A new strategy for construction of artificial miRNA vectors in Arabidopsis

MicroRNAs are a class of small RNAs that specifically suppress their target genes by transcript cleavage or/and translation repression. Natural miRNA precursors have been used for the backbones of artificial miRNA precursors, which can give rise to expected artificial miRNAs with which to repress specific target genes. Artificial miRNA technology is a powerful tool to silence genes of interest. However, it is costly and time-consuming to construct artificial miRNA precursors by the use of an overlapping PCR method. We describe a new strategy to construct artificial miRNAs. A miRNA gene consists of three components (upstream, stem-loop, and downstream regions). Upstream and downstream regions of a natural miRNA transcript were amplified in conjunction with the introduction of two suitable restriction sites in the amplicons, which were inserted into a plasmid to form a median vector. Production of an artificial miRNA vector was easily achieved by insertion of an artificial stem-loop into the median vector. The artificial miRNAs produced by this method efficiently repressed their target genes in Arabidopsis. In addition, two artificial miRNA constructs were expressed as one polycistron driven by the CaMV 35S promoter and their targets were suppressed simultaneously in Arabidopsis. Thus, artificial miRNAs are a powerful tool with which to analyze rapidly the functions of not only a single gene or multiple homologous genes, but also multiple non-homologous genes.