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

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 confirm the biological function of the key protein molecules.Implementation of the project will be provide important theoretical basis for clarifying the molecular mechanism of conidia heat-resistance and creating heat-resistant engineering strains of Metarhizium by molecular design.

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。

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。本研究的前期工作，通过iTRAQ（isobaric Tags for Relative and Absolute Quantitation）技术分析了正常培养绿僵菌纯孢子（作为对照组）和38℃热胁迫处理（恒温水浴2h）孢子的蛋白质表达特征，结果仅发现36个差异表达蛋白且没有差异表达的热激蛋白被发现。在此基础上，本研究将绿僵菌孢子以38℃热胁迫处理2 h，进一步着手从转录水平即采用高通量数字基因表达谱技术（RNA-Seq）分析热胁迫处理前后差异表达基因；进而筛选绿僵菌孢子中耐高温胁迫相关的差异表达基因；其次，结合生物信息学网络分析并确认孢子中耐高温胁迫的特异基因所参与的生物学通路；最后，利用基因敲除等实验手段对关键分子进行生物学功能验证。研究结果如下：(1)高温胁迫下绿僵菌孢子与正常培养孢子（作为对照组）的转录组表达谱存在显著差异，分别发现数千个上、下调相关基因，其中，数十个热激蛋白和海藻糖积累相关基因的表达被发现显著改变；(2)进一步的pathway分析发现大量参与蛋白酶体、内吞作用和自体吞噬代谢通路的基因表达上调，而参与核糖体代谢通路的基因表达下调；这两部分工作已在Applied microbiology and biotechnology正式发表；(3)本研究选择了数个在热胁迫处理前后显著差异表达的基因利用基因敲除方法进行功能分析，其中GPCR（G-protein coupled receptor, EFY96386）基因被敲除后，与野生型和基因回补株相比，突变株对逆境（包括热、UV 、NaCl、SDS、Congo red、H2O2和多菌灵抗性）的敏感度下降；此外，突变株的产孢量也显著下降。这些结果的获得拓展了对孢子热胁迫应答分子机制的理解，也为进一步通过分子设计创制耐热工程菌株提供了重要的理论基础。