miR-155与CEBPB的靶向验证及繁殖性能相关基因的检测  

Targeting Validation of miR-155 and CEBPB and Detection of Reproductive Performance-Related Genes

作　　者：李军义 刘娣 马红[2] 汪亮[2] 霍秀鹏 郝丽[1] LI Junyi;LIU Di;MA Hong;WANG Liang;HUO Xiupeng;HAO Li(College of Wildlife and Protected Area,Northeast Forestry University,Harbin,150040,China;Animal Husbandry Institute of Heilongjiang Academy of Agricultural Sciences,Harbin,150086,China)

机构地区：[1]东北林业大学野生动物与自然保护地学院,哈尔滨150040 [2]黑龙江省农业科学院畜牧研究所,哈尔滨150086

出　　处：《野生动物学报》2023年第2期305-312,共8页CHINESE JOURNAL OF WILDLIFE

基　　金：国家自然科学基金项目(31271324,U20A2052);黑龙江省科学基金项目(C2017011);中央高校基本科研业务费专项资金项目(2572014EA05-03,2572015CA18);国家生猪技术创新中心先导科技项目(NCTIP-XD/C16)。

摘　　要：CEBPB具有调节黄体生成促进排卵的作用,对动物繁殖性能极为重要,试验旨在通过构建CEBPB的3′端非翻译区(3′UTR)双荧光素酶报告基因载体,验证MicroRNA-155(miR-155)调控CEBPB的分子机制。应用在线生物信息学软件预测miR-155与CEBPB基因3′UTR的靶向结合区,PCR扩增获取民猪组织基因组的CEBPB的3′UTR,将其插入psi-Check-2载体,构建野生型(wt-3′UTR)和突变型重组表达载体(mut-3′UTR),鉴定正确后,分别与miR-155模拟物(mimics)/抑制物(inhibitor)/阴性对照物(NC)共转染到PK15细胞,检测荧光素酶活性及CEBPB表达情况。结果表明:成功构建了CEBPB的3′UTR野生型和突变型表达载体,双荧光素酶报告基因检测显示wt-3′UTR/miR-155 mimics组的萤火虫荧光素酶活性受到显著抑制(p<0.05);而mut-3′UTR/miR-155 inhibitor组的萤火虫荧光素酶相对活性与对照组相比变化不显著(p>0.05)。实时荧光定量结果显示,miR-155 mimics组CEBPB、BMP1和PPARG表达水平显著低于miR-155 NC组(p<0.05),而miR-155 inhibitor组CEBPB表达水平高于miR-155 NC组(p<0.05)。综上表明,miR-155可以靶向负调控CEBPB的表达。CEBPB has the effect of regulating luteinization and promoting ovulation,which is extremely important for ani⁃mal reproductive performance.The experiment aims to verify MicroRNA-155(miR-155)Molecular mechanisms regulating CEBPB.In this study,the online bioinformatics software was used to predict the targeted binding region of miR-155 and the 3′UTR of CEBPB gene,and the 3′UTR of CEBPB was obtained by PCR amplification and inserted into the psi-Check-2 vector to construct wild-type(wt-3′UTR)and mutant recombinant expression vector(mut-3′UTR),then they were com⁃bined with miR-155 mimics(mimics)/inhibitors(inhibitor)/negative controls(NC)co-transfected into PK15 cells after correc⁃tion,and luciferase activity and CEBPB expression were detected.The results showed that the 3'UTR wild-type and mutant expression vectors of CEBPB were successfully constructed,and the dual-luciferase reporter gene assay showed that the relative luciferase activity expression of the wt-3′UTR/miR-155 mimics group was significantly inhibited(p<0.05);how⁃ever,the relative luciferase activity expression of the mut-3′UTR/miR-155 inhibitor group was not significantly inhibited.Real-time fluorescence quantitative results showed that the expression levels of CEBPB,BMP1,and PPARG in the miR-155 mimics group were significantly lower than those in the miR-155 NC group(p<0.05),while the expression levels of CEBPB in the miR-155 inhibitor group were higher than those in the miR-155 NC group(p<0.05).It was shown that miR-155 targets and negatively regulates the expression of CEBPB.

关 键 词：繁殖性能 CEBPB基因 MIR-155 

分 类 号：Q492[生物学—生理学]