Neoseiulus Barkeri 's Expression Pattern Of The Toll Pathway Induced By Beauveria Bassiana And Functional Verification Of Spz Gene

Neoseiulus barkeri Hughes,as the domestic phytoseitic mite commercially produced in China,has the advantages of wide feeding range and strong diffusion.Beauveria bassiana is one of the most widely used Bio-control fungi and has good control effect.The combined application of the two is a new way of comprehensive prevention and control.This research group has screened out the Bb025 strain of Beauveria bassiana which has high pathogenicity to the Tetranychus citrus but low infectivity to the N.barkeri,so it is clear that the two strains have the potential of joint prevention and control.However,the mechanism of resistance to Bb025 is still unclear,so it is urgent to explore.Therefore,in this study,N.barkeri and Bb025 were taken as research objects,and RT-qPCR technology was used to study the Toll pathway response mechanism of N.barkeri induced by Bb025,and Spz gene that plays a key role in the Toll pathway was identified by cloning technology.After Spz was silenced by RNAi technology,the expression of other genes and antimicrobial peptides in the Toll pathway was detected,and the role of Spz in the Toll pathway was identified.The main research results are as follows:1 Effect of the infection of Beauveria bassiana on the Toll pathway of N.barkeriAfter spraying or feeding on the mite for 30 min,PCR was used to detect the bacteria?Beauveria bassiana?in the mite,and a negative control group and a blank control group were set up.Only the spraying and feeding groups detected Beauveria bassiana fragments,indicating that the mite can feed on the mite within 30 min.After clear Bb025 intrusive way,RT-qPCR technology was used to study the Bb025 infection after N.barkeri Toll access mode of response,the results show that processing after 2 h,PGRP,Spz,Toll3,Myd88,Tube and Pelle rise significantly expression of different level,antibacterial peptide defensin also raised expression,show that the Bb025 infection after being feeding N.barkeri and induced cause N.barkeri Toll pathways in the immune response.After 4 h,except for Spz and Toll1,all the other genes were down-regulated,but defensin was significantly up-regulated,and the expression level was higher than that at 2 h,indicating that the Toll pathway gene had played its role and activated more defensin production.After 96 h,most of the genes in the Toll pathway and defensin were up-regulated to different degrees again,indicating that Bb025 invaded the body wall of N.barkerii and induced the immune response of the Toll pathway.After 120 h,except for PGRP,Spz and Toll1,the expression of other genes tended to be stable,while defensin showed a higher expression,indicating that the Toll pathway gene had basically completed the response and induced more defensin to resist infection.To sum up,the N.barkeri Toll pathway plays an important role in the immune response induced by Bb025feeding or body wall invasion,and it was also found that Spz gene plays an important role in the Toll pathway response.2 Cloning of Spz gene and expression profile analysis of different mite statesSpz gene was screened out based on transcriptome database,and its cDNA was cloned to obtain its full length.After bioinformatics analysis,it was found that Spz gene existed as an extracellular protein in the body of N.barkeri.According to multiple sequence alignment,Nbspz contains 7 conserved cysteine domains,which are highly conserved in the evolutionary process.Through the construction of phylogenetic tree,it was found that Nbspz was closely related to the species of the same arachnida,and that Nbspz was closely related to Spz5 of different species,indicating that Nbspz may belong to the Spz type5 family.The results showed that the expression levels of Nbspz were higher in nymph mite,female adult mite and male adult mite,indicating that Spz gene played an important role in resisting pathogen infection in nymph,female adult mite and male adult mite.3 Functional verification of the Spz gene of N.barkeri in the Toll pathwayRNAi was obtained by feeding N.barkeri by feeding method,and a silence efficiency of 61%was obtained.At the same time,Toll2,Toll3,Myd88,Tube,and Pelle all had down-regulated expressions.Toll2,Toll3,and Myd88 had significant differences compared with the control group,and defensin was also affected by it and down-regulated.This shows that Spz plays an irreplaceable role in the immune response to the Toll pathway of N.barkeri.After Spz was silenced and treated with Bb025,the mortality rate of N.barkeri significantly increased,and LT₅₀0 and LT₉₅5 were shortened.On the 7th day after treatment,when the cumulative mortality rates of the negative control group and the blank control group were 15%and 15.6%respectively,the cumulative mortality rate of the treatment group was as high as 50.1%,indicating that the sensitivity of the mite to Bb025 was significantly increased after RNAi.It is concluded that Spz gene and the Toll pathway where Spz is located play an important role in the response to Bb025 infection.