Cloning And Expression Analysis Of Sex-related Gene Dax1 In Paralichthys Olivaceus

Sex determination and gonadal differentiation are integral parts of reproduction and the essential process for the survival and evolution of fish species. Dax1 is an unusual member of the orphan nuclear receptor superfamily of transcription factors, and it is a strong candidate gene for a sex determination and gonadal differentiation role in fish. In this study, we have proceeded to the isolation of the full sequence cDNA and 5' flanking region of olive flounder (Paralichthys olivaceus) dax1, and then its expression patterns in adult tissues were analyzed using RT-PCR. Furthermore, the expression of these genes during early embryonic development, and gonadal differentiation and development were studied using real-time RT-PCR. Meanwhile, the methylation patterns of dax1 5'flanking region CpG island in testis and ovary tissues were analyzed. Then, the Dax1 recombination proteint prokaryotic expression vector was constructed and in vitro expressed of using E.coli Transetta(DE3) system.After RT-PCR and subsequent 5- and 3-RACE, the full-lengthy daxl cDMA was obtained from olive flounder gonad tissues. The dax1 cDNA was 1446bp, including a 330bp 3-UTR, a 219 bp 5-UTR, and 897bp ORE which eneodes a predicted 296-amino acid (aa) protein that has a predicted mol wt of 33.08kDa. The 3'UTR is approximately 350 bp and contains a putative poly adenylation signal and a poly(A) tail. As with all daxl genes described so far, the olive flounder dax1 gene lacks the characteristic zinc finger motif in the DNA-binding domain (DBD). Similar to other non-mammal species Dax1, there is only one of the three conserved LXXLL-like motifs that present in the DBD of the mammal Daxl. Multiple sequence alignment results revealed that the predicted olive flounder Dax1 protein shared highest overall sequence identity and similarity to those of other fish followed by, in descending order, bird, amphibia and mammals.The 2Kb sequence of P.olivaceus dax1 5'flanking region was also isolated by genome walking. The putative promoter of olive flounder daxl was described, and putative transcription factor binding sites (TFBS) that have been shown to regulate the other vertebrate dax1 transcription were also identified. Moreover, we have compared the promoter conservation of fish DAX1 putative promoters to those described in other vertebrate, and have identified different promoter modules and frameworks that are present in most of the dax15'flanking regions, within the same order and distances.The tissue specific expression patterns show that daxl mRNA can be detected in many tissues of adult olive flounder. This gene is a sex-related gene with sexual dimorphic expression. The daxl was higher expressed in ovary than that in testis. As for other tissues, the expression level is relatively high in spleen and brain, and is relatively low in liver and muscle. During embryogenesis, its expression was detected in most developmental stages, although the level of expression was different at different stages. Results of real-time RT-PCR indicated that olive flounder dax1 was expressed in primitive gonad and during following periods of gonadal differentiation. Its expression went up rapidly in both testis and ovary before gonad differentiation (total lenghth 2 cm), and expressed relatively low during following periods of gonadal differentiation.This gene is also differentially expressed during five developmental stages of gonad. Generally, the expression quantities in all the five stages of ovary were always higher than those of testis(?). The dax1 expression quantities difference between ovary and testis?? was not significant in the stageâ… . And the daxl expression quantity went up rapidly in both testis and ovary of stageâ…¡. After that, the quantity went down in the stageâ…¢, but the expression in testis was still lower than in ovary. Its expression quantity reached the peak in the ovary of stage IV. Then its expression quantity went down in the stage IV.Analysis results of genes promoter CpG islands methylation patterns showed that there were no sexual dimorphic differences in the CpG methylation level of olive flounder dax1 promoter. For control flounder, there were 1.4%(2/140) CpG Island methylation of dax1 promoter in testis and its 2%(3/140) in ovary. As for gynogenetic and sex reversal flounder, there were 2.8%(4/140) CpG Island methylation of this gene promoter in testis and 2.1%(3/140) methylation in ovary, respectively.Olive flounder dax1 ORF was amplified by RT-PCR using specific primers, and then was inserted into vector pProEXTMHTa to construct the prokaryotic recombinant express vector. The recombinant vector was transducted into E.coli Transetta(DE3) and the protein expression was detected by SDS-PAGE and western blot analysis. The results showed that most recombinant protein expressed in the form of inclusion bodies, and the optimal induction time and IPTG concentration were 4h and 1mM, respectively.