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

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.

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。

硝酸盐还原酶是反硝化过程中第一步关键酶。Geobacillus thermodenitrificans GFX-5具有高温条件下进行厌氧和好氧反硝化的菌株，本项目完成了该菌株硝酸盐还原酶基因narGHJI和narGHJI2及其上游序列和下游相关基因（modB、modA、dnrN、moeA、mobB以及MPT1和MPT2等基因）的克隆；以及 narGHJI2及其上游序列和下游相关基因（Hemerythrin、porA和porG）的克隆；通过对调控基因和调控元件序列的比对分析和预测以及EMSA实验，明确硝酸盐还原酶基因转录不受特下游 Crp、DnrN、Hemerythrin的调控； narG作用的钼辅因子合成受厌氧条件下Crp（cAMP受体蛋白）调控以及可能受到某种细菌趋化性的信号传导的影响，从而间接调控硝酸盐还原酶；发现DnrN可能受Crp（cAMP受体蛋白）调控，参与Fe-S修复； Hemerythrin可能参与O2胁迫解毒，丙酮酸：铁氧还蛋白氧化还原酶Por参与低氧压条件的硝酸盐还原的同时偶联丙酮酸的氧化。本研究揭示了菌株GFX-5中硝酸盐还原的过程中有别于已报道的酸盐还原的调控机制，是一个新的硝酸盐还原代谢调控机制。