Structure And Expression Of SoxE And DMRI1 Gene In Two Loaches

We obtained the full length cDNA of SoxE from two loaches and DMRT1 of Paramisgurnus dabryanus by RACE. Semi-quantitative RT-PCR and fluorescent real-time RT-PCR were conducted to determine the expression profiles. We also examined the expression patterns of the corresponding genes in gonad and early embryonic gonads of two loaches by in situ hybridization. The major research results of this study are as follow:1. Isolation and Expression of SoxE Genes in Misgurnus anguillicaudatusIn this article, to amplify genomic DNA of Misgurnus anguillicaudatus, degenerate primers were designed according to the conservative sequences in HMG-box of Sox% and Sox10 genes. Based on these results, the full-length cDNA of above five fragments were isolated from the brain of Misgurnus anguillicaudatus by using 5’-and 3’-rapid amplification of cDNA ends (RACE). The full-length cDNA of MaSox8a was 2555bp (GenBank accession no. GU166139), containing the 53bp 5’-untranslated region, 1212bp 3’-untranslated region [including poly(A)] and encoding a putative protein of 429 amino acids with a characteristic HMG-box DNA-binding domain of 79 amino acids (aa:86-164);The full-length cDNA of MaSox8b was 1725 bp (GenBank acc. no. GU166140), containing the 85bp 5’-untranslated region,395bp 3’-untranslated region [including poly(A)] and encoding a putative protein of 414 amino acids with a characteristic HMG-box DNA-binding domain of 79 amino acids (aa:89-167); The full-length cDNA of MaSox9a was 3192bp, containing the 326bp 5’-untranslated region,1408bp 3’-untranslated region [including poly(A)] and encoding a putative protein of 485 amino acids with a characteristic HMG-box DNA-binding domain of 79 amino acids (aa:105-183);The full-length cDNA of MaSox9b was 1782bp, containing the 217bp 5’-untranslated region,179bp 3’-untranslated region [including poly(A)] and encoding a putative protein of 462 amino acids with a characteristic HMG-box DNA-binding domain of 79 amino acids (aa:97-175); The full-length cDNA of MaSox10 was 2096bp, containing the 311bp 5’-untranslated region,312bp 3’-untranslated region [including poly(A)] and encoding a putative protein of 490 amino acids with a characteristic HMG-box DNA-binding domain of 79 amino acids (aa:105-183).Semi-quantitative RT-PCR and fluorescent real-time RT-PCR reactions were performed to analyze expression profiles using the glyceraldehyde-3-phosphate dehydrogenase(GAPDH) gene as the reference. During early embryonic development, the expression of MaSox8a and MaSox8b were opposite, and both begain from the gastrulae to the yolk-sac absorption phase. MaSox9a、MaSox9b and MaSox10 all showed a notable up-regulation and highest expression in hatched stage; In adult tissues, the expression level of SoxE varied among tissues, MaSox8a and MaSox8b with stronger expression detected in brain, MaSox9a and MaSox9b with stronger expression detected in testis, MaSox10 with stronger expression detected in heart.In site hybridization results demonstrated that SoxE genes express in premature germ cells (ogania, primary oocytes in ovaries, spermatogonias and spermatocytes in testes) of gonads during gonadal development, suggesting roles of SoxE in Misgurnus anguillicaudatu gonads development and differentiation; The zygotic expression of SoxE 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 SoxE include notochord, somites and eyes.2. Isolation and Expression of SoxE Genes in Paramisgurnus dabryanusIn this article, to amplify genomic DNA of Paramisgurnus dabryanus, degenerate primers were designed according to the conservative sequences in HMG-box of Sox8 and Sox10 genes. Based on these results, the full-length cDNA of SoxE were isolated from the brain of Paramisgurnus dabryanus by RACE. The full-length cDNA of PdSox8a was 2467bp, containing the 53bp 5’-untranslated region,1127bp 3’-untranslated region [including poly(A)] and encoding a putative protein of 428 amino acids with a characteristic HMG-box DNA-binding domain of 79 amino acids (aa:86-164); The full-length cDNA