甜瓜叶片蛋白的提取和双向电泳体系的优化

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800 μg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。

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

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 two different extraction methods leaf protein, and optimized two-dimensional electrophoresis of two important aspects of technology that is 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 acetone extracting leaves protein,and after SDS-PAGEF analyzed, The protein content is higher, Rubisco enzyme compared removed good, bands were clear. When using pH3 ~ 10,18 cm IPG strips of two-dimensional electrophoresis, increasing the low voltage focusing time, get a more clear 2-D map. The protein sample volume 800ug, the maximum number of points were obtained in protein, low protein kurtosis clearer. This experiment established suitable for the melon leaf protein analysis system of two-dimensional electrophoresis, got more than number of protein and high resolution two-dimensional electrophoresis.For the next step in the protein group level analysis of melon disease resistance, yield, properties and related protein provides technical support.