抗根肿病基因PbBa8.1的克隆及其编码蛋白介导的抗病机制初探

Clubroot disease caused by, Plasmodiophora brassicae is the most destructive disease affecting rapeseed (Brassica napus) production in China. Since it emergence, production losses have increased by 60% and more than 8 million mu of farm land have been abandoned because of clubroot disease infestation. Due to the lack of resistant varieties, this disease has become a major threat to the safety of China's rapeseed industry. In the last 7 years, our group at HZAU, took the lead in developing the first Clubroot resistant variety "Hua Shuang 5R", however, due to complex pathogenicity of clubroot disease and long-term cultivation of the same resistant variety is likely to lead to loss of resistance; and the need for developing broad spectrum durable resistant varieties to sustain China’s rapeseed industry. This study aims to clone the "Hua Shuang 5R" resistance gene PbBa8.1 by fine mapping approach, and on that basis further explore the relationship between PbBa8.1 and clubroot disease pathogen by looking for their differential protein interactions in order to identify key effector proteins secreted by pathogens and provide important evidence to support the study of Clubroot disease resistance mechanisms mediated by the disease resistance R proteins and to systematically dissect the mechanism underlying breakdown of Clubroot resistance genes. Overall this study has the potential to provide important theoretical basis for the future engineering of new rapeseed varieties resistant to clubroot disease.

根肿病是由鞭毛菌亚门根肿菌属芸薹根肿菌（原生生物）侵染引起并专性危害十字花科作物的世界性土传病害，我国油菜根肿病发病面积近千万亩且发展迅速。由于生产上缺乏抗病品种，该病已成为威胁我国油菜产业安全的头号杀手。本课题组率先培育了我国首个对根肿病具免疫抗性的新品种“华双5R”，但考虑到长期种植同一抗性品种容易导致抗性丧失，从长远来看生产上需要持久抗性品种这一重大需求，本研究拟进一步通过精细定位克隆“华双5R”的抗性基因PbBa8.1，并在此基础上发掘接种与不接种条件下在抗病反应中与PbBa8.1互作的差异蛋白，鉴定出一些病原菌分泌的效应蛋白（effector），为深入解析该抗病R蛋白所介导抗病机制以及为系统理解抗性基因抗性丧失的机制提供重要支撑，同时这些结论的获得可为通过分子设计培育对根肿病具持久抗性油菜新品种提供坚实的理论依据。

根肿病是由根肿菌（Plasmodiophora brassicae Wor.,Pb）造成的世界分布广泛专性寄生十字花科的土传病害，随着机械化水平的不断提高，逐渐成为困扰我国油菜生产的重大难题，挖掘有效的根肿菌抗性基因，解析根肿菌与植物互作的分子机制具有重大理论意义。我们从头组装了抗源材料欧洲芜菁ECD04全基因组，，将欧洲芜菁抗根肿病材料ECD04与感病甘蓝型油菜材料华双5号通过远缘杂交的方式创建出抗根肿病甘蓝型油菜材料华双5R（H5R），利用BC3F4世代进行抗根肿菌位点PbBa8.1的精细定位。通过重组单株17CZ0566的后代接种中国根肿菌4号生理小种表型结合基因型分析，可将PbBa8.1分为CRA8.1和CRA8.2两个位点，其中CRA8.1为抗根肿菌的主效位点，而CRA8.2为抗根肿菌的微效位点。我们进一步克隆了PbBa8.1区间内的主效抗病基因CRA8.1.5和微效抗病基因CRA8.2.4并初步明确了其抗病功能。我们还针对根肿菌利用效应因子作为标记进行分类，候选效应因子在不同地区菌群中存在单碱基差异，针对效应因子的特异性标记区分不同菌株具有一定的可行性。本研究从根肿菌中成功分离了23个根肿菌效应因子，利用烟草叶片瞬时表达系统鉴定出10个根肿菌效应因子与抗病基因CRa共表达能使烟草叶片出现坏死。我们对筛选到的10个候选根肿菌效应因子进行表达量分析，结果有7个候选效应因子有较高表达，分别为PBRA6449、PBRA1987、PBRA0804、PBRA8597、PBRA8333、PBRA2550、PBRA0619。我们以效应因子PBRA6449为诱饵筛选油菜酵母cDNA文库，最终筛选出23个与诱饵PBRA6449效应因子互作的潜在油菜植物内源蛋白，其中pGADT7-1(XM_022711195.1)、pGADT7-2 (XM_013805263.2)、pGADT7-3(XM_013855863.2)、pGADT7-4（XM_013801130.2），4个具有完整的全长ORF。通过将其与效应因子PBRA6449点对点验证，结果表明其互作关系具备一定真实性。综上所述，本研究针对根肿菌抗性基因的挖掘、根肿菌效应因子与油菜植物内源蛋白的互作进行抗病机制初探，为十字花科芸苔属植物根肿菌抗性研究提供一定的基础支撑。