Expression Of A Phenoloxidase Inhibitor Enhances The Virulence Of Beauveria Bassiana Against Helicoverpa Armigera

As an important pathogenic fungus,Beauveria bassiana has been developed into an environmentally friendly microbial insecticide for the control of various agricultural pests.However,in the production practice,the acceptance of Beauveria bassiana is not high,and the market share is low.The main reasons for this phenomenon are slow insecticidal speed and unstable control effect of Beauveria bassiana.In order to solve this problem,some researchers try to improve the virulence of Beauveria bassiana by genetic modification,so that it can be better applied in production practice.In order to make sure the Beauveria bassiana also suppress the immune system of host insect by the inhibition of phenol oxidase activation reaction,using the method of Agrobacterium-mediated transformation,the exogenous gene-phenoloxidase inhibiting factor Egfl.0 carried by DNA virus(Microplitis demolitor bracovirus)of Habrobracon was transformed into the wild type Beauveria bassiana.we got an engineering strains with high expression of Egf1.0 and observed the insecticidal effect and growth traits.The research show that:1.Some immune responses of Beauveria bassiana to Helicoverpa armigera were detected,and the results showed that after infected with Beauveria bassiana,the prophenoloxidase activation reaction and the hemocyte spreading of the H.armigera was inhibited;2.Egfl.0 was expressed in prokaryotes and recombinant proteins were obtained.In vitro functional analysis of the protein showed that Egf1.0 protein could inhibit the activation of prophenoloxidase activation reaction of Helicoverpa armigera.3.Using the pCAMBIA1300 as the skeleton plasmid,we constructed a binary expression vector pCAMBIA13 00-BAR apply to the genetic transformation of Beauveria bassiana.4.Replace the signal peptide sequence of Egf1.0 with the signal peptide sequence of Beauveria bassiands chitinase gene,and synthesize this fusion gene artificially.The fusion gene was inserted into the pCAMBIA1300-BAR vector and transferred into Beauveria bassiana by agrobacterium-mediated method.After screening and PCR and Western blotting analysis,the transformed strain egfl.0-1 was obtained.5.We analyzed the effect of Egfl.0-1 strain on the phenol oxidase activation of Helicoverpa armigera.The results showed that Egfl.0-1 strain could inhibit the phenol oxidase activation of Helicoverpa armigera to a greater extent.6.The virulence analysis showed that compared with the wild-type,the lethal concentration of Egfl.0-1 was 62.43%lower and the lethal time was 24.33h shorter after infection with Helicoverpa armigera.The results showed that high expression of Egf1.0 in Beauveria bassiana could significantly improve the virulence of the Beauveria bassiana.7.We analyzed the growth of Egf 1.0-1 strain.The results showed that there were no significant differences in growth rate,sporulation rate and spore germination rate between the transgenic strains and the wild-type strains.This result indicated that the high expression of Egf1.0 in Egfl.0-1 strain did not affect the growth of the fungus.In summary,this study imported phenol oxidase inhibiting gene-Egf1.0 into the wild Beauveria bassiana,so that it could inhibit the activation of phenolic oxidase in host insects to a greater extent,thus improving the virulence of Beauveria bassiana.The results provide references for the application of Beauveria bassiana in biological control.