Abstract: 【Aim】 The objective of this study is to further investigate the role of ame-miR-79 in regulating the expression of target genes in the larval guts of Apis mellifera ligustica workers by performing overexpression and knockdown of ame-miR-79 in the larval guts by feeding with mimic and inhibitor of ame-miR-79, respectively. 【Methods】 Molecular cloning and Sanger sequencing were conducted to confirm the sequence authenticity of ame-miR-79 in the larval guts of A. m. ligustica workers. Overexpression and knockdown of ame-miR-79 in the 4-6-day-old larval guts of A. m. ligustica workers were performed through feeding with mimic-miR-79 and inhibitor-miR-79, respectively. Prediction and analysis of target genes of ame-miR-79 were carried out with related bioinformatic software. The overexpression and knockdown effects of ame-miR-79 and the relative expression levels of target genes after overexpression and knockdown of ame-miR-79 were examined by RT-qPCR. 【Results】 ame-miR-79 truly exists in the larval gut of A. m. ligustica workers. Compared with the nonsense mimic (mimic-NC) group, the mimic-miR-79 group showed extremely significantly up-regulated expression of ame-miR-79 in the 4-6-day-old larval guts. In comparison with the nonsense inhibitor (inhibitor-NC) group, the inhibitor-miR-79 group showed significantly down-regulated expression of ame-miR-79 in the 4- and 5-day-old larval guts and extremely significantly down-regulated expression of ame-miR-79 in the 6-day-old larval gut. ame-miR-79 can target 303 genes, which are associated with 27 GO terms and 179 KEGG pathways. In comparison with the mimic-NC group, the mimic-miR-79 group showed extremely significantly down-regulated expression of the cytochrome P450 gene CYP450 in the 4- and 5-day-old larval guts and up-regulated expression of CYP450 in the 6-day-old larval gut, while showed downregulated expression of fringe glycosyltransferase (FG) gene in the 4-day-old larval gut, significantly down-regulated expression of FG in the 5-day-old larval gut, and up-regulated expression of FG in the 6-day-old larval gut. Compared with the inhibitor-NC group, the inhibitor-miR-79 group exhibited extremely significantly up-regulated expression of CYP450 in the 4-day-old larval gut, up-regulated expression of CYP450 in the 6-day-old larval gut, and downregulated expression of CYP450 in the 5-day-old larval gut, while significantly up-regulated expression of FG in the 4-day-old larval gut and extremely significantly up-regulated expression of FG in the 5- and 6-day-old larval guts. 【Conclusion】 ame-miR-79 truly exists in the larval guts of A. m. ligustica workers. Effective overexpression and knockdown of ame-miR-79 in the larval guts of A. m. ligustica workers can be achieved via feeding with mimic and inhibitor, respectively. ame-miR-79 negatively regulates the expression of CYP450 and FG in the larval guts of A. m. ligustica workers.

Key words: Apis mellifera ligustica, miRNA, ame-miR-79, larva, gut, development, target gene