东北七鳃鳗TRAF6基因克隆、表达分析及亚细胞定位研究
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摘要
为研究TRAF6在TLR信号介导的先天免疫中的调控作用,研究通过克隆技术获得了东北七鳃鳗(Lethenteron morii)TRAF6基因的cDNA全长,命名为LmTRAF6。利用实时荧光定量方法(qPCR)分析了LmTRAF6在幼鱼和成鱼中各组织的表达情况以及在铜绿假单胞菌(Pseudomonas aeruginosa)感染后脂肪体、鳃、肠和肾组织在不同时间点的表达量变化。利用双酶切技术构建pEGFP-TRAF6重组质粒并转染HEK293T细胞, 48h后进行荧光观察并拍照。结果表明, LmTRAF6的cDNA全长为2751 bp,开放阅读框(ORF)为1785 bp,编码594个氨基酸。其蛋白结构域高度保守,具有RING结构、两个锌指结构、环-环(Coiledcoil)α螺旋结构域和MATH结构域。通过系统进化树分析,发现LmTRAF6与哺乳类以及鱼类TRAF6的亲缘关系较近,与果蝇、中国对虾TRAF6的亲缘关系较远。qPCR结果显示, LmTRAF6在各组织中均有表达,在幼鱼的心脏、皮肤、鳃、肝中的表达量相对较高,而在肠中表达量相对较低。在成鱼的肾、鳃、肌肉中的表达量相对较高,而在心脏中表达量相对较低。成鱼LmTRAF6在铜绿假单胞菌感染后,鳃、肠和肾的表达量在24h达到峰值。细胞定位显示, LmTRAF6在HEK293T的细胞质和细胞核中均有表达。以上结果为进一步探究七鳃鳗中TRAF6在TLR信号通路中的作用奠定了理论基础。
In order to investigate the role of TRAF6 in Lethenteron morii with signal pathway of TLR, the full-length cDNA of LmTRAF6 was cloned. The distribution of tissues(supraneural body, gill, intestine, kidney) and related gene expression in both juvenile and adult were studied with real-time quantitative PCR. Additionally, the change of gene expression in those tissues from adult L. morii was also examined after challenged by Pseudomonas aeruginosa. A recombinant plasmid pEGFP-TRAF6 was constructed by the double digestion method, and transformed into the HEK293 T cells. Fluorescence observation was conducted after 48 hours, and the results were photographed. The results showed that the length of LmTRAF6 cDNA was 2751 bp, including 1785 bp open reading frame(ORF) and 594 amino acids. The structure of LmTRAF6 was highly similar to that in other mammal and fish species, containing one RING domain, one coiled-coil region, one MATH domain and two zinc fingers. Phylogenetic tree showed that LmTRAF6 was evolutionarily closer to that in mammals and fishes, but not to that in Drosophila melanogaster and Penaeus chinensis. LmTRAF6 was expressed in all tested tissues and their developmental stages. However, higher expression was detected in heart, skin, gill, liver of juvenile, as well as in kidney, gill, and muscle of adult. When adult L.morii was challenged by Pseudomonas aeruginosa, the expression of LmTRAF6 in gill, intestine and kidney reached to the peak after 24 h. Subcellular localization showed that LmTRAF6 was expressed in both cytoplasm and nucleus.
In order to investigate the role of TRAF6 in Lethenteron morii with signal pathway of TLR, the full-length cDNA of LmTRAF6 was cloned. The distribution of tissues(supraneural body, gill, intestine, kidney) and related gene expression in both juvenile and adult were studied with real-time quantitative PCR. Additionally, the change of gene expression in those tissues from adult L. morii was also examined after challenged by Pseudomonas aeruginosa. A recombinant plasmid pEGFP-TRAF6 was constructed by the double digestion method, and transformed into the HEK293 T cells. Fluorescence observation was conducted after 48 hours, and the results were photographed. The results showed that the length of LmTRAF6 cDNA was 2751 bp, including 1785 bp open reading frame(ORF) and 594 amino acids. The structure of LmTRAF6 was highly similar to that in other mammal and fish species, containing one RING domain, one coiled-coil region, one MATH domain and two zinc fingers. Phylogenetic tree showed that LmTRAF6 was evolutionarily closer to that in mammals and fishes, but not to that in Drosophila melanogaster and Penaeus chinensis. LmTRAF6 was expressed in all tested tissues and their developmental stages. However, higher expression was detected in heart, skin, gill, liver of juvenile, as well as in kidney, gill, and muscle of adult. When adult L.morii was challenged by Pseudomonas aeruginosa, the expression of LmTRAF6 in gill, intestine and kidney reached to the peak after 24 h. Subcellular localization showed that LmTRAF6 was expressed in both cytoplasm and nucleus.
