魁蚶C型凝集素基因cDNA的克隆及表达分析
|
摘要
通过cDNA末端快速扩增(Rapid amplification of c DNA ends,RACE)技术克隆得到魁蚶(Scapharca broughtonii)C型凝集素(C-type lectin,Sb-Lec1)基因,该基因全长为700 bp,其中,5′-UTR为29 bp,3′-UTR为167 bp,开放阅读框长度为504 bp,编码167个氨基酸,包括长度为23个氨基酸的信号肽序列、129个氨基酸的糖识别结构域(CRD)以及参与二硫键形成的6个半胱氨酸。预测蛋白分子量为19.11 k Da,理论等电点为4.74。多序列比对结果显示,Sb-Lec1基因CRD编码的氨基酸序列与长牡蛎(Crassostrea gigas)、紫贻贝(Mytilus galloprovincialis)和海湾扇贝(Argopecten irradians)C型凝集素的同源性分别为38%~40%、34%~35%和38%~39%,Sb-Lec1基因编码的氨基酸序列与其他物种的凝集素基因具有相似的结构,均含有形成二硫键的4个保守半胱氨酸。系统进化分析结果显示,魁蚶先与贝类聚为一支,再与脊椎动物聚在一起,表明魁蚶Sb-Lec1基因在进化树上的位置与其传统分类所处位置一致。采用荧光定量PCR技术,检测了Sb-Lec1基因在组织中的表达情况,发现其在肝胰腺、血淋巴、鳃、外套膜、闭壳肌、斧足中均有表达,其中,肝胰腺表达量最高。同时,分析了Sb-Lec1基因在鳗弧菌(Vibrio anguillarum)刺激下的mRNA表达量变化情况。结果显示,与对照组相比,菌刺激组Sb-Lec1基因mRNA在各检测组织中的表达量均显著上调(P<0.05),随着刺激时间的延长,表达量呈先升高后降低的趋势。本研究表明,魁蚶Sb-Lec1基因在机体免疫防御方面发挥重要功能。
The current study cloned the full-length cDNA of C-type lectin(Sb-Lec1) using RACE(Rapid amplification of c DNA ends) method from Scapharca broughtonii with 700 bp that includes a 5′ UTR of 29 bp and 3′ UTR of 167 bp. The 504 bp open reading frame(ORF) encodes a polypeptide of 167 amino acids, including a signal peptide of 23 amino acids, one carbohydrate-recognition domain(CRD) motif of 129 amino acids and 6 cysteines involved in the formation of disulfide bond. The predicted protein molecular weight is 19.11 k Da, with a theory isoelectric point of 4.74. Multiple sequences alignment and phylogeny analysis showed that the identity of Sb-Lec1 gene shared with Crassostrea gigas, Mytilus galloprovincialis, and Argopecten irradians was 38%~40%, 34%~35%, and 38%~39%, respectively. The amino acids of CRD motif had many similarities with other species such as 4 conserved Cys. Phylogenetic analysis revealed two main branches including all C-type lectin of molluscs and the C-type lectin of vertebrate, and that the deduced polypeptide of Sb-Lec1 had the characteristics of the C-type lectin family. Quantitative real-time PCR(q RT-PCR) was used to assess the m RNA expression in all tested tissues, including hemocytes, foot, adductor muscle, mantle, gill, and hepatopancreas. The highest and lowest Sb-Lec1 m RNA were in hepatopancreas and adductor muscle, respectively. Vibrio anguillarumchallange induced Sb-Lec1 m RNA expression in all tested tissues(P<0.05). These results showed that Sb-Lec1 gene may play an important role in immune defense.
The current study cloned the full-length cDNA of C-type lectin(Sb-Lec1) using RACE(Rapid amplification of c DNA ends) method from Scapharca broughtonii with 700 bp that includes a 5′ UTR of 29 bp and 3′ UTR of 167 bp. The 504 bp open reading frame(ORF) encodes a polypeptide of 167 amino acids, including a signal peptide of 23 amino acids, one carbohydrate-recognition domain(CRD) motif of 129 amino acids and 6 cysteines involved in the formation of disulfide bond. The predicted protein molecular weight is 19.11 k Da, with a theory isoelectric point of 4.74. Multiple sequences alignment and phylogeny analysis showed that the identity of Sb-Lec1 gene shared with Crassostrea gigas, Mytilus galloprovincialis, and Argopecten irradians was 38%~40%, 34%~35%, and 38%~39%, respectively. The amino acids of CRD motif had many similarities with other species such as 4 conserved Cys. Phylogenetic analysis revealed two main branches including all C-type lectin of molluscs and the C-type lectin of vertebrate, and that the deduced polypeptide of Sb-Lec1 had the characteristics of the C-type lectin family. Quantitative real-time PCR(q RT-PCR) was used to assess the m RNA expression in all tested tissues, including hemocytes, foot, adductor muscle, mantle, gill, and hepatopancreas. The highest and lowest Sb-Lec1 m RNA were in hepatopancreas and adductor muscle, respectively. Vibrio anguillarumchallange induced Sb-Lec1 m RNA expression in all tested tissues(P<0.05). These results showed that Sb-Lec1 gene may play an important role in immune defense.
