中华绒螯蟹C-型凝集素基因组和启动子的克隆与分析
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摘要
为探究中华绒螯蟹(Eriocheir sinensis)凝集素(Lectin)基因启动子在免疫应答中的转录调控机制,本研究从中华绒螯蟹的足部肌肉组织提取基因组DNA,构建Genome Walker Libraries,克隆C-型凝集素(EsCTL)的基因组和启动子序列。结果显示,EsCTL的基因组全长为2 932bp,其中结构基因由5个外显子(分别为33bp,33bp,165bp,57bp和157bp)和4个内含子(分别为299bp,542bp,199bp和453bp)构成;启动子区域包含4个启动子核心序列,6个TBP(TATA框结合蛋白),11个C/EBPα(CCAAT增强子结合蛋白)和1个SRF(血清应答因子)等真核生物的典型启动子结构特征,此外还包括15个SP1(特异性蛋白1),2个AP1(激活蛋白1),8个GATA-1和10个Oct-1等参与免疫调节的作用元件。EsCTL基因组及其启动子的克隆与特征分析,为深入研究EsCTL基因在免疫应答中表达调控奠定了基础。
In order to explore the transcriptional regulation mechanisms of lectin gene promoter from Chinese mitten crab Eriocheir sinensis in immune response,the Genome Walker Libraries from extracting genomic DNA of E.sinensis foot muscle tissue were constructed for cloning genome and promoter sequences of E.sinensislectin(EsCTL)in this study.The results showed that the full-length EsCTL genomic DNA was of 2 932 bp.The structural gene contained five exons(33 bp,33 bp,165 bp,57 bp and 157 bp)and four introns(299 bp,542 bp,199 bp and 453 bp);the flanking sequence of 976 bp of EsCTL contained several typical transcription regulation elements,such as 4 promoter core sequences,6 TBPs(TATA-box binding protein),11 C/EBPαs(enhancer binding protein)and 1 SRF(serllm response factor),and several transcription regulation elements of immune-related gene,suchas 15 SP1 s(Specificity Protein 1),2 AP1 s(activity protein 1),8 GATA-1 s,and 10 Oct-1 s.The cloning and characterization of EsCTL genome and promoter provide references for the further study on expression regulation of EsCTL gene and its function analysis in the immune response.
In order to explore the transcriptional regulation mechanisms of lectin gene promoter from Chinese mitten crab Eriocheir sinensis in immune response,the Genome Walker Libraries from extracting genomic DNA of E.sinensis foot muscle tissue were constructed for cloning genome and promoter sequences of E.sinensislectin(EsCTL)in this study.The results showed that the full-length EsCTL genomic DNA was of 2 932 bp.The structural gene contained five exons(33 bp,33 bp,165 bp,57 bp and 157 bp)and four introns(299 bp,542 bp,199 bp and 453 bp);the flanking sequence of 976 bp of EsCTL contained several typical transcription regulation elements,such as 4 promoter core sequences,6 TBPs(TATA-box binding protein),11 C/EBPαs(enhancer binding protein)and 1 SRF(serllm response factor),and several transcription regulation elements of immune-related gene,suchas 15 SP1 s(Specificity Protein 1),2 AP1 s(activity protein 1),8 GATA-1 s,and 10 Oct-1 s.The cloning and characterization of EsCTL genome and promoter provide references for the further study on expression regulation of EsCTL gene and its function analysis in the immune response.
引文
[1]段利朋,黄贝,周立红,等.拟穴青蟹两种新C-型凝集素基因的克隆与表达分析[J].水生生物学报,2015,39(2):321-330.DUAN Lipeng,HUANG Bei,ZHOU Lihong,et al.Molecular cloning,characterization and expression of two novel lectins in mud crab,Scylla pramamosain[J].Acta Hydrobiologica Sinica,2015,39(2):321-330.
[2]郝贵杰,王春琳,林锋,等.三疣梭子蟹血清凝集素的生物学特性[J].江苏农业科学,2015,43(1):220-223.HAO Guijie,WANG Chunlin,LIN Feng,et al.Biological characteristics of serum lectin from Portunus trituberculatus[J].Jiangsu Agricultural Sciences,2015,43(1):220-223.
[3]RUNSAENG P,THEPNARONG S,RATTANAPORN O,et al.Cloning and the mRNA expression of a C-type lectin with one carbohydrate recognition domain fromFenneropenaeus merguiensis in response to pathogenic inoculation[J].Molecular and Cellular Probes,2015(29):365-375.
