| Bacillus is a genus of gram-positive, rod-shaped bacteria and a member of the division Firmicutes. It has been widely used in industry, medicine and agricultural field because of its powerful secretion system and production of remarkably large amounts of secondary metabolites such as antibiotics, phytase and extracellular proteases. Previously, a biocontrol agent named as "MaiFengning", containing Bacillus amyloliquefaciens B3strain was developed by our lab. Polyphasic taxonomy method, suppression subtractive hybridization, mass spectrometry and whole genome sequencing technology were performed in this study. The main contents include:1) molecular identification of antagonistic strain B3;2) the mechanism of biocontrol and plant growth promotion of strain B3;3) sequenced and characterize endogenous cryptic plasmid pBSG3;4) construction of engineered strain FZBHarpin which combining Harpin elicitor and nonribosomal peptide antibiotics together. The main results were summarized as follows:1Previously, screening of antagonistic isolates against different plant pathogens from plant rhizosphere soil was performed by our lab members.11antagonistic strains were obtained and classified as Bacillus genus with morphological tests. In this study, the11Bacillus antagonistic isolates and the reference strains were characterized using BOX-PCR genomic fingerprinting with primers BOXAIR, sequence analysis of16S rRNA and gyrB gene fragment, and FAME (Fatty acid methyl esters) tests. There was a high degree of diversity, both phenotypic and genotypic among the isolates of Bacillus.11strains were identified as members of species:Bacillus pumilus (1), Bacillus cereus group (2), Bacillus amyloliquefaciens (4), Bacillus atrophaeus (1), and Bacillus subtilis (3). Our results demonstrate the usefulness of gyrB sequence and BOX-PCR genomic fingerprinting for characterizing the Bacillus genus. In addition, MALDI-TOF-MS was performed for the detection of nonribosomal peptide antibiotics produced by11isolates. The results showed that11strains are able to produce at least one or more lipopeptide and polyketide compounds (such as Fengycin, Bacillomycin D, Difficidin, and Bacillaene), which play important roles in biocontrol.2The plant rhizosphere isolate Bacillus amyloliquefaciens B3is distinguished from the related model organism Bacillus subtilis168by its ability to stimulate plant growth and to suppress plant pathogenic organisms. In order to clear the mechanisms of biocontrol and plant growth promotion, suppressive subtractive hybridization (SSH) was performed between strains of B. amyloliquefaciens B3and B. subtilis168. Totally,49unique fragments of B3were obtained. Three NRPS, one PKS and a cryptic plasmid were found in the unique fragments. A new nonribosomal peptide synthetases (NRPS) fragment was unique insertions in the B3genome and is not present in any bacterial strain so far. In addition, we sampled sequenced the genome of FZB42and identified more than3268genes with>90%identity on the amino acid level to the corresponding genes of Bacillus amyloliquefaciens FZB42. Nine large gene clusters encoding nonribosomal peptide synthetases (NRPS) and polyketide synthases (PKS) occupied about8%of the whole genome. The responsibility of the eight gene clusters for the production of the corresponding secondary metabolites was demonstrated by Matrix-assisted laser desorption ionization-time of flight mass spectrometry. But, the new was unclear now. There are some genes involved in biofilm formation (such as EPS, PGA operon) and promotion of plant growth such as IAA biosynthetic genes,2,3-butanediol synthetic genes.3In this work, we have sequenced and characterized a cryptic plasmid called pBSG3from wild-type Bacillus amyloliquefaciens B3, which is an8439bp circular molecule, with G+C content of40.3%. We provide evidence that pBSG3replicates via the rolling-circle mechanism and belongs to group Ⅲ of the rolling-circle replication plasmid family. The plasmid contains7putative open reading frames (ORFs) including genes repB3, mobB3, rapQ, phrQ, pgsR, and2unknown ORFs (orf1c and orf2). Our observations revealed that the RapQ-PhrQ (response regulator aspartate phosphatase-phosphatase regulator) system is involved in sporulation and RapQ can delay the onset of sporulation.2Escherichia coli and Bacillus potential shuttle vectors, pTRD (containing the minimal replicon) and pTRDS (containing the minimal replicon and the single-strand origin) were developed from pBSG3and tested the stability. Moreover, HpaGxooc protein, which can induce disease and insect resistance in plants, was tried to express with the stable vector pTRDS in Bacillus. In summary, the pBSG3plasmid containing various genes is not only a candidate tool for vector development in Bacillus genus research but also a potential vehicle for the exchange of genetic elements among Bacillus populations that contributes into the survival of bacilli in natural environments.4HpaGxooc, from rice pathogenic bacterium Xanthomonas oryzae pv. oryzicola, is a member of the harpin group of proteins, eliciting hypersensitive cell death in non-host plants, inducing disease and insect resistance in plants and enhancing the plant growth. Bacillus amyloliquefaciens FZB42has been used as powerful weapons for the suppression of plant pathogenic organisms and plant growth promotion in the world. In this study, two recombinant Harpin protein expression vectors pGAprEHS and pUNprEKHS were constructed and integrated into the aprE, nprE site of FZB42. These two vectors were composed of powerful promoter P43and nprB signal peptide, which leads to the expression of Harpin protein continuously and secrete into the medium. The result of RT-PCR analysis reveals the hpal gene can be transcribed normally. Subsequently, we performed greenhouse and field experiments for evaluation of the biocontrol activity and plant growth promotion of the engineering strain FZBHarpin. The results show that FZBHarpin is the best in biocontrol activity. Both FZBHarpin and FZBAN which is a mutant of aprE and nprE are the same in plant growth promotion, and are better than FZB42. |
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