MicroRNAs (miRNAs) can regulate specific gene expression by binding to target mRNA further involution to diverse biological processes. Our previous miRNA sequencing showed that pol-miR-26a and pol-miR-26b have a sex-biased expression in ovary and testis of Paralichthys olivaceus (P. olivaceus). And the gene empty spiracles homeobox 2 (emx2) was proposed to be a candidate target by bioinformatics prediction. In this study, we cloned the P. olivaceus emx2 cDNA, including a coding region of 741 bp and a 3'-untranslated region (UTR) of 912 bp and the 5'-UTR of 12 bp. The Emx2 protein is highly conserved and especially its homeodomain region is 100% identical from teleosts to mammals. Real-time PCR results showed that the emx2 is not only highly expressed in embryonic neurula stage and adult brain but also has abundant expression in adult gonad, moreover, it exhibits higher expression in ovary than testis. To determine the relationship between emx2 and miRNAs, a luciferase reporter assay was performed and verified that the emx2 is a common target gene of pol-miR-26a and pol-miR-26b. These data thus helps further clarify that miR-26a and miR-26b are involved in regulating gonad development partially through its target on emx2 expression in P. olivaceus.
