Sf1、Foxd1 And Yy1 Regulate The Promoters Of Amh In Paralichthys Olivaceus

AMH(Anti-Müllerian hormone),also known as Müllerian inhibiting substance,is a member of the transforming growth superfamiy β.It is highly expressed in the gonads of mammals,birds,scleractinians,amphibians,etc.It exerts its biological effects mainly by acting on its type II receptor(AMHR2)and has an important role in sex determination,gonadal differentiation and development,and has been a hot research target for scholars at home and abroad in recent years.A promoter is a DNA sequence located upstream of the transcription start site that can be specifically recognized and bound by RNA polymerase,and some promoters,after binding to RNA polymerase,often require the assistance of protein cofactors to participate in the transcriptional regulation process.The initiation,efficiency and subsequent biological functions of gene expression are inseparable from the regulation of promoters,so the study of promoters becomes particularly important.More studies have shown that the amh gene is involved in sex determination,gonadal differentiation and development in vertebrates as a sex regulator,but the transcription factors that bind directly to its promoter region and their regulatory relationships have been less reported.In this study,the potential transcription factor binding sites in the promoter region of Paralichthys olivaceus amh were predicted by online software,screened for gene conservation and identified by a dual luciferase reporter gene assay system,followed by identification of key binding sites by progressive deletion of binding sites and targeted mutation of binding sites.Finally,the regulation of amh expression by transcription factors was verified at the cellular level.The main results were as follows:(1)The transcription factor binding sites of Sf1,Foxd1 and Yy1 on the amh promoter of Paralichthys olivaceus were predicted by online prediction software.The amino acid sequences of Sf1,Foxd1,Yy1 a and Yy1 b were found to be conserved compared with other fishes or vertebrates,and the phylogenetic tree analysis revealed that Sf1,Foxd1,Yy1 a and Yy1 b are most closely related to other scleractinian fishes and far from mammals and birds,which is consistent with the taxonomic status of Paralichthys olivaceus,among them,Yy1 a and Yy1 b each clustered into a separate branch in the scleractinian fish.(2)The dual luciferase reporter gene results showed that Sf1,Foxd1,Yy1 a,and Yy1 b all had regulatory effects on the amh promoter region of Paralichthys olivaceus,while Sf1 and the other three transcription factors did not have synergistic or antagonistic effects on the regulation of amh promoter activity.(3)The presence of three binding sites of transcription factor Sf1(-80 bp～-69 bp,-1444 bp～-1434 bp,-1802 bp～-1791 bp),three binding sites of transcription factor Foxd1(-527 bp～-513 bp,-797 bp～-790 bp,-1889 bp～-1882 bp)and four binding sites of transcription factor Yy1(-201 bp～-189 bp,-488 bp～-477 bp,-769 bp～-757 bp,-1330 bp～-1316 bp).The results of the progressive deletion fragment experiment showed that the key binding site of transcription factor Sf1 might exist in the amh promoter region-1791 bp～-1434 bp,the key binding site of transcription factor Foxd1 might exist in the fragment of amh promoter region-790 bp～-513 bp,and the key binding site of amh promoter region-790 bp～-513 bp.amh promoter region-1316bp～-757 bp and-477 bp～-189 bp,the key binding site of Yy1 a in amh promoter region-1941 bp～-1316 bp and-477 bp～-189 bp,and the key binding site of Yy1 b in amh promoter region-1941 bp～-1316 bp and-477 bp～-189 bp;The results of targeted mutation of the amh promoter region binding site showed that the amh promoter regions-1444 bp～-1434 bp,-527 bp～-513 bp,-769 bp～-757 bp and-201 bp～-189 bp which are the key binding sites for transcription factors Sf1,Foxd1,Yy1 a and Yy1 b,respectively.(4)The expression patterns of sf1 and foxd1 were identified by q PCR in various tissues of Paralichthys olivaceus,and both were interfered at the level of the primary cells of the testis of Paralichthys olivaceus.The fluorescence quantification results found that sf1 and foxd1 had similar expression patterns with amh,and both were highly expressed in the testes of Paralichthys olivaceus;The fluorescence quantification results showed that sf1 was mainly expressed in the gonads of Paralichthys olivaceus,and mainly in the testes,where the expression was 1.5 times higher than that in the ovaries,and weakly expressed in other tissues;foxd1 was widely expressed in all tissues of Paralichthys olivaceus,with the highest expression in the brain tissue,followed by the testes,and some expression in the ovaries.The pre-interference experiment found that sf1-936 and foxd1-372 interference chains had the best interference effect,and the interference effect was best at 24 h and 48 n M interference conditions;The interference test found that the decrease of sf1 and foxd1 expression down-regulated the expression of amh in Paralichthys olivaceus,the inhibitory effect of foxd1 interference on amh expression was more pronounced than that of sf1.The results showed that both sf1 and foxd1 had a positive effect on the expression of amh.