[4]DAI Y J,WANG Y Q,ZHAO L L,et al.A novel L-type lectin was required for the multiplication of WSSV in red swamp crayfish(Procambarus clakii)[J].Fish&Shellfish Immunology,2016(55):48-55.
[5]LI C H,YU S X,ZHAO J M,et al.Cloning and characterization of a sialic acid binding lectins(SABL)from Manila clam Venerupis philippinarum[J].Fish&Shellfish Immunology,2011(30):1202-1206.
[6]曹剑香,简纪常,吴灶和.虾类体液免疫研究进展[J].湛江海洋大学学报,2006,26(1):89-93.CAO Jianxiang,JIAN Jichang,WU Zaohe.Shrimp humoral immunity research advances[J].Journal of Zhanjiang Ocean University,2006,26(1):89-93.
[7]孙杰,安利国,王雷,等.甲壳纲动物凝集素的结构特征和分离纯化方法[J].海洋科学,2006,30(6):73-76.SUN Jie,AN Liguo,WANG Lei,et al.Characterizations and purification methods of Lectins of crustaceans[J].Marine Scienes,2006,30(6):73-76.
[8]罗展,张继泉,李富花,等.凡纳滨对虾C-型凝集素LvLec2对不同刺激的免疫应答[J].海洋科学,2010,34(11):103-110.LUO Zhan,ZHANG Jiquan,LI Fuhua,et a1.Cloning of a novel C-type lectin LvLev2from the shrimp Litopenaeus vannamei and its immune response to different challenges[J].Marine Sciences,2010,34(11):103-110.
[9]HUANG Y,AN L,HUI K M,et a1.An LDLa domain-containing C-type lectin is involved in the innate immunity of Eriocheir sinensis[J].Developmental and Comparative Immunology,2014,42(2):333-344.
[10]JIN X K,GUO X N,LI S,et al.Association of a hepatopancreas-specific C-type lectin with the antibacterial response of Eriocheir sinens[J].PLOS One,2013,8(10):1-6.
[11]GUO H Z,ZOU P F,FU J P,et al.Characterization of two C-type lectin-like domain(CTLD)-containing proteins from the cDNA library of Chinese mitten crab Eriocheir sinensis[J].Fish and Shellfish Immunology,201l,30(2):515-524.
[12]GUO X N,JIN X K,LI S,et a1.A novel C-type lectin from Eriocheir sinensis functions as a pattern recognition receptor with antibacterial activity[J].Fish and Shellfish Immunology,2013,35(5):1554-l565.
[13]SUN Y M,LI F H,XIANG J H.Analysis on the dynamic changes of the amount of WSSV in Chinese shrimp Fenneropenaeus chinensis during infection[J].Aquaculture,2013(376/377/378/379):l24-132.
[14]LAI X F,GAO H,KONG J,et al.Genomic organization and promoter characterization of FcCTL,a C-type lectin-like protein in Fenneropenaeus chinensis[J].Gene,2013,518(2):376-380.
[15]王显伟.十足目甲壳动物C型凝集素功能研究[D].济南:山东大学,2012.WANG Xianwei.Functional study of C-type lectins from decapod crustaceans[D].Jinan:Shandong University,2012.
[16]HUANG X,LI W,JIN M,et al.Single CRD containing lectin from Macrobrachium rosenbergii(MrLec)participates in innate immunity against pathogen infections[J].Fish&Shellfish Immunology,2016(51):282-290.
[17]WANG H,SONG L S,LI C H,et al.Cloning and characterization of a novel C-type lectin from Zhikong scallop Chlamys farreri[J].Molecular Immunology,2007(44):722-731.
[18]WANG L L,WANG L L,ZHANG D X,et al.A novel C-type lectin from crab Eriocheir sinensis functions as pattern recognition receptor enhancing cellular encapsulation[J].Fish&Shellfish Immunology,2013(34):832-842.
[19]ZHANG HAO,CHEN L Q,QIN J G,et al.Molecular cloning,characterization and expression of a C-type lectin cDNA in Chinese mitten crab,Eriocheir sinensis[J].Fish&Shellfish Immunology,2011(31):358-363.
[20]赖晓芳,高焕,孔杰,等.中国明对虾FcSGBP-HDL基因和启动子的克隆及序列特征分析[J].水产学报,2013,37(2):175-183.LAI Xiaofang,GAO Huan,KONG Jie,et al.Analysis of genomic structure and the promoter characterization of FcβGBPHDL fromFenneropenaeus chinensis[J].Journal of Fisheries of China,2013,37(2):175-183.
