酵母拮抗菌细胞形态转变的分子调控机制

Biological control using yeast antagonists has been proven to be effective to manage postharvest diseases of fruits and vegetables. To understand the basic aspect of yeast biology is essential for uncovering the biocontrol mechanism. We recently found that when certain yeast strains grew to some density level, their morphology could change from single cell-type to biofilm-type (embedded within a self-produced matrix of extracellular polymeric substance) on certain media surfaces. In this proposal, two common antagonistic yeasts serve as materials. Cell biology, transcriptomics, proteomics and bioinformatics are employed to investigate the mechanism involved in the regulation of morphology change in the yeasts, and the effects on their biocontrol efficacy. This study includes the following parts: (1) comparison of cell structure, component and biocontrol efficacy between single cell-type and biofilm-type yeasts; (2) utilization of RNA-Seq and iTRAQ-based proteomic analysis to investigate gene expression profiles at transcriptome and proteome levels; (3) cluster analysis of data from RNAseq and proteomics, combined with gene knockout and overexpression technique, to discover the signaling pathway involved in yeast morphology change.

基于酵母拮抗菌的生物防治技术是控制果蔬采后病害的有效措施之一。对酵母拮抗菌生物学特性的研究是了解其生物防治机理的前提。本实验室近期发现，当特定的酵母拮抗菌达到一定种群密度，在特定的介质表面上会产生群体效应由单细胞转变为生物膜（由胞外多聚物基质包围形成的结构群落）形态。本项目拟以两种常用的酵母拮抗菌为实验材料，采用细胞生物学、转录组学、蛋白质组学和分子生物学等多学科的方法，研究拮抗菌生物膜的形成机制以及对生防效力的影响。主要研究内容包括：（1）分析酵母拮抗菌发生形态转变之后（从单细胞到生物膜形态），细胞结构、成分和生防效力的变化；（2）使用RNA-Seq技术和iTRAQ蛋白质组学技术，从转录组和蛋白质组水平分析酵母拮抗菌形态转变过程中相关基因和蛋白的表达差异；（3）对组学的结果进行聚类和关联分析，结合基因敲除和过表达的方法挖掘拮抗菌形态转变相关的分子信号途径。

随着人们对食品安全和环境保护等问题关切度的增加，基于酵母拮抗菌的生物防治技术逐渐成为替代化学农药防治果蔬采后病害的有效措施。对酵母拮抗菌生物学特性的研究是认知其生防机理的前提。本项目的研究发现，酵母拮抗菌Pichia kudriavzevii和Candida diversa在琼脂含量为0.3%的YPD培养基上常温培养2天后，会形成生物膜；而在琼脂含量为2%的YPD培养基上培养，则是单细胞状态。处于生物膜形态的酵母菌对于水果寄主（苹果、猕猴桃和梨）采后病害的防治效果更佳。进一步研究表明：生物膜形态的酵母拮抗菌对于高温、氧化、pH和高盐等多种环境逆境的抗性更强，其原因在于处于生物膜形态的酵母菌的抗氧化系统活性更高。对生物膜形态与单细胞形态的拮抗酵母菌进行了蛋白组和转录组学等组学的分析。其中，通过转录组检测分别得到了25009573条（生物膜形态）和20991425条（单细胞形态）的clean reads，共鉴定出了3000多个差异表达基因。通过qPCR随机挑选验证了其中部分差异表达基因的表达，发现与转录组检测的结果相一致。通过生物信息分析发现，这些基因参与了酵母代谢、发育、信号传递、跨膜运输以及胁迫抗性等生物学过程。