黄瓜雌花花冠蛋白质双向电泳技术体系的建立

Two-dimensional Electrophoresis System for Cucumber Female Flower's Corolla

为建立适合黄瓜（Cucumis sativus L.）雌花花冠蛋白质双向电泳技术体系,以开花当天的黄瓜雌花花冠为试验材料,从蛋白质提取方法、IPG胶条梯度、等电聚焦程序、蛋白质上样量、染色方法等方面进行了探索。结果表明：酚抽提法得到的黄瓜雌花花冠蛋白质纯度高、产量高。17 cm p H 4~7 IPG胶条得到的双向电泳图谱蛋白质点分布均匀、清晰。优化后的等电聚焦程序适当增加了低压除盐步骤和时间,得到的双向电泳图谱蛋白质点圆且多。300μg上样量的双向电泳图谱蛋白质点可达400多个,低丰度蛋白质点清晰。硝酸银染色的双向电泳图谱蛋白质点较多。该试验获得了蛋白质点分布均匀、背景清晰、分辨率高、质量高的双向电泳图谱,为研究黄瓜雌花花冠蛋白质组学提供了参考。

An appropriate two- dimensional electrophoresis proteome analysis system of cucumber female flower＇s corolla was established by optimizing the parameters including the protein extraction method, the pH gradient of IPG strip, isoelectric focusing conditions, sample application, and dyeing method. The result showed that phenol extraction method could obtain more protein with higher purity. Two- dimensional electrophoresis maps using 17 cm p H 4-7 IPG strip had no overlapping and protein points distributed evenly.Good protein points were obtained in the two-dimensional electrophoresis maps using optimized isoelectric focusing condition with added low-pressure desalination step and time. When the sample application increased to 300 μg, the number of protein points reached 400 and the low abundance protein points were also clear.More protein points were obtained in the two- dimensional electrophoresis maps using silver staining than colloid Coomassie brilliant blue staining. This study obtained clear background, high resolution, and high quality two-dimensional electrophoresis maps with protein points distributed evenly. It laid a foundation for the research on cucumber female flower＇s corolla proteomics.