| Paenibacillus polymyxa SC2, a strain of plant growth-promoting rhizobacteria (PGPR) had significantly antagonistic to many soil-borne plant pathogenic fungi such as Phytophthora capsici. This has a broad-spectrum antimicrobial activity and can antagonize Fusarium solani, Fusarium oxysporum f. sp. cucumerinum, Pseudoperonospora cubensis, Botrytis cinerea Pers and Botrytis cinerea.In the present study, We obtained a 497bp conserved DNA fragment of NRPSs gene, with the primers TGD and LGG detecting non-ribosomal peptide synthetases concerved domain, with genomic DNA of P. polymyxa SC2 as template. According to the conserved DNA sequence, we designed specific primers of TAIL-PCR, amplified the DNA sequences flanking on the conserved DNA fragment by the method of TAIL-PCR with genomic DNA of P. polymyxa SC2 as template. Subsequently, the TAIL-PCR of upstream or downstream DNA sequences of the last amplified DNA fragments were continually carried out for several times. Alternatively, depending on the identity of the NRPS gene to bamC, mycC, ituC sequences of Bacillus subtilis strains and bmyC of Bacillus amyloliquefaciens FZB42, degenerate primers were designed to amplify the aimed DNA fragments. Then, we obtained a resulting DNA sequence with the length of 19844bp by assembling all amplified DNA fragments.BLAST analysis revealed the ORF is identical to the homologous fus gene cluster sequence of P. polymyxa PKB1, so, depending on the identity of the DNA fragments has obtained to fus gene cluster of P. polymyxa PKB1, degenerate primers were designed to amplify the aimed DNA fragments. Finally, we obtained a resulting DNA sequence with the length of 32680bp by assembling all amplified DNA fragments.Finally, the fus gene cluster was cloned and sequenced (the accession number of GenBank is EU431181), and it spans 32680 bp, including 8 open reading frames (the fus gene cluster), they are fusG, fusF, fusE, fusD, fusC, fusB, fusA, fusTE. Similarity of the 8 ORFs in the fusaricidin biosynthetic gene cluster of P. polymyxa SC2 to the corresponding gene fusG, fusF, fusE, fusD, fusC, fusB, fusA, fusTE of the P. polymyxa PKB1 is 90.61%, 93.42%, 91.75%, 85.54%, 83.29%, 89.95%, 88.97%, 82.88%, and similarity of the deduced amino acid sequences to FusG, FusF, FusE, FusD, FusC, FusB, FusA, FusTE of the strain P. polymyxa PKB1 is 98.77%, 99.15%, 98.28%, 90.64%, 96.31%, 97.78%, 96.81%, and 76.35%, respectively. Then,wo do the primary analysis to the fus gene cluster by NCBI-BLAST.The fusA is 23727 bp, encoding a six-module nonribosomal peptide synthetase. The domain organization for these six modules are C-A-T, C-A-T-E, C-A-T, C-A-T-E, C-A-T-E and C-A-T-TE, respectively. The predicted substrate specificities of the top five A domains within FusA were consistent with the five amino acids, L-Thr, D-Val, L-Tyr, D-Thr and D-Asn. The amino acid substrate for FusA-A6 could not be predicted because its signature sequence shows no similarity to A domains with assigned specificities, including those activating L- and D-Ala. We have build up the phylogenetic tree of FusA-A6 of the P. polymyxa SC2 using the MEGA4 software. Finally, we have predicted that the amino acid substrate for FusA-A6 may be D-Ala. The predicted amino acid composition and sequence of the P. polymyxa SC2 was exactly consistent with the fusaricidin C produced by Bacillus polymyxa KT-8 and LI-F03a produced by Bacillus polymyxa L-1129. Accordingly, P. polymyxa SC2 may be responsible for the synthesis of fusaricidin C (LI-F03a) which has the antifungal activity. Similarity of the six A domains to the corresponding domain A1, A2, A3, A4, A5 and A6 of FusA of P. polymyxa PKB1 are 94.13%, 94.82%, 91.52%, 94.55%, 95.34% and 96.73%, respectively. Accordingly, identity of their DNA sequences are 86.95%, 89.00%, 82.18%, 89.38%, 87.31% and 89.49%. We deduce that fusA is a key gene involved in biosynthesis of nonribosomal peptide fusaricidin C from P. polymyxa SC2. |
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