基于多聚半乳糖醛酸酶抑制蛋白（PGIP）诱导西藏芜菁根肿病抗性的分子机制研究

Clubroot, caused by the obligate parasite Plasmodiophora brassicae Woronin, is one of the most important diseases of crops belonging to the Brassicaceae, causing annual losses of 10%-15% worldwide. It is controlled most effectively by the use of resistant cultivars. At present, resistance genes from stubble turnips (Brassica rapa L.) are most effective and most widely used in resistance breeding of different Brassica crops. Unfortunately, knowledge is limited on the resistant reaction of clubroot which is induced by polygalacturonase- inhibiting proteins (PGIPs) in the resistant cultivars of turnip so far. In this study, the native turnip landraces and semi wild races will be collected and identified in Tibet region, and the core collection will subsequently be constructed through the diversity analysis with the SSR and EST-SSR methods. After identification of core collection plants, the resistant cultivars will be employed to analyze the temporal and spatial expression pattern of BrPGIPs derived from these resistant plants. Moreover, the response of BrPGIPs expression will be carried out during the secondary infection phase, and the biological function of BrPGIPs will also be analyzed by artificial microRNA method. These results can provide some useful clues to illuminate whether there is redundant function among 5 BrPGIPs in turnip or not. Moreover, it is helpful for searching the molecular mechanism of resistant reaction of clubroot induced by the BrPGIPs proteins during the root cortex injection and will also benefit for us to improve the basic research of resistant biology in plant as well as for the genetic resource innovation. Understanding how PGIP works in this capacity holds great potential in regard to the breeding for disease resistance, genetic engineering, and genomic research in Brassica crops.

十字花科植物根肿病由芸薹根肿菌Plasmodiophora brassicae侵染所致，该病现被认为是十字花科作物生产中经济损失最严重的病害。目前对PGIPs诱导芜菁根肿病抗性反应的分子机制还知之甚少。本项目研究拟在西藏地区广泛搜集整理芜菁地方原始栽培种及其半野生种，利用SSR和EST-SSR技术对种质资源开展遗传多样性研究，并构建其核心种质。随后，筛选获得高抗根肿病的种质材料，阐明抗病芜菁品种PGIPs的时空表达模式及其抑制P. brassicae质配合子发生皮层侵染的响应特征，分析芜菁BrPGIP1-BrPGIP5基因的生物学功能，明确PGIPs是否存在功能冗余，进一步揭示芜菁基于PGIPs蛋白诱导根肿病抗性反应的遗传基础与分子机制。研究结果能为充分理解芸薹属作物根肿病诱导抗性的分子机理，完善经济作物的抗病生物学研究基础，开展芸薹属作物抗性种质的创新研究提供新的理论依据和技术支持。

十字花科作物根肿病是由芸薹根肿菌Plasmodiophora brassicae侵染所致，该病现被认为是十字花科作物生产中经济损失最严重的病害，正在世界范围内流行危害，每年因此病造成的减产达20%~30%，严重的地块甚至绝收。目前对PGIPs诱导芜菁根肿病抗性的分子机制还知之甚少。本项目研究在西藏地区广泛搜集整理了172份芜菁地方品种，利用植物形态学形状观察，SSR和EST-SSR技术对上述种质资源开展遗传多样性研究，并构建了包含45份材料的初级核心种质。随后，根肿病抗性鉴定试验获得高抗ECD16/0/0生理小种的芜菁材料4份。随后，对主要芸薹属作物的PGIP基因家族进行了鉴定，分析了芜菁12个BrPGIPs基因的时空表达特性，及其对P. brassicae侵染的响应特征。利用生物信息学工具HMMER对芸薹根肿菌蛋白数据库进行BLAST，获得1个候选PG序列PBRA_006621，其Genebank对应的核酸序列为CEO98507.1。进一步的Y2H和BiFC实验结果显示，9个PGIPs与CEO98507.1形成的所有组合均没有产生互作信号。由此我们认为，芸薹根肿菌中没有编码PGs的基因。最后，分析了BrPGIP4和BrPGIP8的生物学功能，异源过表达BrPGIP4和BrPGIP8拟南芥明显提升了对菌核病的抗性，转基因植株对根肿病未表现出抗性，进一步印证了芸薹根肿菌对寄主的侵染并不依赖于PGs乃至CWDEs。综上，芜菁BrPGIPs可能通过非PGIP—PG的互作方式间接参与根肿病的抗性形成过程。研究结果能为我们充分理解芸薹属作物根肿病诱导抗性的分子机制，完善经济作物的抗病生物学研究基础，开展芸薹属作物抗性种质的创新研究提供新的理论依据和技术支持。