基于高通量测序发现荔枝中新的类病毒及其致病性鉴定

As we all known, viroids are covalent，closed，small circular RNA, which induce different diseases in potato, fruit, flowers and lots of horticultural crops and lead to heavy losses in horticultural production. There is still a lot of viroid undetected or reported. Identifying the pathogenicity of a new discovered pathogens is the premise and foundation of disease control. With the development of modern technology, high-throughput sequencing technology has become a new way to discover the virus and viroid. During analyzing the transcriptome data of litchi, which was come from other research institute, we accidentally discovered several contigs that were suspected belonging to viroid. For further study, it was not only considered as a novel viroid, but also was a species of the genus Apscaviroid by RT-PCR using universal primers in this genus. In this research, we plan to amplify the full sequence by designing the adjacent primers. According to the full sequence information, it will be executable to construct its infectious clone in the pGEM-T and pCAMBIA1305.1 plasmids containing a dimeric head-to-tail transcript of this viroid. The infective transcripts RNA and the agrobacterium liquid can be used to inoculate various herbal plants (Tobacco, Tomato, Cucumber, Chrysanthemum, Leguminosae) and its natural host litchi respectively, so that demonstrate its host range and pathogenicity. In addition to this, we will plan to investigate the viroid in litchi orchards and the herbaceous plants near to litchi plants for the purpose to identify its distribution and pathogenicity. It is not only the first report for this new viroid, but also the first time to find a new viroid that classified to the genus Apscaviroid and that parasitized in subtropical litchi trees. Clarifying its host range and pathogenicity not only strengthen the pathogenic diagnose of litchi viroid, but also provide groundwork for the prevention and control of this viroid disease. It has a profound historical significance to analyze the models of epidemic spreading and protection against to this viroid occurring in other subtropical fruit trees.

类病毒是共价、闭合环状小RNA分子，能引起马铃薯、果树及花卉等多种园艺作物发病并导致重大经济损失。高通量测序(NGS)技术已经成为发现和鉴定病毒及类病毒病原的一条新途径，对新发现的类病毒病原物进行致病性鉴定是防控类病毒病害的前提和基础。本研究室在前期工作中，偶然从荔枝转录组数据中发现疑似类病毒序列，并用RT-PCR方法证实了荔枝中确实存在一个迄今为止未见报道的类病毒新种。本研究将通过设计背向背引物，扩增该类病毒全长序列并构建其侵染性克隆及植物表达载体，用摩擦接种和农杆菌侵染两种方法接种自然寄主和草本指示植物，确定其寄主范围并鉴定其致病性。同时调查该类病毒在田间荔枝及荔枝树周边草本植物上的发生分布情况，确定该类病毒和田间症状之间的对应关系。这是首次在荔枝上发现类病毒，明确其寄主范围及致病性不仅为荔枝类病毒病害的诊断和防控奠定基础，而且对今后进一步研究类病毒的流行传播规律也具有重要意义。

类病毒是共价、闭合、环状的小RNA分子，能侵染马铃薯、果树及花卉等园艺作物，引起严重病害，导致重大经济损失。近些年来，随着高通量测序（NGS）技术的发展和应用，生物学研究进入了大数据时代。因此，数据分析和挖掘显得尤为重要。目前，公共数据库中登录了大量的不同物种的测序数据，包括基因组、转录组以及小RNA等。然而，对这些数据的分析和使用仍比较有限，仅一小部分的信息被分析和提取出来，大部分的信息仍有待于挖掘。从植物病原物检测和鉴定的角度看，很多物种的测序数据中一般也包含病原物的序列，通过数据分析和挖掘，应该可以从中发现新的病原物。本项目的研究验证了这一猜测。通过对公共数据库中的一些转录组数据进行重新分析，分别从荔枝和铁皮石斛中发现了两种新类病毒——荔枝类病毒（LVd）和铁皮石斛类病毒（DVd）。LVd基因组大小为304 nt，含有苹果锈果类病毒属的中央保守区（CCR）和末端保守区（TCR）序列，与已知类病毒的序列相似性最高为62%。LVd在荔枝上的检出率较高。值得指出的是，市售的一年生或两年生荔枝苗能够检出LVd，说明LVd可能通过种子传播。将构建的侵染性克隆接种常见的几种草本植物包括烟草、番茄、菊花和黄瓜，但均未能成功。DVd基因组大小为324 nt，含有苹果锈果类病毒属的中央保守区（CCR）和末端保守区（TCR）序列，与已知类病毒的序列相似性最高仅为56%。LVd在铁皮石斛上的检出率很低，不到1%。总之，本项研究成功从公共数据库中的转录组数据中发现了两种新类病毒，这不仅丰富了类病毒的种类，而且为以后的数据挖掘提供了借鉴。