Cloning DMRT1 CDNA Of Oreochromis Aurea And Study Of Sex-Determining Mechanis In Tilapia

Among the vertebrates, sex determination mechanism of fishes is the most diversified. Infishes, genetic components are still the basis of sex determination, as in higher vertebrates, but the sex-determining genes are not distinctly concentrated on the sex chromosomes because many autosomal genes are also involved in sex determination. In addition, sexuality of fishes is more evidently exhibited as bi-potential during early stageso In different degrees, some environmental factors, such as temperature, exogenous hormones etc., can influence the process of sex differentiation, making the mechanisms of fish sex determination even more complex. Through molecular biology and gene study, we can reveal sex-determining mechanism in tilapia to provide the foundation for improving the masculinity rate.The DM-domain is a DNA-binding motif shared between Drosophila doublesex and C.elegans mab-3.Both doublesex and mab-3 play key roles in sex deterrnination.DMRTl (doublesex and mab-3 related transcription factor l)expression correlates with testicular differentiation gene. In this study, we analyse DMRT1 gene for further studying sex correlative determination gene in tilapia.1. amplification of DM-domainBased on DM-domain of the known fishes, I designed upstream primer P1 and downstream primer P3 and amplified a sequence about 158bp. Sequence blast analysis revealed that the homology of DM-domain in DMRT1 of Oreochromis aurea and Oreochromis niloticus is 96%. Because of the sequence conservation, DM-domain of Oreochromis aurea has highly similarity with Acanthopagrus schlegelii, Oncorhynchus mykiss, Odontesthes bonariensis, Tetraodon nigroviridis, Fugu rubripes, Monopterus albus, Oryzias latipes.2. amplification of male specific motifBased on DM-domain of in DMRT1 of Oreochromis aurea, I designed upstream primer P5. Based on male specific motif of the known fishes, I designed downstream primer P4 and amplified a sequence about 481bp, including male specific motif 72bp. male specific motif sequence blast analysis revealed that the homology of male specificmotif in DMRT1 of Oreochromis aurea and Oreochromis niloticus is 100%. Because of the sequence conservation, male specific motif of Oreochromis aurea has highly similarity with Acanthopagrus schlegelii, Oncorhynchus mykiss, Odontesthes bonariensis, Tetraodon nigroviridis, Fugurubripes, Monopterus albus, Oryziaslatipes. 3. 3'RACEBased on DM-domain of in DMRT1 of Oreochromis aurea, I designed upstream primer P5 andP6. A rapid amplification of cDNA ends (RACE) was used for the isolation of the length cDNA of DMRT1 (DM-domain gene in tesis) from tesis of Oreochromis aureao We amplified a fragment about 1086bp by 3' RACE-PCR , encoding 254 amino acido Sequence analysis revealed that the homology of DMRT1 in Oreochromis aurea and Oreochromis niloticus is 99% . However, we compare the alignment of deduced amino acid between DMRT1 cDNA from Acanthopagrus schlegelii, Monopterus albus, Xiphophorus maculatus, Oncorhynchus mykiss, Oryzias latipes, Oreochromis aurea and DMO cDNA from Oreochromis aurea, Oreochromis niloticus. The score is 99%, 80%, 78%, 73%, 62%, 41% ,26%, 26% respectively . These results indicated that there is great difference in sex-determining mechanism of milter and spawner.