Fig. 9From: Small RNA and degradome sequencing used to elucidate the basis of tolerance to salinity and alkalinity in wheatIn 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 showedBack to article page