Effect Of Spodoptera Exigua MiR-2763 On Se301 Cell And Spodoptera Exigua Nucleopolyhedrovirus(SeMNPV)infection

Spodoptera exigua is an omnivorous pest that can harm many kinds of vegetables such as solanaceous fruits,melons,and green leafy vegetables.It has the characteristics of large food intake,strong destructiveness,fastreproduce.It brings about heavy losses in agricultural production around the world every year.At present,the control of S.exigua is mainly carried out by chemical pesticides.The chemical pesticide residues have caused huge pollution to the ecological environment.At the same time,the extensive use of chemical pesticides also poses a huge threat to human food safety and stockbreeding.As people realize the importance of the ecological environment,there is an urgent need for new safe,efficient agricultural pest management methods.Since baculovirus can specifically infect insects,is safe for humans,poultry,livestock,environment,and can cause large-scale infectious diseases in insect populations,people have developed a variety of baculovirus insecticides for control of agricultural pests such as S.exigua.Spodoptera exigua nucleopolyhedrovirus(SeMNPV)is a baculovirus that can specifically infect S.exigua.It has a good application prospect in the field of biological control of S.exigua.However,we need to understand the mechanism of the viral infection process and the virus-host interaction before the widespread use of SeMNPV insecticides.So far,many studies have found that viral infection and host immune response are regulated by many miRNAs.In recent years,a lot of studies have shown that miRNAs play an important regulatory role in baculovirus infection and insect immune response.In this study,we get a S.exigua miRNA,i.e.,miR-2763,from miRNA high-throughput sequencing data of Se301 cell,and explored the effect of miR-2763 on Se301 cells and SeMNPV infection.The main research results are as follows.1.The bioinformatics analysis of miR-2763 shows that its mature sequence is 23 nt,and the expression site is located at 1243028 nt-1243050 nt of chromosome 21 of the S.exigua genome.Pre-miR-2763 was predicted by theRNAfold website.Its length is 83 nt,and the expression site is located at 1242976 nt-1243058 nt of chromosome 21 of the S.exigua genome.Moreover,pre-miR-2763 is highly conserved in lepidopteran insects.In addition,the U6 snRNA of S.exigua was cloned,which is highly conserved among different species.2.After transfected 1000 nmol/L miR-2763 mimic to Se301 cells,cell viability was decreased and some cells rounded and shrunk.However,after transfected 1000 nmol/L miR-2763 inhibitor to Se301 cells,Neither cell morphology nor cell viability showed significant changes.Apoptosis detection experiments showed that the rounding,shrinkage and decreased cell viability of Se301 cells caused by miR-2763 mimic transfection were not caused by cell apoptosis.3.Transcriptome sequencing found that after transfected 1000 nmol/L miR-2763 mimic into Se301 cells,we screened 389 differentially expressed genes,of which 179 and 210 genes were up-regulated or downregulated,respectively.These 389 differentially expressed genes were annotated to 83 GO terms.And,the 8 most enriched GO terms are nucleus,intracellular membrane-bounded organelle,membrane-bounded organelle,intracellular organelle,organelle,cytoskeletal protein binding,calcium ion binding,actin binging.These 389 differentially expressed genes are annotated to 170 KEGG pathways.The 4 most enriched KEGG pathways are viral carcinogenesis,human papillomavirus infection,PI3K-Akt signaling pathway,protein processing in endoplasmic reticulum.4.After SeMNPV infects S.exigua,the expression of miR-2763 is upregulated.Transfection of miR-2763 mimic in Se301 cells has no significant effect on the total BV(budded virion)production after SeMNPV infection,but,inhibits viral DNA replication and polyhedra production.Moreover,inhibited the expression of miR-2763 in Se301 cells will also lead to the decrease of SeMNPV DNA replication after SeMNPV infection.5.The target genes prediction indicated that miR-2763 might regulate the Aurora B kinase,three prime repair exonuclease 1-like,esterase CXE3.Then,the ORFs of these 3 S.exigua genes were cloned.The dual-luciferase report experiment and expression detection experiment showed that miR-2763 can down-regulate the expression of Aurora B kinase,three prime repair exonuclease 1-like,esterase CXE3.Interfering with the Aurora B kinase of Se301 cells will cause a significant decrease in cell viability and inhibit viral DNA replication after SeMNPV infection.The study of miR-2763′s effect on Se301 cells and its role in SeMNPV infection has improved our understanding of the function of miRNA in the baculovirus-insect interaction.Enriched our understanding of insect antiviral mechanisms.It provides a theoretical basis for the development of more efficient baculovirus insecticides and baculovirus expression systems.