Analysis Of Spodoptera Litura Multicapsid Nucleopolyhedrovirus Polyhedrin And Gp41 Gene And Construction Of Transfer Vector Containing GFP

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 ,the polyhedrin and gp41 gene was cloned and the novel baculovirus transfer vector containing gfp was constructed.The main conclusions are as followed:(1)The polyhedrin gene of three strains (A, B and C) of SpltMNPV from Japan is composed of 747 nucleic acids, which codes a peptide of 249 amino acids. These genes sequences have been submitted to the NCBI GenBank and the accession numbers are AY552474(A type), AY549963(B type), AY549964 (C type), respectively.(2)The gp41 gene was cloned from SpltMNPV Japan-C3 strain genome. This gene, which has been submitted to the NCBI GenBank and the accession numbers is AY605063, is 993bp long and potential to encode a polypeptide with predicted molecular mass of 36.9 kD. By costructing E. coli expression vector, gp41 gene was highly expressed with the induction of IPTG.(3) To obtain the novel baculovirus transfer vector pSplt-gfp, the fragment of gfpgene was inserted into this vector between the 5' end and 3' end-flanking fragments of SpltMNPV ph gene tandem linked into pUC18. The spli cells were cotransfected with pSplt-gfp and the wild SpltMNPV genome DNA. The recombinant virus containing gfp was selected with the limited dilution method. The fluorescence can be observed in the spli cells and the 3rd instar larvae after 24 and 48 hours by infection of the recombinant virus, respectively. The result showed that the recombinant virus was obtained successfully.