高盐胁迫下大弹涂鱼MHC Iα基因对病毒拟似物poly(I:C)的免疫反应
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摘要
主要组织相容性复合体(major histocompatibility complex,MHC)是一类编码细胞表面糖蛋白的基因,在所有硬骨鱼的适应性免疫系统中起着至关重要的作用,而关于MHC基因的研究一直是鱼类分子免疫学和鱼类抗病辅助育种的研究热点之一。本研究首次分析了大弹涂鱼(Boleophthalmus pectinirostris)MHCIα基因的cDNA序列特征,构建了系统发生树,评估了大弹涂鱼MHC Iα基因mRNA在健康个体不同组织中的表达差异,研究了注射病毒拟似物poly(I:C)后MHC Iα基因在机体主要免疫器官肝和脾的表达情况。结果显示,大弹涂鱼MHC Iα基因具有由1101个碱基组成的开放阅读框(ORF),共编码366个氨基酸残基,具有3个蛋白激酶C-磷酸化位点、1个酪蛋白激酶Ⅱ磷酸化位点和1个N-糖基化位点。系统发育分析显示与大弹涂鱼MHCIα基因亲缘关系最密切的是河川沙塘鳢(Odontobutis potamophila)。RT-PCR分析显示,MHCIα基因mRNA在不同组织中均有表达,其中肾和脾组织中表达量最高,鳃和肠组织中表达次之。大弹涂鱼在腹腔注射poly(I:C)后,肝和脾组织中mRNA表达量明显上升,在12 h时, MHC Iα基因mRNA表达量在肝和脾中均达到峰值。本研究结果表明, MHC Iα基因参与了大弹涂鱼在高盐胁迫下的免疫应答。
The major histocompatibility complex(MHC) is a large group of genes that encode cell surface glycoproteins and play a crucial role in the acquired immune systems of all teleosts. The study of the MHC is a major focus in the field of fish molecular immunology, and for the auxiliary breeding of disease resistant fish. The great blue-spotted mudskipper(Boleophthalmus pectinirostris) is special for its unique behavioral, physiological, and biochemical characteristics suitable for amphibian life. This study firstly analyzed the sequence characteristics of the Boleophthalmus pectinirostris MHC Iα gene, then reconstructed the phylogenetic tree and explored the possible molecular evolution. Finally, the mRNA spatial expression pattern of the MHC Iα gene was assessed in healthy tissues, and hepatic and splenic expression patterns were examined after viral mimetic poly(I:C) injection. The results showed that the open reading frame(ORF) was 1,101 bases in length, encoding 366 amino acid residues,with a molecular mass of 41.1 kD and theoretical isoelectric point(pI) of 4.83. The gene included five domains:the leader peptide, α1 domain, α2 domain, α3 domain, and the transmembrane domain. The following secondary protein structure was predicted: ? helix accounted for 37.16%, ?-turn for 7.65%, extended strand for 21.31%, and random coils for 33.88%. There were three protein kinase C phosphorylation sites, one casein kinase II phosphorylation site, and one N-glycosylation site. Phylogenetic analysis showed that the MHC Iα gene of Boleophthalmus pectinirostris was most closely related to the MHC Iα gene of Odontobutis potamophila. The spatial expression analysis showed that MHC Iα was expressed in all tissues examined, particularly in the kidneys and spleen, followed by the gills and intestines. At 0 h, 6 h, 12 h, 24 h, 36 h, 48 h, and 72 h after stimulation there were no significant changes(P>0.05) in expression levels in the control group. During poly(I:C) challenges which were intraperitoneally injected into the liver, MHC Iα mRNA expression level was increased at 6 h(1.63-fold) and the expression increased significantly and was at its maximum at 12 h(P<0.01, 5.30-fold). Then, the expression level decreased at 24 h, but was still significantly higher than the control group(P<0.05, 2.98-fold). At 36 h, expression level increased again, and was still significantly higher than the control group(P<0.01, 4.68-fold). Expression gradually decreased to 2.22-fold and 1.46-fold the control values at 48 h and 72 h, respectively. In spleen tissues,the expression level of MHC Iα mRNA in the experimental group was significantly increased at 6 h(P<0.01,6.25-fold), and the expression level of the experimental group increased significantly to its maximum(P<0.01,9.71-fold) at 12 h. The maximum value expression level was reached earlier than in liver tissue. At 24 h, the expression level also decreased, but was still higher than the control group(P<0.05, 3.25-fold). At 36 h, the expression level increased again and was significantly higher than that in the control group(P<0.01, 5.81-fold), expression levels gradually returned to normal levels at 48 h and 72 h(1.21-fold and 1.38-fold, respectively). The results suggested that MHC Iα plays a crucial role in the immune response of Boleophthalmus pectinirostris against poly(I:C) challenges.
