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为了获得玉米籽粒双向电泳中蛋白质提取的最佳方法,建立最佳的玉米籽粒蛋白双向电泳技术体系,对酚抽提法、三氯乙酸/丙酮沉淀法(TCA/丙酮沉淀法)和可溶性蛋白提取法3种不同提取方法得到的蛋白质进行了分析,并在相同条件下进行双向电泳比对分析,结果表明:可溶性蛋白提取法获得的蛋白纯度高、浓度适中、双向电泳蛋白质点最丰富,最适合双向电泳研究;利用可溶性蛋白提取法获得的蛋白,对第一向等电聚焦参数等条件,以及不同p H值范围的IPG预制凝胶条进行了比较分析,结果表明:采用p H值3~10的IPG预制凝胶条时,采取电压梯度上升的方式,总聚焦70 000 Vhs、上样量800μg效果最佳,采用p H值4~7的IPG预制凝胶条时,总聚焦80 000 Vhs、上样量900μg效果最佳,并且采用p H值4~7的IPG预制凝胶条相比p H值3~10的凝胶条能分离到更多的点,最后结合蛋白质点MALDI-TOF-TOF质谱鉴定分析,建立了一套适用于玉米籽粒的蛋白质双向电泳技术方法,即采用可溶性蛋白提取法提取蛋白,采用24 cm、p H值4~7的IPG干胶条,上样900μg电泳效果最佳,等点聚焦程序:500 V、1 h,1 000V、1 h,2 000 V、1 h,4 000 V、2 h,8000 V、4 h,8 000 V、80 000 Vhs,500 V保持。试验研究为后续玉米籽粒发育差异蛋白研究奠定了技术基础。
In order to obtain the best method of protein extraction in two-dimensional electrophoresis of maize grain, the best two-dimensional electrophoresis system for maize grain protein was established. The phenol extraction, trichloroacetic acid / acetone precipitation (TCA / acetone precipitation) and soluble protein extraction The results showed that the protein obtained by the soluble protein extraction method was of high purity and moderate concentration, and the two-dimensional electrophoresis protein spots were the most abundant and most suitable Two-dimensional gel electrophoresis (PAGE) was used to study the IPG pregelatinized gel strips with different p H value. The results showed that the pG value of 3% 10 IPG preformed gel strips, the voltage gradient of the increase of the way, with a total focus of 70 000 Vhs, the amount of 800 μg sample best results, the use of p H value of 4 to 7 IPG preformed gel strips, the total focus 80 000 Vhs , The amount of 900μg sample was the best, and using IPG pre-gel strips with p H value of 4 ~ 7 could separate more spots than gel strips with p H value of 3 ~ 10, finally binding protein spots MALDI-TOF -TOF mass spectrometry identification points , A set of two-dimensional protein electrophoresis method for corn grain was established, that is, the protein was extracted by soluble protein extraction method. The 24-cm IPG strips with p H of 4-7 were used. Spot-focusing procedures: 500 V, 1 h, 1 000 V, 1 h, 2 000 V, 1 h, 4 000 V, 2 h, 8000 V, 4 h, 8 000 V, 80 000 Vhs, 500 V hold. The experimental research laid the technical foundation for the subsequent research on differential proteins of maize grain development.