| Paenibacillus polymyxa SC2 was screened from the rhizosphere of pepper,which could improve the vegetative environment of plants and secrete antimicrobial substances to inhibit the growth of pathogenic microorganisms and growth hormone and other active substances needed for plant growth.As a kind of rapid and efficient mutagenesis technology,atmospheric and room temperature plasma has been widely used in prokaryotes(bacteria,actinomycetes)and eukaryotes(mold,yeast,microalgae)because of its high mutation rate,simple operation,high safety,rapid mutagenesis and so on.Using atmospheric and room temperature plasma to treat the Paenibacillus polymyxa SC2 to obtain different mutants.The mutagenic conditions were as follows: 120 W,10slm,25 s and mortality rate 92%.Using Escherichia coli as the indicator bacteria and combineing with the plate confrontation method and the oxford cup method,a mutant strain named PM3 that loss the ability to product polymin was obtained and 5 mutant strains which enthance the ability to product polymin.The diameter of the inhibition zone of mutant C520,F373,I31,I135 and I300 were respectively increased 2.0mm,2.54 mm,2.32 mm,2.3mm and 1.95 mm.In addition,two extracellular polysaccharide mutants were obtained.The production of exopoly saccharides of mutant EM1 was significantly reduced and became transparent.The production of exopoly saccharides of mutant EM2 increased significantly and the exogenous polysaccharide has fluidity.Each mutant showed difference in colony morphology,spore formation,antagonize fungi,motility and growth ability.Genomic resequencing of each mutant was found that a large number deletion and insertion variation of large fragments of mutations.There are two SNP,two Small InDel and eighty-five SV including seventy-four DEL and one INS of mutant PM3.A total of 903 differential genes(log2FC>2)were found by transcriptome sequencing of mutant PM3 comparing with SC2 which includes 416 genes up-regulated and 487 genes down-regulated.There are no variation of the gene cluster of polymyxin including pmxA,pmxB,pmxC,pmxD and pmxE and the genes up-regulated at the transcriptional level except gene pmxD.This phenomenon indicated that the synthetic gene of polymyxin is still intact and transcription is normal,but the synthesis or activity of polymyxin is affected.The bioinformatics analysis of transcriptome revealed that the gene ectB was significantly down-regulated(log2FC=-11.61),which was necessary for the synthesis of the 2,4-diaminobutyric acid of the polymyxin precursor.In addition,the genes related to fatty acid metabolic synthesis including acpS,accB,fabG5,fabH3,fabD5,fabD3,fabG13,bio B1,bioB3 were down-regulated.The synthesis of fatty acids has been affected.Because polymyxin is the main substance of Paenibacillus polymyxa to inhibits bacteria composed of amino acid peptide ring and fatty acid composition.So the down-regulation of synthesis-related genes of 2,4-diaminobutyric acid and fatty acid may affect the synthesis of polymyxin,resulting in the mutant PM3 lossing the ability to inhibite bacterial.By adding exogenous L-2,4-diaminobutyric acid dihydrochloride,the mutant PM3 produced a bacteriostatic ring and restored the ability to synthesize polymyxin.Therefore,the deficiency of 2,4-diaminobutyric acid is the main cause of PM3 to unproduce polymyxin.In conclusion,different types of functional mutants were obtained of Paenibacillus polymyxa SC2 by atmospheric and room temperature plasma.The synthesis mechanism of secondary metabolites was studied by whole genome sequencing and transcriptome sequencing.The results showed that ARTP has certain potential for the breeding of Paenibacillus polymyxa and the studying of the mechanism of metabolic synthesis.And it may be a new method for studying on the functional genomics of SC2. |
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