The major histocompatibility complex(MHC) is a large group of genes that encode cell surface glycoproteins and play a crucial role in the acquired immune systems of all teleosts. The study of the MHC is a major focus in the field of fish molecular immunology, and for the auxiliary breeding of disease resistant fish. The great blue-spotted mudskipper(Boleophthalmus pectinirostris) is special for its unique behavioral, physiological, and biochemical characteristics suitable for amphibian life. This study firstly analyzed the sequence characteristics of the Boleophthalmus pectinirostris MHC Iα gene, then reconstructed the phylogenetic tree and explored the possible molecular evolution. Finally, the mRNA spatial expression pattern of the MHC Iα gene was assessed in healthy tissues, and hepatic and splenic expression patterns were examined after viral mimetic poly(I:C) injection. The results showed that the open reading frame(ORF) was 1,101 bases in length, encoding 366 amino acid residues,with a molecular mass of 41.1 kD and theoretical isoelectric point(pI) of 4.83. The gene included five domains:the leader peptide, α1 domain, α2 domain, α3 domain, and the transmembrane domain. The following secondary protein structure was predicted: ? helix accounted for 37.16%, ?-turn for 7.65%, extended strand for 21.31%, and random coils for 33.88%. There were three protein kinase C phosphorylation sites, one casein kinase II phosphorylation site, and one N-glycosylation site. Phylogenetic analysis showed that the MHC Iα gene of Boleophthalmus pectinirostris was most closely related to the MHC Iα gene of Odontobutis potamophila. The spatial expression analysis showed that MHC Iα was expressed in all tissues examined, particularly in the kidneys and spleen, followed by the gills and intestines. At 0 h, 6 h, 12 h, 24 h, 36 h, 48 h, and 72 h after stimulation there were no significant changes(P>0.05) in expression levels in the control group. During poly(I:C) challenges which were intraperitoneally injected into the liver, MHC Iα mRNA expression level was increased at 6 h(1.63-fold) and the expression increased significantly and was at its maximum at 12 h(P<0.01, 5.30-fold). Then, the expression level decreased at 24 h, but was still significantly higher than the control group(P<0.05, 2.98-fold). At 36 h, expression level increased again, and was still significantly higher than the control group(P<0.01, 4.68-fold). Expression gradually decreased to 2.22-fold and 1.46-fold the control values at 48 h and 72 h, respectively. In spleen tissues,the expression level of MHC Iα mRNA in the experimental group was significantly increased at 6 h(P<0.01,6.25-fold), and the expression level of the experimental group increased significantly to its maximum(P<0.01,9.71-fold) at 12 h. The maximum value expression level was reached earlier than in liver tissue. At 24 h, the expression level also decreased, but was still higher than the control group(P<0.05, 3.25-fold). At 36 h, the expression level increased again and was significantly higher than that in the control group(P<0.01, 5.81-fold), expression levels gradually returned to normal levels at 48 h and 72 h(1.21-fold and 1.38-fold, respectively). The results suggested that MHC Iα plays a crucial role in the immune response of Boleophthalmus pectinirostris against poly(I:C) challenges.
引文
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[3]Bernatchez L,Landry C.MHC studies in nonmodel vertebrates:what have we learned about natural selection in 15years?[J].Journal of Evolutionary Biology,2003,16(3):363-377.
