| 细毛羊皮肤毛囊miR-1298-5p的靶基因预测及验证 |
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| 引用本文： | 徐晶,姜怀志,张桂山,孙丽敏,白曼,项露杰.细毛羊皮肤毛囊miR-1298-5p的靶基因预测及验证[J].中国畜牧杂志,2018(4). |
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| 作者姓名： | 徐晶 姜怀志 张桂山 孙丽敏 白曼 项露杰 |
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| 作者单位： | 长春科技学院动物科学技术学院;吉林农业大学动物科学技术学院 |
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| 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。
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| 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 |
Target Gene Prediction and Verification of MiR-1298-5p from Fine Wool Sheep Skin and Hair Follicles |
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| Institution: | ,College of Animal Science and Technology, Changchun Sci-Tech University,College of Animal Science and Technology, Jilin Agricultural University |
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| Abstract: | 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 FGF_2 were determined by RT-PCR and Western blot. Fragment of FGF_2 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 HEK293 T/17 cells to uncover the regulation between MiR-1298-5p and its candidate target genes FGF_2 by dual luciferase reporter system experiment. Results: Bioinformatics analysis showed 3'UTR of FGF_2 presented a binding site of MiR-1298-5p. The results of fluorescence quantitative PCR and western blot showed that between MiR-1298-5p and FGF_2 presented a negative correlation. The results of dual luciferase reporter system experiment showed that MiR-1298-5p inhibited wild type FGF_2. After mutation of target sites, dual luciferase activity of FGF_2 could recover, which indicated FGF_2 was target gene of MiR-1298-5p. Conclusion:MiR-1298-5p regulated hair follicles development of fine wool sheep by negative regulation FGF_2. |
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| Keywords: | Qianhua mutton merino Skin and hair follicles MiR-1298-5p FGF2 Luciferase activity analysis |
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