太平洋牡蛎幼体附着变态差异蛋白质组研究
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摘要
太平洋牡蛎(Crassostrea angulata)是中国主要的海洋养殖经济物种之一,具有重要的经济价值和商业价值,附着变态是牡蛎幼体从受精卵发育到成体所需的重要阶段,其分子机制的研究对牡蛎养殖育苗具有重要的指导意义.本研究利用双向电泳技术分离筛选牡蛎幼体附着变态过程中的差异表达的蛋白,进一步利用质谱技术鉴定幼体附着变态过程中发育相关的关键蛋白,共筛选出128个差异蛋白,鉴定并注释了39种蛋白.通过对差异表达蛋白功能分析,筛选获得了与纤毛组建相关的筑丝蛋白和与稳定钙离子胞内浓度相关的钙网蛋白,为揭示牡蛎幼体面盘组织及纤毛的退化消失和钙离子有效诱导幼体附着变态的机制提供分子数据.同时还获得了调节脂肪酸β-氧化、三羧酸循环和糖酵解重要生理功能的关键酶蛋白:乙酰辅酶A脱氢酶、异柠檬酸脱氢酶和3-磷酸甘油醛脱氢酶,以及调节无脊椎动物机体能量存储和释放的酶蛋白:精氨酸激酶和ATP合成酶,进一步阐明了牡蛎幼体在附着变态过程中能量调节的分子机制.
The Pacific oyster,Crassostrea gigas,is an economically important mariculture species in China,settlement and metamorphosis is an essential process for the larval development of the species.The study of molecular mechanism on larval settlement and metamorphosis is helpful for the oyster growing and seeding.In our studies 128 proteins in different expression were screened and 39 proteins identified during the settlement and metamorphosis with two-dimension gel electrophoresis and mass-spectrometric technique,respectively.The function analysis of the different expressed proteins screened and resulted fibroin related to the formation of cilia and the calcium net proteins related to stabilize the intracellular concentration of calcium ions.These provide the molecular data that reveals the degeneration and disappearance of the veliger tissue and cilia of the oyster larvae and the mechanism of calcium ion to effectively induce the attachment and metamorphosis of the larvae.In the meanwhile,we obtained three key protein enzymes as acetyl coenzyme A dehydrogenase,isocitrate dehydrogenase and 3-phosphate glyceraldehyde dehydrogenase,which respectively regulate the important physiological functions of fatty acid β-oxidation,tricarboxylic acid cycle and glycolysis,and two other enzymes,arginine kinase and ATP synthetase that regulate the energy storage and release in the invertebrate.These observations furtherly clarify the molecular mechanism of energy regulation in oyster larvae during the settlement and metamorphosis.
The Pacific oyster,Crassostrea gigas,is an economically important mariculture species in China,settlement and metamorphosis is an essential process for the larval development of the species.The study of molecular mechanism on larval settlement and metamorphosis is helpful for the oyster growing and seeding.In our studies 128 proteins in different expression were screened and 39 proteins identified during the settlement and metamorphosis with two-dimension gel electrophoresis and mass-spectrometric technique,respectively.The function analysis of the different expressed proteins screened and resulted fibroin related to the formation of cilia and the calcium net proteins related to stabilize the intracellular concentration of calcium ions.These provide the molecular data that reveals the degeneration and disappearance of the veliger tissue and cilia of the oyster larvae and the mechanism of calcium ion to effectively induce the attachment and metamorphosis of the larvae.In the meanwhile,we obtained three key protein enzymes as acetyl coenzyme A dehydrogenase,isocitrate dehydrogenase and 3-phosphate glyceraldehyde dehydrogenase,which respectively regulate the important physiological functions of fatty acid β-oxidation,tricarboxylic acid cycle and glycolysis,and two other enzymes,arginine kinase and ATP synthetase that regulate the energy storage and release in the invertebrate.These observations furtherly clarify the molecular mechanism of energy regulation in oyster larvae during the settlement and metamorphosis.
引文
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[2]MURALIDHARAN S,THOMPSON E,RAFTOS D,et al.Quantitative proteomics of heavy metal stress responses in Sydney rock oysters[J].Proteomics,2012,12:906-921.
[3]WANG H,ZHANG H,WONG Y H,et al.Rapid transcriptome and proteome profiling of a non-model marine invertebrate,Bugula neritina[J].Proteomics,2010,10(16):2 972-2 981.
[4]TAY T L,LIN Q,SEOW T K,et al.Proteomic analysis of protein profiles during early development of the zebra fish,Danio rerio[J].Proteomics,2006,6:3 176-3 188.
