Construction And Biological Activity Analysis Of 20 Recombinant AcMNPV

To develop the green ecological agriculture is one of the important policies for the agricultural development in China.Therefore,it is important to construct and apply biological pesticides in plant protection and pest control.To improve insecticidal activity of baculovirus,20 per os infectious recombinant baculoviruses were constructed by inserting ascovirus encoded genes into AcMNPV via Bac-to-Bac system,and the biological activity of these constructed baculoviruses were analyzed in this study.The results are as follows:1.Constructing the bidirectional expression vector?pFB-ph?:The synthesized fragment,which contained polyhedrin with its promoter and terminator as well as p10 promoter and HSV-tk terminator in an opposite transcriptional orientation of polyhedrin,was inserted to pFastBac HTb vector.As a result,a pFB-ph which could express two foreign genes was constructed finally.2.Obtaining the recombinant AcMNPV:Twenty ascovirus encoded genes were inserted into pFB-ph vectors to generate 20 bacmids by transposition,respectively.Then those recombinant bacmids were transfected into Sf9 to perform the morphological observation of host cells and electron microscopy observation of viral polyhedral.The results indicated that both recombinant viruses and the control AcMNPV?wt-Ac?could assembled normally in Sf9 cell.3.Detecting the biological activity of the recombinant AcMNPV:The lethal dosage detection showed that among the 20 recombinant viruses,Ac-73?Ac-111?Ac-165 had a higher virulence than wt-Ac;the lethal time detection indicated that Ac-151 can speed the death of host larvae.The daily average weight and the daily diet consumption tests of Spodoptera exigua larvae at different viral concentrations(LD₂₀,LD₅₀,LD₉₀)showed that the weight and diet consumption of S.exigua larvae were inhibited by virus infection,and the inhibitions were more obvious accompany to the dosage increasing.On the other hand,the recombinant virus can inhibit larval feeding in various degrees.Daily mortality investigation of S.exigua larvae infected with different dosage baculoviruses showed that a higher mortality was detected in the higher viral concentration infected larvae.In this study,several recombinant baculovirus with eminent properties were obtained by genetic engineering technical,which provided new thought to construct efficiency insect viral insecticide.