Studies On The Function Of Secrt In Spodoptera Exigua Cells Infected By Spodoptera Exigua Multiple Nucleopolyhedrovirus

The Spodoptera exigua（Lepidoptera:Noctuidae）is considered to be one of the most destructive polyphagous herbivorous pests,infesting over130 crop species of more than 30 families grown in fields and greenhouses worldwide and causing significant economic impacts.Overuse of chemical insecticides can lead to increased resistance of Spodoptera exigua and ecological problems.In this background,Spodoptera exigua multiple nucleopolyhedrovirus（SeMNPV）,which caused natural periodic epidemics in insect populations,has received increased attention.At present,SeMNPV has been successfully used as a large-scale commercial biocide for pest control of Spodoptera exigua.However,SeMNPV infection does not all lead to the death of the host but also establishes a latent infection to achieve the goal of virus-host coexistence.The latent infection causes the virulence of SeMNPV to decrease and affects the efficiency of SeMNPV biological control.Investigating the molecular mechanism of SeMNPV infection Spodoptera exigua cells helps to improve the efficiency of bioinsecticides and provides new insight into the resistance mechanism of pests to bioinsecticides.Investigate have shown that Calreticulin（CRT）not only participates in the virus replication process but also regulates intracellular calcium([Ca²⁺]_i),and[Ca²⁺]_i participates in every stage of virus infection.The laboratory established a latent infection cell line P8-Se301-C1,transcriptome data indicated that Spodoptera exigua calreticulin（Secrt）was down-regulated in P8-Se301-C1 cells.Elucidated the role of Secrt in the infection of Spodoptera exigua cells by SeMNPV,and great significance to further explore the molecular mechanism of latent infection.The results of this study are as follows:1.Real-time quantitative PCR showed that the Secrt gene in P8-Se301-C1 cells was down-regulated 1.6 times compared with Se301 cells;through seamless cloning of Secrt gene from Se301 cells,the Se CRT protein encoded by Secrt was endoplasmic reticulum protein with a signal peptide according to bioinformatics analysis.2.After knocking down the Secrt transcript in Se301 cells by RNA interference,the cell proliferation was significantly reduced by 40%,[Ca²⁺]_i concentration was significantly decreased by 27.2%,but cell viability was not affected.These results suggest that Secrt can regulate cell[Ca²⁺]_i concentration and proliferation.3.Real-time quantitative PCR showed that Secrt was differentially expressed in SeMNPV infected cells within 2-24 hours post-infection.After knocking down the Secrt transcript in Se301 cells by RNA interference,viral DNA replication decreased by 44.76%,but it did not affect the BV and polyhedrin production after SeMNPV infection.The above results indicate that Secrt gene is involved in the replication stage of SeMNPV infected Se301 cells.4.Fluo3-AM fluorescent labeling showed that compared with Se301cells,[Ca²⁺]_i in P8-Se301-C1 cells was significantly reduced by 33.73%.Se301 cells were treated with[Ca²⁺]_i chelating agent（BAPTA-AM）,which affected SeMNPV DNA replication,but did not affect budded virion and polyhedrin production.Among them,after treating Se301 cells with 0.25μM and 0.5μM BAPTA-AM,the viral DNA replication decreased by 26%and 52%,respectively.After treating P8-Se301-C1 cells with 100 n M[Ca²⁺]_i promoter（TG）the viral DNA replication increased by 65%.These results suggest that[Ca²⁺]_i plays a role in SeMNPV replication,indicating that[Ca²⁺]_i may play a role in the superinfection exclusion.