Genetic Engineering And Insecticidal Evaluation Of Beauveria Bassiana By Overexpression Of Different Virulence Genes

Along with the development of economy and improvement of people's awareness of environmental protection,the environmentally friendly mycoinsecticides have received great attention.However,all fungal biocontrol agents inevitably experience a latent period for infection development in host hemocoel and always kill insects more slowly than chemical insecticides.This disadvantage has often discouraged the use of fungal formulations in insect pest control.In order to increase mycoinsecticide products in market, it is urgently needed to improve the strain performance by genetic engineering.The entomopathogenic fungus Beauveria bassiana plays a significant role in the bio-control against pests.In this paper,Beauveria bassiana was used for genetical modification with an insect-specific scorpion neurotoxin AaIT and an insect cuticle degradation protease Pr1A by two step transformations using different selective markers.In order to obtain dual engineering strains,the plasmids pGPS3Ben-Pr1A and pGPET1 were constructed with different selective markers and virulent genes.The optimal inhibitory concentrations were selected by drug resistance assays with conidia.The best inhibitory concentration for Glufosinate Ammonium was 100ug/ml,and the best inhibitory concentration for Benomyl was 2ug/ml.Through two different genetic transformation methods,the singly transgenic isolates of Bb13T,Bb202T and the double transgenic strain Bb13TPr1A were acquired.RT-PCR and Western blot results show that pr1A and AaIT are successfully transcribed and expressed by recombinant strains.PR1A activity assays showed that the PR1A activity of double transformants had been greatly improved in comparison to the wild-type strains.Through the initial injection of Bornbyx mori larvae, the transgenic isolates with relatively high virulence were chose for further analysis.The selected AaIT singly transgenic isolates were Bb13T-4-1,Bb13T-10-3 and Bb202T-7.And the double transgenic isolates were Bb13TPr1A-1,Bb13TPr1A-2 and Bb13TPSr1A-3.To evaluate the insecticidal efficacy of transgenic isolates,the wide type Bb202 and the AaIT singly transgenic transformant Bb202T-7 were bioassayed against Monochamus alternatus.The data revealed that the singly transgenic transformant received an improved pathogenic effect by reducing the time of insect death up to 11.1%.At the same time,the wild type and the transformants were bioassayed against the larvae of Masson's pine caterpillar Dendrolimus punctatus and the wax moth Galleria mellonella,respectively.As compared with the wild type,engineered isolates took fewer spores to kill 50%of pine caterpillars,i.e.15-fold less for AaIT single transformant Bb13T and 8-fold less for double transformant Bb13TPr1A,respectively.The median lethal time for Bb13T and Bb13TPr1A were reduced by 40%and 36.7%,respectively against D.punctatus and 24.4%and 20.9 %,respectively against G.mellonella.It is evident in this study that AAIT could be degraded by protease PR1A when both present in samples,recommending that protein interactions should be evaluated before future fungal transformation with multiple genes.