Isolation And Purification Of Antibiotic Substances From The Antarctic Psychrotrophic Bacterium Pseudomonas Sp. C Arid Study On Relevant Functional Genes

Polar region is a microorganism resource treasury which has germplasm noveltyand diversity. Due to the distinct environment, it is more possible to discover novelbioactive substances from polar microorganisms. Study on the relevant functional genesof antimicrobial strains can predict the types of compounds produced and reduce theprobability of the same compounds screened.Using the familiar plant pathogen Fusarium oxysporum as indicator, antimicrobialstrains were isolated from the seawater which was collected during27th ChineseAntarctic Scientific Expedition and marine sediment BN05which was collected during4th Chinese Arctic Scientific Expedition. Molecular identification and phylogeneticanalysis indicated that among the25antimicrobial strains screened,2strains weremembers of Psychrobacter,4strains were members of Pseudomonas, and19strainswere members of Pseudoalteromonas. The study on antimicrobial spectrum of4strainswhich had higher antimicrobial activity indicated that three strains, namely312,83-1and195had obvious antimicrobial effects on Verticillium dahliae, Thanatephoruscucumeris, Alternaria solani, Rhizoctonia solani Kühn, Phomopsis asparagi(Sacc),Phytophthora capsici Leonian and Fusarium oxysporum. Except Rhizoctonia solaniKühn, strain312-1had obvious antimicrobial effect on the other six plant pathogenlisted above. The antimicrobial-validating experiment of antimicrobial strains stored inthe laboratory showed that the Antarctic psychrotrophic strain C, LX-4, LX-2and1-Z18exhibited higher and stable antimicrobial activity. The Antarctic psychrotrophicPseudomonas sp. C which had stronger and stable activity was selected for furtherstudy.Studies on temperature and pH stability of bioactive substances of fermented liquidof the Antarctic psychrotrophic Pseudomonas sp. C showed that it had better stability at40-70℃and pH5-9. According to the active tracking, bioactive substances wereisolated and purified by silica gel column chromatography and HPLC. Compoundstructure was identified by EI-MS/MS and nuclear magnetic resonance (13CNMR,11CNMR). The results showed that10kinds of compounds were isolated and identified,which belonged to cyclic dipeptide, dipeptide, nonylphenol polyoxyethylene ether,acetyl hydrazine derivatives, ester compound, sterides, indole diketone derivativesrespectively. Cycle (Phe-Pro) and14-Nonylphenoxy-3,6,9,12-tetraoxatetradecan-1-ol(nonylphenol polyoxyethylene ether) had higher antimicrobial activity on Fusarium oxysporum and Bacillus subtilis;9,19-cyclocholestan-3-one,4,14-dimethyl-(sterides)has antimicrobial activity on Fusarium oxysporum.Functional genes were amplified from6antimicrobial strains (C,1-Z18, LX-2,LX-4,195and83-1) against plant pathogen fungi by three degenerate primers whichwere designed according to the different biosynthetic pathway. of bioactive substances.The results showed that PKS genetic fragments were amplified from strain C and1-Z18,NRPS genetic fragments were amplified from all of the6antimicrobial strains andhalogenase genetic fragments was amplified only from strain1-Z18. The unknownsequences of two ends of the NRPS genetic fragments of Pseudomonas sp. C wereamplified by genomic walking technology.