适用于大豆叶片蛋白质组分析的双向电泳最佳条件研究

A Two-Dimensional Electrophoresis Protocol Suitable for Proteomic Analysis of Soybean Leaves

针对大豆叶片中蛋白含量较低,含有大量色素、酚类、醌类等次生代谢产物干扰蛋白质双向电泳分离效果的问题,以大豆叶片为材料,对样品制备、蛋白质裂解液组分、等电聚焦(IEF)参数等条件进行研究。结果表明:采用TCA/丙酮法提取大豆叶片总蛋白,裂解缓冲液为9mol·L-1尿素,2mol·L-1硫脲,4%CHAPS,100mmol·L-1DTT,0.5%Bio-Lyte,IEF等电聚焦条件为24000Vh时,2-DE图谱分离到的蛋白点效果最好。初步建立了一套适用于大豆叶片蛋白质组分析的双向电泳(2-DE)体系。

Proteomics is becoming a necessity in plant biology, as it is in medicine, zoology and microbiology.. This study aimed at solving the problem of low content of protein in soybean leaf and the disturbance of secondary metabolites such as chromatophore, phenols and quinones to the separation of two- dimensional electrophoresis. Using leaves of soybean as material, index such as protein extraction, lysis buffer and IEF parameter were modified and improved . Results showed that, soybean leaves protein was extracted with TCA/acetone, then resolubilized in lysis buffer（9 mol·L^-1urea,2 mol·L^-1 thiourea, 100 mmol·L^-1 DTr,0.5% Bio-Lyte） , Isoelectric focusing was performed at 17℃ under the 24000Vh conditions. A 2-DE （ two- dimensional electrophoresis） protocol suited for the separation of proteins from soybean leaves was elementary established.