茶树叶片蛋白双向电泳体系建立与应用

为开展茶树Camellia sinensis 低温和干旱胁迫下差异蛋白的分离和鉴定,以抗逆性较强的茶树品种‘迎霜’为试材,通过对提取方法、IPG 胶条pH 范围、上样量、分离胶浓度、染色方法的比较,筛选适用于茶树叶片的蛋白质双向电泳体系。结果表明,采用TCA-丙酮法或Tris-HCl 法提取叶片总蛋白,选用17 cm pH 4~7IPG 胶条用于等电聚焦,选择1.6~2.2 mg 上样量、13.5%聚丙烯酰胺凝胶进行分离,随后通过高敏考马斯亮蓝R-250 法染色;最终,叶片各分子量的蛋白充分分离,获得的双向电泳图谱分辨率高、背景清晰、重复性好,适用于‘迎霜’低温和干旱胁迫下叶片差异蛋白分析。

Establishment and Application of Two-dimensional Gel Electrophoresis Technology System for Total Protein of Camellia sinensis Leaf

In order to carry out the isolation and identification of the differential proteins under the cold and drought stress of tea plants, using the strong stress-resistant tea tree variety ‘Yingshuang’ as experimental material. The two-dimensional gel electrophoresis technology system suitable for leaf was screened by extraction method, IPG strip pH range, protein content, separation gel concentration and staining method. The results showed that total protein was extracted by TCA-acetone or Tris-HCl method, using 17 cm pH 4—7 IPG strip and 1.6—2.2 mg protein for isoelectric focusing, using 13.5% polyacrylamide gel for electrophoresis, subsequently staining by high sensitivity Coomassie Brilliant Blue R-250 method. Finally, the protein of each molecular weight of the leaf was sufficiently separated, and a 2-DE pattern with high resolution, clear background and good reproducibility was obtained. The system was suitable for the analysis of differential proteins in leaves of ‘Yingshuang’ under cold and drought stress.