Construction And Characterization Of A Recombinant Nucleopolyhedrovirus Which Contains Enhancin From Granulovirus

Baculoviruses are highly selective pathogens for several pest insects. Currently anumber of baculoviruses is used for the protection of crops, vegetables, forests andpastures. However, wider application of baculoviruses has been limited by their slowspeed of action and narrow host specificity. Several genetic approaches are known toimprove the insecticidal properties of baculoviruses. Here, we constructed arecombinant baculovirus contained Agrotis segeturn granulovirus enhancin which mightenhance the oral infectivity of baculoviruses in various lepisopteran insects; we alsoconstruct a DNA-based vector which could generate long double-stand RNA whichwere able to suppress the expression of a P450 gene in Helicoverpa armigera midgut.Chapter 1 is a general introduction composing of four parts. Partâ… is mainly aboutbiology, molecular biology and application of baculoviruses. Partâ…¡focuses on theproperties and application of enhancin from baculoviruses. In partâ…¢, cytochrome P450from insect and its molecular mechanisms of resistance to chemical insecticides arereviewed. In partâ…£, the molecular mechanisms and biological function of RNAi arementioned.A number of enhancing genes which encoded by baculoviruses has been identified.Enhancin, a metalloprotease, might significantly increase the infectivity ofnucleopolyhedrovirus (NPV) and Bacullis thuringiensis. In Chapter 2, the enhancingene from Agrotis segeturn granulovirus (AgseGV) was cloned, sequenced andbioinformatics analyzed. The AgseGV enhancin encodes an 1004 amino acid proteinwith a predicted molecular mass of 115 kDa and contains a conserved zinc-bingingdomain common to metalloproteases. The enhancin gene of AgseGV showsapproximately 25% amino acid identity with other baculovirus enhancins. By usingBac-to-Bac system, a recombinant H. armigera single-nucleocapsidNPV (HearSNPV) expressing the AgseGV enhancin gene trader control of p10 promoter of HearSNPV,nominated HearBacPolhEn, has been constructed. Meanwhile, a recombinantHearSNPV where the polyhedron gene was introduced back, nominated HearBacPolh,was built as a control. In laboratory bioassays, median lethal dose (LD₅₀) ofHearBacPolhEn against 3rd instar of H. armigera larvae was not significantly differentfrom wild-type HearSNPV and HearBacpolh.Double-stranded RNA-mediated interference (RNAi) has recently emerged as apowerful reverse genetics tool to silence gene expression in multiple organisms,including plants, nematodes and insects. In Chapter 3, a DNA vector,pFastBacCYP-EGFP, which contains duplicated 490bp fragments of P450 gene derivedfrom midgut of H. armigera larvae at reversed directions has been constructed. Thisvector would be used to construct a HearNPV Bacmid which could supress theexpression of P450 gene in vivo by synthesizing long hairpin dsRNA.