Molecular identification of dmrt1 and its promoter CpG methylation in correlation with gene expression during gonad development in Culter alburnus.

Abstract

Dmrt1, a member of the Dmrt family, is an important transcription regulator of gender determination. To study the biological function of dmrt1 in sexual differentiation and its potential implication in breeding technology, we obtained the full-length cDNA and proximal promoter sequence of dmrt1 in Culter alburnus, and analyzed the impact of promoter CpG methylation on the gene expression pattern of dmrt1 during gonad development. Dmrt1 was 922bp in length and consisted a 150bp 5'-UTR, a 28bp 3'-UTR, and a 744bp open reading frame (ORF). Based on the coding sequence of the dmrt1 gene, the deduced amino acid sequence was detected, and the protein structure of this gene was predicted in C. alburnus. The results indicate that the structure and function of dmrt1 were highly conservative compared to other vertebrates. The expression level of dmrt1 mRNA in different tissues was explored by qRT-PCR, which was only highly expressed in the testes and almost undetectable in other tissues. The CpG methylation pattern of the dmrt1 promoter was studied using DNA sequencing of sodium bisulfite in adult testes and ovaries, and it was found that dmrt1 promoter CpGs were not methylated in the testes, whereas hypermethylated in the ovaries. These findings demonstrate that DNA methylation can regulate sexual dimorphic expression of dmrt1, and therefore epigenetic modifications may play a critical role in the gonad differentiation of C. alburnus.