番茄嫁接辣椒介导的RNAi抗CMV机制研究

Cucumber mosaic virus (CMV) disease is one of the most widely distributed, most popular and most important diseases on pepper and tomato in China. At present, there are still lack of safe and effective prevention measures to control CMV. The RNAi signal induced by dsRNA can be transmitted through grafting. Grafting-mediated resistance by biotechnology has shown considerable application prospects on pepper because of stubborn transform. In this study, inverted-repeat dsRNA plant expression vectors with different lengths of CMV CP gene, MP gene will be constructed respectively, and then transformed each vector into model tomato (Moneymaker) by agribacterium-mediated transformation. Transgenic tomato (Moneymaker) with resistance to CMV will be obtained. The grafting will be performed using transgenic tomato (Moneymaker) and pepper. Analyses the RNAi signal’s conduction properties and accumulation level in grafted plant. Clarify the influence of different genetic background scion for RNAi signal systematic movement. Explore the relationship between RNAi signal’s features and grafted plant resistance to CMV. Reveal the molecular mechanism of RNAi in plant antiviral defense. Establish the technology system of resistance to viruses by grafting combined with RNAi. The results of this study have important guiding significance and reference value for controlling viruses in production. And can also provide a technical platform for more research on green prevention and control of plant virus diseases.

黄瓜花叶病毒（cucumber mosaic virus，CMV）病是我国辣椒、番茄生产上分布最广、最流行、最重要的病害之一。目前生产上仍缺乏安全有效的防治措施。基于dsRNA诱导的RNAi信号可经嫁接传导，对于辣椒等难转化植物，利用生物技术进行嫁接介导抗病性展现了可观的应用前景。本项目拟构建CMV CP和MP基因不同片段长度的dsRNA载体转化番茄Moneymaker，筛选阳性番茄与辣椒进行嫁接，分析RNAi信号分子在不同嫁接植物体内的传导特性、积累水平，明确不同遗传背景接穗对RNAi信号分子系统运动的影响及其与病毒抗性的关系，深入揭示RNAi在植物抗病防御中的作用机制，建立利用嫁接技术结合RNAi的抗病技术体系。研究结果对防治植物病害具有重要的理论指导意义和参考价值，同时也为更多植物病毒病的绿色防控研究提供技术平台。

黄瓜花叶病毒（cucumber mosaic virus，CMV）病分布广泛，危害严重。目前生产上仍缺乏安全有效的防治措施。基于dsRNA诱导的RNAi信号可经嫁接传导已被证实，利用生物技术进行嫁接介导抗病性的机制值得深入研究。本项目利用RNA沉默原理，选取CMV CP和MP基因不同片段长度（300 bp和500 bp左右），分别构建了4个反向重复dsRNA表达载体（dsC3-5941、dsC5-5941、dsM3-5941、dsM5-5941）转化番茄并进行嫁接，鉴于番茄-辣椒间的嫁接属于种间嫁接，嫁接成活率低，给嫁接植株体内开展RNAi信号传导特性的研究带来困难，故选择阳性转基因番茄-非转基因番茄间嫁接植株为研究对象，建立了番茄嫁接体系，番茄嫁接成活率高达95%以上；明确了siRNA信号可在嫁接植株体内进行扩散传输且可双向传导，向上传导的速率大于向下传导的速率；确定了在几种不同类型的抗性嫁接植株体内RNAi信号的稳定存在性；分析了嫁接植株体内CMV不同基因的相对表达量，发现抗病型嫁接植株体内不同基因的相对表达量较对照植株显著减少；嫁接植株较对照未嫁接番茄植株的CMV发病率低，分别为60%和100%，病情指数分别为37.33%和68.57%。本研究结果表明，siRNA信号可通过嫁接进行双向传输并介导植物对CMV的抗病性，为阐述嫁接介导的RNAi抗病毒机制提供了重要的理论基础。