企鹅珍珠贝Dmrt2基因的克隆及表达分析
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摘要
本研究利用RACE-PCR技术获得企鹅珍珠贝(Pteria penguin)一个Dmrt2基因cDNA的全长序列,通过荧光定量PCR分析Dmrt2基因在各组织中的表达特征,以及在早期卵巢、成熟期卵巢、早期精巢、成熟期精巢和排放期精巢中的表达变化。结果表明,Dmrt2基因全长1 257bp,其中开放阅读框(open reading frame,ORF)为951 bp,编码316个氨基酸,5′非编码区(untranslated region,UTR)为52 bp,3′UTR为254 bp,第20位到第73位氨基酸为DM结构域。预测其分子质量为36.61ku,等电点为9.80。氨基酸序列比对显示该企鹅珍珠贝Dmrt2基因与黑蝶真珠蛤(Pinctada margaritifera)和马氏珠母贝(Pinctada martensii)的Dmrt2基因同源性(identity)最高,分别为46.0%和45.7%。其中在DM结构域高度同源。荧光定量PCR分析组织表达特征显示,Dmrt2在企鹅珍珠贝的外套膜、鳃、消化盲囊、足、精巢和卵巢均有表达,其中在精巢中表达量最高(P<0.05),足为其次,在闭壳肌中没有检测到Dmrt2表达。对性腺发育不同时期的表达量分析发现,Dmrt2在发育早期和成熟期卵巢中表达量都很低,在发育早期精巢中表达量较高,在成熟期精巢检测到最大表达量(P<0.05),到精巢排放期表达显著下降,推测Dmrt2可能与企鹅珍珠贝精巢的发育有关,可能参与了企鹅珍珠贝雄性性别分化和性腺发育这一生理过程。
Dmrt2(doublesex and mab-3-related transcription factor 2) is an important member of the Dmrt gene family with a highly conserved zinc finger-like DM domain and plays an important role in somite formation,organ formation,and skeletal patterning.This study was conducted to investigate the role of Dmrt2 in sex determination and gonadal development in Pteria penguin.The full-length cDNA of Dmrt2 gene was characterized from P.penguin by RACE-PCR,and real-time PCR was performed to assess the expression of Dmrt2 in different tissues and gonads of different growth periods.The results showed that the full-length cDNA of Dmrt2 was 1257 bp,including a 5′UTR of 52 bp,a 3′UTR of 254 bp,and an open reading frame of 951 bp,which encoded a deduced protein of 317 amino acids.The DM domain was from 20 to 73 amino acids.The predicted molecular weight was 36.61 ku,and the isoelectric point was 9.80.The amino acid sequence alignment showed that Dmrt2 of P.penguin shared 46.0% and 45.7% sequence identity with Dmrt2 of Pinctada margaritifera and Pinctada martensii,respectively.The real-time PCR results showed that Dmrt2 was constitutively expressed in the mantle,gill,digestive diverticulum,foot,testis,and ovary,except in the adductor muscle.The expression level was highest in the testis(P < 0.05),followed by the foot.The expression analysis in gonads of different growth periods revealed that Dmrt2 had lower expression in early ovary and mature ovary,higher expression in early testis and emission testis,and the highest expression in mature testis(P < 0.05).This study showed that pp Dmrt2 might play a role in sex determination and gonadal development in P.penguin.
Dmrt2(doublesex and mab-3-related transcription factor 2) is an important member of the Dmrt gene family with a highly conserved zinc finger-like DM domain and plays an important role in somite formation,organ formation,and skeletal patterning.This study was conducted to investigate the role of Dmrt2 in sex determination and gonadal development in Pteria penguin.The full-length cDNA of Dmrt2 gene was characterized from P.penguin by RACE-PCR,and real-time PCR was performed to assess the expression of Dmrt2 in different tissues and gonads of different growth periods.The results showed that the full-length cDNA of Dmrt2 was 1257 bp,including a 5′UTR of 52 bp,a 3′UTR of 254 bp,and an open reading frame of 951 bp,which encoded a deduced protein of 317 amino acids.The DM domain was from 20 to 73 amino acids.The predicted molecular weight was 36.61 ku,and the isoelectric point was 9.80.The amino acid sequence alignment showed that Dmrt2 of P.penguin shared 46.0% and 45.7% sequence identity with Dmrt2 of Pinctada margaritifera and Pinctada martensii,respectively.The real-time PCR results showed that Dmrt2 was constitutively expressed in the mantle,gill,digestive diverticulum,foot,testis,and ovary,except in the adductor muscle.The expression level was highest in the testis(P < 0.05),followed by the foot.The expression analysis in gonads of different growth periods revealed that Dmrt2 had lower expression in early ovary and mature ovary,higher expression in early testis and emission testis,and the highest expression in mature testis(P < 0.05).This study showed that pp Dmrt2 might play a role in sex determination and gonadal development in P.penguin.
