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欧阳淑, 刘晓莉, 王亚坤, 李伟, 朱新平. 黄喉拟水龟Dmrt1基因克隆及其表达特征[J]. 水生生物学报, 2020, 44(4): 749-755. DOI: 10.7541/2020.090
引用本文: 欧阳淑, 刘晓莉, 王亚坤, 李伟, 朱新平. 黄喉拟水龟Dmrt1基因克隆及其表达特征[J]. 水生生物学报, 2020, 44(4): 749-755. DOI: 10.7541/2020.090
OUYANG Shu, LIU Xiao-Li, WANG Ya-Kun, LI Wei, ZHU Xin-Ping. CLONING AND EXPRESSION CHARACTERISTICS OF DMRT1 GENE OF MAUREMYS MUTICA[J]. ACTA HYDROBIOLOGICA SINICA, 2020, 44(4): 749-755. DOI: 10.7541/2020.090
Citation: OUYANG Shu, LIU Xiao-Li, WANG Ya-Kun, LI Wei, ZHU Xin-Ping. CLONING AND EXPRESSION CHARACTERISTICS OF DMRT1 GENE OF MAUREMYS MUTICA[J]. ACTA HYDROBIOLOGICA SINICA, 2020, 44(4): 749-755. DOI: 10.7541/2020.090

黄喉拟水龟Dmrt1基因克隆及其表达特征

CLONING AND EXPRESSION CHARACTERISTICS OF DMRT1 GENE OF MAUREMYS MUTICA

  • 摘要: 为研究环境依赖型性别决定的分子机制, 研究把温度依赖型性别决定(Temperature sex determination, TSD)的黄喉拟水龟(Mauremys mutica)Dmrt1基因作为研究对象, 通过黄喉拟水龟雌雄性腺转录组数据筛选和克隆了Dmrt1 cDNA全长序列, RACE-PCR结果显示该序列全长1811 bp, 包括3′端非编码区572 bp, 开放阅读框1110 bp, 共编码370个氨基酸。氨基酸序列多重比对显示, 黄喉拟水龟Dmrt1基因与红耳龟(Trachemys scripta)同源性最高, 达97.57%; 与中华鳖(Pelodiscus sinensis)Dmrt1同源性达75.54%。与小鼠(Mus musculus)Dmrt1同源性达63.25%, 与中华鲟(Acipenser sinensis)Dmrt1同源性最低, 为21.62%。定量PCR(RT-qPCR)结果显示, Dmrt1在成年黄喉拟水龟卵巢中表达较少, 在精巢中表达较为明显, 但其他体组织中表达量极少, 且Dmrt1在25℃产雄温度下胚胎发育的19—22期表达量逐步增加且显著高于32 ℃产雌温度下表达量。原位杂交分析显示, 黄喉拟水龟Dmrt1主要在支持细胞中表达, 在精原细胞、初级精母细胞、次级精母细胞中有少量表达。以上研究结果表明, Dmrt1基因可能参与黄喉拟水龟性别决定和雄性性腺分化过程, 参与雄性性腺的发育过程, 研究为进一步揭示黄喉拟水龟温度依赖型性别调控机制奠定了基础。

     

    Abstract: The molecular mechanism of temperature-dependent sex determination of Mauremys mutica remains unclear. To investigate the role of Dmrt1 in male sexual differentiation in Mauremys mutica, we cloned the full-length cDNA of Dmrt1 by RACE PCR and investigated its expression. The sequence of Dmrt1 was 1811 bp in length, containing a 572 bp 3′ untranslated region (UTR) and an 1110 bp open reading frame (ORF) encoding 370 amino acid residues. The maximum homology between the derived Dmrt1 of Mauris and Mauremys reevesi Dmrt1 was 97.57%, and the maximum homology to mouse Dmrt1 was 63.25%, and the maximum to Acipenser sinensis Dmrt1 was 21.62%. The reverse transcription quantitative real-time PCR (RT-qPCR) revealed that Dmrt1 was abundantly expressed in adult testis, low in adult ovary and hardly expressed in all other somatic tissues. Importantly, the expression levels of Dmrt1 at 16—25 phase of androgenetic temperature increased gradually and were significantly higher than those of gynogenetic temperature. Chemical in situ hybridization showed that Dmrt1 mainly expressed in the sertoli cells, and was detected modestly in spermatogonia, primary spermatocytes and secondary spermatocytes, but was hardly detected in sperm cells and somatic cells in testis. These findings indicated that Dmrt1 gene may be involved in sex determination and male gonadal differentiation in Mauremys mutica. This study provides a foundation for understanding the mechanism of sex determination in Mauremys mutica.

     

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