中文摘要：

【目的】阐明水稻白叶枯病菌（Xanthomonasoryzaepv．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．NTAResin亲和层析，进行了c加zoo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en鲋xoo-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。

英文摘要：

[ 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.