Gene Function Analysis Of Half-smooth Tongue Sole (Cynoglossus Semilaevis) Csdazl And CSFR2Genes

The half-smooth tongue sole (Cynoglossus semilaevis) is an importantcultured marine fish species in China; its sex determination system isZZ/ZW, with a huge W chromosome in the genetic female karotype.Notably, females grow two to four times faster than males, and thephenomenon of natural sex reversal from genetic female to phenotypicmale is observed. Therefore, the development of all-female stock orimproving the ratio of females would be of significant benefit foraquaculture．Our research group has cloned half-smooth tongue solesex-related genes Cyp19a, Foxl2and Sox9a, the results showed that theexpression levels of Cyp19a and Foxl2were higher in the ovary than inthe testis, while the expression of Sox9a was higher in the testis than inthe ovary.The dazl gene is a member of the DAZ (Deleted in Azoospermia)family, which plays important roles in gametogenesis. In this study, weidentified a dazl homolog in half-smooth tongue sole (Cynoglossussemilaevis), termed csdazl. The full-length cDNA of csdazl was2,026bp,containing a636bp open reading frame (ORF), a170bp5′UTR and a1,220bp3′UTR. Phylogenetic analysis indicated that csdazl clusteredwith that of other fishes. Real-time quantitative PCR showed that, in adultfish, csdazl was expressed strictly in the gonads (testis and ovaries), andthe gene expression was significantly higher in male than in female or inneo-male. In situ hybridization results showed that csdazl gene wasexpressed in primary spermatocyte, secondary spermatocyte and sperm inmale and neo-male fish; however, it was almost not expressed in female. The results indicate that the csdazl gene may play an important role inspermiogenesis of half-smooth tongue sole.To determine the function of csdazl, we constructed a prokaryoticexpression plasmid, pET-32a-CSDAZL, expressed and purified the fusionprotein. We injected the protein into fish by protein transfection. Theresults showed that the expression levels of Cyp19a and Foxl2significantly decreased between6to24h; however, both genes’expressions returned to their normal levels72h after injection, indicatingthat recombinant CSDAZL downregulated the expression offemale-related genes, Foxl2and Cyp19a genes, implying that CSDAZLplays a role in sex differentiation by regulating sex related genesexpression.After genome and transcriptome sequencing of half-smooth tonguesole (Cynoglossus semilaevis), we identified a female specific gene ofhalf-smooth tongue sole, termed CSFR2. The full length cDNA of CSFR2was2042bp, containing a669bp open reading frame (ORF), a223bp5’UTR and an1140bp3’UTR. Compared the sequence by BLASTX, wefound a BicD domain in CSFR2, there were no high homologous genes inNCBI database, indicated that it was a half-smooth tongue sole specificgene. Gonad slice RNA in situ hybridization results showed that CSFR2was expressed in oogonium. Real-time quantitative PCR showed that, inadult fish, CSFR2was expressed in ovary, pituitary and brain. The featurethat CSFR2was only expressed in the gonad axis predicted that CSFR2was related with sex determination and gonad development. In order toresearch gene function of CSFR2, we constructed a prokaryoticexpression plasmid pET-32a-CSFR2, expressed and purified the fusionprotein. We injected the protein into fish by protein transfection. Theresults showed that the expression levels of Cyp19a and Foxl2significantly increased between6to72h, then both genes’ expressions returned to their normal levels72h after injection, indicating thatrecombinant CSFR2upregulated the expression of female-related genes,Foxl2and Cyp19a genes, implying that CSFR2plays a role in sexdifferentiation by regulating sex related genes expression and provided anew strategy in the gene function study.