番鸭VIPR1和miR-317以及MAP3K7和miR-244靶向关系的研究

Verification of Targeting Relationship Between VIPR1 and miR-317 and MAP3K7 and miR-244 in Muscovy Duck

试验旨在验证番鸭就巢相关miRNA与靶基因靶向关系。试验利用miRanda、PITA和RNAhybrid三个软件的交集预测miR-317和miR-244靶基因,构建VIPR1-pmirGLO/MAP3K7-pmirGLO野生型和突变型双荧光素酶重组载体,将miR-317/VIPR1-Wt和miR-244/MAP3K7-Wt分别共转染至鸭胚成纤维细胞,检测双荧光素酶活性。结果显示:与miR-NC组相比,miR-317/VIPR1-Wt共转染组相对荧光值显著降低(P<0.05);VIPR1突变后,miR-NC组与miR-317组的相对荧光值不显著(P>0.05),说明突变后完全抑制miR-317对VIPR1的结合作用,VIPR1与miR-317之间存在相互结合作用。与miR-NC组相比,miR-244/MAP3K7-Wt共转染组相对荧光值无显著性差异(P>0.05);MAP3K7突变后,miR-NC组与miR-244组的相对荧光值差异不显著(P>0.05),表明MAP3K7与miR-244之间不存在相互结合作用。研究提示,miR-317与VIPR1存在确切靶向结合位点,即VIPR1是miRNA miR-317的靶基因,而miR-244与MAP3K7不存在靶向关系。

The aim was to verify the relationship between broodiness-related miRNA and target gene targeting in Muscovy ducks.The intersection of miRanda,PITA and RNAhybrid software were used to predict the target genes of miR-317 and miR-244.Double luciferase recombinant vectors of VIPR1-pmirGLO/MAP3K7-pmirGLO wild-type and mutant were constructed,and miR-317/VIPR1-Wt and miR-244/MAP3K7-Wt were co-transfected into duck embryo fibroblasts to detect the double lucifer⁃ase activity.The results showed that the relative fluorescence value of miR-317/VIPR1-WT co-transfection group decreased sig⁃nificantly(P<0.05)compared with the miR-NC group,while there was no significant change in the relative fluorescence value of miR-NC group and miR-317 group after VIPR1 mutation(P>0.05),which suggested that the binding effect of miR-317 on VIPR1 was completely inhibited after mutation,indicating an interaction between miR-317 and VIPR1.The results showed that there was no significant difference in the relative fluorescence values of miR-244/co-transfection group compared with the miRNC group(P>0.05).Moreover,the relative fluorescence values of miR-NC group and miR-244 group were not significant after MAP3K7 mutation(P>0.05),indicating that there was no interaction between miR-244 and MAP3K7.According to the findings,exact targeting binding site was identified between VIPR1 and miR-317,indicating that VIPR1 was the target gene of miR-317.However,MAP3K7 and miR-244 did not have a targeting relationship.