酿酒酵母孢子萌发的激光镊子拉曼光谱研究

【目的】在生理状态下利用激光镊子拉曼光谱系统对单个酿酒酵母孢子萌发过程进行实时监测,探讨掩盖在群体平均信息下的个体生命信息。【方法】将葡萄糖溶液加入酿酒酵母孢子悬液中诱导孢子萌发,在孢子萌发过程中每隔30min取样并利用激光镊子拉曼光谱系统测定单个酵母孢子的拉曼光谱。【结果】单细胞实时平均拉曼光谱可显示孢子萌发过程中细胞内生物分子的变化:萌发期内分别归属于DNA、蛋白质的722cm-1,1006cm-1峰的峰高基本不变,随后在生长期上升明显,说明生长期胞内开始大量复制DNA,并合成蛋白质;归属脂类的1751cm-1峰的减弱趋势明显,可能是胞内脂类物质消耗造成的。整个萌发生长过程中,源自葡糖糖和海藻糖的858cm-1,908cm-1,1084cm-1,1118cm-1等峰强度先下降后上升,表明在适宜的生长条件下,海藻糖可能转化为单糖被细胞吸收利用,当营养物质逐渐被消耗时,细胞会再次累积海藻糖以抵抗外界不利条件。【结论】激光镊子拉曼光谱技术可反映胞内生物大分子的活动规律,获知单个酵母孢子萌发过程中物质变化的丰富信息,是探索单个活细胞实时生化变化的有效工具。

Study on Saccharomyces cerivisiae Spore Germination Using Laser Tweezers Raman Spectroscopy

Objective] The germination process of Saccharomyces cerivisiae spores under physiological conditions is monitored real-time using laser tweezers Raman spectroscopy to obtain information on metabolic activity of individual spores which could be masked by bulk measurement.Methods] The germination was initiated by addition of glucose solution to spore suspension and samples were harvested at a 30 min interval during germination process.Raman spectra of individual spores at different time points were recorded.Results] The Raman spectra of the single spore reflected the change in bio-molecule content during the process of spore germination:The intensities of band at 722 cm^-1and 1006 cm^-1 (assigned to DNA and protein, respectively) did not change significantly during germination period, whereas they increased dramatically during the outgrowth period.This indicated the DNA replication and protein biosynthesis occurred during outgrowth period.The intensities of 1751 cm^-1 band (associated with lipids) decreased substantially, which might be as the result of consumption of lipids.During the whole germination and outgrowth process, the intensities of bands at 858 cm^-1, 908 cm^-1, 1084 cm^-1and 1118 cm^-1 originated from glucose and trehalose decreased at first and then iecreased.This phenomenon suggested that under the favorable growing conditions, trehalose may be converted into monosaccharide and then be utilized.When nutritional materials are gradually consumed, the cells would accumulate trehalose again in order to resist adverse condition.Conclusion] The experimental results show that the LTRS technique can reveal the metabolic activity of bio-molecules and provides rich information on the change in compound content within single spores during the germination process.Therefore, the LTRS can serve as a valuable tool for the real time monitoring of dynamic changes in bio-molecules at single cell level.