Overexpression of miR1885 in B. napus led to down-regulation of its target genes Bn.TIR.A09 andBn.TNL.A03
Genomic synteny analysis using TBtools (https://github.com/CJ-Chen/TBtools) suggested that bna-miR1885 inB. napus is inherited from the genome of B. rapa through genome merging (Figure S5), and bna-miR1885 and bra-miR1885 shared the same mature sequence. To further verify the function of miR1885, we constructed a vector with the bra-MIR1885 gene under the control of the AA6 promoter (Patent WO 2007/069894) and transformed it intoB. napus cv. K407 (Figure 5A). The strategy using B. napussystem for functional analysis of B. rapa genes have been used in several studies (Liu et a li., 2014; Tian et al., 2018; Cui et al., 2020). In total, seven positive transgenic lines (T0) were screened by PCR (Figure 5B and C). The expression level of pri-miR1885 was confirmed by qRT-PCR. We found that pri-miR1885 was overexpressed in the leaves of X2, X8, X11, X12, and X15 lines (Figure 5D). Compared with WT, there were no obvious morphological abnormalities among miR1885-OE lines at seeding stage (Figure 5B). We then further checked the abundance of mature miR1885 in the transformed lines by qRT-PCR and northern blotting analyses. As expected, mature miR1885 was found to be over-accumulated in the X2, X8, X11, X12, and X15 lines (Figure 5E and 5F). And then, we conducted qRT-PCR analyses to validate the influence of the miR1885 on the expression level of the two target genes in the transgenic lines. As expected, transcript levels ofBn.TIR.A09 and Bn.TNL.A03 were lower in the transgenic lines than in wild type (Figure 5G and 5H).
Next, we checked the RNA levels of miR1885 and its targets in miR1885-OE lines and WT with or without cold treatment by qRT-PCR. As expected, the abundance of miR1885 was increased in miR1885-OE lines and WT after cold treatment (Figure 6A). In addition, the RNA levels of Bn.TIR.A09were further decreased in miR1885-OE under cold stress (Figure 6B), but those of Bn.TNL.A03 were unchanged (Figure 6C), suggesting that there was additional cold-triggered regulation of Bn.TNL.A03 at the transcriptional level, while the cold response of Bn.TIR.A09was majorly contributed by miR1885-mediated post-transcriptional regulation.
Given that CBF/CORs played critical roles in plant response to low temperature, we investigated whether miR1885 can affect the cold induction of CBF/COR factors. To do this, we analyzed the expression levels of Bn.CBF1.CO3 , Bn.CBF2.Ann and Bn.COR15.A03in the miR1885-OE under cold treatment. However, there were no significant differences at the expression levels of Bn.CBF1.CO3 ,Bn.CBF2.Ann and Bn.COR15.A03 after cold treatment between WT and miR1885-OE lines (Figure 6D-F). This result suggested that miR1885 regulated low temperature tolerance in Brassica through CBF/CORs-independent pathway.