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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17 cm pH 3.0~10.0与17 cm pH 4.0~7.0两种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pH 4.0~7.0之间的点数达到621个,因此在双向电泳中,采用pH 4.0~7.0的胶条具有较好的分辨率。
Using proteomics research methods to analyze the extracellular proteins of Magnaporthe grisea can better understand the biological characteristics of the pathogen and pathogenic mechanism. Protein sample preparation and two-dimensional electrophoresis are the basis of proteomics research. The extracellular proteins obtained from liquid culture of Magnaporthe grisea were extracted by freeze-drying, dialysis combined with sodium trichloroacetate-sodium deoxycholate precipitation method (TCA-DOC precipitation method) 7.0 two kinds of pH gradient IPG strips of the same sample for the first isoelectric focusing, 12% SDS-PAGE gel for the second electrophoresis, silver staining with PDQuest software (V7.4) electrophoresis patterns were analyzed. The results showed that TCA-DOC precipitation method had good extraction efficiency in the above two methods for extracting extracellular proteins of Magnaporthe grisea, and the number of protein distributions obtained by this experimental method reached 621 points between pH 4.0 and 7.0 , So in the two-dimensional electrophoresis, the use of strips of pH 4.0 to 7.0 has better resolution.