Study On Recombinant Helicoverpa Armigera Nucleaopolyhdrovirus (HearNPV) With RjAa17f Neurotoxin Gene

Helicoverpa armigera nucleopolyhedrovirus (HearNPV) is an important pathogen incotton bollworm, which has been successfully developed as biological pesticides ofcommercialization in China. However, wider application of baculoviruses has been limiteddue to its disadvantages, such as slow speed of kill, large dosage for older instar larvae, andhigh cost of baculovirus insecticide. To overcome these drawbacks, a variety of geneticapproaches have been applied to improve the insecticidal properties of baculoviruses. Here, arecombinant bacmid RjAa17f-HaBacHZ8expressing the insect-selective neurotoxin (RjAa17f)from Cuban scorpion Rhopalurus junceus was constructed by replacing theUDP-glucosyltransferase gene (egt) using λ-red homologous recombination system. Then, thepolyhedra gene (ph) was repaired to HaBacHZ8-RjAa17f genome by transposition technique,and gained a new recombinant baculovirus RjAa17f-HearNPV with infection activity. At thecellular level, we explored the growth and replication of recombinant virus. In the expressionlevels, we researched the toxin gene transcription of RjAa17f. We compared the differencebetween the HZ8-HearNPV and RjAa17f-HearNPV in submicroscopic structure. Throughbioassays, we studied the insecticidal efficacy of RjAa17f-HearNPV compared to wild typevirus. The main conclusions of this study are as following:1. The ph was repaired successfully to the HaBacHZ8-RjAa17f and HaBacHZ8bytransposition technique to generate artificial genomes of RjAa17f-HearNPV andHZ8-HearNPV, respectively.2. HZ8-HearNPV and RjAa17f-HearNPV were transfected into HzAM1cells,respectively, and confirming that both were able to form normal virus particles and both alsohave infection in intercellular propagation.3. RjAa17f-HearNPV and HZ8-HearNPV have similar viral growth curve and DNAreplication curve, indicated that the insertion of exogenous neurotoxin gene RjAa17f was notaffect viral replication and propagation.4. We used transmission electron microscopy to compare RjAa17f-HearNPV polyhedral with those from HZ8-HearNPV. The results showed that the ODV and the shape ofRjAa17f-HearNPV are similar to HZ8-HearNPV.5. The bioassay results of3rdinstars larvae infected with recombinant virus and wildtype virus respectively, showed that the LD50and LD90of RjAa17f-HearNPVwere reduced by50.9%and19.6%compared to wild-type HearNPV. Besides, the ST50and ST90ofRjAa17f-HearNPV were shorter than wt-virus (13.4%and17.4%decreased, respectively).These results demonstrated that the new recombinant virus has a higher insecticidal activitythan wild-type HearNPV.In this study, the new recombinant virus RjAa17f-HearNPV has a higher virulence andfaster speed in killing bollworm compared with wild-type HearNPV, while there was nosignificant differences in BV production, DNA replication and the morphology of polyhedronbetween RjAa17f-HearNPV and wild-type HearNPV.