三疣梭子蟹NF-κB家族基因Relish和Dorsal的克隆及表达特征
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摘要
为研究Relish和Dorsal在三疣梭子蟹免疫过程中所起到的作用,研究利用RACE技术克隆获得三疣梭子蟹Relish(Pt-Rel)、Dorsal基因(Pt-Dor) cDNA全长,并通过实时荧光定量PCR技术分析了Pt-Rel和Pt-Dor基因在健康蟹不同组织及其原代培养的血淋巴细胞在感染不同微生物后的表达情况。结果显示,Pt-Rel cDNA长3254 bp,ORF长2949 bp,编码983个氨基酸,Pt-Dor cDNA长2348 bp,ORF长1911 bp,编码637个氨基酸;蛋白结构预测分析发现Pt-Rel和Pt-Dor均包含RHD (Rel homology domain)及IPT (Immunoglobulin-like fold,Plexins,TranscriPtion factors)Rel/NF-κB家族蛋白经典结构域。Pt-Rel和Pt-Dor与其他节肢动物Relish、Dorsal氨基酸序列具有很高的相似性;在系统进化分析中Pt-Rel和Pt-Dor分别与中华绒螯蟹等甲壳动物的Relish、Dorsal聚在一支,而昆虫类聚在另一支。Pt-Rel和Pt-Dor在检测的6种组织中均有表达,且2个基因均在血淋巴细胞中表达量最高。蟹血淋巴细胞体外感染实验结果表明,不同病原微生物对2个基因表达的影响非常相似,假丝酵母在2h明显诱导了Pt-Rel和Pt-Dor基因的表达,而金黄色葡萄球菌及溶藻弧菌在感染4h后使Pt-Rel和Pt-Dor基因的表达显著上调。上述研究结果表明Pt-Rel和Pt-Dor基因很有可能参与了三疣梭子蟹的抗感染免疫过程。
Relish and Dorsal,members of nuclear factor-κB(NF-κB) family protein,play crucial role in immune response in Arthropoda.In this study,the cDNA sequences of Relish and Dorsal were cloned by rapid amplification of cDNA ends(RACE).There are 3254 nucleotides in Pt-Rel with a putative open reading frame(ORF) of 2949 bp that encodes a polypeptide of 983 amino acid residues.The Pt-Dor cDNA was 2348 bp in length including an ORF encoding a polypeptide of 637 amino acid residues.Prediction of protein domains revealed that both Pt-Rel and Pt-Dor contained typical NF-κB family structures RHD(Rel homology domain) and IPT(Immunoglobulin-like fold,Plexins,Transcription factors).Alignment of amino acid sequences for Pt-Rel and Pt-Dor revealed high identities with other arthropodas orthologs.Phylogenetic tree analysis based on amino acid sequences showed that the Pt-Rel and Pt-Dor were distinctly clustered with crustaceans Relish and Dorsal,respectively,but separated from insect orthologs.Pt-Rel and Pt-Dor were expressed in all tested tissues with the highest level in hemocytes.Three different pathogenic microorganism significantly induced both Pt-Rel and Pt-Dor expression in primary cultured crab hemocytes at 2 h by Candida lusitaniae infection and 4 h by Staphylococcus aureus and Vibrio alginolyticus challenge.These data suggest that Pt-Rel and Pt-Dor might be involved in host defense against to microbial pathogens in Portunus trituberculatus.
Relish and Dorsal,members of nuclear factor-κB(NF-κB) family protein,play crucial role in immune response in Arthropoda.In this study,the cDNA sequences of Relish and Dorsal were cloned by rapid amplification of cDNA ends(RACE).There are 3254 nucleotides in Pt-Rel with a putative open reading frame(ORF) of 2949 bp that encodes a polypeptide of 983 amino acid residues.The Pt-Dor cDNA was 2348 bp in length including an ORF encoding a polypeptide of 637 amino acid residues.Prediction of protein domains revealed that both Pt-Rel and Pt-Dor contained typical NF-κB family structures RHD(Rel homology domain) and IPT(Immunoglobulin-like fold,Plexins,Transcription factors).Alignment of amino acid sequences for Pt-Rel and Pt-Dor revealed high identities with other arthropodas orthologs.Phylogenetic tree analysis based on amino acid sequences showed that the Pt-Rel and Pt-Dor were distinctly clustered with crustaceans Relish and Dorsal,respectively,but separated from insect orthologs.Pt-Rel and Pt-Dor were expressed in all tested tissues with the highest level in hemocytes.Three different pathogenic microorganism significantly induced both Pt-Rel and Pt-Dor expression in primary cultured crab hemocytes at 2 h by Candida lusitaniae infection and 4 h by Staphylococcus aureus and Vibrio alginolyticus challenge.These data suggest that Pt-Rel and Pt-Dor might be involved in host defense against to microbial pathogens in Portunus trituberculatus.
