细毛羊皮肤毛囊miR-1298-5p的靶基因预测及验证  

Target Gene Prediction and Verification of MiR-1298-5p from Fine Wool Sheep Skin and Hair Follicles

作　　者：徐晶 姜怀志[2] 张桂山[2] 孙丽敏[2] 白曼[2] 项露杰 XU Jing;JIANG Huai-zhi;ZHANG Gui-shan;SUN Li-min;BAI Man;XIANG Lu-jie(College of Animal Science and Technology,Changchun Sci-Tech University,Jilin Changchun 130060,China;College of Animal Science and Technology,Jilin Agricultural University,Jilin Changchun 130118,China)

机构地区：[1]长春科技学院动物科学技术学院,吉林长春130600 [2]吉林农业大学动物科学技术学院,吉林长春130118

出　　处：《中国畜牧杂志》2018年第4期79-82,共4页Chinese Journal of Animal Science

基　　金：吉林省教育厅项目(2015566);吉林省教育厅科技发展计划项目(2015186);吉林省科技发展计划项目(20160204018NY)

摘　　要：为探讨miR-1298-5p及其靶基因与细毛羊毛囊生长发育的关系,本实验以乾华肉用美利奴羊不同发育时期皮肤毛囊组织为材料,通过生物信息学方法预测miR-1298-5p的靶基因,采用RT-PCR和Western Blot对miR-1298-5p及其靶基因进行定量检测;扩增靶基因3'UTR区,构建野生型和突变型靶基因表达载体后与miR-1298-5p mimics共同转染到HEK293T/17细胞中,利用双荧光素酶报告基因实验验证miR-1298-5p与靶基因的靶向关系。结果表明:FGF_2基因3'UTR区含有miR-1298-5p结合位点,FGF_2为miR-1298-5p潜在靶基因,通过核酸水平和蛋白水平定量表达规律发现miR-1298-5p与靶基因FGF_2呈负相关,初步确定FGF_2为miR-1298-5p的靶基因。双荧光素酶报告基因实验显示,miR-1298-5p mimics能够抑制野生型FGF_2的靶位点报告载体的荧光素酶活性;但当靶基因结合位点突变后,FGF_2被抑制的荧光素酶活性能够得到恢复。综上,miR-1298-5p与FGF_2有靶向关系,miR-1298-5p能通过负调控FGF_2的表达调控羊毛囊发育。To investigate the relationship among hair follicles development,MiR-1298-5p and its target genes in fine wool sheep,potential target genes of MiR-1298-5p from different stage skin and hair follicles of Qianhua mutton merino were predicted by bioinformatics analysis,and relative expression of MiR-1298-5p and its potential target gene FGF2 were determined by RT-PCR and Western blot.Fragment of FGF2 3’UTR was cloned,and then wild type and mutant expression vectors were constructed.The expression vectors and MiR-1298-5p mimics were used to transfect into HEK293T/17 cells to uncover the regulation between MiR-1298-5p and its candidate target genes FGF2 by dual luciferase reporter system experiment.Results:Bioinformatics analysis showed 3'UTR of FGF2 presented a binding site of MiR-1298-5p.The results of fluorescence quantitative PCR and western blot showed that between MiR-1298-5p and FGF2 presented a negative correlation.The results of dual luciferase reporter system experiment showed that MiR-1298-5p inhibited wild type FGF2.After mutation of target sites,dual luciferase activity of FGF2 could recover,which indicated FGF2 was target gene of MiR-1298-5p.Conclusion:MiR-1298-5p regulated hair follicles development of fine wool sheep by negative regulation FGF2.

关 键 词：乾华肉用美利奴 羊皮肤毛囊 miR-1298-5p FGF2 双荧光素酶报告基因实验 

分 类 号：Q78[生物学—分子生物学] S826.2[农业科学—畜牧学]