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2018, 08, v.37 3296-3303
斑马鱼tnfb启动子的克隆和报告基因载体的活性分析
基金项目(Foundation): 教育部留学回国人员科研启动基金(D-8002-15-0042);; 上海市教委重点创新项目(13ZZ127);上海市教委水产高峰学科项目共同资助;; 中美海洋研究中心基金(A1-0209-15-0806)
邮箱(Email): qhzhang@shou.edu.cn;
DOI: 10.13417/j.gab.037.003296
发布时间: 2017-11-20
出版时间: 2017-11-20
网络发布时间: 2017-11-20
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摘要:

本研究通过采用生物信息学软件预测细胞因子tnfb基因5'上游2 000 bp左右的序列,并预测其相关的转录因子,克隆启动子,构建双荧光素酶报告基因表达载体,经双酶切和测序鉴定,最后通过双荧光素酶报告基因系统检测重组载体的活性。研究发现tnfb的转录因子结合位点为:Oct-1、TBP、GATA-1、RSRFC4、GLO、C/EBPα、NF-资B、ER、Pit-1a、Oct-2、Oct-2.1、RAP1。tnfb的启动子区没有发现Cp G岛,其5'侧翼序列含有与转录密切相关的TATA Box和CAAT Box转录元件。将tnfb的启动子片段插入p GL3-enhancer构建p GL3-tnfb-promoter-enhancer质粒后,质粒经酶切测序显示酶切片段大小一致,序列正确;转染了p GL3-tnfbpromoter的Raw264.7细胞的相对荧光素酶活性高于对照组细胞,双荧光素酶报告基因检测系统证实构建的p GL3-tnfb-promoter-enhancer具有启动子活性。为研究tnfb参与的免疫相关的信号通路之间的调控提供了有力的研究工具。

Abstract:

In this study,the cytokine tnfb gene 5' upstream sequence of about 2 000 bp and its related transcription factors were predicted by using bioinformatics software.The promoter was cloned and the dual luciferase reporter gene expression vector was constructed.The sequence was identified by the double enzyme digestion and sequencing method,and finally,the activity of the recombinant vector was detected by the double luciferase reporter gene system..It was found that the transcription factor binding sites of tnfb included Oct-1,TBP,GATA-1,RSRFC4,GLO,C/EBPα,NF-κB,ER,Pit-1 a,Oct-2,Oct-2.1,and RAP1.The Cp G island was not found in tnfb promoter region,and its 5' flanking sequence contained TATA Box and CAAT Box transcriptional elements closely related to transcription.The promoter fragment tnfb was inserted into p GL3-enhancer to construct p GL3-tnfb-promoter enhancer plasmid.The enzyme cut sequencing showed that thesize of fragments was consistent and the sequence was correct.Relative luciferase activity of Raw264.7 cells transfected by p GL3-tnfb-promoter was higher than that of control cells.Dual luciferase report gene detection system proved that p GL3-tnfb-promoter enhancer had promoter activity.It would provide a powerful tool for studying tnfb involved in the regulation of immune related signaling pathways in zebrafish.

参考文献

Bates J.M.,Akerlund J.,Mittge E.,and Guillemin K.,2007,Intestinal alkaline phosphatase detoxifies lipopolysaccharide and prevents inflammation in zebrafish in response to the gut microbiota,Cell Host&Microbe,2(6):371-382

Beutler B.A.,Greenwald D.,Hulmes J.D.,Chang M.,Pan Y.C.E.,Mathison J.,Ulevitch R.,and Cerami A.,1985,Identity of tumor necrosis factor and the macrophage-secreted factor cachectin,Nature,316:552-554

Chen X.Y.,and Hou Y.Y.,2004,Research advances of fish cytokines,Shuisheng Shengwu Xuebao(Acta Hydrobiologica Sinica),28(6):668-673(陈旭衍,侯亚义,2004,鱼类细胞因子研究进展,水生生物学报,28(6):668-673)

Duan H.Q.,and Wang L.,2017,Cloning and Expression profile of interleukin-1βgene inSchizothorax prenanti,Shengwu Jishu Tongbao(Biotechnology Bulletin),33(2):97-101(段荟芹,王利,2017,齐口裂腹鱼白介素-1β基因的克隆及表达谱,生物技术通报,33(2):97-101)

