一个缺乏P1基因的马铃薯Y病毒科病毒运动蛋白的鉴定及其功能研究

Yam chlorotic necrotic mosaic virus (YCNMV) is a new tentative member of the genus Macluravirus in the family Potyviridae. It lacks of the P1 coding region which was known for playing an important role in the cell-to-cell movement and transmission process. It was still unknow what effects will be taken to the movement process if lack of P1 protein. Today, the movement proteins (MPs) in most genus of the family Potyviridae have been well studied in the past few years. However, there is still no movement protein was identified from the members of the genus Macluravirus, and the mechanism of cell-to-cell movement in members of this genus is also ambiguous. To promote the understanding of the infection mechanism of YCNMV, we attempt to identify the MPs of YCNMV by applying the trans-complementation system with the movement-defective PVX infectious clone. Mutation analysis may also be conducted by employing the infectious clone of YCNMV. It was presumed that three to five MPs may be involved in the movement process of YCNMV due to the properties of multifuction protein, and we will investigate the basical function of at least one movement protein by bimolecular fluorescence complementation (BiFC), yeast two-hybrid screen (YTHS) and gene mutant assay in this study. Based on the results mentioned above, we believe this research will promote our understanding on the infection mechanism of members in genus Macluravirus and the function of P1 region in family Potyviridae.

薯蓣褪绿坏死花叶病毒(YCNMV)是一个缺乏P1基因的马铃薯Y病毒科柘橙病毒属病毒。P1基因与病毒细胞间移动和系统传播有密切关系。缺乏P1基因是否会对病毒传播中具有重要作用的移动蛋白功能有影响。目前，马铃薯Y病毒科其它属病毒的运动蛋白已有报道。然而，对于缺乏P1基因的柘橙病毒属病毒的运动蛋白仍没有被鉴定，也没有该属细胞到细胞间运动的相关分子机制信息。为了探索YCNMV的侵染机制，本项目拟利用运动缺陷型PVX载体和YCNMV可能运动蛋白进行反向互补实验(以构建该病毒侵染性克隆进行突变分析为补充)鉴定出YCNMV的运动蛋白。因Potyviridae病毒蛋白是多功能蛋白，预期可能会有3-5个蛋白与病毒的运动有关。同时，利用双分子荧光互补、酵母双杂交和突变分析等研究方法初步了解至少一个运动蛋白的基本功能。通过这些研究将为我们理解柘橙病毒属病毒的侵染机制和马铃薯Y病毒科P1基因的功能提供有用的数据。

薯蓣褪绿花叶病毒(Yam chlorotic mosaic virus,YCMV)是一个缺乏P1基因的马铃薯Y病毒科柘橙病毒属病毒。HC-Pro，P3N-PIPO，CI，VPg和CP基因在马铃薯Y病毒科病毒中都有报道可能具有运动蛋白功能，但因病毒不同其运动蛋白也可能不同。到目前为止，在柘橙病毒属中还没有相关病毒运动蛋白的研究报道，也没有该属病毒细胞到细胞间运动的相关分子机制信息。本项目开展了YCMV可能运动蛋白的鉴定及功能研究和YCMV侵染性克隆的构建。. 亚细胞定位实验表明，HC-Pro，VPg和CP蛋白主要分布于细胞核和细胞质中，无明显的分布趋向性，与运动蛋白相关性较小。而P3N-PIPO和CI在细胞中没有检测到表达信号，说明他们细胞中可能没有表达或表达量较低。然而，当P3N-PIPO和CI分别与沉默抑制子P19共浸润后观察到P3N-PIPO定位于细胞膜，CI主要定位于细胞质中，呈现出胞内聚集体结构。当P3N-PIPO、CI和P19共浸润后可在胞间连丝观察到点刻状结构。这些结果暗示了P3N-PIPO和CI可能是YCMV的运动蛋白。BiFC实验结果发现P3N-PIPO与CI有明显的互作，并在胞间连丝处呈现出典型的点刻状结构。这些结果表明，P3N-PIPO和CI是通过相互作用来共同完成YCMV运动蛋白功能的。另外，BiFC实验表明，P3N-PIPO和CI均能与P19有相互作用。. 开展了YCMV HC-Pro和VPg沉默抑制子的鉴定，结果表明，HC-Pro和VPg未表现出沉默抑子的功能特性。沉默抑制子的鉴定还需开展更多的研究。. 构建了T7和35S启动子下YCMV的侵染性克隆，经验证均无侵染性。. 蚜传实验表明，YCMV可通过桃蚜进行传播，但传播效率低。