小麦小花细胞核蛋白质双向电泳体系的优化

Optimization of Two-Dimensional Electrophoresis for Nucleus from Wheat Floret

为建立适用于小麦小花细胞核的分离及其蛋白质双向电泳(two-dimensional electrophoresis,2-DE)体系,以小麦品种西农1376三核期小花为供试材料,采用叶绿素含量测定、荧光显微镜观察和组蛋白免疫印迹检测等方法对分离和富集到的细胞核进行了纯度评价,并在SDS-PAGE胶浓度和蛋白质上样量等方面对细胞核蛋白质双向电泳体系进行了探索和优化,确立了一套适用于小麦小花细胞核的分离及其蛋白质双向电泳的技术方法。结果表明,获得的细胞核完整且纯度较高;经TCA/丙酮法提取其蛋白质,采用17cm、pH 4~7IPG胶条和12%SDS-PAGE分离胶,上样量为230μg的双向电泳体系,更适合于小麦小花细胞核蛋白质组分离,经PDQuest 2DE 8.0.1软件统计分析,可在2-DE图谱上分辨出约264个蛋白点,蛋白点清晰呈圆形,无横条纹干扰。

Using 2-DE to analyze the nuclear proteomic at subcellular level,the chlorophyll content,microscope and western blot were used to evaluate the purity of nucleus from wheat floret at trinucleate stage.The SDS-PAGE gel concentration and the loading quantity of sample were optimized for 2-DE analysis system.The results showed that,it was an efficient method for isolating intact nucleus using differential centrifugation.The nuclear proteins were extracted by the TCA/acetone method,and about 264 protein spots could be detected on the map,with 230μg proteins of nucleus loaded onto 17 cm,pH 4~7 IPG strip for 2-DE followed by silver staining.The system was suitable for isolation of intact nucleus and establishment of two-dimensional electrophoresis.