Fig. 9

In planta visualization of the cleavage of target genes by miRNAs in tobacco leaves. a GUS activity associated with a miRNA/target pair. miRNAs or cleavage contiguous fragments of target genes were cloned to down-stream of CaMV35S promoter in pStart-GUS vector. For each miRNA/target pair, the GUS stain assay was made by five parts, including miRNA (marked by 246, 27, 84, 1120 or 9664, indicating novel_246, novel_27, novel_84, miR1120c and miR9664, respectively), target fragment (marked by 246 t, 27 t, 84 t, 1120 t or 9664 t), mutant target fragment (marked by 246 m, 27 m, 84 m, 1120 m or 9664 m), co-expressed miRNA/target (marked by 246 + 246 t, 27 + 27 t, 84 + 84 t, 1120 + 1120 t or 9664 + 9664 t) and co-expressed miRNA/mutant target (246 + 246 m, 27 + 27 m, 84 + 84 m, 1120 + 1120 m or 9664 + 9664 m). The pStart-GUS vector was used as the positive control, while a wild type tobacco leaf served the negative control. b Quantification of GUS activity in transformed tobacco leaves calculated from mean pixel densities. c Relative expression level of GUS gene in transformed tobacco leaves with a miRNA/target pair. d RLM 5’-RACE to validate the target mRNA cleavage sites for five miRNAs. The red line show the cleavage sites by miRNA. 10 clones were sequenced after RLM 5’-RACE for every miRNA/Target pair and the number of sequences found at the exact cleavage site was showed