RESUMEN
20-Hydroxyecdysone (20E) plays an essential role in coordinating developmental transitions in insects through responsive protein-coding genes and microRNAs (miRNAs). However, the interplay between 20E and miRNAs during insect metamorphosis is unknown. In this study, using small RNA sequencing, a comparative miRNA transcriptomic analysis in different development stages, and 20E treatment, we identified ame-bantam-3p as a key candidate miRNA involved in honeybee metamorphosis. Target prediction and in vitro dual-luciferase assays confirmed that ame-bantam-3p interacts with the coding region of the megf8 gene and promotes its expression. Meanwhile, temporal expression analysis revealed that the expression of ame-bantam-3p is higher in the larval stage than in prepupal and pupal stages, and that this expression pattern is similar to that of megf8. In vivo, we found that the mRNA level of megf8 was significantly increased after the injection of ame-bantam-3p agomir. A 20E feeding assay showed that 20E downregulated the expression of both ame-bantam-3p and its target gene megf8 on larval days five, six, and seven. Meanwhile, the injection of ame-bantam-3p agomir also reduced the 20E titer, as well as the transcript levels of essential ecdysteroid synthesis genes, including Dib, Phm, Sad, and Nvd. The transcript levels of 20E cascade genes, including EcRA, ECRB1, USP, E75, E93, and Br-c, were also significantly decreased after ame-bantam-3p agomir injection. However, ame-bantam-3p antagomir injection and dsmegf8 injection showed the opposite effect to ame-bantam-3p agomir injection. Ame-bantam-3p agomir treatment ultimately led to mortality and the failure of larval pupation by inhibiting ecdysteroid synthesis and the 20E signaling pathway. However, the expression of 20E signaling-related genes was significantly increased after megf8 knockdown, and larvae injected with dsmegf8 showed early pupation. Combined, our results indicate that ame-bantam-3p is involved in the 20E signaling pathway through positively regulating its target gene megf8 and is indispensable for larval-pupal development in the honeybee. These findings may enhance our understanding of the relationship between 20E signaling and small RNAs during honeybee development.
Asunto(s)
MicroARNs , Animales , Abejas/genética , Larva/metabolismo , MicroARNs/genética , MicroARNs/metabolismo , Ecdisteroides/metabolismo , Pupa , Ecdisterona/farmacología , Ecdisterona/metabolismo , Metamorfosis Biológica/genética , Familia de Proteínas EGF/metabolismo , Proteínas de Insectos/genética , Proteínas de Insectos/metabolismoRESUMEN
The small GTPase Ran is a member of the Ras superfamily of small GTP-binding proteins, which plays a key role in the translocation of RNA and proteins through the nuclear pore complex. In this study, the full-length cDNA sequence of LmRan gene was obtained, which consists of 648-nucleotides open reading frame (ORF) and encodes 215 amino acids. RT-qPCR results revealed that LmRan was expressed in all developmental days and tissues investigated. Injection of dsLmRan into 4th and 5th instar nymphs, resulted in a significant down-regulation of LmRan transcripts, respectively. All dsLmRan-injected nymphs died before molting. Further hematoxylin and eosin staining of the integument showed that there was no apolysis occurred after silencing LmRan. In addition, the weight of dsLmRan-injected nymphs was significantly lower than that of the control group, and the gastric caecum and midgut was severely smaller. Especiallly, the mRNA level of LmCYP302a1, LmCYP315a1 and LmCYP314a1 responsible for 20E synthesis, LmE75 and LmE74 genes involved in the 20E signaling pathway, LmGfat, LmUAP1 and LmCHT10 genes involved in chitin metabolism pathway were dramatically decreased in the dsLmRan-injected nymphs. Together, the results indicated that LmRan participate in the 20E signaling pathway, which is essential for the growth and development of locusts.