猪miR-192对DLG5和ALCAM基因的靶向作用关系验证

发布时间：2018-03-05 09:09

  本文选题：猪　切入点：miRNA-　出处：《农业生物技术学报》2017年09期 　论文类型：期刊论文

【摘要】：在生理病理过程中,micro RNAs(miRNAs)通过作用于相应靶基因发挥着重要的作用。通过高通量测序结合荧光定量实验验证,本课题组前期初步确定miR-192靶向调控Discs大同源物(discs large homolog 5,DLG5)和活化白细胞黏附分子(activated leukocyte cell adhesion molecule,ALCAM)基因在断奶仔猪抗大肠杆菌(Escherichia coli)感染过程中发挥了重要的作用。本研究采用双荧光素酶报告系统和Western blot方法,验证DLG5和ALCAM基因是否受到miR-192的特异性调控。构建含有靶基因位点的荧光素酶报告基因重组载体,与PRL-TK和miRNA-192模拟物mimics、inhibitor或阴性对照共转染293细胞,24 h后收集细胞检测荧光素酶活性和靶基因蛋白表达变化。同时检测3种大肠杆菌感染肠上皮细胞(intestinal epithelial cells,IPEC-J2)后靶基因的表达变化。本研究成功获得荧光素酶报告基因重组载体,荧光结果显示,miRNA-192 mimics显著抑制2个报告重组载体的荧光素酶活性(P0.05),而miRNA-192inhibitor则显著促进2个报告重组载体的荧光素酶活性(P0.05)。同时,miRNA-192模拟物处理组靶基因DLG5和ALCAM蛋白水平极显著低于阴性对照组,再次验证了上述结果。3种大肠杆菌感染后,2个靶基因的表达均显著或极显著上升。由结果可知,猪miR-192对DLG5和ALCAM基因具有靶向抑制作用,且DLG5和ALCAM基因的表达确实与大肠杆菌感染有关。本研究结果为miR-192及其靶基因在断奶仔猪抵抗F18大肠杆菌感染过程中的功能和调控机制相关研究提供了一定的实验基础和理论依据,进一步为猪抗大肠杆菌病有效遗传标记的筛选提供了科学依据。
[Abstract]:Micro RNAsmiRNAsM plays an important role in physiological and pathological processes by acting on the corresponding target gene, which is verified by high throughput sequencing and fluorescence quantitative experiments. Our team initially identified that miR-192 targeting regulation of Discs large homolog 5 (DLG5) and activated leukocyte adhesion molecule leukocyte cell adhesion (ALCAM) gene play an important role in the resistance of weaned piglets to Escherichia coli infection. Double luciferase report system and Western blot method were used. To verify whether DLG5 and ALCAM genes were specifically regulated by miR-192, we constructed a recombinant vector of luciferase reporter gene containing target gene sites. The luciferase activity and target gene protein expression were detected in 293 cells cotransfected with PRL-TK and miRNA-192 mimicsl inhibitor or negative control for 24 h. The target genes were detected after three kinds of Escherichia coli infected intestinal epithelial cells were infected with intestinal epithelial cells IPEC-J2. The recombinant vector of luciferase reporter gene was successfully obtained in this study. The fluorescence results showed that miRNA-192 mimics significantly inhibited the luciferase activity of two reporter recombinant vectors, while miRNA-192inhibitor significantly promoted the luciferase activity of the two recombinant vectors. At the same time, the target gene DLG5 and ALCAM protein levels in the treatment group of miRNA-192 mimics were significantly increased. It was significantly lower than that in the negative control group. The results showed that the expression of two target genes was significantly or extremely significantly increased after infection of E.coli. The results showed that porcine miR-192 could inhibit the expression of DLG5 and ALCAM genes. The expression of DLG5 and ALCAM genes is indeed related to E. coli infection. The results of this study provide a certain experimental study on the function and regulation mechanism of miR-192 and its target genes in the process of weaning piglets resisting F18 E. coli infection. Basic and theoretical basis, It provides a scientific basis for screening effective genetic markers for resistance to Escherichia coli in pigs.
【作者单位】： 扬州大学动物科学与技术学院/江苏省动物遗传繁育与分子设计重点实验室;扬州大学教育部农业与农产品安全国际合作联合实验室;
【基金】：国家自然科学基金(No.31372285和No.31572360) 江苏省科技支撑计划(No.BE2014357和No.BE2015329)
【分类号】：S828
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本文编号：1569602