MicroRNAs (miRNAs) are a class of small RNAs that arise from an endogenous transcript. Like natural miRNAs, artificial miRNAs can specifically repress their target genes, thus artificial miRNAs are a powerful tool for the suppression of gene expression in plants. However, constructing artificial miRNA is often costly and time-consuming by the use of an overlapping PCR method.
Prof. Yu Diqiu and his team of Xishuangbanna Tropical Botanical Garden (XTBG) described a new strategy to produce an artificial miRNA construct.
Two median vectors (pAMIR319a and pAMIR395a) were constructed that can be used as the backbones of artificial miRNAs in Arabidopsis. Both vectors contain two restriction sites (EcoRI and EcoRV), which allowed the insertion of a stem-loop that includes an artificial miRNA sequence. Because upstream and downstream regions of a natural miRNA transcript have been cloned into the median vectors, only one stem-loop sequence from one PCR reaction was required for the production of an artificial miRNA construct. The method improved the efficiency of construction of artificial miRNA vectors.
Their experiments confirmed that artificial miRNAs constructed by the use of pAMIR319a or pAMIR395a can be expressed successfully and the transgenic plants phenocopied the mutants of target genes, which implied that these two vectors are effective for the processing of mature artificial miRNAs in Arabidopsis. Artificial miRNAs as a tool of gene suppression are convenient and rapid for the reverse genetics research.
In addition, when two non-homologous genes are closely linked, two artificial miRNAs can be coexpressed to repress the expression of these two genes, which mimics the double mutants. Therefore, the application of artificial miRNAs will contribute to progress in gene function analysis by reverse genetics.
The study entitled “A new strategy for construction of artificial miRNA vectors in Arabidopsis” has been published in Planta, 235(6):1421-1429, DOI: 10.1007/s00425-012-1610-5