Molecular Cloning And Expression Analysis Of Dmrt3in Goldfish, Carassius Auratus

Goldfish, Carassius auratus, plays an important role in fields of edible and ornamental fish in China, and it has sexual reproduction of diploid subspecies and natural gynogenetic polyploid subspecies, which makes C. auratus the good and unique model in study of fish genome evolution and the mechanism of reproductive control. The DMRT(doublesex and mab-3related transcription factor)family is a type of transcription factors involved in regulation of sex determination. In order to investigate the function of Dmrt3, a member of the Dmrt family, in regulation of embryogenesis and sex differentiation, as well as its potential implication in breeding of C. auratus, we used degenerate PCR and genome walking (GenomeWalker of ClonTech) in cloning full length cDNA of Dmrt3, and analyzed its expression patterns during the early embryogenesis and in various adult tissues in C. auratus using RT-PCR and nested PCR.The entire Dmrt3cDNA of goldfish is2179bp long, including a408bp long5’-UTR (Untranslated Regions), a424bp long3’-UTR and a1347bp open reading frame (ORF), which encoded a protein with448amino acids. Protein structural analysis based on the putative amino acid sequence of DMRT suggested that C. auratus DMRT3contained both a common DM domain and a DMA domain, suggesting that it was more phylogenetically related to DMRT4and DMRT5. Nested PCR examination indicated that the expression of Dmrt3could not be detected until bud stage, the amount of expression obviously increased within a limit of low level at15-somite stage. In adult tissues, it was only detected in the testes. This expression pattern suggested that Dmrt3might be involved in regulation of organogenesis and gonad development of male. Bisulfite sequencing analysis showed that the Dmrt3promoter CpG-island, which in the1207bp of the promoter region, was not methylated in gametes and in various tissues, suggesting that the sex-specific and tissue-specific expression of Dmrt3might be not mediated by different methylation. More than that, we identified a pseudogene, named pDmrt3, derived from reverse transcript of Dmrt3. These results provided essential materials for studying the function of Dmrt3in the sex differentiationand potential application in breeding of C. auratus, as well as the evolutionary relationship of Dmrt genes.