和田羊FGF5基因RNA干扰载体构建及筛选  

Construction and screening of RNA interference vector aimed at Hotan sheep FGF5 gene

作　　者：曹少奇 陈岩 高新梅 喻恒彬 王静[2] 胡广东 Cao Shaoqi;Chen Yan;Gao Xinmei;Yu Hengbin;Wang Jing;Hu Guangdong(Xinjiang Animal Husbandry Station,Xinjiang Urumqi 830000;College of Animal Science and Technology,Shihezi University,Xinjiang Shihezi 832061)

机构地区：[1]新疆维吾尔自治区畜牧总站,新疆乌鲁木齐830000 [2]石河子大学动物科技学院,新疆石河子832061

出　　处：《现代畜牧兽医》2023年第9期32-36,共5页Modern Journal of Animal Husbandry and Veterinary Medicine

基　　金：新疆维吾尔自治区多浪羊品种选育推广技术体系专项(2021DLY-23);兵团重点领域科研攻关计划项目肉羊健康养殖模式关键技术集成创新与示范(2021AB014)。

摘　　要：试验旨在构建并筛选出高效的和田羊成纤维细胞生长因子5(FGF5)基因的RNA干扰载体,根据和田羊FGF5基因序列,设计4条短发夹RNA(shRNA)干扰片段和1条阴性对照片段。扩增后克隆至pGPU6载体,获得4个重组质粒pGPU6-shRNA-FGF1、pGPU6-shRNA-FGF2、pGPU6-shRNA-FGF3、pGPU6-shRNAFGF4和阴性对照质粒pGPU6-shRNA-FGF5。通过琼脂糖凝胶电泳和DNA测序对质粒进行酶切鉴定和测序鉴定。鉴定后的质粒通过电刺激法转染至和田羊成纤维细胞,通过qRT-PCR和Western Blotting(WB)分析FGF5基因的表达情况。结果显示,与阴性载体相比,4个RNA干扰表达载体均可显著降低FGF5 mRNA和蛋白的表达量(P<0.05),其中pGPU6-shRNA-FGF3干扰效率最高,为85.20%。研究表明,FGF5干扰表达载体构建成功并可有效干扰和田羊FGF5基因表达。The aim of the experiment was to construct and screen out efficient RNA interference vectors of Hotan sheep FGF5 gene.According to the FGF5 gene sequence of Hotan sheep,four shRNA interference fragments and one negative control fragment were designed.Four recombinant plasmids pGPU6-shRNA-FGF1,pGPU6-shRNA-FGF2,pGPU6-shRNA-FGF3,pGPU6-shRNA-FGF4 and negative control plasmid pGPU6-shRNA-FGF5 were obtained.The plasmid was identified by enzyme digestion and DNA sequencing by agarose gel electrophoresis.The identified plasmids were transfected into Hotan sheep fibroblasts by electrical stimulation,and the expression of FGF5 gene was analyzed by qRT-PCR and WB.The results showed that compared with the negative vector,the four RNA interference expression vectors could significantly reduce the expression levels of FGF5 mRNA and protein(P<0.05),and pGPU6-shRNA-FGF3 had the highest interference efficiency(85.20%).The study indicates that the FGF5 interference vector is successfully constructed and can effectively interfere with FGF5 gene expression in Hotan sheep.

关 键 词：和田羊 RNA干扰 FGF5基因 载体构建 成纤维细胞 

分 类 号：S813.1[农业科学—畜牧学]