柑橘叶片和果皮总蛋白质提取及双向电泳体系的建立

Extraction of Total Proteins from Leaves and Peels of Citrus reticulata and Establishment of Two-dimensional Electrophoresis System

摘要:

以春甜橘(Citrus reticulata Blanco ‘Chuntianju’)果皮和叶片为材料,分别用Tris-HCl、尿素/硫脲(Thi/Urea)、三氯乙酸/丙酮(TCA)和酚(Phe)等4种方法提取柑橘总蛋白质,从蛋白质产量、单向SDS-PAGE和双向电泳等方面进行比较。结果表明,4种方法的分离效果存在较大差异,不论是以柑橘叶片还是果皮为材料,均以TCA法最好,且双向电泳图谱分辨率较好,蛋白点清晰、均匀、基本没有条纹,且蛋白点多。这说明TCA法不仅能很好地去除柑橘果皮、叶片中存在的大量干扰物质,而且还能得到稳定的蛋白点。

Abstract:

The total proteins in peels and leaves of Citrus reticuiata Blanco ‘Chuntianju’ were extracted by four methods, such as Tris-HCl, Thi/Urea, trichloroacetic acid/pacetone precipitation (TCA) and phenol (Phe) extraction methods, respectively, and their effects were compared by protein production, one-way SDS-PAGE and two-dimensional electrophoresis (2-DE). The results showed that TCA method was the best for both leaves and peels of C. reticuiata ‘Chuntianju’. The 2-DE map of proteins extracted by TCA method had high resolution, the protein spots were clear and uniform without stripes, and the number of protein spots were large. It suggested that the TCA method could not only remove the interfering substances in peels and leaves of C. reticuiata ‘Chuntianju’, but also get more stable protein spots.