棉花叶片双向电泳体系的研究

 以棉花叶片为材料，比较分析了不同叶位棉花叶片总蛋白提取方法、线性固相pH梯度(Immobilized pH gradient,IPG)胶条pH值、上样量、水化上样缓冲液中还原剂和两性电解质等对电泳结果的影响。结果表明，三氯乙酸-丙酮沉淀法结合甲醇等有机溶剂洗涤纯化样品能获得纯度较高的棉花叶片蛋白样品。24 cm pH 5～8的线性IPG胶条，300 μg上样量，水化上样缓冲液中用还原剂TBP(三丁基膦)代替DTT(二硫苏糖醇)，两性电解质pH 3～10和pH 5～8按1∶1（V/V）的比例混合使用，并结合适宜的双向电泳参数，可获得背景清晰、重复性较好、蛋白点数目多且分辨率高的双向电泳图谱。从而建立了一套适合于棉花叶片蛋白组学分析的双向电泳技术体系。

Development of Two-dimensional Electrophoresis Protocol Suitable for Proteomic Analysis of Cotton Leaves

We have developed a two-dimensional gel electrophoresis method to analyze the proteomics of cotton Gossypium hirsutum L.leaves.We investigated and compared five processes for the extraction of total proteins from cotton leaves.Two different ranges of a linear immobilized pH gradient(IPG),four different sample loading,reductant gradients and carrier ampholytes in IPG buffer solution were compared.A high purity of cotton protein was extracted by TCA-acetone precipitation,and by cleaning the protein sample with solvent methanol or acetone.The parameters for obtaining a high resolution of cotton 2-D electrophoresis maps were optimized as follows: 24 cm strip with linear(IPG) in pH 5-8,300 μg sample loading,Tributyl phosphate(TBP) substituting for DL-Dithiothreitol(DTT) in the IPG buffer,and a combination of two carrier ampholytes in the pH range of 5-8 and 3-10,with a volume ratio of 1∶1.This optimal 2-D electrophoresis system gave a higher resolution,protein yield,and spot intensity in 2-D electrophoresis analysis.