基于蛋白组学的甜瓜响应蔓枯病菌侵染关键基因的挖掘与功能分析

Melon is one of the most important fruits for summer season in our country, and plays important roles in agriculture structure adjustment and wealth increasing for farmers. However, with the dramatic increasing planting area of melons in protected facilities, gummy stem blight (GSB) is now becoming the most serious disease in melon production. The study of the interaction between melon and Didymella bryoniae will help us to identify important defense genes and to apply them into breeding. This study is firstly to observe the infection process of a susceptible melon cultivar Honeydew and a resistant cultivar PI14071 infected by a green fluorescent protein (GFP) tagged D. bryoniae strain. Then，proteomic, phosphoproteomic and glycoproteomic changes of the two melon cultivars before and after inoculation at different time points are analyzed by 2D-PAGE combined with post-transcriptional modification (PTMs) staining dye, and specific proteins after D. bryoniae inoculation will be identified by MALDI-TOF-MS. Functional analysis including gene cloning, expression analysis, protein abundance and subcellular localization will be performed. This study is to unravel the molecular mechanism during melon in response of D. bryoniae infection, and finally it provides theoretical foundation for melon breeding against gummy stem blight.

甜瓜是我国最重要的夏令水果之一，在农业产业结构调整与农民增收创富中占有重要的地位。设施甜瓜种植面积的急剧增加使甜瓜蔓枯病已成为保护地厚皮甜瓜栽培中发生最普遍、危害最严重的病害。研究甜瓜与蔓枯病菌之间的互作机制对于发掘新的抗病基因并应用到抗病育种中具有重要指导价值。本研究首先拟通过构建携带GFP标记基因的蔓枯病菌，以感病甜瓜Honeydew与抗病甜瓜PI14071为材料，进行蔓枯病菌侵染甜瓜动态过程的组织病理学观察；在此基础上，通过2D-PAGE结合PTMs分子染料探针得到蔓枯病菌侵染不同抗性甜瓜叶片差异表达量蛋白图谱，磷酸化、糖基化蛋白差异图谱，结合MALDI-TOF-MS技术鉴定获得甜瓜响应蔓枯病菌侵染特异蛋白；并对特异蛋白进行基因克隆、表达量分析、亚细胞定位等功能研究。研究有助于阐明甜瓜响应蔓枯病菌侵染应答机制，最终为甜瓜抗蔓枯病育种提供理论依据。

本项目成功建立甜瓜蔓枯菌株农杆菌高效转化体系，成功转化得到携带GFP蔓枯病菌株3株,并用于监测蔓枯病菌在甜瓜中的侵染过程。运用激光共聚焦方法直观的获得了不同时期蔓枯菌株侵染甜瓜的侵染过程，初步明确了蔓枯病菌通过表皮进行侵入并最终导致甜瓜发病的侵染机理。为获得早期甜瓜响应蔓枯病菌关键基因，对不同抗性两个甜瓜材料（感病材料43，抗病材料T9）分别选取1dpi，3dpi的叶片进行双向电泳，以43-DB/CK，T9-DB/CK的差异倍数(>1.5 fold, p<0.05)进行计算，一共获得164个差异蛋白点，其中成功鉴定得到150个差异蛋白，非冗余蛋白数为118个。根据KEGG及文献报道分为代谢途径相关（30个，25.4%）、光合作用相关（13个，11.0%）、生物合成（8个，6.8%）、逆境胁迫相关（22个，18.6%）、氧化还原（7个，5.9%）、转录/翻译（14个，11.9%）、蛋白加工（11个，9.3%）、蛋白酶体（5个，4.2%）、功能未知（8个，6.8%）9大类。本研究鉴定获得几个可能作为早期甜瓜抵御蔓枯病菌胁迫响应相关的重要基因，如P21，heat shock 70 kDa Proteins, 14-3-3 proteins, autophagy-related protein 18g 和 MRGH21等。另外，本研究开发了一种可以广谱并特异性检测蔓枯病菌的LAMP检测方法，并对不同来源的西甜瓜蔓枯病菌进行了抗药性研究。本研究为甜瓜抗蔓枯病育种提供了理论指导，并对蔓枯病的防治具有十分重要的指导意义。