龙湾火山湖放线菌的分离鉴定及活性代谢产物研究

发布时间：2018-01-14 07:14

本文关键词：龙湾火山湖放线菌的分离鉴定及活性代谢产物研究　出处：《东北农业大学》2016年博士论文　论文类型：学位论文

【摘要】：放线菌次级代谢产物丰富,作为微生物天然药物的一项重要来源,其资源的开发与利用一直以来备受科研人员们的广泛关注。随着人类社会的发展,人们对高活性、新构型天然药物的需求愈加强烈,然而,伴随常规生态系统中放线菌资源研究的深入,获得新型天然药物产生菌的难度愈来愈大,这极大地影响了新药的开发效率。因此,寻找新的分离源,从特殊生境中分离放线菌是提高新型天然药物产生菌分离效率的有效途径。三角龙湾火山湖,坐落于中国吉林省辉南县,由20万年前火山喷发后地下水上升聚集而成的火山口湖,以其独特的地貌特征形成了特异的生态系统,为丰富放线菌物种资源,发掘新型天然化合物提供了新的分离来源。因此,本研究以龙湾火山湖土壤沉积物为分离对象,对其进行放线菌的分离、多相鉴定、物种组成分析以及拮抗菌株活性产物研究。研究结果如下:(1)分离获得火山湖放线菌158株。通过培养和形态特征观察后对菌株进行初步归类。选择形态特征具有代表性的菌株进行16S rRNA基因测序。经过Ez Taxon服务器在线分析与典型菌株的序列进行相似性比对,确定菌株的归属。分析结果显示,火山湖放线菌分布于放线菌目的8个属内:链霉菌属(Streptomyces,76%),小单孢菌属(Micromonospora,14%),野野村氏菌属,(Nonomuraea,4%),游动放线菌属(Actinoplanes,2%),小链孢菌属(Catellatospora,1%),地嗜皮菌属(Geodermatophilus,1%),拟诺卡氏属(Nocardiopsis,1%),红球菌属(Rhodococcus,1%)。对候选新种放线菌进行评估分析,通过多相分类学鉴定了3株放线菌(NEAU-C3、NEAU-JM1和NEAU-JM2)为新种放线菌。(2)菌株NEAU-C3的多相分类学鉴定:16S rRNA基因测序结果分析显示,菌株NEAU-C3从属于链霉菌菌属,并与菌株Streptomyces hygroscopicus subsp.ossamyceticus JCM4965T(97.7%)和Streptomyces torulosus JCM 4872T(97.7%)相似性最高。系统发育分析结果显示,菌株NEAU-C3在Neighbour-joining和Maximum-likelihood两种建树图谱中形成了一独立分支,并且与最高相似性菌株S.hygroscopicus subsp.ossamyceticus JCM 4965T和S.torulosus JCM 4872T的DNA同源性均小于新菌种鉴定阈值70%,结合表型特征及生理生化特征上的差别,确定菌株NEAU-C3为链霉菌菌属的一个新种,根据分离源信息,将菌株NEAU-C3命名为Streptomyces vulcanius。(3)菌株NEAU-JM1的多相分类学鉴定:16S r RNA基因测序结果分析显示,菌株NEAU-JM1从属于小链孢菌属,并与菌株Catellatospora parii NEAU-CL2T(99.5%),Catellatospora methionotrophica JCM 7543T(99.4%),Catellatospora coxensis DSM 44901T(99.4%),Catellatospora citrea DSM 44097T(99.3%)和Catellatospora chokoriensis JCM 12950T(99.2%)相似性最高。系统发育分析结果显示,菌株NEAU-JM1在Neighbour-joining和Maximum-likelihood两种建树图谱中与菌株C.parii NEAU-CL2T形成了一独立分支,并与菌株C.coxensis DSM 44901T,C.aurea NEAU-SH16T,C.citrea DSM 44097T,C.methionotrophica JCM 7543T和C.chokoriensis JCM 12950T在系统发育图谱中最相近。DNA同源性结果显示菌株NEAU-JM1与所有相似菌的同源性均小于新菌种鉴定阈值70%,结合表型特征及生理生化特征上的差别,确定菌株NEAU-JM1为小链孢菌属的一个新种,根据分离源信息,将菌株NEAU-JM1命名为Catellatospora vulcania。(4)菌株NEAU-JM2的多相分类学鉴定:16S r RNA基因测序结果分析显示,菌株NEAU-JM2从属于小单孢菌属,并与菌株Micromonospora maoerensis NEAU-MES19T(99.5%),Micromonospora zeae NEAU-gq9T(98.9%),Micromonospora saelicesensis Lupac 09T(98.8%)和Micromonospora matsumotoense JCM 9104T(98.8%)相似性最高。系统发育分析结果显示,菌株NEAU-JM2在Neighbour-joining和Maximum-likelihood两种建树图中,均与菌株M.maoerensis NEAU-MES19T和M.matsumotoense JCM 9104T建在同一分支。菌株NEAU-JM2的gyrB基因相似性比对分析显示,M.maoerensis NEAU-MES19T(94.5%),M.zeae NEAU-gq9T(94.0%),M.saelicesensis Lupac 09T(94.8%)和M.matsumotoense JCM 9104T(92.9%)为其相似性最高的菌株,进一步系统发育分析显示,菌株NEAU-JM2形成一独立分支,不与其任何最高相似性菌株邻近。通过系统发育分析、形态特征、生理生化特征比较及DNA同源性分析确定菌株NEAU-JM2为小单孢菌菌属的一个新种,根据分离源信息,将菌株NEAU-JM2命名为Micromonospora vulcania。(5)抑菌活性测试实验结果显示,菌株LW1-3对10种病原真菌均表现出了良好的抗菌活性,结合细菌抑制实验结果,对菌株LW1-3进行后续的化合物分离,得到2个单体化合物(compound 1和compound 2),经结构鉴定分别为巴佛洛霉素A1(bafilomycin A1)和巴佛洛霉素K(bafilomycin K)。
[Abstract]:Actinomycetes with abundant secondary metabolites, as an important source of natural microbial drugs, development and utilization of the resources of the attention has attracted more and more researchers. With the development of human society, people of high activity, the new configuration of natural drug demand is stronger, however, with the research on Actinomycetes Resources routine the ecological system in depth, to obtain new natural drug producing bacteria become more and more difficult, which greatly affects the efficiency