Abstract
Simple SummaryLncRNAs are a class of non-coding RNAs longer than 200 nt that are involved in a variety of biological processes. Studies on lncRNAs in Bombyx mori have shown that some lncRNAs are involved in brain development, silk production and the response to virus infection of the host. However, the roles of lncRNAs are still largely unknown in the silkworm. In this study, we analyzed the function of lncRNAs Bmdsx-AS1 in silkworm by transgenic overexpression, which not only affects the development of male silkworm external genitalia, but also participates in the regulation of EGFR signaling pathway. Moreover, we studied the upstream promoter of Bmdsx-AS1 and found that the BmAbd-B transcription factor of the Hox gene family can negatively regulate the expression of Bmdsx-AS1. These results laid a substantial foundation for in-depth study of the function of lncRNAs in the silkworm.Long non-coding RNAs (lncRNAs) have been suggested to play important roles in some biological processes. However, the detailed mechanisms are not fully understood. We previously identified an antisense lncRNA, Bmdsx-AS1, that is involved in pre-mRNA splicing of the sex-determining gene Bmdsx in the silkworm. In this study, we analyzed the changes in the male external genitalia of transgenic overexpressed Bmdsx-AS1 silkworm lines and analyzed downstream and upstream responses. We found that Bmdsx-AS1 transgenic silkworms, compared with wild type, showed more claspers in the male external genitalia. Quantitative real-time PCR (qPCR) results indicated that overexpression of Bmdsx-AS1 decreased the expression of genes in the EGFR signaling pathway. Knockdown of Bmdsx-AS1 increased the activity of the EGFR pathway. Through promoter prediction, promoter truncation and electrophoretic mobility shift assay (EMSA) analyses, we found that the protein encoded by the Hox gene BmAbd-B specifically binds to the promoter of Bmdsx-AS1. Moreover, overexpression of BmAbd-B in the silkworm BmE cell line indicated that BmAbd-B negatively regulates the mRNA expression of Bmdsx-AS1. Our study provides insights into the regulatory mechanism of the lncRNA in the silkworm.
Highlights
Total RNA was extracted with TRIzol reagent (Invitrogen, Carlsbad, CA, USA) from the individuals of transgenic silkworms at the first day of the fifth instar larva to detect the expression of Bombyx mori doublesex (Bmdsx)-AS1 with quantitative real-time RT-PCR
In order to detect the biological effects of Bmdsx-AS1, the piggyBac-Bmdsx-AS1 vector was mixed with the helper vector pHA3PIG and injected into 400 silkworm eggs within
DsRed was expressed in compound eyes of transgenic silkworm, but not in wild-type silkworm
Summary
LncRNAs are defined as transcripts of more than 200 nucleotides in length that do not encode functional proteins. They are classified as intergenic transcripts, sense or antisense transcripts and enhancer transcripts [1]. Zhang et al have used genome-wide transcriptome analysis to identify the differentially expressed lncRNAs in silkworm cells with or without BmNPV infection and found that these lncRNAs participate in the host response to BmNPV infection via interactions with their target genes and miRNAs [17]. We identified the antisense lncRNA Bmdsx-AS1, whose genomic sequence is within Bombyx mori doublesex (Bmdsx) gene [19]. We carried out transgenic overexpression of Bmdsx-AS1 to detect its biological roles and explore its regulatory mechanism
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