鲫Dmrt3基因的克隆和表达分析
MOLECULAR CLONING AND EXPRESSION ANALYSIS OF DMRT3 IN GOLDFISH, CARASSIUS AURATUS
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摘要: DMRT家族是一个与性别决定相关的转录因子家族。为了研究家族成员之一的Dmrt3在我国重要养殖鱼类鲫胚胎发育、性别分化中的功能以及在育种中的作用，我们克隆了鲫Dmrt3基因的cDNA全长，并对Dmrt3基因在发育早期和不同组织中的表达进行了分析。结果显示：鲫Dmrt3基因cDNA全长为2182 bp，其中5￠端非编码区408 bp，3'端非编码区427 bp，开放阅读框1347 bp，编码448个氨基酸。蛋白结构预测显示DMRT3除了正常的DM结构域外，还有DMA结构域，在进化上与DMRT4和DMRT5的亲缘关系更近。巢式RT-PCR分析结果表明Dmrt3直到尾芽期才开始有微量表达，表达量在15体节期有明显增加但仍然处在一个较低的水平；在成体组织中只在精巢中检测到表达。这种表达时空模式提示Dmrt3可能在早期器官发生和雄性性腺发育调控中起作用。对Dmrt3启动子CpG岛的甲基化分析表明所检测的组织和配子中并不发生甲基化，说明这种雌雄特异性和组织特异性差异表达并不是通过对该基因启动子的差异甲基化修饰来调控的。此外，我们还发现了鲫Dmrt3的一个由逆转录产物形成的假基因pDmrt3。这些结果为进一步研究鲫Dmrt3在性别分化中的作用和评估其在鲫性别控制育种中的价值，以及分析DMRT家族的进化关系提供了基础资料。Abstract: The DMRT family of transcription factors play a pivotal role in sex determination. In order to investigate the function of Dmrt3, a member of the DMRT family, in the embryogenesis and sex differentiation, as well as its potential implication in breeding of goldfish, we cloned the full length cDNA of Dmrt3 and analyzed its expression during the early embryogenesis and in various adult tissues in goldfish, Carassius auratus. The entire goldfish Dmrt3 cDNA is 2182 bp long, including a 408 bp 5￠-UTR, a 427 bp 3'-UTR and a 1347 bp open reading frame (ORF); the latter encoded a protein with 448 amino acids. Protein structural prediction based on the putative amino acid sequence of DMRT3 revealed that goldfish DMRT3 contained a common DM domain and a DMA domain, suggesting that it was more phylogenetically related to DMRT4 and DMRT5. Nested PCR examination indicated that the expression of Dmrt3 was undetectable until bud stage, and its expression obviously increased but still at a low level at 15-somite stage. In adult tissues, DMRT3 was expressed exclusively in the testes. This expression pattern suggested that Dmrt3 might regulate organogenesis and gonad development of male. No methylation of the Dmrt3 promoter CpG-island in gametes and in various tissues was observed using bisulfite sequencing analysis, suggesting that DNA methylation did not regulate the sex-specific and tissue-specific expression of Dmrt3. Moreover, we identified a pseudogene, named pDmrt3, from the reverse transcript of Dmrt3. These results provide essential materials for studying the function of Dmrt3 in the sex differentiation and potential application in breeding of goldfish, as well as the evolutionary relationship of Dmrt genes.
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Keywords:
- Goldfish /
- Dmrt3 /
- Gene cloning /
- Gene expression
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[1] Raymond C S, Shamu C E, Shen M M, et al. Evidence for evolutionary conservati-on of sex-determining genes [J]. Nature, 1998, 391(6668): 691695
[2] Burtis K C, Baker B S. Drosophila doublesex gene controls somatic sexual differen-tiation by producing alternatively spliced mRNAs encoding related sex-specific poly-peptides [J]. Cell, 1989, 56(6): 9971010
[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): 527535
[4] Smith C A, McClive P J, Western P S, et al. Evolution: Conservation of a sex-deterining gene [J]. Nature, 1999, 402(6762): 601602
[5] Raymond C S, Kettlewell J R, Hirsch B, et al. Expression of Dmrt1 in the genital ridge of mouse and chicken embryos suggests a role in vertebrate sexual developme[J]. Developmental Biology, 1999, 215(2): 208220
[6] Alam M A, Kobayashi Y, Horiguchi R, et al. Molecular cloning and quantitative expression of sexually dimorphic markers Dmrt1 and Foxl2 during female-to-male sex change in Epinephelus merra [J]. General and Comparative Endocrinology, 2008, 157(1): 7585
[7] Wen A Y, You F, Sun P, et al. Cloning of dmrt1 gene and its tissue expression analyses compared with that of P450arom gene in olive flounder (Paralichthys olivaceus) [J]. Marine Sciences, 2010, 34(11): 97102 [文爱韵, 尤锋, 孙鹏, 等. 牙鲆dmrt1基因的克隆及其与P450arom基因的组织表达分析. 海洋科学, 2010, 34(11): 97102]
[8] Deng S P, Chen S L. Molecular cloning, characterization and RT-PCR expression analysis of Dmrt1 from half-smooth tongue-sole, Cynoglossus semilaevis [J]. Journal of Fishery Sciences of China, 2008, 15(4): 577584 [邓思平, 陈松林. 半滑舌鳎Dmrt1基因的cDNA克隆及其表达. 中国水产科学, 2008, 15(4): 577584]
