基于染色体片段代换系的苦瓜白粉病抗性主效基因的克隆与功能分析

The powdery mildew resistance of bitter gourd is an important character for bitter gourd cultivation and disease-resistant breeding. The clone of disease-resistant genes and the study about resistant molecular mechanism are still the important problems which are still not solved.In the earlier stage of this research, the bitter gourd strains which are strongly powdery mildew-susceptible and high powdery mildew resistant were selected to construct F2 and F2:3 family population, the major genes of powdery mildew resistant/QTL were localized at the end domain of the first linkage group accroding to the inoculation results of two seasons in two years and the constructed genetic map. Based on these, using multiple generations backcross and Marker-assisted selection(MAS) to construct the Chromosome Segment Substitution Lines(CSSL) of target domain (major genes/QTL domain) and segregation populations of advanced backcross; then fine mapping the powdery mildew resistant genes, chromosome walking and identifying candidate genes to clone the resistant genes at last. Through genetic transformation functional complementation to proof the accuracy of cloning genes and sequencing the target genes, via subcellular localization, analysis of the temporal spatial expression characteristics, transcriptome analysis and so on to further analyse the biological function of resistant genes. The research results about resistant genes’ localization, clone and function would not only illuminate the molecular mechanism of powdery mildew resistance’s formation but also could be used for molecular marker-assisted breeding , promote the application of molecular design breeding in bitter gourd resistance breeding.

苦瓜白粉病抗性是苦瓜生产及抗病育种的重要性状，其抗病基因的克隆和抗性分子机制是尚未解决的重要科学问题。本项目组前期选取高感和高抗的苦瓜亲本构建了F2及F2:3家系群体，根据两年两季的接种鉴定结果和构建的遗传图谱初步将白粉病抗性主效基因/QTL定位在第1条连锁群的末端区域。在此基础上利用多代回交的方法结合分子标记辅助选择，构建目标区域（主效基因/QTL区域）的染色体片段代换系及高代回交分离群体，对白粉病抗性基因进行精细定位，对候选基因鉴定并最终将其克隆。通过遗传转化功能互补验证克隆基因的准确性，并对目的基因运用序列分析，亚细胞定位，时空表达特征分析，转录组分析等方法进一步分析抗性基因的生物学功能。本项目对苦瓜白粉病抗性基因的定位、克隆和功能的研究结果，不仅可阐明苦瓜白粉病抗性形成的分子机制，也可以用于苦瓜白粉病抗性的分子标记辅助育种，促进分子设计育种在苦瓜抗性育种中的应用。

苦瓜白粉病抗性是抗病育种的重要性状，白粉病生理小种演替速度快，防治难度大，挖掘抗性基因辅助抗病育种具有重要意义。本项目明确了海南苦瓜白粉病的优势菌单囊壳白粉菌(Sphaerotheca fuliginea)的生理小种2（2France）的分生孢子萌发的最适温度（25 ℃）和致死温度（48 ℃,10 min），其喜高湿、黑暗和中性环境，好氧，喜葡萄糖，萌发不需氮源，长波光质较短波更有利于苦瓜白粉病菌分生孢子萌发；此白粉病菌在侵染苦瓜叶片时有4个关键时期：接种后4h为分生孢子萌发高峰期，8h为附着孢形成高峰期，16-24h为次生菌丝形成高峰期，5d为分生孢子梗形成高峰期；明确了苦瓜对生理小种2的抗性为数量性状，抗病相对感病为不完全隐性；通过F1和hai09-2多代回交后自交构建了目标区域的染色体片段代换系（CSSL1.1）和大规模分离群体，精细定位了抗病主效基因McPm1.1；在挖掘抗病相关基因中发现苦瓜MLO基因有组织表达特异性，McMLO1、McMLO6和McMLO12与苦瓜感白粉病相关，McMLO1介导的抗感差异与SA途径无关，JA和ABA分别对McMLO6和McMLO8介导的感病有一定作用；为进一步研究抗性基因功能，建立了高效、稳定的苦瓜离体再生体系，下胚轴不定芽发生需要较高水平的CTK、IAA、ETH、GA和一定量的SA、JA、BR、SLS、ABA及可溶性蛋白和可溶性糖；转录组分析表明抗、感苦瓜品系在响应白粉菌侵染中，抗病能力在时间上的差异体现在36-72hpi，抗病苦瓜hai09-2能快速的调动更多基因抵御白粉菌侵染，在代谢通路上抗性信号转导、自噬等方面，前期抗病苦瓜比感病苦瓜能更迅速的响应白粉菌侵染，后期感病苦瓜在以上途径被显著抑制；抗病苦瓜能更迅速传递Ca2+信号，诱导植物细胞壁强化与超敏等防御反应，白粉菌胁迫下合成抵御病原物的病程相关蛋白PR1的能力抗感苦瓜存在差异。本研究结果为苦瓜白粉病菌培养、保存及高效防治提供了重要参考，为后续苦瓜白粉病的遗传、抗性基因功能研究和解析白粉病抗性机制奠定了良好基础，为分子标记辅助品种改良提供了重要依据。