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

作　　者：宋归华 张迎新 侯泽豪 孙坤坤 方正武 马东方 张改生 王书平 SONG Guihua;ZHANG Yingxin;HOU Zehao;SUN Kunkun;FANG Zhengwu;MA Dongfang;ZHANG Gaisheng;WANG Shujjing(College of Agriculture,Yangtze University/Hubti Key Laboratory of Waterlogging Disaster and Agricultural Use of Wetland,Jingzhou,Hubei 434025,China;Institute of Genetics and Developmental Biology,Chinese Academy of Sciences,Beijing 100101,China;Northwest Agriculture and Forestry University/Wheat Breeding Engineering Research Center,Ministry of Education,Yangling,Shaanxi 712100,China)

机构地区：[1]长江大学农学院/湖北省涝渍灾害与湿地农业重点实验室,湖北荆州434025 [2]中国科学院遗传与发育生物学研究所,北京100101 [3]西北农林科技大学农学院/小麦育种教育部工程研究中心,陕西杨凌712100

出　　处：《麦类作物学报》2018年第6期686-692,共7页Journal of Triticeae Crops

基　　金：国家重点研发计划项目(2017YFD0100800);湖北省自然科学基金项目(2017CFB234;2016CFB478);湿地生态与农业利用教育部工程研究中心开放基金项目(KF201708);长江青年科技创新团队基金项目(2016cqt05/7011802111);长江大学青年科研支持计划基金项目(2016cqn37);长江大学博士启动基金项目(801180010145)

摘　　要：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.

关 键 词：小麦 小花 细胞核 蛋白质组 2-DE 

分 类 号：S512.1[农业科学—作物学] S330