Molecular cloning and characterization of cyp19a1b gene and the effect of Letrozole on its expression in Carassius auratus

In the present study, brain aromatase gene (cyp19a1b) was cloned by using reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA 5′ and 3′ ends (RACE) in Qihe crucian carp (Carassius auratus). We also examined the expression level of cyp19a1b in different development stages and different tissues by real-time PCR, and at the same time, expression profile of cyp19a1b after hCG injection and sex reversal induced by Letrozole treatment was also examined. The results showed that the full-length cDNA of cyp19a1b was 2 894 bp, including a 132 bp 5′- untranslated region (UTR), a 1 319 bp 3′UTR, and a 1 533 bp open reading frame (ORF) encoding a deduced protein with 510 amino acids. Sequence analysis revealed that Cyp19a1b from C. auratus showed the highest amino acid similarity with Cyprinidae fish, while has low similarity with other vertebrates include mammalian and reptiles. Tissue distribution by real-time PCR revealed that cyp19a1b was abundantly expressed in the brain, while barely detectable in other tissues. The expression of cyp19a1b was gradually upregulated from blastula stage, and peaked at neurula stage, then declined during the embryonic development stage. Ontogenic analysis demonstrated that cyp19a1b was expressed abundantly from 30 days after hatching (dah) in the brain, but kept a low level in the trunks. However, cyp19a1b decreased in the brain of sex-reversed males by Letrozole treatment. Intraperitoneal injection of human chorionic gonadotropin experiments showed that expression of cyp19a1b was significantly elevated at 8 h and persisted until 24 h after injection in the brain. These results suggested that cyp19a1b might be involved in the course of nervous system formation, gonad development and sex determination in C. auratus.