金钱鱼Dmrt4基因的克隆及表达分析
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摘要
【目的】研究金钱鱼(Scatophagus argus)Dmrt4(Doublesex and Mab-3 related transcription factors 4)基因在性腺和胚胎发育中的表达。【方法】克隆金钱鱼Dmrt4,采用逆转录PCR检测Dmrt4的组织表达,用实时荧光定量PCR检测Dmrt4在性腺和胚胎发育过程中的表达。【结果与结论】金钱鱼Dmrt4的开放阅读框(ORF)全长1 260 bp,编码蛋白全序列为419个氨基酸。金钱鱼Dmrt4氨基酸序列含有DM和DMA结构域。金钱鱼Dmrt4与欧洲海鲈(Dicentrarchus labrax)和大黄鱼(Larimichthys crocea)的同源性最高,为87.6%;与非洲爪蟾(Xenopus laevis)的同源性最低,为47.1%。金钱鱼Dmrt4与其他鲈形目鱼类在系统进化树上聚为一支。金钱鱼Dmrt4在精巢、卵巢和鳃中高表达,在Ⅲ和Ⅳ期精巢中的表达显著高于V期精巢和Ⅱ、Ⅲ和Ⅳ期卵巢。胚胎发育过程中Dmrt4均有表达,原肠胚的表达水平最高。
【Objective】To evaluate the Dmrt(Doublesex and Mab-3 related transcription factors) gene family which is important for the regulation of reproduction and development in animals.However,the information of Dmrt4 in Scatophagus argus is lacking.【Method】 The Dmrt4 gene of S.argus is cloned,and its tissue distribution and expression profiles at different gonad and embryo developmental stages were analyzed by reverse transcription PCR(RT-PCR) and real-time quantitative PCR(qPCR).【Result and Conclusion】The spotted scat Dmrt4 full-length ORF is 1260 bp and encodes 419 amino acids.Sequence analysis revealed that Dmrt4 consists of DM and DMA domains.Multiple sequence alignment showed that S.argus Dmrt4 was highly homologous to Dmrt4 from Dicentrarchus labrax and Larimichthys crocea(87.6%),but it was less homologous to frog Xenopus laevis(47.1%) at amino acid level.Phylogenetic analysis revealed that Dmrt4 is closest to D.labrax and L.crocea,which is consistent with the taxonomy that indicates the conservation of Dmrt4.In adult spotted scat,Dmrt4 is highly expressed in the testis,ovary and gill.In gonads,Dmrt4 was expressed significantly higher in phase Ⅲ and Ⅳ testes than those of phase V testis and phase Ⅱ,Ⅲ and Ⅳ ovaries.Dmrt4 is expressed during the embryo developmental stages with the highest level at the gastrula stage.
【Objective】To evaluate the Dmrt(Doublesex and Mab-3 related transcription factors) gene family which is important for the regulation of reproduction and development in animals.However,the information of Dmrt4 in Scatophagus argus is lacking.【Method】 The Dmrt4 gene of S.argus is cloned,and its tissue distribution and expression profiles at different gonad and embryo developmental stages were analyzed by reverse transcription PCR(RT-PCR) and real-time quantitative PCR(qPCR).【Result and Conclusion】The spotted scat Dmrt4 full-length ORF is 1260 bp and encodes 419 amino acids.Sequence analysis revealed that Dmrt4 consists of DM and DMA domains.Multiple sequence alignment showed that S.argus Dmrt4 was highly homologous to Dmrt4 from Dicentrarchus labrax and Larimichthys crocea(87.6%),but it was less homologous to frog Xenopus laevis(47.1%) at amino acid level.Phylogenetic analysis revealed that Dmrt4 is closest to D.labrax and L.crocea,which is consistent with the taxonomy that indicates the conservation of Dmrt4.In adult spotted scat,Dmrt4 is highly expressed in the testis,ovary and gill.In gonads,Dmrt4 was expressed significantly higher in phase Ⅲ and Ⅳ testes than those of phase V testis and phase Ⅱ,Ⅲ and Ⅳ ovaries.Dmrt4 is expressed during the embryo developmental stages with the highest level at the gastrula stage.
