桃树类病毒与病毒的协生作用及其与寄主互作的分子机制

The synergism between viriod and virus occur in natural situations and have biological and epidemiological implications.Double infection of peach with Peach latent mosaic viroid(PLMVd) and Cherry green ring mottle virus(CGRMV) frequently occurs in the field.In this work,the synergism of double infection of peach with PLMVd and CGRMV and the molecular mechanism of their interactions with host will be studied.Using microarray technology,in combination with gene ontology(GO)enrichment analysis and metabolic pathway annotator software,we will perform microarray profiling to explore changes in the expression profile of field-grown peach,upon single and double infection with PLMVd and CGRMV.The study will reveal a impact of the double infection with PLMVd and CGRMV on host gene expression among defense/stress responses,chloroplast function,hormone signaling,silencing and diverse other functions comparing a single infection with PLMVd and CGRMV, and find key role genes associated with symptoms development,such as the chlorophyll biosynthetic gene CHl1,the chloroplastic heat-shock protein 90(cHSP90) and hormone metabolism regulating factor in signal transduction. With deep sequencing,semi-quantitative RT-PCR,RLM-RACE(RNA ligase-mediated rapid amplification of cDNA ends),Northern hybridization method,we study the viroid-derived small RNAs(vd-sRNAs) in double infection with PLMVd and CGRMV targeting host cHSP90 mRNA and we aim to reveal symptoms could be produced in synergism between viriod and virus by the RNA silencing machinery.The results of this research will help to further understand synergistic phenomenon occur between viriod and virus.Furthermore,These studies can help to fully understand plant-pathogen interactions and to develop appropriate crop protection strategies.

本项目针对类病毒与病毒协生作用这一科学问题，以桃潜隐花叶类病毒(PLMVd)和樱桃绿环斑驳病毒(CGRMV)为研究对象，通过果园普查，转录组水平分析等方法，研究PLMVd与CGRMV复合侵染引发的协生作用及其与寄主互作的分子机制。通过微阵列技术，配合基因功能富集分析，研究PLMVd/CGRMV复合侵染的寄主基因表达变化（对防御/应激反应，叶绿体功能、激素信号传导、基因沉默等的影响），揭示与协生作用症状变化相关的关键基因（叶绿素热激蛋白90（cHSP90）,叶绿素生物合成基因（CHll）及与激素代谢调节相关因子等）；明确类病毒与病毒的协生作用。用高通量测序，RLM-RACE,Northern杂交方法研究vd-sRNAs对cHSP90 mRNA的作用，明确RNA沉默在类病毒协生作用中的作用。研究结果不仅对PLMVd/CGRMV的防治提供理论依据，而且对其他类病毒或病毒协生作用的研究有借鉴意义。

类病毒/病毒在自然条件下常复合侵染，类病毒/病毒间的协生作用在自然条件下具有生物学和流行病理学意义。本项目首先，通过大量田间普查和室内RT-PCR检测，siRNA高通量测序，明确了包含PLMVd，CGRMV与其他病毒的协生作用；其次，使用转录组分析，配合基因功能富集分析（GO分析和代谢通路Pathway分析），研究病毒复合侵染的寄主基因表达变化，鉴定了一些与协生作用症状变化相关的关键基因，包括与叶绿体相关的cHSP90，及在信号传导中与激素代谢调节相关因子，防御机制相关因子；进一步，用siRNA高通量测序，半定量RT-PCR，荧光定量PCR方法研究vd-sRNAs对cHSP90 mRNA的作用和转录水平，表明复合侵染寄主的cHSP90 mRNA转录水平较低，揭示了病毒介导的RNA沉默的在复合侵染中的可能作用。本项目对类病毒、病毒间协生作用的研究具有重要理论意义，为探索不同病毒属间的协生现象和分子机制的深入研究提供了参考价值，对病毒病的防控具有重要指导意义。