为探究微小RNA(miR-200b)与血管内皮生长因子A(VEGFA)基因之间的靶向关系，采用miRanda和Targetscan软件预测牦牛miR-200b与VEGFA可能存在结合位点，构建了VEGFA基因3′UTR区的野生型和突变型双荧光素酶载体。利用与目的miRNA靶种子序列相结合基因的突变序列，构建该靶基因的突变型重组质粒(pGL3-promoter-mut VEGFA)，再进行重组质粒和目的miRNA的miRNA mimic的共转染实验。结果表明，预测到的miR-200b与VEGFA基因的3′UTR区存在结合位点。PCR进行扩增和测序之后，VEGFA基因3′UTR区的野生型、突变型载体构建成功。共转染VEGFA野生型载体和miR-200b mimic的双荧光素酶活性极显著低于对照组(P < 0.05)，VEGFA突变型载体和miR-200b mimic共转染的双荧光素酶活性与对照组无显著差异(P ＞ 0.05)。说明VEGFA基因的3′UTR区能与miR-200b结合并抑制双荧光素酶活性，验证了VEGFA是miR-200b的靶基因。

In order to investigate the targeting relationship between miR-200b related to yak reproduction and vascular endothelial growth factor A(VEGFA) gene, miRanda and Targetscan were used to predict the possible binding site between miR-200b and VEGFA in yak. The wild-type and mutant dual-luciferase vectors of VEGFA 3′UTR region was constructed. The mutant plasmid pGL3-Promoter-mut VEGFA of the target gene was constructed by using the mutant sequence of the target gene and the target seed sequence of the target miRNA. Then the co-transfection experiment of the target miRNA mimic and the recombinant plasmid was carried out. The results showed that the predicted binding site between miR-200b and the 3′UTR region of VEGFA gene existed. After PCR amplification and sequencing, the results showed that the wild-type and mutant vectors of VEGFA 3′UTR region were successfully constructed. The double luciferase activity of co-transfected VEGFA wild-type vector and miR-200b mimic was significantly lower than that of the control group (P < 0.01), while the double luciferase activity of co-transfected VEGFA mutant vector and miR-200b mimic was not significantly different from that of the control group(P > 0.05). These results indicated that the 3′UTR region of VEGFA gene could bind to miR-200b and inhibit dual luciferase activity, which verified that VEGFA was a target gene of miR-200b.

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甘肃省农业科学院科研条件建设及成果转化项目(2021GAAS51)。