山定子(Malus baccata Borkh.)根系蛋白质提取及双向电泳技术体系的建立  

Establishment of Extraction Methods and Two-dimensional Gel Electrophoresis System for Proteomic Analysis of Malus baccata Borkh. Roots

作　　者：李丽杰[1] 苏宏[1] 马怀宇[1] 吕德国[1] 

机构地区：[1]沈阳农业大学园艺学院/辽宁省果树品质发育与调控重点实验室,沈阳110161

出　　处：《沈阳农业大学学报》2015年第5期532-537,共6页Journal of Shenyang Agricultural University

基　　金："十二五"科技计划项目(2014BAD16B02-1);国家现代苹果产业技术体系项目(CARS-28);辽宁省农业科技攻关产业化项目(2014204004);辽宁省高等学校果树栽培与生理生态创新团队项目(LT2014014)

摘　　要：为开展山定子（Malus baccata Borkh.）根系蛋白质组学研究,以当年生15片真叶的山定子实生苗白色幼嫩根系为试材,采用TCA-丙酮沉淀法、酚抽提法和Trizol抽提法3种方法提取其总蛋白质,并对样品上样量、IPG胶条的pH值范围和等电聚焦参数进行优化,建立适用于山定子根系的双向电泳技术体系。结果表明：采用Trizol抽提法提取的蛋白质产率最高,可达3.15mg·g^-1 FW,且其SDS-PAGE图谱明显优于其他两种方法 ;选用800μg的上样量,17 cm pH值4～7的IPG胶条和改进后的等电聚焦程序所得到的二维电泳图谱蛋白点最多,最清晰,分辨率最高。For the proteomic analysis of Malus baccata Borkh. roots, the roots of 15-leaves′ Malus baccata Borkh. seedlings were used as materials. Total proteins of roots were extracted by three methods including TCA-acetone, phenol and Trizol extraction methods. pH gradient of IPG strip, sample loading quantity and isoelectric focusing parameters of two-dimensional electrophoresis（2-DE） system were also optimized. The results showed that the protein yield and the SDS-PAGE map of total proteins extracted by Trizol extraction method were significantly better than that of other two methods. The protein yield of total proteins extracted by Trizol extraction method was 3.15 mg·g^-1 FW.In addition, the 2-DE map with the combination of 17 cm pH 4-7 IPG strips, 800 μg sample loading quantity and optimized procedure of IEF could obtain the most number of protein spots, high resolution and clear background.

关 键 词：山定子 根系 蛋白质 提取 双向电泳 

分 类 号：S661.1[农业科学—果树学]