| With the vigorous development of green agriculture in China,the use of biological fertilizers in agriculture is becoming more and more extensive.However,the main bottleneck restricting the development of Chinese bio-fertilizer industry is the instability of field application effects.When the fertilizer functional bacteria have been applied into the soil,they faced the discrimination and competition of local microorganisms.It is difficult to make bacteria play a stable role in microbial fertilizer.Kin recognition is the identification between related individuals,which can guide and stabilize the cooperative relationship of the group by secreting and feeling the unique signals between them.Kin recognition is the basis for microorganisms to distinguish between friends and enemies and then further carry out social behaviors.Bacillus strains are commonly used in microbial fertilizers.It is great significance to study the molecular mechanism of the kin recognition and kin discrimination of Bacillus to further understand the mechanism of Bacillus microbial fertilizer.In this study,we constructed a mutant library of Bacillus amyloliquefaciens SQR9 and used molecular biology methods to explore the molecular mechanism of kin recognition and discrimination of SQR9.Our conclusion are as follows.1.By using the existing mutants of B.amyloliquefaciens SQR9 to conduct the swarm meeting assay with the wild strain SQR9 and B.amyloliquefaciens FZB42,we found global regulation genes(spoOA and sinR),flagellar protein synthesis genes fliD potentially involved in kin recognition and discrimination system in B.amyloliquefaciens SQR9.2.Using the transposon TnYLB-1 to construct a mutant library of B.amyloliquefaciens SQR9,and selected mutants whose phenotypes of swarming changed with wide type SQR9.The mutation sites analysis of the selected mutants revealed that in B.amyloliquefaciens SQR9 global regulation genes degU,flagellar protein synthesis genes flhO,spore germination genes(gerAA and gerAB),antibiotic synthesis gene srfAA and other genes(patB and yugK)potentially involved in kin recognition and discrimination system.Using gene knockout technology,the flagella synthesis gene flhO was knocked out,which further verified that the flagellin protein FlhO participated in the kin recognition process of strain SQR9.3.We constructed the protein expression system in E.coli,then successfully expressed and purified the cell surface receptor protein(9-DltA)and flagellum protein(9-FlhO,9-FliD)from SQR9,and flagellum protein(42-FliD)from FZB42.Through using the technology of Biolayer Interferometry(BLI),we tested the affinity between those proteins.The results show that the cell expression receptor protein 9-DltA and flagellin 9-FlhO,9-FliD,42-FliD have a strong affinity;flagellin synthesis protein FliD can bind itself.This phenomenon indicates that flagellin proteins FliD and FlhO are involved in the kin recognition process of strain SQR9.4.The mixed biofilm of SQR9-rfp and FZB42-gfp was observed by Stereo Fluorescence Microscope,and it was found that only SQR9 could form biofilm.The biofilm formation of the non-kin(The swarm interaction phenotype is boundary)strain mixed combination(SQR9&FZB42)is slower and less wrinkled than single bacteria,the ability to antagonize FOC(Fusarium.oxysporum f.sp.Cucumerinum)and promote the growth of cucumber are also weaker than single bacteria.The ability of the kin(The swarm interaction phenotype is merge)bacteria combination(FZB42&mutant AspoOA)to antagonize FOC and the promoting effect on cucumber growth were stronger than single bacteria but its biofilm formation ability is only stronger than the single bacterial mutant AspoOA and weaker than the single bacterial FZB42.Therefore,using the kin recognition theory to analyze the ecological relationship between functional microorganisms helps to build a functionally stable synthetic flora and improve the agricultural application effect of plant probiotic Bacillus. |
PDF Full Text Request