油菜素内酯诱导黄瓜疫病抗性的蛋白质组学研究

Phytophthora blight, caused by Phytophthora melonis Leonian, is one of the most important soil-borne fungal diseases of cucumber plants in China.High temperature and humidity conditions in southchina are favourable for disease development.Brassinosteroids are essential steroidal plant hormones, which have shown to overcome several kinds of pathogens.In this study, cucumber plants were inoculated by adding suspension of zoospores to the soil, and 24-epibrassinolide and its inhibitor brassinazole were also injected to the soil.To study the alleviating effect of 24-epibrassinolide on phytophthora blight in cucumber plants and its mechanism of the biolgical action, we compared the high resolution two-dimensional gel electrophoresis (2-DE) protein profiles in leaves and roots among different treatments, and the main differentially expressed protein spots were determined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and liquid chromatography electro-spray ionization tandem mass spectrometry (LC-ESI-MS/MS). Furthermore, RT-PCR and Real-time PCR approach based on peptide sequences were used to compare transcript and protein accumulation patterns for candidate proteins. In conclusion, our study could provide the theory basis for plant-pathogen interaction model and application of 24-brassinolide to control phytophthora blight, which has important theoretical and actual value.

瓜类疫霉（Phytophthora melonis）引起的疫病是危害我国黄瓜生产的毁灭性土传病害，华南地区高温高湿的气候条件尤其适宜该病的发生。油菜素甾醇类化合物是一类对植物的生长发育有重要影响的新型激素，能提高植物体对多种病害的抗性。本研究通过表油菜素内酯（EBR）及其抑制剂brassinazole预处理2d，灌根接种P.melonis游动孢子悬浮液，利用以双向电泳和质谱分析为基础的蛋白质组技术，分离和鉴定响应P.melonis侵染和EBR诱导的差异蛋白；结合生物信息学方法并采用Western blot和实时荧光定量PCR技术分析差异蛋白，明确差异蛋白的特性和生理功能，探讨P.melonis和黄瓜植株的互作机制以及EBR诱导黄瓜植株疫病抗性的分子机制。该研究可为通过EBR等外源物质的施用防治黄瓜疫病提供理论依据。

瓜类疫霉（Phytophthora melonis）引起的疫病是危害我国黄瓜生产的毁灭性土传病害，华南地区高温高湿的气候条件尤其适宜该病的发生。油菜素甾醇类化合物是一类对植物的生长发育有重要影响的新型激素，能提高植物体对多种病害的抗性。本研究通过表油菜素内酯（EBR）预处理2d，灌根接种P.melonis游动孢子悬浮液，利用iTRAQ体外标记技术分离和鉴定了黄瓜根系中响应P.melonis侵染和EBR诱导的差异蛋白，利用Lable free非标记技术和鉴定了黄瓜茎中差异蛋白，结合生物信息学方法明确差异蛋白的特性和生理功能，探讨P.melonis和黄瓜植株的互作机制以及EBR诱导黄瓜植株疫病抗性的分子机制。.研究结果表明：① 尚未发现黄瓜疫病免疫品种，总体上华南型黄瓜抗性高于华北型黄瓜，可能归因于华南型黄瓜对高湿或者涝害的抗性较高。② 不同浓度的EBR预处理、病原菌接种的同时进行EBR处理、病原菌接种后进行EBR处理三种方式在黄瓜疫病中的防治效果表明，EBR处理提高黄瓜疫病抗性主要归因于EBR可激活黄瓜免疫相关基因表达，诱导黄瓜免疫抗性，但是对疫霉菌本身孢子囊形成、诱导孢子释放以及菌丝生长并无直接抑制作用。③ 研究发现，黄瓜幼苗灌根接种0.5*104 Phytophthora melonis，1 μmol/L EBR预处理可显著诱导黄瓜茎中ca2+-依赖的蛋白激酶、MAPK3等免疫相关基因上调表达，进而诱导植物免疫反应，增强对病原菌的抗性。④ 研究发现，1 μmol/L EBR预处理可显著促进黄瓜茎中木质素的生物合成，激活细胞壁防御，阻止病原菌侵染。⑤ 研究发现，1 μmol/L EBR预处理可显著根系中乙烯生物合成，但是未能激活下游信号表达，有待于进一步验证。该研究可为通过EBR等外源物质的施用防治黄瓜疫病提供理论依据。