Molecular Cloning And Expression Analysis Of DAZL And WT1a Gene In The Half-smooth Tongue-sole, Cynoglossus Semilaevis

Half-smooth tongue sole (Cynoglossus semilaevis) belongs to Pleuronectiformes,Soleidae，Cynoglossus，which is one of the most valuable fishes and a rare marineflatfish in Chinese coastal. However, Females grow two to three times faster thanmales in Half-smooth tongue sole and the phenomenon of natural sex reversal infemale half-smooth tongue sole is universal. In crosses between sex reversal femalesand normal females, the ratio of sex reversal females in F1progeny is higher than F1progeny of normal males and females. Thus, the development of all-female stockwould be of significant benefit for aquaculture．To develop all-female stock ofCynoglossus semilaevis and investigate sex determination mechanism is necessary.The DAZL and WT1a gene were obtained by RACE cloning in Cynoglossussemilaevis, we found that both of them are related to sex.Above all, along with thegenes expression, gonadal development and differentiation of Cynoglossussemilaevisfry (16d,26d,36d,46d,56d,66d,86d,116d) were observed usinghistological techniques. These are not only beneficial to clarify the function of DAZLand WT1a gene in ZZ/ZW fish and the role in sex reversal, but also providetheoretical basis and basis materials for female parthenogenesis of Cynoglossussemilaevis.Combining the geneticsex identification with histological techniques, the wholeglands developing process of Cynoglossus semilaevis from16days to116days afterhatch was followed. The result showed that the presumptive ovary proliferated morerapidly than testis. It was at46days after hatch the female formed big cells, but gonadremained undifferentiated in males. At56days after hatch, the female formed primaryovarian cavity, at this time, it can be say that the gonad begins to differentiate. Inaddition, we observed that the gonads of sex reversal females can produce sperm byobserving gonad slices of sex reversal female fish, which is similar to that of normalmales.The Deleted in Azoospermia (DAZ) gene plays an important role in the process of gametogenesis. It was frequently deleted in infertile men. The DAZL gene wasisolated from the AZFc region on the long arm of the human Y chromosome originally.Here, we obtained the full-long DAZL gene by cloning in Cynoglossus semilaevis.The full-length cDNA of DAZL was2026bp containing636bp open reading frame(ORF),170bp5′UTR and1220bp3′UTR. This sequence encoded211amino acidscontaining RNA recognition motif (RRM) and DAZ repetitive element. Alignmentanalysis showed that DAZL gene of Cynoglossus semilaevis had a relatively highhomology with that of Lates calcarifer. NJ-phylogenetic tree indicated that DAZLgene of Cynoglossus semilaevis is together with that of other fishes. Inaddition,Real-time quantitative PCR results showed that DAZL was almost notexpressed in the tissues except gonads. Thus, we can know that DAZL is agender-specific gene. During the embryonic period, the DAZL only4h-24h after hatchwas expressed. It is remarkable that the expression rises rapidly at the56days whenthe gonads begin to different. These results indicate that DAZL gene is agender-specific gene and may play an important role in the gonad differentiation ofCynoglossus semilaevis.The Wilms’ tumor suppressor gene (WT1) encodes a zinc-finger transcriptionfactor that plays an essential role in organ development, most notably in the kidneyand gonad. Despite its importance for sex determination, knowledge of the regulationof WT1expression is scarce. Here, the WT1a gene was obtained by cloning inCynoglossus semilaevis. Gonadal development and differentiation of Cynoglossussemilaevis (16-66d) were observed by using histological techniques. Sequenceanalysis revealed that the full-length cDNA was1886bp containing1470bp openreading frame (ORF),245bp5′UTR and171bp3′UTR. This sequence encoded489amino acids containing KTS alternative splice. Two SSR sequences and twotransmembrane helix regions are found in the end of the OFR. We infer that they arespecific sequences only in Cynoglossus semilaevis．Alignment analysis showed thatWT1a gene of Cynoglossus semilaevis had a high homology with that of Danio rerioand Oncorhynchus mykiss. NJ-phylogenetic tree indicated that WT1a gene of fish isidenticaler to WT1gene of other species than the WT1b gene of fish. Gonadaldevelopment and differentiation of Cynoglossus semilaevis were observed by usinghistological techniques. Gonad remained undifferentiated46days (31.2mm) afterhatching. When larva fishes grow to56days(36.0mm), the presumptive ovary proliferated more rapidly than testis. Real-time quantitative PCR results showed thatWT1a was expressed in six tissues and extremely higher in gonad. Thus, we can knowthat WT1a is sex-related gene. It is remarkable that it was expressed highest in normaltestis and lowest in gonad of sex-reversal female fish. These show that WT1a may benot important in the process of sex reversal in cynoglossus semilaevis. The geneexpressed in all the analyzed developmental stages, from early embryo to66dayjuveniles. Its expression level is the highest at neurula stage (20h) and gently in16-66days. These results indicate that WT1a gene is a sex related gene and mayindispensable for gonad differentiation of Cynoglossus semilaevis.