三疣梭子蟹含硒谷胱甘肽过氧化物酶基因克隆及其表达分析
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摘要
为探究三疣梭子蟹的免疫机制以及谷胱甘肽过氧化物酶(GPx)在甲壳动物响应寄生虫感染免疫过程中的作用,本研究采用RACE方法从三疣梭子蟹中克隆获得硒半胱氨酸-谷胱甘肽过氧化物酶基因的全长cDNA序列,其cDNA序列全长为696bp,5′-UTR长度为102bp,3′-UTR长度为87bp,开放阅读框长度为507bp,编码168个氨基酸,其中含有一个典型的由蛋白石终止密码子(220TGA222)编码的硒半胱氨酸(40U)。预测了该基因编码的氨基酸序列及其中的保守结构域,包括GPx家族签名序列(64LAFPCNQF71)、活性位点序列(152WNFEKF157)以及与酶催化活性相关的氨基酸位点包括谷氨酰胺(74Q)、精氨酸(90R和168R)和色氨酸(142W)。相似性比对和系统发育分析结果表明,三疣梭子蟹硒半胱氨酸-谷胱甘肽过氧化物酶(SeGPx)与甲壳动物中的SeGPxs相似性较高,其中与拟穴青蟹SeGPx相似性最高,并与其在系统发育树中聚为一支。经血卵涡鞭虫侵染后(0—192h),SeGPx基因在三疣梭子蟹的血细胞、肝胰腺和鳃组织中的转录水平均发生显著性升高。该结果表明,SeGPx在三疣梭子蟹应对血卵涡鞭虫的免疫反应中发挥重要作用,可通过调控甲壳宿主体内被病原扰乱的氧化还原状态进而起到宿主组织保护作用。
Glutathione peroxidase(GPx) is an essential antioxidant enzyme that associated with the immune defense of aerobic living organisms against invading pathogens.To investigate the potential role of GPx genes in crustacean immune response against parasitic infection,we isolated and characterized a novel selenium-dependent glutathione peroxidase gene(PtSeGPx) in Portunus trituberculatus.The open reading frame(ORF) encoded 168 amino acids,with a characteristic selenocysteine residue(40 U) encoded by an opal codon(220 TGA222).The conserved domains including the GPx signature motif 2(64 LAFPCNQF71) and the active site motif(152 WNFEKF157),as well as the catalytically related amino acid residues consisting of Gln(74 Q),Arg(90 R),Trp(142 W),and Arg(168 R),were identified in the deduced PtSeGPx protein sequence.Amino acid comparison and phylogenetic analyses indicated that the PtSeGPx was closely related to and clustered with the crustacean SeGPxs.The PtSeGPx transcripts were extensively and induced significantly in multiple major tissues(e.g.hemocytes,hepatopancreas,and gills) of P.trituberculatus during its host immune response against the invasion of the parasitic dinoflagellate Hematodinium,implying its involvement in the crustacean innate immunity against parasitic infection.
Glutathione peroxidase(GPx) is an essential antioxidant enzyme that associated with the immune defense of aerobic living organisms against invading pathogens.To investigate the potential role of GPx genes in crustacean immune response against parasitic infection,we isolated and characterized a novel selenium-dependent glutathione peroxidase gene(PtSeGPx) in Portunus trituberculatus.The open reading frame(ORF) encoded 168 amino acids,with a characteristic selenocysteine residue(40 U) encoded by an opal codon(220 TGA222).The conserved domains including the GPx signature motif 2(64 LAFPCNQF71) and the active site motif(152 WNFEKF157),as well as the catalytically related amino acid residues consisting of Gln(74 Q),Arg(90 R),Trp(142 W),and Arg(168 R),were identified in the deduced PtSeGPx protein sequence.Amino acid comparison and phylogenetic analyses indicated that the PtSeGPx was closely related to and clustered with the crustacean SeGPxs.The PtSeGPx transcripts were extensively and induced significantly in multiple major tissues(e.g.hemocytes,hepatopancreas,and gills) of P.trituberculatus during its host immune response against the invasion of the parasitic dinoflagellate Hematodinium,implying its involvement in the crustacean innate immunity against parasitic infection.
