The Regulation Of Silkworm Larvae Edysis By Bmo-miR-1

MicroRNAs (miRNAs) are small RNAs with the size of～22nt which are transcripted by RNA Pol II and processed by Drosha and Dicer. MiRNAs regulate target genes via post-trancriptianal repression. To date, more than20thousand miRNAs were discovered to play important roles in almost all the physiological activities among different organisms. In recent years, study of miRNAs in silkworm, bombyx mori was also being gradually carried out, scientists from different countries identified a total of562silkworm mature miRNAs, and conducted a profiling of spatial and temporal expression via microarray and deep sequencing technology. All the efforts built a good fundation of further study.MiR-1is an evolutionarily conserved miRNA which is mainly involved in multiple processes including the development and proliferation and differentiation of muscle tissue, the regulation of ion channels, inhibition of tumor and viral replication and other physiological processes. Bombyx mori miR-1, with the mature sequence length is22nt, expressed mainly in the head, body wall, etc. what kind of biological function did bmo-miR-1involved? Which genes are bmo-miR-1's targets? In order to answer these two questions, we designed the following research programs:Raise the level of miR-1in silkworm by miR-1mimics to observe the abnormal phenotype may arise; Survey differentially expressed genes in miR-1mimics injected silkworm by gene chip technology; Predict silkworm miR-1target genes, combined with microarray data to find possible target genes; Detect target gene mRNA and protein levels, and use dual luciferase reporter system to verify the the target genes of miR-1. Through this program, the main findings were as follows:(1) Extrinsic bmo-miR-1causes molting defect of silkwormSilkworm larvae body was shorten and became prepupae after spinning finished. In2-3days pupa skin formed and old cuticle was moulted. In the pre-pupal stage, silkworm injected with miR-1mimics could not slough off the old epidermis, through the old translucent epidermis we can see the formation of the internal pupae epidermis was slightly affected. After Stripping the old epidermis, a deformated pupa can be found, due to the bondage of the old epidermis, there was a distinct constriction in the pupa cephalothoraxes, and formation of wings was uncompleted. A small amount of liquid may contain a variety of enzymes was found between old and new epidermis, which could cause the pupa skin damage. The pupa with the outer epidermis wasmanually peeled were still be able to survive for several days, but died before eclosion, presumably caused by the water loss due to incomplete pupa epidermis.(2) Extrinsic bmo-miR-1changes expression profile of numerous genesMolting of silkworm was consisted with multiple biochemical and physiological processes which involved hundreds of genes. A total of1450genes were detected differentially expressed, including814up-regulated genes and636down-regulated genes. GO classification results showed that614genes had more than one GO annotation, involving a total of19GO sub-categories, cellular processes, physiological processes, the catalytic activity and metabolism were four most enriched GO categories, indicating a large number of genes related to basal metabolic and physiological processes were affected. Further analysis found that differentially expressed genes are more enriched in molecular structure of activity, immune system process, the extracellular region. The categories of growth and migration only contained down-regulated genes, indicating that the growth and the formation of the epidermal structure maintenance are affected. Meanwhile, the damage to the epidermis led to the surface barrier defects, which induce the activation of the immune system.132out of255cuticle proteins were detected,85%and75%of the RR-1and RR-2family members which involved in chitin binding were down-regulated, while CPG, Tweedle and CPFL family, which were not considered to be chitin-related, showed no obvious down-regulation. CPH family had more diversity in structure and function, the numbers of down-and up-regulated genes were relatively close. It could be speculated that the causes of molt abnormal phenotype may be partially attributed to the abnormal chitin-protein interactions caused by altered expression pattern of cuticle proteins.(3) Some down-regulated genes are potential bmo-miR-1targetsThe prediction of the target gene is an essential part of miRNA functional research.87potential bmo-miR-1target genes were predicted based on available silkworm3'UTR data. Many miR-target relationships were conserved in the silkworm and other species, such as vATPase family members (BmATP6v0c1and BmATP6v0d1), and adhesive transfer protein TAGLN, etc. Combined with differential gene expression data, we found18potential target genes were transcriptionally down-regulated, including the above three conserved bmo-miR-1target genes. B. mori chitinase gene BmChi, a bmo-miR-1potential target, was5-times trancriptionally down-regulated; we speculate that it might be suppressed by bmo-miR-1. (4) BmChi is a target of bmo-miR-1To investigate the relationship of BmChi and miR-1, BmCHI was expressed in prokaryotic cells, purified to prepare monoclonal antibody. Microarray, RT-PCR and western blotting result showed that the expression of BmChi in the silkworms injected with bmo-miR-1was significantly suppressed. Constructs for dual-luciferase reporter gene assay were also generated. The results showed that bmo-miR-1regulated the expression of reporter genes through the3'UTR of BmChi. Of those potential target sites, Site1which located at the356nt had a less contribution to the miRNA mediated suppression. All the evidences aforementioned demonstrated that BmChi was a target gene of bmo-miR-1.(5) BmChi is critical for molting of silkworm8chitinase genes, termed as Bmchi, BmChiR1,BmCHT2,BmCHT6,BmCHT6b,BmCHTl1, BmIDGF and BmCHT12, belonging to GH18family were predicted using homologous alignment and system anagenesis analysis. All8genes had their unique characteristics. Based on the functions of the homologous genes in other organisms, we prospect that these genes may envolved in the molting, eclosion and degradation of peritrophic membrane in silkworm. The temporal expression profile showed that BmChi was highly expressed before molting, while the expression was hard to detect during the feeding stage, thus we prospect that BmChi may involves in the degradation of chitin during molting. Furthermore, the silkworms injected with bmo-miR-1were arrested during molting, mainly because of the disability to degradate the old cuticle.