甜瓜叶片蛋白的提取和双向电泳体系的优化

The Extraction and Optimization of Two-Dimensional Electrophoresis for Leaf Protein of Melon

为建立适合甜瓜（Cucumis melo L.）叶片蛋白质组分析的双向电泳体系（2-D）,以甜瓜植物叶片为试验材料,比较了2种不同植物叶片蛋白的提取方法,并对双向电泳的2个重要的环节技术即聚焦参数和上样量进行了优化。结果表明：与Tirs-酚提取法相比,使用改良后的Mg/NP-40/PEG3350/TCA丙酮提取法提取叶片蛋白,经过SDS-PAGEF分析之后,所得的蛋白含量较高,Rubisco酶去除的较好,条带清晰。采用pH 3-10,18 cm的IPG胶条进行双向电泳时,适当增加低电压聚焦时间,得到了较为清晰的2-D图谱。蛋白上样量为800μg时,得到的蛋白点数最多,低峰度蛋白较为清晰。研究建立了适用于甜瓜叶片蛋白分析的双向电泳体系,得到了蛋白点数目多且分辨率高的双向电泳图谱。为下一步在蛋白组学水平上分析与甜瓜抗病、产量、性状等相关蛋白提供了技术支持。

To establish suitable two-dimensional electrophoresis proteome analysis system（2-D） of melon（Cucumis melo L.） leaves. In this experiment, we used melon leaves as experimental material, comparing twodifferent extraction methods leaf protein, and optimized two-dimensional electrophoresis of two importantaspects of technology that was focused on the parameters and sample volume. The results showed that：compared with Tirs- phenol extraction, using an improved method Mg/NP- 40/PEG3350/TCA acetoneextracting leaves protein, and after SDS-PAGEF analysis. The protein content was higher, Rubisco enzymecompared removed good, bands were clear. When using pH 3-10, 18 cm IPG strips of two-dimensionalelectrophoresis, increasing the low voltage focusing time, get a clearer 2-D map. The protein sample volume800 μg, the maximum number of points was obtained in protein, low protein kurtosis clearer. This experimentestablished suitable for the melon leaf protein analysis system of two-dimensional electrophoresis, got morethan number of protein and high resolution two-dimensional electrophoresis. For the next step in the proteingroup level analysis of melon disease resistance, yield, properties and related protein provides technical support.