嗜水气单胞菌Hfq蛋白K56位点酰基化修饰响应环境胁迫的机制研究

细菌全局性调控因子Hfq蛋白在转录后水平调控mRNA翻译响应外界环境胁迫中起重要作用。申请人前期研究发现嗜水气单胞菌Hfq蛋白K56位点具有乙酰化和琥珀酰化修饰，在响应不同环境胁迫下表型有明显差异，但其作用机制尚不明确。在此基础上，本课题拟首先利用RIP-Seq技术，比较环境胁迫下与Hfq系列K56位点突变蛋白特异结合的sRNA和mRNA序列，并通过Northern与 Western blotting技术对其结合进行验证；其次结合DIA定量蛋白质组学技术进行多组学联合分析，筛选关键差异基因/蛋白；然后通过qPCR、PRM与基因缺失等方法，验证关键RNAs在环境胁迫响应中的变化与生物学功能；最后，利用EMSA和SPR等技术，验证关键RNAs与Hfq系列蛋白结合的稳定性，阐明酰基化修饰Hfq蛋白介导细菌响应环境胁迫的分子机制。该研究将有助于深化对蛋白翻译后修饰介导细菌生理功能调控机理的认识。

As a global regulatory factor, Hfq protein plays an important role in post-transcriptional regulation of mRNA translation in response to environmental stresses. In our previously research, we have found acetylation and succinylation modifications on the K56 site of Hfq in Aeromonas hydrophila and their phenotypes were significantly different in response to different environmental stresses, but the mechanism was still unknown. On this basis, this project first uses RIP-seq technology to compare the sequence of sRNA and mRNA specifically binding to Hfq K56 mutation site under different environmental stresses, and then through the Hfq-RNAs binding was verified by Northern blotting and Western blotting technology; Secondly, the key differential genes / proteins were screened by DIA quantitative proteomics technology, then the biological functions of key RNAs in response to environmental stress were verified by qPCR, PRM and gene deletion; Finally, the binding stability of key Hfq-RNAs was verified by EMSA and SPR technologies. In this way, we try to elucidate the molecular mechanism of acylation modified Hfq protein in response to environmental stress. This study will be helpful to deepen the understanding of the mechanism of protein post-translational modification mediated regulation of bacterial physiological function.