马氏珠母贝PmFAMeT基因的克隆及表达
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摘要
【目的】克隆马氏珠母贝(Pinctada martensii)法尼酸甲基转移酶(FAMe T)基因,并分析其在各组织中的表达。【方法】利用cDNA末端快速扩增技术(RACE)克隆获得马氏珠母贝FAMeT(PmFAMeT)基因的cDNA全长序列,利用实时荧光定量PCR(qPCR)方法分析PmFAMeT在马氏珠母贝不同组织中的表达模式。【结果与结论】PmFAMeT包含5′非编码区120 bp,3′非编码区968 bp和开放阅读框(ORF)1 536 bp,编码511个氨基酸。序列分析表明,PmFAMeT含有信号肽序列和跨膜结构域,并有WSC结构域、TSP1结构域和2个Methyltransf_FA结构域。将推导的PmFAMeT氨基酸序列与其他物种的FAMeT序列进行比对发现,不同物种的Methyltransf_FA序列同源性较高。PmFAMeT在马氏珠母贝的各个组织中均有表达,且在边缘膜、肝胰腺和性腺中的表达量显著高于其他组织。
【Objective】To clone the Farnesoic acid O-methyltransferase gene(PmFAMeT) and analyze its expression patterns in tissues of Pinctada martensii.【Method】The full-length cDNA sequence of PmFAMeT was cloned by rapid amplification of cDNA ends(RACE) and the expression of this gene was analyzed by RT-PCR.【Result and Conclusion】The PmFAMeT full-length cDNA contained 5′ UTR(120 bp),the 3′UTR(968 bp) and open reading frame(1 536 bp) which encoded 511 amino acids.Analysis of deduced amino acids showed that it contained a signal peptide and transmembrane domain,WSC domain,TSP1 domain and 2 Methyltransf_FA domain.Results from multi-sequence comparisons showed that the Methyltransf_FA domain of PmFAMeT has high homology compared with other species.PmFAMeT gene was expressed in the hepatopancreas,gonad,adductor muscle,marginal zone,pallial zone and central zone of P.martensii with higher expression level in the marginal zone,gonad and hepatopancreas(P<0.05).
【Objective】To clone the Farnesoic acid O-methyltransferase gene(PmFAMeT) and analyze its expression patterns in tissues of Pinctada martensii.【Method】The full-length cDNA sequence of PmFAMeT was cloned by rapid amplification of cDNA ends(RACE) and the expression of this gene was analyzed by RT-PCR.【Result and Conclusion】The PmFAMeT full-length cDNA contained 5′ UTR(120 bp),the 3′UTR(968 bp) and open reading frame(1 536 bp) which encoded 511 amino acids.Analysis of deduced amino acids showed that it contained a signal peptide and transmembrane domain,WSC domain,TSP1 domain and 2 Methyltransf_FA domain.Results from multi-sequence comparisons showed that the Methyltransf_FA domain of PmFAMeT has high homology compared with other species.PmFAMeT gene was expressed in the hepatopancreas,gonad,adductor muscle,marginal zone,pallial zone and central zone of P.martensii with higher expression level in the marginal zone,gonad and hepatopancreas(P<0.05).
引文
[1]GUNAWARDENE Y,TOBE S,BENDENA W,et al.Function and cellular localization of farnesoic acid O-methyltransferase(FAMeT)in the shrimp,Metapenaeus ensis[J].European Journal of Biochemistry,2002,269(14):3587-3595.
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[5]TAMONE S L,CHANG E S.Methyl farnesoate stimulates ecdysteroid secretion from crab Y-organs in vitro[J].General and Comparative Endocrinology,1993,89(3):425-432.
[6]HUI J,TOBE S,CHAN S M.Characterization of the putative farnesoic acid O-methyltransferase(LvFAMeT)c DNA from white shrimp,Litopenaeus vannamei:evidence for its role in molting[J].Peptides,2008,29(2):252-260.
[7]谢熙,朱冬发,崔晓雨,等.三疣梭子蟹FAMeT基因克隆及其在蜕皮周期中的表达水平[J].水产学报,2013,37(7):994-1001.
[8]谢熙.三疣梭子蟹卵巢发育过程中FAMeT和MIH基因表达变化[D].宁波:宁波大学,2013.
[9]夏西超.Kk-42对凡纳滨对虾生长发育相关基因时空表达的影响[D].新乡:河南师范大学,2011.