[9] Deng S P, Wang J J, Wu T L, et al. cDNA cloning and expression analysis of Dmrt1 in Clarias fuscus [J]. Acta Hydrobiologica Sinica, 2012, 36(4): 610617 [邓思平, 王静杰, 吴天利, 等. 胡子鲇Dmrt1基因全长cDNA克隆及其表达分析. 水生生物学报, 2012, 36(4): 610617]
[10] Cao M, Duan J, Cheng N, et al. Sexually dimorphic and ontogenetic expression of dmrt1, cyp19a1a and cyp19a1b in Gobiocypris rarus [J]. Comparative Biochemistry and Physiology-Part A: Molecular Integrative Physiology, 2012, 162(4): 303309
[11] Xia W, Zhou L, Yao B, et al. Differential and spermatogenic cell-specific expressionof DMRT1 during sex reversal in protogynous hermaphroditic groupers [J]. Molecular and Cellular Endocrinology, 2007, 263(1): 156172
[12] 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): 9971005
[13] Zhou X, Li Q, Lu H, et al. Fish specific duplication of Dmrt2: Characterization of zebrafish Dmrt2b [J]. Biochimie, 2008, 90(6): 878887
[14] Meng A, Moore B, Tang H, et al. A Drosophila doublesex-related gene, terra, is involved in somitogenesis in vertebrates [J]. Development, 1999, 126(6): 12591268
[15] Liu S, Li Z, Gui J F. Fish-specific duplicated Dmrt2b contributes to a divergent fun-ction through Hedgehog pathway and maintains left-right asymmetry establishment f-unction [J]. PLoS One, 2009, 4(9): e7261
[16] 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 and Proteomics, 2006, 1(1): 5968
[17] Li Q, Zhou X, Guo Y, et al. Nuclear localization, DNA binding and restricted expression in neural and germ cells of zebrafish Dmrt3 [J]. Biology of the Cell, 2008, 100(8): 453463
[18] Lu Y, Li Z, Zhu X L, et al, Molecular cloning and characterization of Dmrt3 during xenopus tropicalis embryogenesis [J]. Journal of Zhejiang University (Science Edition), 2012, 39(5): 564570 [陆怡, 李政, 朱晓龙, 等. 热带爪蟾Dmrt3基因的克隆及在早期胚胎发育中的表达. 浙江大学学报 (理学版), 2012, 39(5): 564570]
[19] Andersson L S, Larhammar M, Memic F, et al. Mutations in DMRT3 affect locomotion in horses and spinal circuit function in mice [J]. Nature, 2012, 488(7413): 642646
[20] Balciuniene J, Bardwell V J, Zarkower D. Mice mutant in the DM domain gene Dmrt4 are viable and fertile but have polyovular follicles [J]. Molecular and Cellular Biology, 2006, 26(23): 89848991
[21] Huang X, Hong C S, O'Donnell M, et al. The doublesex-related gene, XDmrt4, is required for neurogenesis in the olfactory system [J]. Proceedings of the National Academy of Sciences of the United States of America, 2005, 102(32): 1134911354
[22] Xu S, Xia W, Zohar Y, et al. Zebrafish dmrta2 regulates the expression of cdkn2c in spermatogenesis in the adult testis [J]. Biology of Reproduction, 2013, 88(1): 112
[23] Kim S, Kettlewell J R, Anderson R C, et al. Sexually dimorphic expression of multiple doublesex-related genes in the embryonic mouse gonad [J]. Gene Expression Patterns, 2003, 3(1): 7782
[24] 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): 9599
[25] Brunner B, Hornung U, Shan Z, et al. Genomic organization and expression of the doublesex-related gene cluster in vertebrates and detection of putative regulatory regions for DMRT1 [J]. Genomics, 2001, 77(1): 817
[26] Gui J F, Zhou L. Genetic basis and breeding application of clonal diversity and dual reproduction modes in polyploid Carassius auratus gibelio [J]. Science China Life Sciences, 2010, 53(4): 409415
[27] Ge W, Jiang Y G. The natural gynogenesis of fish [J]. Acta Hydrobiologica Sinica, 1989, 13(3): 274286 [葛伟, 蒋一珪. 鱼类的天然雌核发育. 水生生物学报, 1989, 13(3): 274286]
[28] Volff J N, Zarkower D, Bardwell V J, et al. Evolutionary dynamics of the DM domain gene family in metazoans [J]. Journal of Molecular Evolution, 2003, 57(1): S241S249
[29] Force A, Lynch M, Pickett F B, et al. Preservation of duplicate genes by complementary, degenerative mutations [J]. Genetics, 1999, 151(4): 15311545
[30] Kleinjan D A, Bancewicz R M, Gautier P, et al. Subfunctionalization of duplicated zebrafish pax6 genes by cis-regulatory divergence [J]. PLoS Genetics, 2008, 4(2): e29
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