引文
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[23]Liu C J,Liu X,Wu Y,et al.Separation and cytological character of peripheral blood lymphocytes in Japanese lamprey[J].Chinese Journal of Zoology,2008,43(1):82-87[刘岑杰,刘欣,吴毓,等.日本七鳃鳗类淋巴细胞的分离及细胞学特征.动物学杂志,2008,43(1):82-87]
[24]H?cker H,Vabulas R M,Takeuchi O,et al.Immune cell activation by bacterial CpG-DNA through myeloid differentiation marker 88 and tumor necrosis factor receptorassociated factor(Traf)6[J].Journal of Experimental Medicine,2000,192(4):595-600
[25]Poole E,King C A,Sinclair J H,et al.The UL144 gene product of human cytomegalovirus activates NFkappaBvia a TRAF6-dependent mechanism[J].The EMBOJournal,2006,25(18):4390-4399
[26]Baud V,Liu Z G,Bennett B,et al.Signaling by proinflammatory cytokines:oligomerization ofTRAF2 and TRAF6 is sufficient for JNK and IKK activation and target gene induction via an amino-terminal effector domain[J].Genes&Development,1999,13(10):1297-1308
[27]Lamothe B,Campos A D,Webster W K,et al.The RINGdomain and first zinc finger of TRAF6 coordinate signaling byinterleukin-1,lipopolysaccharide,and RANKL[J].Journal of Biological Chemistry,2008,283(36):24871-24880
[28]Wooff J,Pastushok L,Hanna M,et al.The TRAF6 RINGfinger domain mediates physical interaction with Ubc13[J].FEBS Letters,2004,566(1):229-233
[29]Yang K,Zhu J M,Sun S G,et al.The coiled-coil domain of TRAF6 is essential for its auto-ubiquitination[J].Biochemical&Biophysical Research Communications,2004,324(1):432-439
[30]Ye H,Arron J R,Lamothe B,et al.Distinct molecular mechanism for initiating TRAF6 signalling[J].Nature,2002,418(6896):443-447
[31]Basu M,Swain B,Maiti N K,et al.Inductive expression of toll-like receptor 5(tlr5)and associated downstream signaling molecules following ligand exposure and bacterial infection in the Indian major carp,mrigal(Cirrhinus mrigala)[J].Fish&Shellfish Immunology,2012,32(1):121-131
[32]Wei J G,Guo M L,Gao P,et al.Isolation and characterization of tumor necrosis factor receptor-associated factor6(TRAF6)from grouper,Epinephelus tauvina[J].Fish&Shellfish Immunology,2014,39(1):61-68
[33]Li Y W,Li X,Xiao X X,et al.Molecular characteriza-tion and functional analysis of TRAF6 in orange-spotted grouper(Epinephelus coioides)[J].Developmental&Comparative Immunology,2014,44(1):217-225
[34]Lemaitre B,Hoffmann J.The host defense of Drosophila melanogaster[J].Annual Review of Immunology,2007,25(1):697-743
[35]Qian Y,Commane M,Ninomiya-Tsuji J,et al.IRAK-mediated translocation of TRAF6 and TAB2 in the interleukin-1-induced activation of NFkappa B[J].Journal of Biological Chemistry,2001,276(45):41661-41667
[36]Bai S T,Zha J,Zhao H B,et al.Tumor necrosis factor receptor associated factor 6 is an intranuclear transcriptional coactivator in osteoclasts[J].Journal of Biological Chemistry,2008,283(45):30861-30867
[37]Pham L V,Zhou H J,Lin-Lee Y C,et al.Nuclear tumor necrosis factor receptor-associated factor 6 in lymphoid cells negatively regulates c-Myb-mediated transactivation through small ubiquitinrelated modifier-1 modification[J].Journal of Biological Chemistry,2008,283(8):5081-5089
[2]Magnadottir B.Innate immunity of fish(overview)[J].Fish&Shellfish Immunology,2006,20(2):137-151
[3]Medzhitov R.Toll-like receptors and innate immunity[J].Nature Reviews Immunology,2001,1(2):135-145
