Study On The Effect Of Bmlipase-1 On BmNPV Replication-Related Gene Dbp

Bmlipase-1 has been identified as a protein with activity against Bombyx mori nuclear polyhedrosis virus(BmNPV)in silkworm.However,there is no report on how the protein specifically exerts antiviral function.In this study,the effect of Bmlipase-1 on viral replication-related genes during viral replication and proliferation was discussed in order to resolve the antiviral mechanism of Bmlipase-1,and the following results were obtained:1.Successfully constructed recombinant virus: Recombinant vector capable of simultaneously expressing the viral polyhedrin gene and the Bmlipase-1 gene(experimental group),the viral polyhedrin gene and the green fluorescent protein gene(egfp)(control group).Recombinant baculoviruses were then constructed by Bac-to-Bac baculovirus expression system.Green fluorescence and viral polyhedra were observed by microscope in the control group;Western Blot verified the expression of the Bmlipase-1 gene except for the viral polyhedra was observed in the experimental group.2.Bmliapse-1 has an effect on the expression of BmNPV replication related gene of dbp: After the constructed recombinant virus is expanded and the virus titer is measured,the same amount of virus are infected into the silkworm BmN cells and the cells are collected at different times after infection,then the collected cells are reverse-transcribed into cDNA,the expression level of the virus replication-related gene and the difference in the viral genome copy value were detected by quantitative real-time polymerase chain reaction(qRT-PCR).The results of qRT-PCR showed that there was no significant difference in the expression of all the virus replication related genes except dbp.The difference of dbp expression was the most significant at 48 h after infection,and the viral genome copy value was the most significant at 24 h after infection.Northern Blot verification results are consistent with qRT-PCR results.3.Bmlipase-1 interacts with the dbp promoter: It was proved by gel electrophoresis mobility shift assay that the dbp promoter can bind to Bmliapse-1;the position at which the Bmlipase-1 protein binds to the dbp promoter is the 10 base sequence(CACTTCAATT).By constructing recombinant virus with a promoter(dbp-75 bp)containing the binding site and another promoter(dbpL-65 bp)which the binding site was removed.Moreover,using egfp as a reporter gene,differences in green fluorescence were observed,and Western Blot results showed significant differences in activity between the two promoters.Promotertranscription factor analysis revealed that there is a 6 base(TTCAAT)motif in the 10 base sequences,and it is speculated that this motif plays a particularly important role in the role of the dbp promoter.In summary,it is preliminarily believed that the antiviral mechanism of Bmlipase-1 is that Bmlipase-1 inhibits the expression of dbp,the BmNPV virus replication-related gene,by binding to the dbp promoter,thereby inhibiting the replication and proliferation of BmNPV and achieving antiviral effect.This paper provides a new theoretical basis for better research on the prevention and control of lepidopteran insect viruses,and provides new ideas for further research on the inhibition mechanism of silkworm genes on viruses.