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 .