Ewbank J.J.,2002,Tackling both sides of the host-pathogen equation with Caenorhabditis elegans,Microbes Infect,4:247-256

Feng X.R.,Chen Y.L.,Zhao X.,Wang W.D.,Zhang J.H.,Yang Z.G.,Sun Z.,Jia S.M.,and Lu Q.,2012,Cloning and sequence analysis of interleukin 10(IL-10)full length c DNAfrom Cyprinus carpio,Anhui Nongye Kexue(Agricultural Science&Technology),40(22):11314-11316(冯祥汝,陈义龙,赵晓,王文东,张俊辉,杨振国,孙真,贾生美,卢强.,2012,鲤鱼白细胞介素10(i L-10)基因c DNA克隆及序列分析,安徽农业科学,40(22):11314-11316)

Frederickson R.M.,Micheau M.R.,Iwamoto A.,and Miyamoto N.G.,1989,5'flanking and first intron sequences of the human beta-actin gene required for efficient promoter activity,Nucleic Acids Research,17(1):253-270

Hassa P.O.,Covic M.,Hasan S.,Imhof R.,and Hottiger M.O.,2001,The enzymatic and DNA binding activity of PARP-1are not required for NF-资B coactivator function,Journal of Biological Chemistry,276(49):45588-45597

Hassa P.O.,and Hottiger M.O.,1999,A role of poly(adp-ribose)polymerase in nf-kappab transcriptional activation,Biological Chemistry,380(7-8):953

Hoffmann J.A.,2003,The immune response of Drosophila,Nature,426:33-38

Howe K.,Clark M.D.,Torroja C.F.,Torrance J.,Berthelot C.,Muffato M.,Collins J.E.,Humphray S.,Mc Laren K.,Matthews L.,Mc Laren S.,Sealy I.,Caccamo M.,Churcher C.,Scott C.,Barrett J.C.,Koch R.,Rauch G.,White S.,Chow W.,Kilian B.,Quintais L.T.,Guerra-Assun觔o J.,Zhou Y.,Gu Y.,Yen J.,Voge J.,Eyre T.,Redmond S.,Banerjee R.,Chi J.X.,Fu B.Y.,Langley E.,Maguire S.F.,Laird G.K.,Lloyd D.,Kenyon E.,Donaldson S.,Sehra H.,Almeida-King J.,Loveland J.,Trevanion S.,Jones M.,Quail M.,Willey D.,Hunt A.,Burton J.,S ims S.,Mc Lay K.,Plumb B.,Davis J.,Clee C.,Oliver K.,Clark R.,Riddle C.,Eliott D.,Threadgold G.,Harden G.,Ware D.,Mortimer B.,Kerry G.,Heath P.,Phillimore B.,Tracey A.,Corby N.,Dunn M.,Johnson C.,Wood J.,Clark S.,Pelan S.,Griffiths G.,Smith M.,Glithero M.,Howden P.,Barker N.,Stevens C.,Harley J.,Holt K.,Georgios Panagiotidis G.,Lovell J.,Beasley H.,Henderson C.,Gordon D.,Auger K.,Wright D.,Collins J.,Raisen C.,Dyer L.,Leung K.,Robertson L.,Ambridge K.,Leongamornlert D.,Mc Guire S.,Gilderthorp R.,Griffiths C.,Manthravadi D.,Nichol S.,Barker G.,Whitehead S.,Kay M.,Brown J.,Murnane C.,Gray E.,Humphries M.,Sycamore N.,Barker D.,Saunders D.,Wallis J.,Babbage A.,Hammond S.,Mashreghi-Mohammadi M.,Barr L.,Martin S.,Wray P.,Anrew Ellington A.,Matthews N.,Ellwood M.,Woodmansey R.,Clark G.,Cooper J.,Tromans A.,Grafham D.,Skuce C.,Pandian R.,Andrews R.,Harrison E.,Kimberley A.,Garnett J.,Fosker N.,Hall R.,Garner P.,Kelly D.,Bird C.,Palmer S.,Gehring I.,Berger A.,Dooley C.M.,Ersan-譈r俟n Z.,Eser C.,Geiger H.,Geisler M.,Karotki L.,Kirn A.,Konantz J.,Konantz M.,Oberl覿nder M.,Rudolph-Geiger S.,Teucke M.,Osoegawa K.,Zhu B.,Rapp A.,Widaa S.,Langford C.,Yang F.T.,Carter N.P.,Harrow J.,Ning Z.,Herrero J.,Searle S.M.J.,Enright A.,Geisler R.,Plasterk R.H.A,Lee C.,Westerfield M.,de Jong P.J.,Zon L.I.,Postlethwait J.H.,N俟sslein-Volhard C.,Hubbard T.J.P.,Crollius H.R.,Rogers J.,and Stemple D.L.,2013,The zebrafish reference genome sequence and its relationship to the human genome,Nature,496(7446):498-503

