Anti-insect Mechanism Analysis Of AcMNPV-mediated Expression Of A Chinese Scorpion Neurotoxin Under The Early And Late Promoter

Baculoviruses are rod-shaped,enveloped viruses with circular double-stranded DNA genomes ranging in size from 80 to 180 kbp that specifically infect arthropods of the insect orders Lepidoptera,Hymenoptera and Diptera.Autographa californica multiple nucleopolyhe-drovirus(AcMNPV),the archetype species of the a-baculoviruses of the baculoviridae,is advantageous over traditional chemical insecticides since they have longer anti-pest effects in the field and the pests are less likely to develop resistance.However,the slow anti-insect action and the narrow killing range of insects restrict the further application of AcMNPV.Buthus martensii Karsch(BmK)is a kind of scorpions which venom contains excitatory insect toxins.BmK IT is a polypeptide of 69-72 amino acid residues,acting specifically on insects and inducing a fast excitatory contraction paralysis upon injection.Previously,we engineered BmK IT gene into the genome of AcMNPV and BmK IT was inserted to the downstream of immediate early(IE 1),late(P10)and very late(Polh or PH)promoters,respectively.Based on our previous work,this study intends to disclose anti-insect mechanism and regulation analysis of these promoters in AcMNPV-BmK IT at cellular and insect level.This study mainly divided into three parts:In the first part,results from western blot and fluorescence microscopy assay showed that AcMNPV-mediated expression of BmK IT under the IE 1,P10 and PH promoter had an effect on the expression and nuclear polymerization of P53 in the nuclei.Sf9 cells were infected with AcMNPV,AcMNPV-BmK IT(IE1),AcMNPV-BmK IT(P10)and AcMNPV-BmK IT(PH)at the concentration of 2.28×1012 vp/mL(10 MOI)for 6-45 h,respectively.Western blot analysis confirmed the time-effect relationship between the expression level and regulation of promoters.Fluorescence microscopy analysis showed that AcMNPV-mediated expression of BmK IT under the IE1,P10 or PH promoter could promote the nuclear polymerization of P53 and accelerate the apoptosis of Sf9 cells.In the second part,effect of AcMNPV-mediated expression of BmK IT under the IE 1,P10 or PH promoter on the nuclear polymerization of actin in Sf9 cells was analyzed.Sf9 cells were infected with AcMNPV,AcMNPV-BmK IT(IE1),AcMNPV-BmK IT(P10),AcMNPV-BmK IT(PH)at the concentration of 2.28×1012 vp/mL(10 MOI)for 6-46 h,respectively.The results from immunofluorescence assay and western blot showed that cells infected with these four viruses for 6-21 h,microfilaments were formed on the ventral surface and actin filaments appeared in nuclei in both treatment groups.At 37 h.p.i.(hour post infection),nuclear actin decreased in the AcMNPV-BmK IT(P10)and AcMNPV-BmK IT(PH)treatment groups.However,nuclear actin decreased in the AcMNPV-BmK IT(IE1)treatment group at 46 h.p.i..These findings showed that AcMNPV-mediated expression of BmK IT under the IE1,P10 or PH promoter delayed the nuclear polymerization of F-actin and accelerated the clearance of actin in the nuclei.We suspected that this phenomenon was due to the change of cellular microenvironment induced by the effect of BmK IT on the cellular sodium ion channels.To adapt to this change,recombinant virus adjusted its proliferation progress through regulation of actin rearrangement.In the third part,regulation analysis of AcMNPV-mediated expression of BmK IT under the IE1,P10 and PH promoter was performed in vivo.Larvae of Heliothis armigera were infected with AcMNPV,AcMNPV-BmK IT(IE1),AcMNPV-BmK IT(P10),AcMNPV-BmK IT(PH)for 1-12 h at the concentration of 3.16×108 PIBs/mL,respectively.In midgut tissue,the transcription level of BmK IT reached the top value at 4,8,12 h.p.i.in AcMNPV-BmK IT(IE1),AcMNPV-BmK IT(P10)and AcMNPV-BmK IT(PH)treatment group,respectively.Moreover,the top transcription level of BmK IT in AcMNPV-BmK IT(P10)treatment group was 1.4 and 3.2 times of that inAcMNPV-BmK IT(IE1)and AcMNPV-BmK IT(PH)group.The transcription of BmK IT was also detected in the epidermal tissue of infected larvae.The low transcription level in three treatment groups at 1 h.p.i.was detected,which suggested that a small quantity of budded viruses in the sample infected the epidermal tissue directly.In the AcMNPV-BmK IT(P10)and AcMNPV-BmK IT(PH)treatment groups,high transcription level of BmK IT was detected at 8 h and 12 h.p.i.,respectively.TUNEL assay showed that the degree of midgut tissue apoptosis in the AcMNPV-BmK IT(IE 1),AcMNPV-BmK IT(P10)and AcMNPV-BmK IT(PH)treatment groups was higher than that in the AcMNPV treatment group.The degree of midgut tissue apoptosis in the AcMNPV-BmK IT(PH)treatment group was the highest.These results suggested that the recombinant AcMNPV improved the expression of BmK IT,which would further induce apoptotic cell death in the midgut tissue and thus promoted the insecticidal efficacy.The phenoloxidase activity in haemolymph of larvae infected with AcMNPV-BmK IT(IE1)reached the top level at 4 h.p.i.,which was 1.5,1.2 and 1.3 times of that in AcMNPV,AcMNPV-BmK IT(P10)and AcMNPV-BmK IT(PH)treatment groups,respectively.Phenoloxidase activity in the AcMNPV-BmK IT(P10)and AcMNPV-BmK IT(PH)treatment groups was maximal at 8 h.p.i.,and the phenoloxidase activity in AcMNPV-BmK IT(PH)group was higher than that in AcMNPV-BmK IT(P10)group.The time-effect relationship between the insect's humoral immunity and regulation of promoters was confirmed.It was noteworthy that the changes of phenoloxidase activity were correlated with the transcription level of BmK IT which was regulated by the IE1,PH or P10 promoter.The results from western blot showed that the expression level of P53 reached the top at 4,8 and 8 h.p.i.in AcMNPV-BmK IT(IE1),AcMNPV-BmK IT(P10)and AcMNPV-BmK IT(PH)treatment group,respectively.In this study,the effect and mechanism analysis of AcMNPV-mediated expression of BmK IT under the IE1,P10 and PH promoter on Sf9 cells and larvae of Heliothis armigera were performed.Besides uncovering the universal principles mediating baculovirus's capacity in infecting lepidoptera pests,these results provide a theoretical basis for the development and mechanism analysis of recombinant virus biopesticides.