| Autographa california multiple nuclear polyhedrosis virus(AcMNPV)is a kind of model baculovirus and also a potential microbial insecticide.BmK IT is an insect-specific toxin derived from Buthus martensii Karsch,and we constructed recombinant baculovirus AcMNPV-BmK IT previously.We found that AcMNPV-BmK IT could accelerate the expression of P53 protein in host cells at the early stage of infection and improve the transcription level of apoptosis inhibitor gene p35.And we also found that AcMNPV-BmK IT showed higher cytotoxicity and insect resistance.In order to clarify the anti-insect mechanism of AcMNPV-BmK IT,we analyzed the transcriptome of AcMNPV-BmK IT infected Sf9 cells by Solex sequencing method.Baculovirus infection can activate antiviral response of host cells by reducing total protein synthesis to inhibit virus proliferation.In short,e IF2?kinase was activated after infection to phosphorylate e IF2?,which could inhibit translation initiation,thereby reducing total protein synthesis and progeny virus production.Ac-PK2 protein can inhibit the activation of eIF2?kinase to rescue translation initiation.In order to determine whether overexpression of Ac-PK2 protein can improve the insecticidal activity of AcMNPV,we constructed recombinant virus AcMNPV-PK2-EGFP/RFP and performed further studies.This study is divided into four parts:PART ? Transcriptome sequencing was performed to screen the differentially expressed genes in AcMNPV-BmK IT infected Sf9 cellsIn order to explore the anti-insect mechanism of AcMNPV-BmK IT and analyze the impact of AcMNPV-BmK IT infection on the transcription level of Sf9 cells,we extracted the RNA of Sf9 cells 72 h after AcMNPV and AcMNPV-BmK IT infection and conducted reverse transcription.The transcription level was analyzed by Solexa sequencing.A total of 61269 transcripts were identified in the three treatment groups: control group(C),AcMNPV treatment group(AT)and AcMNPV-BmK IT treatment group(ABT).54189,57979 and 56575 transcripts were detected in the controlgroup(C),AcMNPV treatment group(AT),and AcMNPV-BmK IT treatment group(ABT),respectively.A total of 7711 proteins were annotated with 26 kinds of KOG.For KEGG annotation on the proteins,2853 genes annotated with 339 pathways.GO functional classification predicted that10443 proteins were annotated with GO classification.A total of 957 differentially expressed genes were identified: 1)In the first group(ABT V.S.AT),38 up-regulated genes were analyzed,while 135 down-regulated genes were identified;2)In the second group(ABT V.S.C),129 up-regulated genes and 84 down-regulated genes were analyzed;3)In the final group(AT V.S.C),459 genes were up-regulated and 112 genes were down-regulated.We selected 11 genes from these differentially expressed genes to verify the sequencing data using q PCR method,and the results of q PCR were basically consistent with the results of transcriptome sequencing.The results of KEGG and GO functional analysis of differentially expressed genes showed that AcMNPV and AcMNPV-BmK IT significantly affected the cell processes,such as DNA replication and protein translation in host cells.Literature investigation found that host cells could resist virus infection by reducing total protein synthesis,and Ac-PK2 protein expressed by AcMNPV could rescue protein translation,so whether overexpression of Ac-PK2 protein can improve the cytotoxicity and anti-insect activity of AcMNPV was further studied.PART ? Effect of AcMNPV-PK2-EGFP on host cell energy metabolism and progeny virus productionWe constructed recombinant AcMNPV-PK2-EGFP which could overexpress Ac-PK2 protein,using bac-to-bac baculovirus recombinant expression system.5 MOI recombinant virus AcMNPV-PK2-EGFP was used to infect Sf9 cells.The results of fluorescence microscopy and western blot showed that PK2-EGFP fusion protein was overexpressed from 24 h and reached the maximum level at 72 h p.i..Western blot results showed that phosphorylated e IF2? in AcMNPV-PK2-EGFP infected Sf9 cells decreased from 48 h p.i.,compared with AcMNPV treatment group.Glucoseconsumption analysis