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[11]WANG Q H,HAO R J,ZHAO X X,et al.Identification of EGFR in pearl oyster(Pinctada fucata martensii)and correlation analysis of its expression and growth traits[J].Bioscience Biotechnology and Biochemistry,2018,82(7):1073-1080.
[12]HAO R J,ZHENG Z,WANG Q H,et al.Molecular and functional analysis of PmCHST1b in nacre formation of Pinctada fucata martensii[J].Comparative Biochemistry and Physiology.Part B,Biochemistry&Molecular Biology,2018,225:13-20.
[13]ZHENG Z,LIANG J L,HUANG R L,et al.Identification of a novel miR-146a from Pinctada martensii involved in the regulation of the inflammatory response[J].Fish&Shellfish Immunology,2016,54:40-45.
[14]HUANG J L,LI S G,LIU Y J,et al.Hemocytes in the extrapallial space of Pinctada fucata are involved in immunity and biomineralization[J].Scientific Reports,2018,8(1):4657.DOI:10.1038/s41598-018-22961-y
[15]郝瑞娟,郑哲,王庆恒,等.马氏珠母贝磺基转移酶Pm CHST9基因的分子特征与表达分析[J].基因组学与应用生物学,2015,34(11):2387-2394.
[16]TONG S M,CHEN Y,ZHU J,et al.Subcellular localization of five singular WSC domain-containing proteins and their roles in beauveria bassiana responses to stress cues and metal ions[J].Environmental Microbiology Reports,2016,8(2):295-304.
[17]ADAMS J.Thrombospondins:multifunctional regulators of cell interactions[J].Annual Review of Cell and Developmental Biology,2001,17:25-51.
[18]LAWLER J,HYNES R.The Structure of human thrombospondin,an adhesive glycoprotein with multiple calcium-binding sites and homologies with several different proteins[J].The Journal of Cell Biology,1986,103(5):1635-1648.
[19]SMITH K F,NOLAN K F,REID K B,et al.Neutron and X-ray scattering studies on the human complement protein properdin provide an analysis of the thrombospondin repeat[J].Biochemistry,1991,30(32):8000-8008.
[20]VIEIRA C U,BONETTI A M,SIMOES Z L P,et al.Farnesoic acid O-methyl transferase(FAMeT)isoforms:conserved traits and gene expression patterns related to caste differentiation in the stingless bee,Melipona Scutellaris[J].Archives of Insect Biochemistry and Physiology,2008,67(2):97-106.
[21]NAGARAJU G,REDDY P,REDDY P.In vitro methyl farnesoate secretion by mandibular organs isolated from different molt and reproductive stages of the crab Oziotelphusa senex senex[J].Fisheries Science,2006,72(2):410-414.
[22]NAGARAJU G,REDDY P,REDDY P.Mandibular organ:It's relation to body weight,sex,molt and reproduction in the crab,Oziotelphusa senex senex Fabricius(1791)[J].Aquaculture,2004,232:603-612.
[23]李琛,郝瑞娟,王庆恒,等.马氏珠母贝磺基转移酶Pm CHST11基因的分子特征与表达分析[J].基因组学与应用生物学,2017,36(1):150-157.
[24]傅郁.海湾扇贝(Argopecten irradians)外套膜组织学组织化学研究[D].青岛:中国海洋大学,2007.
[25]孙虎山,王宜艳,王平,等.栉孔扇贝外套膜和鳃粘液细胞的类型与分布[J].中国水产科学,2002,9(4):315-317.
[26]刘雅,郑哲,王庆恒,等.马氏珠母贝Pm-CTSC基因的克隆与表达分析[J].基因组学与应用生物学,2018,37(11):4723-4729.
[27]曹艳飞,曾森林,焦钰,等.马氏珠母贝(Pinctada fucata martensii)细胞粘附分子3(Pm-CAM3)基因的克隆与表达分析[J].基因组学与应用生物学,2017.[2017-09-25].http://kns.cnki.net/kcms/detail/45.1369.Q.20170924.1531.010.html
[28]吴羽媛,郭志颖,梁海鹰,等.马氏珠母贝Tlr6基因的克隆、序列分析与表达[J].水产学报,2017,41(11):1687-1698.
[29]雷倩楠,崔少峰,梁海鹰,等.马氏珠母贝TRAF7基因c DNA的克隆及表达分析[J].基因组学与应用生物学,2016,35(1):79-86.
[30]焦钰,杜晓东,王庆恒,等.马氏珠母贝IKKε同源基因的克隆及表达分析[J].基因组学与应用生物学,2014,33(2):266-272.
