Study On Biocontrol Function Of Antagonistic Protein From Bacillus Atrophaeus SF1

In this paper,the fungal cell wall degrading enzymes produced by a biocontrol Bacillus atrophicus SF1 isolated in the previous laboratory were studied.Three protein purifications were obtained by heterologous expression.The antagonistic mechanism of three proteins against pathogenic bacteria was analyzed at macro and micro levels.The main contents and results of this paper are as follows:1.The serine protease gene aprX,?-1,3-glucanase gene bglS and chitinase gene chiA were cloned from SF1,the full length of which were 1332 bp,1086 BP and 1781 bp,respectively.The recombinant plasmids pEASY~?-Blunt-E1-aprX,pEASY~?-Blunt-E1-bglS,pEASY~?-Blunt-E1-chiA were successfully constructed and transformed into E.coli.PCR Identification,SDS-PAGE detection and western blot analysis showed that the recombinant strains A2-6,B2-4 and C1-4 were successfully constructed The activity of serine protease,beta-1,3-glucanase and chitinase reached 5.77 U/mL,1.70 U/mL and 7.93 U/mL respectively by optimizing the induction conditions of recombinant strains.2.The expressed proteins of the recombinant strain were purified by salting out,dialysis and nickel column affinity chromatography.Three kinds of antagonistic proteins were obtained respectively.SDS-PAGE analysis showed that they had reached electrophoretic purity.3.The average bacteriostasis rates of the three proteins were 30.8%,31.2% and 32.5%respectively.Optical microscopy,confocal laser scanning microscopy(LSCM)and scanning electron microscopy(SEM)showed that the three proteins could destroy the mycelial cell wall and inhibit the growth of pathogenic bacteria,but the destruction sites and modes of each enzyme were different4.The average bacteriostasis rates of the three proteins were 32.4%,34.8% and 36.2%respectively.Optical microscopy,confocal laser scanning microscopy(LSCM)and scanning electron microscopy(SEM)showed that the inhibition rate was significantly higher than that of single enzyme?5.The average bacteriostasis rate of pathogenic bacteria treated with three proteins was 37.8%.Optical microscopy,confocal laser scanning microscopy(LSCM)and scanning electron microscopy(SEM)showed that the synergistic effect of the three proteins was significantly higher than that of single enzyme.