酸胁迫下乳酸乳球菌非编码小RNAs042的高转录机制研究

前期工作表明，多个非编码小RNA在乳酸乳球菌酸响应及酸耐受体系中起重要的调控作用。其中s042在酸胁迫下转录量明显上调，且能通过作用于精氨酸代谢调控因子ArgR等提高菌株的耐酸能力。然而s042在酸胁迫下的高转录机制尚不清楚。我们采用生物信息学软件预测了s042的启动子区及可能与之结合的转录因子。本课题拟进一步利用DNA pull down技术调取酸应激过程中与s042启动子区结合的蛋白，经EMSA体外验证获得s042的上游转录因子。构建该转录因子过表达、缺失及回补菌株，利用Northern blot、报告基因系统以及ChIP-qPCR的方法进行体内验证。最后利用分子模拟、DNase I足迹以及点突变结合EMSA实验确定该转录因子与s042启动子区的结合位点。本课题不仅对阐明酸胁迫下乳酸乳球菌sRNA的响应机制具有重要意义，而且为进一步构建酸胁迫下转录因子-sRNA协同调控网络奠定基础。

Previous investigation has proved the regulatory role of several small non-coding RNAs in the acid response and acid tolerance system of Lactococcus lactis. Among them, the transcriptional level of s042 under acid stress was significantly up-regulated. s042 can improve acid resistant capability of the strain by acting on arginine metabolism regulator ArgR and other target genes. However, the response mechanism of s042 to acid stress is still unclear. We applied bioinformatics software to predict the promoter region of s042 and the related transcriptional regulators. In this research, DNA affinity chromatography-pull down technique will be used to attain the proteins which can bind to s042 promoter under acid stress. EMSA experiment will be applied to screen and verify the upstream transcriptional regulators of s042 in vitro. The regulator overexpressing, deficient and complementary strains will be constructed. The binding ability between s042 promoter and the transcriptional regulators will be verified in vivo by Northern blot, reporter gene system and ChIP-qPCR technique. Finally, the binding sites of the transcriptional regulator and s042 promoter will be determined by molecular simulation, DNase I footprint assay and site mutation combined with EMSA experiment. Not only is this research essential for elucidating the response mechanism of L. lactis sRNAs under acid stress, but it lays foundation for further constructing the transcriptional regulator-sRNA synergistic regulation network.