為闡明克氏原螯蝦(Procambarus clarkii) Doublesex(PcDsx)的功能,采用RACE技術克隆獲得PcDsx cDNA序列,利用qRT-PCR檢測該基因的表達情況。生物信息學分析表明該基因cDNA全長1584 bp,包括243 bp 5′ UTR、765 bp ORF(編碼254 aa)和576 bp 3′ UTR；PcDsx蛋白包含一個保守的DM結構域,與東方刺龍蝦(Sagmariasus verreauxi)SvDsx的DM結構域相似性較高?；虮磉_分析結果顯示,PcDsx廣泛表達于成年雌雄克氏原螯蝦的各組織中,其中雌雄克氏原螯蝦觸角腺中該基因的相對表達含量最高,性腺和肌肉中的相對表達含量也較高,并且發現該基因在成年雌蝦多種組織中的表達與相應雄蝦組織中的表達存在顯著性差異；克氏原螯蝦早期發育不同時期表達分析結果表明,PcDsx的表達水平在出膜后3 d達到一個高峰,而雌雄幼蝦中PcDsx的最高表達分別出現在出膜后41 d和115 d,此外,該基因在出膜后期雌雄幼蝦中的表達亦表現出顯著性差異。
Doublesex (Dsx) and its homologous genes are important regulators of sex differentiation in the animal kingdom, which determine the differentiation of animal somatic and germ cells. To elucidate the function of Doublesex(PcDsx)gene in Procambarus clarkii, the cDNA sequence was cloned by the rapid amplification of cDNA ends (RACE) and the expression of PcDsx was determined by quantitative real-time PCR (qRT-PCR). The full-length PcDsx cDNA is 1584 bp, with a 243 bp 5′ untranslated region, a 765 bp open reading frame (ORF) (coded 254 aa) and a 576 bp 3′ untranslated region. The deduced protein of PcDsx cDNA was 254 aa. The predicted PcDsx protein was found to contain a conserved DM domain. The phylogenetic analysis revealed that the PcDsx DM domain has a high similarity to the SvDsx DM domain of Sagmariasus verreauxi. The results of gene expression analysis showed that PcDsx was widely expressed in various tissues of the adult crayfish. The relative expression of PcDsx was the highest in antennary glands, followed by muscle and gonads. It was found that the expression of PcDsx in various tissues of adult female were significantly different from that of male corresponding tissues. Within the early development of P. clarkii, the expression level of PcDsx reached a peak at the third day after day after hatching, while the highest expression of PcDsx in male and female juveniles appeared at 41 and 115 day after hatching, respectively. Besides, the expression of PcDsx in male and female juveniles also showed significant differences.