Cloning and expression profile of Bmyan in the silkworm (Bombyx mori) and experimental validation as one target of microRNA 7

Abstract:

microRNAs (miRNAs) are an extensive class of ~22-nucleotide (nt) endogenous noncoding RNAs regulating life activities of metazoans through binding to 3′-untranslated regions (3′-UTRs) of their target genes. This work aimed to identify yan gene in the silkworm, reveal its expression profile and confirm if it is one target of bmo-miR-7 and, as such, have potential for contributing to better understanding of the molecular mechanisms involved in the metamorphosis of silkworm. Based on homolog searching and PCR amplification, we cloned the coding sequence (CDS) of Bmyan, which encodes 476 amino acid residues and contains SAM-PNT and ETs domains. Quantitative PCR (q-PCR), RT-PCR and microarray data revealed high expression of Bmyan in the head, body wall and ovary of day-3 fifth instar larval silkworm, low or no expression in other tissues. It was lowly expressed in the early larval stages, but highly expressed from late spinning to day 4 pupa. The 3′-UTR of Bmyan was obtained by rapid-amplification of cDNA ends (3′RACE) and predicted to contain two potential recognition sites of bmo-miR-7. The luciferase reporter vector containing the 3′-UTR of Bmyan was constructed and co-transfected into BmE cell line with the mimic of bmo-miR-7 and the decreased relative activity of luciferase showed that Bmyan is one target of bmo-miR-7. This work helps further functional analysis of bmo-miR-7 and Bmyan in the silkworm.