苹果属植物树皮组织总蛋白提取及分离方法的建立

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800 μg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。

Extraction and Separation Analysis of Total Protein from the Bark of Apple Branches

We aimed to establish a suitable extraction method for analyzing total protein in the bark of apple branches. The branches of an 8-year-old cultivar (Huayue) were used as material. We compared 3 different extraction methods, including TCA-acetone precipitation (A), methanol/ammonium acetate precipitation (B), and improved Tris-phenol extraction (C). We optimized the extraction procedures and established an ideal method for analyzing the total protein in the bark of apple branches. The improved Tris-phenol extraction method could reveal a good map of 993 protein spots, significantly more than the other two methods: 418 and 674 protein spots were obtained with TCA-acetone precipitation and methanol/ammonium acetate precipitation, respectively. Furthermore, the gel background was clear, and the protein spots were better focused than with the two other methods. After comparing sample loading quantity, we selected 800 μg as the ideal loading quantity for our study. Furthermore, we selected several proteins for MALDI-TOF-TOF/MS analysis and database searching, and the protein spots were finally identified. In this study, we optimized the extraction procedures and sample loading quantity for total protein and finally obtained an ideal 2-D electrophoresis map. Our study may provide a good basis for further proteome analysis of apple branches.