香鱼HSP90β的克隆、序列特征及表达分析
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摘要
热休克蛋白(Heat Shock Proteins,HSPs)是一种分子伴侣,具有广泛的生物学活性。从香鱼(Plecoglossus altivelis)肝c DNA文库获得HSP90β亚型(Pa HSP90β)c DNA全序列。Pa HSP90β全长2726 bp,包含1个由2175 bp组成的开放阅读框,编码724个氨基酸,预测相对分子质量为83.30 ku,等电点为4.83。氨基酸序列分析显示,Pa HSP90β含有HSP90的5个保守信号区,氨基酸序列第34~188处具有HATPase(histidine kinase-like ATPases)结构域;与白鲑(Coregonus lavaretus)HSP90β亲缘关系最近,氨基酸序列同源性高达95.8%。实时荧光定量PCR(Quantitative real-time PCR,q PCR)结果表明,Pa HSP90βmRNA主要在香鱼中表达量最高,脾脏次之。镉胁迫后,香鱼肝、脾、肾、鳃和肠组织中Pa HSP90βmRNA表达显著上调;盐度胁迫后,除肠组织外,其他组织中Pa HSP90βmRNA表达总体显著上调。综上,Pa HSP90β与重金属及盐度胁迫密切相关,可能参与鱼类抵抗环境胁迫的生理过程。
Heat shock proteins( HSPs) are one type of molecular chaperones,and have a wide range of biological activities. The fulllength c DNA sequence of ayu HSP90β gene( Pa HSP90β) was obtained from the liver tissue c DNA library of ayu( Plecoglossus altivelis). The c DNA sequence of Pa HSP90β was 2726 nucleotides,containing a 2175 bp open reading frame that encoded a protein of 724 amino acids with a deduced molecular weight( MW) of 83. 30 ku and a isoelectric point( p I) of 4. 83. Multiple sequence alignment of complete amino acid sequences showed that Pa HSP90β contained five conserved HSP90 family signatures,and a typical HATPase( histidine kinase-like ATPases) domain,which was located at positions 34 to 188. Pa HSP90β shared the highest amino acid sequence identity( 95. 8%) with that of European whitefish( Coregonus lavaretus). Quantitative real-time PCR( q PCR) analysis showed that Pa HSP90β mRNA had the highest expressional level in ayu liver,followed by the spleen. Upon Cd exposure,Pa HSP90β transcripts were significantly up-regulated in liver,spleen,kidney,intestine and gill of ayu. After the salinity stress,Pa HSP90β mRNA expression was significantly up-regulated in all the tissues except the intestine. In summary,Pa HSP90β had a close relationship with heavy metal and salinity stress,and may be involved in physiological processes against environmental stresses in fish.
Heat shock proteins( HSPs) are one type of molecular chaperones,and have a wide range of biological activities. The fulllength c DNA sequence of ayu HSP90β gene( Pa HSP90β) was obtained from the liver tissue c DNA library of ayu( Plecoglossus altivelis). The c DNA sequence of Pa HSP90β was 2726 nucleotides,containing a 2175 bp open reading frame that encoded a protein of 724 amino acids with a deduced molecular weight( MW) of 83. 30 ku and a isoelectric point( p I) of 4. 83. Multiple sequence alignment of complete amino acid sequences showed that Pa HSP90β contained five conserved HSP90 family signatures,and a typical HATPase( histidine kinase-like ATPases) domain,which was located at positions 34 to 188. Pa HSP90β shared the highest amino acid sequence identity( 95. 8%) with that of European whitefish( Coregonus lavaretus). Quantitative real-time PCR( q PCR) analysis showed that Pa HSP90β mRNA had the highest expressional level in ayu liver,followed by the spleen. Upon Cd exposure,Pa HSP90β transcripts were significantly up-regulated in liver,spleen,kidney,intestine and gill of ayu. After the salinity stress,Pa HSP90β mRNA expression was significantly up-regulated in all the tissues except the intestine. In summary,Pa HSP90β had a close relationship with heavy metal and salinity stress,and may be involved in physiological processes against environmental stresses in fish.
引文
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[19]CHEN Y M,KUO C E,WANT T Y,et al.Cloning of an orangespotted grouper Epinephelus coioides heat shock protein 90AB(HSP90AB)and characterization of its expression in response to nodavirus[J].Fish&Shellfish Immunology,2010,28(5-6):895-904.
[20]MANCHADO M,SALAS-LEITON E,INFANTE C,et al.Molecular characterization,gene expression and transcriptional regulation of cytosolic HSP90 genes in the flatfish Senegalese sole(Solea senegalensis Kaup)[J].Gene,2008,416(1-2):77-84.
