沙门氏菌中伴侣蛋白Hfq与小RNA的相互作用方式以及对靶基因的调控机理分析

Salmonella is one of the important pathogens that infect both Human and Animals, and it is a threat to human health and animal husbandry development. Small RNA (sRNA) is an important regulator in Salmonella, involved in regulating a variety of life activities. The regulation of small RNAs usually requires the chaperone protein Hfq, without Hfq, the regulate function of small will be lost. Right now, the study of small RNAs is mainly focused on the binding of small RNA to target genes, and study of Hfq mainly focuses on the structure of proteins. In fact, it is important to elucidate the interaction between Hfq and small RNA, to analyze the regulatory region of Hfq to target genes, and to reveal the mechanism of Hfq regulation. Applicant has done several research about regulatory site between Hfq on small RNA and Hfq on target gene. Basis on previous study, in this project, Toeparinting assay and 'Scarless' DNA recombineering and other technology will be used to analyze the core region of Hfq pairing with small RNA and target gene, furthermore the regulation mechanism of Hfq will be clarified. Basis on the discovery from this study, the theory of small RNA regulation will be enriched, and hoping the theoretical of Salmonella prevention will be constructed, also a new molecular genetic tool would be developed.

沙门氏菌是临床上常见的人兽共患的致病菌，是威胁人类健康和畜牧养殖业发展的重要病原菌之一。小RNA(sRNA)是沙门氏菌中的重要调控因子，参与调节多种生命活动。小RNA行使调节功能通常需要伴侣蛋白Hfq的协同作用。现在对小RNA的研究主要集中在小RNA与靶基因的结合方式上，对Hfq的研究主要集中在蛋白结构上。实际上，阐明Hfq与小RNA的相互作用，分析Hfq对靶基因的调节区域，揭示Hfq的调控机理也十分重要。申请人前期开展了Hfq对小RNA和靶基因的调节位点分析研究，本项目拟在此基础上，应用Toeparinting assay和 ‘Scarless’ DNA recombineering等方法，分析和揭示Hfq分别与小RNA和靶基因的结合核心区域，进而阐明Hfq对小RNA和靶基因的调控机理，希望能进一步丰富小RNA调控理论，为沙门氏菌的防治以及分子遗传学工具的开发，提供理论基础。

沙门氏菌中小RNA(sRNA)是一类重要调控因子，参与调节多种生命活动。小RNA行使调节功能通常需要伴侣蛋白Hfq的协同作用，通过在后转录时期对靶基因表达的调控，调节多种生命进程，但是，Hfq、sRNA及靶基因之间的相互作用方式还不清晰。本研究通过对多个已知的Hfq、sRNA靶基因的研究，发现Hfq与sRNA结合位点位于sRNA的3’ 末端的富U序列，富U序列对于Hfq维持sRNA的稳定性以及协助其调控靶基因的表达是必要的，同时，Hfq本身通过与靶基因mRNA 5’ UTR区域的富A序列相结合，对mRNA在后转录水平有调控作用，并且Hfq的C末端氨基酸对于维持其对靶基因的调控有重要的作用。项目结果确定了Hfq，sRNA与靶基因三者之间的作用区域，推导了基因调控的模型，进一步补充了Hfq及sRNA的调控理论。项目实施过程中，课题参与硕士研究生9名，完成项目相关硕士学位论文4篇，撰写发表科研论文11篇。