靶向猪CLTC基因miRNA的预测与验证

发布时间：2018-04-01 08:07

  本文选题：猪　切入点：CLTC　出处：《浙江农林大学学报》2017年03期

【摘要】：网格蛋白介导的胞吞是病毒侵入细胞的重要途径,网格蛋白重链(clathrin heavy chain,CLTC)是形成网格蛋白小窝结构的重要组成部分。针对CLTC基因的转录后调控特别是调控猪Sus scrofa CLTC的miRNA目前还不太清楚。本研究旨在筛选出调控猪CLTC基因的miRNA。利用生物信息学方法预测出6个靶向猪CLTC基因的miRNA,将猪CLTC基因3′UTR克隆至双荧光素酶报告基因载体psi CHECK2中获得双荧光素酶报告基因重组载体psi CHECK2-CLTC-3′UTR。将预测得到的miRNA分别和重组载体psi CHECK2-CLTC-3′UTR共转染到细胞中,以乱序序列作为阴性对照(NC),检测miRNA对重组质粒荧光素酶活性的影响。结果发现miR-205,miR-1,miR-129-5p和miR-206均能够显著抑制荧光素酶活性(P0.05)。在猪肾上皮细胞系PK15细胞中超表达miR-1和miR-129-5p后,定量PCR(q-PCR)结果显示:猪CLTC基因的表达量显著下调。突变了psi CHECK2-CLTC-3′UTR载体中这4个miRNA的种子序列的结合位点发现:miR-1对突变质粒中的荧光素酶无显著抑制作用。表明miR-1与猪CLTC基因有直接的靶向关系,并通过其种子序列抑制CLTC基因的表达。
[Abstract]:Grid protein mediated endocytosis is an important pathway for virus invasion into cells, and grid protein heavy chain clathrin heavy chain1 (CLTCc) is an important component of the formation of grid protein nest structure.The posttranscriptional regulation of CLTC gene, especially the miRNA that regulates porcine Sus scrofa CLTC, is unclear.The aim of this study was to screen out the miRNA that regulates the porcine CLTC gene.Six miRNAs targeting porcine CLTC gene were predicted by bioinformatics. The porcine CLTC gene 3'UTR was cloned into double luciferase reporter gene vector psi CHECK2 to obtain double luciferase reporter gene recombinant vector psi CHECK2-CLTC-3 UTR.The predicted miRNA and the recombinant vector psi CHECK2-CLTC-3'UTR were cotransfected into the cells respectively, and the sequence sequence was used as negative control to detect the effect of miRNA on the luciferase activity of the recombinant plasmid.The results showed that both miR-205 miR-1 miR-129-5p and miR-206 could significantly inhibit luciferase activity (P0.05).After overexpression of miR-1 and miR-129-5p in porcine renal epithelial cell line PK15, the results of quantitative PCRQ-PCRR showed that the expression of porcine CLTC gene was significantly down-regulated.The binding sites of the four miRNA sequences in the psi CHECK2-CLTC-3'UTR vector were mutated. It was found that the luciferase in the mutant plasmid was not significantly inhibited by 1: miR-1.The results showed that miR-1 had direct targeting relationship with porcine CLTC gene, and the expression of CLTC gene was inhibited by its seed sequence.
【作者单位】： 浙江农林大学动物科技学院;
【基金】：国家自然科学基金青年基金资助项目(31501921) 浙江省自然科学基金青年基金资助项目(LQ15C170001) 浙江农林大学科研发展基金资助项目(2014FR068)
【分类号】：Q78
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本文编号：1694668