麒麟鸡Frizzled4基因的miRNA筛选及验证载体构建

Screening of miRNA and Verification Vector Constructing for Frizzled4 gene of Kirin Chicken

研究利用生物信息学方法选出调控Fzd4基因的miRNAs,且利用q RT-PCR方法检测了卷羽麒麟鸡和片羽怀乡鸡中miRNAs和Fzd4基因表达水平,进一步构建了Fzd4基因双荧光素酶报告基因pmir Glo载体,为后期在细胞水平验证miRNA和Fzd4靶标关系提供条件。结果显示:Fzd4基因的CDS区存在miRNA-1458(mi R-1458)和miRNA-1623(mi R-1623)靶标结合位点,与经典的调控机制有所不同,其3′UTR存在miRNA-184-3p(mi R-184-3p)结合位点;相对于怀乡鸡,麒麟鸡皮肤中mi R-1458、mi R-1623和Fzd4基因表达水平极显著升高(P<0.01),而mi R-184-3p表达量极显著降低(P<0.01);构建了Fzd4基因双荧光素酶报告载体,为细胞水平研究其具体调控机制奠定基础。研究表明Fzd4基因的关键miRNA为mi R-184-3p以及Fzd4基因双荧光素酶报告载体的构建为进一步研究其在毛囊生长发育过程的作用奠定基础。

In this paper,miRNAs of regulating Fzd4 using bioinformatics tools were selected,and the expression level of miRNAs and Fzd4 in Kirin chicken and Huaixiang chicken using q RT-PCR was detected. Furthermore,the target relationships between the miRNA and the Fzd4 gene were verified at the cell level via constructing Fzd4 gene dual luciferase pmir Glo vector. The results showed as follows：there were target binding sites of miRNA-1458（mi R-1458）and miRNA-1623（mi R-1623）in Fzd4 CDS,while being different from classical regulatory mechanism,the binding site of miRNA-184-3p（mi R-184-3p）was existed in 3′UTR;Compared with Huaixiang chicken,the expression level ofmi R-1458,mi R-1623 and Fzd4 was significantly increased（P〈0.01）;Fzd4 dual luciferase reporter vector was successfully constructed,which would lay the foundation for research specific regulatory mechanisms at the cellular level. The results showed that the key miRNA was mi R-184-3p for Fzd4 gene,Fzd4 dual luciferase reporter vector were constructed,which paving the way for further study its role on hair follicle growth and development.