引文
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[19]Zhou S M,Li M,Yang N,et al.First description and expression analysis of tumor necrosis factorreceptor-associated factor 6(TRAF6)from the swimming crab,Portunus trituberculatus[J].Fish&Shellfish Immunology,2015,45(2):205-210
[20]Belvin M P,Anderson K V.A conserved signaling pathway:the Drosophila Toll dorsal pathway[J].Annual Review of Cell and Developmental Biology,1996,12(1):393-416
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[22]Wen R,Li F,Sun Z,et al.Shrimp MyD88 responsive to bacteria and white spot syndrome virus[J].Fish&Shellfish Immunology,2013,34(2):574-581
[23]Yan H,Li F H,Wang B.Expression of Rel/NF-κB family FcRel gene in vibrio-challenged Chinese shrimp Fenneropenaeus chinensis[J].Oceanologia et Limnologia Sinica,2008,39(6):628-633[阎慧,李富花,王兵,等.中国明对虾Rel/NF-κB家族基因在弧菌刺激下的表达.海洋与湖沼,2008,39(6):628-633]
[24]Ma J W,Lv J J,Liu P,et al.Na+/H+-exchanger in swimming crab(Portunus trituberculatus):cloning,characterization and mRNA expression under salinity stress[J].Acta Hydrobiologica Sinica,2016,40(5):902-907[马金武,吕建建,刘萍,等.三疣梭子蟹Na+/H+-exchanger基因克隆鉴定及在盐度胁迫下的表达分析.水生生物学报,2016,40(5):902-907]
[2]Sen R.Multiple nuclear factors interact with the immunoglobulin enhancer sequences[J].Journal of Immunology,2006,177(11):7485-7496
[3]Ma Z P,Zhang F S,Tong J K.Overview and graph theory of the immune system of Crustacean[J].Journal of Anhui Agricultural Sciences,2011,39(1):291-294[马寨璞,张繁霜,佟霁坤.甲壳动物免疫系统概述及其图论表示.安徽农业科学,2011,39(1):291-294]
[4]Yan H.Cloning and Expression of Nuclear Transcription Factors Rel/NF-κB family genes in Chinese shrimp Fenneropenaeus chinensis[D].Thesis for Master of Science.Institute of Oceanology,Chinese Academy of Sciences,Beijing.2008[阎慧.中国明对虾核转录因子NF-kB家族基因的克隆与表达分析.硕士学位论文,中国科学院海洋研究所,北京.2008]
[5]Tanji T,Hu X,Weber A N R,et al.Toll and IMD pathways synergistically activate an innate immune response in Drosophila melanogaster[J].Molecular&Cellular Biology,2007,27(12):4578-4588
[6]Wang G L,Jin S,Chen Y E,et al.Study on pathogens and pathogenesis of emulsification disease of Portunus trituberculatus[J].Advances In Marine Science,2006,24(4):526-531[王国良,金珊,陈寅儿,等.三疣梭子蟹肌肉乳化病的病原及其致病性研究.海洋科学进展,2006,24(4):526-531]
[7]Yu A Q,Jin X K,Li S,et al.Molecular cloning and expression analysis of a dorsal homologue from Eriocheir sinensis[J].Developmental&Comparative Immunology,2013,41(4):723-727
[8]Shi Y R,Jin M,Ma F T,et al.Involvement of relish gene from macrobrachium rosenbergii,in the expression of anti-microbial peptides[J].Developmental&Comparative Immunology,2015,52(2):236-244
[9]Suwattana V,Premruethai S,Ikuo H,et al.Activation of PmRelish from Penaeus monodon by yellow head virus[J].Fish&Shellfish Immunology,2015,42(2):335-344
[10]Li F,Yan H,Wang D,et al.Identification of a novel relish homolog in Chinese shrimp Fenneropenaeus chinensis and its function in regulating the transcription of antimicrobial peptides[J].Developmental&Comparative Immunology,2009,33(10):1093-1101
