Molecular characterization of Dkkl1 gene and its response to exogenous hormone treatment in Pelodiscus sinensis

Dkkl1 gene plays an important role in mammalian testicular development and spermatogenesis, but the research of Dkkl1 gene in turtles is still limited. Therefore, in order to explore its potential function and mechanism of action in turtles, we cloned a cDNA fragment of Dkkl1 gene from Chinese soft-shelled turtle (Pelodiscus sinensis), and analyzed its sequence characteristics, expression pattern and response to exogenous hormone treatment. The cDNA of Dkkl1 gene was 823 bp in length, with 3' UTR of 67 bp, 5' UTR of 90 bp, open reading frame of 666 bp, and encoded 222 amino acids in total. Dkkl1 protein is a kind of alkaline protein with poor stability and high hydrophilicity. The secondary and tertiary structures of Dkkl1 protein are mainly dominated by α-helix and irregular coil. Amino acid sequence homology comparison showed high similarity with Chinemys reevesii Dkkl1 protein (81%), but low homology with Dermochelys coriacea Dkkl1 protein (70%). RT-PCR and RT-qPCR analyses reveal that Dkkl1 mRNA was significantly highly expressed in spermathecae of 3-winter-age P. sinensis adults (P<0.001), while it was hardly expressed in the rest of somatic tissues. Moreover, the expression of Dkkl1 gene in P. sinensis spermathecae gradually increased with age and peaked at 3-winter-age. In addition, both 17β-estradiol and 17α-methyltestosterone treatments significantly inhibited the expression of Dkkl1 gene in adult P. sinensis spermathecae (P<0.05). The results suggest that the Dkkl1 gene might play an important role in the process of testicular development and spermatogenesis in P. sinensis.