引文
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[14]兰国宝,阎冰,廖思明,等.金钱鱼生物学研究及回顾[J].水产科学,2005,24(7):39-41.
[15]MUSTAPHA U F,JIANG D N,LIANG Z H,et al.Male-specific Dmrt1 is a candidate sex determination gene in spotted scat(Scatophagus argus)[J].Aquaculture,2018,495:351-358.
[16]蔡泽平,胡家玮,王毅.金钱鱼早期发育的观察[J].热带海洋学报,2014,33(4):20-25.
[17]蔡泽平,王毅,胡家玮,等.金钱鱼的繁殖生物学及诱导产卵试验[J].热带海洋学报,2010,29(5):180-185.
[18]LI G L,ZHANG M Z,DENG S P,et al.Effects of temperature and fish oil supplementation on ovarian development and foxl2 m RNA expression in spotted scat Scatophagus argus[J].Journal of Fish Biology,2015,86(1):248-260.
[19]崔丹,刘志伟,刘南希,等.金钱鱼性腺发育及其组织结构观察[J].水产学报,2013,37(5):696-704.
[20]张克伟,陈华谱,江东能,等.金钱鱼IGF-1和IGF-2的克隆及其在胚胎发育过程的表达[J].广东海洋大学学报,2018,38(2):7-14.
[21]YANG W,CHEN H P,CUI XF,et al.Sequencing,de novo assembly and characterization of the spotted scat Scatophagus argus,(Linnaeus 1766)transcriptome for discovery of reproduction related genes and SSRs[J].Chinese Journal of Oceanology and Limnology,2017(2):1-13.
[22]GUAN G,KOBAYASHI T,NAGAHAMA Y.Sexually dimorphic expression of two types of DM(Doublesex/Mab-3)-domain genes in a teleost fish,the Tilapia(Oreochromis niloticus)[J].Biochem Biophys Res Commun,2000,272(3):662-666.
[23]苏利娜,丁祝进,李鸿,等.团头鲂Dmrt4基因的克隆与表达分析[J].华中农业大学学报,2013,32(6):110-116.
[24]HUANG X,HONG CS,O'DONNELL M,et al.The doublesex-related gene,XDmrt4,is required for neurogenesis in the olfactory system[J].Proc Natl Acad Sci USA,2005,102(32):11349-11354.
[25]VEITH AM,SCH?FER M,KLüVER N,et al.Tissue-specific expression of dmrt genes in embryos and adults of the platyfish Xiphophorus maculatus[J].Zebrafish,2006,3(3):325-337.
[26]董晓丽,陈松林.半滑舌鳎Dmrt4基因DNA全序列的克隆与表达分析[J].中国水产科学,2013,20(3):499-505.
[2]SHEN M M,HODGKIN J.Mab-3,a gene required for sex-specific yolk protein expression and a male-specific lineage in C.elegans[J].Cell,1988,54(7):1019-1031.
[3]BURTIS KC,BAKER BS.Drosophila doublesex gene controls somatic sexual differentiation by producing alternatively spliced m RNAs encoding related sex-specific polypeptides[J].Cell,1989,56(6):997-1010.
[4]汪海.DMRT家族生物信息学分析及Dmrt4/Dmrt6表达与功能初步研究[D].重庆:西南大学,2013.
[5]ZHANG X,WANG H,LI M,et al.Isolation of doublesexand mab-3-related transcription factor 6 and its involvement in spermatogenesis in tilapia[J].Biol Reprod,2014,91(6):1-10.
[6]SHENG Y,CHEN B,ZHANG L,et al.Identification of Dmrt genes and their up-regulation during gonad transformation in the swamp eel(Monopterus albus)[J].Mol Biol Rep,2014,41(3):1237-1245.
