酿酒酵母蛋白激酶A催化亚基细胞定位对其功能特异性的调控机制研究

This study mainly focuses on the mechanism of protein localization of Protein Kinase A catalytic subunits in regulating their distinct functions in the yeast Saccharomyces cerevisiae. The results will not only be helpful to understand the regulation mechanism of Protein Kinase A in functioning on cell growth and stress resistances, but also with theoretical importance for the breeding of industrial strains with improved stress resistances.In this work, we will uncover the regulation mechanism of Tpk localization in its functions via the following three points: Firstly, we will study the effect of Tpks in regulating cell growth rates and stress resistances, revealing the distinct biological functions of Tpks; Secondly, through the GFP tagged with Tpks, we will trace the localization of Tpks in cells released from the negative regulation of Bcy1 regulatory subunit, as well as in glucose-induced cells, whose cAMP/PKA signalling was acivated by glucose signals. This part of work will find the distinctions of Tpk subcellular localization. Finally, using fil1 mutant as the background, we will study the molecular mechanism of Tpks in regualting cell growth and stress resistance, which are incompatible in wildtype cells, exploring the connection between the Tpks subcellular localization and their divers functions, and finally demonstrating the molecular regulation mechanism of the biological functions of Protein Kinase A catalytic subunits in regulating cell growth and stress resistances.

研究酿酒酵母蛋白激酶A催化亚基细胞定位对其功能特异性的调控机制,既有助于完善蛋白激酶A对调控细胞周期和胁迫响应机的生理功能，又可为高耐受性酵母工业菌种分子育种提供理论指导，具有重要的理论意义和应用价值。本项目拟从三个方面探索催化亚基蛋白定位对细胞生长和胁迫耐受性调控机制。首先从分析各催化亚基分别对细胞生长与胁迫耐受性的特异性调控作用出发，发现各亚基调控细胞生长和耐受性的分工情况；其次，通过绿色荧光蛋白标签及其成像技术，观察游离态以及环腺苷酸信号通路激活过程中各催化亚基胞内的静态定位以及动态追踪，研究各催化亚基自身胞内定位属性，以及在激活过程中胞内动态迁移的差异性；最后，以fil1突变株为材料，揭示细胞生长和耐受性矛盾双方相互协调的分子机制，建立蛋白激酶A催化亚基胞内定位对其功能之间的关联, 从分子水平上阐述蛋白激酶A催化亚基分别对细胞生长速率和胁迫耐受性的特异性调控作用和调控机制。

酿酒酵母菌种的发酵速率和胁迫耐受性是酿酒酵母工业菌种发酵性能的关键因素，提高菌种发酵速率和耐受性是酵母工业菌种改良的重要目标。本项目首先分析了酿酒酵母蛋白激酶A的三个催化亚基分别对细胞生长和耐受性的异性性调控作用，以及催化亚基协同作用方式，阐述了三个催化亚基的分工情况；通过绿色荧光蛋白标记技术，观察了各个催化亚基以及调剂亚基的细胞定位属性，并且研究了催化亚基调节亚基细胞定位迁移情况，结合已有的调节亚基功能特异性，研究了蛋白质细胞定位对功能的调控机制；将fil1点突变整合，进一步研究腺苷酸环化酶基因点突变对耐受性的影响；构建了适用于酿酒酵母工业菌种的基因无痕修饰的方法和体系，并运用该体系精细调控环腺苷酸信号通路的中心元件腺苷酸环化酶编码基因CYR1的启动子，构建得到了具有优良发酵性能和高耐受性的工业菌种。本研究成果既完善了蛋白激酶A对调控细胞周期和胁迫响应的生理功能理论研究，又为高耐受性酵母工业菌种育种提供了技术方法，具有重要的理论意义和应用价值。