Cloning and transcriptional regulation of tf and tfr1a promoters in Megalobrama amblycephala

In order to explore the transcriptional regulation mechanism of the tf and tfr1a genes in Megalobrama amblycephala, the genomic sequences of tf and tfr1a were obtained from whole genome sequence database. Transcription factor binding sites and CpG islands in the promoter regions of tf and tfr1a genes were predicted by bioinformatics methods. Fragments of different length of the predicted promoter region of tf and tfr1a were cloned by PCR amplification. The amplified different fragments were ligated to the pGL3-Basic/pEGFP-1 vector. Subsequently, the recombinant plasmids were transiently transfected into Hela cells for fluorescence detection by the Dual-Luciferase Reporter System. Bioinformatics analysis showed that there was no CpG island site in the tf promoter, and there were two CpG island sites in the tfr1a promoter. A total of 9 tf and 10 tfr1a recombinant plasmids containing promoter fragments of different lengths were successfully constructed. The detection of Dual-Luciferase Reporter System showed that the core region of the tf promoter was −268—+56 bp, and the −1 308—−1 102 bp fragment may have a transcription factor binding site that positively regulates the gene expression. The core region of the tfr1a promoter was −224—+48 bp, and the +48—+92 bp region may contain negative regulatory elements that inhibit the transcription of this gene, while the −1 229—−1 219 bp region might contain positive regulatory transcription factor binding sites that promote the tfr1a gene expression.