Figure 4. qRT-PCR analysis of the abundance of miR1885 and its targets under cold stress in B. napus.
(A) qRT-PCR analysis of the abundance of miR1885 in B. napus under 22 °C and 4 °C condition. U6 was used as endogenous reference gene. (B) Alignment of miR1885 mature sequence between B. rapa and B. napus . Short black line represents the perfect matches. (C) Validation of miR1885 target genes and cleavage sites using 5’-RACE PCR. Predicted TIR, NBS, and LRR domains encoded by target genes are labeled (Left). Reverse complementary matches of miR1885 and their target sites were showed and vertical arrows indicated 5 termini of miRNA-guided cleavage products, as identified by 5’-RACE, with frequency of clones shown (Right). (D ) The transcript levels of Bn.TIR.A09 andBn.TNL.A03 in B. napus under cold treatment. “D” represents days. (E) Relative transcript levels of cold-related genes in wild-type B. napus under 22 °C and 4 °C condition.ACT7 was used as endogenous reference gene. Error bar represents mean ± SE; n = 3. * indicates P < 0.05; ** indicates P < 0.01; Student’s t-test.