SF-1、FOXL2 Regulate The Promoters Of Aromatase In Cheilinus Undulatus

Sex differentiation is an important acdamic issue in biological development.Due to the diverse mechanisms of fish development,it is good material for studying the sex problems of vertebrates.The mechanism which environmental factors regulate fish reversal is a complex and interesting problem in the gender development mechanism.Cheilinus undulatus is a kind of sexual reversal fish,and the expression regulation of aromatase directly affects gonadal reversal.For socially sexually reversible fish,finding an upstream regulator of Cyp19al can reveal the mechanism of the sex-regulatory pathway.Steroid-producing factor 1(SF-1/Ad4BP)is a member of the nuclear receptor superfamily and affects gender development by regulating aromatase expression.The forkhead transcription factor(FOXL2)is involved in the maintenance of ovarian development and its function.In this study,we investigated the regulatory activities of FOXL2 and SF-1 on the Cheilinus undulatus aromatase promoter by dual luciferase reporter technology and identified its key components.1.The ORF of FOXL2 and SF-1 were cloned by PCR,which were 1467 bp and 921 bp.The eukaryotic expression vector was constructed by double enzyme digestion.Three transcription sites of SF-1 present on the Cyp19a1a promoter were predicted,and the Cypl9alb promoter contained five and three FOXL2 transcriptional binding sites of SF-1.2.The eukaryotic expression vector of SF-1 and FOXL2 were co-transfected into the cell lines HEK293T and COS-7 by constructing the luciferase reporter system of the Cyp19a1a and Cyp19a1b promoters,and the results showed that SF-1 And FOXL2 can enhance the activity of the lipoamarin aromatase promoter,respectively,and the co-transfection of the two transcription factors can significantly up-regulate the activity of the Cyp19a 1 promoter.3.By constructing promoter progressive deletion,fusion PCR,site-directed mutagenesis and other techniques,three Cypl9ala promoter progressive deletions and site mutants were constructed,and the results showed that the-120 bp to-112 bp mutation significantly reduced SF-1.Transcriptional activity of the Cyp19a1a promoter.Five Cypl9alb promoter progressive deletion vectors were constructed,and the predicted sites in the region with a large influence on the transcriptional activity of the promoter were selected for mutation.It was found that the potential binding site of the mutant SF-1 could not significantly affect the activity of the Cypl9alb promoter.Three locus mutants of the Cypl9alb promoter progressive deletion vector were constructed.The results showed that the-890bp to-872bp mutation significantly reduced the transcriptional activity of the brain aromatase Cyp19a1b.This study found that both SF-1 and FOXL2 can regulate the Cypl9ala promoter and Cypl9alb promoter.SF-1 has a key binding element on the Cypl9ala promoter..FOXL2 has a key binding element on the Cypl9alb promoter.The combination of SF-1 and FOXL2 enhances the activity of the Cheilinus undulatus aromatase promoter.This study laid a theoretical foundation for the identification of the aromatase gene transcriptional regulatory pathway in Cheilinus undulatus.