生物技术通报2016，Vol.32Issue(9)：72-82,11.DOI:10.13560/j.cnki.biotech.bull.1985.2016.09.011
茅苍术质膜蛋白的制备及其双向电泳蛋白质组学平台的构建
Establishment of Membrane Proteomics Platform with Two-dimensional Electrophoresis for Preparing Identifying Plasma Membrane Proteins from Atractylodes lancea
摘要
Abstract
In order to understand related metabolic pathways of secondary metabolites and carry on metabolic regulation of active medical ingredients in endangered medicinal plant Atractylodes lancea,the method of plasma membrane proteomics was studied in this paper. Purified plasma membrane proteins were respectively extracted and purified from root,stem,and leaf of A. lancea using ultracentrifugation combined with aqueous two-phase partitioning. Moreover,the conditions for two-dimensional electrophoresis(2DE)analysis were optimized,and partial proteins in the latter were identified. The results showed that treating the ultra-centrifuged crude membrane compositions with the aqueous two-phase system of 6.4%,6.3%,and 6.1% Dextran T-500/PEG 3350(w/w)respectively,the high- purity plasma membrane proteins of 92.1%(root),91.5%(stem)and 90.8%(leaf)were obtained. The plasma membrane proteins were decomposed by 2% CHAPS and 2%Triton X-100 as the comprehensive detergents,loaded in the amount of 100 μg and resolved by IEF of 80 000 Vhs and 12.5%-15% SDS-PAGE gradient gel,and total 267,297 and 248 protein spots in the plasma membrane protein profiles of root,stem and leave respectively were detected by the Image Master 2D Platinum 7.0 software. And similarities and differences of 3 tissue proteins were further comparatively analyzed.At last,5 proteins were selected and successfully identified by MALDI-TOF-MS. Conclusively,a complete proteomics technology platform for preparing high-purity plasma membrane proteins from varied tissues of root,stem and leave of A. lancea and identifying the mass spectrometry of membrane protein is established.关键词
茅苍术/质膜蛋白纯化/双向电泳/膜蛋白质组Key words
Atractylodes lancea/purification of plasma membrane proteins/two-dimensional electrophoresis/membrane proteomics引用本文复制引用
陈飞,周彤,魏宇佳,杨晶,戴传超..茅苍术质膜蛋白的制备及其双向电泳蛋白质组学平台的构建[J].生物技术通报,2016,32(9):72-82,11.基金项目
国家自然科学基金项目（21406119，31070443），江苏省高校自然科学基金项目 （）
