The Function Study Of AcMNPV-BmK IT In Regulation Of Baculovirus Ac109 And Ac30

The genome of baculovirus is a double stranded,ring and super helical DNA,which is about 80-180 kbp.Their hosts are Lepidoptera,Hymenoptera and Diptera insects.At present,Autographa californica multiple nucleopolyhedrovirus(AcMNPV)is used widely for pest control.Its genome is about 130 kbp and contains 154 ORFs.It was found that AcMNPV had the advantages of high insecticidal rate and biosafety,but its application was limited due to its slow insecticidal speed and narrow insecticidal spectrum.ac109 and ac30 are two genes related to the proliferation of AcMNPV.Ac109 core gene encodes a later period structural protein,which is necessary in the process of nucleocapsid assembly.The ac30 gene is a non-essential gene for the virus replication,but it could provide a selective growth advantage for the virus to facilitate the virus multiplication.BmK IT was an excitatory insect specific neurotoxin that was cloned from the Chinese scorpion Buthus martensii Karsch,which contained69 amino acids and 4 disulfide bonds.Previous experiments proved that the recombinant baculovirus AcMNPV-BmK IT(IE1)could significantly enhance the virus anti-insect activity.At the same time,we obtained the transcriptome data of three samples,control group(Sf9 cells),AcMNPV treatment group and AcMNPV-BmK IT(IE1)treatment group,at 72 h p.i..There were 173 differentially transcripted genes in the recombinant virus treatment group compared with the wild virus treatment group,213differentially transcripted genes in the recombinant virus treatment group compared with the control group,and 611 differentially transcripted genes between the wild virus treatment group and the control group.According to the validation,we found that,compared with that in AcMNPV treatment group,the transcriptional level of ac109 and ac30 had a significantly reduced in the recombinant virus AcMNPV-BmK IT treated Sf9 cells at 72 h p.i..Therefore,this study focused on the function of Ac109 and Ac30 during the process of virus infection and how AcMNPV-BmK IT(IE1)regulated the expression of Ac109 and Ac30 and then affected the proliferation of progeny virus.This study was divided into three parts:Part I:Construction of recombinant virus AcMNPV-Ac109-EGFP and AcMNPV-Ac30-EGFPAccording to the data from transcriptome sequencing,we selected some differentially expressed genes to analyze the transcription level by qRT-PCR.Compared with the AcMNPV treatment group,ac109 gene and ac30 gene in the recombinant virus AcMNPV-BmK IT(IE1)treatment group were significantly down-regulated in 72 h p.i..In order to analyze the effect of Bm K IT on ac109 and ac30,Bac-to-Bac baculovirus expression system was used to construct recombinant virus AcMNPV-Ac109-EGFP and AcMNPV-Ac30-EGFP.AcMNPV-mediated overexpression of Ac109 and Ac30 were used to analyze how AcMNPV-BmK IT(IE1)regulated these two proteins and then affected the progeny virus proliferation.ac109 and ac30 gene fragments were amplified from the AcMNPV genome and inserted into the intermediate vector pFastBacDual-EGFP.The resulting plasmids(pFB D-Ac109/Ac30-EGFP),namely,were transformed into the Escherichia coli strain DH10 Bac and transposed into the AcMNPV genome and bacmid-Ac109/Ac30-EGFP were obtained.Then,cells were transfected with bacmid-Ac109/Ac30-EGFP to get the first generation recombinant viruses AcMNPV-Ac109/Ac30-EGFP.Continuous culture of the first generation recombinant viruses got the two and three generations of recombinant virus.The virus titer of AcMNPV-Ac109-EGFP and AcMNPV-Ac30-EGFP was 2×10~7 and 1×10~7 pfu/ml,respectively.Then,Sf9cells were infected with AcMNPV-Ac109/Ac30-EGFP at 5 MOI(Multiplicity of infection),and fluorescence microscopy detection and Western blot analysis showed that the number of Sf9 cells harboring green fluorescence and intracellular green fluorescence intensity were significantly enhanced and the expression level of Ac109/Ac30-EGFP increased with the prolongation of infection time and reached the maximum value at 72 h p.i..Part II:The funcation study of AcMNPV-BmK IT in regulation of Ac109 and Ac30The previous results showed that the transcription levels of ac109/ac30gene were significantly down-regulated when AcMNPV-BmK IT(1.5 MOI)infected Sf9 cell for 72 hours,so how does BmK IT regulate the expression of Ac109/Ac30 and how do over expression of Ac109 and Ac30 affect the progeny virus and the insecticidal efficiency of the virus?Sf9 cells were infected by the recombinant virus AcMNPV-Ac109/Ac30-EGFP+AcMNPV-BmK IT,and western blot and fluorescence location analysis showed that BmK IT could accelerate the aggregation of Ac109-EGFP in nucleus,and delay the aggregation of Ac30-EGFP