克氏原螯虾Dmrt1基因克隆及表达特征分析

Gene cloning and expression characteristics of Dmrt1 gene in Procambarus clarkii

  • 摘要: 【目的】克隆克氏原螯虾(Procambarus clarkii)Dmrt1基因(PcDmrt1),分析其在雌性和雄性克氏原螯虾不同组织中及不同发育时期性腺中的表达特征,为研究克氏原螯虾性别分化与发育提供理论依据。【方法】克隆PcDmrt1基因开放阅读框(ORF),并对其进行生物信息学分析,通过实时荧光定量PCR检测PcDmrt1基因在雌性和雄性克氏原螯虾不同组织及不同发育时期性腺中的表达特征。【结果】PcDmrt1基因ORF长1752 bp,共编码583个氨基酸残基。PcDmrt1蛋白分子量64.9 kD,理论等电点(pI)为9.51,包含2个DM结构域。同源比对分析表明,克氏原螯虾PcDmrt1氨基酸序列与红螯螯虾(Cherax quadricarinatus)Dmrt氨基酸序列同源性最高,为61.2%。基于Dmrt氨基酸序列相似性构建的系统发育进化树表明,克氏原螯虾与东澳岩龙虾(Sagmariasus verreauxi)、红螯螯虾和美洲螯龙虾(Homarus americanus)亲缘关系较近。PcDmrt1基因在克氏原螯虾性腺、肝胰腺、鳃、脑、眼柄、肌肉和肠道7个组织中均有表达,性腺中相对表达量最高,肝胰腺和眼柄次之,在其他组织中相对表达量较低,在性腺、肝胰腺和眼柄3个组织中,雌性和雄性克氏原螯虾Pcdmrt1基因相对表达量存在显著(P<0.05)或极显著(P<0.01,下同)差异。在克氏原螯虾不同发育时期,PcDmrt1基因相对表达量在出膜后5 d出现高峰,最高表达出现在出膜后40 d,此时雄性幼虾PcDmrt1基因相对表达量极显著高于雌性幼虾。【结论】克氏原螯虾PcDmrt1蛋白含有2个DM结构域,属于典型的Dmrt家族成员,在进化上保守。PcDmrt1基因广泛表达于克氏原螯虾各组织中并在性腺中高表达,基因相对表达量在发育早期呈上升趋势,表明PcDmrt1基因可能在早期参与克氏原螯虾性别分化与组织发育。

     

    Abstract: 【Objective】The Dmrt1 gene of Procambarus clarkii(PcDmrt1) was cloned, and its expression patterns were analyzed in different tissues of female and male P. clarkii, as well as in gonads during different developmental periods. This study provided a theoretical basis for the investigation of sex differentiation and development in P. clarkii.【Method】The open reading frame(ORF) of PcDmrt1 gene was cloned, and bioinformatics analysis was performed on it.The expression characteristics of the PcDmrt1 gene in various tissues and gonads of female and male P. clarkii during different developmental periods were analyzed by real-time fluorescence quantitative PCR. 【Result】The ORF of PcDmrt1gene was 1752 bp in length, encoding 583 amino acid residues. The PcDmrt1 protein had a molecular weight of 64.9 kD, a theoretical isoelectric point(pI) of 9.51 and contained two DM domains. Homology alignment analysis revealed that the amino acid sequence of PcDmrt1 had the highest homology with the Dmrt amino acid sequence of Cherax quadricarinatus(61.2%). The phylogenetic evolutionary tree constructed based on the similarity of Dmrt1 amino acid sequence showed that P. clarkii was closely related to Sagmariasus verreauxi, Cherax quadricarinatus and Homarus americanus. The PcDmrt1 gene was expressed in seven tested tissues of gonad, hepatopancreas, gills, brain, eyestalks, muscles and intestine of P. clarkii. The relative expression level in gonad was the highest, followed by hepatopancreas and eyestalk, and the relative expression level in other tissues was low. Significant(P<0.05) or extremely significant(P<0.01, the same below) differences were observed in the relative expression of the PcDmrt1 gene in gonad, hepatopancrea and eyestalk between female and male P. clarkii. At various developmental periods of P. clarkii, the relative expression of PcDmrt1 gene reached its peak at 5 d after hatching, with the highest expression observed in juvenile shrimp at 40 d after hatching. At this time, the relative expression of PcDmrt1 gene in male P. clarkii was found to be significantly higher than that in female P. clarkii. 【Conclusion】The PcDmrt1 protein of P. clarkii contains two DM structural domains, which is typical Dmrt family member and is evolutionarily conserved. The PcDmrt1 gene is widely expressed in various tissues of P.clarkii, with the highest expression observed in gonads. The relative expression level of the gene appears to increase during the early stages of development, suggesting a potential role for the PcDmrt1 gene in sex differentiation and tissue development in P. clarkii during this period.

     

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