Purification,identification And Antifungal Mechanism Analysis Of Extracellular Protein Produced By Bacillus Atrophaeus XW2

Poplar anthracnose caused by Colletotrichum gloeosporioides can lead to tree weakness,leading to serious restriction on the growth,development and ecological functions of poplar trees,and caused a series of economic and ecological loss.At present,there are a lot of researches related to the epidemiology and management of poplar anthracnose disease.However,no efficient control method has been concluded.Therefore,to discover and clarify antifungal compounds against Colletotrichum gloeosporioides growth and appressorium formation,and study on its gene regulatory network is of great importance giving a theoretical basis on developing new fungicides and control poplar anthracnose.In this study,antifungal protein produced by Bacillus atrophaeus XW2,one endophytic bacteria isolated from healthy poplar leaves,were purified and identified,and the antifungal mechanism against C.gloeosporioides was analyzed.Main results are as follows（1）In this study,one antifungal protein,which can suppress appressorium formation was obtained from the fermentation broth of strain XW2.According to salting-out,column chromatography and polyacrylamide gel electrophoresis purification methods,one electrophoretic pure antifungal protein was obtained,of which the molecular weight is about 20 kDa and pI 4.89 This antifungal protein was named as BaAFP（2）We characterized the category of BaAFP is one 2-Cys peroxiredoxin.According to liquid chromatography-tandem mass chromatography（LC-MS/MS）method,11 specifically matched peptide sequences were obtained after identification and comparison with UniProt database.The nucleic acid sequence of BaAFP was cloned from the genome of strain XW2.The sequence was determined by bioinformatics analysis.As a result,a 543 bp ORF that codes a 180 amino acid sequence was analyzed.The sequence contains a conserved domain of PRX_Typ2cys.The molecular formula is C₉₁₂H₁₄₀₉N₂₄₁O₂₈₁S₈.（3）The pETM30-BaAFP expression vector was constructed,and the biologically active recombinant BaAFP was expressed by BL21（DE3）E.coli.The optimal Ni+column purification eluate was determined to have an imidazole concentration of 150 mM.（4）We ensured BaAFP have efficient effect on appressorium formation,germ tube formation,hypha branching,intracellular lipid distribution,septum formation,and ROS gathering.Cloned BaAFP sequence was inserted and expressed in Escherichia coli prokaryotic expression system.After exposed to BaAFP,spores of C.gloeosporioides germinated normally but appressorium formation was suppressed.During 3-8h,lipid compounds were increased in germinated spores and germ tubes but didn’t decrease with germ tube elongation.ROS in fungi cells didn’t gather on tips of germ tubes too.After exposed to BaAFP for 12-20h,mycelial became bold and branches increased.At this point,BaAFP over 0.1 mg/mL completely suppressed the formation of appressorium,well less than 0.1 mg/mL BaAFP lead to a few appressorium formatting.After 40h cultivation under BaAFP,a few of appressorium increased but most are malformed ones,which damaged after PEG4000 treatment.Spores exposed to BaAFP can’t normally infect onion epidermis,showing decreased infection capacity.On poplar leaves,2 mg/mL BaAFP treated position suppressed the infection of C.gloeosporioides,and ROS gathered at the same place of treatment position.