不同失水状态发菜类囊体膜蛋白的分离及鉴定
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摘要
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 Nostocflagelliforme 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 6h(water contenting 51.2%)and on dehydration for 24h(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 25kD to 60kD.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,F1F0ATP synthase subunit alpha,F1F0ATP 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.
The method of liquid nitrogen grinding and ultrasonic were applied to crush Nostocflagelliforme 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 6h(water contenting 51.2%)and on dehydration for 24h(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 25kD to 60kD.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,F1F0ATP synthase subunit alpha,F1F0ATP 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.
引文
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[3]BIBBY T S,MARY I,NIELD J,et al.Low-light-adapted prochlorococcus species possess specific antennae for each photosystem[J].Nature,2003,424(6 952):1 051-1 054.
[4]HU F(胡锋),HUANG J L(黄俊丽),QIN F(秦峰,)et al.Progress in chloroplast thylakoid membrane and membrane proteins[J].Chinese Bulletin of Life Sciences(生命科学),2011,23(3):291-297(in Chinese).
[5]KASHINO Y,LAUBER W M,CARROLL J A,et al.Proteomic analysis of a highly active photosystem II preparation from the cyanobacteriumSynechocystis sp.PCC 6803reveals the presence of novel polypeptides[J].Biochemistry,2002,41(1):8 004-8 012.
[6]LIANG W Y,ZHOU Y W,WANG L X,et al.Ultrastructural,physiological and proteomic analysis of Nostoc flagelliformein response to dehydration and rehydration[J].Journal of Proteomics,2012,75:5 604-5 627.
[7]WANG M(王梅),ZHONG Z P(钟泽璞),WANG K F(王可玢),et al.Isolation of chlorophyll protein complexes from the thylakoid membrane in Nostoc flagelliforme and their spectroscopic characterization[J].Journal of Integrative Plant Biology(植物学报),2000,42(7):684-688(in Chinese).
[8]GAO Z Q,WANG G C,TSENG C K.An improved method for isolation of photosystemⅡfrom marine algaPorphyra yezoensis Udea[J].Indian Journal of Biochemistry and Biophysics,2005,42:41-47.
[9]LIANG W Y(梁文裕),NISHIFUJI T K.Purification and characterization of C-phycocyanin from Nostoc flagelliforme[J].Acta Bot.Boreal.-Occident.Sin.(西北植物学报),2008,28(2):303-309.
[10]ADEWOYE L O,WOROBEC E A.Multiple environmental factors regulate the expression of the carbohydrate-selective OprB porin of Pseudomonas aeruginosa[J].Canadian Journal of Microbiology,1999,45(12):1 033-1 042.
[11]WYLIE J L,WOROBEC E A.The OprB porin plays a central role in carbohydrate uptake in Pseudomonas aeruginosa[J].Journal of Bacteriology,1995,177(11):3 021-3 026.
[12]LEFEBVRE-LEGENDRE L,VAILLIER J,BENABDELHAK H,et al.Identification of a nuclear gene(FMC1)required for the assembly/stability of yeast mitochondrial F1-ATPase in heat stress conditions[J].Journal of Biological Chemistry,2001,276:6 789-6 796.
[13]ZHANG X X(张欣欣),LIU SH K(柳参奎).Identification and characterization of mitochondrial ATP synthase small subunit gene in rice(Oryza sativa L.)[J].Molecular Plant Breeding(分子植物育种),2003,1(5/6):605-612(in Chinese).
[14]CAPALIDI R A,AGGELER R,WILKENS S,et al.Structural changes in theγandεsubunits of the Escherichia coli.F1F0-type ATPase during energy coupling[J].Journal of Bioenergetics and Biomembranes,1996,28:397-401.
[15]SHEN J R,INOUE Y.Binding and function of two new extrinsic components,cytochrome c-550and a 12kD protein in cyanobacterial photosystemⅡ[J].Biochemistry,1993,32:1 825-1 832.
[16]BRICKER T M.Oxygen evolution in the absence of the 33kD manganese-stabilizing protein[J].Biochemistry,1992,31:4 623-4 628.
[17]BRICKER T M,FRANKEL LK.The structure and function of 33kD extrinsic protein of photosystemⅡ:A critical assessment[J].Photosynthesis Research,1998,56:157-173.