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稻瘟菌胞外蛋白的提取及电泳方法初探
  • 期刊名称:云南农业大学学报,2009(5):661-667
  • 时间:0
  • 分类:S435.111[农业科学—农业昆虫与害虫防治;农业科学—植物保护]
  • 作者机构:[1]云南农业大学农业生物多样性与病虫害控制教育部重点实验室,云南昆明650201
  • 相关基金:国家“973”项目(2006CB100202);国家自然科学基金资助(30860161).
  • 相关项目:云南水稻地方品种稻瘟病菌信号分子关键受体及其在互作中的作用
中文摘要:

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。

英文摘要:

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.

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