Functional analysis of the promoter of the dmrt1 gene in Chinese tongue sole, Cynoglossus semilaevis

The dmrt1 (doublesex and mab-3 related transcription factor 1) gene is considered to play an essential role in testis differentiation and development among metazoan species. As an economically important marine fish in China, Chinese tongue sole (Cynoglossus semilaevis) possess a ZW/ZZ sexdetermining system, and the females usually grow two to four times bigger than males. Previous studies have confirmed that Z-linked dmrt1 gene is the male determining gene in C. semilaevis and TALENs-mediated knocking-out individuals showed increased growth phenotype. In order to investigate the function of dmrt1 regulatory regions and its potential application, we have cloned the promoter of C. semilaevis dmrt1. The acquired sequence consisted of a 5'-upstream sequence and a partial exon1; functional analysis showed that there were two positive regulatory regions in the promoter, as well as one negative region and one neutral region. These positive regions were then combined to obtain several newly constructed promoters. Both in vitro and in vivo analysis confirmed one of them (dmrt1 - Δ3+Δ4) showed elevated promoting activity compared to the full-length promoter and could drive exogenous gene expression in tongue sole gonads. Our results will provide useful information for understanding the regulatory mechanism of dmrt1 gene during C. semilaevis sex determination, and the endogenous promoter will facilitate the transgenic research for analyzing sex- and growth-related genes.