不同传代次数的酿酒酵母细胞壁蛋白组学分析

Proteomic analysis of cell wall proteins from Saccharomyces cerevisiae with different generation numbers

摘要:

【目的】探讨酿酒酵母衰老过程中细胞壁蛋白变化, 从蛋白水平上解释酿酒酵母衰老的原因。【方法】以酿酒酵母FFC2146为研究对象, 采用显微镜观察法比较了经2、10、15次连续传代酿酒酵母的细胞形态; 用计算细胞沉降速率的方法考查酵母凝絮性; 通过3,5-二硝基水杨酸法测定降糖速率来表征酵母代谢活力; 采用二硫苏糖醇溶解法结合苯酚萃取法抽提不同传代次数的酿酒酵母细胞壁蛋白; 并且通过双向电泳进行差异性分析。【结果】结果显示随着传代次数的增加酿酒细胞个体表面变得粗糙, 凝絮能力明显增强, 降糖能力明显减弱, 表明多次传代后的酵母体现出衰老现象。双向电泳结果共得到309个胞壁蛋白点, 其中11个蛋白质点存在明显差异。6个蛋白质点在第15代丰度小于第2代丰度2倍以上, 4个蛋白质点只在第15代酵母细胞壁中出现, 1个蛋白质点只在第2代酵母细胞壁中出现。【结论】酿酒酵母FFC2146经过15次连续传代培养后11个细胞壁蛋白丰度发生明显变化, 此11个细胞壁蛋白的表达水平与酿酒酵母衰老相关。

Abstract:

[Objective] To find out the mechanisms of yeast aging. [Methods] We performed continuous inoculation of Saccharomyces cerevisiae FFC2146 from generation to generation. We also observed the morphogenesis of Saccharomyces cerevisiae which has different generation numbers (2, 10, 15) through microscope, and measured absorbance in 600 nm to signify flocculation after sedimentation and determined residual glucose by DNS. Proteomic analysis of yeast cell wall proteins from the 2th and 15th generation was observed by Two-dimensional electrophoresis. [Results] Results showed that, the 15th generation yeast has rougher cell surface, higher flocculating velocity and lower metabolic level than the 2th and 10th generation, indicated that continuous inoculation accelerates yeast recession. Proteins were obtained by dithiothreitol and phenol abstraction. 309 proteins were detected and presented 11 changes responded to cell recession, 6 spots reduced by more than 2-fold in the 15th gel, 4 spots appeared in the 15th gel only and 1 spot appeared in the 2th only. [Conclusion] 11 Proteins from Saccharomyces cerevisiae cell wall changed after continuous inoculation 14 times, the changes of expression levels among these proteins were connected to yeast aging.