半滑舌鳎(Cynoglossus semilaevis)双链RNA依赖的蛋白激酶R(PKR)基因克隆和实时定量表达分析
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摘要
采用RACE方法克隆了半滑舌鳎(Cynoglossus semilaevis)PKR基因(CsPKR),获得了CsPKR全长cDNA序列为2907bp,其中包含1959bp的开放阅读框,100bp的5′非编码区和848bp的3′非编码区。保守结构域分析显示推导的CsPKR氨基酸在N端存在两个特异的dsRBD(dsRNA binding domain dsRBD)结构域,在C端具有多个保守的激酶活性位点,包括ATP结合位点和底物配体结合位点,以及活性环结构A-loop。系统进化树分析显示鱼类、两栖类、鸟类及哺乳类的PKR具有共同的进化起源,CsPKR与牙鲆PKR的亲缘关系最为密切。荧光定量PCR结果显示,CsPKR基因在健康鱼多个组织中广泛表达,在脑中的表达最高,头肾中表达最低。经鳗弧菌和淋巴囊肿病毒分别感染后,CsPKR基因在免疫相关组织中呈现上调表达趋势,其中感染鳗弧菌12h后在血液中表达量较对照组上升了9.28倍,在感染淋巴囊肿病毒24h后,在肝脏中的表达达到最大,为对照组的9.97倍。以上结果暗示CsPKR基因在半滑舌鳎响应细菌和病毒免疫应答中起重要作用。
In this report,the full length cDNA of half smooth tongue sole(Cynoglossus semilaevis)PKR(CsPKR)was cloned by RACE method.It was of 2907bp,including 100bp 5′ UTR,848bp 3′ UTR and an open reading frame(ORF)of1959bp encoding 652 animo acids.The putative CsPKR protein contains two N-terminal conserved dsRNA binding domain(dsRBD)and some active sites ATP binding sites and A-loop structures on the C-terminal.Phylogenetic analysis showed that fish PKR shared a common origin with birds and mammals PKRs.Tissue expression profile analysis by quantitative real-time reverse transcription PCR showed that CsPKR mRNA was constitutively expressed in all examined healthy tissues with the predominant in blood and the lowest in head kidney.After infection with Vibrio anguillarum,CsPKR gene expression had a varying degree of increase in blood,intestine,spleen,head kidney,and liver from 12h to 7d.The expression of CsPKR was up-regulated to 9.28-fold higher than PBS control at 12h in blood.After infection with Lymphocystis disease virus(LCDV),the expression of CsPKR increased gradually and reached a peak at 24h up to 9.97-fold than PBS control in the live.All of the results suggested CsPKR plays a potential role in response to bacterial and virus infections in half smooth tongue sole.
In this report,the full length cDNA of half smooth tongue sole(Cynoglossus semilaevis)PKR(CsPKR)was cloned by RACE method.It was of 2907bp,including 100bp 5′ UTR,848bp 3′ UTR and an open reading frame(ORF)of1959bp encoding 652 animo acids.The putative CsPKR protein contains two N-terminal conserved dsRNA binding domain(dsRBD)and some active sites ATP binding sites and A-loop structures on the C-terminal.Phylogenetic analysis showed that fish PKR shared a common origin with birds and mammals PKRs.Tissue expression profile analysis by quantitative real-time reverse transcription PCR showed that CsPKR mRNA was constitutively expressed in all examined healthy tissues with the predominant in blood and the lowest in head kidney.After infection with Vibrio anguillarum,CsPKR gene expression had a varying degree of increase in blood,intestine,spleen,head kidney,and liver from 12h to 7d.The expression of CsPKR was up-regulated to 9.28-fold higher than PBS control at 12h in blood.After infection with Lymphocystis disease virus(LCDV),the expression of CsPKR increased gradually and reached a peak at 24h up to 9.97-fold than PBS control in the live.All of the results suggested CsPKR plays a potential role in response to bacterial and virus infections in half smooth tongue sole.