indicated that the energy consumption of both treatment groups was increased,and the glucose consumption rate in AcMNPV-PK2-EGFP treatment group was significantly higher than that in the wild type group at 36,48 and 60 h p.i.,which were 1.11,1.2 and 1.34 times of AcMNPV group,respectively.The lactic acid accumulation in culture medium of AcMNPV-PK2-EGFP treatment group were significantly lower than that of AcMNPPV treatment group at 48,60,72 h p.i.,but there was no significant difference between AcMNPV-PK2-EGFP group and control group.The content of cellular ATP in AcMNPV-PK2-EGFP group increased significantly at 48,60 and 72 h p.i.,which was 1.13,1.14,1.10 times of that in the wild-type treatment group,respectively.The activity of hexokinase(HK)in AcMNPV-PK2-EGFP treatment group was significantly higher than that in control group at 48 and 60 h,which was 1.21 and 1.16 times respectively.The results of plaque assay showed that the production of progeny virus in the AcMNPV-PK2-EGFP treatment group were significantly higher than that in AcMNPV treatment group at 48 and 72 h p.i..And we also found that deletion of ac-pk2 gene reduced the production of progeny virus at 48 and 72 h p.i.These results suggested that recombinant AcMNPV-PK2-EGFP could affect the energy metabolism of the host cells to create a more favorable environment for the replication of progeny virus.Next,we further studied whether the infection of AcMNPV-PK2-EGFP will accelerate the apoptosis of host cells.PART ? Apoptosis mechanism analysis in AcMNPV-PK2-EGFP infected Sf9 cellsFlow cytometry analysis showed that the apoptosis rate of Sf9 cells infected by the recombinant virus AcMNPV-PK2-EGFP were significantly higher than that of AcMNPV treatment group at 48 and 72 h p.i..In order to further investigate the apoptosis mechanism of Sf9 cells infected by recombinant virus,we constructed AcMNPV-PK2-RFP.The results of ROS activity detection indicated that the active oxygen level in AcMNPV-PK2-RFP treatment group was significantly higher than that in wild treatment group at 48 and 72 h p.i..Western blot analysis showed that the expression of Sf P53 increased at 48,60 and 72 h p.i in AcMNPV-PK2-RFP infected Sf9 cell,which was 1.16,1.39,1.79 times of that in AcMNPV group,respectively.The results of mitochondrial membrane potential detection showed that the green fluorescence intensity of AcMNPV-PK2-RFP treatment group was higher than that of AcMNPV treatment group at 24,48 and 72 h p.i.,indicating that mitochondria membrane potential of AcMNPV-PK2-RFP infected Sf9 cell was lower than that of the AcMNPV treatment group.Then,we separated the mitochondria and detected the distribution of cytochrome c in mitochondria and cytoplasm by western blot.The results showed cytochrome c released from mitochondria to cytoplasm in two treatment group.Compared with AcMNPV group,mitochondrial cytochrome c in AcMNPV-PK2-RFP infected Sf9 cell reduced obviously from 24 h p.i..According to the above results,we speculated that AcMNPV and AcMNPV-PK2-RFP affected the apoptosis of Sf9 cell by stimulating mitochondrial apoptosis signaling pathway.Because AcMNPV-PK2-RFP increased the production of progeny virus,and newly generated progeny viruses would conduct secondary infection on Sf9 cells,then accelerated the activation of mitochondrial apoptosis pathway in AcMNPV-PK2-RFP treatment group.PART ? The anti-insect activity and mechanism of AcMNPV-PK2-EGFP against Spodoptera exigua larvaeThe results from previous two parts suggested that AcMNPV-PK2-EGFP/RFP could regulate energy metabolism of host cells,help progeny virus replication,and accelerate apoptosis of host cells.So does the recombinant AcMNPV-PK2-EGFP have higher anti-insect activity? We did further experiments.The result of q PCR showed that the recombinant baculovirus AcMNPV-PK2-EGFP could increase the expression of Ac-pk2 gene in intestinal tissue and nerve cord tissue of infected Spodoptera exigua larvae.The result of q PCR also showed that co-infection of AcMNPV-PK2-EGFP and AcMNPV-BmK IT could increase the expressionof BmK IT gene in midgut