黄瓜叶微卷、白粉病抗性突变体PMrl基因的克隆及其抗性机理研究

Powdery mildew (PM) is an important disease in cucumber production in China. Very few resistance genes or QTL have been cloned and the molecular mechanisms of PM resistance in cucumber are largely unknown. From an EMS mutagenesis population, we identified a leaf rolling mutant C1561 that also showed high level of PM resistance. Our preliminary work revealed that this leaf rolling/PM resistance phenotype was due to a single dominant gene PMrl. In this project, we propose to conduct map-based cloning of the PMrl gene with the fine mapping method. The function of candidate gene(s) will be validated with allelism test and association analysis in cucumber natural populations . We will also examine the temporal and spatial expression patterns of the candidate gene through qPCR and in situ hybridization analysis. The regulatory network for PM resistance will be investigated through bulked segregant RNA-Seq and Yeast Two-hybrid System and BIFC. Confocal and electron microscopy will be performed to understand the process of pathogenesis in mutant and wild type near isogenic lines before and after PM pathogen inoculation. This proposed research will provide insights into the genetic and molecular basis of leaf rolling and its link with PM resistance. The results will not only provide new knowledge on molecular mechanisms of PM resistance, but also have practical significance in using this new mutant in cucumber molecular breeding for PM resistance or development.

白粉病是黄瓜的三大病害之一，挖掘抗性相关基因并解析其抗性机制，有助于了解黄瓜白粉病抗性反应的途径和机理，对确定更有效的抗病育种途径，培育高抗白粉病的黄瓜新品种具有重要意义。但由于黄瓜遗传基础狭窄且白粉病抗性多由隐性多基因控制，使抗性基因挖掘困难，对抗性分子机制了解甚少。突变体是难得的资源，可大大提高对白粉病抗性的研究效率。课题组创制了一个叶微卷、白粉病抗性显著提高的优异突变体，并将其显性突变基因（暂命名PMrl）定位在含4个基因的区间内，与目前预测或克隆的白粉病抗性基因不同。本项目拟利用精细定位、位点多样性检测等方法克隆和验证PMrl；通过显微观察、寄主细胞化学分析、PMrl原位杂交及时空表达等分析，揭示PMrl在白粉病抗性中的作用；利用BSR-Seq和酵母双杂等解析PMrl参与的调控网络。研究结果不仅可增加抗病相关基因的多态性，而且也可从一个新的视角诠释黄瓜白粉病的抗性机制。

白粉病是黄瓜的三大病害之一，挖掘抗性相关基因并解析其抗性机制，对确定更有效的抗病育种途径，培育高抗白粉病的黄瓜新品种具有重要意义。突变体可提高对白粉病抗性的研究效率。课题组创制了一个叶微卷、白粉病抗性显著提高的突变体，通过精细定位和MutMap相结合的方法，筛选到控制显性突变性状的候选基因CsHDZ，其CDS上第581个碱基发生点突变，该位点位于与miRNA165/166的结合位点上；通过群体连锁分析、位点多样性检测、另一个表型类似突变体基因的定位和验证、拟南芥和黄瓜遗传转化等证据，说明CsHDZ是控制突变性状的目标基因，该基因定位于细胞核且具有转录激活活性。白粉菌生长发育观察发现，CsHDZ基因影响白粉菌的萌发率和产孢量。寄主组织化学分析等表明CsHDZ基因不通过胼胝质、木质素、H2O2等影响白粉病抗性。转录组数据表明，SA或JA等未参与到突变体对白粉病的抗性提高上，接种白粉菌后差异基因主要富集在蜡质等通路上。叶片蜡质的GC-MS/GC-FID分析发现，突变体叶片蜡质含量显著提高。甲苯胺蓝染色表明突变体叶片角质层厚度增加。白粉菌孢子体外萌发试验发现叶片蜡质可抑制白粉菌萌发。烟草和拟南芥中异源过表达CsHDZ基因，转基因植株的蜡质含量增加。对CsHDZ基因的调控网络进行分析，发现CsHDZ基因受miRNA165/166的调控；通过酵母单杂筛选发现CsHDZ正向调控了蜡质合成基因CER1；通过酵母双杂、BIFC及pull-down等证实CsHDZ与CsMYB96互作。综上提出了CsHDZ基因参与调控白粉病抗性的初步机制：CsHDZ点突变后与cs-miRNA166 结合和剪切效率下降，使CsHDZ累积，引起CsCER1和MYB96上调，导致叶片蜡质增加进而增强了突变体对白粉病的抗性。研究结果从一个新的视角诠释黄瓜白粉病的抗性机制。