山葡萄花序蛋白质双向电泳技术体系的建立

Establishment of two-dimensional electrophoresis platform for Vitis amurensis flower

【目的】为开展山葡萄花序蛋白质组学研究。【方法】以山葡萄(Vitis amurensis Rupr)花序为试材,通过对蛋白质提取方法、裂解液成分、胶条pH梯度的改进,建立了适于山葡萄花序蛋白质组学研究的最佳双向电泳技术体系。【结果】采用酚提取法提取花序蛋白质,用含有硫脲和DTT的裂解液Ⅱ裂解,选用17 cm pH 4～7的IPG胶条在适宜的双向电泳条件下分离,可以获得分辨率高、背景清晰且重复性好的双向电泳图谱,可以检测出1 088个清晰的蛋白点。【结论】酚提取法适于山葡萄花序蛋白质的提取,硫脲和DTT可增强花序蛋白质的溶解性。

[Objective]This work was performed to start the study of proteomic research using Vitis amurensis flower tissue. [Method]Vitis amurensis flower tissue proteome was extracted by two protein extraction protocols and lysis buffers, and various types of gel strips were evaluated. [ResultlThe results showed that phenol protein extraction method is superior than the TCA-acetone. The lysis buffer 11 with thiourea and DrI＇F is efficient in resolubilizing the protein pellet. 17em immobilized pH gradient strips with pH 4 to 7 could perform well in terms of protein separating in the first dimension. Finally, 1 088 total protein spots were detected in the 2-DE gel map, which suggested that an efficient 2-DE platform with high repeatability and resolution for Vitis amurensis flower had been established. [Conclusion]Phenol protein extraction method was suitable for Vitis amurensis flower protein extract, thiourea and DTT enhanced flower protein solubility.