金钱鱼Dmrt5基因的克隆及表达分析
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摘要
为了解金钱鱼Dmrt5在性腺中的作用,克隆了金钱鱼Dmrt5,采用逆转录PCR检测了它的组织分布,并采用荧光定量PCR检测了Dmrt5在性腺和胚胎期中的表达模式.实验结果表明,金钱鱼Dmrt5的ORF全长为1347 bp,编码蛋白含448个氨基酸残基.同源性分析发现,金钱鱼Dmrt5与橙线雀的相似性最高为96. 0%,与人的相似性最低为68. 0%.通过系统进化分析可知金钱鱼Dmrt5与鲈形目鱼类Dmrt5为一类,与其进化地位一致.组织分布表明,金钱鱼Dmrt5在下丘脑和脑垂体中高表达.对性腺不同发育时期的表达分析发现,Dmrt5在Ⅲ期和Ⅳ期精巢中的表达显著高于Ⅴ期精巢,而在Ⅱ、Ⅲ、Ⅳ期卵巢间的表达无显著差异. Dmrt5在胚胎期各阶段都有表达,但差异不显著.金钱鱼Dmrt5可能具有性别调控和神经发生等功能.
In order to study the function of Dmrt5 in gonad,Dmrt5 gene in Scatophagus argus was cloned,then its expression in different tissues was examined by reverse-transcription(RT-PCR),and its expression in gonads and embryos at different developmental stages was examined as well by quantitative Real-time PCR(q PCR). The full-length ORF of Dmrt5 in S. argusis1 is 347 bp,encoding 448 amino acids. Homology analysis indicated that Dmrt5 in Acanthochromis polyacanthus is with the highest similarity of 96. 0% to that in S. argus,while human Dmrt5 is with the lowest similarityof 68. 0% to that in S. argus. Phylogeneticanalysis revealed that S. argus Dmrt5 is clustered with Dmrt5 in perciform fishes,which is consistent with their evolutionary status. Tissue distribution indicated high expression of Dmrt5 in the hypothalamus and pituitary gland. Dmrt5 was expressed higher in testis at stage Ⅲ and IV than at stage V,while its expression in ovaries showed no significant difference at stage Ⅱ,Ⅲ and Ⅳ. Meanwhile,Dmrt5 was expressed evenly in embryos at various stages. Dmrt5 may functionas a regulator of sex differentiation and neurogenesis in S. argus.
In order to study the function of Dmrt5 in gonad,Dmrt5 gene in Scatophagus argus was cloned,then its expression in different tissues was examined by reverse-transcription(RT-PCR),and its expression in gonads and embryos at different developmental stages was examined as well by quantitative Real-time PCR(q PCR). The full-length ORF of Dmrt5 in S. argusis1 is 347 bp,encoding 448 amino acids. Homology analysis indicated that Dmrt5 in Acanthochromis polyacanthus is with the highest similarity of 96. 0% to that in S. argus,while human Dmrt5 is with the lowest similarityof 68. 0% to that in S. argus. Phylogeneticanalysis revealed that S. argus Dmrt5 is clustered with Dmrt5 in perciform fishes,which is consistent with their evolutionary status. Tissue distribution indicated high expression of Dmrt5 in the hypothalamus and pituitary gland. Dmrt5 was expressed higher in testis at stage Ⅲ and IV than at stage V,while its expression in ovaries showed no significant difference at stage Ⅱ,Ⅲ and Ⅳ. Meanwhile,Dmrt5 was expressed evenly in embryos at various stages. Dmrt5 may functionas a regulator of sex differentiation and neurogenesis in S. argus.
引文
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[15]杨秀荣,蒋和生,杨宁.鸟类性别决定与性别分化机制[J].遗传,2012,34(4):407-411.
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[19]URQUHART J E,BEAMAN G,BYERS H,et al. DMRTA2(Dmrt5)is mutated in a novel cortical brain malformation[J]. Clin Genet,2016,89(6):724-727.
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[22]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.
[23]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.
[24]陈华谱,杨尉,王耀嵘,等.泰国斗鱼igf2基因c DNA的克隆及表达分析[J].海南热带海洋学院学报,2018,25(5):1-7.
[25]王妹,邓思平,陈华谱,等.金钱鱼Amhr2基因的克隆及表达分析[J].广东海洋大学学报,2018,38(3):17-24.
[26]王妹,陈华谱,江东能,等.饥饿和复投喂对金钱鱼四种igfbp基因表达的影响[J].海南热带海洋学院学报,2017,24(5):1-7.
[27]李斌,梁华芳,陈兆明,等.波纹龙虾C-型凝集素Ph Lec A的基因克隆与表达[J].热带海洋学报,2016,35(5):29-37.
[28]王富轩,肖述,向志明,等.香港牡蛎(Crassostrea hongkongensis)Commd1基因的分子克隆及其在盐度胁迫下的表达分析[J].热带海洋学报,2017,36(1):48-55.
