水稻纹枯病菌dsRNA真菌病毒的基因组结构与功能分析

Genome organizations and functional analyses of dsRNA mycoviruses in Rhizoctonia solani In the previous research conducted in my laboratory, some dsRNA virus-containing strains of rice sheath blight pathogen, Rhizoctonia solani AG-1 IA, have been selected. In order to elucidate the taxonomic taxa and the relationships of their genome organizations and functions of these dsRNA viruses harbored by these strains, this study is carried out. Through this research, in the biological characteristics of this pathogen, to understand the differences in the growth rate, colony morphology and pathogenicity of dsRNA virus-containing and dsRNA virus-free strains; in the viral genome organization and sequence analysis, the full length sequences of 2-3 dsRNA viral genomes were obtained by cDNA cloning, and assembled by using DNAMAN software, the full length sequences were obtained and then submitted to NCBI database for comparative analysis, and their phylogenetic trees were constructed using Mega 5.0 software, so as to elucidate the taxonomic taxa and genome organizations of these dsRNA viruses; in terms of the interaction between mycovirus and host fungus, R. solani AG-1 IA, experiments of virus removal, transmission and transfection were conducted to get the virus-free and virus-containing isogenic strains, and then the traditional methods of plant pathology, as well as the technologies of genomics and proteomics were used to analyse the biological properties, differentially expressed genes and proteins of these isogenic strains, so as to understand the exact effect of hypovirulence-related dsRNA virus on the virulence of R. solani, and thus to elucidate the interaction mechanism of mycovirus and its host fungus at the molecular level.

为了阐明水稻纹枯病菌dsRNA病毒的分类地位及其基因组结构与功能的关系，开展本研究。通过本研究，在病菌生物学特性方面，明确这些含dsRNA病毒的菌株与不含病毒的菌株在生长速率、菌落形态和致病力等方面的差异；在病毒基因组结构与序列分析方面，拟对2～3个dsRNA病毒基因组的全长序列进行cDNA克隆，通过DNAMAN软件进行序列拼接，获得全长序列后提交到NCBI数据库进行比对分析，使用Mega 5.0软件构建其系统发育树，阐明这些dsRNA病毒的分类地位及其基因组结构；在病毒与寄主真菌互作方面，拟通过脱毒、传毒和转染试验，获得带毒和不带毒的等基因菌系，然后利用传统的植物病理学方法以及基因组学和蛋白质组学技术，分析这些等基因菌系在生物学特性、基因和蛋白质差异表达方面的差异，以明确弱毒力相关的dsRNA病毒对纹枯病菌致病力的确切影响，从而从分子水平上阐明真菌病毒与寄主真菌的互作机制。

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。真菌病毒是一类侵染真菌并能在真菌细胞内复制的病毒，一些真菌病毒能够削弱植物病原真菌的致病力，因而具有生防潜力。为了发现更多的侵染水稻纹枯病菌的dsRNA真菌病毒，明确其基因组结构与功能的关系，阐明真菌病毒与寄主真菌的互作机制，开展了本研究。.主要研究内容和重要研究结果如下：（1）水稻纹枯病菌真菌病毒的多样性。从43个供试菌系中发现了17个菌系含有分子量大小为1.7～19 Kb的dsRNA真菌病毒，表明了水稻纹枯病菌真菌病毒具有多样性。（2）新真菌病毒的快速筛选。以水稻纹枯病菌已报道的真菌病毒RdRp保守区为基础，设计特异引物，建立了快速筛选新病毒的RT-PCR方法，并筛选出携带新真菌病毒的菌株4个。（3）新型真菌病毒的基因组结构与功能的关系。对分别来源于水稻纹枯病菌4个菌系（A105、C24、D122和GD-2）的4个新型真菌病毒的全基因组序列进行了克隆，通过DNAMAN软件进行序列拼接，获得全长序列后提交到NCBI数据库进行比对分析；使用Mega 5.0软件构建其系统发育树，对其序列进行分析，明确了这4个真菌病毒的分类地位，将它们分别命名为RsRV3、RsPV5、RsRV5和RsEV1，并阐明了其基因组结构与功能的关系。（4）真菌病毒与水稻纹枯病菌的互作机制。通过传毒试验和脱毒试验，获得了2对带毒与不带毒的等基因菌系D122/ D122-P和GD-118-P-V1/GD-118 P；利用传统的植物病理学方法比较了这些等基因系在生物学性状方面的变化，发现D122和GD-118-P-V1菌系的真菌病毒分别会增强和减弱水稻纹枯病菌的致病力；利用转录组学和代谢组学技术，分别分析了这2对等基因菌系在基因表达和代谢物谱方面的差异，从在生物学、转录组学和代谢组学水平上阐明了真菌病毒与水稻纹枯病菌的互作机制。.通过这些研究，明确了水稻纹枯病菌真菌病毒的多样性，阐明了新型真菌病毒的基因组结构与功能的关系，为将来应用弱毒病毒对水稻纹枯病进行生物防治奠定了基础。