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中国农学通报

中国农学通报 ›› 2012, Vol. 28 ›› Issue (34): 211-218.

所属专题： 生物技术

• 林学 园艺 园林 • 上一篇    下一篇

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   

  • 收稿日期:2012-06-01 修回日期:2012-07-23 出版日期:2012-12-05 发布日期:2012-12-05

Establish the two-dimensional electrophoresis system and preliminary proteomics analysis for Fusarium oxysporum f. sp. Cubense

  • Received:2012-06-01 Revised:2012-07-23 Online:2012-12-05 Published:2012-12-05

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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次能有效的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，成功的鉴定了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: 优化, 优化

Abstract:

The aims were to optimize the extraction methods of Fusarium oxysporum 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 obtained 8 protein spots which represented 8 unique proteins. These results suggested that the extracted proteins and method of in-gel digestion had good MS compatibility, and could also provide foundation for further investigating the differentially expressed proteins between the difference thalli.