引文
Chen ZL.Mammalian C-type lectin superfamily.Progress in Biochemistry and Biophysics,1997,24(6):491-496[陈政良.哺乳类C型凝集素超级家族.生物化学与生物物理进展,1997,24(6):491-496]
Drickamer K,Taylor M.Biology of animal lectins.Annual Review of Cell Biology,1993,9:237-264
Hu YT,Zhang DC,Cui SG,et al.Seauence features and functional analysis of the C-type lectin gene(Po LEC1)from pearl oyster Pinctada fucata.Journal of Fisheries of China,2011,35(9):1327-1336[胡钰婷,张殿昌,崔淑歌,等.合浦珠母贝C-型凝集素基因的序列特征和功能分析.水产学报,2011,35(9):1327-1336]
Huang YH,Liu ZH,Wu B,et al.Gene cloning and expression analysis of catalase in Scapharca broughtonii.Journal of Fisheries of China,2016,40(6):856-866[黄永欢,刘志鸿,吴彪,等.魁蚶过氧化氢酶基因克隆及表达分析.水产学报,2016,40(6):856-866]
Kolatkar AR,Weis WI.Structural basis of galactose recognition by C-type animal lectins.Journal of Biological Chemistry,1996,271:6679-6685
Li M,Zhou SM,Liu L,et al.Molecular clone and expression of C-type lectin in Meretrix meretrix.Oceanologia et Limnologia Sinica,2015,46(5):1186-1192[李猛,周素明,刘璐,等.文蛤(Meretrix meretrix)C-型凝集素基因的分子克隆及表达分析.海洋与湖沼,2015,46(5):1186-1192]
Li M,Zhu L,Zhou C Y,et al.Molecular characterization and expression of a novel big defensin(Sb-BDef1)from ark shell,Scapharca broughtonii.Fish and Shellfish Immunology,2012,33(5):1167-1173
Liu HM,Wu B,Liu ZH,et al.Genetic diversity and geographic population structures of Scapharca broughtonii.Progress in Fishery Sciences,2017,38(6):92-99[刘寒苗,吴彪,刘志鸿,等.魁蚶(Scapharca broughtonii)不同地理群体的遗传多样性及种群结构.渔业科学进展,2017,38(6):92-99]
Livak KJ,Schmittgen TD.Analysis of relative gene expression data using real-time quantitative PCR and the 2-(35)(35)CTmethod.Methods,2001,25(4):402-408
Mao YZ,Zhou CY,Zhu L,et al.Identification and expression analysis on bactericidal permeability-increasing protein(BPI)/lipopolysaccharide-binding protein(LBP)of ark shell,Scapharca broughtonii.Fish and Shellfish Immunology,2013,35(3):642-652
Robinson MJ,Sancho D,Slack EC,et al.Myeloid C-type lectins in innate immunity.Nature Immunology,2006,7(12):1258-1265
Tamplin ML,Fosher WS.Occurrence and characteristics of agglutination of Vibrio cholerae by serum from the eastern oyster,Crassostrea virginica.Applied and Environmental Microbiology,1989,55(11):2882-2887
Tang QS,Qiu XY,Wang J,et al.Resource enhancement of arkshell(Scapharca(Anadara)broughtonii)in Shandong offshore waters.Chinese Journal of Applied Ecology,1994,5(4):396-402[唐启升,邱显寅,王俊,等.山东近海魁蚶资源增殖的研究.应用生态学报,1994,5(4):396-402]
Wang H,Song LS,Li CH,et al.Cloning and characterization of a novel C-type lectin from Zhikong scallop Chlamys farreri.Molecular Immunology,2007,44(5):722-731
Wang S.Study on the selection of reference genes and the expression regulation of C-type lectin gene in Haliotis discus hannai.Master′s Thesis of Shanghai Ocean University,2015,43-45[王圣.皱纹盘鲍内参基因筛选及C型凝集素基因表达规律的研究.上海海洋大学硕士研究生学位论文,2015,43-45]
Xing JX,Liu YJ,Lu YP,et al.Cloning and functional analysis of C-type lectin from Musca domestica(Diptera:Muscidae).Acta Entomologica Sinica,2016,59(1):16-24[邢建晓,刘艳娟,路永鹏,等.家蝇C-型凝集素的克隆与功能分析.昆虫学报,2016,59(1):16-24]
Xu W,Wang H,Song LS,et al.c DNA Cloning and m RNAexpression of C-type lectin from scallop Chlamys farreri.Chinese High Technology Letters,2005,15(1):83-88[胥炜,王昊,宋林生,等.栉孔扇贝C型凝集素基因的克隆与表达研究.高技术通讯,2005,15(1):83-88]
