还原型谷胱甘肽在酒酒球菌SD-2a抗乙醇胁迫中的作用及机制研究

Oenococcus oeni is the main lactic acid bacteria responsible for conducting malolactic fermentation (MLF) in wines. Despite the inability of O. oeni to synthesize GSH, this bacterium can capture it from the media. GSH enrichment could improve the resistance to acid or (and) ethanol stress to O. oeni. Using the specific functions of GSH to improve the cell physiological activity and stress resistance is one of the important strategies to improve its ethanol stress resistance. However, its mechanism has yet been unclear. In the project, O. oeni SD-2a was selected as experimental material. First, to study the effect of GSH on the physiological and biochemical indexes and cell shape of O. oeni SD-2a at intracellular microenvironment and membrane level in ethanol stress environment. Then, protein expression profiles of O. oeni SD-2a during ethanol stress environment with GSH and the control one were constructed using isobaric tags for relative and absolute quantification (iTRAQ)-coupled two-dimensional liquid chromatography tandem mass spectrometry (2D-LC MS/MS) proteomics approach, so as to gain differential expression proteins and related biological indicators. Finally, the expression levels of the selected key proteins genes were evaluated by qRT-PCR technology. The aim of this study was to achieve this objective it was illustrate its possible mechanism of GSH in protection of O. oeni against the ethanol stress. Then it can lay the foundation for the successful modification and selection of O. oeni in the future.

酒酒球菌是启动葡萄酒苹果酸-乳酸发酵最重要的乳酸菌。尽管酒酒球菌不能合成谷胱甘肽（glutathione，GSH），但它能够吸收外源GSH来提高其酸或（和）乙醇胁迫抵抗能力，而利用GSH的特定功能来改善酒酒球菌细胞生理活性和胁迫抗性成为提高其乙醇胁迫抗性重要的策略之一，但其作用机制尚不十分清楚。据此，本项目以酒酒球菌SD-2a为试验材料，首先从细胞内环境和细胞膜水平上研究乙醇胁迫环境下GSH对酒酒球菌SD-2a细胞生理生化指标和细胞形态的影响。然后应用iTRAQ结合2D-LC-MS/MS技术寻找GSH在保护酒酒球菌SD-2a抵御乙醇胁迫过程中的差异表达蛋白质和相关生物学指标。最后利用qRT-PCR技术对筛选出的关键蛋白质基因表达量进行验证分析。本研究旨在揭示GSH保护酒酒球菌抵御乙醇胁迫的调控机制，进而为成功改造和选育优良酒酒球菌奠定基础。

酒酒球菌是启动葡萄酒苹果酸-乳酸发酵最重要的乳酸菌。尽管酒酒球菌不能合成谷胱甘肽（glutathione，GSH），但它能够吸收外源GSH来提高其酸或（和）乙醇胁迫抵抗能力，而利用GSH的特定功能来改善酒酒球菌细胞生理活性和胁迫抗性成为提高其乙醇胁迫抗性重要的策略之一，但其作用机制尚不十分清楚。据此，本项目从细胞内环境和细胞膜水平上研究乙醇胁迫环境下GSH对酒酒球菌SD-2a细胞生理生化指标的影响。然后应用DIA结合2D-LC-MS/MS技术寻找GSH在保护酒酒球菌SD-2a抵御乙醇胁迫过程中的差异表达蛋白质和相关生物学指标。并且利用qRT-PCR技术研究了GSH对酒酒球菌SD-2a在不同胁迫条件下关键基因表达情况的影响。主要研究结果如下：在乙醇胁迫环境中，GSH的添加能够促进细胞的生长，降低乙醇胁迫引起的细胞膜通透性和细胞膜流动性的增加，防止菌体细胞膜的损伤，在一定程度上有利于保护细胞膜完整性，还有助于提高胞内ATP的含量，提高菌体在乙醇胁迫中的存活率。通过DIA技术建立了未进行乙醇胁迫、乙醇胁迫以及添加谷胱甘肽进行乙醇胁迫的酒酒球菌SD-2a在短期和长期乙醇胁迫过程中的蛋白表达图谱。蛋白质组比较分析获得了279个差异表达蛋白，这些蛋白质与氨基酸运输与代谢、碳水化合物转运与代谢、能量产生与转化、胁迫响应、菌体生物被膜和细胞壁合成、群体感应等代谢途径及生理过程密切相关。生物信息学结果表明GSH的添加对于丙酮酸代谢、糖酵解/糖异生途径代谢影响显著。GSH提高（或抑制）了酒酒球菌中多种与胁迫相关蛋白的表达量（或下调），并作为应对乙醇胁迫诱导损伤的细胞保护物质或是胁迫响应蛋白表达的调节物质发挥作用，维持菌体结构的完整以抵御乙醇胁迫。研究结果为优良酒酒球菌的改造和选育提供了理论支持并有助于揭示GSH保护酒酒球菌抵御乙醇胁迫的调控机制。