| The Protaetia brevitarsis seriously endangers the production of cotton,fruit trees and other cash crops,and endangers the ecological environment.Because of its strong flying ability and lack of natural enemies,it is difficult to prevent and control the beetles.So the biological control of the beetles is being gradually focused on.Preliminary laboratory studies have found that Bacillus thuringiensis Cry 8 proteins can effectively destroy the intestinal structure of grubs,but ultimately did not cause their death.Preliminary laboratory studies showed that the haemolymph and intestinal isolates of Scarab inhibited the growth of Bt strain.It was speculated that the reason for the unsatisfactory insecticidal efficacy of Bt strain might be related to grub immunity and intestinal endophytic bacteria.In this paper,the influence of immune related genes on the community structure of endophytic bacteria in the midgut of grubs was analyzed by interfering with immune related genes,and the effect of immune related genes on the resistance of Bt strains was further analyzed.1.Screening of Immune Genes of Scarabs from White Star FlowerFour immune-related genes,pgrp1,gnbp,tab and pgrp-lb,were cloned and their ds RNA fragments were selected by bioinformatics combined with ds RNA design score.2.Establishment of immune interference system for Scarab of White Star Flower(1)The ds RNA fragment was expressed and enriched in HT115 strain by L4440 plasmid,and the expression system of ds RNA was optimized.It was found that the expression effect was better when the concentration of inoculation was OD6002 and IPTG was 5 mmol/L.(2)The interference system was constructed by screening the concentration and sampling time of ds RNA injection.The results showed that the interference efficiency was higher when the injection volume was 0.9 g/L,and the interference efficiency was the highest when sampling 48 hours after injection.3.Effect of immune gene interference on bacterial community structure in midgut contents of Scarab larvae(1)The interference efficiency of four immune genes was detected by Real-Time PCR.The average interference efficiency of gnbp-ds RNA injection group was 70.4%;that of tab-ds RNA injection group was 76.2%;that of pgrp1-ds RNA injection group was 72.2%;and that of pgrp-lb-ds RNA injection group was 31.4%.(2)The 16 S r RNA gene of midgut contents in pgrp1 i treatment group,gnbpi treatment group,Tabi treatment group and gfpi control group was sequenced.The results showed that the immune gene interference treatment affected the community structure of midgut bacteria in grubs,and the expression level of peritrophic Matrix Protein(PMP)was affected by immune gene interference.Pgrp-lbi treatment group and Tabi treatment group,while pgrp1 treatment group decreased the expression of peritrophic protein compared with the control group.4.Effect of immune gene interference on the susceptibility of Bt strainThe results showed that the half lethal concentration(LC50)of G03 was significantly lower than that of the control group(p < 0.01),suggesting that the lethal efficiency of Bt strain was improved by the interference of immune genes.The above data show that the response of immune genes plays an important role in inhibiting the insecticidal activity of Bt strains against grubs.The insecticidal effect of Bt strains is indirectly affected by the way of influencing the expression of peritrophic membrane proteins and maintaining the balance of endophytic bacterial communities in the midgut of grubs.However,the immune pathway involved and the mechanism of indirectly enhancing the virulence of Bt strain remain to be further studied. |
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