甜瓜白粉病广谱抗性基因CmMlo6的克隆与功能解析

Powdery mildew is a devastating diseases of melon which seriously aspects yields and fruit quality. The large number of physiological races of the powdery mildew and highly variable in their virulence make that control melon powdery mildew by using monogenic resistance is quite difficult. Clone and function study of the gene conferring broad-spectrum resistance is not only necessary for elucidating the mechanisms of broad-spectrum powdery mildew resistance, but also for creating new powdery mildew resistance germplasm resources. Recently evidences indicated that loss-of-function mutations in Mlo genes confer high levels of broad-spectrum resistance. Based on the melon whole genome sequence and RT-PCR, we found a Mlo gene CmMlo6, which is the potential target for melon broad-spectrum powdery mildew resistance. In this study, we plan to apply the further functional resolution of CmMlo6 gene at transcriptome level, cellular level and transgenosis level. RT-qPCR strategy will be applied to detect the expression profiles of CmMlo6 after inoculate the P.xanthii and different kinds of hormones. The electrophoretic mobility shift assays will be performed to determine the binding capability of the recombinant CmMlo6 and subcellular localization of CmMlo6 will be performed to clarify the spatical distribution in cell level. Then the vector of RNAi and overexpression of CmMlo6 will be constructed to verify their biological function.The results will provide technical support for melon efficient precision resistance breeding and lay a solid foundation for elucidating the mechanisms of melon broad-spectrum resistance to powdery mildew.

白粉病是甜瓜（Cucumis melo L.）生产中重要病害之一，严重影响甜瓜的产量和品质。由于甜瓜白粉病菌生理小种多、分化快，利用专化型抗性基因防控甜瓜白粉病困难较大。开展甜瓜白粉病广谱抗性基因研究，对于揭示白粉病广谱抗病机制、创制甜瓜白粉病新抗源具有重要意义。本研究在充分挖掘甜瓜基因组信息、筛选得到甜瓜白粉病广谱抗性候选基因CmMlo6的基础上，进一步从表达、细胞和转基因水平对该基因进行功能解析。利用RT-qPCR分析CmMlo6基因的时空表达模式，通过阻滞凝胶和亚细胞定位研究CmMlo6基因在细胞中的作用部位和起抗性作用的关键元件，利用RNAi沉默和转基因超表达验证CmMlo6基因的生物学功能，研究结果将为甜瓜定向精准抗病分子育种提供技术支撑，为深入解析甜瓜白粉病广谱抗性分子机制奠定理论基础。

本项目在甜瓜基因组测序完成的情况下，基于基因组信息全面挖掘参与甜瓜抗白粉相关Mlo基因，经过筛选获得甜瓜白粉病广谱抗性候选基因CmMlo6的基础上，并进一步对该基因的表达模式、生物学特性及功能进行全面解析。首先，根据基因组预测结果设计引物从抗感材料里扩增得到了CmMlo6基因cDNA全长，该基因cDNA全长均为1314bp，抗感材料在抗感材料中存在两个SNP分别是299 bp处的G-A和531bp T-C突变位点。该基因在抗性材料中编码437个氨基酸，该蛋白存在7个MLO蛋白特有的跨膜螺旋；通过基因枪介导的洋葱表皮瞬时表达实验发现CmMlo6蛋白的定位于细胞膜上起作用。RT-QPCR结果显示该基因在甜瓜不同组织部位均表达，但表达丰度不同，在子叶和真叶中表达量较高，其次是茎和幼果，根和花中表达量较低。接种白粉病菌不同时间点CmMlo6基因的表达量进行分析发现，CmMlo6基因的表达量在接种后3h和6h与对照差别不大，接种12h达到高峰，随后24h迅速降低，36h表达量再次升高，48h和72h表达量低于对照。转基因过表达和RNAi互补实验初步验证了该基因的具有抗白粉病功能，以上研究结果将为甜瓜定向精准抗病分子育种提供技术支撑，为深入解析甜瓜白粉病广谱抗性分子机制奠定理论基础。