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A novel approach for the construction of plant amiRNA expression vectors

机译：构建植物amiRNA表达载体的新方法

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Artificial microRNA (amiRNA) technology has been applied in Arabidopsis thaliana and other plants to efficiently silence target genes of interest. Here we described a novel approach to construct plant amiRNA expression vectors with seamless enzyme-free cloning (SEFC) and mating-assisted genetically integrated cloning (MAGIC). Two pairs of primers were designed when the loop of amiRNA precursor was longer than 60bp while three oligonucleotides were used to amplify the linearized vector containing the amiRNA precursor whose loop was smaller than 60bp. The PCR products were transformed into Escherichia coli to generate the donor plasmid containing the amiRNA expression cassette through homologous recombination in vivo. The amiRNA expression cassette was then transferred to the recipient plasmid via MAGIC and an amiRNA expression plasmid was created. More than 200 amiRNA expression vectors were generated with this approach, three of which have been transformed into A. thaliana and successfully silence the target genes. Given its low-cost and simplicity, this novel approach of plant amiRNA expression vectors construction will benefit the study of individual gene function and establishment of plant amiRNA libraries.
机译：人工microRNA（amiRNA）技术已应用于拟南芥和其他植物中，以有效地沉默目标基因。在这里，我们描述了一种新颖的方法来构建具有无缝无酶克隆（SEFC）和交配辅助基因整合克隆（MAGIC）的植物amiRNA表达载体。当amiRNA前体的环长超过60bp时，设计了两对引物，而三个寡核苷酸用于扩增包含环小于60bp的amiRNA前体的线性化载体。通过体内同源重组将PCR产物转化到大肠杆菌中以产生含有amiRNA表达盒的供体质粒。然后将amiRNA表达盒通过MAGIC转移至受体质粒，并创建amiRNA表达质粒。用这种方法产生了200多个amiRNA表达载体，其中三个已转化为拟南芥并成功沉默了靶基因。鉴于其低成本和简单性，这种构建植物amiRNA表达载体的新方法将有利于研究单个基因功能和建立植物amiRNA文库。

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