引用本文:	周伟,何林文,陆勤勤,朱建一,高山,杨睿灵,杨芳,王广策.条斑紫菜类囊体膜蛋白质组双向电泳研究方法[J].海洋科学,2014,38(3):63-68.

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条斑紫菜类囊体膜蛋白质组双向电泳研究方法
周伟^1,2,3, 何林文¹, 陆勤勤⁴, 朱建一⁴, 高山^1,2, 杨睿灵⁵, 杨芳⁵, 王广策¹
1.中国科学院海洋研究所实验海洋生物学重点实验室;2.中国科学院大学;3.江苏省海洋水产研究所;4.常熟理工学院生物与食品工程学院;5.天津科技大学海洋科学与工程学院

摘要:

以蓝绿温和胶电泳为工具, 首次在国内用于条斑紫菜(Porphyra yezoensis)类囊体膜色素蛋白质复合物的研究。结果显示: (1)利用非离子型去污剂十二烷基麦芽糖苷(DM)增溶条斑紫菜类囊体膜,DM/Chla(w/w)15︰ 1 产生的效果较好, 在BN-PAGE 胶中可以分离到较多较清晰的条带。(2)选取蔗糖密度层50%条带制备得到的类囊体膜样品进行第一向BN-PAGE 和第二向SDS-Urea-PAGE 电泳实验。第一向电泳分离出4 个蛋白复合物, 对第二向电泳胶上的15 个蛋白点切取做质谱鉴定, 检测到PSⅡ 47ku, PSⅡ 44ku, cytochrome f , PSⅡ D2, PSⅡ D1 等蛋白。本实验证实了温和胶电泳与SDS 电泳结合对于条斑紫菜这种原始红藻的类囊体膜研究方面应用的可行性。

关键词: 条斑紫菜(Porphyra yezoensis) 类囊体膜蓝绿温和胶电泳 SDS 尿素电泳

DOI：10.11759/hykx20130131002

分类号:

基金项目:国家自然科学基金项目(31272664); 国家海洋局项目(201105023); 国家863 科技计划项目(2012AA10A406); 江苏省科技支撑计划项目(BE2012420, BE2011375); 江苏省农业科技自主创新项目(Cx(12)2039); 江苏省三新工程项目(2011-2012, 2012-2013)

Two-dimensional electrophoresis of thylakoid membrane proteome in Porphyra yezoensis

Abstract:

The blue-native polyacrylamide gel electrophoresis was used for the first time in investigation of thylakoid membrane proteome of Porphyra yezoensis. The results showed that: (1) dodecyl-β-maltoside was a suitable detergent for the solubilization and stabilization of super-complexes of thylakoid membrane proteins. The detergent/Chla ratio of 15︰1 gave the best solubilizing effect and a series of clear protein bands were obtained using BN-PAGE assay; (2) The sample collected at 50% sucrose was separated by BN-PAGE in the first dimension and then SDS-Urea-PAGE in the second dimension. Four protein complexes were separated by 1-D BN-PAGE gels. 15 protein dots separated by 2-D SDS-Urea-PAGE were analyzed by MALDI-TOF MS and a series of proteins including PSⅡ, 47ku, PSⅡ, 44ku, cytochrome f, PSⅡ D2, PSⅡ and D1 were confirmed. These data demonstrated that combination of BN-PAGE with SDS-Urea-PAGE can be well used in study of thylakoid membrane proteome of P. yezoensis.

Key words: Porphyra yezoensis thylakoid membrane BN-PAGE SDS-Uera-PAGE