Cloning And Genome-walking Of PKS And NRPS Gene From Polar Bacteria With Antifungal Activity Against Plant Pathogenic Fungi

Polar region is a microorganism resource treasury which has germplasm noveltyand diversity. Due to the distinct environment, various novel bioactive substances maybe discovered from the polar microorganisms. Study on the relevant functional genes ofantimicrobial strains potentially provide evidence for in-depth studies of the polarmolecular biology of microbial secondary metabolites from the biosynthetic pathway,and that also could provide genetic resources for future development.The antifungal activity against plant pathogenic fungi Fusarium oxysporum of38strains of polar bacteria stored in the laboratory was validated by method of agardiffusion. The results showed that13strains had distinct antimicrobial activity. Theantimicrobial-validating experiment of antimicrobial strains showed that the Antarcticpsychrotrophic strain C-1、P499、83-1、P406and205exhibited higher and more stableantimicrobial activity. Molecular identification and phylogenetic analysis on the basis of16S rRNA gene indicated that11strains belonged to genus of Pseudomonas,1strainbelonged to genus of Psychrobacter and1strain belonged to genus of Arthrobacter.PKS and NRPS genes were amplified from the13antimicrobial strains againstplant pathogen fungi by PKS-D/PKS-U and NRPS-D/NRPS-U primers which weredesigned according to the different biosynthetic pathway of bioactive substances. Theresults showed that PKS genetic fragments were amplified from5antimicrobial strainsand the positive rate was38.5%; NRPS genetic fragments were amplified from10antimicrobial strains and the positive rate was76.9%. PKS and NRPS genetic fragmentswere simultaneously amplified from strain C-1、P499、83-1、P406and205; two differentNRPS genetic fragments were respectively amplified from strain C-1and P499whichdeserved further study.The unknown sequences of two ends of the PKS and NRPS genetic fragments ofPseudomonas sp.83-1were amplified by the genomic walking technology. A5118bpcontinuous DNA sequence was obtained by four stepwise walking of NRPS geneticfragments and a1622bp continuous DNA sequence was obtained by two stepwisewalking of PKS genetic fragments, respectively. The open reading frames (ORFs) werepredicted and screened large multi-domain megasynthase involved in the biosynthesisof NRPS. The results showed that three complete ORFs were found in the NRPSmodule. ORF1encoded a adenylation (A) domain, ORF2encoded a condensation (C) domain and ORF3encoded one A domain and two C domain.The experiment on PKS and NRPS genetic of polar activity against plantpathogenic fungi strains has made some progress, which can be conducive to furtherunderstanding of the molecular mechanisms of metabolites, predicting possiblecompound types and providing strong evidence that marine bioactive bacteria canproduce natural produets through PKS and NRPS pathways.