Cloning, Molecular Characterization And Function Of DMO And DMT In Oreochromis Aurea

Tilapia is native to Africa but introduced elsewhere as a valuable food fish. It is very popular all over the world. In aquaculture, the male grows more quickly than the female by 40-50%, so it is very important to improve the male rate. In the hybrid combinations of various cichlid fishes of the genus Tilapia, the male rate of offspring of Oreochromis aurea(♂)×Oreochromis niloticus(♀) is the highest(95%), but it should be 100% according to the chromosomal theory. Sex determination and differentiation in fish is highly variable and complicated, so molecular biology methods are used necessarily to reveal the sex regulatory mechanism and improve the male rate in tilapia at gene level.The DMRT genes constitue a new gene family related to sex-determination. Like the Double-sex gene of Drosophila melanogaster and the Mab-3 gene of Caenor habditis elegents, they encode transcription factors characterized by a conserved zinc-finger like DNA-binding motif, the DM domain, which is thought to bind DNA in the process of sex differentiation and development. In 1998, DMRT1 genes were found to regulate sex determination and differentiation in vertebrates extensively. So far, the DMRT genes have been discovered in a wide range of animal species, such as fish, amphibian, reptiles, birds and mammals. These evidently reveal the evolutionary conservation of DMRT gene family.The DM domain gene family has multiple members in both invertebrates and vertebrates. For example, up to date, there are at least seven DM domain gene found in mouse, eight in human, four in Drosophila, eleven in C. elegans, and six in fish (DMRT1-5 and DMRT2b). All of them encode putative transcription factors related to the sexual regulator Dsx of Drosophila and Mab-3 of C. elegans. Multiple DM domain genes have been suggested to be involved in mouse sexual development. At least three mouse DM domain genes in addition to DMRT1 are expressed in embryonic gonad, including DMRT3, DMRT4, and DMRT7. However, DMRT2 is expressed in presomitic desoderm and development somites, while DMRT5 and DMRT6 are expressed primarily in the brain, suggesting a role in other developmental precesses.Although some of the DM genes are involved in sexual development, function of most of these genes remains unclear, and we know remarkable little about the evolution of the DM genes. It is still the question that the male-specific role of the DM gene is primordial during their evolution, or independently evolved by convergence. Furthermore, it is essential to understand the roles of the DM genes in regulatory pathway of sex determination, or if any, the roles in other developmental processes.There is also few structural and functional analysis concerning DM domain genes of the model fish Oreochromis aurea. We report here cloning, characterization and expression of DMO and DMT of Oreochromis aurea.In this study, RT-PCR and RACE were used for the cloning of DMO (DM-domain gene in ovary) and DMT (DM-domain gene in testis) full length cDNA from ovary and testis of Oreochromis aurea, respectively. DMO and DMT genes were sequenced and analyzed by bioinformatics methods. Sequence analysis revealed a 1571 bp cDNA full-length sequence of DMO containing 148 bp 5'-untranslated region, 193 bp 3-untranslated region and 1230 bp ORF encoding 409 amino acid. Homology of DMO from Oreochromis aurea and Oreochromis niloticus was 96.3%. However, we compared the alignment of deduced amino acid sequences between DMO cDNA from Oreochromis aurea and DMRT1 cDNA from Oreochromis niloticus, fugu, rainbow trout, medaka, rat to human. The score was 25.7%, 25.8%, 24.3%, 29.7%, 22.5% and 22.0%, respectively. A 1260 bp cDNA full-length sequence of DMT encoded 292 amino acids, which contained 74 bp 5'-untranslated region, 307 bp 3'-untranslated region and 879 bp ORF. The deduced amino acid sequence aligned with those of DMRT1 genes from different species, high sequence homologies were obtained as revealed in phylogenic tree constructed.The amino acid sites of DM domains may form C2/H2 model zinc-finger structure to bind specific DNA sequence and regulate sex differentiation and development. DMT contained a male specific motif, which was well conserved among numerous DMRT1 genes, but was absent in DMO indicating that DMT represented a male-type DM-domain gene and played an important role in sex differentiation and development.The bioinformatics analysis revealed that DMO had two helical segments that were 97-112 amino acid sequence and 155-168 amino acid sequence, and did not contained signal peptide. It was a transmembrane and hydrophilic protein. DMT had not helical segment and