[4]Rodgers J R,Cook R G.MHC class Ib molecules bridge innate and acquired immunity[J].Nature Reviews Immunology,2005,5(6):459-471.
[5]Hughes A L,Yeager M.Natural selection at major histocompatibility complex loci of vertebrates[J].Annual Review of Genetics,1998,32(1):415-435.
[6]K?nig R,Huang L Y,Germain R N.MHC class II interaction with CD4 mediated by a region analogous to the MHCclass I binding site for CD8[J].Nature,1992,356(6372):796-798.
[7]Ohta Y,Okamura K,McKinney E C,et al.Primitive synteny of vertebrate major histocompatibility complex class I and class II genes[J].Proceedings of the National Academy of Sciences of the United States of America,2000,97(9):4712-4717.
[8]Bingulac-Popovic J,Figueroa F,Sato A,et al.Mapping of mhc class I and class II regions to different linkage groups in the zebrafish,Danio rerio[J].Immunogenetics,1997,46(2):129-134.
[9]Wang D Q,Zhong L,Wei Q W,et al.Evolution of MHCclass I genes in two ancient fish,paddlefish(Polyodon spathula)and Chinese sturgeon(Acipenser sinensis)[J].FEBS Letters,2010,584(15):3331-3339.
[10]Hansen J D,La Patra S.Induction of the rainbow trout MHCclass I pathway during acute IHNV infection[J].Immunogenetics,2002,54(9):654-661.
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[12]Terado T,Okamura K,Ohta Y,et al.Molecular cloning of C4 gene and identification of the class III complement region in the shark MHC[J].The Journal of Immunology,2003,171(5):2461-2466.
[13]Livingstone D R.Contaminant-stimulated reactive oxygen species production and oxidative damage in aquatic organisms[J].Marine Pollution Bulletin,2001,42(8):656-666.
[14]Brown J A,Moore W M,Quabius E S.Physiological effects of saline waters on zander[J].Journal of Fish Biology,2001,59(6):1544-1555.
[15]Nolan D T,Op’t Veld R L J M,Balm P H M,et al.Ambient salinity modulates the response of the tilapia,Oreochromis mossambicus(Peters),to net confinement[J].Aquaculture,1999,177(1-4):297-309.
[16]Yin F,Peng S M,Sun P,et al.Effects of low salinity on antioxidant enzymes activities in kidney and muscle of juvenile silver pomfret Pampus argenteus[J].Acta Ecologica Sinica,2011,31(1):55-60.
[17]Rubino J G,Zimmer A M,Wood C M.Intestinal ammonia transport in freshwater and seawater acclimated rainbow trout(Oncorhynchus mykiss):evidence for a Na+coupled uptake mechanism[J].Comparative Biochemistry and Physiology Part A:Molecular&Integrative Physiology,2015,183:45-56.
[18]Tamura K,Peterson D,Peterson N,et al.MEGA5:Molecular evolutionary genetics analysis using maximum likelihood,evolutionary distance,and maximum parsimony methods[J].Molecular Biology and Evolution,2011,28(10):2731-2739.
[19]Srisapoome P,Ohira T,Hirono I,et al.Cloning,characterization and expression of cDNA containing major histocompatibility complex class I,IIαand IIβgenes of Japanese flounder Paralichthys olivaceus[J].Fisheries Science,2004,70(2):264-276.
[20]Zhang X H,Dai Z X,Zhang G H,et al.Molecular characterization,balancing selection,and genomic organization of the tree shrew(Tupaia belangeri)MHC class I gene[J].Gene,2013,522(2):147-155.
[21]Pinto R D,Randelli E,Buonocore F,et al.Molecular cloning and characterization of sea bass(Dicentrarchus labrax,L.)MHC class I heavy chain andβ2-microglobulin[J].Developmental&Comparative Immunology,2013,39(3):234-254.
[22]Scharsack J P,Kalbe M,Schaschl H.Characterization of antisera raised against stickleback(Gasterosteus aculeatus)MHC class I and class II molecules[J].Fish&Shellfish Immunology,2007,23(5):991-1002.
