THE EACYP19A1A PROMOTER OF RED-SPOTTED GROUPER DIRECTS TH EXPRESSION OF GFP IN EARLY PRIMARY GONAD CELLS OF ZEBRAFISH

Cytochrome P450 aromatase (P450arom) is the key enzyme responsible for estrogen biosynthesis in many species, and is believed to participate in the regulation of sexual differentiation of teleost fishes. However, the regulation mechanisms of aromatase genes remain unknown. In this study, we examined the role of EaCyp19a1a promoter in ovary specific expression of EaCyp19a1a. Firstly, nine 5′-deletion promoter region were obtained by PCR amplification using primers designed according to the accurate localizations of the special positive regulatory elements in the isolated 5′-flanking sequence, such as SOX5, CREB, SF-1, and so on. Then, these nine deletion mutants covering EaCyp19a1a promoter were constructed into the recombinant pEGFP-N1 (4.1 kb) vector, and examined their transcription activities. The construct of pEaCyp19a1a-EGFP7 vector was proved to possess the highest activity by transient transfection in COS-7 cells. Subsequently, the linearized pEaCyp19a1a-EGFP7 DNA was microinjected into fertilization eggs of zebrafish and the embryos were observed termly with fluorescent microscopy for the green fluorescent protein (GFP) expression. Specific green fluorescence was found to be restricted in the early primary gonad cells as early as zebrafish embryos at the hatching stage (about 48 hpf) and extend gradually along with the direction of germinal ridge development in larvae. These results indicated that some cis-acting elements were binding to EaCyp19a1a promoter and had the ability to regulate EaCyp19a1a gene expression specificly in the gonad. This research laid a foundation for further study on the role of aromatase in sex differentiation and sex inversion of fish.