Analysis Of Differential Expressed Sporulation Protein Of Beauveria Bassiana Induced By Cicada Exuviae

Beauveria bassiana is Broad-Spectrum Entomopathogenic Fungi,Compared with other microbial pathogens of insect,entomopathogenic fungi can invade host actively,and can infect pest circularly.Moreover, host can rarely developresistance to entomopathogneic fungi.Therefore,mycoinsecticides are being considered as alternative and supplement to chemical pesticides.However,the acceptance of fungal products for pest control is very limited.What make limited acceptance of fungal insecticides is that they are not as fast acting as chemical products,sensitive to adverse environment,lose their effectiveness more rapidly.Therefore, entomoapathogenic fungal strain improvement is necessary.With the elucidation of molecular basis of fungal entomopathogenicity,gene engineering might overcome these drawbacks and could lead to an ideal biocontrol microorganism.The studies about molecular nosogenesis of entomopathogenic fungus were from virulence related proteins and diseases geneclonging mostly,which analysis little from proteomics.This research using Beauveria bassiana induced by cicada exuviae,Analysis of differential expressed protein,try to find virulence related proteins.We construct the two dimensional electrophoresis method according to the material Two-dimensional gel electrophoresis which is important in proteomics was improved.Some important steps,such as sample prepare,isoelectric focusing,balance,dyeing were improved.For sample preparation,the funge sample was grounded in liquid nitrogen and then homogenized in the lysis buffer(0.5M Tris-HCl pH 8.3,2%(v/v) Nonidet P-40,20 mM MgCl₂,2%(v/v)β-mercaptoethanol,1 mM PMSF),then centrifuged at 16 000g for 20 minute at 4℃,was carefully recovered,avoiding the unhomogenized material at the bottom of the centrifuge tube.For the preparative gel staining,we tried the "blue silver" method to improve the resolution of the two dimensional electrophoresis map.The two-dimensional gel electrophoresis(2-DE) profiles were performed using statistical analysis software(PDQuest),which out of more than 750 protein spots reproducibly detected and analyzed,38 spots showed significant changes under induced,18 proteins were up-regulated,20 proteins down-regulated.There were 4 obvious spots of specific expression,No.2,No,8 and No.9 were down-regulated,No.22 was up-regulated.This showed that the expressed regulation system was changed complexly under the inducing of cicada exuviae.Using two dimensional electrophoresis and Peptide Mass Fingerprint(PMF) analysis,which the differential expressed protein:No.26 was sporulation protein and it was up-regulated.The sporulation protein may be involved in the induce of Beauveria bassiana,which effected the time of mitosis and meiosis in sporulation.No.19 and No.39 were uracil-DNA glycosylase and up-regulated(DNA replication, recombination,and repair),and it may be involved in sporulation.Meanwhile it also analysis other proteins in this study,which may be have direct and indirect effect in sporulation.