引文
许文军,施慧,徐汉祥等,2007.养殖梭子蟹血卵涡鞭虫感染的初步研究.水生生物学报,31(5):637-642
李才文,许文军,2014a.海水甲壳类寄生性病原血卵涡鞭虫(Hematodinium spp.)研究进展.海洋与湖沼,(1):1-10
李才文,宋书群,刘云等,2014b.寄生性病原血卵涡鞭虫(Hematodinium sp.)感染山东半岛养殖梭子蟹的初步研究.海洋与湖沼,(4):776-782
王金凤,李才文,李蒙等,2015.血卵涡鞭虫感染三疣梭子蟹的病原形态学及组织病理学变化.海洋与湖沼,46(4):748-757
农业部渔业渔政管理局,2017.中国渔业统计年鉴.北京:中国农业出版社,1-157
Allmang C,Krol A,2006.Selenoprotein synthesis:UGA does not end the story.Biochimie,88(11):1561-1571
Arthur J R,2001.The glutathione peroxidases.Cell Mol Life Sci,57(13-14):1825-1835
Aumann K D,Bedorf N,Brigelius-FlohéR et al,1997.Glutathione peroxidase revisited-simulation of the catalytic cycle by computer-assisted molecular modelling.Biomed Environ Sci,10(2-3):136-155
Brigelius-FlohéR,Maiorino M,2013.Glutathione peroxidases.Biochim Biophys Acta,1830(5):3289-3303
Duan Y F,Liu P,Li J T et al,2013.Expression profiles of selenium dependent glutathione peroxidase and glutathione S-transferase from Exopalaemon carinicauda in response to Vibrio anguillarum and WSSV challenge.Fish Shellfish Immunol,35(3):661-670
Duan Y F,Zhang J S,Dong H B et al,2015.Oxidative stress response of the black tiger shrimp Penaeus monodon to Vibrio parahaemolyticus challenge.Fish Shellfish Immunol,46(2):354-365
Fu M J,Zou Z H,Liu S F et al,2012.Selenium-dependent glutathione peroxidase gene expression during gonad development and its response to LPS and H2O2 challenge in Scylla paramamosain.Fish Shellfish Immunol,33(3):532-542
Herbette S,Roeckel-Drevet P,Drevet J R,2007.Seleno-independent glutathione peroxidases.More than simple antioxidant scavengers.FEBS J,274(9):2163-2180
Li C W,Song S Q,Liu Y et al,2013.Hematodinium infections in cultured Chinese swimming crab,Portunus trituberculatus,in northern China.Aquaculture,396-399:59-65
Li M,Li C W,Wang J F et al,2015a.Immune response and gene expression in hemocytes of Portunus trituberculatus inoculated with the parasitic dinoflagellate Hematodinium.Mol Immunol,65(1):113-122
Li M,Wang J F,Song S Q et al,2015b.Early transcriptional response to the parasitic dinoflagellate Hematodinium in hepatopancreas of Portunus trituberculatus.J Invertebr Pathol,130:28-36
Li M,Wang J F,Song S Q et al,2016.Molecular characterization of a novel nitric oxide synthase gene from Portunus trituberculatus and the roles of NO/O2.--generating and antioxidant systems in host immune responses to Hematodinium.Fish Shellfish Immunol,52:263-277
Liu C H,Tseng M C,Cheng W,2007.Identification and cloning of the antioxidant enzyme,glutathione peroxidase,of white shrimp,Litopenaeus vannamei,and its expression following Vibrio alginolyticus infection.Fish Shellfish Immunol,23(1):34-45
Liu K F,Yeh M S,Kou G H et al,2010.Identification and cloning of a selenium-dependent glutathione peroxidase from tiger shrimp,Penaeus monodon,and its transcription following pathogen infection and related to the molt stages.Dev Comp Immunol,34(9):935-944
Liu Y,Cui Z X,Li X H et al,2013.A newly identified anti-lipopolysaccharide factor from the swimming crab Portunus trituberculatus with broad spectrum antimicrobial activity.Fish Shellfish Immunol,34(2):463-470
Livak K J,Schmittgen T D,2001.Analysis of relative gene expression data using real-time quantitative PCR and the2-ΔΔCt method.Methods,25(4):402-408
Margis R,Dunand C,Teixeira F K et al,2008.Glutathione peroxidase family-an evolutionary overview.FEBS J,275(15):3959-3970
Rasmussen R,2001.Quantification on the LightCycler.In:Meuer S,Wittwer C,Nakagawara K I eds.Rapid Cycle Real-time PCR.Berlin,Heidelberg:Springer Press,21-34
