黄瓜绿斑驳花叶病毒（CGMMV）的运动蛋白与寄主西瓜蛋白互作研究

Cucumber green mottle mosaic virus (CGMMV) is quarantine pests of agricultural plants prescribed by the Ministry of Agriculture of China. It causes huge economic losses to the production of watermelon. The CGMMV encodes 4 proteins and one of them is movement protein（MP）, which help the virus to shuttle between plant cells. It is showed that MP can induce abiotic stress-related gene expression in the host during infection. In previous work the MP gene was cloned from CGMMV and the expression vector of plant was constructed. The constructed vector was transferred into Agrobacterium stain and then transgenic plant was obtained by Agrobacterium-mediated transformation method. A yeast expression library had been constructed using leaf tissue of watermelon induced by CGMMV. Based on these studies, our objectives were: (1) to evaluate the anti-virus capabilities of transgenic plants and clarify the mechanism of resistance of watermelon to MP by detect DNA methylation, siRNA and so on. (2) Obtain target protein of watermelon interactions with MP by screening yeast expression library. To preliminarily analyze the identified interacting proteins with MP by bioinformatics, gene expression, and so on, and further investigate one or two interacting proteins with MP as potential targets for disease resistance. Subcellular localization of target gene in the cell was determined using GFP transient expression vector. Interaction of two proteins will be verified by test of biomolecular fluorescence complementation (BiFC); Number of copies, expression level, and location of interacting proteins in cell will be researched. (3) The functional gene will be over-expression and reverse inhibit expression to understand its effects on disease resistance and the role in resistance signaling pathway. This study will lay the foundation for control of CGMMV and deepen the understanding of signaling pathways regulating disease resistance in watermelon- CGMMV interactions.The germplasm of transgenic watermelon resistance to CGMMV will also be obteined.

黄瓜绿斑驳花叶病毒（CGMMV）是被农业部划为检疫对象的有害生物，给我国西瓜生产造成巨大损失。该病毒编码4个蛋白，其中运动蛋白（MP）协助病毒在细胞间移动。已有研究表明，MP与寄主互作后诱发寄主抗逆相关基因表达。前期，项目组克隆了CGMMV的MP基因并构建了植物表达载体，通过农杆菌介导的遗传转化获得了转基因西瓜植株；构建了CGMMV诱导西瓜叶片组织后的酵母表达文库。据此，本研究计划：1、对转基因植株进行抗病毒能力评价,并对基因组甲基化、siRNA等进行检测，以明确西瓜对MP的抗性机理；2、筛选酵母表达文库寻找与MP互作的靶蛋白。对靶蛋白进行初步分析，筛选其中1-2个抗病相关蛋白进行功能研究，如对靶蛋白进行亚细胞定位和双分子荧光互作等研究；3、将功能基因进行过表达和反向抑制表达，探明其对西瓜抗病性及信号传导途径的影响。该研究将为了解西瓜抗病毒机理及防治CGMMV提供理论支持和抗病毒种质材料。

黄瓜绿斑驳花叶病毒（CGMMV）是被我国政府和国际上几十个国家政府列为检疫对象的有害生物，给西瓜以及整个葫芦科作物的生产都造成巨大损失。该病毒编码4个蛋白，其中运动蛋白（MP）协助病毒在细胞间移动。研究表明，MP与寄主蛋白互作后诱发寄主抗病相关基因表达。构建了CGMMV诱导西瓜叶片组织后的酵母表达文库，利用酵母双杂交技术筛选的到多个MP互作的功能蛋白。我们对与MP互作的西瓜蛋白的功能进行了研究。MP蛋白能够与西瓜热激蛋白HSP20发生互作，并首次证实西瓜热激蛋白HSP20与CGMMV运动蛋白互作，在抗病毒侵染时发挥重要作用。.MP蛋白还能够与西瓜ClMTB甲基转移酶发生互作。ClMTB甲基转移酶含有一个保守MT-70结构域，同源分析发现他是一个m6A修饰的甲基转移酶基因。.m6A修饰是RNA的腺嘌呤上的第6为N原子上的H被甲基所取代。他是RNA内部最重要的内部修饰之一，调控RNA的转运、降解、稳定及翻译效率。这种修饰在胚胎发育、幼苗生长、花发育及逆境响应中发挥重要的作用。研究发现，拟南芥m6A去甲基酶AtALKBH9B能够降低AMV病毒的甲基化水平从而抑制病毒复制和侵染。而当TMV病毒侵染烟草后，烟草中m6A修饰水平逐渐降低。那么，m6A修饰是否在CGMMV响应中发挥作用？作用机理又是怎样的呢?为了探明这一问题，我们主要进行了以下3方面的工作：1、鉴定了西瓜中的m6A原件，并对它们在CGMMV侵染后的响应模式进行了研究。2、基于 m6A-seq的西瓜响应CGMMV侵染的m6A甲基化谱分析。3、 ClMTB在西瓜CGMMV抗病响应中的功能。研究表明以MP-ClMTB蛋白互作所调控的m6A修饰很可能在西瓜CGMMV抗病响应中发挥重要作用。.在DNA水平上研究了基因组甲基化对抗病毒的影响，研究发现西瓜基因组CHH区段甲基化水平的降低，在抗病毒侵染过程中发挥重要作用。.在转录后调控层面进行了转录组、micRNA测序、代谢组测序和分析，发现西瓜miR164b及靶基因ClNAC21协同，在抗病毒侵染过程中发挥重要作用。