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水稻白叶枯病菌已成为研究植物-病原物互作模式的病菌。要利用蛋白质组学方法深入研究其生物学特性和致病机理,人们面临着其胞内蛋白提取和有效分离方法尚未建立的问题。本文采用TCA丙酮沉淀法和裂解液沉淀法提取白叶枯病菌胞内蛋白,利用两种等点聚焦程序(IEFⅠ,IEFⅡ)进行一向等电聚焦,用三种蛋白上样浓度(400μg,600μg,800μg)进行双向电泳,G-250烤染后,通过Imagemaster软件对双向电泳图谱进行比较分析。结果表明,裂解液沉淀法、等电聚焦程序Ⅱ(IEFⅡ)、600μg上样浓度三者相结合的方法是提取和分离白叶枯病菌胞内蛋白的理想方法,能获得较好的实验效果。该方法的建立为白叶枯病菌蛋白质组学研究奠定基础。
Xanthomonas oryzae has become a pathogen that studies plant-pathogen interaction patterns. To exploit the biological characteristics and pathogenic mechanism of proteome in depth, people face the problem that their intracellular protein extraction and effective isolation methods have not been established. In this paper, TCA acetone precipitation and lysis solution precipitation were used to extract the intracellular proteins of B.cereosporioides. The two isofocusing procedures (IEFⅠ, IEFⅡ) were used for isoelectric focusing. The three protein concentrations (400μg, 600μg, 800μg) for two-dimensional electrophoresis. After G-250 was grilled and dyed, the two-dimensional electrophoresis patterns were comparatively analyzed by Imagemaster software. The results showed that the combination of lysate precipitation method, isoelectric focusing program Ⅱ (IEFⅡ), and 600 μg loading concentration is an ideal method for extracting and isolating the intracellular proteins of bacterial leaf blight, and better experimental results can be obtained. The establishment of this method laid the foundation for the proteomics research of X. oryzae.