Overexpression of miR1885 in B. napus led to
down-regulation of its target genes Bn.TIR.A09 andBn.TNL.A03
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 into B. napus cv. K407
(Figure 5A). 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 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 between WT and miR1885-OE
lines after cold treatment (Figure 6D-F). This result suggested that
miR1885 regulated low temperature tolerance in Brassica through
CBF/CORs-independent pathway.