基于蛋白质组学方法的绿僵菌孢子热胁迫响应分子机制研究

项目名称： 基于蛋白质组学方法的绿僵菌孢子热胁迫响应分子机制研究

项目编号： No.31201568

项目类型： 青年科学基金项目

立项/批准年度： 2013

项目学科： 植物保护学、园艺学与植物营养学

项目作者： 汪章勋

作者单位： 安徽农业大学

项目金额： 23万元

中文摘要： 绿僵菌是一类广泛应用于生物防治的昆虫病原真菌，孢子耐热性差限制了其商品化程度和大田应用效率。然而，关于绿僵菌及其他生防真菌孢子耐热的分子机理，至今仍不甚明确。本项目以虫生真菌模式生物金龟子绿僵菌为研究对象，从分子水平上探讨该菌孢子高温胁迫响应的机制。首先，实验拟将绿僵菌孢子以38℃热胁迫处理2 h，运用定量蛋白质组技术（iTRAQ）分析高温胁迫下绿僵菌孢子与正常培养孢子（作为对照组）的蛋白质组表达谱，并筛选出绿僵菌孢子中耐高温胁迫相关的差异表达蛋白，同时利用实时荧光定量PCR和Western 技术进行耐高温特异蛋白表达的验证；其次，结合生物信息学网络分析，进一步确认孢子中耐高温胁迫的特异蛋白及其相应基因所参与的生物学通路；最后，利用基因过表达和沉默等实验手段对关键蛋白分子进行生物学功能验证。该项目的实施将为阐明绿僵菌孢子耐热的分子机理和通过分子设计创制耐热工程菌株提供重要的理论基础。

中文关键词： 罗伯茨绿僵菌；热胁迫；RNA-seq；iTRAQ；功能基因组学

英文摘要： Metarhizium is an insect pathogenic fungi widely used as a biocontrol agent,low thermotolerance of conidia is the main impediment to the level of commercialization and the efficiency of field applications. However, the mechanisms involved in the thermotolerance of Metarhizium and other fungal biocontrol agents are poorly understood. In the present study,we used model organisms of entomogenous fungi M.anisopliae as object of study to investigate the mechanism of high temperature stress response of conidia at the molecular level. Firstly, we intend to treat with conidia of M.anisopliae by heat stress at 38℃ for 2 h, proteomic expression profiles of conidia under high temperature stress and normal cultured conidia (as control group) were analyzed by the use of quantitative proteomic technology (iTRAQ),and then high-temperature stress-related protein of conidia were further screened, at the same time, Real-time PCR and Western technology was employed to validate the expression of specific proteins concerned with the high temperature stress; Secondly, specific proteins concerned with the high temperature stress and their corresponding genes involved in biological pathways were further confirmed by bioinformatics network analysis;Finally,experimental methods,such as gene overexpression and silencing, were employed to

英文关键词： Metarhizium robertsii；heat stress；RNA-seq；iTRAQ；Functional genomics