tae-miR399 targets TaUBC24
To verify the target relationship between tae-miR399 and TaUBC24, the psRNATarget software analysis showed that there are six potential tae-miR399 response elements/binding sites (MREs) in the 5’ untranslated region (UTR) of TaUBC24 (Fig. 3A). The sequence of MRE (2) was the same as that of MRE (3) and (4). The sequence of MRE (1) had the highest basal complementarity (Fig. 3A). Using RNA ligase-mediated 5’ RACE (RLM-5’ RACE), we found that TaUBC24 was cleaved at 15 bp downstream of MRE (4) (Fig. 3B-(a)). However, no cleavage was detected at the other five predicted target sites in TaUBC24 . To independently examine the relationship between tae-miR399 and the potential target site, we performed transient coexpression assays inNicotiana benthamianaleaves. Scince the full length of TaUBC24 mRNA is 4230bp, it is difficult to clone and vector construction. The partial TaUBC24 containing the six potential MREs (pTaUBC24 ) was cloned with a length of 678 bp. The pBI121-GUS vector (harboring the GUS gene) was introduced into cells of tobacco leaves using the Agrobacterium-mediated transformation system. Control leaves infiltrated with pBI121-GUS exhibited the GUS phenotype revealed by histochemical staining, while leaves infiltrated with pBI121-pTaUBC24 , in which the target sequence was fused upstream of the GUS gene, showed a similar phenotype. In leaves infiltrated with pBI121-tae-miR399, however, in which the GUS gene was replaced by the precursor of tae-miR399, the GUS phenotype was not observed. Meanwhile, GUS staining was markedly decreased in leaves co-transformed with the strain mixture pBI121-tae-miR399 and pBI121-pTaUBC24 (Fig. 3C). This was considered to be additional evidence that tae-miR399 could targetTaUBC24 .
To confirm the results of these histochemical observations, we carried out RLM-5’ RACE assay using RNA from tobacco leaves infiltrated with the strain mixture pBI121-tae-miR399 and pBI121-pTaUBC24 to determine the predicted cleavage site in TaUBC24 . The PCR products were cloned and sequenced. The results showed the cleavage site following at 21 bp downstream of MRE (3) (Fig. 3B-(b)). Taken together, these results show that tae-miR399 targets TaUBC24 .