引文
龙华,郑英,2006.鱼类抗病基因的研究进展.中国医学研究与临床,4(1):32—47
李景芬,刘莉,曹访,2011.PolyI:C体外诱导草鱼PKR基因的克隆与表达.安徽农业科学,39(33):20514—20516
沙珍霞,王娜,王启龙等,2010.半滑舌鳎髓样分化因子的克隆和表达分析.中国水产科学,17(4):659—669
Barber G N,2005.The dsRNA-dependent protein kinase,PKR and cell death.Cell Death Differ,12:563—570
Chen L,Zhang M,Sun L,2011.Identification and expressional analysis of two cathepsins from half-smooth tongue sole(Cynoglossus semilaevis).Fish Shellfish Immunol,31(6):1270—1277
del Castillo C S,Hikima J,Ohtani M et al,2012.Characterization and functional analysis of two PKR genes in fugu(Takifugu rubripes).Fish Shellfish Immunol,32(1):79—88
Meurs E,Chong K,Galabru J et al,1990.Molecular and characterization of the human double-stranded RNA-activated protein kinase induced by interferon.Cell,62:379—390
Rothenburg S,Deigendesch N,Dey M et al,2008.Double-stranded RNA-activated protein kinase PKR of fishes and amphibians:varying the number of double-stranded RNA binding domains and lineage-specific duplications.BMC Biol,6(12):1—19
Rothenburg S,Deigendesch N,Dittmar K et al,2005.PKR-like eukaryotic initiation factor-alpha kinase from zebrafish contains Z-DNA binding domains instead of dsRNA binding domains.Proc Natl Acad Sci USA,102:1602—1607
Sha Z X,Wang Q L,Liu Y et al,2012.Identification and expression analysis of goose-type lysozyme in half-smooth tongue sole(Cynoglossus semilaevis).Fish Shellfish Immunol,32(5):914—921
Su J,Zhu Z,Wang Y et al,2008.Molecular cloning,characterization and expression analysis of the PKZ gene in rare minnow Gobiocypris rarus.Fish Shellfish Immunol,25(1—2):106 —113
Tamura K,Peterson D,Peterson N et al,2011.MEGA5:molecular 421 evolutionary genetics analysis using maximum likelihood,evolutionary distance,and maximum parsimony methods.Mol Biol Evol,28:2731—2739
Toch A M,Zhang P,Das S et al,2006.Interferon action and the double-stranded RNA-dependent enzymes ADARI adenosine deaminase and PKR protein kinase.Prog Nucleic Acid Res Mol Biol,81:369—434
Wang N,Yang C G,Sun Z Z et al,2011.Signal transducer and activator of transcriotions(STAT3)homologue in turbot(Scophthalmus maximus):Molecular characterization and expression analysis.Fish and Shellfish Immunology,30:255—262
Xu T J,Chen S L,Ji X S et al,2009.Molecular cloning,genomic structure,polymorphism and expression analysis of major histocompatibility complex class IIA and IIB genes of half-smooth tongue sole(Cynoglossus semilaevis).Fish Shellfish Immunol,27(2):192—201
Zenke K,Nam Y K,Kim K H,2009.Molecular cloning and expression analysis of double-stranded RNA-dependent protein kinase(PKR)in rock bream(Oplegnathus fasciatus).Vet Immunol Immunopathol,133(2—4):290—295
Zhang P,Samuel C E,2007.Protein kinase PKR plays a stimulus-and virus-dependent role in apoptotic death and virus multiplication in human cells.Virol,81:8192—8200
Zhu R,Zhang Y B,Zhang Q Y et al,2008.Functional domains and the antiviral effect of the double-stranded RNAdependent protein kinase PKR from Paralichthys olivaceus.Virol,82:6889—6901
Zhu R,Zhang Y B,Zhang Q Y et al,2009.Subcellular localization and inductive expression of the dsRNA-dependent protein kinase PKR from Japanese flounder,Paralichthys olivaceus.Progress in Nature Science,19:1227—1234
