水稻纹枯病菌菌核形成过程中基因和蛋白质差异表达分析

The differentially expressed genes and proteins during sclerotium formation of Rhizoctonia solani were investigated at the genome and proteome levels by using the sclerotia of R. solani at three various developmental stages as the tested materials. In respect to genomics, the differentially expressed genes were screened by means of cDNA-AFLP technique, and the genes were verified by molecular cloning, sequencing and semi-quantitative RT-PCR, so that the full-length sequences of the genes were obtained. The genes were aligned with the sequences in the NCBI database to search for the homologous related genes, so as to infer their preliminary functions, and then the functions of the genes were verified by means of RNAi and gene overexpression techniques, so as to clarify the roles of these differentially expressed genes during sclerotium formation. In respect to proteomics, the differentially expressed proteins were screened by the use of two-dimensional electrophoresis and mass spectrometry analysis, and the results of protein identification were submitted to the NCBI database for the alignment, so as to analyze the functions of the proteins, the differences of the differentially expressed proteins were verified at the mRNA level by using real-time quantitative PCR, and finally, the gene Ontology function and signaling pathway were analysed. The findings will reveal the regulation mechanism of sclerotia formation of R. solani at the genome and proteome levels, and will lay the foundation for developing a new strategy for the control of rice sheath blight.

以水稻纹枯病菌菌核形成三个不同阶段的菌体为供试材料，从基因组和蛋白质组水平对菌核形成各阶段差异表达的基因和蛋白质进行研究。在基因组方面，利用cDNA-AFLP技术，筛选菌核形成过程中差异表达的基因，并通过分子克隆、测序和半定量RT-PCR的验证，获得差异表达基因的全长序列，并提交NCBI数据库进行比对，查找同源的相关基因，推断其初步功能，再通过RNAi和基因过表达技术验证差异基因的功能，从而明确这些差异表达基因在菌核形成过程中的作用。而在蛋白组方面，采用双向电泳和质谱分析技术，筛选菌核形成相关的差异表达蛋白，将蛋白鉴定结果提交NCBI数据库进行比对，分析获得的蛋白质的功能，通过实时定量PCR验证差异表达蛋白在mRNA水平上的差异，最后进行基因本体功能和信号通路分析。研究结果将从基因和蛋白质水平上揭示水稻纹枯病菌菌核形成的调控机制，为制定新型的水稻纹枯病防控策略奠定基础。

由立枯丝核菌（Rhizoctonia solani）引起的水稻纹枯病是世界性的水稻三大病害之一，给水稻生产造成了巨大的经济损失。该真菌不产生无性孢子，菌核在病害循环中起着重要作用。.本项目以水稻纹枯病菌菌核发育三个代表性阶段的菌体（菌丝体、白色菌核和成熟菌核）为材料，开展了菌核发育过程中差异表达基因和蛋白质研究。内容包括：总RNA提取方法的优化、菌核发育过程中差异表达基因的cDNA-AFLP分析、菌核发育过程中的转录组和蛋白质组学分析以及菌核发育相关基因Rsbtb和Rspmm的克隆与功能分析。.在基因组和转录组方面，本研究首先优化和建立了一种从水稻纹枯病菌菌核中提取总RNA的高效和简便的方法；随后利用cDNA–AFLP技术筛选了水稻纹枯病菌菌核发育过程中差异表达的基因，通过基因克隆技术，最终获得了256个转录衍生片段（transcript-derived fragment, TDF），并将这些TDF提交到NCBI GenBank数据库，获得了相应的登录号；接着采用BLST2GO软件对这些差异表达基因进行了生物信息学分析，结果表明：这些基因主要参与了代谢进程 (38.1%) 、细胞进程 (21.4%) 、定位 (5.2%) 和应激 (2.2%) 等过程；荧光定量PCR（qRT-PCR）表明，多数差异表达基因的表达量与cDNA-AFLP的表达谱相一致；根据基因的功能，选取了与菌核发育相关的基因Rsbtb和Rspmm，通过构建其基因沉默和过表达载体，对它们的功能进行了分析。.在蛋白组方面，运用iTRAQ技术分析了菌核发育过程中差异表达的蛋白质，结果表明，共鉴定得到2 269个蛋白质；采用双向电泳和质谱分析技术，筛选菌核形成过程中差异表达的蛋白质，将蛋白鉴定结果提交NCBI数据库进行比对；本研究分析、鉴定了5个差异表达蛋白质的功能，并通过qRT-PCR验证差异表达蛋白在mRNA水平上的表达量差异，并进行了GO功能分析和信号通路分析。.通过以上研究，获得了一批与菌核发育有关的差异表达基因和蛋白质，为菌核发育分子机理的进一步研究提供了大量的候选基因和蛋白质资源，这些基因和蛋白质中有的可能是菌核发育所必须的关键基因和蛋白质。对这些基因和蛋白质功能的研究，有助于发现水稻纹枯病菌新的作用靶标，对于新型的、抑制菌核形成和发育的水稻纹枯病菌高效杀菌剂的开发具有重要的科学意义和应用前景。