| The abuse of antibiotics has led to an increasing number of multi-drug resistant strains worldwide,which has had a huge impact on agriculture,animal husbandry,aquaculture,the environment and human health.It is extremely urgent to find high-efficiency,low-toxicity,green and safe antibiotic substitutes.A variety of secondary metabolites produced by Bacillus are considered to be an important source of natural antibacterial drugs.Biosynthetic gene clusters of these metabolites can be obtained through genome sequencing and bioinformatics analysis.This study is conducted on the basis of a strain of Lysinibacillus boronitolerans,which was isolated from the bulk solf of tomato and rice rotation system and showing antagonistic activity against many bacterial and fungal pathogens.This study aimed to identify the genes encoding the antibacterial substance Bacteriocin and to test its growthpromoting effect.The results of this stuy provided theoretical basis and technical support for the further development and application of L.boronitolerans.The main results obtained are as follows:1.The genome of L.boronitolerans was sequenced and assembled.Results showed the complete genome sequence of the strain is characterized by a circular chromosome of 4.68 Mb,with a 37.27% G+C content,containing 4885 protein-coding genes,the total length of which was 4,174,044 bp,with the average length of 854 bp.Besides,7 Pseudogenes genes,34 r RNA genes,107 t RNA genes,94 other nc RNA genes,22 CRISPR sequences,18 gene islands,and 6 prophages were predicted.2.Comparative Genomic Analysis was conducted between L.boronitolerans and two strains of Bacillus,i.e.,Bacillus amyloliquefaciens and Bacillus subtilis subsp.subtilis str.168.The results showed that the genome of L.boronitolerans contains the largest number of unique genes(2614),most of which were related to transport,two-component system,synthesis and metabolism of amino acid,and carbon metabolism according to GO and KEGG analysis.The gene cluster responsible for the biosynthesis of the secondary metabolites were predicted in these three strains using anti SMASH,and a gene cluster encoding a potential bacteriocin antibacterial substance was selected.3.The function of the selected gene cluster involved in the bacteriocin biosynthesis were predicted using bioinformatics methods;According to the inhibitory effect on Bacillus pumilus,recombinant E.coli BL21 stains expressing the gene clusters(GE00661-GE00670)and(GE00664-GE00669)were successfully obtained;GE00667 and GE00669 are identified as the key genes for the synthesis of bioactive Bacteriocin using the method of amino aciddirected mutation;Finally,in view of the results of Liquid Chromatography Tandem Mass Spectrometry(LC-MS/MS)analysis,it was identified that the protein sequence of Bacteriocin has an identitiy of 24.1% with the protein sequence of Piricyclamide 7005 E4 Pir E4 of cyanobacteria.4.The heterologously expressed bacteriocin was extracted and enriched using ammonium sulfate with different saturations,and the results showed that ammonium sulfate with 70% saturation can obtain the extract with the most siginificant inhibitory effect.The antibacterial spectrum of bacteriocin was tested and the results showed that the growth of Bacillus pumilus、Bacillus velezensis、Pseudomonas syringae pv.tomato DC3000 and Xanthomonas axonopodis pv.manihotis were inhibited by bacteriocins,while the inhibitory effect on Staphylococcus、Bacillus subtilis、Neoscytalidium dimidiatum、Botryodiplodia theobromae、Magnaporthe oryzae and Rhizoctonia solani.was not observed. |
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