Mass Spectrometry Of SpltMNPV â…¡ BV And ODV's Protein Composion And Analysis Of Four ORFs

Spodoptera litura(Spli) is an omnivorous agricultural and forestry pests of Lepidoptera Noctuidae, mainly against cruciferous vegetables, beans, melons, onions and other crops. As the Spodoptera litura feeding miscellaneous, generations overlap, and strong resistance, it is difficult to control. Chemical synthesis of various pesticides are serious pollution of the environment and pest resistance to chemical synthetic pesticides growing circumstances, biological control is more and more important and urgent. From natural search and selection of new highly virulent virus strain is another important way to develop biological pesticides NPV. SpltMNPVâ…¡is a kind of reproduction rate and the highly virulent new virus variants. The whole genome sequence has been determined complete. However, the composition of the BV and ODV were unknown, to determine the composition of the BV and ODV, we analyse BV and ODV by mass spectrometry, the BV and ODV structutal proteins were determined, and the promoter activity analysis, transcription phase analysis and expression phase analysis have been done on the ORF6, ORF13, ORF57 and ORF146 genes,as well,the function of the ORF57 gene was identified. The major findings are as follows:1, The constitutive protein of SpltMNPVâ…¡BV and ODV were determined by mass spectrometry .The SpltMNPVâ…¡BV was composed of 42 different protein, compared with known homology baculovirus gene, 26 species of which gene function were identified; SpltMNPVâ…¡ODV was composed of 46 kinds of proteins, compared with known homology baculovirus gene, 24 species of which gene function were identified.2, SpltMNPVâ…¡ORF6, ORF13, ORF57 and ORF146 gene structure analysis have been done, ORF6 gene was 1509bp, encoding 502aa, predicted protein molecular weight of 55.0kDa, both early promoter motif CAGT and late promoter motif ATAAG were found, suggesting that the gene may be a gene that both express in early and late phase in the viral life cycle. ORF13 gene opening reading frame has a length of 333 bp, encoding 110 amino acids, predicted protein molecular weight of 12.2kD. A TATA box was found upstream of Start codon ATG, late promoter motif ATAAG was found upstream of Start codon ATG, there is no obvious early promoter motif. ORF57 gene is 1098bp, encoding 365aa, predicted protein molecular weight of 42.6kDa. A TATA box was found upstream of Start codon ATG, we did not find significant early and late promoter motifs upstream of the start codon. ORF146 gene covers 1383 bp open reading frame, encoding 460 amino acids, predicted protein molecular weight of 50.2kD. A TATA box and a late promoter motif ATAAG were found upstream of the gene start codon ATG.3, SpltMNPVâ…¡ORF6, ORF13, ORF57 and ORF146 gene promoter activity and transcription phase were analyzed. Both early and late gene transcription were found in the virus life cycle,. ORF13 and ORF146 genes which could be activated by SpltMNPVâ…¡trans-acting factors in the late stage of infection, which can increase the transcription level. The ORF6 gene can also be activated by SpltMNPVâ…¡trans-acting factors, but the transcription level in the late stage of infection was decreased significantly.4, Part of SpltMNPVâ…¡ORF6, ORF13 and ORF146 gene sequences were expressed by prokaryotic expression system,which products were purified and idenified by mass spectrometry,.The immunization of guinea pigs produced a polyclonal antibody., antibody titers were determined by ELISA, antibody titers of polyclonal antibodies which produced from the three gene expression product were high, more than in 1:2500.5, Using polyclonal antibodies as first antibody, products from SpltMNPVâ…¡ORF6, ORF13 and ORF146 gene expression in the host cell were analyzed. 0RF6 gene expressed in infected cells as early as 8 h after infection, 0RF146 gene expressed as early as 4 h after infection, these two genes were expressed both early and late phase, and their Promoter analysis and special libraries is consistent with the results. The ORF13 first infected cells in the event of 12h, and continued until the late phase,which shown ORF13 was the late gene. But the ORF13 promoter activity analysis and transcription phase analysis demonstrated that the gene was expressed both early and late phase. This estimate is due to the gene product, in the initial stage of protein synthesis was low which not enough to detect.6, We cloned and expressed the SpltMNPVâ…¡ORF57 gene complete ORF, the expression product was purified and identified using mass spectrometry. With the isotope method and specific detection of trace phosphorus (malachite green reagent method) to determine the ORF57 gene encoding a protein has both polynucleotide kinase function and 3 'phosphatase function. ORF57 gene is a bifunctional enzyme gene.