小麦小花高纯度线粒体的分离及其蛋白质双向电泳体系建立

Isolation of High Purity Mitochondria from Wheat Floret for Two-dimensional Electrophoresis

应用差速离心和Percoll不连续密度梯度法分离纯化小麦三核期小花线粒体.在裂解液选择、IPG胶条pH值范围、SDS-PAGE胶浓度及蛋白质上样量等方面对线粒体蛋白质双向电泳体系进行探索和优化,确立了一套适用于小麦小花高纯度完整线粒体的分离方法及其蛋白质双向电泳的技术体系.结果表明,采用20%、24%和40%Percoll密度梯度和28%Percoll自形成密度高速离心体系,获得了有活性、高纯度且较完整的线粒体;经TCA-丙酮法提取蛋白,以7 mol/L尿素,2mol/L硫脲,4%CHAPS(W/V),65 mmol/L DTT,0.5%IPG缓冲液(V/V),0.001%溴酚蓝(W/V)裂解液溶解蛋白,采用17 cm,pH 4～7 IPG胶条和11%SDS-PAGE分离胶,上样量为160μg,硝酸银染色法,更适合小麦小花线粒体蛋白质组双向电泳分离.经PDQuest 2DE 8.0.1软件包统计分析,在2-DE图谱上分辨出约150个蛋白点,蛋白点清晰呈圆形,无横条纹干扰,这为利用双向电泳技术在亚细胞水平对线粒体进行蛋白质组学研究与分析奠定了基础,更为进一步分析研究线粒体与雄性不育的关系提供了理论与技术支撑.

The mitochondria from the trinucleate stage wheat floret were separated and purified by differential centrifugation and discontinuous Percoll density gradient methods. Subsequently, two- dimensional electrophoresis of mitochondrial proteins were optimized with different conditions of the lysis liquid, pH of IPG strip, SDS-PAGE gel concentration, and protein loadings. High purity and integrity mitochondria were obtained with a high speed centrifugal system of 20 % , 24 % and 40 % Percoll or 28 % Percoll to form the density gradient. The buffer solution （V/V） containing 7 mol/L urea, 2 mol/L thiourea, 4% CHAPS （W/V） , 65 mmol/L DTT, 0.5% IPG, and 0. 001% bromophenol blue （W/V） lysate solution was used to treat the extracted proteins following TCA-acetone method. The 17 cm IPG strip of pH 4 -7 and 11% SDS-PAGE was used for the separation of 160 μg samples and the silver nitrate staining was performed prior to PDQuest 2DE analysis. The results showed that about 150 protein spots could be identified.