did not contained signal peptide. It was a non-transmembrane and hydrophilic protein. DMO and DMT included two same functional domains, which played the roles of sex control, dimerising and binding palindromic DNA, respectively. DMO and DMT both included several phosphorylation sites implicating that they could play some roles during cellular signal conduct and their activities might be related to the regulation of many signals during signal route. DMO and DMT had similar advanced structures including two a-helix regions. Moreover the B-cell epitopes possibly localized in or nearby the DMO protein's N-termianl No.1-5, 41-51, 65-67, 86-89, 98-110,154-170,183-203, 205-248, 258-264, 284-291, 293-298, 270-375, 389-392 and No.402-410, and DMT protein's N-termianl No.1-9,17-28, 77-84,114-123,131-139,157-184 and No.96～207.The temporal and spatial expression patterns were analyzed by Real-time Quantitative RT-PCR at cellular level. Their transcripts appeared from early gastrulae stage during embryonic development, and maintained a considerable high level till the one day's fry, but level of DMO was higher than that of DMT. Treatment with hormone not noly improved remarkably the female or male rate, but also changed level of DMO and DMT during sex differentiation, implicating that they could be related to hormone regulation. No DMO and DMT transcripts were found in liver, kidney, spleen, heart and muscle, but unequal amount of DMO transcripts were detected in both brains tissues of female and male Oreochromis aurea, which suggested that mRNA expression of DMO was specific in central nervous system (CNS). In addition, we also found the abundant transcripts of DMO in ovary and DMT in testis. This supports that the DMO and DMT genes play important roles not only on the sex determination, but also on the development processes of early embryogenesis. Based on these results, we suggest that DMO should play a key role in CNS and ovary development of Oreochromis aurea and DMT in testis. Study on DMO and DMT expression facilitates the elucidation of the roles of them and the understanding of sex differentiation and development in fish.Their mRNA expression was further analyzed at cellular levels using in situ hybridization. The results were as follows: DMO was expressed only in ovary and DMT was observed in testis exclusively. DMO mRNA uniformly dispersed throughout the cytoplasm of oocytes at all stages. With the development of oocytes in oogonia and stage I,II oocytes, the expression signal became stronger. In stage of III oocyte, DMO mRNA was uniformly observed in cytoplasm between grains of yolk. In the later stage, the signals of DMO mRNA significantly decreased in most of the regions while remained strong at cortical region. DMO mRNA signals were not detected throughout the oocytes after the yolk was full of the cytoplasts. During spermatogenesis, the positive signals of DMT mRNA could only be detected in spermatogenia and primary spermatocytes while the signals in the former were much stronger than in the latter. Howerer, no signals could be detected in spermatids. The results suggest that DMO and DMT may play an important role in maintenance and functioning of the gernline stem cell-oogonia and spermatogonia.To further inquire into the function of DMO and DMT, the intact regions encoding DMO and DMT obtained by RT-PCR were sub-cloned into the vector pMAL-c2x prokaryotic expression system and introduced into the Escherichia coli TB1 cell for efficient fusion expression. It was found that the expression level was about 50% of total protein in the engineered bacteria after IPTG induction for 4h. Immunoblotting proved the immunogenicity of DMO and DMT. After purification and cleavage, DMO and DMT proteins were used to immunize the adult rabbits following standard protocols. Consequently, we found that polyclonal antibodies against DMO and DMT had high specificity by Western blot analysis.The expression of DMO and DMT protein was also analyzed using the purified antibodies through Western blot and immunohistochemistry. We found DMO was exclusively expressed in ovary and DMT in testis, but specificy by immunohistochemistry is lower than by in situ hybridization. Study on DMO and DMT expression facilitates the elucidation of the roles of them and the understanding of sex differentiation of fish.In general, this is the first time to clone DMO and DMT genes related to sex control from Oreochromis aurea. The cloning, molecular characterization and function of DMO and DMT in Oreochromis aurea provide not only new materials for researches on DMRT molecular evolution and similarity comparison, but also theoretical basis for better understanding the sex control and DMRT in fish.