罗伦隐球酵母Rho1对逆境胁迫的响应机制及其在生防中的作用

The control of postharvest decay by using antagonistic yeasts, as a safe and environment-friendly novel biocontrol means, has become one of the most promising non-fungicidal approaches in recent years. However, the antagonistic yeasts provide a level of disease control equivalent to synthetic fungicides only when supplemented with low doses of synthetic fungicides. Therefore, the efficacy of biocontrol yeasts must be enhanced so that it can be developed into a really economically feasible alternative. In order to explore the role of the Rho1, which is considered to be the master regulator of the cell wall integrity (CWI) signaling pathway, in adaptation of the environmental stress and the biocontrol efficacy of C. laurentii, this study was designed (1) to analyze the relationship between the expression of the rho1 and survival rate and biocontrol efficacy of postharvest fungal diseases of a well known antagonistic yeast Cryptococcus laurentii by monitor the responses of rho1 to various stress condition including high and low temperature stress, oxidative stress and osmotic stress and vibration stress; and (2) to investigate the effect of overexpression of the rho1 in Pichia pastoris GS115 on the resistance to stress conditions and the biocontrol efficacy; and (3) to discover intracellular protein-protein interactions between Rho1 and the regulators of Rho1, such as Guanosine nucleotide exchange factors and GTPase-activating proteins, or between Rho1 and the effectors for Rho1 by two-hybrid experiments, and (4) to compare the proteomic profile of C. laurentii under normal and stress conditions by LC-MS/MS. The results of this study will provide the new and fundamental information on the molecular mechanisms by which C. laurentii suffers from the stress conditions and the function of the Rho1 related pathways in the biocontrol of C. laurentii as well as to develop the novel technology to enhance the ability to adaptation of the stress and the biocontrol efficacy of antagonistic yeasts.

利用拮抗酵母抑制果实采后病害，作为一种安全而且环境友好的新型生物保鲜技术，受到了国内外广泛的关注。但其抑菌效力目前与化学杀菌剂相比仍有较大的差距。为探索细胞壁完整性信号途径的主调节因子Rho1在拮抗酵母逆境适应和生防效力中的作用，本项目拟在分析一株著名的采后生防酵母-罗伦隐球酵母rho1对温度胁迫、氧化胁迫、渗透压和振动等胁迫条件下的响应规律的基础上建立rho1与该酵母逆境适应之间的相关性；通过在毕赤酵中过表达rho1研究其在抑制果实采后病害中的作用；利用酵母双杂交技术筛选、鉴定与Rho1互作蛋白，通过LC-MS/MS技术在蛋白组层面上系统观测Rho1及其互作蛋白在逆境胁迫下的表达规律，从而进一步分析Rho1相关的生理代谢途径和所涉及的逆境反应信号网络。研究结果不仅将为明确罗伦隐球酵母Rho1及相关的代谢途径在其逆境适应中的分子机理，还将为开展增强酵母逆境适应和生防活性的技术提供新的信息。

利用拮抗酵母抑制果实采后病害，作为一种安全而且环境友好的新型生物保鲜技术，受到了国内外广泛的关注。但其抑菌效力目前与化学杀菌剂相比仍有较大的差距。为探索细胞壁完整性信号（CWI）途径的主调节因子Rho1在拮抗酵母逆境适应和生防效力中的作用，本项目在分析罗伦隐球酵母rho1及CWI信号途径相关因子对胁迫条件下的响应规律的基础上建立了rho1与该酵母逆境适应之间的相关性；通过在模式酵中过表达rho1研究其在逆境适应和抑制果实采后病害中的作用；利用酵母双杂交技术筛选、鉴定与Rho1互作蛋白；通过定量荧光PCR技术、转录组技术和蛋白组学技术系统观测Rho1及CWI途径相关因子在逆境胁迫下的表达规律。研究结果对明确罗伦隐球酵母Rho1及CWI信号途径在逆境和生防中的作用具有重要作用，并将为开展增强酵母逆境适应和生防活性的技术提供新的信息。