引文
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[8]Matsui T,Sasaki A,Akazawa N,et al.Celf1 regulation of dmrt2a is required for somite symmetry and left-right patterning during zebrafish development[J].Development,2012,139(19):3553-3560.
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[10]Matsushita Y,Oshima Y,Nakamura M.Expression of DMRT genes in the gonads of Rana rugosa during sex determination[J].Zoological Science,2007,24(1):95-99.
[11]Yu F F,Wang M F,Zhou L,et al.Molecular Cloning and Expression Characterization of Dmrt2 in Akoya Pearl Oysters,Pinctada martensii[J].Journal of Shellfish Research,2011,30(2):247-254.
[12]蒙钊美,李有宁,邢孔武.珍珠养殖理论与技术[M].北京:科学出版社,1996:39-41.Meng Zhaomei,Li Youning,Xing Kongwu.The theory and technoligy of the pearl cultivation[M].Beijing:Science Press,1996:39-41.
[13]冯永勤,梁盛,梁晓春,等.企鹅珍珠贝游离有核珍珠培育技术[J].水产科技情报,2016,43(1):10-14.Feng Yongqin,Liang Sheng,Liang Xiaochun,et al.The cultivation technology of round pearl in Pteria penguin[J].Fisheries Science&Technology Infomation,2016,43(1):10-14.
[14]Wexler J R,Plachetzki D C,Kopp A.Pan-metazoan phylogeny of the DMRT gene family:a framework for functional studies[J].Development Genes and Evolution,2014,224(3):175-181.
[15]Hong C S,Park B Y,Saint J P.The function of Dmrt genes in vertebrate development it is not just about sex[J].Developmental Biology,2007,10(1):1-9.
[16]汪海,王婷茹,袁静,等.脊椎动物Dmrt基因的研究进展[J].贵州农业科学,2012,40(5):148-152.Wang Hai,Wang Tingru,Yuan Jing,et al.Advances in Dmrt gene family of vertebrate[J].Guizhou Agricultural Sciences,2012,40(5):148-152.
[17]Wang Z,Miyake T,Edwards S V,et al.Tuatara(Sphenodon)genomics BAC library construction,sequence survey,and application to the DMRT gene family[J].Journal of Heredity,2006,97(6):541–548.
[18]Bewick A J,Anderson D W,Evans B J.Evolution of the closely related,sex-related genes DM-W and DMRT1in African clawed frogs(Xenopus)[J].Evolution,2011,65(3):698-712.
[19]曹谨玲,陈剑杰,甘西.鱼类DMRT基因的研究进展[J].广东海洋大学学报,2011,31(01):94-98.Cao Jinling,Chen Jianjie,Gan Xi.Advances in the DMRT genes research of fishes[J].Journal of Guangdong Ocean University,2011,31(1):94-98.
[20]李法君,付春鹏,罗永巨.Dmrt基因在水生生物中的研究进展[J].水生生物学报,2016,40(5):1068-1077.Li Fajun,Fu Chunpeng,Luo Yongju.Research progress of Dmrt genes in hydrobiontes[J].Acta Hydrobiologica Sinica,2016,40(5):1068-1077.
[21]Wang F,Yu Y,Ji D,et al.The DMRT gene family in amphioxus[J].Journal of Biomolecular Structure and Dynamics,2012,30(2):191-200.
[22]许宝红,肖真明,肖调义,等.大鲵Dmrt2基因c DNA的克隆与表达分析[J].水产学报,2012,36(8):1179-1184.Xu Baohong,Xiao Zhenming,Xiao Diaoyi,et al.Molecular cloning and expression analysis of Dmrt2 gene in Andrias davidianus[J].Journal of Fisheries of China,2012,36(8):1179-1184.
[23]Yamaguchi A,Lee K H,Fujimoto H,et al.Expression of the DMRT gene and its roles in early gonadal development of the Japanese pufferfish Takifugu rubripes[J].Comparative Biochemistry and Physiology Part D:Genomics Proteomics,2006,1(1):59-68.
[24]Zhou X,Li Q,Lu H,et al.Fish specific duplication of Dmrt2:Characterization of zebrafish Dmrt2b[J].Biochimie,2009,90(6):878-887.
[25]Winkler C,Hornung U,Kondo M,et al.Developmentally regulated and non-sex-specific expression of autosomal dmrt genes in embryos of the Medaka fish(Oryzias latipes)[J].Mechanisms of Development,2004,121(7-8):997-1005.
