与瓜类褪绿黄化病毒抑制子蛋白P22互作的寄主因子鉴定及其功能研究

Cucurbit chlorotic yellows virus(CCYV) is a newly identified virus in China and transmitted by whiteflies. It infects many economically important cucurbit plants and its infection seriously affects the yields and quality of cucurbits. RNA silencing serves as an important antiviral role in plants. As a counter-defense, many plant viruses encode suppressors that block silencing. Though the components involved in RNA silencing are clear, the mechanism of action of viral suppressors is not well understood and the role of host factors in the process is just beginning to emerge. Screening host factors that interact with viral suppressor will contribute to the identification of host factors involved in the process. In this project, firstly, a melon cDNA library will be used to identify host proteins that physically interact with CCYV P22 through combined verification of yeast co-transformation, bimolecular fluorescence complementation (BiFC) and co-immunoprecipitation, and secondly, the key interacting domain and the function of interacting melon proteins will be elucidated. With these data, this study will lay the foundation for elucidating the mechanism of P22 during infection, provide new ideas for the function of host regulators in RNA silencing pathway, and supply new theoretical evidence for cultivating resistant breed of melon with gene regulation measures.

瓜类褪绿黄化病毒（Cucurbit chlorotic yellows virus, CCYV）作为一种在国内新发现的病毒，通过烟粉虱传播侵染许多重要瓜类作物，严重影响瓜类的产量和品质。RNA沉默现象是植物体内的一种抗病毒机制，病毒通过编码沉默抑制子来对抗RNA沉默，目前有关沉默途径中相关组分的研究较为清楚，而有关调控沉默抑制过程的寄主因子的研究才刚刚起步，筛选与沉默抑制子互作的寄主因子有利于鉴定参与沉默抑制的其他寄主蛋白。本项目通过构建甜瓜cDNA文库，进行文库筛选寻找与CCYV沉默抑制子P22互作的甜瓜蛋白，分别通过酵母共转化、双分子荧光互补技术和免疫共沉淀技术进行体外和体内互作验证，进而确定互作蛋白与P22互作的关键功能域及其对病毒侵染和沉默抑制的影响，该项目的顺利实施以期为阐明P22蛋白在病毒侵染寄主过程中的作用机制奠定基础，为寻找参与RNA沉默抑制调控的寄主因子提供新的思路。

本研究以提取甜瓜叶片总RNA为供试材料，应用SMART技术构建甜瓜cDNA酵母文库，文库的滴度为2.05×106 CFU /mL，插入片段大小范围为0.5～2 Kb，重组率为100%。将P22303的基因片段构建到诱饵质粒pGBKT7以获得重组克隆pGBKT7p22303，对其进行自激活和毒性检测表明该蛋白无自激活活性和毒性。将诱饵质粒进行文库筛选获得98个克隆，测序比对后选择同源性比较高且阅读框正确的5个基因，分别为Rpol、SKP1A、SKP1B、TF1和TRF26，分别扩增其全长阅读框，将其构建到pGADT7载体上，与P22进行酵母共转化验证。结果表明其中有3个蛋白可以与P22互作，它们分别是Cucumis sativus ribosomal prtoein，（Rpo1），Cucumis sativus SKP1-like protein 1A-like，Cucumis sativus SKP1-like protein 1B-like。通过蛋白分析软件对P22303蛋白进行功能域分析发现其84—101aa为跨膜区，通过构建诱饵质粒pGBKT71-252，将其分别与pGADT7SKP1A，pGADT7RPol, pGADT7SKP1B进行共转验证，结果表明Rpol能与pGBKT71-252互作，SKP1A和SKP1B均不能互作。通过BiFC进行体内互作验证结果表明RpoI和SKP1A能够与P22发生体内互作，而SKP1B不能与P22在烟草中互作。为了进一步研究互作蛋白的基因功能，本研究通过分两段扩增CCYV RNA1和RNA2全长的方式分别构建了两者的35S启动子驱动的侵染性克隆，侵染性验证试验表明侵染性克隆能够在黄瓜上引起典型的褪绿黄化症状，进一步通过Western blot和RT-PCR验证确定了CCYV侵染性克隆的侵染性，侵染性克隆的构建为后续的基因功能研究奠定了基础。