[7]SU L,ZHOU F,DING Z,et al.Transcriptional variants of Dmrt1 and expression of four Dmrt genes in the blunt snout bream,Megalobrama amblycephala[J].Gene,2015,573(2):205-215.
[8]MASUYAMA H,YAMADA M,KAMEI Y,et al.Dmrt1mutation causes a male-to-female sex reversal after the sex determination by Dmy in the medaka[J].Chromosome Res,2012,20:163-176.
[9]WEBSTER KA,SCHACH U,ORDAZ A,et al.Dmrt1 is necessary for male sexual development in zebrafish[J].Dev Biol,2017,422:33-46.
[11]BACHTROG D,MANK JE,PEICHEL CL,et al.Sex determination:why so many ways of doing it?[J].PLoSBiol,2014,12:1001899.
[12]HEULE C,SALZBURGER W,B?HNE A.Genetics of sexual development:an evolutionary playground for fish[J].Genetics,2014,196:579-591.
[13]BALCIUNIENE J,BARDWELL VJ,ZARKOWER D.Mice mutant in the DM domain gene Dmrt4 are viable and fertile but have polyovular follicles[J].Mol Cell Biol,2006,26(23):8984-8991.
[14]兰国宝,阎冰,廖思明,等.金钱鱼生物学研究及回顾[J].水产科学,2005,24(7):39-41.
[15]MUSTAPHA U F,JIANG D N,LIANG Z H,et al.Male-specific Dmrt1 is a candidate sex determination gene in spotted scat(Scatophagus argus)[J].Aquaculture,2018,495:351-358.
[16]蔡泽平,胡家玮,王毅.金钱鱼早期发育的观察[J].热带海洋学报,2014,33(4):20-25.
[17]蔡泽平,王毅,胡家玮,等.金钱鱼的繁殖生物学及诱导产卵试验[J].热带海洋学报,2010,29(5):180-185.
[18]LI G L,ZHANG M Z,DENG S P,et al.Effects of temperature and fish oil supplementation on ovarian development and foxl2 m RNA expression in spotted scat Scatophagus argus[J].Journal of Fish Biology,2015,86(1):248-260.
[19]崔丹,刘志伟,刘南希,等.金钱鱼性腺发育及其组织结构观察[J].水产学报,2013,37(5):696-704.
[20]张克伟,陈华谱,江东能,等.金钱鱼IGF-1和IGF-2的克隆及其在胚胎发育过程的表达[J].广东海洋大学学报,2018,38(2):7-14.
[21]YANG W,CHEN H P,CUI XF,et al.Sequencing,de novo assembly and characterization of the spotted scat Scatophagus argus,(Linnaeus 1766)transcriptome for discovery of reproduction related genes and SSRs[J].Chinese Journal of Oceanology and Limnology,2017(2):1-13.
[22]GUAN G,KOBAYASHI T,NAGAHAMA Y.Sexually dimorphic expression of two types of DM(Doublesex/Mab-3)-domain genes in a teleost fish,the Tilapia(Oreochromis niloticus)[J].Biochem Biophys Res Commun,2000,272(3):662-666.
[23]苏利娜,丁祝进,李鸿,等.团头鲂Dmrt4基因的克隆与表达分析[J].华中农业大学学报,2013,32(6):110-116.
[24]HUANG X,HONG CS,O'DONNELL M,et al.The doublesex-related gene,XDmrt4,is required for neurogenesis in the olfactory system[J].Proc Natl Acad Sci USA,2005,102(32):11349-11354.
[25]VEITH AM,SCH?FER M,KLüVER N,et al.Tissue-specific expression of dmrt genes in embryos and adults of the platyfish Xiphophorus maculatus[J].Zebrafish,2006,3(3):325-337.
[26]董晓丽,陈松林.半滑舌鳎Dmrt4基因DNA全序列的克隆与表达分析[J].中国水产科学,2013,20(3):499-505.
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