[11]Wang D D.Functional studies on the regulation of immunity in Fenneropenaeus chinensis by the nuclear transcription factor NF-κB family genes[D].Thesis for Doctor of Science.Institute of Oceanology,Chinese Academy of Sciences,Beijing.2012[王东东.中国明对虾核转录因子NF-κB家族基因在免疫调控中的功能研究.博士学位论文,中国科学院海洋研究所,北京.2012]
[12]Liu T,Luo K J.Immunity-related domains of Toll and IMD signaling pathways in Drosophila[J].Journal of Environmental Entomology,2011,33(3):388-395[刘甜,罗开珺.果蝇Toll和IMD信号通路中的功能结构域.环境昆虫学报,2011,33(3):388-395]
[13]Uvell H,Engstr?m Y.A multilayered defense against infection:combinatorial control of insect immune genes[J].Trends in Genetics,2007,23(7):342-349
[14]Ge Q Q,Li J,Liang J P,et al.Cloning of Imd immune signal pathway relating Gene of Fenneropenaeus chinensis and their expression analysis[J].Periodical of Ocean University of China,2014,44(1):46-51[葛倩倩,李健,梁俊平,等.中国明对虾Imd免疫信号通路相关基因克隆及表达分析.中国海洋大学学报,2014,44(1):46-51]
[15]Zhao Y Z,Chen X L,Zeng D G,et al.Cloning and analysis of full length cDNA of prophenoloxidase activating factor in Litopenaeus vannamei[J].Journal of Fisheries of China,2014,38(8):1068-1078[赵永贞,陈秀荔,曾地刚,等.凡纳滨对虾酚氧化酶原激活因子基因的克隆及表达分析.水产学报,2014,38(8):1068-1078]
[16]Lin X Y,Yang J,Li H M,et al.Dorsal root ganglion compression as an animal model of sciatica and low back pain[J].Neuroscience Bulletin,2012,28(5):618-630
[17]Huang X D,Liu W G,Guan Y Y,et al.Molecular cloning and characterization of class I NF-κB transcription factor from pearl oyster(Pinctada fucata)[J].Fish&Shellfish Immunology,2012,33(3):659-666
[18]Zhang J,Lv J J,Liu P,et al.Cloning of HMGBa in Portunus trituberculatus and viral infections[J].Acta Hydrobiologica Sinica,2017,41(6):1193-1199[张杰,吕建建,刘萍,等.三疣梭子蟹HMGBa基因克隆及其应答不同病原入侵的表达特征.水生生物学报,2017,41(6):1193-1199]
[19]Zhou S M,Li M,Yang N,et al.First description and expression analysis of tumor necrosis factorreceptor-associated factor 6(TRAF6)from the swimming crab,Portunus trituberculatus[J].Fish&Shellfish Immunology,2015,45(2):205-210
[20]Belvin M P,Anderson K V.A conserved signaling pathway:the Drosophila Toll dorsal pathway[J].Annual Review of Cell and Developmental Biology,1996,12(1):393-416
[21]Cui X Y,Zhu D F,Tang J,et al.Cloning and expression analysis of ecdysteroid receptor(EcR)in Portunus trituberculatus[J].Journal of Fisheries of China,2013,37(11):1645-1654[崔晓雨,朱冬发,汤洁,等.三疣梭子蟹EcR基因的克隆及表达分析.水产学报,2013,37(11):1645-1654]
[22]Wen R,Li F,Sun Z,et al.Shrimp MyD88 responsive to bacteria and white spot syndrome virus[J].Fish&Shellfish Immunology,2013,34(2):574-581
[23]Yan H,Li F H,Wang B.Expression of Rel/NF-κB family FcRel gene in vibrio-challenged Chinese shrimp Fenneropenaeus chinensis[J].Oceanologia et Limnologia Sinica,2008,39(6):628-633[阎慧,李富花,王兵,等.中国明对虾Rel/NF-κB家族基因在弧菌刺激下的表达.海洋与湖沼,2008,39(6):628-633]
[24]Ma J W,Lv J J,Liu P,et al.Na+/H+-exchanger in swimming crab(Portunus trituberculatus):cloning,characterization and mRNA expression under salinity stress[J].Acta Hydrobiologica Sinica,2016,40(5):902-907[马金武,吕建建,刘萍,等.三疣梭子蟹Na+/H+-exchanger基因克隆鉴定及在盐度胁迫下的表达分析.水生生物学报,2016,40(5):902-907]