[31]WU Y Y,LIANG H Y,WANG Z X,et al.A novel toll-like receptor from the pearl oyster Pinctada fucata martensii is induced in response to stress[J].Comparative Biochemistry and Physiology B:Biochemistry&Molecular Biology,2017,214:19-26.
[32]孟勐,程道军,彭健,等.家蚕法尼酸甲基转移酶(FAMe T)基因的鉴定及表达模式分析[J].蚕业科学,2013,39(4):680-688.
[33]杜俊俏,刁晓平,郑鹏飞,等.芘暴露对马氏珠母贝鳃和肝胰腺抗氧化酶活性的影响[J].生态环境学报,2013,22(10):1711-1716.
[34]王志新,梁海鹰,杜晓东,等.马氏珠母贝热休克蛋白Hsp60基因的克隆与表达分析[J].广东海洋大学学报,2013,33(6):14-23.
[35]李嘉尧.红螯光壳螯虾(Cherax Quadricarinatus)卵黄发生的研究[D].上海:华东师范大学,2008.
[36]李志敏,李健,李吉涛,等.脊尾白虾(Exopalaemon carinicauda)FAMeT基因在卵巢发育周期中的表达分析[J].渔业科学进展,2016,37(1):46-51.
[2]NAGARAJU G.Is Methyl farnesoate a crustacean hormone?[J].Aquaculture,2007,272(1-4):39-54.
[3]LAUFER H,DEMIR N,PAN X J,et al.Methyl farnesoate controls adult male morphogenesis in the crayfish,Procambarus clarkii[J].Journal of Insect Physiology,2005,51(4):379-384.
[4]RODRIGUEZ E M,GRECO L,MEDESANI D A,et al.Effect of methyl farnesoate,alone and in combination with other hormones,on ovarian growth of the red swamp crayfish,Procambarus clarkii,during vitellogenesis[J].General and Comparative Endocrinology,2002,125(1):34-40.
[5]TAMONE S L,CHANG E S.Methyl farnesoate stimulates ecdysteroid secretion from crab Y-organs in vitro[J].General and Comparative Endocrinology,1993,89(3):425-432.
[6]HUI J,TOBE S,CHAN S M.Characterization of the putative farnesoic acid O-methyltransferase(LvFAMeT)c DNA from white shrimp,Litopenaeus vannamei:evidence for its role in molting[J].Peptides,2008,29(2):252-260.
[7]谢熙,朱冬发,崔晓雨,等.三疣梭子蟹FAMeT基因克隆及其在蜕皮周期中的表达水平[J].水产学报,2013,37(7):994-1001.
[8]谢熙.三疣梭子蟹卵巢发育过程中FAMeT和MIH基因表达变化[D].宁波:宁波大学,2013.
[9]夏西超.Kk-42对凡纳滨对虾生长发育相关基因时空表达的影响[D].新乡:河南师范大学,2011.
[10]DU X D,FAN G Y,JIAO Y,et al.The pearl oyster Pinctada fucata martensii genome and multi-omic analyses provide insights into biomineralization[J].Giga Science,2017,6(8):1-12.
[11]WANG Q H,HAO R J,ZHAO X X,et al.Identification of EGFR in pearl oyster(Pinctada fucata martensii)and correlation analysis of its expression and growth traits[J].Bioscience Biotechnology and Biochemistry,2018,82(7):1073-1080.
[12]HAO R J,ZHENG Z,WANG Q H,et al.Molecular and functional analysis of PmCHST1b in nacre formation of Pinctada fucata martensii[J].Comparative Biochemistry and Physiology.Part B,Biochemistry&Molecular Biology,2018,225:13-20.
[13]ZHENG Z,LIANG J L,HUANG R L,et al.Identification of a novel miR-146a from Pinctada martensii involved in the regulation of the inflammatory response[J].Fish&Shellfish Immunology,2016,54:40-45.
[14]HUANG J L,LI S G,LIU Y J,et al.Hemocytes in the extrapallial space of Pinctada fucata are involved in immunity and biomineralization[J].Scientific Reports,2018,8(1):4657.DOI:10.1038/s41598-018-22961-y
[15]郝瑞娟,郑哲,王庆恒,等.马氏珠母贝磺基转移酶Pm CHST9基因的分子特征与表达分析[J].基因组学与应用生物学,2015,34(11):2387-2394.
