FIGURE 5.

MiR169a represses the expression of its target genes at transcriptional or translational level. (A) Comparison of the relative mRNA levels of the indicated miR169’s target genes in the indicated lines. RNA was extracted in the indicated lines for qRT-PCR analysis. The mRNA level was normalized to that in the control plants. Values are means of three replications. Error bars indicate SD. The letters above the bars indicate significant differences (P < 0.01) as determined by a one-way ANOVA followed by post hoc Tukey HSD analysis. Western blotting analysis (B) and confocal images (C,D) show that miR169a suppressed the protein accumulation of Os03g29760 but did not affect the protein level of YFP control. The indicated YFP-3′-UTR_Os03g29760 and YFP-based reporter constructs were transiently expressed alone or co-expressed with miR169a (C) or/and the miR169 target mimicry MIM169a (D) in Nicotiana benthamiana leaves using Agrobacterium-mediated infiltration at the indicated optical density (O.D.) concentration. Protein extracts from the same amount of infiltrated leaves were subjected to Western blot analysis using anti-GFP sera. The Coomassie Blue stained Rubisco indicated the equal sample loading (B). The alignments of miR169a with Os03g29760 target sequence (C) and MIM169a with miR169a (D) were listed below the images, respectively. Size bars, 40 μm. Similar results were obtained in at least two independent experiments.