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【目的】获得银杏叶绿体蛋白质的提取方法,在蛋白质水平探讨银杏光合作用对环境的响应机制。【方法】建立一种适合银杏叶绿体蛋白双向电泳分离的实验体系,采用Tris-平衡酚抽提法对银杏叶绿体蛋白质进行提取,并与三氯乙酸(TCA)-丙酮沉降法进行对比分析。【结果】Percoll密度梯度离心法适用于银杏叶绿体的提取,提取的叶绿体平均完整率在85%左右。单向电泳结果显示,与TCA-丙酮沉降法相比,在用Tris-平衡酚抽提法分离的叶绿体蛋白质泳道中,低分子质量蛋白质条带更多、更清晰。进一步的双向电泳结果表明,用Tris-平衡酚抽提法对银杏叶绿体蛋白质进行提取,蛋白质产量更高,图谱清晰,所分离的蛋白点更多,形态更好,条纹影响相对较小。【结论】Tris-平衡酚抽提法可有效地提取高质量的叶绿体蛋白质并进行双向电泳,可用于银杏叶绿体的蛋白质组学分析。
【Objective】 The objective of this study is to obtain the extraction method of chloroplast protein from Ginkgo biloba and to explore the mechanism of the response of Ginkgo biloba to photosynthesis. 【Method】 An experimental system suitable for the separation of chloroplast protein from Ginkgo biloba by two-dimensional electrophoresis was established. Chloroplast protein was extracted by Tris-balance phenol extraction method and compared with trichloroacetic acid (TCA) -acetone precipitation method. 【Result】 Percoll density gradient centrifugation was applied to the extraction of chloroplast in Ginkgo biloba. The average chloroplast extraction rate was about 85%. The results of one-dimensional electrophoresis showed that the band of low molecular weight protein was more and clearer in the chloroplast protein tract separated by Tris-equilibrium phenol extraction than the TCA-acetone sedimentation method. Further two-dimensional electrophoresis results showed that the extraction of chloroplast protein from Ginkgo biloba by Tris-balance phenol extraction showed higher protein yield, clear chromatogram, more protein spots separated, better morphology and less influence of streaks. 【Conclusion】 Tris-equilibrium phenol extraction method can effectively extract high quality chloroplast proteins and perform two-dimensional electrophoresis, which can be used for proteome analysis of chlorophyll of Ginkgo biloba.