Identification of miR1885 target genes and their cold
responsivity in B. napus
Because bra-miR1885 and bna-miR1885 shared the same mature sequence
(Figure 4B), we used miR1885 to represent the two miRNAs inBrassica crops hereafter. We first predicted the candidate target
genes of miR1885 in B. napus using a plant small RNA target
analysis tool (psRNATarget). Two R genes, Bn.TIR.A09(BnaA09g14980D) and Bn.TNL.A03 (BnaA03g56180D), were identified
as highly credible targets, and the miR1885 target sites were within the
TIR domain. To verify the miR1885 cleavages of the predicted target
genes, we performed a 5′ rapid amplification of cDNA ends (5′-RACE)
assay in B. napus and detected miR1885 cleavage sites frequently
within Bn.TIR.A09 and Bn.TNL.A03 (Figure 4C and Figure
S4), indicating that they were targeted by miR1885 in B. napus .
Next, we examined the transcript levels of these two target genes under
cold stress, using three low temperature-responsive marker genes
(Bn.CBF1.C03 , Bn.HSP70.A01 and Bn.ERF105.C09 ) as
positive controls (Wang and Hua, 2009; Bolt et al., 2017; Liu et al.,
2018). The target genes were down-regulated under cold stress inB. napus (Figure 4D), while the expression of Bn.CBF1.C03 ,Bn.HSP70.A01 were induced and Bn.ERF105.C09 were repressed
with cold treatment, consistent with those reported in previous studies
(Figure 4E).