柑橘衰退病毒与寄主植物间蛋白质互作研究

Citrus tristeza virus (CTV) is the most serious viral pathogen of citrus. The virus has a highly complicated genome, and shows differentiation in its molecular population and biological phenotypes. Previously, many studies have focused on its gene functions and pathogenetic mechanism. However, the specific interaction between a virus and its host proteins is the foundation for a successful viral infection. In this project, we will perform a large-scale mapping of protein-protein interactions between a severe CTV strain and its sensitive host Mexican lime proteins by a high-throughput yeast two-hybrid (Y2H) screens of Mexican lime cDNA library. The purpose is to provide a comprehensive view of the interactions among CTV proteins and the interactions between CTV and its host proteins. A set of proteins involved in those interactions will be identified and their functions will be analyzed. The strain-specific interaction between differental CTV strain and citrus proteins will be compared and its relationships to the viral pathogenecity will be analyzed. Eventually, a network of protein-protein interactions will be constructed by connecting proteins involved in those interactions. When the project is finished, a cDNA library of Mexican lime with a high titer will be obtained. For the first time, the interaction mechanism between CTV and citrus will be revealed at a protein level. The obtained results will provide useful information for further understanding the molecular mechanism of RNA virus infection in plants. In practice view, it can make a fundamental base for discovering new resistant genes to the virus and exploring novel gene inferring strategies for the control of plant virus diseases. Therefore, the performance of the project is theoretically and practically important.

柑橘衰退病毒（CTV）是柑橘上危害最严重的病毒，该病毒基因组结构复杂、存在明显的分子特性及致病性分化，有关该病毒的基因功能及其致病机理是目前的研究热点，而病毒与寄主间蛋白互作是病毒侵染寄主并致病的基础。本项目拟采用高通量的酵母双杂交技术，明确该病毒编码蛋白间及其与寄主蛋白的互作特点，筛选和鉴定直接参与互作的病毒和寄主蛋白，深入分析其功能；通过对CTV不同株系候选基因编码蛋白与寄主蛋白互作的比较分析，探寻CTV与寄主互作的株系特异性及其与该病毒致病性的关系；采用生物信息学方法构建CTV与寄主蛋白互作图谱。项目的完成将获得高滴度的墨西哥莱檬cDNA文库，首次从蛋白质水平上解析CTV与柑橘寄主的互作机理，在理论上可为进一步揭示RNA病毒侵染植物的分子机制提供重要信息，在实践上可为发掘新的抗病毒基因资源、探索新的基因干扰策略控制病毒病提供重要依据并奠定基础，具有十分重要的理论意义和潜在应用价值。

柑橘衰退病毒（CTV）是严重危害柑橘的病原物，有关该病毒基因功能及其致病机理是目前的研究热点，而病毒与寄主蛋白间互作是病毒建立侵染关系的基础。本研究结果表明中国柑橘CTV具有明显的株系及基因型分化，该病毒CPm和CP基因具高度的遗传多样性，基因漂移、基因重组和负向选择压力在CTV复杂种群形成中起重要作用。对该病毒CP抗原表位进行作图，鉴定出3个线性表位及1个决定血清学反应的重要氨基酸位点。这些研究成果为开展该病毒与寄主互作研究奠定了材料基础，并为进一步研究基于抗原结构的CTV诊断技术奠定了理论基础。酵母双杂交分析发现CTV不同株系的CP 和p20均可发生自身互作，首次鉴定出CTV的P20与不含跨膜结构域的P61和P65互作及CPm与P18、P61和P65。构建了墨西哥梾檬和枳壳cDNA文库，采用酵母双杂交技术筛选到8个可能与CTV p20和/或p23互作的寄主蛋白。双分子荧光互补分析确认p20与4个寄主蛋白（编号181、201、213和281）及p23与3个寄主蛋白（编号181、 213和281）具明显互作，且互作的荧光信号在烟叶片表皮细胞中的分布具有明显的差异，其中p20与181和281-C互作信号主要在细胞膜或细胞质，而p20与201、213和281-N互作信号在细胞质中，呈颗粒状；p23与181互作信号分布在烟叶片表皮细胞的气孔周围及细胞膜或细胞质；p23与213和281互作信号在细胞质中，呈颗粒状，推测发生这些互作的组织分布特点可能与不同生物学功能有关。沉默抑制作用分析的结果显示，P20与181互作对P20沉默抑制作用无明显影响。该研究首次鉴定出与CTV互作寄主蛋白质，为进一步揭示植物RNA病毒侵染的分子机制提供了重要信息，并为发掘新的抗病毒基因资源奠定基础，具有重要的科学意义和潜在应用价值。