不同方法提取的小麦籽粒蛋白双向电泳凝胶分离效果的分析比较

Analysis and Comparison of Two-Dimensional Electrophoresis Gel Separation of Wheat Grain Protein Extracted by Different Methods

为探索适于小麦籽粒蛋白质组学研究的样品制备最优方法,本研究以Rht10鲁麦15开花后第31天的籽粒为材料,采用4种不同蛋白质提取方法:TCA/丙酮法、尿素/硫脲法、酚提取法Ⅰ和酚提取法Ⅱ,对不同提取方法得到的蛋白量以及蛋白分离结果进行比较分析。结果显示:TCA/丙酮法和尿素/硫脲法获得的蛋白点较少,分别为484个和489个,蛋白质损失较大,蛋白质在碱性端(pH7附近)堆积严重;酚提取方法Ⅰ得到的蛋白点709个,部分蛋白点弥散,碱性端蛋白轻微堆积;酚提取方法Ⅱ得到的蛋白点866个,蛋白点清晰,无碱性蛋白堆积。研究结果表明,在小麦籽粒蛋白质组的研究中,酚提取方法更为适合小麦籽粒蛋白的提取,同时方法Ⅱ优于方法Ⅰ。

In order to find the most efficient method for sample preparation of wheat grain on two-dimensional electrophoresis（2-DE）, four different methods including TCA/acetone, urea/thiourea, phenol extraction methodⅠ and phenol extraction method Ⅱ were compared by protein content and separation effect of 2-DE gels. The grain of wheat line Rht10 lumai15 of 31 days after anthesis was as material. The results indicated that by using TCA/acetone method and urea/thiourea method, 484 and 489 protein spots could be detected respectively on2-DE gels, which alkaline protein（near pH 7） accumulated seriously. The phenol extraction method Ⅰproduced709 protein spots on 2-DE gels with slight point dispersion and alkaline protein accumulation phenomena. Phenol extraction method Ⅱ was the best with more clear protein spots as many as 866 spots. These results suggested that the phenol extraction method was optimal and efficient method for 2D separation of wheat grain protein, and method Ⅱ was better than method Ⅰ.