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

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。

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.