香蕉枯萎病菌双向电泳体系的建立及蛋白质组学初步研究.PDF

中国农学通报 2012,28(34):211-218 Chinese Agricultural Science Bulletin 香蕉枯萎病菌双向电泳体系的建立及蛋白质组学初步研究 孙 勇，王旭初，易小平 （中国热带农业科学院热带生物技术研究所，海口571101） 摘 要：旨在对香蕉枯萎病菌胞内蛋白的提取方法进行优化，建立菌体胞内蛋白质双向电泳体系。在原 有的基础上优化菌体胞内蛋白提取方法，提取蛋白质进行双向电泳，并应用质谱技术鉴定一些蛋白质来 验证提取蛋白质的质谱兼容性。通过扩大培养的方法获得足够的生物量，采用过滤法收集菌体用来提 取蛋白质，涡旋振荡30 min 能有效破胞，提高蛋白质提取效率。甲醇和丙酮各清洗2 次能有效地清除蛋 白质中的杂质，得到了高质量的蛋白质图谱，选取了10 个蛋白质点进行质谱鉴定，成功地鉴定了8 个蛋 白质点，代表了8 种蛋白质。这些结果表明，提取的蛋白质和采用的胶内酶解方法具有良好的质谱兼容 性，能为进一步研究不同菌体之间的差异表达蛋白质提供技术支持。 关键词：香蕉枯萎病；质谱；蛋白质组学；双向电泳 中图分类号：Q-94 文献标志码：A 论文编号：2012-2067 EstablishmentoftheTwo-dimensionalElectrophoresisSystemandPreliminary ProteomicsAnalysisofFusariumoxysporumf.sp.Cubense Sun Yong, Wang Xuchu, Yi Xiaoping (InstituteofTropicalBioscienceandBiotechnology, CATASA, Haikou 571101) Abstract: The aims were to optimize the extraction methods of Fusarium oxy sporum f. sp. Cubense intracellular proteins, and establish the two-dimensional electrophoresis system for intracellular proteins. The extraction methods of intracellular proteins were optimize based on the past reports, proteins for two-dimensional electrophoresis were extracted, and mass spectrum was used to identify the proteins for verify the mass spectrum compatibility. The sufficient mass was obtained through the expansion culture methods, and the bacteria were collected with filtration for protein extraction. Vortex 30 min could damage the cell effectively; the efficiency of protein extraction was improved. The protein was washed twice by methanol and acetone to remove protein impurities effectively. The high quality 2-DE profiles were obtained. 10 protein spots were selected and identified by MS, successfully