酸土脂环酸芽孢杆菌响应酸热胁迫的分子调控机制研究

酸土脂环酸芽孢杆菌具有嗜酸耐热的双重生理特性，能引起巴氏灭菌果汁腐败变质，给果汁加工业造成经济损失，但其所含的耐酸热稳酶在食品工业中应用潜力巨大，解析该菌响应酸热胁迫的分子调控机制是其危害控制和有效利用的关键。本课题利用Label-free技术筛选酸土脂环酸芽孢杆菌在酸热胁迫下的差异表达蛋白并进行生物信息学分析，研究差异蛋白的分子功能及其参与的代谢途径，进而利用qRT-PCR从富集大量差异蛋白的主要通路中筛选响应酸热胁迫的关键调控蛋白；通过关键蛋白基因过表达、基因敲除、基因回补，验证关键蛋白调控该菌嗜酸耐热特性的作用；利用免疫共沉淀技术筛选关键蛋白的互作蛋白，构建关键蛋白调控网络、预测互作模式，进一步阐明关键蛋白的功能及其在酸热应答中的分子调控机制，从蛋白水平上全面揭示酸土脂环酸芽孢杆菌嗜酸耐热生理适应机制，为食品保藏、微生物抗逆特性研究及抗高温高酸的特殊工业菌研发等提供理论和实验依据。

Alicyclobacillus acidoterrestris is a genus of spoilage bacteria causing contamination in low pH fruits due to its acidophilic and thermophilic resistance, which led to economic losses to fruit juice industry, but its acid-resistant thermostable enzymes have the great potential application in the food industry. Therefore, to investigate the molecular regulatory mechanism how A. acidoterrestris responded to heat and acid stress is the key to hazard control and effective utilization of this microbe. In this study, a Label-free quantification will be used to analyze differentially expressed proteins in A. acidotresstris at the heat and acid stress conditions and bioinformatics analysis will be used to determine the molecular function of the differential protein and metabolic pathways involved; And then, qRT-PCR will be performed to screen key regulating proteins responding to heat and acid stress from main pathways which enriched most differential protein. Gene overexpression, gene knockout and retro-complementation of key proteins will be used to further verify the regulation function of the key proteins in the thermo-acidophilic resistance of A. acidoterrestris; Co-Immunoprecipitation will be used to identify interacting proteins with key proteins, establish the key protein regulation network and the model of proteins interaction to further illuminate the function of key proteins and molecular regulatory mechanism in responding to heat and acid stresses; The results will fully reveal the physiological adaptation mechanism of A. acidoterrestris from the protein level, which will provide a theoretical and experimental basis for acidic beverages preservation, microbial resistance research and the development of special industrial strains resisting the heat and acid conditions.