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【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】 The objective of this study is to elucidate the mechanism of transcriptional regulation of glucanase gene (engAxoo) by the cpx-receptor signaling and Clpxoo of Xanthomonas oryzae pv.oryzae (Xoo). 【Method】 Prokaryotic expression and purification of clpxoo gene were carried out by construction and transformation of gene expression vector, protein induced expression and Ni-NTA Resin affinity chromatography. The binding activity of Clpxoo purified protein to the dextranase gene promoter (engAxoo-p) and the inhibition of its c-diGMP signaling molecule were investigated by fluorescein (FAM) probe labeling and gel blocking assay (EMSA) Determination and analysis. 【Result】 Clpxoo purified protein was successfully obtained under the conditions of optimized induction and purification. Clpxoo blocked with the engAxoo-p sequence, indicating that it has an activity that specifically binds to the promoter. Addition of c-di-GMP to the reaction system can result in the elimination of the binding. 【Conclusion】 Clpxoo could inhibit the binding of engAxoo-p through conformational change after receiving c-di-GMP signal. The optimized Clpxoo protein purification and EMSA method can be used for subsequent large-scale identification of regulatory elements .