团头鲂NOS2基因的克隆和组织表达分析
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摘要
为了探究诱导型一氧化氮合酶(NOS2)在嗜水气单胞菌(Aeromonas hydrophila)感染团头鲂(Megalobrama amblycephala)时发挥的作用,克隆并分析了团头鲂NOS2基因。结果显示,NOS2基因的cDNA序列全长4 215 bp,编码1 080个氨基酸。氨基酸序列同源性分析表明,团头鲂NOS2基因与草鱼NOS2基因的相似性最高,达95%。进化关系分析表明,团头鲂NOS2基因与鲤科鱼类NOS2基因聚在一支。实时荧光定量PCR检测的结果表明,在嗜水气单胞菌感染团头鲂后,NOS2基因在肝脏和头肾中被诱导表达;在脾脏中NOS2基因在3 h被诱导表达,其余时间点表达量显著下调;在肠道组织中NOS2基因在3 h下调表达,在6 h有微弱的上调,之后趋于稳定。研究结果为进一步探索NOS2基因在嗜水气单胞菌感染团头鲂后免疫应答中发挥的作用打下基础。
Inducible nitric oxide synthase(NOS2)gene of Megalobrama amblycephala was cloned and characterized in order to study the function of inducible NOS2 gene from M.amblycephala after challenge of Aeromonas hydrophila.The full-length cDNA of NOS2 gene was 4 215 bp,encoding 1080 amino acids.Multiple sequence alignment revealed that NOS2 gene of M.amblycephala shared 95% similarity with that of Ctenopharyngodon idellus.Phylogenetic analysis showed that NOS2 amino acid sequence of M.amblycephala was relatively conserved in fish,and more closely related to Cypriniformes.The mRNA expression levels of the NOS2 gene were induced in the liver and head kidney after challenge of A.hydrophila.The expression in the spleen was up-regulated at 3 h post injection(hpi).Subsequently,it was significantly down-regulated.The expression in the intestine was up-regulated at 6 hpi of A.hydrophila after a down-regulation at 3 hpi,and then tended to stabilization.Our findings will lay a foundation for the study of the potential role of the NOS2 gene in the host defense against A.hydrophila.
Inducible nitric oxide synthase(NOS2)gene of Megalobrama amblycephala was cloned and characterized in order to study the function of inducible NOS2 gene from M.amblycephala after challenge of Aeromonas hydrophila.The full-length cDNA of NOS2 gene was 4 215 bp,encoding 1080 amino acids.Multiple sequence alignment revealed that NOS2 gene of M.amblycephala shared 95% similarity with that of Ctenopharyngodon idellus.Phylogenetic analysis showed that NOS2 amino acid sequence of M.amblycephala was relatively conserved in fish,and more closely related to Cypriniformes.The mRNA expression levels of the NOS2 gene were induced in the liver and head kidney after challenge of A.hydrophila.The expression in the spleen was up-regulated at 3 h post injection(hpi).Subsequently,it was significantly down-regulated.The expression in the intestine was up-regulated at 6 hpi of A.hydrophila after a down-regulation at 3 hpi,and then tended to stabilization.Our findings will lay a foundation for the study of the potential role of the NOS2 gene in the host defense against A.hydrophila.
引文
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[23]李洁,王虹,周洋,等.团头鲂Toll样受体Ⅱ基因的克隆与表达分析[J].淡水渔业,2017,47(1):22-29.
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[26]Iwakiri Y.Nitric oxide in liver fibrosis:The role of inducible nitric oxide synthase[J].Clin Mol Hepatol,2015,21(4):319-325.
[2]Knowles R G,Moncada S.Nitric oxide synthases in mamals[J].Biochem J,1994,298:249-258.
[3]Kolios G,Valatas V,Ward S G.Nitric oxide in inflammatory bowel disease:a universal messenger in an unsolved puzzle[J].Immunology,2004,113:427-437.
[4]Kr?ncke K D,Fehsel K,Kolb-Bachofen V.Inducible nitric oxide synthase in human diseases[J].Clin Exp Immunol,1998,113:147-156.