[23]Shen T,Lei M L,Wang J R,et al.Molecular cloning,organization,expression and 3D structural analysis of the MHC class Ia gene in the whitespotted bamboo shark(Chiloscyllium plagiosum)[J].Veterinary Immunology and Immunopathology,2014,157(1-2):111-118.
[24]Pang J C,Gao F Y,Lu M X,et al.Major histocompatibility complex class IIA and IIB genes of Nile tilapia Oreochromis niloticus:Genomic structure,molecular polymorphism and expression patterns[J].Fish&Shellfish Immunology,2013,34(2):486-496.
[25]Dominguez M,Takemura A,Tsuchiya M,et al.Impact of different environmental factors on the circulating immunoglobulin levels in the Nile tilapia,Oreochromis niloticus[J].Aquaculture,2004,241(1-4):491-500.
[26]Cuesta A,Laiz-Carrión R,del Río M P M,et al.Salinity influences the humoral immune parameters of gilthead seabream(Sparus aurata L.)[J].Fish&Shellfish Immunology,2005,18(3):255-261.
[27]Liu G Z,Zheng W B,Chen X H.Molecular cloning of proteasome activator PA28-βsubunit of large yellow croaker(Pseudosciana crocea)and its coordinated up-regulation with MHC class Iα-chain andβ2-microglobulin in poly I:C-treated fish[J].Molecular Immunology,2007,44(6):1190-1197.
[28]Xu T J,Sun Y N,Cheng Y Z,et al.Genomic sequences comparison and differential expression of miiuy croaker MHC class I gene,after infection by Vibrio anguillarum[J].Developmental&Comparative Immunology,2011,35(4):483-489.
[2]Chen S X,Hong W S,Su Y Q,et al.Microhabitat selection in the early juvenile mudskipper Boleophthalmus pectinirostris(L.)[J].Journal of Fish Biology,2008,72(3):585-593.
[3]Bernatchez L,Landry C.MHC studies in nonmodel vertebrates:what have we learned about natural selection in 15years?[J].Journal of Evolutionary Biology,2003,16(3):363-377.
[4]Rodgers J R,Cook R G.MHC class Ib molecules bridge innate and acquired immunity[J].Nature Reviews Immunology,2005,5(6):459-471.
[5]Hughes A L,Yeager M.Natural selection at major histocompatibility complex loci of vertebrates[J].Annual Review of Genetics,1998,32(1):415-435.
[6]K?nig R,Huang L Y,Germain R N.MHC class II interaction with CD4 mediated by a region analogous to the MHCclass I binding site for CD8[J].Nature,1992,356(6372):796-798.
[7]Ohta Y,Okamura K,McKinney E C,et al.Primitive synteny of vertebrate major histocompatibility complex class I and class II genes[J].Proceedings of the National Academy of Sciences of the United States of America,2000,97(9):4712-4717.
[8]Bingulac-Popovic J,Figueroa F,Sato A,et al.Mapping of mhc class I and class II regions to different linkage groups in the zebrafish,Danio rerio[J].Immunogenetics,1997,46(2):129-134.
[9]Wang D Q,Zhong L,Wei Q W,et al.Evolution of MHCclass I genes in two ancient fish,paddlefish(Polyodon spathula)and Chinese sturgeon(Acipenser sinensis)[J].FEBS Letters,2010,584(15):3331-3339.
[10]Hansen J D,La Patra S.Induction of the rainbow trout MHCclass I pathway during acute IHNV infection[J].Immunogenetics,2002,54(9):654-661.
[11]Grimholt U,Olsaker I,de Vries Lindstr?m C,et al.A study of variability in the MHC class IIβ1 and class Iα2 domain exons of Atlantic salmon,Salmo salar L.[J].Animal Genetics,1994,25(3):147-153.
[12]Terado T,Okamura K,Ohta Y,et al.Molecular cloning of C4 gene and identification of the class III complement region in the shark MHC[J].The Journal of Immunology,2003,171(5):2461-2466.
