白菜eIF(iso)4E基因与TuMV互作机制解析

The virus disease is the main disease affecting the production of Chinese cabbage. Turnip mosaic virus (TuMV) is the main pathogen causing virus disease. eIF(iso)4E is a kind of very important resistance gene which can interact with protein VPg and initiate the translation and propagation of the virus when TuMV infects the plant host. Our project team has cloned the resistant gene retr02, revealed the resistant mechanism, and found that the different TuMV strains could interact with the various eIF(iso)4E copies. In addition, we have cleared that four amino acid points have played an important role in the interaction process, but the function was unclear. This project is aimed at clearing the key amino acid points of the eIF(iso)4E and TuMV by BiFC, Co-IP and transfecting the Arabidopsis Thaliana. Meanwhile, the three-dimensional structure of the eIF(iso)4E and TuMV VPg would be analyzed to reveal how the key points influenced the structure and function, which would be helpful to build the interaction model and uncover the interaction mechanism. And the project would provide the theoretical basis for improving the resistant hereditary feature and it also has the important scientific significance and application value for molecular design breeding in Chinese cabbage.

病毒病是影响白菜生产的主要病害，芜菁花叶病毒（TuMV）是引起该病害的主要病原。eIF(iso)4E是一类非常重要的TuMV抗性基因，在TuMV侵染植物寄主时与VPg蛋白互作，起始病毒的翻译增殖。项目组在白菜中已克隆了eIF(iso)4E抗病基因retr02，揭示抗性机制，发现TuMV不同株系互作不同的eIF(iso)4E基因，明确了互作过程中的4个差异位点，但是这些互作位点的功能尚不明确。本项目拟围绕白菜eIF(iso)4E与TuMV互作机制这一科学问题，开展BiFC、Co-IP，转染拟南芥等实验，进行互作位点功能分析，同时对eIF(iso)4E和TuMV VPg二者蛋白复合物空间结构解析，构建互作模型，揭示互作的分子机制，为白菜抗TuMV性状的遗传改良提供理论依据，对白菜抗TuMV分子育种具有重要的科学意义及应用价值。

白菜翻译起始因子是一类重要的隐性抗TuMV基因。本研究从8份抗/感TuMV的白菜高代自交系材料中克隆eIF4E和eIF(iso)4E基因，通过酵母双杂交试验（Y2H）和双分子荧光互补试验（BiFC）分析，TuMV不同株系与白菜eIF4E不同拷贝互作方式不同；通过SOE-PCR技术，定点突变TuMV C4/CDN1 VPg间5个差异氨基酸位点，并鉴定了TuMV VPg蛋白的第I52L和L186F位氨基酸变异、白菜BraA.eIF(iso)4E.c蛋白的第L36F位氨基酸变异，在TuMV与白菜eIF(iso)4E互作中起关键作用；利用Phyre2软件对白菜BraA.eIF(iso)4E.c-1蛋白空间结构进行同源模建，该蛋白包含8个β折叠，3个α螺旋和3个延伸环状结构。根据TuMV与白菜eIF(iso)4E的互作关系，以及互作中关键氨基酸位点分析结果，构建二者的互作模型。白菜eIF(iso)4E与TuMV VPg互作，促进病毒在植株内繁殖、传播。当TuMV VPg蛋白氨基酸发生变异（例如I52L），TuMV VPg蛋白空间结构发生变化，不能和白菜eIF(iso)4E蛋白相互作用；当白菜eIF(iso)4E蛋白氨基酸发生变异（L36F），白菜eIF(iso)4E蛋白空间结构发生变化，不能和TuMV VPg蛋白相互作用，此时，TuMV遗传物质RNA不能在植物体内进行自身复制和传播，此时植株对TuMV产生抗性。该研究为精准筛选抗TuMV的白菜种质资源奠定基础。