Construction Of Replication Controllable AcMNPV Vector And Its Application In The Study Of Gene Function Of Mythimna Separata

Autographa californica multiple nucleopolyhedrovirus(AcMNPV)is the most studied virus in baculovirus,it can infect a variety of lepidopteran pests,and can efficiently carry foreign genes into cells for expression,it has the potential to develop as a gene transmission vector,because of its high pathogenicity to host insects,it is difficult to apply it to the study of the gene function of lepidopteran pests.In this study,the induced regulatory expression system was used to control the expression of essential gene for AcMNPV replication to reduce its pathogenicity to host insects,make it a vector carrying foreign genes into insect cells for gene silencing,knock-out,over-expression and protein labeling.And use the constructed vector to study gene function in Mythimna separata.To use AcMNPV as a tool for gene function research,we first need to produce virus.At present,baculovirus is often packaged through Bac-to-Bac system.In mammals,bacterial infection can mediate gene transfer to mammalian cells and tissues for expression.In this study,the factors reported in the literature that are infectious to mammalian cells were cloned into E.coli for expression,screening the factors that have infectivity to insect cells,and finally screening yad A from Yersinia pseudotuberculosis can improve the bacterial infection ability to Sf9 cells.When using yad A expressing bacteria to transfer AcMNPV to Sf9 cells to prepare virus,the efficiency of yad A expressing strain to transfer baculovirus to Sf9 cells is 4.9 times higher than that of ordinary strain.In order to reduce the pathogenicity of AcMNPV to its host,this study used the Red recombination system to knock out the replication essential gene ie1 of AcMNPV,and then used the Tet-On 3G system to control the expression of the ie1 gene.This AcMNPV has a small amount of background replication in sf9 cells.However,the replication in M.separata is completely uncontrolled,indicating that the background expression level is too high when the Tet-On 3G system is used alone to control the replication of baculovirus,and it cannot achieve the purpose of controlling the replication of baculovirus.Since the background expression level is still too high when using Tet-On 3G system to control virus replication,this study uses a variety of strategies to reduce the background expression level of the Tet-On 3G system.One is to use the Csy4 system to reduce the TetOn 3G system at the post-transcriptional level.Another strategy is to construct a transgenic armyworm to integrate the Tet-On 3G gene into the M.separata genome.When the optimized Tet-On 3G-Csy4 system with a lower background expression level is used to control the expression of the ie1 gene of AcMNPV,the replication of the virus in sf9 cells can be regulated by the inducer doxycycline(Dox).Inject the virus into M.separata,and start adding antiviral drug abacavir to the feed 24 h to 48 h after feeding Dox,the infection rate of M.separata can reach 60.2%-70.1%,while the survival rate is maintained at 58.9%-82.2%,and the virus replication can be controlled in M.separata.The virus can infect M.separata without killing it.In constructing a way to reduce the background expression level of the transgenic M.separata,although we successfully inserted the Tet-On 3G gene into the M.separata genome,this transgenic strain could not support the virus replication of the ie1 gene knockout.In order to use the replication-controllable AcMNPV vector for the study of the gene function of M.separata,we load the ds RNA gene expression cassette and CRISPR/Cas9 system into the AcMNPV genome,selected the M.separata Mschs B gene as the target,and carried out gene silencing and knockout experiments respectively,however,target gene silencing and knockout were not detected.In this study,the factors with infection ability to Sf9 cells were screened,and baculovirus was produced by bacterial mediated gene transfer to Sf9 cells,which simplified the virus packaging process,reduced the production cost,and provided convenience for high-throughput production of baculovirus.In this study,the replication controllable AcMNPV vector was constructed to reduce the pathogenicity of AcMNPV to the host,and foreign genes could be carried into insects for silencing,knock-out,over-expression and protein labeling,which laid a foundation for the further study of gene function using virus vector.