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为了建立适于苹果属植物树皮组织总蛋白提取的技术方法,以8年生华月苹果(Malus domestica)树枝条为试材,通过比较不同提取方法(TCA-丙酮沉淀法(A)、甲醇/醋酸铵沉淀法(B)和改良的Tris-酚抽提方法(C))并优化提取条件,确立了最适提取及分离方法为改良的Tris-酚抽提方法。在2-DE分离时,该方法所获得的样品图谱中蛋白点总数为993个,明显多于TCA-丙酮沉淀(418个)和甲醇醋酸铵沉淀(674个)方法,并且与其它两种方法相比,该方法获得的图谱背景更清晰,蛋白点聚焦效果更好。此外,经过3个梯度上样量的图谱分离效果比较,确定了800μg为本研究中2-DE分析的理想上样量。另外,为了验证该提取及分离方法的可行性,进一步对蛋白质表达谱中的部分蛋白点进行了质谱分析,且这些蛋白点均得到了成功鉴定。该研究通过优化总蛋白提取方法及样品上样量等条件,获得了理想的双向电泳分离图谱,为苹果属植物树皮组织材料的蛋白质组学研究奠定了基础。
In order to establish the technical method for extracting total protein from the bark of apple tree, 8-year-old Malus domestica branches were used as materials to compare the different extraction methods (TCA-acetone precipitation method, methanol / Ammonium acetate precipitation method (B) and modified Tris-phenol extraction method (C)) were optimized and the extraction conditions were optimized. The optimum extraction and separation methods for Tris-phenol were established. At the 2-DE separation, the total number of protein spots in the sample obtained by this method was 993, which was significantly higher than that of TCA-acetone precipitation (418) and methanol ammonium acetate precipitation (674). Compared with the other two methods Compared with this method, the background obtained by the method is clearer and the protein spots are more focused. In addition, 800 μg was determined as the ideal sample for 2-DE analysis in this study after comparing the results of three gradient loading samples. In addition, in order to verify the feasibility of the extraction and separation methods, some protein spots in the protein expression profile were further analyzed by mass spectrometry, and these protein spots were successfully identified. In this study, the ideal two-dimensional electrophoresis separation pattern was obtained by optimizing the methods of total protein extraction and sample loading, which laid the foundation for the proteomics research of the bark tissue material of apple genus.