山楂属植物中苹果褪绿叶斑病毒全基因组解析及其致病机制研究

Apple chlorotic leaf spot virus (ACLSV) is one kind of RNA virus impairing the plants of Rosaceae family. ACLSV is one of the most economically important viral pathogens, but its pathogenic mechanism is unknown. Hawthorn (Crataegus spp.), originated in China, is not only one important economic forest tree, but also a good ornamental plant in Rosaceae family . In our previous study, we found that the hawthorn trees were infected by ACLSV. In this project, de novo assembly of the whole genome of ACLSV in hawthorn trees will be obtained by high-throughput sequencing. The effects of ACLSV on gene expression levels in hawthorn trees will be elucidated by transcriptome analysis. The structure and character of vsiRNAs originated from ACLSV will be carried out by high-throughput sequencing of small RNAs form the hawthorn plant infected with ACLSV. And then the target genes of vsiRNAs will be predicted by analysis the expression of genes of hawthorn with the complementary sequence of vsiRNA. The splitting site on predicted target gene will be validated by degradome sequencing. The effect of vsiRNA on target genes in hawthorn and functions of target genes will be validated by transferring the target gene and vsiRNA into plants. The aim of this project is to elucidate the pathogenic mechanism of ACLSV with vsiRNA. The results of this project will lay a foundation for ACLSV prevention.

苹果褪绿叶斑病毒（Apple chlorotic leaf spot virus, ACLSV）是一种广泛侵染蔷薇科植物的RNA病毒，对经济性状具有严重影响，但其致病机制并不明确。山楂原产我国，是蔷薇科中重要的经济林植物和良好的园林观赏植物。前期研究结果表明，我国的山楂属植物中宿有ACLSV。本项目拟以"国家果树种质沈阳山楂圃"保存的山楂种质资源为试材，首先利用高通量测序技术解析山楂属植物中ACLSV的全基因组序列；然后对感染ACLSV的山楂植株和健康植株进行转录组、小RNA组、降解组分析，明确ACLSV对山楂基因表达的影响，揭示源自ACLSV的病毒小干扰RNA（vsiRNA）组成和特性，预测山楂基因组中vsiRNA的潜在靶基因；最后通过转基因验证vsiRNA对山楂靶基因的转录后沉默作用及靶基因的生物学功能。本项目旨在从vsiRNA角度揭示ACLSV的致病机制，为ACLSV的防治奠定基础。

苹果褪绿叶斑病毒（Apple chlorotic leaf spot virus, ACLSV）是一种广泛侵染蔷薇科植物的RNA病毒，对经济性状具有严重影响，但其致病机制并不明确。山楂原产我国，是蔷薇科中重要的经济林植物和良好的园林观赏植物。前期研究结果表明，我国的山楂属植物中宿有ACLSV。本项目以“国家果树种质沈阳山楂圃”保存的山楂种质资源为试材，首先利用高通量测序技术解析山楂属植物中3个ACLSV分离物的全基因组序列，并与其它蔷薇科树种中的ACLSV分离物进行了序列比较和进化关系分析。建立了山楂属植物中ACLSV的RT-PCR检测体系，对80份山楂属植物携带ACLSV的情况进行检测，结果表明18份山楂资源携带ACLSV（带毒率为22.5%）。然后对感染ACLSV的山楂植株和健康植株进行转录组、小RNA组测序。转录组结果表明，两种材料之间有9910个差异表达。这些差异基因的KEGG功能聚类分析表明，“植物病原菌互作”是上调差异基因富集最显著的途径，“类黄酮生物合成”是下调差异基因富集最显著的途径。小RNA组测序表明，感病材料中存在源自ACLSV的病毒小干扰RNA（vsiRNA），对这些vsiRNA的组成和特性进行分析，并预测了vsiRNA靶向山楂的基因。通过qRT-PCR定量，RLM-5’RACE和烟草瞬时转化试验验证了vsiR1360(-)能够作用于山楂基因CpLRR-RLK1。从山楂中克隆出CpLRR-RLK1的全长编码区，构建了CpLRR-RLK1基因的过量表达载体，并实现了CpLRR-RLK1基因在苹果的过量表达，初步揭示了CpLRR-RLK1基因表达水平与抗病性的关系。本项目已发表论文7篇，其中在SCI源期刊发表论文3篇。