β-miR-33a特异调控HADHB基因3′UTR靶标位点验证  

Validation ofβ-miR-33a specific regulating HADHBgene 3′UTR target site

作　　者：崔志倩 芦春艳[2] 郭佳[1] 赵尧璐 邢沈阳[1] 赵志辉[1] 

机构地区：[1]吉林大学动物科学学院,吉林长春130062 [2]吉林大学农业实验基地,吉林长春130062

出　　处：《中国兽医学报》2017年第2期282-286,共5页Chinese Journal of Veterinary Science

基　　金：吉林省省级产业创新专项资金资助项目(2016C032);国家科技重大专项资助项目(2016ZX08009003-006-002);国家"863"计划资助项目(2013AA102505);国家自然科学基金资助项目(31372278)

摘　　要：为验证β-miR-33a与候选基因HADHB3′UTR是否存在特异性识别的靶标关系,本试验将β-miR-33amimics、β-miR-33ainhibitor及NC质粒分别转染至奶牛乳腺上皮细胞,通过Real-time(qPCR)验证β-miR-33a与HADHB基因mRNA水平表达量的关系。利用PCR获得奶牛HADHB3′UTR 201bp DNA片段,将其克隆到pmiR-RB-REPORTTM双荧光素酶报告载体中,获得野生型载体,利用定点突变技术将HADHB3′UTR种子区-CAATGCA定点突变-CATGTAGC,获得突变型载体。将野生型载体和突变型载体分别与β-miR-33a-mimics组成双荧光素酶报告系统,转染至奶牛乳腺上皮细胞,48h后测定其荧光活性。结果显示,奶牛乳腺上皮细胞中转染的野生型与突变型载体的双荧光活性存在显著性差异(P<0.05),由此证明,在奶牛乳腺上皮细胞中β-miR-33a可以与HADHB3′UTR的靶位点-CAATGCA特异性结合。The purpose of this experiment was to verify the specific identification relationship between β-miR-33a and candidate gene of HADHB 3＇UTR,mimics β-miR-33a,inhibitor NC and β- miR-33a were transfected into bovine mammary epithelial cells, and the relationship between β- miR-33a and HADHB mRNA expression was verified by real-time （qPCR）. Then,the cow gene HADHB 3＇UTR （201 bp） was amplified by PCR. And, HADHB 3＇UTR was cloned into the pmiR-RB-REPORTTM dual luciferase reporter vector to obtain the wild-type vector. The seed zone （CAATGCA） of HADHB 3＇UTR was mutated as CATGTAGC using site directed mutation to obtain the mutant vector. The double luciferase reporter systems were composed of the wild type and mutant vectors with β-miR-33a mimics, respectively, which were transfected into bovine mammary epithelial cells. After 48 h,the fluorescence activity was determined. The results showed that there was a significant difference between the wild-type and the mutant carriers in the trans- fection of the mammary epithelial cells （P〈0.05）, β-miR-33a can be combined with the target site （CAATGCA） of HADHB 3＇UTR in mammary epithelial cells of dairy cow.

关 键 词：HADHB基因3’UTR β-miR-33a 双荧光素酶报告基因 乳腺上皮细胞 

分 类 号：S853.91[农业科学—临床兽医学] Q78[农业科学—兽医学]