大豆Rag_P746基因抗蚜性的功能鉴定及其信号传导途径的研究

Identification of resistant gene and characterization of resistant mechanism is critical for aphid controlling. Resistant gene Rag_P746 has been obtained by map-based cloning based on recombinant inbred lines (RILs) generated by the cross between resistance germplasm P746 and susceptible variety Dongnong47.We intend to identify the ability of aphid resistance in both Rag_P746 overexpression and RNA interference lines, and the accumulation of callose, the amount of reactive oxygen species (ROS) and the activity of key enzymes involved in its metabolism for aphid infestation will be tested. With these approaches the foundation of Rag_P746 would be identified and characterized. Furthermore, the mechanism of aphid resistance related to Rag_P746 will be revealed by differential regulated genes in overexpression lines and transgenic acceptors before and after aphid infestation with RNA-seq and sequence analysis. Yeast two-hybrid system will be applied to screen the factors interacting with Rag_P746, and the results will be verified with co-immunoprecipitation (Co-IP) and forster resonance energy transfer - fluorescence lifetime imaging microscopy (FRET-FLIM) assay. Thus, the aphid resistance mechanism involved in Rag_P746 will be disclosed and the progress of soybean breeding for aphid resistance will be accelerated.

明确抗蚜大豆资源中抗性基因功能及其信号传导途径是有效防治大豆蚜虫的前提和基础。前期利用抗蚜大豆资源P746及感蚜品种东农47构建重组自交系群体，通过图位克隆获得蚜虫抗性基因Rag_P746。本项目拟对已转Rag_P746基因及其RNAi的大豆转化株系进行加代、分子验证及遗传分析；筛选出稳定遗传的纯合转化株系进行蚜虫抗性分析，并对蚜虫接种不同时期植株叶片中胼胝质的积累、活性氧含量及其代谢途径中关键酶活性进行分析，揭示Rag_P746基因的抗性功能特点；利用转录组测序，基因共表达及关联分析等方法，分析Rag_P746基因转化材料及受体品种在蚜虫接种前后分子表达差异；通过酵母双杂交筛选Rag_P746互作因子并通过免疫共沉淀及荧光共振能量转移-荧光寿命成像等手段进行验证，揭示Rag_P746基因参与的大豆蚜虫抗性形成机理，为培育抗蚜大豆新品种奠定基础。

为了明确抗蚜大豆资源中抗性基因功能及其信号传导途径，为有效防治大豆蚜虫提供理论基础，对前期克隆的大豆抗蚜基因Rag_P746的功能和抗性机理进行研究。分析发现Rag_P746提供的蚜虫抗性是由单碱基突变造成的。通过在感虫材料东农47中过表达该抗性基因以及在大豆毛状根中瞬时表达Rag_P746的方法，发现Rag_P746正调控对大豆蚜虫的抗性。通过在拟南芥突变体稳定表达该基因发现能够提高拟南芥对烟蚜的抗性，说明Rag_P746能够提供广谱的蚜虫抗性。通过蚜虫取食前后植物体内激素含量及相关基因表达分析发现，水杨酸在大豆抗蚜虫方面发挥重要作用。通过对Rag_P746互作蛋白的筛选及后期分子验证发现GmWRKY40能够与Rag_P746发生互作。由此推测Rag_P746能够通过与GmWRKY40互作，影响水杨酸的合成，并最终导致对蚜虫抗性的产生。