稻瘟菌胞外蛋白的提取及电泳方法初探

Study on Extracting and 2-D Electrophoresis of Extracellular Protein in Magnaporthe grisea

利用蛋白质组学研究方法对稻瘟菌的胞外蛋白进行分析，可以更好地了解该病原菌的生物学特性和致病机制。蛋白质样品制备和双向电泳，都是蛋白质组学研究的基础。采用冷冻干燥，透析结合法与三氯乙酸-脱氧胆酸钠沉淀法（TCA-DOC沉淀法）提取液体培养稻瘟菌得到的胞外蛋白，利用17cm pH3.0-10.0与17cm pH4.0-7.0两种pH梯度IPG胶条对同一样品进行第一向等电聚焦，12％ SDS-PAGE凝胶进行第二向电泳，银染后用PDQuest软件（V7.4）对电泳图谱进行分析。结果表明，在上述两种提取稻瘟菌胞外蛋白的方法中，TCA-DOC沉淀法具有较好的提取效果，应用该实验方法得到的蛋白质分布在pH4.0-7.0之间的点数达到621个，因此在双向电泳中，采用pH4.0-7.0的胶条具有较好的分辨率。

Proteomics analysis of Magnaporthe grisea is crucial for understanding of its biological char- acteristics and pathogenetic mechanisms. Protein extraction and two-dimensional gel electrophoresis （2-D） are basical methods for proteomics study. In this study, extracellular proteins of rice blast fungus, M. grisea, extracted from liquid cultured infiltration were concerntrated-dialysis through freeze drying system and TCA-DOC precipitation method, on first dimensional gel electrophoresis using 17 cm respectively. The extracted proteins were separated pH 3.0 - 10.0 and 17cm pH 4.0 - 7.0 IPG strips and visualized the proteins by using silver staining follow second-dimensional electrophoresis. And protein maps were analyzed by PDQuest （V7.4） software. The results showed that the electrophoresis effect of proteins prepared by TCA-DOC precipitation is better than one by freeze drying combing with dialysis; totally 621 protein spots distributed in gel of pH 4.0 -7.0 were detected. And 2-D results showed that the distinguishability of protein spots on gel used pH 4.0 - 7.0 IPG strips showed better than one used pH 3.0-10.0 IPG strips.