Study On The Mechanism Of MicroRNA-34-5p,Encoded By Spodoptera Frugiperda,in Participating In Resistance To AcMNPV Infection

Spodoptera frugiperda is one of the significant migratory pests issued by the Food and Agriculture Organization of the United Nations in the Global Alert.Spodoptera frugiperda invaded in Yunnan Province of China in 2019,and rapidly spread to many provinces across the country,posing a serious threat to various cash crops such as corn in China.Compared with the environmental and drug resistance problems caused by traditional chemical insecticides,baculovirus,as an insect-specific microbial insecticide,shows stronger advantages and is used to control a variety of agricultural pests.microRNA（miRNA）plays an important role in the antiviral immunity of insects.Baculovirus infection to host insects caused differential expression of miRNA in the host.To clarify the regulatory role and molecular mechanism of the miRNA encoded by Spodoptera frugiperda in the process of regulating the replication of baculovirus,we analyzed the effect of Autographa californica multiple nuclear polyhedrosis virus（AcMNPV）infection with Sf9 cells on the host miRNA expression profile through high-throughput sequencing.At the same time,we analyzed the effect of AcMNPV infection on the miRNA pathway of host cells.microRNA-34-5p（miR-34-5p）,which regulated AcMNPV infection,was screened out from the differentially expressed miRNAs.Subsequently,the regulatory function of miR-34-5p on the proliferation of AcMNPV was clarified by transfection of miR-34-5p mimic and inhibitor in vitro and construction of recombinant bacmid which over-expressed miR-34-5p.Dual-luciferase reporter assay confirmed that odv-e66,ac78and ie2 of AcMNPV were the target genes of miR-34-5p,revealing the molecular mechanism of miR-34-5p inhibiting AcMNPV replication.This study is divided into four parts,and the main results are as follows:1.The microRNA pathway participates in the antiviral response in the host insects of Spodoptera frugiperda Sf9 cellsFirst,s RNA sequencing was performed on the uninfected cells and AcMNPV-EGFP infected Sf9 cells by high-throughput sequencing,and 91 known miRNAs and 104 novel miRNAs were analyzed.A total of 49 differentially expressed miRNAs were obtained in Sf9 cells after AcMNPV-EGFP infection,of which 10 miRNAs were significantly up-or down-regulated.At the same time,we selected 17 differentially expressed miRNAs for RT-qPCR verification.The results showed that the differentially expressed trend of miRNAs was the same as that of high-throughput sequencing,except miR-278-3p.At the same time,The target genes of significant differential expressed miRNAs（10 of all）were predicted,and they were analyzed through GO and KEGG pathways.The results showed that the potential genes played important roles in multiple pathways.Second,the transcription level of key genes Dicer-1,Argonaute1,Exportin-5 and Ran in miRNA pathway after AcMNPV-EGFP infection in Sf9 cells were tested.Results showed the transcription level of Exportin-5,Dicer-1 and Argonaute1 in Sf9 cells increased after AcMNPV-EGFP infection,indicating that the microRNA pathway was involved in the antiviral response in Spodoptera frugiperda insect host.2.miR-34-5p participates in anti-virus infection by regulating the proliferation of AcMNPV progeny virus and the energy metabolism in host cellsFirst,the effect of miR-11-3p,miR-2a-3p,miR-34-5p,miR-92a-3p and miR-278-3p on AcMNPV BV titer was analyzed using TCID₅₀ end-point dilution method after transfection of miRNA mimics and inhibitors in vitro.The results showed that the lg(TCID₅₀/m L)value of miR-34-5p mimic group was significantly reduced,and miR-34-5p inhibited the proliferation of AcMNPV progeny virus.Second,the effects of miR-11-3p,miR-2a-3p,miR-34-5p,miR-92a-3p and miR-278-3p on the energy metabolism in host cells after AcMNPV infection were analyzed.The results showed that miR-11-3p increased the energy metabolism level of host cells after AcMNPV infection,miR-2a-3p inhibited the energy metabolism of host cells after AcMNPV infection,miR-92a-3p and miR-278-3p had no effect on energy metabolism of host cells,and miR-34-5p inhibited the energy metabolism of host cells after AcMNPV infection.Third,the miR-34-5p expression level in Sf9 cells which infected by AcMNPV was detected.The results showed the expression level of miR-34-5p decreased to 50%at 12 and 