Determination of Expression Patterns of Plant miRNAs by Transcriptional Fusion Using GUS and Fluorescent Reporters.

MicroRNAs are noncoding RNAs of 20-24 nucleotides (nt) in length that act as repressors of genes and are important in key developmental processes in the entire life cycle of plants. The MIR genes encode miRNAs; MIRs are transcribed by RNA polymerase II and, in their promoter region, have signals that respond to development and stress processes. Therefore monitoring the activity of the promoter using reporters is the first step to know its expression pattern and thus explore a specific function. Then, to determine the function of a microRNA, the first step is to resolve its expression pattern; this can be achieved by in situ hybridization, RNA blot assays, or quantitative PCR. However, the study of the expression of a MIR gene is straightforward with the use of reporter proteins such as β-D-glucuronidase (GUS) or fluorescent proteins such as GFP or mCherry. To do this, it is necessary to clone the promoter/regulatory region of the MIR gene and place it upstream of the reporter gene; in this way the activity of the promoter will be a direct reflection of the expression of the MIR gene. Here, we indicate step-by-step instructions on how to make transcriptional fusion constructions to clone a promoter region of a MIR gene fused to the classical reporter proteins GUS and mCherry in Arabidopsis thaliana. This method is particularly useful to dissect the promoter region of a MIR gene and find its expression pattern in space and time.