Molecular cloning and sexually dimorphic expression of DMRT4 gene in Oreochromis aureus

Abstract

The DM-domain gene family has at least eight members with conserved DNA-binding DM-domain, which encodes putative transcription factors related to the sexual regulator Dsx of Drosophila and Mab-3 of C. elegans. Although some of the DM genes are involved in sexual development, the function of most of these genes remains unclear. In this study, rapid amplification cDNA ends (RACE) was used for the isolation of DMRT4 full-length cDNA from the ovary of the blue tilapia Oreochromis aureus. The full-length of DMRT4 cDNA was 1,571 bp, containing the 148 bp 5'-untranslated region, 193 bp 3'-untranslated region and 1,230 bp open reading frame. The deduced amino acid sequence of the open reading frame (ORF) encoded a protein of 409 amino acids with a theoretical pI of 8.492 and a calculated molecular weight of 44.12 kDa. One conserved functional domain, DM-domain was identified in blue tilapia DMRT4. The DMRT4 full-length gene obtained from the blood was 1,741 bp, containing a 156 bp intron. Phylogenetic analysis indicated that the amino acid sequences encoded by DMRT4 genes from different species had a high degree of sequence identity as revealed in phylogenetic tree constructed. Real-time quantitative Reverse-Transcription Polymerase Chain Reaction (RT-PCR) was used to analyze the expression patterns of DMRT4 in different developmental stages and different tissues in Oreochromis aureus. DMRT4 mRNA was detected from early gastrulae stage during embryonic development, and maintained a considerable high level until 1 day post hatching. With the increase of age, enhanced DMRT4 mRNA was observed in ovary and brain. After 15 and 30 days, fries treated with 17beta-estradiol had a significant increase in DMRT4 mRNA levels compared with the control fries (P < 0.05). DMRT4 was found to be expressed in the ovary and endbrain, thalamencephalon, pituitary, not detected in the liver, kidney, spleen, heart and muscle of adult fish. These results showed that the DMRT4 gene have potentially important roles in gonadal development and may have contributed to the functional endocrine axis. Our study provides fundamental understanding to the structure and functions of DMRT4 protein and the mechanisms of sex control in Oreochromis aureus.