李景芬,刘莉,曹访,2011.PolyI:C体外诱导草鱼PKR基因的克隆与表达.安徽农业科学,39(33):20514—20516
沙珍霞,王娜,王启龙等,2010.半滑舌鳎髓样分化因子的克隆和表达分析.中国水产科学,17(4):659—669
Barber G N,2005.The dsRNA-dependent protein kinase,PKR and cell death.Cell Death Differ,12:563—570
Chen L,Zhang M,Sun L,2011.Identification and expressional analysis of two cathepsins from half-smooth tongue sole(Cynoglossus semilaevis).Fish Shellfish Immunol,31(6):1270—1277
del Castillo C S,Hikima J,Ohtani M et al,2012.Characterization and functional analysis of two PKR genes in fugu(Takifugu rubripes).Fish Shellfish Immunol,32(1):79—88
Meurs E,Chong K,Galabru J et al,1990.Molecular and characterization of the human double-stranded RNA-activated protein kinase induced by interferon.Cell,62:379—390
Rothenburg S,Deigendesch N,Dey M et al,2008.Double-stranded RNA-activated protein kinase PKR of fishes and amphibians:varying the number of double-stranded RNA binding domains and lineage-specific duplications.BMC Biol,6(12):1—19
Rothenburg S,Deigendesch N,Dittmar K et al,2005.PKR-like eukaryotic initiation factor-alpha kinase from zebrafish contains Z-DNA binding domains instead of dsRNA binding domains.Proc Natl Acad Sci USA,102:1602—1607
Sha Z X,Wang Q L,Liu Y et al,2012.Identification and expression analysis of goose-type lysozyme in half-smooth tongue sole(Cynoglossus semilaevis).Fish Shellfish Immunol,32(5):914—921
Su J,Zhu Z,Wang Y et al,2008.Molecular cloning,characterization and expression analysis of the PKZ gene in rare minnow Gobiocypris rarus.Fish Shellfish Immunol,25(1—2):106 —113
Tamura K,Peterson D,Peterson N et al,2011.MEGA5:molecular 421 evolutionary genetics analysis using maximum likelihood,evolutionary distance,and maximum parsimony methods.Mol Biol Evol,28:2731—2739
Toch A M,Zhang P,Das S et al,2006.Interferon action and the double-stranded RNA-dependent enzymes ADARI adenosine deaminase and PKR protein kinase.Prog Nucleic Acid Res Mol Biol,81:369—434
Wang N,Yang C G,Sun Z Z et al,2011.Signal transducer and activator of transcriotions(STAT3)homologue in turbot(Scophthalmus maximus):Molecular characterization and expression analysis.Fish and Shellfish Immunology,30:255—262
Xu T J,Chen S L,Ji X S et al,2009.Molecular cloning,genomic structure,polymorphism and expression analysis of major histocompatibility complex class IIA and IIB genes of half-smooth tongue sole(Cynoglossus semilaevis).Fish Shellfish Immunol,27(2):192—201
Zenke K,Nam Y K,Kim K H,2009.Molecular cloning and expression analysis of double-stranded RNA-dependent protein kinase(PKR)in rock bream(Oplegnathus fasciatus).Vet Immunol Immunopathol,133(2—4):290—295
Zhang P,Samuel C E,2007.Protein kinase PKR plays a stimulus-and virus-dependent role in apoptotic death and virus multiplication in human cells.Virol,81:8192—8200
Zhu R,Zhang Y B,Zhang Q Y et al,2008.Functional domains and the antiviral effect of the double-stranded RNAdependent protein kinase PKR from Paralichthys olivaceus.Virol,82:6889—6901
Zhu R,Zhang Y B,Zhang Q Y et al,2009.Subcellular localization and inductive expression of the dsRNA-dependent protein kinase PKR from Japanese flounder,Paralichthys olivaceus.Progress in Nature Science,19:1227—1234