Zelensky AN,Gready JE.The C-type lectin-like domains superfamily.FEBS Jounal,2005,272(24):6179-6217
Zhang H.Study on the genes and their functions of the complement-like components from scallops.Doctoral Dissertation of Institute of Oceanology,Graduate School of Chinese Academy of Sciences,2010,17-20[张峘.扇贝补体样成分的基因及其功能研究.中国科学院研究生院(海洋研究所)博士研究生学位论文,2010,17-20]
Zheng LB,Liu ZH,Wu B,et al.Ferritin has an important immune function in the ark shell Scapharca broughtonii.Developmental and Comparative Immunology,2016,59:15-24
Zheng LB,Wu B,Liu ZH,et al.A manganese superoxide dismutase(Mn SOD)from ark shell,Scapharca broughtonii:Molecular characterization,expression and immune activity analysis.Fish and Shellfish Immunology,2015,45(2):656-665
Zheng LB,Wu B,Liu ZH,et al.Cloning and expression analysis of galectin from Scapharca broughtonii(Sb Gal).Oceanologia et Limnologia Sinica,2015,46(5):1061-1070[郑利兵,吴彪,刘志鸿,等.魁蚶(Scapharca broughtonii)半乳糖凝集素(Sb Gal)基因c DNA的克隆及表达分析.海洋与湖沼,2015,46(5):1061-1070]
Zhou LQ,Yang AG,Wang QY,et al.Studies on the hemocytes types and their immunological functions in bloody clam(Scapharca broughtonii).Journal of Fisheries of China,2013,37(4):599-606[周丽青,杨爱国,王清印,等.魁蚶血细胞分类及其免疫功能的初步分析.水产学报,2013,37(4):599-606]
Zhu L,Song LS,Xu W,et al.Identification of a C-type lectin from the bay scallop Argopecten irradians.Molecular Biology Reports,2009,36(5):1167-1173
Zhu L,Song LS,Xu W,et al.Molecular cloning and immune responsive expression of a novel C-type lectin gene from bay scallop Argopecten irradians.Fish and Shellfish Immunology,2008,25(3):231-238
Drickamer K,Taylor M.Biology of animal lectins.Annual Review of Cell Biology,1993,9:237-264
Hu YT,Zhang DC,Cui SG,et al.Seauence features and functional analysis of the C-type lectin gene(Po LEC1)from pearl oyster Pinctada fucata.Journal of Fisheries of China,2011,35(9):1327-1336[胡钰婷,张殿昌,崔淑歌,等.合浦珠母贝C-型凝集素基因的序列特征和功能分析.水产学报,2011,35(9):1327-1336]
Huang YH,Liu ZH,Wu B,et al.Gene cloning and expression analysis of catalase in Scapharca broughtonii.Journal of Fisheries of China,2016,40(6):856-866[黄永欢,刘志鸿,吴彪,等.魁蚶过氧化氢酶基因克隆及表达分析.水产学报,2016,40(6):856-866]
Kolatkar AR,Weis WI.Structural basis of galactose recognition by C-type animal lectins.Journal of Biological Chemistry,1996,271:6679-6685
Li M,Zhou SM,Liu L,et al.Molecular clone and expression of C-type lectin in Meretrix meretrix.Oceanologia et Limnologia Sinica,2015,46(5):1186-1192[李猛,周素明,刘璐,等.文蛤(Meretrix meretrix)C-型凝集素基因的分子克隆及表达分析.海洋与湖沼,2015,46(5):1186-1192]
Li M,Zhu L,Zhou C Y,et al.Molecular characterization and expression of a novel big defensin(Sb-BDef1)from ark shell,Scapharca broughtonii.Fish and Shellfish Immunology,2012,33(5):1167-1173
Liu HM,Wu B,Liu ZH,et al.Genetic diversity and geographic population structures of Scapharca broughtonii.Progress in Fishery Sciences,2017,38(6):92-99[刘寒苗,吴彪,刘志鸿,等.魁蚶(Scapharca broughtonii)不同地理群体的遗传多样性及种群结构.渔业科学进展,2017,38(6):92-99]
Livak KJ,Schmittgen TD.Analysis of relative gene expression data using real-time quantitative PCR and the 2-(35)(35)CTmethod.Methods,2001,25(4):402-408