[13]Livingstone D R.Contaminant-stimulated reactive oxygen species production and oxidative damage in aquatic organisms[J].Marine Pollution Bulletin,2001,42(8):656-666.
[14]Brown J A,Moore W M,Quabius E S.Physiological effects of saline waters on zander[J].Journal of Fish Biology,2001,59(6):1544-1555.
[15]Nolan D T,Op’t Veld R L J M,Balm P H M,et al.Ambient salinity modulates the response of the tilapia,Oreochromis mossambicus(Peters),to net confinement[J].Aquaculture,1999,177(1-4):297-309.
[16]Yin F,Peng S M,Sun P,et al.Effects of low salinity on antioxidant enzymes activities in kidney and muscle of juvenile silver pomfret Pampus argenteus[J].Acta Ecologica Sinica,2011,31(1):55-60.
[17]Rubino J G,Zimmer A M,Wood C M.Intestinal ammonia transport in freshwater and seawater acclimated rainbow trout(Oncorhynchus mykiss):evidence for a Na+coupled uptake mechanism[J].Comparative Biochemistry and Physiology Part A:Molecular&Integrative Physiology,2015,183:45-56.
[18]Tamura K,Peterson D,Peterson N,et al.MEGA5:Molecular evolutionary genetics analysis using maximum likelihood,evolutionary distance,and maximum parsimony methods[J].Molecular Biology and Evolution,2011,28(10):2731-2739.
[19]Srisapoome P,Ohira T,Hirono I,et al.Cloning,characterization and expression of cDNA containing major histocompatibility complex class I,IIαand IIβgenes of Japanese flounder Paralichthys olivaceus[J].Fisheries Science,2004,70(2):264-276.
[20]Zhang X H,Dai Z X,Zhang G H,et al.Molecular characterization,balancing selection,and genomic organization of the tree shrew(Tupaia belangeri)MHC class I gene[J].Gene,2013,522(2):147-155.
[21]Pinto R D,Randelli E,Buonocore F,et al.Molecular cloning and characterization of sea bass(Dicentrarchus labrax,L.)MHC class I heavy chain andβ2-microglobulin[J].Developmental&Comparative Immunology,2013,39(3):234-254.
[22]Scharsack J P,Kalbe M,Schaschl H.Characterization of antisera raised against stickleback(Gasterosteus aculeatus)MHC class I and class II molecules[J].Fish&Shellfish Immunology,2007,23(5):991-1002.
[23]Shen T,Lei M L,Wang J R,et al.Molecular cloning,organization,expression and 3D structural analysis of the MHC class Ia gene in the whitespotted bamboo shark(Chiloscyllium plagiosum)[J].Veterinary Immunology and Immunopathology,2014,157(1-2):111-118.
[24]Pang J C,Gao F Y,Lu M X,et al.Major histocompatibility complex class IIA and IIB genes of Nile tilapia Oreochromis niloticus:Genomic structure,molecular polymorphism and expression patterns[J].Fish&Shellfish Immunology,2013,34(2):486-496.
[25]Dominguez M,Takemura A,Tsuchiya M,et al.Impact of different environmental factors on the circulating immunoglobulin levels in the Nile tilapia,Oreochromis niloticus[J].Aquaculture,2004,241(1-4):491-500.
[26]Cuesta A,Laiz-Carrión R,del Río M P M,et al.Salinity influences the humoral immune parameters of gilthead seabream(Sparus aurata L.)[J].Fish&Shellfish Immunology,2005,18(3):255-261.
[27]Liu G Z,Zheng W B,Chen X H.Molecular cloning of proteasome activator PA28-βsubunit of large yellow croaker(Pseudosciana crocea)and its coordinated up-regulation with MHC class Iα-chain andβ2-microglobulin in poly I:C-treated fish[J].Molecular Immunology,2007,44(6):1190-1197.
[28]Xu T J,Sun Y N,Cheng Y Z,et al.Genomic sequences comparison and differential expression of miiuy croaker MHC class I gene,after infection by Vibrio anguillarum[J].Developmental&Comparative Immunology,2011,35(4):483-489.