条斑紫菜类囊体膜蛋白质组双向电泳研究方法  

Two-dimensional electrophoresis of thylakoid membrane proteome in Porphyra yezoensis

作　　者：周伟[1,2,3] 何林文[1] 陆勤勤[3] 朱建一[4] 高山[1,2] 杨睿灵[5] 杨芳[5] 王广策[1] 

机构地区：[1]中国科学院海洋研究所实验海洋生物学重点实验室,山东青岛266071 [2]中国科学院大学,北京100049 [3]江苏省海洋水产研究所,江苏南通226007 [4]常熟理工学院生物与食品工程学院,江苏常熟215500 [5]天津科技大学海洋科学与工程学院,天津300457

出　　处：《海洋科学》2014年第3期63-68,共6页Marine Sciences

基　　金：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)

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PSⅡ47ku,PSⅡ44ku,cytochrome f,PSⅡD2,PSⅡD1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。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.

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 

分 类 号：Q946[生物学—植物学]