[21]陈文波,李文笙,林浩然.鲤胰岛素样生长因子结合蛋白-2和-3基因启动子克隆与序列分析[J].水产学报,2010,34(10):1469-1477.CHEN Wenbo,LI Wensheng,LIN Haoran.Cloning and analysis of insulin-like grow th factor binding protein-2and-3promoters in common carp(Cyprinus carpio)[J].Journal of Fisheries of China,2010,34(10):1469-1477.
[22]陈哲,雷明明,施振旦.猪SIM1基因启动子活性及表达模式分析[J].华北农学报,2015,30(2):6-11.CHEN Zhe,LEI Mingming,SHI Zhendan.Analysis of promoter activity of porcine SIM1gene and its expression pattern[J].Acta Agriculturae Boreali-Sinica,2015,30(2):6-11.
[23]ZAID A,LI R,LUCIAKOVA K,et a1.On the role of the general transcription factor Spl in the activation and repression of diverse mammalian oxidative phosphorylation genes[J].Journal of Bioenergetics and Biomembranes,1999,31(2):129-135.
[24]苏艳芳,付元帅,施志,等.牙鲆SRF基因的克隆及真核表达载体的构建[J].海洋渔业,2015,37(4):331-340.SU Yanfang,FU Yuanshuai,SHI Zhi,et al.Cloning of SRF from Paralichthy solivaceus and eukarytic expression vector construction[J].Marine Fisheries,2015,37(4):331-340.
[25]孟庆祥,王玲,姜容,等.干扰素保守序列结合蛋白在白血病中表达的研究[J].中华血液学杂志,2002,23(7):379-380.MENG Qingxiang,WANG Ling,JIANG Rong,et al.Interferon consensus sequence binding protein expression in leukemia[J].Chinese Journal of Hematology,2002,23(7):379-380.
[2]郝贵杰,王春琳,林锋,等.三疣梭子蟹血清凝集素的生物学特性[J].江苏农业科学,2015,43(1):220-223.HAO Guijie,WANG Chunlin,LIN Feng,et al.Biological characteristics of serum lectin from Portunus trituberculatus[J].Jiangsu Agricultural Sciences,2015,43(1):220-223.
[3]RUNSAENG P,THEPNARONG S,RATTANAPORN O,et al.Cloning and the mRNA expression of a C-type lectin with one carbohydrate recognition domain fromFenneropenaeus merguiensis in response to pathogenic inoculation[J].Molecular and Cellular Probes,2015(29):365-375.
[4]DAI Y J,WANG Y Q,ZHAO L L,et al.A novel L-type lectin was required for the multiplication of WSSV in red swamp crayfish(Procambarus clakii)[J].Fish&Shellfish Immunology,2016(55):48-55.
[5]LI C H,YU S X,ZHAO J M,et al.Cloning and characterization of a sialic acid binding lectins(SABL)from Manila clam Venerupis philippinarum[J].Fish&Shellfish Immunology,2011(30):1202-1206.
[6]曹剑香,简纪常,吴灶和.虾类体液免疫研究进展[J].湛江海洋大学学报,2006,26(1):89-93.CAO Jianxiang,JIAN Jichang,WU Zaohe.Shrimp humoral immunity research advances[J].Journal of Zhanjiang Ocean University,2006,26(1):89-93.
[7]孙杰,安利国,王雷,等.甲壳纲动物凝集素的结构特征和分离纯化方法[J].海洋科学,2006,30(6):73-76.SUN Jie,AN Liguo,WANG Lei,et al.Characterizations and purification methods of Lectins of crustaceans[J].Marine Scienes,2006,30(6):73-76.
[8]罗展,张继泉,李富花,等.凡纳滨对虾C-型凝集素LvLec2对不同刺激的免疫应答[J].海洋科学,2010,34(11):103-110.LUO Zhan,ZHANG Jiquan,LI Fuhua,et a1.Cloning of a novel C-type lectin LvLev2from the shrimp Litopenaeus vannamei and its immune response to different challenges[J].Marine Sciences,2010,34(11):103-110.
[9]HUANG Y,AN L,HUI K M,et a1.An LDLa domain-containing C-type lectin is involved in the innate immunity of Eriocheir sinensis[J].Developmental and Comparative Immunology,2014,42(2):333-344.
