基于蛋白组学研究甜瓜响应MNSV侵染的应答机制

Cucumis.melo is one of the important economic crops in China. Melon infected MNSV show system symptoms, including Leaf, petiole, stem appeared necrotic spots or yellow spots and whole plant dwarfing. The fruit showed cavernous space after infection MNSV, which severely lost commodity value and restrict the development of melon industry. In recent years, the cross protection of attenuated strains has become a means of preventing and curing viral diseases. But it has not been studied that how to resist the attack of virulent strains after the attenuated virus and how many proteins were expressed. Therefore, to investigate the resistant genes in melon infected attenuated MNSV and analysis of response mechanism of melon infected MNSV, the study will obtain the attenuated virus by over-lapping PCR method, then inoculate the melon with MNSV attenuated strain and wild strain.Using the iTRAQ-based quantitative proteomic approach,we analysis the key proteins induced by attenuated virus and explore the genes involved in defense and clarify their functions, understand the response mechanism of melon infected MNSV. It is better understand the molecular mechanism of the plant-pathogen interaction and the molecular mechanism of melon resistance against the pathogen. The results will be to provide theoretical basis and practical value for the new cultivation improvement using attenuated virus strain to resistant MNSV.

甜瓜坏死斑点病毒(Melon necrotic spot virus，MNSV)是我国甜瓜上重要病害，该病害侵染甜瓜后果实内部出现海绵状空隙丧失商品价值，严重威胁甜瓜产业发展且难于防治。近年来弱毒株交叉保护已成为防治病毒病的重要手段，目前国内外对MNSV弱毒株交叉保护及寄主受到弱毒株侵染后启动了哪些途径的蛋白表达、这些蛋白又如何抵抗强毒株攻击的研究还未开展。因此，本项目为了挖掘甜瓜受到弱毒株诱导后参与其抗性反应的基因和分析甜瓜响应MNSV侵染的应答机制，采用over-lapping PCR方法获得MNSV弱毒株，并用MNSV弱毒株和野生株侵染甜瓜，运用iTRAQ技术分析甜瓜受弱毒株诱导的特异蛋白，挖掘与诱导抗病相关的基因并阐明其功能，解析寄主植株甜瓜对MNSV侵染后的应答机制，揭示寄主与病原物的互作及其宿主抗病的机制，为培育抗MNSV的甜瓜新品系提供理论依据。

甜瓜坏死斑点病毒(Melon necrotic spot virus，MNSV)是甜瓜上的重要病毒之一，该病毒严重制约了甜瓜产业的发展。为了解析甜瓜应答MNSV的侵染，揭示寄主与病原物互作的致病机制机制，本研究构建了MNSV的侵染性克隆突变体库，初步筛选了1个发病延迟株系，并在此基础上构建了携带病毒的GFP载体，该载体可实现病毒侵染后在植物体内的可视化，为快速筛选抗病毒品种提供支撑。此外，本研究运用iTRAQ技术分析甜瓜受MNSV侵染前后的蛋白差异，共获得可信的差异蛋白186个，其中3-羟基异丁酸脱氢酶（3-Hydroxyisobutyrate dehydrogenase-like 1，3-HIBADH）在MNSV接种3d后的蛋白水平及mRNA水平的表达量均提高很多。前期的研究表明p42是MNSV编码的致病因子，因此3-HIBADH与致病因子p42的互作将会为解析病毒的致病机制奠定基础，于是我们进行了3-HIBADH与p42的互作研究，结果表明3-HIBADH与MNSV致病因子p42互作，且互作区域为p42-s domain，烟草表皮细胞中共定位发现两者的互作使3-HIBADH在烟草表皮的亚细胞定位发生了变化，且产生了大量的颗粒状物质，推测该物质可能是p42是病毒的沉默抑制子相关，参与了病毒的沉默机制，其互作的生物学意义仍待研究。该结果为病毒与寄主的互作致病机制奠定基础，为培育抗MNSV品系提供新思路。