半野生宽皮柑橘“广柑”抗烟草疫霉菌基因的发掘

Phytophthora spp. causes the most serious diseases including gummosis and root rots in Citrus. Due to lack of resistance genes, breeding for resistance to the pathogen has achieved little progress. Citrus reticulate Blanco cv. Guanggan and Citrus reticulate Blanco cv. Panpugan, which were identified in our previous studies as the most resistant and the most susceptible germplasms to Phytophthora nicotianae, respectively, will be used to cross with each other. Individuals of the F1 generation will be inoculated with the pathogen to assess their resistance, and the segregation data will be used to dissect the mode of inheritance of the resistance genes. Transcriptomes of both parents will be profiled and used to mine for SNPs, from which 200 SNP markers that will cover the whole citrus genome will be developed. Resistant and susceptible individuals of the F1 population will be genotyped at these SNP loci. The odd ratios of the SNPs will be calculated and compared between the resistant and the susceptible populations to obtain the correlations between resistance and SNP odd ratios. The SNPs and the genes closely linked to resistance will thus be identified. Cloning will be followed to isolate up to 3 most tightly linked candidate resistance genes. Over-expression vectors will be constructed and used to transform tobacco and sweet orange. RNAi vectors will also be constructed to transform Guanggan. Transgenic plants will be challenged with the pathogen to verify the function of the genes. Our study will most probably obtain the genes resistant to Phytophthora spp, reveal the underlying genetic mechanisms and lay a basis for resistance breeding.

疫霉菌感染可导致柑橘脚腐病、流胶病等重大病害，而抗病育种却因缺乏抗病基因而裹足不前。本研究拟利用前期从本地柑橘资源中筛选出的高抗烟草疫霉菌材料广柑和高感材料潘溥柑作为亲本互交，通过接种疫霉菌评价F1代个体的抗性，分析抗病性状的遗传规律；分析广柑和潘溥柑的转录组，开发两个亲本间转录本上的SNP标记；挑选在基因组上分布均匀的200个SNP标记，分析F1代抗、感个体的SNP频率，关联分析抗病性状分离结果与SNP频率的相关性，鉴别与抗病紧密连锁的候选SNP标记；克隆1-3个最紧密连锁的候选基因，构建过表达载体，分别转化烟草和甜橙，构建RNAi载体转化广柑，评价转基因植株的抗性，以此来验证候选抗病基因。本研究有望获得柑橘抗疫霉菌基因，解析其抗病机理，为抗疫霉病育种打下分子遗传学基础。

疫霉菌感染可导致柑橘脚腐病、流胶病等重大病害，而抗病育种却因缺乏抗病基因而裹足不前。本研究利用前期从本地柑橘资源中筛选出的高抗烟草疫霉菌材料广柑和高感材料潘溥柑作为亲本互交，通过接种疫霉菌评价F1代个体的抗性，不同抗性等级的杂种后代株数基本上吻合正态分布模型，说明广柑的烟草疫霉菌抗性是多基因控制的数量性状。重测序鉴定了杂交亲本间的SNP标记，共计获得位于外显子的纯合SNP标记位点626个，根据基因注释信息分析功能。qPCR鉴定了参与广柑抗烟草疫霉菌相关的PR蛋白基因，发现PR3基因在广柑中的表达水平远高于枳和感病资源红橘和潘溥柑，且在广柑中受疫霉菌强烈诱导表达，转基因过表达PR3基因显著提高沙田柚转基因后代的烟草疫霉菌抗性。RNA seq研究烟草疫霉菌侵染对广柑基因表达的影响发现，病原菌侵染显著增强细胞壁代谢、次生代谢物质合成、植物防御和信号转导等途径基因的表达；乙烯生物合成相关基因受病原菌诱导表达，推测茉莉酸/乙烯途径参与了广柑的抗病反应；光反应（特别是系统PSII）相关基因的下调表达和淀粉生物合成途径相关基因上调表达能够有效减少广柑叶片中小分子糖的合成，继而限制病原菌获取小分子糖减少其能量来源，限制病原菌的传播；效应子触发的免疫反应中，与RIN4蛋白互作的抗病R基因RPM1和PRS2都上调表达，RIN4基因下调表达，已知RPM1、PRS2及其互作蛋白RIN4在植物抗病反应中起到重要作用，我们首次在柑橘中发现了RPM1、PRS2及其互作蛋白RIN4抗病机制参与了抗烟草疫霉菌反应。本研究有望获得柑橘抗疫霉菌基因，解析其抗病机理，为抗疫霉病育种打下分子遗传学基础。