Establishment Of Genetic Transformation System Of Bacillus Velezensis And Identification Of Antifungal Substances

Bacillus velezensis,a new species of the genus Bacillus,was first discovered by Spanish scholars in a river named"Vélez"in 2005.It was reported that B.velezensis could produce a variety of metabolites including antifungal proteins,lipopeptides and polyketides to inhibit the growth of multiple pathogenic fungi.Thereby B.velezensis has been widely used as a biocontrol agent for crop fungal diseases.In this study,B.velezensis NSZ-YBGJ001 with excellent biocontrol performance was researched,and studies were carried out as follows.The potential antifungal protein was determined by intercepting the fermentation broth and fungistatic experiments,and the proteins were assayed by mass spectrometry.The lipopeptides with anti-fungal effects were identified via methanol extraction as well as HPLC-MS analysis.The genetic transformation system of B.velezensis NSZ-YBGJ001 was established by plasmid construction and electrotransformation condition optimization.In addition,an endogenous plasmid p BV01 in B.velezensis NSZ-YBGJ001 was isolated and be cured by plasmid incompatibility.The specific research content is as follows:Bacillus can produce small molecule peptides,which has obvious inhibitory effect on pathogenic fungi.In this study,molecular interception and Tricine-SDS-PAGE were used to isolate the peptides in the fermentation broth,and two potential antifungal peptides B and D were identified by protein profile.The coding sequences of these two peptides were amplified from B.velezensis NSZ-YBGJ001chromosome,and were expressed in E.coli.However,the recombinant proteins did not present antifungal effects on the test strains.Hence,antifungal substance in fermentation broth of B.velezensis NSZ-YBGJ001 was other substancesIt was reported that some lipopeptides with antifungal effects on many fungi were synthesized in Bacillus,thereby some studies on lipopeptide were performed.Potential lipopeptides were extracted from the fermentation broth of B.velezensis NSZ-YBGJ001,and the lipopeptides presented significant antifungal activities against pathogenic fungi including Rhizoctonia solani.Subsequently,the lipopeptides was analyzed by HPLC-MS,which included C13～C15 Surfactin of the Surfactin family C15-Bacillomycin D,and C13-Bacillomycin D analogues in the Iturin family.Finally,lipopeptides among the metabolites of B.velezensis NSZ-YBGJ001 preliminarily determined as the significant antifugal substances.The electrotransformation system of B.velezensis NSZ-YBGJ001 was then established to manipulate velezensis NSZ-YBGJ001 in molecular level for enhanced production of lipopeptides,further promoting its biological control effect.With the optimal transformation parameters,the new constructed new plasmid p UBC01 achieved a high transformation efficiency,up to 1.2×10~3 CFU/μg.The genetic transformation system provides a strong guarantee for the genetic modification of B.velezensis NSZ-YBGJ001.In addition,the endogenous plasmid p BV01 of B.velezensis NSZ-YBGJ001 was extracted,and the complete map of this plasmid was obtained by DNA sequencing.Through comparison and bioinformatics analysis,the CDS of several genes such as rep A encoding plasmid replication protein,rap A encoding aspartase A and rac A encoding DNA binding protein were predicted.This research provides a theoretical and practical basis for the genetic modification and expansion of B.velezensis NSZ-YBGJ001’s application in biological control.