Gene cloning and expression characteristics of Dmrt1 gene in Procambarus clarkii
[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 pe-riods.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 dif-ferent developmental periods were analyzed by real-time fluorescence quantitative PCR.[Result]The ORF of PcDmrt1 gene 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 Pc-Dmrt1 gene was expressed in seven tested tissues of gonad,hepatopancreas,gills,brain,eyestalks,muscles and intes-tine 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 be-low)differences were observed in the relative expression of the PcDmrt1 gene in gonad,hepatopancrea and eyestalk be-tween 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 fe-male 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 du-ring the early stages of development,suggesting a potential role for the PcDmrt1 gene in sex differentiation and tissue de-velopment in P.clarkii during this period.
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