Identification Of Bmo-miR-7 Target Gene Bmhairy

By now, increasing number of silkworm microRNAs have been identified and spationtemporally profiled based on microarray, solexal sequencing and Northern blot experiments, thus leaving a good foundation to their further functional study in this animal.In Drosophila,miR-7 plays an important role in the development of sensory organs, since its ectopic expression causes wing margin nick, bristle density and the like mutations. Recognized by miR-7 via matching the highly conserved site in its 3'UTR, Hairy inhibits proneural genes in the peripheral nervous system, patterns bristle and informs sensory development. However, it is not determined whether there also exisit similar regulatory relation in Bombyx mori. A preliminary exploration was therefore carried out here to probe into this issue.1. Cloning and expression analysis of BmhairyWe cloned the homologue of Drosophila hairy in Bombyx mori, and named it Bmhairy. It consisits of two introns and three exons;cloned sequence is 1020bp, encoding 217 amino acids, and its 3'UTR is 366bp. The predicted protein comprises three domains, HLH, Orange and WRPW, which have been confirmed to be highly conserved in vertebrates and invertebrates. The sequence of Bmhairy is most similar to that of the homologue in Coleoptera insect Tribolium (Tribolium castaneum). Bmhairy was strongly expressed in embryos from 6 h to 7d and lowly expressed in diverse tissues of fifth-instar day3 lava of silkworm。2. Target site prediction and dual luciferase reporter assayRNAhybrid analysis revealed a target site of bmo-miR-7 in the 3'UTR of Bmhairy, which was supported by manipulation of the amount of bmo-miR-7 and 3'UTR of Bmhairy via co-transfection in the BmE cell lines with bmo-miR-7 mimics and Bmhairy 3'UTR luciferase reporter gene.3. Prokaryotic expression and polyclonal antibody preparation of BmhairyThe recombinant plasmid of the subcloned Bmhairy was transformed into the expression strain Rosetta, followed by inducement with IPTG to produce Bmhairy protein in the form of inclusion bodies. The rotein was then purified by affinity chromatography and elector-elution to generate specific polyclonal antibody. Western blotting analysis confirmed the expression of Bmhairy in the silkworm embryo of 24h,3d,6d and day 1,day 4, day 8 pupa. However, the changing levels of bmo-miR-7 trigured no significant pulse accumulation of Bmhairy protein, suggesting existence of the potential fine-tuned or neutral model in the embryonic development of silkworm.