玉米蛋白质组研究中双向电泳技术条件的优化

Two Dimensional Electrophoresis of Optimization and Extraction of Proteome Research of Maize

为了优化玉米叶片双向凝胶电泳技术条件,通过比较3种总蛋白提取方法对玉米双向电泳结果的影响,并对蛋白质上样量、IEF等电聚焦条件及SDS-聚丙烯酰胺凝胶浓度等参数进行探索、优化;结果发现,与酚提取法和磷酸缓冲液法相比,采用改进的TCA/丙酮法提取总蛋白操作简单、方便,所得的2-DE图谱中蛋白质点数量多、背景清晰,是一种适用于提取玉米苗期叶片总蛋白的有效方法;同时筛选出:第一向IEF等电聚焦样品上样量为800μg,聚焦条件为20 000 V/h,在浓度为12.5%的SDS-聚丙烯酰胺凝胶中进行电泳,能够在17 cm的IPG胶条上获得背景低、分辨率较高、重复性好的双向电泳图谱,该优化体系适用于玉米叶片蛋白质组双向电泳分离,对玉米蛋白质组学研究具有重要参考价值。

In order to establish a set of two dimensional gel electrophoresis technique for maize,the effects of three kinds of total protein extraction methods on the results of two dimensional electrophoresis of maize were studied. Meanwhile,the protein sample volume,condition of isoelectric focusing and the density of polyacrylamide gel were explored and optimized. The results showed that TCA-acetone for total protein extraction was simple and convenient compared with the method of phenol extraction and phosphate Buffer（ PB） solution method. The 2-D maps had abundant protein spots and more clear background,this study established a suitable proteomics methods for forage maize. Meantime,it screened out the first dimension electrophoresis was performed on the sample 800 μg and focus conditions 20 000 V/h; The second electrophoresis was performed on 12. 5% polyacrylamide gels,it could be obtained two-dimensional electrophoresis map at 17 cm of background low and higher resolution. Optimization system would be suitable for two dimensional electrophoresis of total protein of forage maize leaves,the method can be applied to corn leaf proteome analysis of sample extraction. The useful information can be provided for the proteomics research of maize.