牛PSMA5基因RNA干扰载体的构建与筛选及在奶牛乳腺上皮细胞中的表达  

RNA interference vector construction and selection of cattle PSMA5 gene and its expression in cow mammary gland epithelial cells

作　　者：迟玉杨[1] 张晶[1] 徐子娴 高安崇[2] 王秀娜[2] 周长海[1] 牛淑玲[1] 金永成[1] 沈景林[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》2016年第1期112-117,131,共7页Chinese Journal of Veterinary Science

基　　金：国家自然科学基金青年科学基金资助项目(31301996);高等学校博士学科点专项科研基金资助项目(20130061120112)

摘　　要：设计4条待选的shRNA序列以及1条阴性对照序列,构建RNA干扰载体,然后转染到奶牛乳腺上皮细胞(MAC-T)中,最后利用实时荧光定量PCR和Western blotting技术分别检测蛋白酶体α5亚基(PSMA5)在奶牛乳腺上皮细胞(MAC-T)中mRNA和蛋白水平的表达。结果显示:转染shRNA-1组,shRNA-3组和shRNA-4组的PSMA5基因相对表达量显著低于阴性对照组(P<0.05),且shRNA-4组的PSMA5基因相对表达量最低。结果表明:本试验成功构建并筛选出了PSMA5基因RNA干扰载体,并在奶牛乳腺上皮细胞(MAC-T)中成功表达。This study was to explore the role of proteasome alpha 5 subunits （PSMA5） in the period of conjugated linoleic acid （CLA） endogenous synthesis. Firstly, four shRNA sequeaces and one negative control sequence were designed to construct the RNA interference vector. Then,transfected the RNA in- terference vector into cow mammary gland epithelial cells （MAC-T）. Finally, detected the relative expres- sion of mRNA and protein levels of the PSMA5 gene in the cow mammary gland epithelial cells （MAC- T） by real-time fluorescent quantitative PCR and Western blotting technology. The results suggested that in transfected shRNA-1, shRNA-3 and shRNA-4 groups,relative mRNA and protein levels expression of PSMA5 gene was significantly lower than the negative control group（P〈0.05）. In conclusion,we con- structed and selected RNA interference vector of cattle PSMA5 gene successfully,and detected its expres- sion in cow mammary gland epithelial cells （MAC-T）.

关 键 词：PSMA5 共轭亚油酸 MACT RNA干扰载体 

分 类 号：Q78[生物学—分子生物学] S852.23[农业科学—基础兽医学]