亚硝酸盐胁迫下短乳杆菌BS6中GlnR与Nir相互作用及表达调控机制

The utilization of Nitrite reductase (Nir) degradation in fermented food is the ultimate countermeasures to deal with the pollution of the nitrite. In the preliminary studies, we have studied the structure and function of transcriptional regulator glnR of Lactobacillus brevis BS6 and the GAD-control mechanism by the GlnR, built the glnR deletion mutant and the revertant plasmid, and analyzed the characteristic of degradation nitrite by the strain under the nitrite stress and the part conservative fragment of nir gene from L. brevis BS6. Because the glnR is the key transcriptional regulator in the lactic acid bacteria nitrogen metabolism and the nir operon transcription and expression regulation mechanism is unclear, the full-length of nir operon will be amplified by TAIL-PCR and the structure and function of nir operon will be analyzed firstly, then the nir operon transcriptional level and expression regulation by the transcriptional regulator GlnR under the nitrite stress will be studied, the nir promoter with the GlnR binding properties and interactions will be established in this project finally. This study would establish the foundation of the functions of nir operon regulation system, the combination of mode components, and the molecular regulation mechanism of the new regulation model and the signal transduction of lactic acid bacteria for the further study. All these studies would be great significance for looking for a safe and effective control or degradation of nitrite by biodegradable method quickly and for guaranteeing the food security of our country.

利用亚硝酸盐还原酶(Nir)降解发酵食品中产生的亚硝酸盐，是应对亚硝酸盐污染的根本对策。本项目组前期研究了Lactobacillus brevis BS6转录调控子glnR的结构功能和GlnR对GAD调控机制，构建了glnR缺失菌株和回补质粒，并分析了该菌株在亚硝酸盐胁迫下降解亚硝酸盐特性和nir部分保守片段。glnR是乳酸菌氮代谢中的关键转录调控子, 有关乳酸菌nir操纵子的转录和表达调控机制尚不清楚（尚未见报道）。据此，本项目拟从TAIL-PCR克隆nir操纵子全长基因及其结构和功能分析着手，研究亚硝酸盐胁迫下GlnR对nir操纵子转录水平和表达的调控，确立nir启动子与GlnR结合特性和相互作用，为进一步揭示乳酸菌nir操纵子调控系统的功能及模式元件的组合、新的调控模型和信号传导的分子调控机制奠定基础，这对加快寻找安全有效的亚硝酸盐生物降解法，进而保障国家的食品安全具有重要意义。

利用亚硝酸盐还原酶(Nir)降解发酵食品中产生的亚硝酸盐，是应对亚硝酸盐污染的根本对策。本项目组前期研究了短乳杆菌BS6转录调控子glnR的结构功能和GlnR对GAD调控机制，并分析了该菌株在亚硝酸盐胁迫下降解亚硝酸盐特性和nir部分保守片段。glnR是乳酸菌氮代谢中的关键转录调控子, 但有关乳酸菌nir操纵子的转录和表达调控机制尚不清晰。为此，本项目首先研究了亚硝酸盐胁迫下短乳杆菌BS6亚硝酸盐还原酶酶学性质并实现了该酶的食品级高效诱导表达，然后在GlnR与Nir启动子体外相互作用的研究中，通过细菌单杂交和凝胶阻滞探索到了nir操纵子的转录起始位点和全局性转录调控蛋白GlnR与启动子结合的特异性，确定了调控蛋白在启动子序列中特异识别序列，并利用qRT-PCR阐释了亚硝酸盐胁迫下短乳杆菌BS6的GlnR对nir的调控规律和亚硝酸盐胁迫下GlnR对nir操纵子表达和基因转录水平的调控机制。在利用重叠延伸PCR技术敲除了Nir基因后，酵母双杂交实验进一步阐释了Nir和glnr的相互作用机制。本研究旨在阐释乳酸菌nir操纵子的分子调控机制和加快寻找安全有效的亚硝酸盐生物降解法提供理论基础。