Investigation Of SpltMNPV-JP-B Per Os Infectivity Genes And Molecule Identification Of SpltMNPV-JP-C1

Spodoptera litura (Lepidoptera:Noctuidae) multicapsid nucleopolyhedrovirus, SpltMNPV,which,in china, was firstly isolated from cadavers of Spodoptera litura in Guangzhou, belongs to the subgroup A of Nucleopolyhedrovirus.Spodoptera litura, the host of the virus is a kind of polyphagous insect which is harmful to economic crop and vegetables such as alimentary crop,cotton,oil plants and so on. And it is also one of the primary insects pests around the world. Present, SpltMNPV is considered as the most effective insecticide against the insect, therefore, the researches on it are becoming more and more popular. Those years, some of the studies about the virus have been greatly improved, such as the sequence determination, the analysis of its gene structure, its function and regulation of expression. Those progresses, especially the analysis of some important gene structures of it, have contributed to the selecting of the strain with stronger virulence, the improving and developing of this viral insecticide, and the constructing of the baculovirus expression vector.In order to research on Spli-SpltMNPV system, there of per os infectivity gene was cloned and the novel baculovirus transfer vector containing egfp was constructed.The main conclusions are as followed:(1)The per os infectivity factors of B strains of SpltMNPV from Japan are composed of 1581,1278 and 603 nucleic acids, which codes a peptide of 527,426 and 201 amino acids.These genes sequences have been submitted to the NCBI GenBank and the accession numbers are FJ384665(pif) FJ384666(pif-2) FJ384667(pif-3).Proteins, second structure and the molecule phylogeny are analyzed based on these sequences of amino acids.(2)The pif-2 gene was highly expressed in the E.coli and analyzed its phase of transcription.The pif-2 gene was cloned into pET32a(+) and induced in E.coli by IPTG. A specific band was detected at 69 kD,identified by Western Blotting.Collect the cells at the different time,which were infected by the SpltMNPV BV. The phase of transcription was analyzed by the method of RT-PCR.The exist of transcript of pif-2 was found from 3h to 72h.(3)The recombinant virus marked by egfp was constructed, pif-2 was inactive. The fragment of ph-egfp was inserted into this vector between the 5'end and 3'end-flanking fragments of SpltMNPV pif-2 gene tandem linked into pUC19. The spli cells were cotransfected with pSplt-â–³pif-2-egfp and the wild SpltMNPV genome DNA. The recombinant virus containing egfp was selected with the limited dilution method.(4) SpltMNPV-JP-C1 was invested at the level of molecule.The pure genome of C1 was cut by enzyme and constructed the genomic library.8 random clones was sequenced.The sequences were compared with the G2 and C3.From the result we can found that compared with the C3,ll the clones were hightly homologous with the G2.