[10]JIN X K,GUO X N,LI S,et al.Association of a hepatopancreas-specific C-type lectin with the antibacterial response of Eriocheir sinens[J].PLOS One,2013,8(10):1-6.
[11]GUO H Z,ZOU P F,FU J P,et al.Characterization of two C-type lectin-like domain(CTLD)-containing proteins from the cDNA library of Chinese mitten crab Eriocheir sinensis[J].Fish and Shellfish Immunology,201l,30(2):515-524.
[12]GUO X N,JIN X K,LI S,et a1.A novel C-type lectin from Eriocheir sinensis functions as a pattern recognition receptor with antibacterial activity[J].Fish and Shellfish Immunology,2013,35(5):1554-l565.
[13]SUN Y M,LI F H,XIANG J H.Analysis on the dynamic changes of the amount of WSSV in Chinese shrimp Fenneropenaeus chinensis during infection[J].Aquaculture,2013(376/377/378/379):l24-132.
[14]LAI X F,GAO H,KONG J,et al.Genomic organization and promoter characterization of FcCTL,a C-type lectin-like protein in Fenneropenaeus chinensis[J].Gene,2013,518(2):376-380.
[15]王显伟.十足目甲壳动物C型凝集素功能研究[D].济南:山东大学,2012.WANG Xianwei.Functional study of C-type lectins from decapod crustaceans[D].Jinan:Shandong University,2012.
[16]HUANG X,LI W,JIN M,et al.Single CRD containing lectin from Macrobrachium rosenbergii(MrLec)participates in innate immunity against pathogen infections[J].Fish&Shellfish Immunology,2016(51):282-290.
[17]WANG H,SONG L S,LI C H,et al.Cloning and characterization of a novel C-type lectin from Zhikong scallop Chlamys farreri[J].Molecular Immunology,2007(44):722-731.
[18]WANG L L,WANG L L,ZHANG D X,et al.A novel C-type lectin from crab Eriocheir sinensis functions as pattern recognition receptor enhancing cellular encapsulation[J].Fish&Shellfish Immunology,2013(34):832-842.
[19]ZHANG HAO,CHEN L Q,QIN J G,et al.Molecular cloning,characterization and expression of a C-type lectin cDNA in Chinese mitten crab,Eriocheir sinensis[J].Fish&Shellfish Immunology,2011(31):358-363.
[20]赖晓芳,高焕,孔杰,等.中国明对虾FcSGBP-HDL基因和启动子的克隆及序列特征分析[J].水产学报,2013,37(2):175-183.LAI Xiaofang,GAO Huan,KONG Jie,et al.Analysis of genomic structure and the promoter characterization of FcβGBPHDL fromFenneropenaeus chinensis[J].Journal of Fisheries of China,2013,37(2):175-183.
[21]陈文波,李文笙,林浩然.鲤胰岛素样生长因子结合蛋白-2和-3基因启动子克隆与序列分析[J].水产学报,2010,34(10):1469-1477.CHEN Wenbo,LI Wensheng,LIN Haoran.Cloning and analysis of insulin-like grow th factor binding protein-2and-3promoters in common carp(Cyprinus carpio)[J].Journal of Fisheries of China,2010,34(10):1469-1477.
[22]陈哲,雷明明,施振旦.猪SIM1基因启动子活性及表达模式分析[J].华北农学报,2015,30(2):6-11.CHEN Zhe,LEI Mingming,SHI Zhendan.Analysis of promoter activity of porcine SIM1gene and its expression pattern[J].Acta Agriculturae Boreali-Sinica,2015,30(2):6-11.
[23]ZAID A,LI R,LUCIAKOVA K,et a1.On the role of the general transcription factor Spl in the activation and repression of diverse mammalian oxidative phosphorylation genes[J].Journal of Bioenergetics and Biomembranes,1999,31(2):129-135.
[24]苏艳芳,付元帅,施志,等.牙鲆SRF基因的克隆及真核表达载体的构建[J].海洋渔业,2015,37(4):331-340.SU Yanfang,FU Yuanshuai,SHI Zhi,et al.Cloning of SRF from Paralichthy solivaceus and eukarytic expression vector construction[J].Marine Fisheries,2015,37(4):331-340.
[25]孟庆祥,王玲,姜容,等.干扰素保守序列结合蛋白在白血病中表达的研究[J].中华血液学杂志,2002,23(7):379-380.MENG Qingxiang,WANG Ling,JIANG Rong,et al.Interferon consensus sequence binding protein expression in leukemia[J].Chinese Journal of Hematology,2002,23(7):379-380.