Ren Q,Sun R R,Zhao X F et al,2009.A selenium-dependent glutathione peroxidase(Se-GPx)and two glutathione S-transferases(GSTs)from Chinese shrimp(Fenneropenaeus chinensis).Comp Biochem Physiol C:Toxicol Pharmacol,149(4):613-623
Shields J D,Squyars C M,2000.Mortality and hematology of blue crabs,Callinectes sapidus,experimentally infected with the parasitic dinoflagellate Hematodinium perezi.Fish Bull,98:139-152
Small H J,Shields J D,Hudson K L et al,2007a.Molecular detection of Hematodinium sp.infecting the blue crab,Callinectes sapidus.J Shellfish Res,26(1):131-139
Small H J,Shields J D,Neil D M et al,2007b.Differences in enzyme activities between two species of Hematodinium,parasitic dinoflagellates of crustaceans.J Invertebr Pathol,94(3):175-183
Stentiford G D,Shields J D,2005.A review of the parasitic dinoflagellates Hematodinium species and Hematodiniumlike infections in marine crustaceans.Dis Aquat Organ,66(1):47-70
Toppo S,Vanin S,Bosello V et al,2008.Evolutionary and structural insights into the multifaceted glutathione peroxidase(Gpx)superfamily.Antioxid Redox Signal,10(9):1501-1514
Ursini F,Maiorino M,Brigelius-FlohéR et al,1995.Diversity of glutathione peroxidases.Methods Enzymol,252:38-53
Wang J F,Li M,Xiao J et al,2017.Hematodinium spp.infections in wild and cultured populations of marine crustaceans along the coast of China.Dis Aquat Organ,124(3):181-191
Xia X F,Zheng J J,Shao G M et al,2013.Cloning and functional analysis of glutathione peroxidase gene in red swamp crayfish Procambarus clarkii.Fish Shellfish Immunol,34(6):1587-1595
Yeh S P,Liu K F,Chiu S T et al,2009.Identification and cloning of a selenium dependent glutathione peroxidase from giant freshwater prawn,Macrobrachium rosenbergii.Fish Shellfish Immunol,27(2):181-191
李才文,许文军,2014a.海水甲壳类寄生性病原血卵涡鞭虫(Hematodinium spp.)研究进展.海洋与湖沼,(1):1-10
李才文,宋书群,刘云等,2014b.寄生性病原血卵涡鞭虫(Hematodinium sp.)感染山东半岛养殖梭子蟹的初步研究.海洋与湖沼,(4):776-782
王金凤,李才文,李蒙等,2015.血卵涡鞭虫感染三疣梭子蟹的病原形态学及组织病理学变化.海洋与湖沼,46(4):748-757
农业部渔业渔政管理局,2017.中国渔业统计年鉴.北京:中国农业出版社,1-157
Allmang C,Krol A,2006.Selenoprotein synthesis:UGA does not end the story.Biochimie,88(11):1561-1571
Arthur J R,2001.The glutathione peroxidases.Cell Mol Life Sci,57(13-14):1825-1835
Aumann K D,Bedorf N,Brigelius-FlohéR et al,1997.Glutathione peroxidase revisited-simulation of the catalytic cycle by computer-assisted molecular modelling.Biomed Environ Sci,10(2-3):136-155
Brigelius-FlohéR,Maiorino M,2013.Glutathione peroxidases.Biochim Biophys Acta,1830(5):3289-3303
Duan Y F,Liu P,Li J T et al,2013.Expression profiles of selenium dependent glutathione peroxidase and glutathione S-transferase from Exopalaemon carinicauda in response to Vibrio anguillarum and WSSV challenge.Fish Shellfish Immunol,35(3):661-670
Duan Y F,Zhang J S,Dong H B et al,2015.Oxidative stress response of the black tiger shrimp Penaeus monodon to Vibrio parahaemolyticus challenge.Fish Shellfish Immunol,46(2):354-365
Fu M J,Zou Z H,Liu S F et al,2012.Selenium-dependent glutathione peroxidase gene expression during gonad development and its response to LPS and H2O2 challenge in Scylla paramamosain.Fish Shellfish Immunol,33(3):532-542
Herbette S,Roeckel-Drevet P,Drevet J R,2007.Seleno-independent glutathione peroxidases.More than simple antioxidant scavengers.FEBS J,274(9):2163-2180
Li C W,Song S Q,Liu Y et al,2013.Hematodinium infections in cultured Chinese swimming crab,Portunus trituberculatus,in northern China.Aquaculture,396-399:59-65
