Fnr参与植物乳杆菌WU14亚硝酸盐降解的调控机制

The utilization of Nitrite reductase (Nir) degradation in pickled food is the ultimate countermeasures to deal with the pollution of the nitrite. In the preliminary studies, the Nir characteristics and the positive regulation mechanism between the transcription factor GlnR and Nir of Lactobacillus plantarum WU14 have been studieded by the methods of bacterial one hybrid, qRT-PCR and Electrophoretic Mobility Shift Assay (EMSA). The transcription and expression of many genes and other transcription factors could be directly or indirectly regulated by the global transcription factor Fnr, which is on top of bacterial transcriptional regulatory networks. However, the regulatory mechanism in the degradation of nitrite involved the Fnr of lactic acid bacteria is unclear. So the oxygen sensing mechanism of Fnr and Nir and the nir operon expression regulated by Fnr in L. plantarum WU14 will be implemented with the method of gene transcription and expression level analysis under the stress of nitrite. After the Fnr and GlnR interaction is analysed, the nir operon expression regulation mechanism in L. plantarum WU14 will be studied through the regulation of glnR transcription regulation controlled by the Fnr, so as to clarify the regulation mechanism of Fnr involved in the nitrite degradation of L. plantarum WU14. 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)降解腌制食品中的亚硝酸盐是消除亚硝酸盐污染的根本对策。项目组前期利用细菌单杂交，qRT-PCR和凝胶阻滞等技术手段研究了植物乳杆菌WU14的Nir原酶特性及转录因子GlnR对Nir的正调控机制。全局性转录因子Fnr处于细菌转录调控网络顶端，可直接或间接调控许多基因及转录因子的转录和表达。然而乳酸菌Fnr如何参与Nir的表达及调控，目前尚不清楚，故本项目拟从基因转录和表达水平分析亚硝酸盐胁迫下植物乳杆菌WU14 Fnr和Nir对氧感应和Fnr对nir操纵子表达调控机制，分析Fnr与GlnR相互作用，探究Fnr通过调节glnR转录而调控nir操纵子表达机制，阐明Fnr参与亚硝酸盐降解的调控机理，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组合、新调控模型和信号传导的分子调控机制奠定基础，对加快寻找安全有效的亚硝酸盐生物降解法，保障国家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。本项目前期研究了亚硝酸盐胁迫下植物乳杆菌WU14的Nir酶学性质及转录因子GlnR与Nir互作及表达正调控机制，并实现了该酶的食品级高效诱导表达。全局性转录因子Fnr处于细菌转录调控网络顶端，可直接或间接调控许多基因及转录因子的转录和表达，但乳酸菌的Fnr如何参与Nir的表达和调控，目前尚不清楚。为此，本研究首先研究了WU14的Fnr和Nir对氧感应机制，通过分析亚硝酸盐胁迫下WU14对氧的感应及有氧无氧高铁低铁条件下菌体生长和亚硝酸盐降解能力，胞内蛋白和琥珀酸脱氢酶酶活变化规律及Fnr和nir的转录表达，证明在亚硝酸盐浓度逐渐升高情况下Fnr对于Nir具有正调控作用。在利用细菌单杂证明GlnR与nir启动子片段有相互作用和正调控现象后，通过酵母双杂证明了0.1%NaNO2胁迫下Fnr和Nir蛋白在表达调控中的相互作用，从转录水平和蛋白表达水平上阐释了WU14的Fnr对nir操纵子表达和基因转录的调控机制。通过凝胶阻滞实验研究了Fnr和Pnir和Pglnr启动子的结合，证明Fnr能直接调控 Pnir启动子也能调控Pglnr，通过GlnR蛋白再调控Pnir启动子来间接调控Pnir启动子，阐释了亚硝酸盐胁迫下全局性转录调控蛋白Fnr参与WU14亚硝酸盐降解的多层次调控机制和科学问题。在完成基因组测序和分析后利用Cre/loxP系统敲除了WU14的Fnr，检测了基因敲除并进行功能回补实验，并完成了6-磷酸-β-葡萄糖苷酶的基因克隆、蛋白表达以及生物信息学分析。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，最终为探索乳酸菌Nir降解亚硝酸盐的分子调控机制提供了理论依据。