CRISPR/Cas9结合感病基因介导的黄瓜白粉病和霜霉病持久抗性研究

Both powdery mildew (PM) and downy mildew (DM) are very serious diseases in cucumber. Breeding of resistant cucumber varieties has been undertaken for several decennia, but underlying resistance genes have to date not been functionally characterized. The concept on plant susceptible genes (S gene) in recent years has provided a new strategy to generate durable resistance to cucumber powdery and downy mildews by editing S genes. We and our partner have mined most of cucumber S genes using the whole genome sequence information. Also, the constructions of CRISPR/Cas9 vector for Mlo、Pmr and Dmr homologous genes in cucumber were generated. Based on well-rounded CRISPR/Cas9 editing system and genetic transformation system , this project aims to edit cucumber S genes using the CRISP/CAs9 technology to reach potential durable resistance to mildews in cucumber. By an intelligent combination of S gene concept and CRISP technology, this project will provide cucumber mutants with durable resistance to powdery and downy mildews. Protein domains of a S gene that are critical for making mutation will be identified, which can be the target regions for allele mining in natural cucumber germplasm or mutation populations. The mode of action of S genes and their best performing CRISP mutants will be investigated by RNAseq with microscopic study to generate knowledge on durable control of mildews in cucumber.

白粉病和霜霉病是影响黄瓜生产的主要病害。针对这两种病害的抗病育种研究进行了几十年，但时至今日抗病基因的作用机制仍未明确。近年来关于感病基因（S gene）的研究成果，为黄瓜抗病育种研究提供了新思路。通过对S基因进行编辑创新持久抗性材料是防治黄瓜白粉病和霜霉病的新策略。本项目组及合作者已经完成了黄瓜全基因组感病基因的挖掘，以及针对黄瓜Mlo、Pmr和Dmr同源基因多靶点CRISPR/Cas9载体的构建，同时拥有完整的CRISPR/Cas9编辑系统和黄瓜子叶遗传转化体系。本项目以此为基础，旨在利用CRISP/Cas9技术编辑黄瓜白粉病和霜霉病S基因，以期获得对两种病害的持久抗病性，创制具有持久抗性的种质材料；鉴定出导致产生持久抗病性的S基因的关键蛋白结构域，并作为自然种质或变异群体抗病位点挖掘的目标区段；同时揭示寄主和病原物互作过程中S基因的作用模型，解析白粉病和霜霉病持久抗性的分子机理。

白粉病和霜霉病是黄瓜的两种严重的病害，选育抗病品种是控制白粉病和霜霉病的最根本的途径。本研究利用全基因组关联分析技术，在全基因组范围内检测到霜霉病抗性相关的18个位点，并分别在dmG1.4，dmG2.1，dmG4.1，dmG5.2，dmG7.1区域预测到5个抗病相关基因。检测到白粉病抗性相关的12个位点，并分别在pmG2.1，pmG5.2，pmG5.3，pmG4.1区域预测到6个抗病相关基因。在黄瓜基因组中鉴定到62个白粉病感病基因，并根据感病基因的SNP变异进行了单倍型的白粉病抗性分析。结合已知的33个霜霉病感病基因，最终选择APP、PMR4-8、PMR4-9、DMR1、DMR6-1、DMR6-2、CAD1、MLO1、MLO8、MLO11作为候选感病基因。构建了感病基因的CRISPR/Cas9载体系统和RNAi载体系统的质粒载体11个，并通过农杆菌介导的遗传转化体系将载体系统转化黄瓜受体新泰密刺。共获得RNAi载体T0代阳性转化苗120株，通过后代植株的田间抗性调查、候选基因表达分析、苗期接种鉴定发现PMR4-9、DMR1、DMR6-2白粉病抗性表型突出，获得3个抗白粉病的PMR4-9沉默植株；5个抗霜霉病的DMR1沉默植株和4个抗霜霉的DMR6-2沉默植株。此外，共获得CRISPR/Cas9载体T0代阳性转化苗共35株，通过后代植株的抗性鉴定，获得抗白粉病的AAP编辑植株4株，CAD1编辑植株1株，PMR4-9编辑植株4株，DMR1编辑植株1株，MLO编辑植株2株。以上研究获得了具有黄瓜白粉病、霜霉病抗性的材料，对创制抗白粉、霜霉病的优质黄瓜种质具有重要意义。