tae-miR399-mediated cleavage of TaUBC24
To validate tae-miR399 mediated cleavage of TaUBC24 , 5’ RLM-RACE
analysis was conducted using FirstChoice® RLM-RACE Kit
(Thermofisher, USA). 3’ gene specific primers for TaUBC24 were
listed in Table S2. Interactions between tae-miR399 and TaUBC24on potential target genes were demonstrated using transient
co-expression assays in N. benthamiana leaves. The precursor
sequence of tae-miR399 was cloned from Dn1 genomic DNA and then cloned
into pBI121 using Homologous recombination technology. The full-length
of partial TaUBC24 sequence containing MREs was amplified using
primers pTaUBC24 -F: 5’-CTGAACAGTCGTTGGTTGCTCT-3’ and primerspTaUBC24 -R: 5’-CTAAAGAACCGTCTCTGAACAGC-3’, and Taq DNA Polymerase
(CWBIO). The PCR product was cloned into pBI121, which drives expression
using the cauliflower mosaic virus 35S promoter. To co-express
tae-miR399 and MREs, A. tumefaciens (EHA105) strains containing
these plasmids were coinfiltrated into 7-week-old N. benthamianaleaves. β-Glucuronidase (GUS) staining were conducted 2 d after
infiltration. All primers of 5’ RLM-RACE were designed using the
software Primer 6.0 and listed Table S2.