鹌鹑MITF基因的克隆、表达及启动子甲基化研究

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

【目的】研究MITF基因在鹌鹑胚胎发育过程中的表达变化及其与羽色形成的关系,分析启动子甲基化对MITF基因表达的影响。【方法】利用RT-PCR克隆鹌鹑MITF基因mRNA的CDS区域并进行DNA测序,对得到的序列进行BLAST比对等生物信息学分析。利用RT-qPCR分析MITF基因在栗羽朝鲜鹌鹑和北京白羽鹌鹑胚胎发育第6、8、10、12和14天的表达变化。克隆朝鲜鹌鹑和北京白羽鹌鹑MITF基因的启动子区域并进行DNA测序,利用BS-PCR分析启动子CpG岛的甲基化水平。【结果】鹌鹑MITF基因与日本鹌鹑同源基因相似度达99%,包含1476 bp的开放阅读框,编码蛋白由492个氨基酸残基组成,没有信号肽和明显的跨膜区,成熟蛋白位于细胞核中。在胚胎发育的6~12 d,朝鲜鹌鹑MITF基因m RNA表达水平显著或极显著高于北京白羽鹌鹑(P<0.05或P<0.01)。鹌鹑MITF基因启动子区域包含丰富的顺式作用元件和1个346 bp的CpG岛,朝鲜鹌鹑MITF基因启动子区域的平均甲基化水平为18.52%,北京白羽鹌鹑的平均甲基化水平为29.63%,两者差异显著(P<0.05)。【结论】北京白羽鹌鹑MITF基因启动子区域高甲基化状态导致其表达水平下降,这与其羽色白化有一定的关系。

[Purpose]To 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. [Methods]The 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.[Results]The similarity of quail MITF gene with homologous gene in Japanese quail was over99%. 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). [Conclusion]The 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.