大白菜抗软腐突变体功能基因鉴定和表达分析

Soft rot is one of main diseases in Chinese cabbage practice production. The lack of accessions with high resistance to soft rot is one of problems in Chinese cabbage breeding program, which limited the discovery of good resistance genes and breeding selection of new resistance varieties. In this research, we will use soft rot resistant mutants with similar genetic background as materials. Methods of high throughout sequence mapping mutated genes (MutMap)and gene expression profiling (RNA-Seq) will be applied to identify new genes resisted soft rot in Chinese cabbage. The genetic patterns and expression levels of key genes will be clarified and their expression network will be obtained. Furthermore, the molecular regular mechanism to main pathogenic bacteria of soft rot will be explored in Chinese cabbage. The CRISPR system of gene silence technology in Chinese cabbage will be established. Combing with vernalization-infiltration transformation method, genes function and regular patterns will be researched. Through performing this project, useful accessions for functional genomics in Chinese cabbage will be created. And new gene resources will be identified and molecular regular mechanism to soft resistance will be clarified. These finds not only contributed to keep and increase the international position in brassica rapa genomics of China, but also provided new knowledge to breed new resistance varieties adapted well in the market.

软腐病是大白菜生长过程中的主要病害之一，高抗软腐病种质的缺乏限制了优异抗病基因的发掘和抗病品种的培育。本研究在获得同一遗传背景下的大白菜抗软腐病突变体基础上，利用MutMap高通量测序定位突变位点和RNA-Seq基因表达谱方法，鉴定大白菜抗软腐病新基因资源，确定其遗传特点和表达模式，获得大白菜抗软腐病相关基因的表达互作模式图，明确抗主要致病菌的作用机理；建立大白菜CRISPR基因敲除技术，结合真空渗透转基因体系，开展基因的功能和调控方式研究。通过项目实施，创制大白菜功能基因组学研究的优良材料，发现新型基因资源，解析抗软腐病的分子调控机制。保持和提升我国白菜基因组研究的国际地位和竞争力，为定向培育大白菜抗病新品种提供有利条件。

芸薹属蔬菜是重要蔬菜作物，其中大白菜种植面积和消费量皆居蔬菜作物前列。软腐病是芸薹属作物三大病害之一，田间发病会严重影响其产量和品质。目前缺乏严格意义的高抗软腐病的大白菜育种材料，对软腐病的抗性机理知之甚少。本项目通过离体及活体接种软腐病菌筛选，建立大白菜适宜的抗性分级评价体系，筛选出大白菜抗软腐病突变体sr及多份抗/耐软腐病新种质；利用RNA-Seq测序技术，揭示大白菜防御软腐病相关基因的表达调控机制，接种6-12h可能是大白菜对软腐病菌防卫的重要调控期，病原菌模式激发免疫反应在植物对Pcc的防御中扮演着核心防御角色，Pcc可刺激寄主植物增加內源激素茉莉酸的含量，茉莉酸的积累参与了大白菜对Pcc的免疫反应，茉莉酸/乙烯作为正向调节信号可以增强对Pcc的抗性，吲哚族和芳香族硫甙在大白菜抗软腐病突变体sr中积累并发挥防御Pcc的功能；分别以高感病大白菜“A03”抗软腐病突变体sr及耐软腐病小白菜“华冠”为亲本杂交获得F2代分离群体，分析大白菜软腐病抗性可能是由多个基因控制，利用构建的高密度遗传图谱和Mutmap技术，鉴定出抗软腐病候选基因BrWRKY33、BrPGIP1和BrTIFY10B等多个候选基因，并成功克隆其中3个基因; 探索建立大白菜的CRISPR敲除体系和遗传转化体系，为之后抗软腐病候选基因功能验证提供了极大的技术。该研究率先解析大白菜抗软腐病的调控机制，在同一遗传背景下的高抗软腐病突变体在解析多个相似基因的表达差异方面具有优势。挖掘大白菜抗软腐病的关键调控基因，为大白菜抗病分子标记辅助育种提供有利条件。