Imbalzano A.N.,Kwon H.,Green M.R.,and Kingston R.E.,1994,Facilitated binding of TATA-binding protein to nucleosomal DNA,Nature,370(6489):481

Kadonaga J.T.,Jones K.A.,and Tjian R.,1986,Promoter-specific activation of RNA polymerase II transcription by Sp1,Trends in Biochemical Sciences,11(1):20-23

Liu Z.J.,Moav B.,Faras A.J.,Guise K.S.,Kapuscinski A.R.,and Hackett P.B.,1990,Functional analysis of elements affecting expression of the beta-actin gene of carp,Molecular&Cellular Biology,10(7):3432-3440

Li S.Y.,Li J.,Wang L.L.,Zhong J.H.,Chen Q.,Xiong W.F.,Zeng X.W.,and He X.S.,2017,Bioinformatics analysis of s TGC3 gene promoter and construction of dual luciferase report gene expression vector,Zhongnan Yixue Kexue Zazhi(Journal of University of South China),45(1):48-53(李素云,李佳,王莉莉,钟嘉宏,陈青,熊文芳,曾小微,贺修胜,2017,Stgc3基因启动子生物信息学分析及载体构建,中南医学科学杂志,45(1):48-53)

Ma D.M.,Han L.Q.,and Bai J.J.,2016,Sequence and activity analysis of GHRH promoter region from Micropterus salmoides,Haiyang Yuye(Marine Fisheries),38(4):383-390(马冬梅,韩林强,白俊杰,2016,大口黑鲈ghrh基因启动子区域序列分析及其活性检测,海洋渔业,38(4):383-390)

Nedwin G.E.,Naylor S.L.,Sakaguchi A.Y.,Smith D.,Jarrett-Nedwin,J.,Pennica,D.,Goeddel D.,and Gray P.W.,1985,Human lymphotoxin and tumor necrosis factor genes:structure,homology and chromosomal localization,Nucleic Acids Research,13(17):6361-6373

Neumann N.F.,and Belosevic M.,1997,Sequential activation and deactivation of antimicrobial killing mechanisms of cytokine activated goldfish macrophages,Developmental and Comparative Immunology,2(21):188

Ruddle N.H.,2014,Lymphotoxin and TNF:how it all began-a tribute to the travelers,Cytokine&growth factor reviews,25(2):83-89

Semon D.,Kawashima E.,Jongeneel C.V.,Shakhov A.N.,and Nedospasov S.A.,1987,Nucleotide sequence of the murine TNF locus,including the TNF-α(tumor necrosis factor)and TNF-β(lymphotoxin)genes,Nucleic acids research,15(21):9083

Trinchieri G.,2004,Cytokines and cytokine receptors,Immunological Reviews,2(1):5-7

Tzou P.,De Gregorio E.,and Lemaitre B.,2002,How Drosophila combats microbial infection:a model to study innate immunity and host-pathogen interactions,Current Opinion in Microbiology,5(1):102-110

Van der Sar A.M.,Musters R.J.,Van Eeden F.J.,Appelmelk B.J.,Vandenbroucke-Grauls C.M.,and Bitter W.,2003,Zebrafish embryos as a model host for the real time analysis of Salmonella typhimurium infections,Cellular Microbiology,5(9):601-611

