Expression Of Bacillus Thuringiensis Vip3Aa Gene In Entomogenous Fungus Beaveria Bassiana

Beauveria bassiana is one of the most common entomogenous fungi, with widehost range and great convenience for massproduction, and in its natural medium,sometimes it possibly causes epidemic in between the host population which is beneficialto represses pests breeding rapidly or abundantly. However, like other entomogenous fungi,it always kills insects more slowly than chemical insecticides and this disadvantagediscourages the use of it in insect pest control. Therefore, improving its virulence usinggenetic engineering technology to increase mycoinsecticide products in market is ofsignificant application value. In this study, foreign gene Vip3Aa from Bacillusthuringiensis was transformed into two initial strains Bb13and Bb1944, resulting inconstruction of two monocopic chitinase-overexpressive transgenic engineered strains.Based on the sequence of Vip3Aa, gene for a kind of vegetative insecticidal proteinsof Bacillus thuringiensis, primers were designed with appropriate restriction sites, toamplify the full-length Vip3Aa gene. The PCR products digested by XhoI and EcoRI wereligated with the vector pbarGPE1digested by the same restriction enzyme to construct arecombinant plasmid pbarGPE1-vip3Aa. The plasmid expression vector linearized by ScaIwas transformed into Bb13and Bb1944strains of Beauveria bassiana, one of the mostimportant entomopathogenic fungus, by blastospore-based transformation system. ReverseTranscription-PCR detection of Vip3Aa transcription in Bb13V and Bb1944V suggestedthat Vip3Aa gene was consistently expressed in the transformants.To compare the virulence of Bb13and Bb13V on the third instar larvae of theMasson’s pine caterpillar, Dendrolimus punctatus, a bioassay was conducted by treatmentsof spray, ingestion of conidial suspension and both for Bb13V and spray for Bb13.Conidial suspension concentration was settled at1×10~5、1×10~6、1×10~7、1×10~8and2×10~8conidia/ml, with0.05%Tween80as control. The results showed that as compared to thewild-type strain Bb13, the corrected cumulative mortality caused by Bb13V treated withthe3methods increased by over30%for the suspensions of1×10~5、1×10~6、1×10~7、and1×10~8conidia/ml and virulence by spray was increased by33.6~62.1-folds; mortalitycaused by ingestion was higher than by spray for all concentrations. Under theconcentration of1×10~8, mortality by spray was significantly higher than by spray and LT50shortened by6.89days.To compare the virulence of Bb1944and Bb1944V on the second instar larvae of thecorn borer, Pyrausta nubilalis, a bioassay was conducted by treatments of spray, ingestion of conidial suspension and both for Bb1944V and spray for Bb1944. The results showedthat as compared to the wild-type strain Bb1944, virulence by spray of Bb1944V wasincreased by50.5~78.1-folds. Under the concentration of2×10~8, mortality by spray wassignificantly higher than by spray and LT50shortened by1.58days.Consequently, Vip3Aa gene was expressed in the transgenic strain so as to infect hostsmainly through cuticle, and its virulence was raised substantially as a result.