tissue and nerve cord tissue of larvae,and the expression of detoxification genes(sod,p450 and cat)in midgut tissue also be affected.Western blot analysis showed that the phosphorylation of e IF2?in the AcMNPV treatment group and AcMNPV-BmK IT+AcMNPV treatment group increased gradually during the infection process,and the phosphorylation of e IF2? in AcMNPV-PK2-EGFP treatment group reached the maximum level at 4 h p.i.,and decreased at 8 and 12 h p.i..In AcMNPV-BmK IT+AcMNPV-PK2-EGFP treatment group,the phosphorylation of e IF2? reached the maximum level at 8 h p.i.,then decreased at 12 h p.i..These results indicated that,compared with the AcMNPV group,AcMNPV-PK2-EGFP could reduce the phosphorylation of e IF2 a in the midgut tissues of infected Spodoptera exigua larvae,which is beneficial to the replication of progeny virus.At the same time,we observed that the expression of P53 protein began at 1 h p.i.in each viral treatment group,and the expression of P53 protein in AcMNPV-BmK IT+AcMNPV-PK2-EGFP treatment group was significantly higher than that in the AcMNPV,AcMNPV-PK2-EGFP,AcMNPV-BmK IT+AcMNPV treatment group at 4,8 and 12 h p.i.,which indicated that AcMNPV-BmK IT+AcMNPV-PK2-EGFP could accelerate the apoptosis of intestinal tissue cells in infected Spodoptera exigua larvae.Phenoloxidase activity analysis showed that the activity of phenoloxidase in AcMNPV-BmK IT+AcMNPV-PK2-EGFP treatment group reached the high level at 4 h p.i.,which was 1.58,1.25 and 1.18 times of that in AcMNPV,AcMNPV-PK2-EGFP,and AcMNPV-BmK IT+AcMNPV treatment group at4 h,respectively.Next,we calculated the average body weight,mortality rate,pupation rate and eclosion rate of Spodoptera exigua larvae in the control group and the virus treatment group.The mortality rate of AcMNPV,AcMNPV-PK2-EGFP,AcMNPV-BmK IT+AcMNPV and AcMNPV-BmK IT+AcMNPV-PK2-EGFP was 54.78%,76.6%,83.3% and 90%,respectively.The corresponding pupation rate was 33.3%,26.67%,20%,16.67%,and eclosion rate was 20%,16.67%,13.3% and 10%,respectively.Comparedwith AcMNPV,the mortality rate of the AcMNPV-PK2-EGFP treatment group was significantly increased,the pupation rate and eclosion rate were decreased,which indicated that AcMNPV-PK2-EGFP had higher anti-insect activity.Compared with AcMNPV-BmK IT +AcMNPV treatment group,the eclosion process of AcMNPV-BmK IT+AcMNPV-PK2-EGFP treatment larvae was delayed,pupation and eclosion rate were decreased,and the final mortality was significantly increased.Based on the above results,AcMNPV-PK2-EGFP had higher anti-insect activity than wild-type virus,and the co-infection of AcMNPV-PK2-EGFP and AcMNPV-BmK IT further increased the anti-insect activity.The results of this part showed that recombinant baculovirus AcMNPV-PK2-EGFP and AcMNPV-BmK IT increased host insect mortality by influencing the detoxification genes(sod,p450,cat)expression and the activity of phenoloxidase,intestinal tissue e IF2? phosphorylation and the expression of apoptosis related P53 protein.It has also been clear that co-infection of recombinant virus AcMNPV-PK2-EGFP and AcMNPV-BmK IT had higer insect-resistant activity.In this study,the effect of AcMNPV-BmK IT on the transcription level of Sf9 cells was analyzed by transcriptome,and more than 900 differentially expressed genes were identified.Then,we constructed recombinant virus AcMNPV-PK2-EGFP/RFP.And we analyzed the effect of overexpression Ac-PK2 protein on host cell energy metabolism and progeny virus production during infection process,and confirmed that the recombinant virus AcMNPV-PK2-RFP could accelerate the apoptosis of host cells by stimulating mitochondrial apoptosis signaling pathway.Moreover,the anti-insect activity and mechanism of the recombinant virus AcMNPV-PK2-EGFP were analyzed at the larvae level,and we found that co-infection of AcMNPV-PK2-EGFP and AcMNPV-BmK IT could enhance the anti-insect activity of AcMNPV-BmK IT.This study provided experimental basis for study of the anti-insect mechanism of baculovirus,andshowed great significance for the scientific and safty control of lepidoptera pests. |
PDF Full Text Request