海南三亚红树林放线菌多样性及抗菌活性
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摘要
目的研究海南三亚红树林底泥样品中放线菌的多样性及抗菌活性,以期发现可以产生新颖活性物质的药用放线菌资源。方法采用11种分离培养基以稀释涂布法对样品中的放线菌进行选择性分离;对分离菌株的16S rRNA基因序列进行扩增、比对,开展多样性分析;放线菌发酵液经乙酸乙酯萃取,采用纸片法对粗提物进行抗菌活性筛选;同时对放线菌的生物合成基因NRPS、PKS I和PKS II进行筛选。结果共分离到149株放线菌,它们分布于6个目12个科16个属,其中小单孢菌属为优势菌属;菌株MG16072的16S rRNA基因序列与最近有效菌株Actinomadura hallensis H647-1T的相似率为97.94%,为潜在Actinomadura属新种;抗菌活性检测结果显示,有20株放线菌至少对1株检定菌具有抗菌活性;NRPS、PKS I、PKS II基因筛选结果显示,有82株放线菌至少含有1种生物合成基因。结论海南三亚红树林含有丰富的药用放线菌资源,具有从中发现新颖活性物质的潜力。
Objective The research aimed to study the diversity and anti-microbial activity of actinobacteria isolated from sediments of mangrove in Sanya, Hainan. Methods Actinobacteria were isolated using dilution plating technique with 11 different isolation media. Diversity of actinobacteria was analyzed based on 16 S rRNA gene sequence. Fermentation broths of actinobacteria were extracted by ethyl acetate. Antibacterial and antifungal assay of extracts were tested by disk diffusion method. Furthermore, the screening of NRPS, PKS I and PKS II genes was carried out by PCR method. Results The 149 actinobacteria isolates were distributed in 16 genera affiliated with 12 families of 6 orders, among them, Micromonospora are the dominant genus. The 16 S rRNA sequence similarity between strain MG16072 and the nearest strain Actinomadura hallensis(H647-1 T DQ076484) in EzBioCloud database was 97.94%, which indicated that strain MG16072 is a potential new species of Actinomadura. 20 strains showed antibacterial and/or antifungal activities, and 82 strains carried at least one of NRPS, PKS I and PKS II genes. Conclusion Mangrove in Sanya are rich in medicinal actinobacteria, which could be a potential source for the discovery of new bioactive natural products.
Objective The research aimed to study the diversity and anti-microbial activity of actinobacteria isolated from sediments of mangrove in Sanya, Hainan. Methods Actinobacteria were isolated using dilution plating technique with 11 different isolation media. Diversity of actinobacteria was analyzed based on 16 S rRNA gene sequence. Fermentation broths of actinobacteria were extracted by ethyl acetate. Antibacterial and antifungal assay of extracts were tested by disk diffusion method. Furthermore, the screening of NRPS, PKS I and PKS II genes was carried out by PCR method. Results The 149 actinobacteria isolates were distributed in 16 genera affiliated with 12 families of 6 orders, among them, Micromonospora are the dominant genus. The 16 S rRNA sequence similarity between strain MG16072 and the nearest strain Actinomadura hallensis(H647-1 T DQ076484) in EzBioCloud database was 97.94%, which indicated that strain MG16072 is a potential new species of Actinomadura. 20 strains showed antibacterial and/or antifungal activities, and 82 strains carried at least one of NRPS, PKS I and PKS II genes. Conclusion Mangrove in Sanya are rich in medicinal actinobacteria, which could be a potential source for the discovery of new bioactive natural products.
引文
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[28]许敏,李静,戴素娟,等.广东湛江红树林植物内生放线菌资源勘探及生物活性研究[J].中国抗生素杂志,2016,41(1):26-34.
[29]李飞娜,潘臻,庹利,等.澳门红树林植物内生放线菌多样性及新颖性研究[J].中国抗生素杂志,2017,42(4):284-293.
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[32]Maskey R P,Li F C,Qin S,et al.Chandrananimycins A~C:production of novel anticancer antibiotics from a marine Actinomadura sp.isolate M048 by variation of medium composition and growth conditions[J].J Antibiot,2003,56(7):622-629.
[33]徐平,李文均,高慧英,等.聚酮类化合物生物合成途径基因阳性菌株生物多样性研究[J].微生物学报,2005,45(6):821-827.
[34]郑有坤,刘凯,熊子君,等.药用植物内生放线菌多样性及天然活性物质研究进展[J].中草药,2014,45(14):2089-2096.
[2]Holguin G,Vazquez P,Bashan Y.The role of sediment microorganisms in the productivity,conservation,and rehabilitation of mangrove ecosystems:an overview[J].Biol Fert Soils,2001,33(4):265-278.
[3]范航清.红树林[M].南宁:广西科学技术出版社,2000:18-23.
[4]Wu S L,Jiang L M.Recent advances in mangrove actinomycetes[J].Curr Biotechnol,2012,2(5):335-340.
[5]Li M Y,Xiao Q,Pan J Y,et al.Natural products from semimangrove flora:Source,chemistry and bioactivities[J].Nat Prod Rep,2009,26(2):281-298.
[6]Hong K,Gao A H,Xie Q Y,et al.Actinomycetes for marine drug discovery isolated from mangrove soils and plants in China[J].Mar Drugs,2009,7(1):24-44.
