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Marine Sediment-Derived Actinomycetes From Pacific Sector Of The Arctic Ocean: Diversity, Secondary Metabolite Synthase Genes And Biological Activities

Posted on:2015-01-02Degree:MasterType:Thesis
Country:ChinaCandidate:M YuanFull Text:PDF
GTID:2180330431964290Subject:Marine biology
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Actinomycete is an important source of natural products. With the discovery ofactinomycin,33,500kinds of bioactive secondary metabolites have been found frommicroorganisms so far. Among them,13,700kinds are discovered from actinomycetes,which accounts for40%of all the bioactive products. In recent years, separatingactinomycetes and their active substances has become an inevitable trend. It is coldand the water temperature is below0℃in Arctic, in order to survive in such extremeenvironments, the relevant physiological and biochemical characteristics ofactinomycetes have to be generated. Thus actinomycetes from the Arctic Ocean mayproduce special bioactive substances with novel structure.In this paper, actinomycetes from24sediment samples collected from the ArcticOcean were isolated and cultured and phylogenetic analysises were carried outthrough16S rDNA sequences; the isolates were tested for salt tolerance and sea waterrequirement, screening for secondary metabolites synthase and antimicrobial activity;Further research about one strain Y18which showed marvelous antimicrobial activitywas taken, such as molecular identification, phylogenetic analysis, fermentationcondition optimization and stability of fermentation liquid.143actinomycetes were isolated with5kinds of isolation medium through thedispersion and differential centrifugation method from24sediment samples of Pacificsector, Arctic Ocean. BOX-PCR fingerprinting analysis was used to remove samestrains from one station.16S rDNA gene sequence analysis of these70different stainsreveals that they belong to17genera: Agrococcus(1), Microbacterium(3),Salinibacterium(7), Arthrobacter(5), Citricoccus(4), Kocuria(6), Rhodococcus(8),Arsenicicoccus(1), Janibacter(7), Nocardiopsis(6), Dietzia(5), Brevibacterium(3),Saccharopolyspora(1), Streptomyces(8), Corynebacterium(1), Microlunatus(1) and Nocardioides(1), most of them belong to rare actinomycetes. Strain Y284is suggestedas a novel strain of genera Saccharopolyspora with the highest16S rDNA similarityabout97.771%to the closest type strain Saccharopolyspora gregorii NCIMB12823T(X76962) and Y25with the highest16S rDNA similarity about97.804%to theclosest type strain Nocardioides kribbensis KSL-2T(AY835924). The results ofactinomycetes isolation with different medium are quite different. The actinomycetesseparation rate of M7medium is70.97%and this medium has the most variousgenera. So M7medium is more suitable to isolate marine acinobacteria than other4kinds of media.PKSⅠ, PKSⅡ, NRPS, phzE, dTGD, Halogenase and CYP genes are observedin41(58.6%),27(38.6%),25(35.7%),16(22.9%),60(85.7%),27(38.6%) and32(45.7%)of the70strains. The results of antimicrobial activities are as follows:9strains are resistant to Candida albicans, they belong to genera Streptomyces andNocardiopsis;12strains (genera Microlunatus, Nocardiopsis and Streptomyces) cansuppress the fecundity of Bacillus subtilis; one strain can produce substance againstEscherichia coli and one strain can inhibit the growth of Staphylococcus aureus, theyboth belong to genera Streptomyces.The actinomycete strain Y18belongs to genus Nocardiopsis according to16SrDNA sequence analysis. Its closest type strain is Nocardiopsis dassonvillei subsp.Dassonvillei DSM43111Tand the similarity is99.8%. Its fermentation mediumcontains peptone2g, casein hydrolysate2g, yeast extract2g, glucose1g, sea water1L,the seed liquid inoculum for4%, medium initial pH8.0, the culture temperature of28℃, liquid volume20%in1000ml flask, and fermentation time6days. The researchof the stability of heat, acid, alkali and storage we found that it had a stable activity ontemperature, acidity, alkali and stored at4℃.These results indicate that the Arctic marine sediments are rich in actinomycetesresources and have the potential to produce a variety of bioactive secondarymetabolites. This study increases marine actinomycetes resource reserves, enrichesthe understanding of marine actinomycetes, provides a basis and valuable reference of the research, development and utilization of actinomycetes from the polar marinesediments and has a huge scientific value.
Keywords/Search Tags:the Arctic Ocean, marine sediments, actinomycete, diversity, antimicrobial activity, secondary metabolites
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