Abstract: 【Aim】 Juvenile hormone acid methyl transferase (JHAMT) is the key rate-limiting enzyme in the juvenile hormone (JH) synthetic pathway. This study aims to screen and verify the miRNAs targetedly regulating the transcription of JHAMT, and to eveal the important action mechanism of miRNAs in JH biosynthesis in Drosophila melanogaster. 【Methods】 MiRNAs targeting JHAMT in D. melanogaster were predicted using online websites miRanda, TargetScan and microT-CDS, and those predicted by all the three websites were selected as the miRNA candidates targeting JHAMT. The targeted relationship between candidate miRNAs and JHAMT were verified using a dual luciferase assay system. The expression profiles of miRNA and JHAMT during D. melanogaster development were detected by qRT-PCR. The effects of miRNAs on the JHAMT expression and metamorphosis in D. melanogaster were tested by overexpressing miRNA in the corpora allata using qRT-PCR and the fly GAL4-UAS system, respectively. 【Results】 A total of 5, 18 and 16 miRNAs targeting JHAMT were predicted by miRanda, TargetScan and microT-CDS, respectively, and four miRNAs including miR-252-5p, miR-277-3p, miR-1002-5p and miR-987-5p were coincidentally predicted by these three algorithms. Dual luciferase assay results showed that miR-252-5p mimics significantly decreased the luciferase activities of wild-type JHAMT 3′UTR luciferase reporter, and the suppression effect was compromised when the miR-252-5p binding sites within the 3′UTR of JHAMT were mutated. qRT-PCR results showed that miR-252-5p and JHAMT displayed opposite expression patterns in egg, larval and prepupal stages of D. melanogaster, and overexpression of miR-252 in the corpora allata significantly decreased the expression levels of JHAMT and the JH primary response gene Kr-h1. Meanwhile, overexpression of miR-252 resulted in similar phenotypes as JH deletion, such as delayed pupation, reduced pupal size and increased pupal lethality. 【Conclusion】 miR-252-5p affects Drosophila metamorphosis through targeting JHAMT and regulating JH biosynthesis.

Key words: Drosophila melanogaster, miRNA, target gene, juvenile hormone acid methyl transferase, miR-252-5p, metamorphosis