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.

【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-diGMP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。

[ 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-13-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 （engAxoo- p） 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.