[29]雷蕾,李忻怡,张育辉. Dmrt基因在脊椎动物性别决定与分化中的研究进展[J].中国组织化学与细胞化学杂志,2013,22(3):269-276.
[2]VEITH A M,KLATTIG J,DETTAI A,et al. Male-biased expression of X-chromosomal DM domain-less Dmrt8 genes in the mouse[J]. Genomics,2006,88(2):185-95.
[3]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.
[4]郭一清,程汉华,高尚,等.脊椎动物DMRT基因家族的系统发生及同线性分析[J].遗传学报,2004,31(10):1103-1108.
[5]夏伟,周莉,桂建芳.真骨鱼类DMRT基因家族的连锁结构及其系统发生[J].水生生物学报,2009,33(2):254-263.
[6]程子华. Dmrt基因的功能和特点[J].安徽农业科学,2006,34(5):853-856.
[7]ZHULINGYANG,WILKEN J,PHILLIPS N U,et al. Sexual dimorphism in diverse metazoans is regulated by a novel class of intertwined zinc fingers[J]. Genes Devt,2000,14(14):1750-1764.
[8]ELLEGREN H. Hens,cocks and avian sex determination. A quest for genes on Z or W?[J]. EMBO Rep.,2001,2(3):192-196.
[9]KIMS,KETTLEWELLJR,ANDERSON RC,et al. Sexually dimorphic expression of multiple doublesex-related genes in the embryonic mouse gonad[J]. Gene Expr Patterns,2003,3(1):77-82.
[10]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.
[11]RAYMOND S C,SHAMU C E,SHEN M M,et al. Evidence for evolutionary conservation of sex determining genes[J]. Nature,1998,391:691-695.
[12]VEITIA R,NUNES M,BRAUNER R,et al. Deletions of distal 9p associated with 46,XY male to female sex reversal:definition of the breakpoints at 9p23. 3-p24. 1[J]. Genomics,1997,41:271-274.
[13]RAYMOND C S,PARKER E D,KETTLEWELL J R,et al. A region of human chromosome 9p required for testisdevelopment contains two genes related to known sexual regulators[J]. Hum Mol Genet,1999(8):989-996.
[14]SMITH C A,ROESZLER K N,OHNESORG T,et al. The avian Z-linked gene DMRT1 is required for male sex determination in the chicken[J]. Nature,2009,461(7261):267-271.
[15]杨秀荣,蒋和生,杨宁.鸟类性别决定与性别分化机制[J].遗传,2012,34(4):407-411.
[16]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]. Mech Dev.,2004,121(7/8):997-1005.
[17]杨东.尼罗罗非鱼性别决定机制和性别相关的分子标记[D].武汉:华中农业大学,2006.
[18]GUOY Q,CHENG H H,HUANG X,et al. Gene structure,multiple alternative splicing,and expression in gonads of zebrafish Dmrt1[J]. Biochem Biophys Res Commun,2005,330(3):950-957.
[19]URQUHART J E,BEAMAN G,BYERS H,et al. DMRTA2(Dmrt5)is mutated in a novel cortical brain malformation[J]. Clin Genet,2016,89(6):724-727.
[20]GUO Y Q,LI Q,GAO S,et al. Molecular cloning,characterization,and expression in brain and gonad of Dmrt5 of zebrafish[J]. Biochem Biophys Res Commun,2004,324:569-575.
[21] YAMAGUCHI A,LEE K H,FUJIMOTOH,et al. Expression of the DMRT gene and its roles in early gonadal development of the Japanese pufferfish Takifugu rubripes[J]. Comp Biochem Physiol,Part D:Genomics Proteomics,2006(1):59-68.
[22]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.
[23]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.
[24]陈华谱,杨尉,王耀嵘,等.泰国斗鱼igf2基因c DNA的克隆及表达分析[J].海南热带海洋学院学报,2018,25(5):1-7.
[25]王妹,邓思平,陈华谱,等.金钱鱼Amhr2基因的克隆及表达分析[J].广东海洋大学学报,2018,38(3):17-24.
[26]王妹,陈华谱,江东能,等.饥饿和复投喂对金钱鱼四种igfbp基因表达的影响[J].海南热带海洋学院学报,2017,24(5):1-7.
[27]李斌,梁华芳,陈兆明,等.波纹龙虾C-型凝集素Ph Lec A的基因克隆与表达[J].热带海洋学报,2016,35(5):29-37.
[28]王富轩,肖述,向志明,等.香港牡蛎(Crassostrea hongkongensis)Commd1基因的分子克隆及其在盐度胁迫下的表达分析[J].热带海洋学报,2017,36(1):48-55.
[29]雷蕾,李忻怡,张育辉. Dmrt基因在脊椎动物性别决定与分化中的研究进展[J].中国组织化学与细胞化学杂志,2013,22(3):269-276.