桃果实蛋白质双向电泳影响因素的研究

以‘大久保’桃（^{Prunus persica} L.'Okubao'）果实为试材，通过比较不同上样量、不同上样缓冲液、不同平衡缓冲液、不同染色方式等条件下的双向电泳图谱，探讨以上各因素对桃果实双向电泳图谱效果的影响。结果表明考马斯亮蓝染色后的电泳图谱虽然蛋白质点的数量少于银染色后的图谱，但背景较为清晰，适用于蛋白质组分析。采用固相pH梯度17 cm胶条，100 μg蛋白质上样量，样品干粉溶于375 μL含有硫脲、磺基三甲基胺乙内脂3~10的上样缓冲液中，等电聚焦后用含有磷酸三丁酯的平衡缓冲液还原，进行SDS-PAGE，银染色得出的双向电泳图有蛋白点1 209个，纹理较少，适用于桃果实蛋白质组学分析且兼容于下游技术的蛋白质质谱鉴定。

Studies on Influence Factors of Protein Two-dimensional Electrophoresis in Prunus persica Fruit

Peach （Prunus persica L.‘Okubao’）fruit was adopted in this experiment. Comparing different effects of two-dimensional electrophoresis （2-DE）gels that were involved with quantitative loading of sample, sample buffer, equilibration buffer and the type of staining to explore influence factors of 2-DE patterns on peach fruit. Although the two-dimensional gel stained by coomassie brilliant blue had gained less protein spots than the gel stained by silver, it had a clear background intensity and was applicable to the proteomic study. Furthermore, approximately 100 μg of protein was brought to a final volume of 375 μL sample buffer which contained thiourea and sulfphobetaine 3-10 and loaded on 17 cm immobilized pH gradients strip, then the strip was incubated in equilibration buffer with tributyl phosphine after isoelectric focusing. The two-dimensional gel stained by silver detected 1 209 protein spots and had slight streaks. The results demonstrated that the protocol was suited to proteomic analysis of peach fruit and compatibled with protein identification by mass spectrometry of downstream processing.