红三叶抗白粉病qrp-1基因的克隆及功能分析

Trifolium pratense cv. Minshan is a special legume resource in cold and humid area of northwest region with high forage yield, crude protein content and abundant isoflavone in forage. It is a vital crop in the animal husbandry and pharmaceutic industry because of its medical and feeding value. However, it was seriously infected by powdery mildew (Uncinula bicornis (Wallr.: Fr.) Lév) in the long cultivation process due to severe degeneration and slow innovation. In order to solve this problem, this study will clone the powdery mildew resistant gene qrp-1 in red clover selected from our previous research using the map-based method and validate its powdery mildew resistant function via the construction of over expression vector, genetic transformation of red clover and transgenic red clover infection of uncinula bicornis. After this study, the powdery mildew resistant gene qrp-1 in red clover will be cloned and validated. This study will not only establish foundation to the molecular markers-assisted breeding and breeding of new red clover variety with high forage yield and resistance to powdery mildew, but also has important instruction to the mechanism study and exploration of powdery mildew resistance gene in red clover. For this study, it is the first time to clone and validate the genes controlling the powdery mildew disease in red clover breeding and has important innovation.

岷山红三叶是适宜于甘肃高寒阴湿区种植的特有牧草种质资源，其草产量和粗蛋白含量高，而且富含异黄酮，集药用和饲用价值于一体，在保健食品业和草牧业生产中发挥着重要作用。但经过长期种植，该品种退化严重，重度感染白粉病，对草产量和异黄酮提取量影响极大。培育抗白粉病、高产优质红三叶新品种成为草牧业生产亟待解决的问题。为解决此问题，本项目拟对项目组前期定位的抗白粉病主效qrp-1基因进行图位克隆，并通过构建过表达载体、红三叶遗传转化和白粉菌种浸染转基因红三叶植株等方法验证qrp-1基因的抗白粉病功能。通过实施该项目，可以完成红三叶抗白粉病qrp-1基因的克隆和功能分析，不仅可以为红三叶抗白粉病分子标记辅助育种和培育抗白粉病、高产优质红三叶新品种奠定基础，而且对红三叶抗白粉病机理研究和基因挖掘有重要指导意义。本项目拟实施的抗白粉病qrp-1基因克隆和功能分析，在红三叶遗传育种中尚属首次，具有重要创新性。

白粉菌（Erysiphales）作为一类普遍而重要的专性生物营养型病原菌，严重降低红三叶草产量与品质，限制其在草牧业中的应用与发展。为阐明白粉菌对红三叶生理生化、内源激素和细胞结构的影响并验证抗白粉病TpGDSL基因的功能，本项目首先对感白粉病品种（岷山红三叶）和×抗白粉病品种（“甘农RPM1”红三叶）的杂交F2代进行抗病性评价，并建立白粉病抗感分离群体，采用人工接菌的方法，测定白粉菌侵染后不同抗性群体的生理生化变化、内源激素含量和细胞结构变化；利用F2代群体的抗病单株和感病单株作为试验材料，进行转录组分析；克隆由转录组分析得到的红三叶抗白粉病相关候选基因TpGDSL，同时构建pHB-GDSL过表达载体，并遗传转化拟南芥和红三叶。主要结果如下：接菌后抗白粉病红三叶材料内源激素的效应更明显；抗白粉病材料的纤维素、半纤维素、木质素和羟脯氨酸糖蛋白含量略高于感病材料；红三叶GDSL同源基因有较高的本底表达和差异表达倍数，Log2（FC）为10.62，本研究选择GDSL基因进行研究；采用农杆菌介导的花序浸染法将重组过表达载体pHB-GDSL分别转入甘红1号红三叶和野生型拟南芥中，得到11和16个株系的 T1代阳性转基因种子；红三叶白粉病菌具有很强的专一性，用拟南芥转基因植物无法完成抗白粉病TpGDSL基因的功能验证；项目组建立的甘红1号红三叶遗传转化体系验证了TpGDSL基因的抗白粉病功能。本项目将为红三叶转基因研究和抗白粉病分子辅助育种奠定基础。