Mao YZ,Zhou CY,Zhu L,et al.Identification and expression analysis on bactericidal permeability-increasing protein(BPI)/lipopolysaccharide-binding protein(LBP)of ark shell,Scapharca broughtonii.Fish and Shellfish Immunology,2013,35(3):642-652
Robinson MJ,Sancho D,Slack EC,et al.Myeloid C-type lectins in innate immunity.Nature Immunology,2006,7(12):1258-1265
Tamplin ML,Fosher WS.Occurrence and characteristics of agglutination of Vibrio cholerae by serum from the eastern oyster,Crassostrea virginica.Applied and Environmental Microbiology,1989,55(11):2882-2887
Tang QS,Qiu XY,Wang J,et al.Resource enhancement of arkshell(Scapharca(Anadara)broughtonii)in Shandong offshore waters.Chinese Journal of Applied Ecology,1994,5(4):396-402[唐启升,邱显寅,王俊,等.山东近海魁蚶资源增殖的研究.应用生态学报,1994,5(4):396-402]
Wang H,Song LS,Li CH,et al.Cloning and characterization of a novel C-type lectin from Zhikong scallop Chlamys farreri.Molecular Immunology,2007,44(5):722-731
Wang S.Study on the selection of reference genes and the expression regulation of C-type lectin gene in Haliotis discus hannai.Master′s Thesis of Shanghai Ocean University,2015,43-45[王圣.皱纹盘鲍内参基因筛选及C型凝集素基因表达规律的研究.上海海洋大学硕士研究生学位论文,2015,43-45]
Xing JX,Liu YJ,Lu YP,et al.Cloning and functional analysis of C-type lectin from Musca domestica(Diptera:Muscidae).Acta Entomologica Sinica,2016,59(1):16-24[邢建晓,刘艳娟,路永鹏,等.家蝇C-型凝集素的克隆与功能分析.昆虫学报,2016,59(1):16-24]
Xu W,Wang H,Song LS,et al.c DNA Cloning and m RNAexpression of C-type lectin from scallop Chlamys farreri.Chinese High Technology Letters,2005,15(1):83-88[胥炜,王昊,宋林生,等.栉孔扇贝C型凝集素基因的克隆与表达研究.高技术通讯,2005,15(1):83-88]
Zelensky AN,Gready JE.The C-type lectin-like domains superfamily.FEBS Jounal,2005,272(24):6179-6217
Zhang H.Study on the genes and their functions of the complement-like components from scallops.Doctoral Dissertation of Institute of Oceanology,Graduate School of Chinese Academy of Sciences,2010,17-20[张峘.扇贝补体样成分的基因及其功能研究.中国科学院研究生院(海洋研究所)博士研究生学位论文,2010,17-20]
Zheng LB,Liu ZH,Wu B,et al.Ferritin has an important immune function in the ark shell Scapharca broughtonii.Developmental and Comparative Immunology,2016,59:15-24
Zheng LB,Wu B,Liu ZH,et al.A manganese superoxide dismutase(Mn SOD)from ark shell,Scapharca broughtonii:Molecular characterization,expression and immune activity analysis.Fish and Shellfish Immunology,2015,45(2):656-665
Zheng LB,Wu B,Liu ZH,et al.Cloning and expression analysis of galectin from Scapharca broughtonii(Sb Gal).Oceanologia et Limnologia Sinica,2015,46(5):1061-1070[郑利兵,吴彪,刘志鸿,等.魁蚶(Scapharca broughtonii)半乳糖凝集素(Sb Gal)基因c DNA的克隆及表达分析.海洋与湖沼,2015,46(5):1061-1070]
Zhou LQ,Yang AG,Wang QY,et al.Studies on the hemocytes types and their immunological functions in bloody clam(Scapharca broughtonii).Journal of Fisheries of China,2013,37(4):599-606[周丽青,杨爱国,王清印,等.魁蚶血细胞分类及其免疫功能的初步分析.水产学报,2013,37(4):599-606]
Zhu L,Song LS,Xu W,et al.Identification of a C-type lectin from the bay scallop Argopecten irradians.Molecular Biology Reports,2009,36(5):1167-1173
Zhu L,Song LS,Xu W,et al.Molecular cloning and immune responsive expression of a novel C-type lectin gene from bay scallop Argopecten irradians.Fish and Shellfish Immunology,2008,25(3):231-238