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【目的】建立适于番茄叶片蛋白质组分析的双向电泳(2-DE)技术,为开展番茄蛋白质组学研究奠定技术基础。【方法】对番茄叶片蛋白质提取方法、蛋白质裂解液、上样量、胶条p H范围等关键步骤进行优化。【结果】采用三氯乙酸/丙酮法提取番茄叶片总蛋白质,含有7 mol/L尿素,2 mol/L硫脲,4%CHAPS,30 mmol/L Tris-HCl(p H 8.0)裂解缓冲液,上样量为100 mg,以p H 4~7、18 cm的IPG胶条在12%SDS-PAGE凝胶浓度下进行双向电泳,得到了蛋白点均匀分布、低峰度蛋白点较为清晰,蛋白点数目多且分辨率高的2-DE图谱。【结论】建立了一套适于番茄叶片蛋白质分析的双向电泳技术体系,为下一步在蛋白组学水平上分析番茄逆境胁迫等相关蛋白提供了技术支持。
【Objective】 Two-dimensional electrophoresis (2-DE) was developed for the proteomic analysis of tomato leaves and laid the technical foundation for the development of tomato proteomics. 【Method】 The key steps of protein extraction method, protein lysis solution, sample loading, p H range of tapes were optimized. 【Result】 The total protein of tomato leaves was extracted by trichloroacetic acid / acetone method. The total protein was extracted with 7 mol / L urea, 2 mol / L thiourea, 4% CHAPS and 30 mmol / L Tris-HCl The sample volume was 100 mg, and the IPG strips with p H 4 ~ 7,18 cm were electrophoresed at 12% SDS-PAGE gel concentration. The protein spots were evenly distributed and the spots with low kurtosis were clear. The protein spots The number of points and high resolution 2-DE map. 【Conclusion】 A set of two-dimensional electrophoresis system was established for the analysis of tomato leaf protein, which provided technical support for the next step in the analysis of proteomics of stress-related proteins in tomato.