油菜叶片总蛋白质双向电泳样品制备方法的改进

Improvement of Preparation Methods of Two-Dimensional Eleteophoretic Samples of Proteome from Brassica Leaves

以甘蓝型油菜"扬油6号"的叶片为试验材料,分别采用传统的TCA/Acetone(三氯乙酸/丙酮沉淀法)和改进的PEG(polyethylene glycol)分步提取法提取叶片可溶性总蛋白,并利用条件一致的蛋白质双向电泳体系进行比较。TCA/Acetone法提取的蛋白质双向电泳图谱背景中由于高丰度"housekeeping"结构蛋白的存在,特别是叶片中参与光合作用的Rubisco蛋白的干扰,图谱中低丰度调控蛋白受到了高度覆盖和遮蔽现象,影响双向电泳图谱的质量。而PEG分步提取法提取的蛋白质样品,可以剔除Rubisco蛋白,使获得的双向电泳图谱清晰,无斑点间的遮蔽现象,为油菜叶片蛋白质组定量和定性分析提供了丰富的信息。

With leaves of Brassica napus L. cv. ＂ YangYou 6＂ used as experimental materials,the proteins were extracted from leaves by TCA/Acetone precipitation and differential PEG（ Polyethylene glycol）precipitation method and compared by two-dimensional electrophoresis system with uniform conditions of protein. The background noise of two-dimensional eleteophoresis map of protein extrac- ted by TCA/Acetone method was great, and clarity was poor. This was partially attributed to the existence of high-abundant proteins, such as ribulose-1,5-bisphosphate carboxylase/oxygenase（Rubisco） in leaves. They engaged a large proportion of the whole-cell pro- teins and thus preventd low-abundant proteins from being up-taken by immobilized pH gradient （IPG） strip, consequently making the latter poorly detectable by 2-DE. So they formed high coverage, shading on the other protein spots and affected the accurate expression of map information. However, through depletion of the high-abundant protein Rubisco during differential PEG precipitation method, the background noise of electrophoresis map was little, and PEG fractionation improved detection of low-abundant proteins, there was no shading phenomenon among spots. Therefore it provided abundant information for quantitative and qualitative analyses on the proteomes from Brassica leaves.