Validation of cold-responsive mature miRNAs in B. rapa
To validate the cold-responsive miRNAs detected by small RNA sequencing,
we further analyzed the cold responsiveness of mature bra-miR1885,
bra-miR398, bra-miR408, bra-miR161, bra-miR157, bra-miR393, and
bra-miR168 using qRT-PCR. We found that the abundance of bra-miR1885,
bra-miR398 and bra-miR168 were significantly up-regulated by cold
stress, whereas bra-miR161, bra-miR157 and bra-miR393 were significantly
down-regulated under cold stress, as compared to normal temperature at
the same growth stage. These results were consistent with the small RNA
sequencing data (Figure 2A). Among these miRNAs, bra-miR398 and
bra-miR168 were conserved miRNAs that were induced more than three-fold
by cold treatment (Figure 2A). We then performed northern blotting
analyses to further confirm the cold responsiveness of bra-miR398,
bra-miR168 and bra-miR1140. Consistently, the accumulation of bra-miR398
and bra-miR168 was much higher in 4 °C than in 22 °C, andBrassica -specific miR1140 was repressed after cold treatment
(Figure 2B). In addition, the results of qRT-PCR analyses further
confirmed the abundance of mature bra-miR1885 was increased in leaf
under cold stress as compared to normal condition (Figure 2C).
Next, to identify the potential cold-regulated cis -elements in
the bra-MIR1885 promoter, we extracted the promoter sequence ofbra-MIR1885 using Promoter 2.0 Prediction Server
(https://services.healthtech.dtu.dk/service.php?Promoter-2.0), and
analyzed the cis- elements in the putative bra-MIR1885promoter sequence using PlantCare
(http://bioinformatics.psb.ugent.be/webtools/plantcare/html/).
These analyses revealed that the promoter contained light-responsive,
defense-responsive, stress-responsive, and low temperature-responsive
elements (LTR: CCGAAA) (Figure S2). A previous study has revealed that
LTR motif (CCGAAA) within the promoter region of CsINV5 was the
core cis element in response to low temperature (Qian et al.,
2018). This result suggested that the bra-MIR1885 promoter
contained a low temperature-responsive element that potentially
regulated the expression of bra-MIR1885 under cold stress.