[16]TONG S M,CHEN Y,ZHU J,et al.Subcellular localization of five singular WSC domain-containing proteins and their roles in beauveria bassiana responses to stress cues and metal ions[J].Environmental Microbiology Reports,2016,8(2):295-304.
[17]ADAMS J.Thrombospondins:multifunctional regulators of cell interactions[J].Annual Review of Cell and Developmental Biology,2001,17:25-51.
[18]LAWLER J,HYNES R.The Structure of human thrombospondin,an adhesive glycoprotein with multiple calcium-binding sites and homologies with several different proteins[J].The Journal of Cell Biology,1986,103(5):1635-1648.
[19]SMITH K F,NOLAN K F,REID K B,et al.Neutron and X-ray scattering studies on the human complement protein properdin provide an analysis of the thrombospondin repeat[J].Biochemistry,1991,30(32):8000-8008.
[20]VIEIRA C U,BONETTI A M,SIMOES Z L P,et al.Farnesoic acid O-methyl transferase(FAMeT)isoforms:conserved traits and gene expression patterns related to caste differentiation in the stingless bee,Melipona Scutellaris[J].Archives of Insect Biochemistry and Physiology,2008,67(2):97-106.
[21]NAGARAJU G,REDDY P,REDDY P.In vitro methyl farnesoate secretion by mandibular organs isolated from different molt and reproductive stages of the crab Oziotelphusa senex senex[J].Fisheries Science,2006,72(2):410-414.
[22]NAGARAJU G,REDDY P,REDDY P.Mandibular organ:It's relation to body weight,sex,molt and reproduction in the crab,Oziotelphusa senex senex Fabricius(1791)[J].Aquaculture,2004,232:603-612.
[23]李琛,郝瑞娟,王庆恒,等.马氏珠母贝磺基转移酶Pm CHST11基因的分子特征与表达分析[J].基因组学与应用生物学,2017,36(1):150-157.
[24]傅郁.海湾扇贝(Argopecten irradians)外套膜组织学组织化学研究[D].青岛:中国海洋大学,2007.
[25]孙虎山,王宜艳,王平,等.栉孔扇贝外套膜和鳃粘液细胞的类型与分布[J].中国水产科学,2002,9(4):315-317.
[26]刘雅,郑哲,王庆恒,等.马氏珠母贝Pm-CTSC基因的克隆与表达分析[J].基因组学与应用生物学,2018,37(11):4723-4729.
[27]曹艳飞,曾森林,焦钰,等.马氏珠母贝(Pinctada fucata martensii)细胞粘附分子3(Pm-CAM3)基因的克隆与表达分析[J].基因组学与应用生物学,2017.[2017-09-25].http://kns.cnki.net/kcms/detail/45.1369.Q.20170924.1531.010.html
[28]吴羽媛,郭志颖,梁海鹰,等.马氏珠母贝Tlr6基因的克隆、序列分析与表达[J].水产学报,2017,41(11):1687-1698.
[29]雷倩楠,崔少峰,梁海鹰,等.马氏珠母贝TRAF7基因c DNA的克隆及表达分析[J].基因组学与应用生物学,2016,35(1):79-86.
[30]焦钰,杜晓东,王庆恒,等.马氏珠母贝IKKε同源基因的克隆及表达分析[J].基因组学与应用生物学,2014,33(2):266-272.
[31]WU Y Y,LIANG H Y,WANG Z X,et al.A novel toll-like receptor from the pearl oyster Pinctada fucata martensii is induced in response to stress[J].Comparative Biochemistry and Physiology B:Biochemistry&Molecular Biology,2017,214:19-26.
[32]孟勐,程道军,彭健,等.家蚕法尼酸甲基转移酶(FAMe T)基因的鉴定及表达模式分析[J].蚕业科学,2013,39(4):680-688.
[33]杜俊俏,刁晓平,郑鹏飞,等.芘暴露对马氏珠母贝鳃和肝胰腺抗氧化酶活性的影响[J].生态环境学报,2013,22(10):1711-1716.
[34]王志新,梁海鹰,杜晓东,等.马氏珠母贝热休克蛋白Hsp60基因的克隆与表达分析[J].广东海洋大学学报,2013,33(6):14-23.
[35]李嘉尧.红螯光壳螯虾(Cherax Quadricarinatus)卵黄发生的研究[D].上海:华东师范大学,2008.
[36]李志敏,李健,李吉涛,等.脊尾白虾(Exopalaemon carinicauda)FAMeT基因在卵巢发育周期中的表达分析[J].渔业科学进展,2016,37(1):46-51.