啤酒花矮化类病毒不同变体致病性差异的分子机制研究

Viroids are nocoding, circular RNAs that can enter plant cells and establish systemic infection, causing severe disease symptoms via regulation of host gene expression. Molecular mechanism of viroid pathogenesis is still largely unknown. The purpose of this study is to further understand the mechanism how viroid incites diseases, based on the research about molecular base of different pathogenicity between two Hop stunt viroid (HSVd) variants (HSVd-g and HSVd-h). These two variants show five different nucleotides between their genome and cause different symptoms on inoculated cucumber (cv ‘suyo’) which is indicator host plants of HSVd. Firstly, each nucleotide of the five mutations between HSVd-g and HSVd-h will be changed through site-directed mutagenesis to generate various HSVd mutants. Purified cDNA of all the obtained mutants will be ligated themselves, producing head-to-tail dimers of cDNA, namely, infections cDNA clones. RNA transcripts of infections cDNA clones of all HSVd mutants will be inoculated into cucumber plants. Pathogenicity of HSVd mutants will be determined by analyzing replication rate, accumulation of HSVd RNA and symptoms of plants. The results will identify the specify nucleotide that majorly controls HSVd pathogenicity. Secondly, we will carry out transcriptome analysis of cucumber plants infected with HSVd-g and HSVd-h and establish transcriptome database of the infected cucumber plant. From the obtained database, genes show homology with small RNAs derived from HSVd genome will be selected. Moreover, the genes with different accumulation level in the two transcriptome databases of infected cucumber plants will also be found. Thirdly, the two sets of infected cucumber plants which has been analyzed by transcriptome will be conducted secondary metabolites determination and produce two metabolomics databases. Different kinds of secondary metabolites and ones with difference of concentration could be filtered between the two metabolomics databases. Following this, metabolic pathway associated with the filtered secondary metabolites will be determined using bioinformatics. Results of this research will not only determine molecular mechanism of different pathogenicity between HSVd-g and HSVd-h, but also provide more information about viroid pathogenesis.

类病毒是一类非编码、环状RNA 分子。它侵染寄主植物，通过影响寄主的基因表达而引发病症。目前，对该机制仍不清楚。本课题以致病性不同的两个啤酒花矮化类病毒(Hop stunt viroid, HSVd)的变体HSVd-g和HSVd-h为材料，通过研究它们的致病性差异来揭示类病毒的致病机制。首先，利用定点突变技术，系统分析HSVd基因组中碱基的改变对其致病性的影响，从而确认决定HSVd致病性的碱基位点。其次，对感染这两个变体的黄瓜样品进行转录组分析，获得转录组数据库，比较这两个数据库中基因的种类和表达量，筛选出差异表达的黄瓜基因。另外，建立代谢组数据库，比较感染这两个变体的黄瓜体内的次级代谢产物的种类及含量，鉴定差异的次级代谢产物，并且分析它们所参与的代谢路径。研究结果不仅能够明确HSVd不同变体致病性差异的分子机制，而且有助于进一步理解类病毒的致病机制。

类病毒是一类非编码、环状RNA 分子。它侵染寄主植物，通过影响寄主的基因表达而引发病症。目前，对该机制仍不清楚。本课题以致病性不同的啤酒花矮化类病毒(HSVd)的变体HSVd-g和HSVd-h为材料，应用定点突变、转录组测序和代谢组测序等技术，系统分析了这两个变体致病性差异的原因。所取得的主要结果包括：1）利用定点突变技术获得了HSVd-g的5个单碱基突变体（相对于HSVd-h来说）。它们在黄瓜上的致病性明显不同，突变体HSVd-g54的致病性最强。这表明单个碱基的改变就能够影响HSVd-g的致病性。2）获得了HSVd-g54和HSVd-h侵染的黄瓜在接种后2 d，14 d和28 d的高质量的转录组数据，分析发现高达1965个基因在这两个变体侵染的黄瓜之间呈现差异表达。这说明感染这两个变体的黄瓜不仅在症状方面具有明显的差异，而且在基因表达水平方面也具有明显不同。功能注释发现这些差异表达基因与光合作用、植物的防卫反应、激素的合成及代谢等生化过程有关。3）获得了HSVd-g和HSVd-h侵染的黄瓜的代谢组数据，共包含50种不同的代谢产物。比较发现果糖、葡萄糖、谷氨酰胺、谷氨酸、丙氨酸、甘油酸盐、天门冬氨酸和苹果酸等代谢物的含量在这两个变体侵染的黄瓜之间具有明显的差异。这说明HSVd-g和HSVd-h侵染的黄瓜在代谢物水平上具有明显差异。研究结果不仅从类病毒基因组结构、寄主转录组和代谢组水平方面解释了HSVd不同变体致病性差异的原因，而且有助于进一步理解类病毒的致病机制。