Cloning, Expression And Functional Analysis Of BmEm4 Gene From Silkworm, Bombyx Mori

E(spl)m4 gene was identified as a microRNA-targeted gene in Drosophila and involved in the Notch signaling pathway. However, researches about Bombyx mori Notch signaling pathway and its related protein is still deficient.We have cloned a homologue of E(spl)m4 gene from Bombyx mori by RT-PCR and named it BmEm4. The accession number was FJ436408. This ORF contained 477 bp, encoding a polypeptide of 158 amino acids with a predicted molecular weight of 18.01 kDa. Bioinformatics analysis suggested it contained some highly conserved motifs (such as Brd box, GY box, K box and CAAC motif) and two potential microRNAs binding sites in 3′UTR. The ORF of the gene was cloned into the prokaryotic expression vector pET-28a and the recombinant plasmid was transformed into E.coli BL21 (DE3). After being induced with IPTG, this gene was successfully expressed in deposition of the supersonic fragmentation. The molecular weight of this fusion protein was about 24 KDa (His tag was weight of 3.56 kDa, and BmEm4 was weight of 18.01 kDa) and identified by the mass-spectrum. After being purified with Ni2+ affinity chromatography, the purified fusion protein was used to immunize a male New Zealand rabbit, the titer of the polyclonal antibodies reaches 1:25600 measured by ELISA. According to Western blotting and real-time PCR analyses, the BmEm4 appeared noticeable differences between the protein levels and mRNA levels among different developmental stages, the mRNA was highly transcript in the egg but hardly detect the protein in this stage. Moreover, the result showed that BmEm4 was selected expressed in some tissues of the day-3 fifth instar larvae, such as head, epidermis, gut, fatty body, trachea, with the highest in the larval head, which had been confirmed by immunohistochemistry. We also compared the quantity of BmEm4 mRNA in different tissues of the fifth instar larva and found that BmEm4 mainly expressed in silkworm's head, ovary, epidermis and gut. These results were consistent with the translation analysis of BmEm4 by real-time qRT-PCR. Immunostaining indicated that BmEm4 is primarily located at the cytoplasm. We propose that BmEm4 may play similar roles with E(spl)m4 in regulating neurogenesis and is probably be regulated by some microRNAs binding the conserved motif in 3′UTR during the Bombyx mori development. We blocked the Notch signaling pathway withγ-secretase inhibitor in order to examination the relationship between BmEm4 and Notch signaling pathway. The results showed that theγ-secretase inhibitors could reduce the transcription and expression level of BmEm4 in BmN cell. We proposed that BmEm4 was regulated by Notch signaling pathway as a target gene.