寄主诱导软腐病菌DAPL1及DAPL2基因沉默增强猕猴桃抗病性的机理研究

Fruit rot is the most serious fungal disease in kiwifruit storage period, which seriously shackled the healthy development of kiwifruit industry in the world. Develop resistance breeding is critically urgent to kiwifruit industry. On the fundation of explored virulence genes based on the whole genome of Diaporthe actinidiae and strand-specific RNA-Seq of three susceptible kiwifruit cultivars during infection, the project is intended to apply host-induced gene silencing (HIGS) technique to verify the function of pectate lyase virulence genes DAPL1 and DAPL2. Meanwhile, HIGS expression vector will be constructed by Gateway technology, and transgenic resistant cultivars will generated by agrobacterium mediated transformation. The result will lay the foundation for molecular breeding of kiwifruit resistant varieties by HIGS, and provide important theoretical research basis and technical support for solving the threat of soft rot disease to kiwifruit industry in China.

果实软腐病是猕猴桃贮藏期最严重的真菌性病害，严重桎梏了世界猕猴桃产业的健康发展。尽快对猕猴桃软腐病展开抗性育种研究是我国猕猴桃产业的迫切需求。在前期利用主要致病菌Diaporthe actinidiae的全基因组及其侵染3个高感猕猴桃品种的链特异性转录组数据，深入挖掘关键致病基因的研究基础上，本项目拟运用寄主诱导的基因沉默（Host-induced gene silencing, HIGS）技术对果胶酸裂解酶（Pectate lyase）编码基因DAPL1及DAPL2进行反向致病功能验证。同时，使用Gateway技术构建HIGS干扰表达载体，通过农杆菌介导的遗传转化方法获得猕猴桃抗软腐病转基因株系。项目的开展将为后续进一步利用HIGS技术进行猕猴桃分子抗性育种研究奠定基础，并为缓解果实软腐病对我国猕猴桃产业的威胁提供重要理论依据及技术支撑。

果实软腐病是猕猴桃贮藏、转运及销售期间最严重的真菌性病害，给世界猕猴桃产业造成了重大经济损失。开展猕猴桃软腐病抗性育种研究是我国猕猴桃产业发展亟待解决的关键问题。在前期研究基础上，本项目首次构建了D.actinidiae菌株的原生质体介导遗传转化体系及全新的Crisper/Cas9真菌基因编辑载体，得到了果胶酸裂解酶（Pectate lyase）编码致病基因DAPL1及DAPL2的基因编辑突变子。其次，通过野生型菌株及突变子的菌落形态特征观察、生长速度测定、菌丝显微结构观察及菌株致病力等对比分析，确定了DAPL1及DAPL2基因的关键致病基因功能。最后，成功构建了HIGS遗传转化体系，获得猕猴桃抗软腐病转基因株系。本项目的开展为猕猴桃软腐病及其他真菌病害的致病基因功能验证及分子抗性种质创制奠定了理论及技术基础。