| The flacherie disease of silkworm is a serious disease of sericulture. Bombyx mori infectious flacherie virus (BmIFV) is one of the important pathogen to cause flacherie disease, which has paid extensive attention from scholars. BmIFV is a sense single-stranded RNA viruses and especially infects the goblet cells in the midgut of silkworm, It has be classified as Picornavirales, Iflaviridae, Iflavirus.In this study, the sequence of the5’region before the BmIFV structural proteins contains the same frame start codon AUG (with the same stop codon) by bio-informatics analysis. Secondary structure analysis shows:The structural element predicted for the5’upstream region of capsid proteins of IFV genome are mainly simple hairpins in the anterior part (loop1-8) like the5’UTR of PnV that own internal ribosome entry site and the tetraloops in the posterior part (loop9-15) like type Ⅱ IRES of picornavirus. Construction of recombinant baculovirus dual-fluorescent protein gene and dual-luciferase gene expression system confirmed the sequence of BmIFV5’region possesses internal ribosome entry site (IRES) function.The different lengths sequences own different IRES activity. The1-155nt fragment IRES-mediated translation activity of the downstream exogenous gene is the lowest, while the highest activity region of the IRES-mediated exogenous protein is the1-599nt fragment.The recombinant baculovirus (vR-IFV599-G)containing the dual-fluorescent protein infected four Lepidoptera incect cell lines (BmN cell line, Sf9cell line, Tn cell lines, and SL cell lines). BmIFV IRES mediated downstream exogenous gene was expressed in BmN, Sf9and Tn cell lines. The cap structure mediated and downstream BmIFV IRES-mediated exogenous gene was not expressed in SL cell lines. The Dot blotting analysis revealed that there were not hybrid zones band of EGFP mRNA in recombinant virus vR-IFV599-G infected SL cells. Perhaps the recombinant baculovirus can not infect or polyhedrin promoter can not be activated in the SL cells. BmIFV IRES function may not have species-specific, may be able to have activity in mammals, insects and plants.The vR-IFV599-G was infected five-instar silkworm. The full bodies of silkworm infected by the vR-IFV599-G had red and green fluorescence. Infected silkworm tissues (midgut, silk gland, fat body and epidermis) were proceed the western blotting analysis.All of the four kinds of tissues own red fluorescent protein and green fluorescent protein hybrid zones, indicating that the activity of BmIFV IRES mediated downstream exogenous gene expression was no tissue specific. While, there are differences in BmIFV specific infection of the goblet cells of the silkworm midgut. It indicated the characteristics of the BmIFV infection may be not related to the translation specificity, may be related to its infection receptor specificity and replication specificity.The5siRNA sequences were designed according to the sequence of the5’region of BmIFV5by transient transfection of siRNA transfection in BmN cells, recombinant baculovirus vR-IFV599-F infection, discovery of RNAi-of Loop15right BmIFV the IRES mediated the downstream of the firefly luciferase gene expression has a good inhibitory effect. And then we use the piggy Bac, transposition system was successfully constructed containing the shRNA-Loopl5for G418selection of transgenic BmN cell (T-15i-BmN).In summary, we demonstrated the5’regions of BmIFV own the cap-independent IRES function and the function is no species specificity and no tissue specificity. At the same time, RNA interference technology can significantly inhibit BmIFV IRES activity, and transgenic cell was constructed for the IRES sequence. |
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