Construction Of SpltMNPV Recombinant Virus And Clone Of Iap Gene In SpltMNPV-JP-G_1-2

Spodoptera litura((Lepidoptera:Noctuidae)) is one of the most serious pest around the world.They are highly polyphagous,attacking 389 specieses,109 families of agricultural crops and greening plants.Recently,chemical pesticides are frequently used to control pest but has little effect.Considing to protect the environmental and to balance ecological environment,Spodoptera litura multicapsid nucleopolyhedrovirus (SpltMNPV) has been developed as a commercial biopesticide to control the crop inscets in the world.However,SpltMNPV also has definite restrictions in outdoors condition,a slowish speed on killing pest and more quantity and narrow host specificity limit to use in large scale.In order to solve the above problems,one of the most approaches to improve the insecticidal properties of SpltMNPV is by means of genetic engineering and molecular biology to lack a certain gene and express the gene of the insect-selective toxins.The sequence of SpltMNPV-JP-G_1-2 iap gene was cloned and analysed in this research.SpltMNPV-JP-G_1-2 and G_10-3 were recombined and a new recombinant virus was constrcucted by lacking of polh or egt gene and inserting insect-selective toxinBmk ITa1 and egfp under the pph promoter.The main conclusions are as followed:1.The iap gene was cloned from SpltMNPV-JP-G_1-2 strain genome.This gene that has been submitted to the NCBI GenBank and the accession numbers is FJ384668.It's 411bp long and potential to encode 136 amino acids,a polypeptide with predicted molecular weight of 15.67 kD.Analyzing nucleotide and amino acid sequence of gene iap in SpltMNPV Japanese strain(G_1-2)was done at the molecular biological level.2.Genetic engineering and molecular biology approaches have been developed to enhance the insecticidal properties of SpltMNPV-JP.Eight SpltMNPV recombinant transfer vectors have been constructed successfully and to lay the foundation for constructing and filtrating the insecticide of baculovirus.3.Genetic recombinant SpltMNPV has been carried out.The spli cells were cotransfected with ten transfer vectors and the wild SpltMNPV genome DNA.With homologous recombination and limiting dilution assay,three recombinant virus, SpltMNPV-ΔegtG_1-2-p10-egfp-pph-Bt,SpltMNPV-Δegt G_1-2-p10-egfp-pph-BmK ITa1 and SpltMNPV-Δegt G_10-3-pph-egfp,were gained.The results lay the foundation for the improvement and development of this virus insecticide and further study of biological control.