Bombyx Mori L. Nucleopolyhedrovirus (BmNPV) And Proteome Analysis Of Silkworm Proteins Related To BmNPV Infection Resistance

BmNPV(Bombyx mori L.nucleopolyhedrovirus) is a key pathogeny to silkworm with highly infective.And the reciprocity between virus and host is an important issue which was paid many attentions by researchers all the time.In this paper,we stuied the protein composition of baculovirus ODV on proteomic level,and the related proteins of resistance to infection in host through constructed near-isogenic line,wish to find some proteins which are related to resistance or susceptibility of silkworm.We also screened the molecular makers that related to resistance on DNA level.In addition,we evaluated the constructed near-isogenic line from different points of view,and analyzed the method that identified mass spectrometry results using EST database.The results are shown as follows.We obtained the occlusion-derived virus(ODV) virion of Bombyx mori nucleopolyhedrovirus(BmNPV) by sucrose density gradient centrifugation,and used techniques of SDS-PAGE,2-DE and MS to identify the proteins present within or associated with ODV virion for the first time.19 proteins,including 15 major proteins of the virion encoded by BmNPV,Polyhedrin,P78/83,Orf 40,Orf54,GP41/P40,P95,VP39,P25,ODV-EC43,Orf94,Orf109, P74,P49,ODV-E56 and PTP,respectively,and 4 proteins of silkworm,heat shock cognate protein,heat shock protein hsp21.4,glutathione S-transferase 2 and cyclophilin A,were identified with confidence.Two BmNPV proteins, orf40 and PTP,those didn't detected as components of ODV previously,were identified in this study.And we propose a hypothesis that those host-derived proteins maybe important for ODV capsid assembly and maturation.In our study,two silkworm strains,namely NB which is highly resistant to NPV and 306 which is highly susceptible to NPV,and their near-isogenic line BCg,were used.500 RAPD random primers were used to screen molecular markers in DNA mixtures of these strains respectively.Three molecular markers namely OPF-07,OPX-06 and OPX-18 were found linked to major gene resistant to NPV.One molecular marker,OPF-07₂₀₂₃,was validated in some backcrossing generations.We cloned,sequenced and analyzed this special fragment appearing only in the resistant strains.And with bioinformatic analysis, we speculate there is an insertion of DNA fragment,and a retrotransposon gene of the Z chromosome in silkworm was found.In addition,a qualitative method was introduced,which can judge the presentation or not of PCR production by staining directly.To facilitate identification of the proteins in NB that are employed in this prominent anti-virus response,we constructed a near-isogenic silkworm line (NIL) by introgression,which is resistant to BmNPV but is almost genetically identical to the parental strain 306 that is BmNPV-susceptible.In this paper, proteome maps and differences of protein patterns of the midgut,hemolymph and fat body tissues of the highly resistant,highly susceptible and near-isogenic silkworm strains were investigated by two-dimensional gel electrophoresis (2-DE) and matrix-assisted laser desorption/ionization -time of flight (MALDI-TOF) mass spectrometry.A comparison between the different protein expressions of these three silkworm strains led us identified five differently expressed proteins in midgut.Among these protein,proteins 1 and 2 were expressed in NB and BC9 higher than in 306,but proteins 3,4 and 5 were expressed in 306 higher than in others,these protein may involved in the resistance or susceptibility of BmNPV.In hemolymph,there are two differentially expressed proteins,beta-N-acetylglucosaminidase 2 and aminoacylase.We speculate that the excessive expression ofβ-N-acetylglucosaminidase in hemolymph of the resistant silkworm can probably disturb the N-linked glycans of GP64 protein on the cell membrane which is an essential process for initiating second infections,and thus reduce the reproduction of infectious viruses.4 proteins were successfully identified in fat body,and 2 of which are possibly related to energy metabolism.Protein 1 was identified similar to phosphoglycerate kinase,Protein 2 was identified with a silkworm arginine kinase.The third one may have similar function as integrase,and the forth one is completely novel.For evaluating the accuracy of Near-isogenic line that we construct on molecular level,especially the high resemblance of heredity background between the constructed near-isogenic strain and the recurrent parent.We summarized the results of RAPD(Random Amplified Polymorphic DNA) and 2-DE(Two-dimensional polyacrylamide gel electrophoresis),and compared the resemblance between the near-isogenic and parent strains on DNA and protein levels.The results shows,the resemblance level is less than the theoretical value, but still valuable for research of resistance,and for references to other differential genes of silkworm.We collected silkworm EST sequences,each EST sequence was translated in six frames,and the database was transformed to the format which can be recognized by Mascot program.We analyzed the mass spectrometry data through Mascot program,and an example was given to illuminate that this method can analysis novel peptides,which are missing from current protein sequence databases.The result shows this method has some role, and there is reference value to proteins of some species that haven't enough pepetide data.