不同失水状态发菜类囊体膜蛋白的分离及鉴定

Separation and Identification of Thylakoid Membrane Proteins in Nostoc flagelliforme on Dehydration

采用液氮研磨与超声波破碎相结合的方法,破碎不同失水状态的发菜藻体和细胞,用差速离心法制备发菜类囊体膜粗制品,蔗糖密度梯度高速离心纯化类囊体膜,SDS-PAGE电泳分离类囊体膜蛋白,并对膜蛋白进行了MALDI-TOF-TOF/MS质谱分析和鉴定。结果表明:(1)充分吸胀4h后失水6h的发菜(含水量51.2%)和失水24h的发菜(含水量14.9%),经过多步差速离心后再进行蔗糖密度梯度高速离心,可得到纯化的类囊体膜。(2)发菜类囊体膜蛋白SDS-PAGE电泳分离到14个条带,共鉴定出8种蛋白,根据其功能可分为4类——光合作用相关蛋白(光系统Ⅱ锰稳定蛋白PsbO,F1F0 ATP合成酶α亚基和β亚基)、结构域蛋白、选择性通道蛋白OprB、未知蛋白(hypothetical protein Npun_R1321、Npun_R3785、N9414_02186),它们在发菜的光合作用中具有重要作用。

The method of liquid nitrogen grinding and ultrasonic were applied to crush Nostoc flagelliforme colonies and cells.The crudes were isolated by differential centrifugation;thylakoid membranes were purified by high speed centrifugation with gradient density sucrose;the thylakoid membrane proteins were extracted and separated by SDS-PAGE.The results suggested that：（1）Thylakoid membrane of colonies on dehydration for 6 h（water contenting 51.2%） and on dehydration for 24 h（water contenting 14.9%） were purified by many tims differential centrifugation and then by high speed centrifugation with gradient density sucrose.（2）14 bands of thylakoid membrane protein were separated by SDS-PAGE,their mass weight mainly ranged from 25 kD to 60 kD.8 kinds of protein were identified by MALDI-TOF-TOF/MS and database searching.According to their biological functions,these thylakoid membrane proteins were divided into 4 categories,including photosynthesis proteins（photosystem Ⅱ manganese-stabilizing protein PsbO,F1F0 ATP synthase subunit alpha,F1F0 ATP synthase subunit beta）,binding domain protein（hemerythrin HHE cation binding domain-containing protein）,carbohydrate-selective porin（carbohydrate-selective porin OprB）,unknown proteins（hypothetical protein Npun_R1321,Npun_R3785 and N9414_02186）.They might play an important role in photosynthesis of N.flagelliforme.