红树植物秋茄叶片双向电泳技术体系的建立及优化

Establishment and Optimization of 2 - DE Technique System in Leaf Proteome of Kandelia candel

摘要:

对适用于秋茄(Kandelia candel)叶片蛋白质组学研究的双向电泳技术体系进行了优化。结果表明,采用酚抽提法提取的蛋白质浓度较低,约1.7 μg μL^-1,SDS-PAGE电泳后几乎无条带;而采用改良TCA-丙酮沉淀法可显著提高提取液中蛋白质的含量,可达12.4 μg μL^-1。秋茄叶片蛋白质主要分布在pH 4~7的范围内,最适于采用pH 3~10 NL的胶条,17 cm胶条的最适上样量为400 μg,经银染后可得蛋白点约(564±34)个。用TCA-丙酮沉淀法提取秋茄叶片全蛋白质组,按优化的参数进行双向电泳分离,获得了高分辨率、高重复性的图谱,可用于秋茄蛋白质组学研究。

关键词:秋茄;蛋白质;提取;双向电泳(2-DE);技术优化

Abstract:

In order to study the proteome of Kandelia candel leaves, a two-dimensional electrophoresis (2-DE) system was established and optimized. Compared with the low protein content (1.7 μg μL^-1) extracted by phenol (Phe), the improved trichloroacetic acid/acetone precipitation (TCA) method could obviously increase the extracting efficiency up to 12.4 μg μL^-1. The protein spots of Kandelia candel leaves mainly distributed in the range of pH 4-7. The optimal loading quantity of samples were 400 μg gel^-1 for a 17 cm IPG strip (pH 3-10 NL), which can detect 564±34 protein spots after silver staining. Therefore, the high resolution and repeatable 2-DE map could be obtained by using TCA extract method and the optimum system, which could widely use in proteome study of Kandelia candel.

Key words:Kandelia candel;Protein;Extraction;Two-dimensional electrophoresis (2-DE);Optimization