A simplified method for constructing artificial microRNAs based on the osa-MIR528 precursor.
A simplified method for constructing artificial microRNAs based on the osa-MIR528 precursor.
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DOI：
10.1016/j.jbiotec.2012.02.015
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发表时间：
2012-08
影响因子：
4.1
通讯作者：
Fei Yan;Yuwen Lu;Gentu Wu;Jiejun Peng;Hongying Zheng;Lin Lin-Lin;Jianping Chen
中科院分区：
文献类型：
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作者：
Fei Yan;Yuwen Lu;Gentu Wu;Jiejun Peng;Hongying Zheng;Lin Lin-Lin;Jianping Chen
人工miRNAs(AmiRNAs)已在多种植物中成功地用于沉默内源基因或病毒RNA。Schwab等人的方法被广泛用于构建amiRNAs，需要四个PCR。我们描述了一种简化的基于OSA-MIR528骨架的构建amiRNA的方法，该方法是通过在引物中增加延长序列，一步一步完成的。确定了OSA-MIR528前体所需的延长序列长度。利用这种方法，我们构建了针对烟草UPF1基因的amRNA，结果表明，当UPF1基因瞬时表达时，它可以沉默叶片中UPF1的表达。
Artificial miRNAs (amiRNAs) have been used successfully in various plants to silence endogenous genes or viral RNAs. The method of Schwab et al., widely used to construct amiRNAs, requires four PCRs. We describe a simplified method of constructing amiRNA based on the osa-MIR528 backbone using one PCR step by addition of prolonging sequence to the primers. The length of prolonging sequence needed in the osa-MIR528 precursor was determined. Using this method, we constructed amiRNA targeting the Nicotiana benthamiana UPF1 gene and showed that it functioned in silencing UPF1 expression in leaves when expressed transiently.