大豆TCP转录因子GmTRP19响应疫霉菌侵染的分子调控机理研究

Soybean phytophthora root rot is one of the most destructive soil-borne diseases of soybean, and the production of soybean have been caused serious losses. TCP (TEOSINTE-BRANCHED/CYCLOIDEA/PCF) is an unique transcription factors in higher plants, which could bind to the cis- elements, and then regulate the expression of downstream target gene. In previous studies, a TCP transcription factor, GmTRP19 (Glyma05g03610), was localized mainly to nucleus, was found a differential expression in different resistant soybean varieties induced by Phytophthora sojae, and overexpression analysis demonstrated that GmTRP19 enhanced resistance to P. sojae in transgenic soybean roots. In this study, GmTRP19 transgenic plants will be obtained by genetic transformation method using soybean cotyledon node as explant, the function of GmTRP19 will be researched and the downstream target genes will be identified, meanwhile, screening of the GmTRP19 interacting proteins, to further identify the function of the downstream target genes and the interacting proteins. Then, it will be conducted to establish the resistance network of GmTRP19 and the molecular mechanism responsive to infection of Phytophthora sojae. It will also lay the theoretical foundation for disease resistance genetic engineering breeding and shorten the breeding process of soybean phytophthora root rot. Finally, the theoretical basis and genetic resources will be provided to promote the breeding of high yield and quality soybeans varieties.

大豆疫霉根腐病是大豆最具破坏性的土传病害之一，对我国大豆生产造成严重损失。TCP是一类高等植物特有的转录因子，它能够与顺式作用元件结合调控下游靶基因表达。大豆TCP转录因子GmTRP19（Glyma05g03610）是申请人前期验证的受疫霉菌诱导后在不同抗感大豆品种中差异表达的核定位因子，且过表达GmTRP19的大豆根抗病能力增强。本项目将通过以大豆子叶节为外植体的遗传转化方法获得GmTRP19转基因植株，进行GmTRP19功能分析及下游关键基因的预测，同时筛选GmTRP19互作蛋白，并对下游基因和互作蛋白进行功能分析，初步建立GmTRP19参与的抗病调控网络，为揭示大豆抗疫霉根腐病分子机理以及进行抗病基因工程育种奠定理论基础，为加速和缩短我国大豆疫霉根腐病的抗病育种进程提供指导意义，最终为促进大豆高产优质品种选育提供理论依据和遗传资源。

大豆疫霉根腐病是由大豆疫霉菌引起的危害大豆生产的世界范围毁灭性病害，抗病基因工程育种是解决该难题的有希望的途径之一，因此，解析大豆抗病信号转导途径，阐明抗病分子机理及防卫机制，可为从抗病基因工程手段解决该问题奠定一定的理论基础。. 本课题克隆了一个受疫霉菌诱导后在不同抗感大豆品种中差异表达的TCP家族核定位因子GmTRP19，通过基因定量检测和启动子活性分析等方法发现GmTRP19基因受疫霉菌诱导后表达量显著提高且启动子活性显著增强；通过以子叶节为外植体的大豆遗传转化方法获得稳定的GmTRP19基因敲除株系，抗性鉴定结果证实GmTRP19基因正调控大豆对疫霉菌的抗性；通过转录组测序分析、DAP测序分析、酵母单杂交和EMSA等方法筛选获得GmTRP19直接调控的下游靶标基因，结合下游基因表达模式和转基因材料抗性鉴定等证明GmTRP19可以通过抑制过氧化物酶合成相关基因POD29L的表达来降低ROS生成，从而提高大豆对疫霉菌的抗性；同时，通过酵母双杂交、BiFC和转基因材料抗性鉴定等方法证明大豆MYB176蛋白和GmTRP19蛋白之间存在互作关系，且过量表达MYB176基因可以提高大豆对疫霉菌的抗性。. 本课题建立了GmTRP19基因表达和GmTRP19调控下游基因表达网络，同时整合了GmTRP19互作蛋白正调控大豆对疫霉菌抗性的关系，初步建立了GmTRP19参与的响应疫霉菌侵染的抗病调控网络，为解析大豆抗疫霉根腐病机理奠定理论基础；同时，本课题创制了一系列大豆抗疫霉根腐病种质资源，为开展大豆抗病分子育种打下良好基础，具有重要的科学意义和应用价值。