的结合

Binding of transcription regulator Clpxoo to promoter of endoglucanase gene engAxoo was inhibited by c-di-GMP in Xanthomonas oryzae pv． oryzae．

摘要:

摘要:【目的】阐明水稻白叶枯病菌(Xanthomonas oryzae pv．oryzae，Xoo) c-di-GMP信号受体和转录调控因子Clpxoo对葡聚糖酶基因(engAxoo)的转录调控作用机制。【方法】通过基因表达载体的构建和转化、蛋白诱导表达及其Ni-NTA Resin亲和层析，进行了clpxoo基因的原核表达和产物纯化。通过荧光素(FAM)探针标记和凝胶阻滞试验( EMSA)对Clpxoo 纯化蛋白与葡聚糖酶基因启动子(engAxoo-p)的结合活性及其c-di-GMP信号分子的抑制作用进行了测定分析。【结果】在优化的诱导表达和纯化条件下，成功地获得了Clpxoo纯化蛋白。Clpxoo 可与engAxoo-p序列发生阻滞现象，表明它具有与启动子特异性结合的活性。在反应体系中加入c-di-GMP可导致结合作用的消除。【结论】Clpxoo接受c-di-GMP信号后，可能通过构象的改变，从而与engAxoo-p的结合活性受到抑制;优化的Clpxoo蛋白纯化和EMSA方法可以用于后续的调控元大规模鉴定的研究。

Abstract:

Abstract: ［Objective］ To understand the regulatory mechanism by cyclic diguanylate (c-di-GMP) receptor and transcriptional regulator Clpxoo ofexpression of endoglucanase gene (engAxoo) in Xanthomonas oryzae pv．oryzae，the bacterial leaf blight pathogen of rice．［Methods］A plasmid to expressclpxoo gene was constructed and transformed into Escherichia coli for expression by isopropylthio-β-D-galactoside (IPTG) induction． The recombinant protein was purified by Ni-NTA resin． The binding affinity between purified Clpxoo protein and the promoter of endoglucanase gene (engAxoop) was determined by electrophoretic mobility shift assay using fluoresce in (FAM) -labeled probes．The role of c-di-GMP on the binding was also examined．［Results］Under the optimized conditions，Clpxoo was expressed and purified successfully． Mobility shift of engAxoo-p in the presence of Clpxoo was observed，indicating that specific binding occurred between them．Moreover，addition of c-di-GMP molecules in the above reaction system abolished such binding．［Conclusion］ Once interacting with the signal molecule c-di-GMP，Clpxoo conformational structure may change substantially，which results in inhibition of binding to engAxoo-p; The optimized methods for Clpxoo protein purification and electrophoretic mobility shift assay (EMSA) can be used for subsequent identification of Clp regulon in a larger scale．