华根霉蛋白质组双向电泳体系的建立及优化

Construction and Optimization of Two-Dimensional Electrophoresis for Proteome Analysis of Rhizopus chinensis

DOI：10.3969/j.issn.1673-1689.2015.05.007

中文关键词: 华根霉 全蛋白 双向电泳

英文关键词: Rhizopus. chinensis,intracellular proteins,two-dimensional electrophoresis

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中文摘要:

华根霉(Rhizopus chinensis CCTCC M201021)是从我国传统微生物载体--大曲中分离筛选得到的一株丝状真菌,具有重要的工业应用前景。为建立一种适于华根霉胞内蛋白质的双向电泳体系以进行蛋白质组学研究,作者对华根霉胞内蛋白质的提取方法及双向电泳流程相关参数进行了考察和优化.确定采用液氮研磨与高速珠磨相结合的方法对华根霉进行细胞破碎,用TCA/丙酮对提取的蛋白质进行纯化,采用被动水化的上样方式上样,24 cm、pH 4~7的线性IPG胶条,上样量为1 200 μg蛋白质,考马斯亮蓝G-250胶体染色法,最终得到了背景清晰、分辨率高的双向电泳图谱。

英文摘要:

Rhizopus chinensis CCTCCM201021 is an important filamentous fungus,which was isolated from Daqu,a traditional leaven in the production of Chinese liquor. In order to understand this fungal deeply,the conditions of two-dimensional electrophoresis(2-DE) for proteome analysis of R. chinensis were investigated and optimized. Intracellular proteins of Rhizopus chinensis was extracted and separated for 2-DE. Different sample preparation methods,parameters of the gel,sample loading quantity and methods were investigated to optimize 2-DE maps of R. chinensis. Ultimately disrupt cell in liquid nitrogen and mechanical agitation,precipitate proteins with TCA/acetone,passively rehydrate proteins with 24 cm pH 4~7 linear strips were selected. The sample loading quantity for each strip was 1 200 μg,and modified colloidal coomassie brilliant blue G-250 was applied to stain gels. Finally,2D-gel map with no obvious vertical and horizontal stripes,low background,high resolution and good reproducibility was obtained. Approximately 800 protein spots were isolated from the 2-DE protein map after analyzed by PD-Quest software.

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