in nucleus.Then,the effect of BmK IT on the progeny virus production of AcMNPV-Ac109/Ac30-EGFP was analyzed.The results showed that,compared with AcMNPV treatment group,AcMNPV-BmK IT regulated the progeny virus production of AcMNPV-Ac109-EGFP in the trend of decreasing and increasing.At 72 h p.i.,the amount of progeny virus was lower than that in AcMNPV treatment group,which was consistent with the results from transcriptome data.But the amount of progeny virus was higher than that of AcMNPV group at 96 and120 h p.i..For AcMNPV-Ac30-EGFP+AcMNPV-BmK IT treatment groups,the amount of progeny virus was lower than that in AcMNPV treatment group at 72 h p.i.,which was consistent with the results from transcriptome data,too.In the AcMNPV-Ac30-EGFP+AcMNPV-BmK IT treatment group,BmK IT still down-regulated the progeny virus yield of recombinant virus at96 h p.i.,compared with that in AcMNPV treatment group.But,it exceeded the wild treatment group at 120 h p.i..It suggested that Bm K IT affected the yield of the progeny virus by influencing the aggregation of Ac109-EGFP and Ac30-EGFP in the nucleus.In the anti-insect analysis,AcMNPV-Ac109/Ac30-EGFP were used to infect Spodoptera exigua larvae.The mortality rates were 66.67%and 71.67%,the pupae rates were 25%and 28.33%,and the emergence rates were 11.67%and 13.33%,respectively;The mortality rates of Spodoptera exigua larvae in the recombinant virus AcMNPV-Ac109/Ac30-EGFP+AcMNPV-BmK IT treatment group were 78.33%and 63.33%respectively,which were higher than that in AcMNPV-Ac109-EGFP treatment group and lower than that in AcMNPV-Ac30-EGFP treatment group.The pupae rates were 21.67%and 36.67%respectively,which were lower than that in AcMNPV-Ac109-EGFP treatment group and higher than that in AcMNPV-Ac30-EGFP treatment group.The emergence rates were8.33%and 11.67%respectively,which were lower than that in AcMNPV-Ac109-EGFP treatment group and AcMNPV-Ac30-EGFP treatment group.These results showed that BmK IT enhanced the anti-insect activity of AcMNPV-Ac109-EGFP and inhibited the anti-insect activity of AcMNPV-Ac30-EGFP.Part III:Function analysis of baculovirus auxiliary protein Ac30Previous study showed that BmK IT could regulate the proliferation of progeny virus by regulating the expression of Ac109 and Ac30.And,we also found that the AcMNPV-Ac30-EGFP+AcMNPV-BmK IT treatment had higher virus yield and insecticidal efficiency than the AcMNPV+AcMNPV-BmK IT treatment group.Some literatures reported that ac30 gene was an auxiliary gene,and could provide selective growth advantage for virus to facilitate virus proliferation,which showed that Ac30 could help AcMNPV-BmK IT virus proliferation.So,was the assistive function of Ac30broad-spectrum?Does over-expression of Ac30 affect the expression of Ac109?In this section,we took advantage of the two recombinant viruses to analyze the effect of overexpression of Ac30 on the transcriptional of ac109and nuclear accumulation of Ac109.It showed that Ac30 increased the transcription level of ac109 at 24-96 hours,and Ac30 could accelerate the nucleus accumulation of Ac109-EGFP.Further,we studied the effects of different MOIs of recombinant virus AcMNPV-Ac30-EGFP on the nuclear accumulation of Ac109.The results showed that in AcMNPV-Ac30-EGFP and AcMNPV-Ac109-EGFP co-infected Sf9 cells,the nuclear accumulation ofAc109-EGFPwas24haheadofthatinthe AcMNPV-Ac30-EGFP(1MOI)+AcMNPV-Ac109-EGFP(2.5 MOI)treatment group when MOI of AcMNPV-Ac30-EGFP(MOI?2)increased.At the same time,the time of nuclear accumulation of Ac109-EGFP reached the maximum concentration from 120 h p.i.(AcMNPV-Ac30-EGFP(1MOI)+AcMNPV-Ac109-EGFP(2.5 MOI))improved to 72 h p.i.(AcMNPV-Ac30-EGFP(5MOI)+AcMNPV-Ac109-EGFP(2.5 MOI)).Lactic acid content analysis showed that Sf9 cells were co-infected with AcMNPV-Ac30-EGFP(5 MOI)and AcMNPV-Ac109-EGFP(2.5 MOI)at 48 h,72 h,96 h,120 h,the content of lactic acid was 0.85,0.898,0.9,0.97 times of that in the AcMNPV-Ac30-EGFP(2.5MOI)+AcMNPV-Ac109-EGFP(2.5MOI)treatment group,respectively.So,with the increasion of MOI of AcMNPV-Ac30-EGFP,the host cell and recombinant virus AcMNPV-Ac109-EGFP replication required more energy,which resulted in the decreasion of lactic acid content.In this study,we found that ac109 and ac30 were novel target genes during the AcMNPV-BmK IT(IE1)infection process.Recombinant virus AcMNPV-Ac109-EGFP and AcMNPV-Ac30-EGFP were constructed by using Bac-to-Bac expression system.We revealed the regulation mechanism of AcMNPV-BmK IT on Ac109 and Ac30,which provided a reference for exploring new theories and approaches of biological control of insect pests.At the same time,we clarified the function of baculovirus auxiliary gene ac30,which enriched the research data of baculovirus auxiliary gene.