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Micro RNA模拟靶序列(target mimic,TM)通过竞争性结合miRNA,从而干扰miRNA对靶标m RNA的调控.我们前期工作发现:以黄瓜花叶病毒(Cucumber mosaic virus,CMV)作为载体在植物体内表达TM序列有效地抑制了miRNA的活性或稳定性,从而消减了miRNA对靶基因的调控.但是,miRNA与CMV携带的TM序列的结合在一定程度上抑制了病毒的积累.研究分析了miRNA靶向病毒携带的TM序列对病毒抑制作用的内在原因.a.通过RNA印迹分析CMV携带不同miRNA TM序列对病毒积累的影响,进一步明确miRNA靶向病毒携带的TM序列对病毒的抑制作用;b.利用GFP作为报告基因,通过荧光显微镜、蛋白质印迹以及RNA印迹分析TM序列对重组病毒积累的影响;c.以GFP作为报告基因,利用荧光显微镜观察和免疫印迹方法分析模拟靶序列对GFP翻译的影响;d.利用CMV病毒的反式复制系统分析miRNA模拟靶序列对病毒负链RNA合成的影响.结果表明,多种植物内源的miRNA靶向CMV基因组携带的miRNA TM序列,在不同程度上抑制了病毒的积累,miRNA与其TM序列的结合抑制GFP蛋白的翻译和负链的合成.植物内源的miRNA通过与病毒基因组携带的miRNA模拟靶序列结合,通过抑制病毒蛋白的翻译以及病毒负链RNA的合成,从而降低了病毒的积累水平.基于该论文的研究结果有可能建立一种抗病毒的新方法.
MicroRNA mimic (TM) targets interfere with miRNAs' regulation of target m RNA through competitive binding of miRNAs. Our previous work showed that CMV was expressed in plants as a vector TM sequence effectively inhibits the activity or stability of miRNA, thus reducing the regulation of miRNA on the target gene.However, the combination of miRNA and TM sequence carried by CMV to a certain extent, inhibited the virus accumulation.Studies on miRNA target The intrinsic cause of the virus-carrying TM sequence on virus inhibitiona.Analysis of the effect of CMV-carrying different miRNA TM sequences on viral accumulation by Northern blotting to further clarify the virus-inhibitory effect of TM sequences carried by the miRNA-targeted virus; b. GFP as a reporter gene to analyze the effect of TM sequence on recombinant virus accumulation by fluorescence microscopy, Western blotting and Northern blotting; c. Using GFP as a reporter gene, the effect of simulated target sequences on GFP translation was analyzed by fluorescence microscopy and immunoblotting; d. Analysis of the effect of miRNA mimicking the target sequence on the viral negative strand RNA synthesis using the CMV viral trans-replication system The results showed that many Endogenous miRNAs target the miRNA (TM) sequences carried by the CMV genome and inhibit the accumulation of viruses to varying degrees. The binding of miRNAs to their TM sequences inhibits translation and negative strand synthesis of GFP proteins. Genome-carried miRNAs mimic the binding of target sequences and reduce the level of virus accumulation by inhibiting the translation of the viral proteins and the synthesis of viral negative-stranded RNAs. Based on the results of this paper, it is possible to establish a new antiviral approach.