[7]Calcul L,Waterman C,Ma W S,et al.Screening mangrove endophytic fungi for antimalarial natural products[J].Mar Drugs,2013,11(12):5036-5050.
[8]Liu A R,Wu X P,Xu T.Research advances in endophytic fungi of mangrove[J].J Appl Ecol,2007,18(4):912-918.
[9]Manivasagana P,Venkatesana J,Sivakumarc K,et al.Pharmaceutically active secondary metabolites of marine actinobacteria[J].Microbiol Res,2014,169(4):262-278.
[10]Subramani R,Aalbersberg W.Marine actinomycetes:An ongoing source of novel bioactive metabolites[J].Microbiol Res,2012,167(10):571-580.
[11]郑志成,周美英,姚炳新.红树林根际放线菌的组成及生物活性[J].厦门大学学报(自然科学版),1989,28(3):306-310.
[12]洪葵.红树林放线菌及其天然产物研究进展[J].微生物学通报,2013,53(11):1131-1141.
[13]Jensen P R,Dwight R,Fenical W.Distribution of actinomycetes in near-shore tropical marine sediments[J].Appl Environ Microb,1991,57(4):1102-1108.
[14]Lee L H,Zainal N,Azman A S,et al.Diversity and antimicrobial activities of actinobacteria isolated from tropical mangrove sediments in malaysia[J].Sci World J,2014,2014(6):698178-698192.
[15]李文均,唐蜀昆,王栋,等.新疆青海中度嗜盐放线菌生物多样性初步研究[J].微生物学报,2004,44(1):1-7.
[16]Hayakawa M.Studies on the isolation and distribution of rare actinomycetes in soil[J].Actinomycetologica,2008,22(1):12-19.
[17]董艳萍,郭琳,旭格拉·哈布丁,等.塔克拉玛干沙漠南麓土壤放线菌资源勘探及抗菌活性筛选[J].中国抗生素杂志,2013,38(4):241-247.
[18]Takahashi Y,Matsumoto A,Seino A,et al.Rare actinomycetes isolated from desert soils[J].Actinomycetologica,1996,10(2):91-97.
[19]周双清,黄小龙,黄东益,等.Chelex-100快速提取放线菌DNA作为PCR扩增模板[J].生物技术通报,2010(2):123-125.
[20]Kim O S,Cho Y J,Lee K,et al.Introducing EzTaxon-e:a prokaryotic 16S rRNA gene sequence database with phylotypes that represent uncultured species[J].Int J Syst Evol Micr,2012,62(3):716-721.
[21]Tamura K,Peterson D,Peterson N,et al.MEGA5:molecular evolutionary genetics analysis using maximum likelihood,evolutionary distance,and maximum parsimony methods[J].Mol Biol Evol,2011,28(10):2731-2739.
[22]Ayuso-Sacido A,Genilloud O.New PCR primers for the screening of NRPS and PKS-I Systems in Actinomycetes:Detection and distribution of these biosynthetic gene sequences in major taxonomic groups[J].Microb Ecol,2005,49(1):10-24.
[23]Mets?-Ketel?M,Salo V,Halo L,et al.An efficient approach for screening minimal PKS genes from Streptomyces[J].FEMS Microbiol Lett,1999,180(1):1-6.
[24]薛杨,杨众养,王小燕,等.海南省红树林湿地生态系统服务功能价值评估[J].亚热带农业研究,2014,10(1):41-47.
[25]符国瑷,黎军.海南岛古老与原生的三亚红树林[J].热带林业,1999,27(1):12-19.
[26]刘敏,周秋平,黄惠琴,等.八门湾红树林土壤微生物多样性分析及放线菌的分离与鉴定[C].全国第九届海洋生物技术与创新药物学术会议摘要集,2014.
[27]吴家法,吴思婷,李智鸣,等.茅尾海红树林土壤可培养放线菌多样性及其抗尖孢镰刀菌活性分析[J].中国抗生素杂志,2017,42(4):294-301.
[28]许敏,李静,戴素娟,等.广东湛江红树林植物内生放线菌资源勘探及生物活性研究[J].中国抗生素杂志,2016,41(1):26-34.
[29]李飞娜,潘臻,庹利,等.澳门红树林植物内生放线菌多样性及新颖性研究[J].中国抗生素杂志,2017,42(4):284-293.
[30]甄永苏.抗肿瘤药物研究与开发[M].北京:化学工业出版社,2004:608-609.
[31]江宁宇,彭飞,方志锴,等.海洋来源马杜拉放线菌FIM95-F26产生的芳香聚酮类抗菌活性代谢产物的研究[J].中国抗生素杂志,2015,40(3):161-165.
[32]Maskey R P,Li F C,Qin S,et al.Chandrananimycins A~C:production of novel anticancer antibiotics from a marine Actinomadura sp.isolate M048 by variation of medium composition and growth conditions[J].J Antibiot,2003,56(7):622-629.
[33]徐平,李文均,高慧英,等.聚酮类化合物生物合成途径基因阳性菌株生物多样性研究[J].微生物学报,2005,45(6):821-827.
[34]郑有坤,刘凯,熊子君,等.药用植物内生放线菌多样性及天然活性物质研究进展[J].中草药,2014,45(14):2089-2096.