Study on the Cloning, Expression and Promoter Methylation of MITF Gene in Quail

PurposeTo study the expression of MITF gene during embryonic development and the relationship between MITF gene expression levels with feather colors in different quails. The effect of promoter methylation on the gene expression levels was analyzed.

MethodsThe CDS region of MITF gene was cloned by RT-PCR and DNA sequencing. The sequence was analyzed by bioinformatics methods, such as BLAST analysis, and so on. RT-qPCR was used to analyze the expression levels of MITF gene on the 6th, 8th, 10th, 12th and 14th day of embryonic development in Korean quail and Beijing white quail. The promoter region of MITF gene was cloned and DNA sequenced, and the methylation levels of CpG island were analyzed by BS-PCR technology.

ResultsThe similarity of quail MITF gene with homologous gene in Japanese quail was over 99%. Quail MITF gene contained a whole open reading frame (ORF) of 1 476 bp, which encoded a protein, including 492 amino acid residues. MITF protein was without signal peptide and transmembrane region and the mature protein was located in the nucleus. During the 6-12 days of embryonic development, the expression level of MITF gene mRNA in Korean quails was significant or extremely significant higher than that in Beijing white quails (P<0.05 or P<0.01). There were abundant cis-elements and a 346 bp CpG island in the promoter region of MITF gene in quail. The average methylation level of MITF gene promoter region in Korean quail was 18.52%, and that in Beijing white quail was 29.63%, the difference was significant (P<0.05).

ConclusionThe hypermethylation of MITF gene promoter region in Beijing white quail results in the decrease of its expression level, which is related to the white feather color.