[21]ZHANG C N,LI X F,TIAN H Y,et al.Effects of fructooligosaccharide on immune response,antioxidant capability and HSP70 and HSP90 expressions of blunt snout bream(Megalobrama amblycephala)under high ammonia stress[J].Fish Physiology and Biochemistry,2015,41(1):203-217.
[22]李明云,丁天喜,竺俊全,等.我国香鱼的研究现状及增养殖前景[J].宁波大学学报(理工版),1999,12(4):85-90.
[23]TAMURA K,STECHER G,PETERSON D,et al.MEGA6:molecular evolutionary genetics analysis version 6.0[J].Molecular Biology and Evolution,2013,30(12):2725-2729.
[24]LU X J,CHEN J,HUANG Z A,et al.Influence of acute cadmium exposure on the liver proteome of a teleost fish,ayu(Plecoglossus altivelis)[J].Molecular Biology Reports,2012,39(3):2851-2859.
[25]CHEN J,SHI Y H,HU H Q,et al.Apolipoprotein A-I,a hyperosmotic adaptation-related protein in ayu(Plecoglossus altivelis)[J].Comparative Biochemistry and Physiology Part B:Biochemistry and Molecular Biology,2008,152(2):196-201.
[26]黄左安,陈炯,陆新江,等.香鱼凝血因子X基因表达与鳗利斯顿氏菌感染的相关性[J].动物学研究,2011,32(5):492-498.
[27]LI C H,LU X J,LI D F,et al.Passive protective effect of chicken egg yolk immunoglobulins against experimental Vibrio anguillarum infection in ayu(Plecoglossus altivelis)[J].Fish&Shellfish Immunology,2014,37(1):108-114.
[28]LIVAK K J,SCHMITTGEN T D.Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCTmethod[J].Methods,2001,25(4):402-408.
[29]林亚秋,郑玉才,吉红,等.草鱼HSP90基因c DNA序列克隆及其组织表达差异[J].水产科学,2009,28(8):439-442.
[30]ALI A,KRONE P H,PEARSON D S,et al.Evaluation of stressinducible hsp90 gene expression as a potential molecular biomarker in Xenopus laevis[J].Cell Stress&Chaperones,1996,1(1):62-69.
[2]HEIKKILA J J.Heat shock protein gene expression and function in amphibian model systems[J].Comparative Biochemistry and Physiology Part A:Molecular&Integrative Physiology,2010,156(1):19-33.
[3]ROBERTS R J,AGIUS C,SALIBA C,et al.Heat shock proteins(chaperones)in fish and shellfish and their potential role in relation to fish health:a review[J].Journal of Fish Diseases,2010,33(10):789-801.
[4]TAIPALE M,JAROSZ D F,LINDQUIST S.HSP90 at the hub of protein homeostasis:emerging mechanistic insights[J].Nature Reviews Molecular Cell Biology,2010,11(7):515-528.
[5]CHEN B,PIEL W H,GUI L,et al.The HSP90 family of genes in the human genome:insights into their divergence and evolution[J].Genomics,2005,86(6):627-637.
[6]CHEN B,ZHONG D,MONTEIRO A.Comparative genomics and evolution of the HSP90 family of genes across all kingdoms of organisms[J].BMC Genomics,2006,7:156.
[7]PAN F,ZARATE J M,TREMBLAY G C,et al.Cloning and characterization of salmon hsp90 c DNA:upregulation by thermal and hyperosmotic stress[J].Journal of Experimental Zoology,2000,287(3):199-212.
[8]PALMISANO A N,WINTON J R,DICKHOFF W W.Tissue-specific induction of Hsp90 mRNA and plasma cortisol response in chinook salmon following heat shock,seawater challenge,and handling challenge[J].Marine Biotechnology,2000,2(4):329-338.
[9]WU C X,ZHAO F Y,ZHANG Y,et al.Overexpression of Hsp90from grass carp(Ctenopharyngodon idella)increases thermal protection against heat stress[J].Fish&Shellfish Immunology,2012,33(1):42-47.
[10]WEI T,GAO Y,WANG R,et al.A heat shock protein 90βisoform involved in immune response to bacteria challenge and heat shock from Miichthys miiuy[J].Fish&Shellfish Immunology,2013,35(2):429-437.
[11]YANG W K,HSEU J R,TANG C H,et al.Na+/K+-ATPase expression in gills of the euryhaline sailfin molly,Poecilia latipinna,is altered in response to salinity challenge[J].Journal of Experimental Marine Biology and Ecology,2009,375(1-2):41-50.