[5]NOMURA M,NAKAJIMA A,INABA K.Proteomic profiles of embryonic development in the ascidian Ciona intestinalis[J].Developmental Biology,2009,325:468-481.
[6]ZHU B,GAO K S,WANG K J,et al.Gonad differential proteins revealed with proteomics in oyster(Saccostrea cucullata)using alga as food contaminated with cadmium[J].Chemosphere,2012,87:397-403.
[7]CAO A,FUENTES J,COMESANA P,et al.A proteomic approach envisaged to analyse the bases of oyster tolerance/resistance to bonamiosis[J].Aquaculture,2009,295(3/4):149-156.
[8]LOPEZ J L,ABALDE S L,FUENTES J.Proteomic approach to probe for larvae proteins of the mussel Mytilus galloprovincialis[J].Marine Biotechnology,2005,7(4):396-404.
[9]THIYAGARAJAN V,QIAN P Y.Proteomic analysis of larvae during development,attachment,and metamorphosis in the fouling barnacle,Balanus amphitrite[J].Proteomics,2008,8(15):3 164-3 172.
[10]THIYAGARAJAN V,WONG T,QIAN P Y.2D gel-based proteome and phosphoproteome analysis during larvae metamorphosis in two major marine biofouling invertebrates[J].Journal of Proteome Research,2009,8(6):2 708-2 719.
[11]ZHANG H,WONG Y H,WANG H,et al.Quantitative proteomics identify molecular targets that are crucial in larvae settlement and metamorphosis of Bugula neritina[J].Journal of Proteome Research,2011,10(1):349-360.
[12]CHANDRAMOULI K H,MOK F S,WANG H,et al.Phosphoproteome analysis during larval development and metamorphosis in the spionid polychaete Pseudopolydora vexillosa[J].BMC Developmental Biology,2011,11:31-45.
[13]ZHANG Y,SUN J,XIAO K,et al.2D gel-based multiplexed proteomic analysis during larvae development and metamorphosis of the biofouling polychaete tubeworm Hydroides elegans[J].Journal of Proteome Research,2010,9(9):4 851-4 860.
[14]KALYANNSUNDARAM M.Larval development of the edible oyster Crassostrea medrasensis(preston)[J].Mahasagar,1992,25:51-54.
[15]VIDELA J A,CHAPARRO O R,THOMPSON R J.Role of biochemical energy reserves in the metamorphosis and early juvenile development of the oyster Ostrea chilensis[J].Marine Biology,1998,132:635-640.
[16]ELLINGTON W R.Phosphocreatine represents a thermodynamic and functional improvement over other muscle phosphagens[J].Journal of Experimental Biology,1989,143:177-194.
[17]WYSS M,MAUGHAN D,WALLIMANN T.Re-evaluation of the structureand physiological function of guanidino kinases in fruitfly(Drosophila),sea urchin(Psammechinusmilaris),and man[J].Biochemical Journal,1995,309:255-261.
[18]NEWSHOLME E A,BEIS I,LEECH A R,et al.The role of creatine kinase and arginine kinase in muscle[J].Biochemical Journal,1978,172:533-537.
[19]ELLINGTON W R.Evolution and physiological roles of phosphagen systems[J].Annual Review Physiology,2001,63:289-325.
[20]韩贻仁.分子细胞生物学[M].北京:科学出版社,2001:71-75.
[21]KRAUSE K H,MICHALAK M.Calreticulin[J].Cell,1997,88:439-443.
[22]EGGLETON P,REID K B,KISHORE U,et al.Clinical relevance of calreticulin in systemic lupus erythematosus[J].Lupus,1997,6:564-571.
[23]MICHALAK M,BURNS K,ANDRIN C,et al.Endoplasmic reticulum form of calreticulin modulates glucocorticoid-senstive gene expression[J].Journal of Biological Chemistry,1996,271:29 436-29 445.
[24]SONTHEIMER R D,NGUYEN T Q,CHENG S T,et al.The unveiling of calreticulin-A clinically relevant tour of modem cell biology[J].Journal of Investigative Medicine,1995,43:362-370.
[25]BURNS K,DUGGAN B,ATKINSON E A,et al.Modulation of gene expression by calreticulin binding to the glucocorticoid receptor[J].Nature,1994,367:476-480.
[26]祁剑飞,曾志南,宁岳,等.几种阳离子对福建牡蛎幼虫附着和变态的影响[J].福建水产,2013,35:27-31.