苹果和葡萄中环状小RNA分子的鉴定及其致病性研究

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.

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。

到目前为止，发现的类病毒仍不足40种，这极大地限制了人们对类病毒的认识。本项目的目的是利用高通量测序技术发现更多新类病毒。研究使用的材料是表现有苹果锈果病的苹果树以及生长在新疆的100余树龄的老葡萄树。这主要是考虑到之前有人推测苹果中可能存在一种未知类病毒；不同的葡萄类病毒之间会发生重组，可能会产生新类病毒。分别对这两个样本进行了小RNA的高通量测序。首先，对获得的数据进行生物信息学分析，其中最主要的是利用PFOR程序进行拼接，以此获得大量环状RNA。主要研究结果包括：.1）苹果样品中，除ASSVd外，发现了一个长度为434 nt并且与公共数据库中登录的序列均无同源性的新RNA。RT-PCR和Northern-blot分析均证实该RNA为环状。二级结构中包含锤头状核酶结构，生化分析证实其具有核酶活性。生物学接种实验表明，它可以侵染苹果并进行自我复制。总之，该RNA为鳄梨日斑类病毒科的一种新类病毒，将其命名为苹果锤头状类病毒（AHVd）。值得指出的是，AHVd是目前为止发现的基因组最大的类病毒。.2）葡萄样品中，除啤酒花矮化类病毒、葡萄黄斑类病毒和澳大利亚葡萄类病毒，发现了一个长度为328 nt并且与柑橘类病毒6号（CVd-VI）具有同源性的环状RNA。它的环状特性得到了实验证据的支持。其二级结构呈棒状，包含有苹果锈果类病毒属的一些重要特征如中央保守区（CCR）、发夹结构I（HP I）和末端保守区（TCR）。生物学接种实验表明，该RNA可以单独侵染葡萄，能够在葡萄中进行自我复制。由此，将该环状RNA鉴定为苹果锈果类病毒属的一种新类病毒，命名为葡萄潜隐类病毒（GLVd）。.本项目共获得了2种新类病毒，引起了国内外同行的高度关注。这不仅丰富了已知类病毒的种类，而且使人们意识到类病毒的数量还有更多，其基因组还可以更大。这无疑对于促进类病毒的研究具有重要的意义。.

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