苹果和葡萄中环状小RNA分子的鉴定及其致病性研究

项目名称： 苹果和葡萄中环状小RNA分子的鉴定及其致病性研究

项目编号： No.31471747

项目类型： 面上项目

立项/批准年度： 2015

项目学科： 农业科学

项目作者： 李世访

作者单位： 中国农业科学院植物保护研究所

项目金额： 85万元

中文摘要： 类病毒是一类共价闭合的单链环状小RNA分子，可引起多种园艺作物病害；卫星RNA是一类寄生于辅助病毒内的非编码小RNA，完全依赖辅助病毒完成复制，并影响其在寄主上诱发症状。本实验以有锈果症状的苹果和200年历史的葡萄为研究对象，构建其小RNA文库，进行高通量测序并获取小RNA序列，并用PFOR（Progressive Filtering of Overlapping small RNAs）软件发现两种新的环状RNA分子，依其二级结构预测为类病毒或卫星RNA。我们将用分子实验手段验证其存在，明确其结构及生化特性，并构建侵染性克隆，用接种实验研究其自我复制能力和侵染性。对于卫星RNA的鉴定，根据高通量测序结果，分离纯化辅助病毒，并采取环状RNA分子和辅助病毒单独或组合接种实验研究其致病性。此研究对我国苹果和葡萄病害的诊断及防治意义深远，也为类病毒及卫星RNA的分类和进化研究提供重要参考价值。

中文关键词： 类病毒；卫星RNA；苹果；葡萄；高通量测序

英文摘要： Viroids are small self-replicating RNA that spread within and between organisms,which are single-stranded,circular,and noncoding RNA molecules.They can replicate in the nucleus or chloroplast and then traffic cell-cell through plasmodesmata and long-distance through the phloem to establish systemic infection. Currently known viroids are classified into two families : Avsunviroidae and Pospiviroidae, based on their sequences, structures and biological properties. Another kind of pathogenic small RNA is satellite RNA, which is the smallest infectious agent whose replication is thought to be completely dependent on their helper virus. Both of the two kinds of RNA moleculars can lead to important diseases in horticultural plants. Our laboratory choose apple with scar skin symptoms and the 200-year-old grapevine as the research objects, contructing the small RNA libraries, and get the RNA sequence information by the way of high-throughput sequencing . By using the software named Progressive Filtering of Overlapping small RNAs (PFOR) analysising of the information, we obtained two circular RNA moleculars that never found before and predicted that may be viroid or satellite RNA depending on the analysising the secondary structure. For this reason, we plan to certificate the existence by molecular biological technology, so that illuminating the property in structure and biochemistry. At the same time, in order to conform the characteraster of self-replication and infectivity, we decide to construct infectious clone, and inoculate the tobacco plants. For the satellite RNA, dependent on the high-throughput sequencing result,we assemble different inoculating exprement that inoculating the helper virus and satellite RNA,single or both,so that prove whether the RNA is pathogenic.In this research,we can get more information that influences apple and grapevine growing in different kind of diseases.This will make a profound meaning for diagnosising and preventing diseases,and also provide the basis for classification and origin of evolutionary mechanism of viroids and satellite RNA.

英文关键词： viroid;satellite RNA;apple;grapevine;high-throughput sequencing