Study On The Function Of Ac51,ac73 And Ac75 In The Autographa Californica Multiple Nucleopolyhedrovirus

Baculo virus is one of the most beneficial viruses among all known virus types and has been widely used in biological insecticides,eukaryotic expression vector systems,vaccine preparation,surface display systems and gene therapy.Autographa californica multiple nucleopolyhedrovirus(AcMNPV)is the model species of baculoviruses.It is the first to be completely sequenced,and is the most widely studied baculovirus.In order to make better use of baculovirus,it is necessary to reveal the function of each gene encoded by the virus in the life cycle of baculo virus.The functions of ac51,ac73 and ac75 genes in AcMNPV are unknown.The ac51 gene has homo logs in all Group ? and ? lepidoptera NPVs genomes;ac73 gene is only found in the sequenced Group ? lepidoptera NPVs genomes;homo logs of ac75 gene are present in all lepidoptera NPVs,GVs and hymenoptera NPVs genomes,but not in the diptera NPVs genomes.In this thesis,the functions of ac51,ac73 and ac75 genes were analyzed.The effect of each gene on virus proliferation,formation virion and virus infection in insect larva was analyzed.In addition,the subcellular localization of the proteins encoded by the ac51,ac73,and ac75 genes was analyzed to reveal the specific functions of those genes in AcMNPV infectinon.The paper begins with a review of the literature,focusing on baculovirus genome and classification,baculovirus phenotype and infection cycle,expression of baculovirus genes,classification ofbaculovirus genes,and application of baculoviruses.The background knowledge of ac51,ac73,ac75 genes and the research purpose and significance of this paper are expounded.In the thesis,we first used the ac73 gene as the research object to construct the recombinant virus vAcac73-ko-PH-PG(deleted with ac73)and vAcac73-req-PH-PG(virus rescued with ac73)using?-Red homologous recombination technology and Bac-to-Bac system.The two recombinant viruses and vAcPH-PG(wild type virus)were transfected or infected S-f9 cells and cells were observed by light microscope and fluorescence microscope,respectively.It was found that vAcac73-ko-PH-PG still can produce polyhedra and infectious virus particles.A one-step growth curve analysis was performed on the three recombinant viruses.The overall growth curve was similar,showing a steady growth trend,but the titer of the deleted recombinant virus was significantly lower than that of the wild type and the rescue type.Therefore,the yield of the deleted virus is lower than that of rescued and wild type viruses.Electron microscopic observations of the cells infected with vAcac73-ko-PH-PG revealed there were normal nucleocapsids and abnormally long hollow tubular structures.Some of the polyhed a had large cavities in the middle,and some contained vacuoles in which nucleocapsid was not packaged.Biological assay showed that the LT50 of vAcac73-ko-PH-PG was 42 h longer than that of the wild-type virus.The hemolymph extracted from Spodoptera exigua larvae feeded on the polyhedra of vAcac73-ko-PH-PG can infect insect cells,suggesting that the hemolymph contained virus particles.The results of the subcellular localization showed that Ac73-EGFP protein was localized in the nucleus in the early stage of virus infection,and Ac73-EGFP protein was present in both nucleus and cytoplasm of the later infection.In summary,the ac73 gene is a non-essential gene,but deletion of the gene affects the yield of AcMNPV budded virion and the morphology of the polyhedron.For the deleted virus,the infectivity is weakened,and the insecticidal speed is reduced.The Ac73 protein may initially function in the nucleus,and it functions in the cytoplasm in the late stage of infection.Then we used the ac51 and ac75 genes as research objects.The X-Red homologous recombination technology and the Bac-to-Bac system were also used to construct the deleted and rescued recombinant virus plasmids of ac51 and ac75,respectively.Through experiments such as transfection or infection of Sf9 cells,and the drawing of a one-step growth curve,it was found that the loss of either of these two genes caused the virus to lose its infectivity,while the rescued recombinant virus was infectious.Electron microscopy observations revealed that the deletion of the two genes resulted in the inability of the nucleocapsid assembly and the inability to form a polyhedron.Therefore,the ac51 and ac75 genes are all essential genes for AcMNPV,and the virus is not infectious after gene knockout.The results of subcellular localization analysis of Ac51 and Ac75 proteins showed that the Ac51-EGFP protein was localized in the nucleus after viral infection,and accumulated at the nucleus edge of the cell in the later phase;the Ac75-EGFP protein is mainly located in the nuclear zone in the early stage of infection and is in an aggregated form,and enters the cytoplasm to perform its function in the later stage.It suggests that both Ac51 and Ac75 proteins function in nucleus in the early stage of virus infection,and later Ac75 protein also functions in cytoplasm.Ac51 and Ac75 are predicted to be associated with the formation of viral nucleocapsid.The results of the paper indicate that the ac51 and ac75 genes are both essential genes of AcMNPV.The ac73 gene is a non-essential gene,which is not essential for virus propagation and oral infection of insects.However,knockout of the ac73 gene affects the yield of virions and the morphology of polyhedra.For the deleted virus,the virus titer decreases,and the insecticidal speed becomes slow.