Abstract: 【Aim】 To determine the expression level changes of microRNAs (miRNAs) of Apis mellifera during the overwintering period, explore the potential relationships between miRNAs and their target mRNAs and cold resistance, so as to further reveal the molecular mechanism of cold resistance of A. mellifera at the miRNAomics level. 【Methods】 sRNA-seq technology was used to identify miRNAs in four subspecies of A. mellifera including A. m. ligustica, A. m. mellifera, A. m. caucasica and A. m. carnica during the early and middle overwintering periods. The differentially expressed miRNAs (DEmiRNAs) in the four subspecies during different overwintering periods were screened by P≤0.05 and |log₂ fold change|≥1. The screened target mRNAs of miRNAs were predicted using related bioinformatic software, and then annotated by GO and KEGG databases. The regulatory network of DEmiRNAs with target mRNAs was constructed according to the targeted binding relationship, followed by visualization with Cytoscape. Eight DEmiRNAs including ame-miR-263a-5p, ame-miR-184-3p, ame-miR-263b-5p, ame-miR-190-5p, ame-miR-6052-5p, ame-miR-9a-5p, ame-miR-100-5p and ame-miR-306-5p were randomly selected for RT-qPCR validation.【Results】A total of 210 miRNAs were predicted in the four subspecies of A. mellifera during the overwintering period, including 178 conserved miRNAs and 32 new miRNAs. The length of miRNAs in the four subspecies of A. mellifera ranged from 18 to 30 nt, of which the most distributed lengths are 22 and 23 nt, and the number of miRNAs with the first base U is the largest. DEmiRNAs with the highest expression level were ame-miR-1-3p, ame-miR-276-3p and ame-miR-184-3p in the four subspecies of A. mellifera during the early and middle overwintering periods. A total of 22 DEmiRNAs were screened from A. m. ligustica between the early and middle overwintering periods, targeting 394 mRNAs which were annotated to 161 GO functional terms and 16 KEGG pathways. A total of 28 DEmiRNAs were screened from A. m. mellifera between the early and middle overwintering periods, targeting 415 mRNAs which were annotated to 147 GO functional terms and 15 KEGG pathways. A total of 67 DEmiRNAs were screened from A. m. caucasica between the early and middle overwintering periods, targeting 1 021 mRNAs which were annotated to 171 functional GO functional terms and 21 KEGG pathways. A total of 18 DEmiRNAs were screened from A. m. carnica between the early and middle overwintering periods, targeting 330 mRNAs which were annotated to 147 GO functional terms and 13 KEGG pathways. A complicated regulatory network was formed between DEmiRNAs and target mRNAs in the four subspecies of A. mellifera. RT-qPCR results demonstrated that the expression trend of the eight DEmiRNAs was consistent with the data by sRNA-seq, which confirmed the reliability of our sequencing data.【Conclusion】The miRNA expression level changes in the four subspecies of A. mellifera during different overwintering periods were clarified, and several candidate molecular target miRNAs with potential regulation of cold resistance of A. mellifera were obtained, among which ame-miR-14-3p and ame-miR-3786-5p negatively regulate the expression of multiple mRNAs in A. m. mellifera, A. m. caucasica and A. m. carnica, but not in A. m. mellifera. miRNAs are involved in the cold resistance of A. mellifera by regulating the expression of target genes involved in glycerophospholipid metabolism, MAPK signaling pathway and mTOR signaling pathway.

Key words: Apis mellifera, microRNA, target genes, overwintering, cold resistance, regulatory network