[4]Suzuki N,Suzuki S,Duncan G S,et al.Severe impairment of interleukin-1 and Toll-like receptor signalling in mice lacking IRAK-4[J].Nature,2002,416(6882):750-756
[5]Ninomiya-Tsuji J,Kishimoto K,Hiyama A,et al.1999,The kinase TAK1 can activate the NIK-I kappa B as well as the MAP kinase cascadein the IL-1 signalling pathway[J].Nature,1999,398(6724):252-256
[6]Janssens S,Beyaert R.A universal role for MyD88 in TLR/IL-1R-mediated signaling[J].Trends in Biochemical Sciences,2002,27(9):474-482
[7]Kongchum P,Hallerman E M,Hulata G,et al.Molecular cloning,characterization and expression analysis of TLR9,MyD88 and TRAF6 genes in common carp(Cyprinus carpio)[J].Fish&Shellfish Immunology,2011,30(1):361-371
[8]Stockhammer O W,Rauwerda H,Wittink F R,et al.Transcriptome analysis of Traf6 function in the innate immune response of zebrafish embryos[J].Molecular Immunology,2010,48(1-3):179-190
[9]Phelan P E,Mellon M T,Kim C H,et al.Functional characterization of full-length TLR3,IRAK-4,and TRAF6 in zebrafish(Danio rerio)[J].Molecular Immunology,2005,42(9):1057-1071
[10]Zhao F,Li Y W,Pan H J,et al.Grass carp(Ctenopharyngodon idella)TRAF6 and TAK1:molecular cloning and expression analysis after Ichthyophthirius multifiliis infection[J].Fish&Shellfish Immunology,2013,34(6):1514-1523
[11]Qiu L,Song L S,Yu Y D,et al.Identification and expres-sion of TRAF6(TNF receptor-associated factor 6)gene in Zhikong scallop(Chlamys farreri)[J].Fish&Shellfish Immunology,2009,26(3):359-367
[12]He C B,Wang Y,Liu W D,et al.Cloning,promoter analysis and expression of the tumor necrosis factor receptorassociated factor 6(TRAF6)in Japanese scallop(Mizuhopecten yessoensis)[J].Molecular Biology Reports,2013,40(8):4769-4779
[13]Wang P H,Wan D H,Gu Z H,et al.Litopenaeus vannamei tumor necrosis factor receptor-associated factor 6(TRAF6)responds to Vibrio alginolyticus and white spot syndrome virus(WSSV)infection and activates antimicrobial peptide genes[J].Developmental&Comparative Immunology,2011,35(1):105-114
[14]Lu Y L,Li C H,Zhang P,et al.Two adaptor molecules of MyD88 and TRAF6 in Apostichopus japonicus Toll signaling cascade:molecular cloning and expression analysis[J].Developmental and Comparative Immunology,2013,41(4):498-504
[15]Armstrong A P,Tometsko M E,Glaccum M,et al.ARANK/TRAF6-dependent signal transduction pathway is essential for osteoclast cytoskeletal organization and resorptive function[J].Journal of Biological Chemistry,2002,277(46):44347-44356
[16]Park H,Huang X,Lu C,et al.MicroRNA-146a and microRNA-146b regulate human dendritic cell apoptosis and cytokine production by targeting TRAF6 and IRAK1proteins[J].Journal of Biological Chemistry,2015,290(5):2831-2841
[17]Kobayashi N,Kadono Y,Naito A,et al.Segregation of TRAF6-mediated signaling pathways clarifies its role in osteoclastogenesis[J].The EMBO Journal,2001,20(6):1271-1280
[18]Kobayashi T,Walsh P T,Walsh M C,et al.Traf6 is a critical factor for dendritic cell maturation and development[J].Immunity,2003,19(3):353-363
[19]Lomaga M A,Henderson J T,Elia A J,et al.Tumor necrosis factor receptorassociated factor 6(TRAF6)deficiency results in exencephaly and isrequired for apoptosis within the developing CNS[J].Journal of Neuroscience the Official Journal of the Society for Neuroscience,2000,20(19):7384-7393
[20]Naito A,Azuma S,Tanaka S,et al.Severe osteopetrosis,defective interleukin-1 signalling andlymph node organogenesis in TRAF6-deficient mice[J].Genes Cells,1999,4(6):353-362