[12]CARA J B,ALURU N,MOYANO F J,et al.Food-deprivation induces HSP70 and HSP90 protein expression in larval gilthead sea bream and rainbow trout[J].Comparative Biochemistry and Physiology Part B:Biochemistry and Molecular Biology,2005,142(4):426-431.
[13]NI M,WEN H,LI J,et al.Two HSPs gene from juvenile Amur sturgeon(Acipenser schrenckii):cloning,characterization and expression pattern to crowding and hypoxia stress[J].Fish Physiology and Biochemistry,2014,40(6):1801-1816.
[14]GUO R,YOUN S H,KI J S.Heat shock protein 70 and 90 genes in the harmful dinoflagellate Cochlodinium polykrikoides:genomic structures and transcriptional responses to environmental stresses[J].International Journal of Genomics,2015,2015:484626.
[15]SAID ALI K,FERENCZ,NEMCS K J,et al.Expressions of heat shock and metallothionein genes in the heart of common carp(Cyprinus carpio):effects of temperature shock and heavy metal exposure[J].Acta Biologica Hungarica,2010,61(1):10-23.
[16]HERMESZ E,BRAH M M,NEMCS K J.Identification of two hsp90 genes in carp[J].Comparative Biochemistry and Physiology Part C:Toxicology&Pharmacology,2001,129(4):397-407.
[17]LIU H,CHEN H,JING J,et al.Cloning and characterization of the HSP90 beta gene from Tanichthys albonubes Lin(Cyprinidae):effect of copper and cadmium exposure[J].Fish Physiology and Biochemistry,2012,38(3):745-756.
[18]XIE Y,SONG L,WENG Z,et al.Hsp90,Hsp60 and s Hsp families of heat shock protein genes in channel catfish and their expression after bacterial infections[J].Fish&Shellfish Immunology,2015,44(2):642-651.
[19]CHEN Y M,KUO C E,WANT T Y,et al.Cloning of an orangespotted grouper Epinephelus coioides heat shock protein 90AB(HSP90AB)and characterization of its expression in response to nodavirus[J].Fish&Shellfish Immunology,2010,28(5-6):895-904.
[20]MANCHADO M,SALAS-LEITON E,INFANTE C,et al.Molecular characterization,gene expression and transcriptional regulation of cytosolic HSP90 genes in the flatfish Senegalese sole(Solea senegalensis Kaup)[J].Gene,2008,416(1-2):77-84.
[21]ZHANG C N,LI X F,TIAN H Y,et al.Effects of fructooligosaccharide on immune response,antioxidant capability and HSP70 and HSP90 expressions of blunt snout bream(Megalobrama amblycephala)under high ammonia stress[J].Fish Physiology and Biochemistry,2015,41(1):203-217.
[22]李明云,丁天喜,竺俊全,等.我国香鱼的研究现状及增养殖前景[J].宁波大学学报(理工版),1999,12(4):85-90.
[23]TAMURA K,STECHER G,PETERSON D,et al.MEGA6:molecular evolutionary genetics analysis version 6.0[J].Molecular Biology and Evolution,2013,30(12):2725-2729.
[24]LU X J,CHEN J,HUANG Z A,et al.Influence of acute cadmium exposure on the liver proteome of a teleost fish,ayu(Plecoglossus altivelis)[J].Molecular Biology Reports,2012,39(3):2851-2859.
[25]CHEN J,SHI Y H,HU H Q,et al.Apolipoprotein A-I,a hyperosmotic adaptation-related protein in ayu(Plecoglossus altivelis)[J].Comparative Biochemistry and Physiology Part B:Biochemistry and Molecular Biology,2008,152(2):196-201.
[26]黄左安,陈炯,陆新江,等.香鱼凝血因子X基因表达与鳗利斯顿氏菌感染的相关性[J].动物学研究,2011,32(5):492-498.
[27]LI C H,LU X J,LI D F,et al.Passive protective effect of chicken egg yolk immunoglobulins against experimental Vibrio anguillarum infection in ayu(Plecoglossus altivelis)[J].Fish&Shellfish Immunology,2014,37(1):108-114.
[28]LIVAK K J,SCHMITTGEN T D.Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCTmethod[J].Methods,2001,25(4):402-408.
[29]林亚秋,郑玉才,吉红,等.草鱼HSP90基因c DNA序列克隆及其组织表达差异[J].水产科学,2009,28(8):439-442.
[30]ALI A,KRONE P H,PEARSON D S,et al.Evaluation of stressinducible hsp90 gene expression as a potential molecular biomarker in Xenopus laevis[J].Cell Stress&Chaperones,1996,1(1):62-69.