Screening Proteins Binding To Bombyx Mori Baculovirus Polyhedrin Gene Promoter And Functional Study Of 39k Gene

The polyhedrin gene is Bombyx mori nucleopolyhedrovirus(BmNPV) very late expressed genes, it is often regarded as the insect baculovirus expression system starting the exogenous gene expression promoter because of its promoter offer a super-efficient start function. However, its efficient start up mechanism still needs much deeper research. Therefore, in this study, we use the promoter as bait to screen the interacting protein factors by Yeast one-hybrid system, including 38K、39K、LEF5、P6.9、ORF60、ORF61、FP25、GP64、V-CATH. In order to detect the regulation effect of the above protein fators, we constructed a recombinant bacmid with BmNPV polyhedral promoter drove-firefly luciferase and Bombyx mori cytoplasm action A3 promoter drove-renilla luciferase. Quantitative detection of luciferase activity proved that positive regulatory factor LEF5 and negative regulator factor 39 K, ORF61 interact with polyhedral gene promoter. The regulationary functions of the other protein factors were not bovious.BmNPV 39 k gene which encodes a phosphoprotein associated with viral gene expression, exists in genomes of all lepidopteran baculoviruses. The results of existing studies show that the 39 k gene is related to the regulation of viral gene expression, but the specific regulation effects of BmNPV 39 k gene on viral replication and transcription is still unknown. In order to further explore the biological functions of 39 k gene, We detected that 39 k gene was early gene, respectively to delete and recover 39 k gene to construct a knockout bacmid(39k-ko-Bacmid) and 39k-rescued bacmid(39k-re-Bacmid) by red recombination technique and Bac-to-Bac system. Then the constructed bacmids DNA were transfected into BmN cells. The results showed that both types of viruses could yield infectious virion in cells, indicating that 39 k gene was not essential for the replication of BmNPV, however, the deletion of 39 k gene could reduce virus titer significantly. In late transfection, the replication level of the viral genome was related to infectious BV which was generated by virus. Possibly, the reduced BV led to decreased replication capacity of deficient virus in late transfection. Furthermore, the results of qPCR showed that the deletion of 39 k gene had no effect on virus early replication but would decrease the transcription level of viral late genes and cause extremely significant reduction of viral gene transcription level(p<0.01) in different phases.In conclusion, 39 k gene is not essential for the replication of BmNPV, but the deletion could reduce virus gene transcription level in different phases and enhance the activity of polyhedral promoter. We speculate that the 39 K protein which may act as a repressor protein bound to polyhedron gene promoter, so that affect the polyhedron gene promoter combining with other transcription factors, and then result in decreasing of polyhedron gene promoter activity. This study will lay the foundation for further investigating the effect of BmNPV 39 k gene on viral genome replication and transcription, also provide information on the efficient transcription mechanism of Bombyx mori baculovirus expression system.