高温菌GFX-5硝酸盐还原酶基因的克隆及其表达调控机制研究

项目名称： 高温菌GFX-5硝酸盐还原酶基因的克隆及其表达调控机制研究

项目编号： No.31200047

项目类型： 青年科学基金项目

立项/批准年度： 2013

项目学科： 微生物学、植物学

项目作者： 王志春

作者单位： 中国科学院微生物研究所

项目金额： 20万元

中文摘要： 硝酸盐还原酶是反硝化的关键酶，目前对其调控的研究主要集中在厌氧反硝化过程，而关于好氧反硝化的调节机制研究报道很少。Geobacillus thermodenitrificans GFX-5是本实验室分离的能在高温条件下进行厌氧和好氧反硝化的菌株，本项目拟分别克隆该菌株在厌氧和好氧条件下发挥功能的硝酸盐还原酶基因及其上下游序列；通过序列的比对分析初步预测其调控基因和调控元件；对硝酸盐还原酶及调控因子进行表达；通过EMSA、DNase足迹法等方法，明确硝酸盐还原酶基因转录所受特异性结合调控因子及其调控元件的关系；结合荧光定量RT-PCR和响应面设计分析硝酸盐还原酶基因转录水平与调控因子的关系及其影响因素。本研究的结果将阐明菌株GFX-5中参与厌氧、好氧反硝化的硝酸盐还原酶及其基因转录调控的机制，同时也将为高温好氧反硝化菌的筛选及应用提供理论依据。

中文关键词： 硝酸盐还原酶；基因调控；cAMP受体蛋白；钼辅因子；抗氧机制

英文摘要： Nitrate reductase is the key enzyme involving the denitrification.To the present, Regulation of denitrification studies mainly concentrated in anaerobic process, little of it in the aerobic denitrifican process.Geobacillus thermodenitrificans GFX-5 isolated by our lab is a thermophilic denitrifying bacteria involving aerobic and anaerobic progress. This research will clone the nitrate reductase gene including the upstream and downstream sequence of it, get a prediction of the regulator gene and regulatory elements through the blast and analysis of the sequences, and then express the nitrate reductase gene and regulator gene. In this research, we will certify the specific binding regulatory factors of the gene nar through EMSA test and the binding sites by DNase footprinting.And we will analyze the influencing factors of transcription of gene nar with the combination of Real-time fluorescence quantitative RT-PCR and response surface analysis. This study aims to investigate the expression and regulation mechanisms of the nitrate reductase in strain GFX-5 under thermophilic anaerobic and aerobic conditions. Thus the study of the mechanism is not only of scientific significance but also will provide a theoretical basis for the screening and application of thermophilic aerobic denitrifying bacteria.

英文关键词： Nitrate reductase；Gene regulation；Crp (cAMP receptor protein)；Mo-cofactor；Antioxidant Defence