of PdSox8b was 2166bp, containing the 201bp 5’-untranslated region,720bp 3’-untranslated region [including poly(A)] and encoding a putative protein of 414 amino acids with a characteristic HMG-box DNA-binding domain of 79 amino acids (aa:89-167); The full-length cDNA of PdSox9a was 3232bp, containing the 329bp 5’-untranslated region,1439bp 3’-untranslated region [including poly(A)] and encoding a putative protein of 487 amino acids with a characteristic HMG-box DNA-binding domain of 79 amino acids (aa: 105-183); The full-length cDNA of PdSox9b was 1946bp, containing the 219bp 5’-untranslated region, 341bp 3’-untranslated region [including poly(A)] and encoding a putative protein of 461 amino acids with a characteristic HMG-box DNA-binding domain of 79 amino acids (aa:97-175); The full-length cDNA of PdSox10 was 2053bp, containing the 312bp 5’-untranslated region,262bp 3’-untranslated region [including poly(A)] and encoding a putative protein of 492 amino acids with a characteristic HMG-box DNA-binding domain of 79 amino acids (aa:106-184).Semi-quantitative RT-PCR and fluorescent real-time RT-PCR reactions were performed to analyze expression profiles using the glyceraldehyde-3-phosphate dehydrogenase(GAPDH) gene as the reference. During early embryonic development, the expression of PdSox8a、PdSox8b、PdSox9b and PdSox10 showed a downward trend from the gastrulae to the yolk-sac absorption phase, PdSox9a was opposite, with a notable up-regulation; In adult tissues, the expression level of SoxE varied among tissues, and all with strong expression detected in gonad. Moreover, PdSox8a with stronger expression detected in brain, PdSox8b、PdSox9a and PdSox9b with stronger expression detected in liver, PdSox10 with stronger expression detected in heart.The results of in site hybridization in Paramisgurnus dabryanus were same as Misgurnus anguillicaudatus.3. mRNA Spatio-temporal expression analysis and multiple alternative splicing of DMRT1 in Paramisgurnus dabryanusTo isolate DMRT1 gene, we amplified the DM-domain by PCR, using degenerate primers designed on the basis of the conserved region amino acid sequence of the DMRT1 genes registered in Genbank. Based on these results, the full-length cDNA of DMRT1 were cloned from Paramisgurnus dabryanus using 5’-and 3’-RACE. Five isoforms generated by alternative splicing were cloned from Paramisgurnus dabryanus, which all have the DM domain and 3’polyA tail yet.They were named PdDMRT1a1 (1903bp, encode 267aa), PdDMRT1a2(1714bp, encode 267aa), PdDMRT1a3(887bp, encode 267aa), PdDMRT1b(724bp, encode 220aa) and PdDMRT1c(946bp, encode 196aa) respectively. PdDMRT1a1,2 and 3 all have five exons, by the difference between them was only in 3’UTR. PdDMRT1b lacks 9bp of exon4 in its 3’end and the whole exon5. PdDMRT1c lacks both exon4 and exon5 but uses a 63bp region of intron sequence as its exon 4 at the 3’ends.Several technologies, such as Semi-quantitive PCR, fluorescent quantitative RT-PCR and in situ hybridization, were used to analyse the expression pattern of DMRT1. The result revealed that the expression of PdDMRT1 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 Paramisgurnus dabryanus. These results provided the basic data for elucidating DMRT1 role(s) for sex-determination and gonadal differentiation in vertebrates.In conclusion, my study established that 1) Subgroup E, at least including five members in two loaches; 2) In this study, two distinct Sox8 genes were identified in two loaches, so far only one case reported that two duplicates of Sox8 were identified in Takifugu rubripes; 3)We also obtained two distinct Sox9 genes and one copy of Sox10 from two loaches; 4) We identified that alternative splicing have been occured in Paramisgurnus dabryanus; 5) This experiment first used a variety of methods to detect the expression patterns of each objective gene. Results indicated that SoxE have a possible functional redundancy in some domains and they are essential for the differentiation of nervous and reproductive system. DMRT1gene was relevant to the formation and function maintainance of testis.