中华鳖Dmrt1基因的克隆、表达及在性逆转中的变化分析

在大部分脊椎动物中,Dmrt1基因在雄性性别决定和性腺分化中起重要的调控作用.本文从m RNA和蛋白水平分析Dmrt1基因的组织差异性表达、在不同发育阶段性腺中的细胞定位及在性逆转中的表达变化,研究Dmrt1基因在中华鳖性别分化中的调控作用.Rapid-amplification of c DNA ends(RACE)结果显示,Dmrt1基因c DNA序列全长2409 bp,其中5′非编码区为230 bp,3′非编码区为1072 bp,开放阅读框为1107 bp,编码368个氨基酸,具有一个高度保守的DM结构域.荧光定量PCR和免疫组化结果显示,Dmrt1在性腺分化之前的第16期雄性性腺中开始表达,先于Amh和Sox9基因表达.随着性腺的发育,Dmrt1蛋白主要定位于性腺Sertoli细胞的细胞核上,在雌性性腺发育过程中并未见其表达.此外,在雌二醇诱导的雄性转雌性性逆转胚胎性腺中,Dmrt1表达显著下调;在芳香化酶抑制剂诱导的雌性转雄性性腺中,Dmrt1表达则显著上升.上述研究表明,Dmrt1基因是中华鳖雄性特异性基因,参与雄性性腺的发育过程,可能在中华鳖早期性别决定中起重要的调控作用.

Molecular Cloning and Expression Pattern of Dmrtl and Its Expression Change in Sex Reversal in Pelodiscus sinensis

In many vertebrate species, Dmrt1 gene is required for male sexual differentiation. In this study, the mRNA and protein expression of Dmrt1 gene and its cellular localization in gonads of different developmental stages of both sexes were examined to investigate the involvement of Dmrt1 in sexual differentiation in Pelodiscus sinensis. RACE results showed that the complete cDNA sequence of P. sinensis Dmrt1 was composed of 230 bp of 5′ un-translated region, 1072 bp of 3′ un-translated region and 1107 bp of open reading frame, bearing a highly conserved DM domain, which encodes 368 amino acids. Real-Time PCR and immunofluorescence analysis revealed that Dmrt1 gene initiated expression in male embryonic gonad of stage 16 before gonadal differentiation, prior to Sox9 and Amh gene. During the gonadal development, Dmrt1 protein localized in the nuclei of pre-sertoli cells, however, no Dmrt1 signal was detected in female gonad. Moreover, Dmrt1 expression was significantly decreased in estrogen-induced male to female sex reversal. Whereas, Dmrt1 was trigged to express in aromatase inhibitor-induced female to male sex reversal. In conclusion, this study indicates that Dmrt1 gene is a male-specific gene and participates in the male gonadal development, which is likely to play a key role during primary male sex determination in P. sinensis.