Fig. 4

miR-31-5p directly targets acox1. a The relative expression of miR-31-5p, acox1 mRNA, and protein after low-temperature acclimation by qPCR (n = 6) and Western blot (n = 3). The bar graphs show mean ± s.e.m. (**, P < 0.01; ***, P < 0.001; Student’s t test). b The relative expression of miR-31-5p, acox1 mRNA, and protein after injecting agomir-31-5p (Ago-31-5p) and agomir-control (Ago-control) by qPCR (n = 6) and Western blot (n = 3). c The relative expression of miR-31-5p, acox1 mRNA, and protein after injecting antagomir-31-5p (Anta-31-5p) and antagomir-control (Anta-control) by qPCR (n = 6) and Western blot (n = 3). d Luciferase reporter assays in S2 cells co-transfected with agomir-31-5p or agomir-control and psiCHECK2 vectors containing wild (WT) or mutant (MT) sequences of predicted target sites. The bars are means ± s.e.m. and differ significantly from each other with different letters (n = 8, one-way ANOVA with Tukey’s HSD multiple comparison, P < 0.05). AC, low-temperature acclimation (4 °C); Control (25 °C). The last larval beetles used in a, c were collected at 10–15 Nov. 2017, and beetles in b and d were collected at 5 Oct. 2017