芸薹种一个新的生理小种特异性抗根肿病基因PbBa3.2的克隆及功能研究

Clubroot disease, caused by Plasmodiophora brassicae, is one of the most serious diseases in crucifers worldwide, and endangered the production of Brassica crops in China. Identification of clubroot resistance (CR) gene and application of these genes to develop CR cultivars is an effective way to control clubroot disease. Although several CR genes were identified in Brassica rapa, these genes showed resistance to only race 2 or 4 of P. brassicae. Further, less is known about the defence mechanisms such as plant innate immune system involved in clubroot resistance. All these limited the application of molecular breeding of CR cultivars. In the previous study, we identified a novel isolate-specific CR gene which showed resistance to race 10 among tested pathotypes. In addition, we developed a near-isogenic line of Chinese cabbage that only carries the PbBa3.2 locus. This locus was mapped to chromosome A03 of B. rapa, and inherited as a single dominant gene. On the bases of above results, we will focused on the followings in this proposal: i) to clone the PbBa3.2 gene by map-based cloning and annotation of gene structure; ii) to study the function of PbBa3.2 by transformation; iii) to study the defence mechanism of clubroot resistance based on the global transcriptional analysis and in combination of the expression pattern of PbBa3.2. A couple of plants with transgene of PbBa3.2 and its wild type will be used for RNA sequencing. Transcriptional analysis will focus on the genes involved in the plant innate immune system, and genes differentially expressed will be selected for further analysis. The results will provide more valuable information to reveal the defence mechanisms in clubroot resistance, and supply the theoretical foundation for molecular breeding of CR cultivars in Brassica vegetable crops.

根肿病是由根肿菌侵染引起的一种世界性病害，现已严重威胁到我国芸薹属作物的生产。挖掘抗根肿病（CR）基因，选育抗病品种是解决根肿病的有效途径。前人从芸薹种发现了多个CR基因，但这些基因只对生理小种2或4表现抗性，且对其参与的防御机制研究仍然滞后，不能满足育种的需求。前期研究中，我们从芜菁中发现了一个对生理小种10表现特异抗性的新位点PbBa3.2，并获得了含该基因的大白菜近等基因系。该基因位于大白菜A03染色体，表现为单基因显性遗传。本项目拟在此基础上，利用图位克隆技术克隆PbBa3.2基因并诠释其结构；通过转基因分析验证该基因的功能；通过PbBa3.2的转基因和非转基因植株的转录组测序，重点分析参与先天性免疫系统的差异表达基因，并结合PbBa3.2的表达模式初步提出芸薹种植物对根肿菌的防御机制。研究结果不仅对进一步揭示抗根肿病机制奠定基础，也为芸薹种蔬菜作物的抗根肿病分子育种提供理论依据。

十字花科根肿病是由芸薹根肿菌侵染引起的一种世界性土传病害，所有十字花科作物的感病品种都能被侵染。根肿病严重危害着我国大白菜等芸薹属作物的栽培和产量，并引起大量的经济损失。因此，发掘抗病根肿病基因，培育抗根肿病新品种是目前有效，环境友好型的防治根肿病的一种手段。抗病基因是研究植物-病原菌互作过程中的关键因素。基于此，本项目通过对抗根肿病基因CRd的精细定位将其定位在A03染色体上15.02-15.15Mb范围内，通过基因克隆，分析了抗病基因的结构特点；通过转基因对CRd进行基因功能验证；通过构建CRd基因的系统进化树分析其进化关系，发现CRd是不同于其他已知抗根肿病基因的新基因。此外，通过亚细胞定位明确CRd基因在植物体内细胞膜上发挥功能以及通过转录组分析，初步揭示了CRd基因的根肿病抗性机理。该研究结果不仅可以应用于大白菜等芸薹属作物抗根肿病新品种的选育，同时也为揭示CRd基因抗根肿病的机理研究奠定基础。