[21]Naito A,Yoshida H,Nishioka E,et al.TRAF6-deficient mice display hypohidrotic ectodermal dysplasia[J].Proceedings of the National Academy of Sciences of theUnited States of America,2002,99(13):8766-8771
[22]Bajoghli B,Guo P,Aghaallaei N,et al.A thymus candidate in lampreys[J].Nature,2011,470(7332):90-94
[23]Liu C J,Liu X,Wu Y,et al.Separation and cytological character of peripheral blood lymphocytes in Japanese lamprey[J].Chinese Journal of Zoology,2008,43(1):82-87[刘岑杰,刘欣,吴毓,等.日本七鳃鳗类淋巴细胞的分离及细胞学特征.动物学杂志,2008,43(1):82-87]
[24]H?cker H,Vabulas R M,Takeuchi O,et al.Immune cell activation by bacterial CpG-DNA through myeloid differentiation marker 88 and tumor necrosis factor receptorassociated factor(Traf)6[J].Journal of Experimental Medicine,2000,192(4):595-600
[25]Poole E,King C A,Sinclair J H,et al.The UL144 gene product of human cytomegalovirus activates NFkappaBvia a TRAF6-dependent mechanism[J].The EMBOJournal,2006,25(18):4390-4399
[26]Baud V,Liu Z G,Bennett B,et al.Signaling by proinflammatory cytokines:oligomerization ofTRAF2 and TRAF6 is sufficient for JNK and IKK activation and target gene induction via an amino-terminal effector domain[J].Genes&Development,1999,13(10):1297-1308
[27]Lamothe B,Campos A D,Webster W K,et al.The RINGdomain and first zinc finger of TRAF6 coordinate signaling byinterleukin-1,lipopolysaccharide,and RANKL[J].Journal of Biological Chemistry,2008,283(36):24871-24880
[28]Wooff J,Pastushok L,Hanna M,et al.The TRAF6 RINGfinger domain mediates physical interaction with Ubc13[J].FEBS Letters,2004,566(1):229-233
[29]Yang K,Zhu J M,Sun S G,et al.The coiled-coil domain of TRAF6 is essential for its auto-ubiquitination[J].Biochemical&Biophysical Research Communications,2004,324(1):432-439
[30]Ye H,Arron J R,Lamothe B,et al.Distinct molecular mechanism for initiating TRAF6 signalling[J].Nature,2002,418(6896):443-447
[31]Basu M,Swain B,Maiti N K,et al.Inductive expression of toll-like receptor 5(tlr5)and associated downstream signaling molecules following ligand exposure and bacterial infection in the Indian major carp,mrigal(Cirrhinus mrigala)[J].Fish&Shellfish Immunology,2012,32(1):121-131
[32]Wei J G,Guo M L,Gao P,et al.Isolation and characterization of tumor necrosis factor receptor-associated factor6(TRAF6)from grouper,Epinephelus tauvina[J].Fish&Shellfish Immunology,2014,39(1):61-68
[33]Li Y W,Li X,Xiao X X,et al.Molecular characteriza-tion and functional analysis of TRAF6 in orange-spotted grouper(Epinephelus coioides)[J].Developmental&Comparative Immunology,2014,44(1):217-225
[34]Lemaitre B,Hoffmann J.The host defense of Drosophila melanogaster[J].Annual Review of Immunology,2007,25(1):697-743
[35]Qian Y,Commane M,Ninomiya-Tsuji J,et al.IRAK-mediated translocation of TRAF6 and TAB2 in the interleukin-1-induced activation of NFkappa B[J].Journal of Biological Chemistry,2001,276(45):41661-41667
[36]Bai S T,Zha J,Zhao H B,et al.Tumor necrosis factor receptor associated factor 6 is an intranuclear transcriptional coactivator in osteoclasts[J].Journal of Biological Chemistry,2008,283(45):30861-30867
[37]Pham L V,Zhou H J,Lin-Lee Y C,et al.Nuclear tumor necrosis factor receptor-associated factor 6 in lymphoid cells negatively regulates c-Myb-mediated transactivation through small ubiquitinrelated modifier-1 modification[J].Journal of Biological Chemistry,2008,283(8):5081-5089