Cloning, Expression And Alternative Splicing Of DMRT1 In Misgurnus Anguillicaudatus

The DM (Doublesex and Mab-3) domain was identified by the homologous analysis between the sex determining gene doublesex(dsx) in Drosophila melanogaster and Mab-3 in Caenorhabditis.elegans. DM domain-containing genes act as transcription regulators, with the DNA domain conferring sequence-specific zinc finger-like DNA binding. More DM domain-containing genes have been identified from vertebrates including mammals, birds, turtle and fishes. So, a novel family was identified named DMRT (Doublesex and Mab-3 related transcription factor). As one of member in the family, Dmrt1 is an important transcription factor, which research is the most thorough and extensive to date. Dmrt1 is the first conserved gene found among animal phyla for sexual development, which plays an important role during animal sex determination. For fish serves as a link in the phylogenetic evolution of vertebrate, it is an important research carrier of sex determination. So researching it can not only help to reveal the mechanisms of the sex determination and the differentiation system in fish; but also help to expound the evolutional process of the sex determination and the differentiation system in vertebrates.To isolate the Dmrt1 gene, we amplified the Dmrt1 DM-domain by PCR, using the forward and backward primers designed on the basis of the conserved region amino acid sequence of the Dmrt1 genes registered in Genbank. With the testis cDNA of Misgurnus anguillicaudatus as template, one major fragment(about 140bp) was amplified by RT-PCR. Based on these study, the full-length cDNA of Dmrt1 were cloned from Misgurnus anguillicaudatus using 5'-and 3'-rapid amplification of cDNA ends(RACE). The 5′-and 3′-RACE results were jointed to obtain the full-lengths of DMRT1 cDNA sequences with the DNAMAN software respectively. Six novel isoforms generated by alternative splicing were cloned from Misgurnus anguillicaudatu, which all have the DM domain and 3'polyA tail. In terms of general nomenclature rule, they were named MaDmrt1a1 (2162 bp, encode 267aa), MaDmrt1a2(2042 bp, encode 267aa), MaDmrt1a3(1993 bp, encode 267aa), MaDmrt1a4 (848 bp, encode 267aa), MaDmrt1b (1840 bp, encode 219aa) and MaDmrt1c (666 bp, encode 182aa) respectively. By means of genome comparison, we conjecture that MaDmrt1a have all the five exons, MaDmrt1b lacks exon4, and MaDmrt1c lacks both exon4 and exon5 but uses a 36bp region of intron sequence as its exon 4 at the 3'ends. MaDmrt1a2 , MaDmrt1a3 and MaDmrt1a4 have anterior alternative polyadenylation signals in 3'UTR than Dmrt1a1.Several technologies, such as Semi-quantitive PCR, ?uorescent quantitative RT-PCR and in situ hybridization, were used to analyse the expression pattern of Dmrt1. The result revealed that the expression of Dmrt1 was testis-specific in adults, with no or very week expression in ovary. This pattern is in line with that of other species's Dmrt1. Contrary to expectations, all the Dmrt1 transcripts were detectable in all embryos tested. However, take the result by and large, each isoform's expression trend was risie at first and then decline. Taken together, these results provided the basic data for elucidating Dmrt1 role(s) for sex-determination and gonadal differentiation in vertebrates. The differential expression of these transcripts provides new insight into roles of alternative splicing of Dmrt1 in governing sex differentiation and development of Misgurnus anguillicaudatus. In site hybridization results demonstrated that Dmrt1 express in premature germ cells (ogania, primary oocytes in ovaries, spermatogonias and spermatocytes in testes) of gonads during gonadal development,suggesting roles of Dmrt1 in Misgurnus anguillicaudatu gonads development and differentiation. The zygotic expression of Dmrt1 all occure in the middle of segmentation, and persists throughout tail-bud formed, strong positive hybrid signals could be detected in the spine. Till hatched larva, the major tissues expressing Dmrt1 include notochord, somites and eyes. Taken together, these results provided the basic data for elucidating Dmrt1 role(s) for sex-determination and gonadal differentiation in vertebrates.