[5]Bogdan C,Rollinghoff M,Diefenbach A.Reactive oxygen and reactive nitrogen intermediates in innate and specific immunity[J].Curr Opin Immunol,2000,12:64-76.
[6]郭闯.嗜水气单胞菌研究进展[J].水产科学,2003,22(6):48-51.
[7]田甜,胡火庚,陈昌福.团头鲂细菌性败血症病原菌分离鉴定及致病力研究[J].华中农业大学学报,2010,29(3):341-345.
[8]李明之,曲小良,王桂香.细菌性败血症鱼病治疗体会[J].淡水渔业,2001,31(2):41.
[9]Liu L,Zhou Y,Zhao X,et al.Oligochitosan stimulated phagocytic activity of macrophages from blunt snout bream(Megalobrama amblycephala)associated with respiratory burst coupled with nitric oxide production[J].Dev Comp Immunol,2014,47(1):17-24.
[10]Liu X,Tang X,Wang L,et al.Molecular cloning and expression analysis of mannose receptor in blunt snout bream(Megalobrama amblycephala)[J].Mole Biol Rep,2014,41(7):4601-4611.
[11]Livak K J,Schmittgen T D.Analysis of relative gene expression data using real-time quantitative PCR and the 2(-DeltaDeltaC(T))Method[J].Methods,2001,25:402-408.
[12]汪新艳,赵太强,郭嘉聪,等.草鱼诱导型一氧化氮合酶cDNA和启动子的克隆及分析[J].四川动物,2011,30:510-516.
[13]Reddick J I,Goostrey A,Secombes C J.Cloning of iNOS in the small spotted catshark (Scyliorhinus canicula)[J].Dev Comp Immunol,2006,30:1009-1022.
[14]Kein?nen R,Vartiainen N,Koistinaho J.Molecular cloning and characterization of the rat inducible nitric oxide synthase (iNOS)gen[J].Gene,1999,234:297-305.
[15]Yao C L,Ji P F,Wang Z Y,et al.Molecular cloning and expression of NOS in shrimp,Litopenaeus vannamei[J].Fish Shellfish Immunol,2010,28(3):453-460.
[16]Wang H,Chen X P,Qiu F Z,et al.Increased hepatic expression of nitric oxide synthase type II in cirrhotic rats[J].World J Gastroen-terol,2004,10(13):1923-1927.
[17]温振才.鲤鱼iNOS全长cDNA的获得及在感染条件下的差异表达分析[D].长春:吉林大学,2016.
[18]张永安,孙宝剑,聂品.鱼类免疫组织和细胞的研究概况[J].水生生物学报,2000,24(6):648-654.
[19]张胜根.点带石斑鱼组织中一氧化氮合成酶活性及免疫反应性研究[D].天津:天津农学院,2011.
[20]郭建,周开亚,陈泳宏,等.应激大鼠脾脏内诱导型一氧化氮合酶活性变化及其意义[J].应用及环境生物学报,2005,11(4):474-476.
[21]李军华,何小飞,于皆平,等.NF-kB、iNOS在大鼠实验性结肠炎肠组织的表达及意义[J].胃肠病学和肝病学杂志,2003,12(5):436-439.
[22]廖美娟,梁桦,梁幸甜,等.姜黄素抑制诱导型一氧化氮合酶活性减轻大鼠肠缺血再灌注损伤[J].实用医学杂志,2015,31(20):3302-3303.
[23]李洁,王虹,周洋,等.团头鲂Toll样受体Ⅱ基因的克隆与表达分析[J].淡水渔业,2017,47(1):22-29.
[24]Kleinert H,Schwarz P M,Forstermann U.Regulation of the expression of inducible nitric oxide synthase[J].J Biol Chem,2003,384:1343-1364.
[25]Xie Q W,Kashiwabara Y,Nathan C.Role of transcription factor NF-kappa B/Rel in induction of nitric oxide synthasee[J].J Biol Chem,1994,269:4705-4708.
[26]Iwakiri Y.Nitric oxide in liver fibrosis:The role of inducible nitric oxide synthase[J].Clin Mol Hepatol,2015,21(4):319-325.
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