牦牛VEGFA双荧光素酶载体构建及与miR-200b的靶向验证  

Construction of Yak VEGFA Double Luciferase Vectors and Their Targeting against miR-200b

作　　者：谢建鹏 陈平 王斐[1] 何振富[1] XIE Jianpeng;CHEN Ping;WANG Fei;HE Zhenfu(Institute of Animal Husbandry,Pasture and Green Agriculture,Gansu Academy of Agricultural Sciences,Lanzhou Gansu 730070,China)

机构地区：[1]甘肃省农业科学院畜草与绿色农业研究所,甘肃兰州730070

出　　处：《寒旱农业科学》2023年第8期745-749,共5页Journal of Cold-Arid Agricultural Sciences

基　　金：甘肃省农业科学院科研条件建设及成果转化项目(2021GAAS51)。

摘　　要：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.

关 键 词：牦牛 载体构建 双荧光素酶 VEGFA基因 miR-200b 

分 类 号：S823.8[农业科学—畜牧学]