72 h p.t.,and decreased to 70%and 80%at 24 and 48 h p.t.,respectively.Absolute quantitative PCR was used to detect the replication of virus and host genomic DNA in AcMNPV infected Sf9 cells.The results showed that miR-34-5p inhibited the replication of virus DNA and promoted the replication of host genomic DNA.Fourth,we constructed the recombinant Bacmid-4×pre-miR-34-EGFP by serially arranging four pre-miR-34.The expression of miR-34-5p increased to 10.5,13.4,7.9 and 7.1 times of that of mock group after transfection Bacmid-4×pre-miR-34-EGFP into Sf9 cells at 12～72 h p.t.Fifth,TCID₅₀end-point dilution method was used to detect the effect of Bacmid-4×pre-miR-34-EGFP on the proliferation of AcMNPV progeny virus.The results showed that Bacmid-4×pre-miR-34-EGFP inhibited the proliferation of AcMNPV progeny virus.At the same time,Bacmid-4×pre-miR-34-EGFP inhibited AcMNPV genomic DNA replication,promoted the host genomic DNA replication,and inhibited the utilization of glucose of host cells.3.Prediction and validation of miR-34-5p targeting AcMNPV encoded genesFirst,the target sites of miR-34-5p in the CDS region of AcMNPV encoding gene were predicted by miRanda software,RNA22 v2 and RNAhybrid online website,and 17potential target genes were predicted.Secondly,the complementary pairing between miR-34-5p and its 11 potential target genes were proved by dual-luciferase reporter assay.The viral target genes odv-e66,ac78 and ie2 were turned out to be the direct targets of miR-34-5p.Finally,the transcriptional level of AcMNPV genes was detected by RT-qPCR.The results showed that miR-34-5p overexpression inhibited the transcriptional level of odv-e66,ac78,ie2,gp64,lef3,vp80,and ac132,while the transcriptional level of these genes was up-regulated by miR-34-5p inhibitor.It indicated that miR-34-5p targeted odv-e66,ac78,and ie2 genes by inhibiting their m RNA transcription.4.Effect of miR-34-5p on the activity of AcMNPV infected with Spodoptera frugiperdaFirst,the expression and differential expression of miR-34-5p in different instars larvae of Spodoptera frugiperda after AcMNPV-EGFP infection by RT-qPCR.The miR-34-5p expression level was decreased by the increase of the instar of the Spodoptera frugiperda larvae.During the AcMNPV-EGFP infection,miR-34-5p expression was significantly decreased in the second,third and fourth instar larvae.Secondly,the LD₅₀and LT₅₀ values of AcMNPV-EGFP and AcMNPV-4×pre-miR-34-EGFP to the fourth instar larvae were measured.The results showed that the LD₅₀ values of AcMNPV-EGFP and AcMNPV-4×pre-miR-34-EGFP were 4.4×10⁷ and 6.9×10⁸ vp/larva,respectively.The LD₅₀ value of the latter is 15.68 times that of the former.There was no significant difference in the LT₅₀ value between the two viruses.Finally,the effect of AcMNPV-4×pre-miR-34-EGFP on the pupation and emergence of larvae was analyzed.Results showed the pupation rate of AcMNPV-4×pre-miR-34-EGFP group was higher than that of AcMNPV-EGFP group at the injection dose of 2.8×10⁸,2.8×10⁹ and 2.8×10¹⁰vp/larva,and the emergence rate of AcMNPV-4×pre-miR-34-EGFP group was lower than that of AcMNPV-EGFP group at the injection dose of 2.8×10⁴ and 2.8×10⁵ vp/larva.These results showed that miR-34-5p overexpression inhibited the anti-insect activity of AcMNPV against Spodoptera frugiperda larvae,and affected the pupation and emergence of larvae.To sum up,this study analyzed the effect of AcMNPV infection on the miRNA expression profile of Sf9 cells and identified 49 differentially expressed miRNAs.Subsequently,miR-34-5p,which regulates the proliferation of AcMNPV progeny virus,was screened out from the differentially expressed miRNA.The molecular mechanism of miR-34-5p regulating the proliferation of AcMNPV progeny virus was explored.Three viral target genes of miR-34-5p,odv-e66,ac78 and ie2,were identified,and the effect of miR-34-5p on the anti-insect activity of AcMNPV was analyzed at the insect level.It was clear that the overexpression of miR-34-5p reduced the anti-insect activity of AcMNPV.In this study,a regulator of insect-baculovirus interaction was found,which proved the direct regulatory effect of miR-34-5p on AcMNPV progeny viruses proliferation.This study provides a solid theoretical basis for insect antiviral immunity.At the same time,it also provides a basis for the application of RNAi in pest control.It plays an important role in the green control of lepidoptera pests.