Abstract
RNA interference (RNAi) is the small-interfering RNA (siRNA)-mediated post-transcriptional gene silencing. Experimental determination of the RNAi effects on the decrease of mRNA level is a time-consuming work. We here show a method which allows us to evaluate directly the activity of siRNA-mediated mRNA cleavage. The RNAi construct against the tobacco microsome omega-3 fatty acid desaturase gene (NtFAD3) had been introduced into tobacco plants. The resulting RNAi transgenic plant (R11) accumulates the siRNA for the NtFAD3 gene. The partial NtFAD3 genomic region corresponding to the RNAi target region was cloned behind the 35S promoter, and expressed transiently by using particle bombardment. The transcripts from the bombarded gene were detected by RT-PCR in the wild-type plants but only in a trace level in the R11 plants. This result indicates that the siRNA-directed cleavage can be rapidly evaluated by using this assay system.
