Analysis Transcriptomal Features Of WT And SCEV Of Bacillus Amyloliquefaciens PEBA20 And Construction Of CheA Gene Mutants

Bacillus amyloliquefaciens PEBA20 was an endophytic bacterium isolated from from plants,which has strong antimicrobial activity against many plant pathogenic fungi and bacteria.SCEV is a stably inherited phenotypic heterogeneity mutant obtained by continuous subculture of this laboratory in a single environment.In this study,the transcriptomes of SCEV and WT were sequenced and analyzed to differences in gene transcription levels.We also got the mutant strain ?che A using the method of homologous recombination which is unable to form complicated biofilm.The ?che A lost the swarming motility and chemotaxis.The main findings are as follows:About 2.4 G of clean data was obtained by RNA-seq.There were 3,825 and 3,757 expressed genes in WT and SCEV,respectively.There are 2,339 genes with differentially expressed.There is a large of difference expressed genes in TCA cycle pathways which is important pathways of carbon metabolism by the KEGG enrichment analysis.It may lead to differential metabolism of certain intermediates.Spo0A,kin A,sig D,and sig F,the key regulators of sporulation initiation,were significantly up-regulated,although spore coat protein and spore protein-related genes were significantly down-regulated in the sporulation-related pathways.From this we speculated that the spore formation of SCEV may be later than WT.The NRPS/PKS clusters are all up-regulated.There were 34 differentially expressed genes were up-regulated in the chemotaxis and flagellar assembly pathways.But the chemotaxis of WT was stronger than SCEV at 0.3% LB.The quorum sensing regulatory pathway contained 22 up-regulated genes and 12 down-regulated genes.At 0.7% LB,WT exhibited typical population locomotor characteristics while SCEV completely lost this exercise capacity.Although it has not been concluded that SCEV has a loss of motility in the population,the expression level of some quorum sensing genes is still higher than that of WT.We suspect that the quorum sensing may be different in liquid media.The che A gene mutant and che B gene mutation of PEBA20 were constructed by homologous recombination technology.The homologous recombinant fragments che A and che B were amplified by PCR from DNA of PEBA20.And the kanamycin resistance gene was amplified from p EASY-T1.The che AF,che AR and kanamycin resistance were ligated into the p MUTIN4 plasmid to get p MUTIN4-che A.And this circumstance is the same as p MUTIN4-che B.The resulting plasmid was used to transform the PEBA20 wild type strain by electroporation.The ?che A mutant could not form robust and complicated biofilm,which is flat and slick on solid media.The colony collapsed and the edge slightly raised middle part collapsed,and the edge slightly raised with translucent.In air-liquid interface,the ?che A mutant was unable to form a floating biofilm.At 0.7% LB and 0.3% LB,che A mutants lost swarming motility and chemotaxis.