[26]Teaniniaraitemoana V,Huvet A,Levy P,et al.Gonad transcriptome analysis of pearl oyster Pinctada margaritifera identification of potential sex differentiation and sex determining genes[J].Bio Med Central Genomics,2014,15(1):1-20.
[27]杨东,余来宁.鱼类性别与性别鉴定[J].水生生物学报,2006,30(2):221-226.Yang Dong,Yu Laining.Sex and sex identification of fish[J].Acta Hydrobiologica Sinica,2006,30(2):221-226.
[28]Osawa N,Oshima Y,Nakamura M.Molecular cloning of Dmrt1 and its expression in the gonad of Xenopus[J].Zoological Science,2005,22(6):681-687.
[29]Sreenivasulu K,Ganesh S,Raman R.Evolutionarily conserved,DMRT1,encodes alternatively spliced transcripts and shows dimorphic expression during gonadal differentiation in the lizard,Calotes versicolor[J].Mechanisms of Development,2002,119 Suppl 1(7574):S55.
[30]Shetty S,Kirby P,Zarkower D,et al.DMRT1 in a ratite bird:evidence for a role in sex determination and discovery of a putative regulatory element[J].Cytogenetic&Genome Research,2002,99(1-4):245-251.
[31]Pask A J,Behringer R R,Renfree M B.Expression of DMRT1 in the mammalian ovary and testis--from marsupials to mice[J].Cytogenetic&Genome Research,2003,101(3-4):229-236.
[32]梁象秋,方纪祖,杨和荃.水生生物学(形态和分类)[M].北京:中国农业出版社,2002:250-251.Liang Xiangqiu,Fang Jizu,Yang Hequan.Aquatic biology(morphology and classification)[M].Beijing:China Agricuture Press,2002:250-251.
[2]Picard M A,Cosseau C,Mouahid G,et al.The roles of Dmrt(Double sex/Male-abnormal-3 Related Transcription factor)genes in sex determination and differentiation mechanisms:Ubiquity and diversity across the animal kingdom[J].Comptes Rendus Biologies,2015,338(7):451-462.
[3]Erdman S E,Burtis K C.The Drosophila doublesex proteins share a novel zinc finger related DNA binding domain[J].The EMBO Journal,1993,12(2):527-535.
[4]Matson C K,Zarkower D.Sex and the singular DM domain:insights into sexual regulation,evolution and plasticity[J].Nature reviews.Genetics,2012,13(3):163-174.
[5]Ottolenghi C,Veitia R,Barbieri M,et al.The human doublesex-related gene,Dmrt2,Is homologous to a gene involved in somitogenesis and encodes a potential bicistronic transcript[J].Genomics,2000,64(2):179-186.
[6]Sato T,Rocancourt D,Marques L,et al.A Pax3/Dmrt2/Myf5 regulatory cascade functions at the onset of myogenesis[J].PLOS Genetics,2010,6(4):e1000897.
[7]Liu S,Li Z,Gui J F.Fish-specific duplicated dmrt2b contributes to a divergent function through Hedgehog pathway and maintains left-right asymmetry establishment function[J].PLOS One,2009,4(9):e7261.
[8]Matsui T,Sasaki A,Akazawa N,et al.Celf1 regulation of dmrt2a is required for somite symmetry and left-right patterning during zebrafish development[J].Development,2012,139(19):3553-3560.
[9]Quinonez S C,Park J M,Rabah R,et al.9p partial monosomy and disorders of sex development:Review and postulation of a pathogenetic[J].American Journal of Medical Genetics Part A,2013,161(8):1882-1896.
[10]Matsushita Y,Oshima Y,Nakamura M.Expression of DMRT genes in the gonads of Rana rugosa during sex determination[J].Zoological Science,2007,24(1):95-99.
[11]Yu F F,Wang M F,Zhou L,et al.Molecular Cloning and Expression Characterization of Dmrt2 in Akoya Pearl Oysters,Pinctada martensii[J].Journal of Shellfish Research,2011,30(2):247-254.
[12]蒙钊美,李有宁,邢孔武.珍珠养殖理论与技术[M].北京:科学出版社,1996:39-41.Meng Zhaomei,Li Youning,Xing Kongwu.The theory and technoligy of the pearl cultivation[M].Beijing:Science Press,1996:39-41.
[13]冯永勤,梁盛,梁晓春,等.企鹅珍珠贝游离有核珍珠培育技术[J].水产科技情报,2016,43(1):10-14.Feng Yongqin,Liang Sheng,Liang Xiaochun,et al.The cultivation technology of round pearl in Pteria penguin[J].Fisheries Science&Technology Infomation,2016,43(1):10-14.
[14]Wexler J R,Plachetzki D C,Kopp A.Pan-metazoan phylogeny of the DMRT gene family:a framework for functional studies[J].Development Genes and Evolution,2014,224(3):175-181.
