| In recent years,a large number of Spodoptera frugiperda have invaded China,causing threats and hidden dangers to Chinese crops.Baculovirus that specifically infects arthropods is one of the microbial insecticides in the face of this crop disaster.However,the use of wild-type viruses as insecticides has some disadvantages,such as long insecticidal time and narrow insecticidal spectrum.Research and modification of the baculovirus model organism,AcMNPV,genes can make up for the shortcomings of wild-type viruses.Functional studies of each gene are essential before modification of the viral genome.Ac106/107 is an open reading frame on AcMNPV with a total length of 732 bp.Studies showed that the progeny virus didn’t proliferate when bm90,the homologous gene in Bombyx mori nuclear polyhedrosis virus（BmNPV）of ac106/107,was knocked out.The latest research showed that,the infectivity of the progeny virus BV was lost when ac106/107 was knocked out.These studies showed that ac106/107 plays an important role in AcMNPV.In order to clarify and explore the mechanism of Ac106/107in the process of AcMNPV infection of host Sf9 cells,two parts of experiments were performed to analyze the function of Ac106/107.Part Ⅰ:The recombinant viruses vAcac106/107KO-EGFP,vAcac106/107KO-Ac106/107（rep）-EGFP and vAc-Ac106/107-EGFP were constructed and obtainedFirst,we usedλ-red homologous recombination technology to insert chloramphenicol resistance gene into ac106/107 of the original bacmid to achieve the purpose of gene knockout.After that,we used the bac to bac system to transpose the egfp and ac106/107-egfp genes to Bacmidac106/107KO,respectively,to obtain the knockout bacmid(Bacmidac106/107KO-EGFP)and the repair bacmid(Bacmidac106/107KO-Ac106/107（rep）-EGFP),transpose the ac106/107-egfp gene to bacmid,and finally obtain the over-expressed bacmid（Bacmid-Ac106/107-EGFP）.Finally,these obtained bacmids were transfected into Sf9 cells,and the recombinant viruses were obtained at 72 h p.t..By observing the fluorescence of the first,second and third generation progeny viruses of each recombinant virus under fluorescence microscope and TCID50 test on the three recombinant viruses and wide-type virus,we concluded that the progeny virus did not proliferate after knocking out ac106/107,and the progeny virus proliferation ability returned to the wild-type level after supplementing ac106/107.After overexpression of ac106/107,the proliferation ability increased by 14.5%than the wild-type at 72 h p.t.and ac106/107 played an important role in AcMNPV.Part Ⅱ:Functional analysis of knockout,repair and overexpression of ac106/107 in host cells infected by AcMNPVIn order to explore which stage of AcMNPV proliferation in host cells is affected by knocking out ac106/107 and the specific impact mechanism,the expression and intracellular localization of Ac106/107-EGFP protein,viral DNA replication,viral late gene transcription,and viral transcription of RNA polymerase subunits,the glucose consumption of host cells and the accumulation of lactic acid after viral bacmid transfection of cells were carried out experimentally.First,we studied the expression of Ac106/107 during virus replication and observed the localization of Ac106/107 in the host Sf9 cells by fluorescence microscope.The results showed that after the cells were transfected with Bacmid-Ac106/107-EGFP and Bacmidac106/107KO-Ac106/107（rep）-EGFP,the expression of Ac106/107-EGFP was detected at 6-48 h p.t.,and the expression level increased with the transfection.The dyeing time increases with the extension.Fluorescence localization results showed that Ac106/107-EGFP expressed after Bacmidac106/107KO-Ac106/107（rep）-EGFP and Bacmid-Ac106/107-EGFP transfected cells were mainly distributed in the cytoplasm and a small amount entered the nucleus at 6 h p.t..Then it was distributed in the nucleus and cytoplasm;this result proved that Ac106/107 could play some roles in the nucleus.Afterwards,by measuring the virus genome copy number,we found that after Bacmidac106/107KO-EGFP and Bacmid-Ac106/107-EGFP were transfected into cells,the genome copy numbers of the two bacmids were not significantly different from those of the wild type,this showed that ac106/107 did not affect viral DNA replication.Then we measured the transcription levels of the late viral genes 38k,vp39,and gp64 after the four bacmids were transfected into host cells and found that,compared with the wild-type virus,the transcription levels of these three genes were reduced after ac106/107 was knocked out,and the transcription level of these three genes increased significantly after overexpression of ac106/107.38K and VP39 are related proteins of the viral nucleocapsid,GP64 is the viral envelope glycoprotein,indicating that ac106/107 affects the transcription of 38k,vp39 and gp64,thereby affecting the formation of the complete nucleocapsid of the virus and the reinfection of the virus eventually lead to abnormal proliferation of the virus.Transcription of late viral genes does not depend on the host RNA polymerase but on the viral RNA polymerase encoded by the virus itself.Then we explored the transcription levels of the four subunits of viral RNA polymerase after the host cells were transfected with the four bacmids.The experimental results showed that compared with the wild type,the transcription level of these four subunits was reduced after ac106/107 was knocked out,and the transcription level of these four genes was significantly increased after ac106/107 was overexpressed.It showed that ac106/107 could affect the transcription of viral RNA polymerase subunits,and then caused changes in the transcription levels of late genes 38k,vp39 and gp64.Finally,by measuring the glucose utilization and lactic acid accumulation of the host cells after bacmid transfection of the host cells,it was found that transfection of wild-type,over-expression,and repair virus bacmids into cells respectively would lead to increased cellular glucose utilization and accelerated lactic acid accumulation.The overexpression type was higher than that of the wild type and the repair type treatment groups,and there is no significant difference between the repair type and the wild type treatment groups;after knocking out the ac106/107 type bacmid transfected cells,the glucose consumption and lactic acid accumulation in the cells were lower than in the other three experiment groups,but it was still higher than normal cell metabolism,indicating that Ac106/107 affected host cell metabolism.This study started with progeny virus proliferation,DNA replication,RNA polymerase subunit transcription and late gene transcription,and explored the function of Ac106/107 in the process of AcMNPV infecting host Spodoptera frugiperda Sf9 cells,and further enriched the regulation in AcMNPV functional annotation of genes.There are many challenges in creating target-oriented green pesticides that are safe for humans,animals and non-target organisms.The innovation of its core molecular targets is the main bottleneck restricting its development.This study provides an experimental basis for screening insect resistant genes and target analysis of AcMNPV virus insecticides. |
