| This study analyzed the expression and differences of prestin in different tissues by real-time quantitative PCR(RT-qPCR). Gene clone and dual-luciferase activity assay were performed to analyze the transcription activities of different premotors. Subsequently, we predicted the potential cis-regulatory elements by bioinformatics analysis. The main results of this study are as follows:(1) RT-qPCR was performed to quantify the total mRNA of Prestin in cochlea, cerebra and testicle. We found prestin was also expressed in the mouse testis and brain tissue with low levels in addition to highly expression in the cochlea OHC. We also quantified the transcriptional variant 1 in these tissues, and we found different transcriptional variants were expressed, and the expression of transcriptional variant 1 accounted for 58.08%,43.77% and 62.91% respectively in cochlea, brain and testis.(2) We analyzed the differences of the known transcriptional variants, and we designed amplification of the specific fragment for different transcriptional variants. We found transcriptional variant 1 in cochlea, cerebra, testicle and GC-1 spg cells. There was transcriptional variant 2 and/or 3 in cochlea. Then,5’RACE was performed to amplify 5’-DNA sequence of presitn in cochlea and testicle, and we found transcriptional variant 2 and/or 3 in testicle. But there were no unknown transcriptional variants had been identified.(3) Dual luciferase reporter assay was performed and we found high transcriptional activity with promoter 2 and lower promoter 1. It also illustrated that there are two transcriptional activation regulatory elements located in the 280bp to 765bp upstream of the first exon and 952bpupstream of the second exon. There were transcriptional suppression regulatory elements in the 765bp to 1096bp upstream of the first exon and 952bp to 1954bp upstream of the second exon.(4) We also predicted many potentially transcription factors binding sites, and found many transcription factor GATA-3,-2,-1 binding sites, which was expressed highly correlated with prestin in cochlea. We surmised that transcription factor GATA-3,-2,-1 was transcriptional regulator factors in mouse spermatogonia cells.In this study we investigated the expression and regulation of prestin, and these results layed the foundation for clarification of the highly specific expression regulation pattern in the future. |