[15]Hong C S,Park B Y,Saint J P.The function of Dmrt genes in vertebrate development it is not just about sex[J].Developmental Biology,2007,10(1):1-9.
[16]汪海,王婷茹,袁静,等.脊椎动物Dmrt基因的研究进展[J].贵州农业科学,2012,40(5):148-152.Wang Hai,Wang Tingru,Yuan Jing,et al.Advances in Dmrt gene family of vertebrate[J].Guizhou Agricultural Sciences,2012,40(5):148-152.
[17]Wang Z,Miyake T,Edwards S V,et al.Tuatara(Sphenodon)genomics BAC library construction,sequence survey,and application to the DMRT gene family[J].Journal of Heredity,2006,97(6):541–548.
[18]Bewick A J,Anderson D W,Evans B J.Evolution of the closely related,sex-related genes DM-W and DMRT1in African clawed frogs(Xenopus)[J].Evolution,2011,65(3):698-712.
[19]曹谨玲,陈剑杰,甘西.鱼类DMRT基因的研究进展[J].广东海洋大学学报,2011,31(01):94-98.Cao Jinling,Chen Jianjie,Gan Xi.Advances in the DMRT genes research of fishes[J].Journal of Guangdong Ocean University,2011,31(1):94-98.
[20]李法君,付春鹏,罗永巨.Dmrt基因在水生生物中的研究进展[J].水生生物学报,2016,40(5):1068-1077.Li Fajun,Fu Chunpeng,Luo Yongju.Research progress of Dmrt genes in hydrobiontes[J].Acta Hydrobiologica Sinica,2016,40(5):1068-1077.
[21]Wang F,Yu Y,Ji D,et al.The DMRT gene family in amphioxus[J].Journal of Biomolecular Structure and Dynamics,2012,30(2):191-200.
[22]许宝红,肖真明,肖调义,等.大鲵Dmrt2基因c DNA的克隆与表达分析[J].水产学报,2012,36(8):1179-1184.Xu Baohong,Xiao Zhenming,Xiao Diaoyi,et al.Molecular cloning and expression analysis of Dmrt2 gene in Andrias davidianus[J].Journal of Fisheries of China,2012,36(8):1179-1184.
[23]Yamaguchi A,Lee K H,Fujimoto H,et al.Expression of the DMRT gene and its roles in early gonadal development of the Japanese pufferfish Takifugu rubripes[J].Comparative Biochemistry and Physiology Part D:Genomics Proteomics,2006,1(1):59-68.
[24]Zhou X,Li Q,Lu H,et al.Fish specific duplication of Dmrt2:Characterization of zebrafish Dmrt2b[J].Biochimie,2009,90(6):878-887.
[25]Winkler C,Hornung U,Kondo M,et al.Developmentally regulated and non-sex-specific expression of autosomal dmrt genes in embryos of the Medaka fish(Oryzias latipes)[J].Mechanisms of Development,2004,121(7-8):997-1005.
[26]Teaniniaraitemoana V,Huvet A,Levy P,et al.Gonad transcriptome analysis of pearl oyster Pinctada margaritifera identification of potential sex differentiation and sex determining genes[J].Bio Med Central Genomics,2014,15(1):1-20.
[27]杨东,余来宁.鱼类性别与性别鉴定[J].水生生物学报,2006,30(2):221-226.Yang Dong,Yu Laining.Sex and sex identification of fish[J].Acta Hydrobiologica Sinica,2006,30(2):221-226.
[28]Osawa N,Oshima Y,Nakamura M.Molecular cloning of Dmrt1 and its expression in the gonad of Xenopus[J].Zoological Science,2005,22(6):681-687.
[29]Sreenivasulu K,Ganesh S,Raman R.Evolutionarily conserved,DMRT1,encodes alternatively spliced transcripts and shows dimorphic expression during gonadal differentiation in the lizard,Calotes versicolor[J].Mechanisms of Development,2002,119 Suppl 1(7574):S55.
[30]Shetty S,Kirby P,Zarkower D,et al.DMRT1 in a ratite bird:evidence for a role in sex determination and discovery of a putative regulatory element[J].Cytogenetic&Genome Research,2002,99(1-4):245-251.
[31]Pask A J,Behringer R R,Renfree M B.Expression of DMRT1 in the mammalian ovary and testis--from marsupials to mice[J].Cytogenetic&Genome Research,2003,101(3-4):229-236.
[32]梁象秋,方纪祖,杨和荃.水生生物学(形态和分类)[M].北京:中国农业出版社,2002:250-251.Liang Xiangqiu,Fang Jizu,Yang Hequan.Aquatic biology(morphology and classification)[M].Beijing:China Agricuture Press,2002:250-251.