Vilcek J.,and Le J.,1994,Immunology of cytokines:an introduction.,In:Thomson A.W.,ed.,The Cytokine Handbook(2nd edition),Academic Press,London,UK,pp.1-19

Wang X.Y.,Lan N.,Zhang Y.,Wang X.,and Li G.P.,2016,Construction and analysis of the luciferase reporter gene of human MUC5AC promoter,Hainan Yixue(Hainan Medical Journal),27(15):2409-2412(王孝芸,蓝楠,张沄,王星,李国平,2016,人muc5ac启动子荧光报告基因的构建与分析,海南医学,27(15):2409-2412)

Wang Y.F.,and Xue L.Y.,2013,Cloning and characterization analysis of interleukin-8 Gene in Larimichthys crocea,Shengwu Xinxixue(Chinese Journal of Bioinformatics),11(3):233-236(王亚芳,薛良义,2013,大黄鱼白细胞介素8基因克隆及特征分析,生物信息学,11(3):233-236)

Wienholds E.,Van Eeden F.,Kosters M.,Mudde J.,Plasterk R.H.,and Cuppen E.,2003,Efficient target-selected mutagenesis in zebrafish,Genome Research,13(12):2700-2707

Xu R.T.,Zhou H.,Liu W.L.,Wu W.,Liu X.Y.,Zhang W.Q.,Tan J.,and Zhao M.,2016,Construction and identification of human p-selection promoter luciferase report gene vector,Nanfang Yike Daxue Xuebao(Journal of Southern Medical University),36(3):332-338(徐若霆,周辉,刘玮璐,吴炜,刘鲜艳,张文强,谭洁,赵明,2016,人细胞表面黏附分子P选择素启动子报告基因的构建及鉴定,南方医科大学学报,36(3):332-338)

Xue M.,Li X.,and Chen W.,2013,Bioinformatics analysis and identification of transcriptional regulation of human UCA1gene,Nanfang Yike Daxue Xuebao(Journal of Southern Medical University),33(11):1596-1599(薛梅,李旭,陈葳,2013,人UCA1基因转录调控的生物信息学分析与鉴定,南方医科大学学报,33(11):1596-1599)

Yan P.,Jian J.C.,and Wu Z.H.,2013,Cloning and expression analysis of interleukin-6 in grass carp(Ctenopharyngodon idella),Guangdong Haiyang Daxue Xuebao(Journal of Zhanjiang Ocean University),33(3):46-51(颜鹏,简纪常,吴灶和,2013,草鱼白细胞介素6基因克隆与表达分析,广东海洋大学学报,33(3):46-51)

Yang Y.,Li J.J.,Wang X.,and Qiu G.Z.,2011,Progress on reporter gene and its application,Shengmin Kexue Yanjiu(Life Science Research),15(3):277-282(杨宇,李江江,王项,邱冠周,2011,报告基因及其应用研究进展,生命科学研究,15(3):277-282)

Yazawa R.,Hirono I.,Ohira T.,and Aoki T.,2005,Induction of Japanese flounder tnf promoter activity by lipopolysaccha ride in zebrafish embryo,Marine Biotechnology,7(3):231-235

Zhao Z.,Cao Y.,Li M.,and Meng A.,2001,Double-stranded RNA injection produces nonspecific defects in zebrafish,Developmental Biology,229(1):215-223

基本信息:

DOI:10.13417/j.gab.037.003296

中图分类号:S917.4

引用信息:

[1]张秋月,丁安,张哲,等.斑马鱼tnfb启动子的克隆和报告基因载体的活性分析[J].基因组学与应用生物学,2018,37(08):3296-3303.DOI:10.13417/j.gab.037.003296.

基金信息:

教育部留学回国人员科研启动基金(D-8002-15-0042);; 上海市教委重点创新项目(13ZZ127);上海市教委水产高峰学科项目共同资助;; 中美海洋研究中心基金(A1-0209-15-0806)

发布时间:

2017-11-20

出版时间:

2017-11-20

网络发布时间:

2017-11-20

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