Li M,Li C W,Wang J F et al,2015a.Immune response and gene expression in hemocytes of Portunus trituberculatus inoculated with the parasitic dinoflagellate Hematodinium.Mol Immunol,65(1):113-122
Li M,Wang J F,Song S Q et al,2015b.Early transcriptional response to the parasitic dinoflagellate Hematodinium in hepatopancreas of Portunus trituberculatus.J Invertebr Pathol,130:28-36
Li M,Wang J F,Song S Q et al,2016.Molecular characterization of a novel nitric oxide synthase gene from Portunus trituberculatus and the roles of NO/O2.--generating and antioxidant systems in host immune responses to Hematodinium.Fish Shellfish Immunol,52:263-277
Liu C H,Tseng M C,Cheng W,2007.Identification and cloning of the antioxidant enzyme,glutathione peroxidase,of white shrimp,Litopenaeus vannamei,and its expression following Vibrio alginolyticus infection.Fish Shellfish Immunol,23(1):34-45
Liu K F,Yeh M S,Kou G H et al,2010.Identification and cloning of a selenium-dependent glutathione peroxidase from tiger shrimp,Penaeus monodon,and its transcription following pathogen infection and related to the molt stages.Dev Comp Immunol,34(9):935-944
Liu Y,Cui Z X,Li X H et al,2013.A newly identified anti-lipopolysaccharide factor from the swimming crab Portunus trituberculatus with broad spectrum antimicrobial activity.Fish Shellfish Immunol,34(2):463-470
Livak K J,Schmittgen T D,2001.Analysis of relative gene expression data using real-time quantitative PCR and the2-ΔΔCt method.Methods,25(4):402-408
Margis R,Dunand C,Teixeira F K et al,2008.Glutathione peroxidase family-an evolutionary overview.FEBS J,275(15):3959-3970
Rasmussen R,2001.Quantification on the LightCycler.In:Meuer S,Wittwer C,Nakagawara K I eds.Rapid Cycle Real-time PCR.Berlin,Heidelberg:Springer Press,21-34
Ren Q,Sun R R,Zhao X F et al,2009.A selenium-dependent glutathione peroxidase(Se-GPx)and two glutathione S-transferases(GSTs)from Chinese shrimp(Fenneropenaeus chinensis).Comp Biochem Physiol C:Toxicol Pharmacol,149(4):613-623
Shields J D,Squyars C M,2000.Mortality and hematology of blue crabs,Callinectes sapidus,experimentally infected with the parasitic dinoflagellate Hematodinium perezi.Fish Bull,98:139-152
Small H J,Shields J D,Hudson K L et al,2007a.Molecular detection of Hematodinium sp.infecting the blue crab,Callinectes sapidus.J Shellfish Res,26(1):131-139
Small H J,Shields J D,Neil D M et al,2007b.Differences in enzyme activities between two species of Hematodinium,parasitic dinoflagellates of crustaceans.J Invertebr Pathol,94(3):175-183
Stentiford G D,Shields J D,2005.A review of the parasitic dinoflagellates Hematodinium species and Hematodiniumlike infections in marine crustaceans.Dis Aquat Organ,66(1):47-70
Toppo S,Vanin S,Bosello V et al,2008.Evolutionary and structural insights into the multifaceted glutathione peroxidase(Gpx)superfamily.Antioxid Redox Signal,10(9):1501-1514
Ursini F,Maiorino M,Brigelius-FlohéR et al,1995.Diversity of glutathione peroxidases.Methods Enzymol,252:38-53
Wang J F,Li M,Xiao J et al,2017.Hematodinium spp.infections in wild and cultured populations of marine crustaceans along the coast of China.Dis Aquat Organ,124(3):181-191
Xia X F,Zheng J J,Shao G M et al,2013.Cloning and functional analysis of glutathione peroxidase gene in red swamp crayfish Procambarus clarkii.Fish Shellfish Immunol,34(6):1587-1595
Yeh S P,Liu K F,Chiu S T et al,2009.Identification and cloning of a selenium dependent glutathione peroxidase from giant freshwater prawn,Macrobrachium rosenbergii.Fish Shellfish Immunol,27(2):181-191