of drug development. Therefore, to find new sources of separation, from special habitat actinomycete isolation is an effective way to improve the new type of natural drug producing bacteria separation efficiency. The triangle Longwan volcano lake is located. In Huinan County, Jilin province Chinese, by 200 thousand years ago after the volcano rising groundwater gathered from the volcano lake, with its unique features, forming a specific ecological system, abundant actinomycetes species Resources, provides the new sources of discovery of new natural compounds. Therefore, the research on Longwan volcano Lake soil sediments as the separation object, separation of actinomycetes, phase identification, analysis and Research on the species composition of antagonistic strains active products. The results are as follows: (1) isolated from the volcano Lake actinomycetes 158 strains. Through the cultivation and morphology of isolates were primarily classified. Morphological characteristics of representative strains of 16S rRNA gene sequencing. By sequence analysis of Ez Taxon server and the online typical strains similarity comparison, determine the strains belonging. Analysis results show that the volcano Lake actinomycetes in distribution line Objective: 8 bacteria within the genus Streptomyces (Streptomyces, 76%), Micromonospora (Micromonospora, 14%), wild Nomura's genus, (Nonomuraea, 4%), Actinoplanes (Actinoplanes, 2%), microstreptospora (Ca Tellatospora, 1%), to Dermatophilus (Geodermatophilus, 1%), quasi Nocardia (Nocardiopsis, 1%), Rhodococcus (Rhodococcus, 1%). The candidate species of actinomycetes were evaluated by polyphasic taxonomy and identification of 3 strains of actinomycetes (NEAU-C3, NEAU-JM1 and NEAU-JM2) as a new species actinomycete strain NEAU-C3. (2) the identification of Polyphasic Taxonomy: 16S rRNA gene sequencing analysis showed that strain NEAU-C3 belongs to the genus Streptomyces, and strain Streptomyces hygroscopicus subsp.ossamyceticus JCM4965T Streptomyces torulosus JCM (97.7%) and 4872T (97.7%) the highest similarity. Phylogenetic analysis showed that NEAU-C3 strain formed a independent branch in Neighbour-joining and Maximum-likelihood two kinds of achievements, and the highest similarity with subsp.ossamyceticus JCM 4965T and S.torulosus S.hygroscopicus strain JCM 4872T DNA homology Less than 70% new species identification threshold, combined with the characteristics and physiological and biochemical characteristics of phenotype differences, determine a new species of the genus Streptomyces strain NEAU-C3 was isolated, according to sources of information, the strain of NEAU-C3 named Streptomyces vulcanius. (3) NEAU-JM1 strains identification of Polyphasic Taxonomy: 16S R RNA gene sequencing analysis showed that strain NEAU-JM1 according to microstreptospora, and strain Catellatospora parii NEAU-CL2T (99.5%), Catellatospora methionotrophica JCM 7543T (99.4%), Catellatospora coxensis DSM 44901T (99.4%), Catellatospora citrea DSM 44097T Catellatospora chokoriensis JCM (99.3%) and 12950T (99.2%) the highest similarity. Phylogenetic analysis showed that strain NEAU-JM1 in Neighbour-joining and Maximum-likelihood the two contribution of NEAU-CL2T and strain C.parii formed a separate branch, and strain C.coxens Is DSM 44901T, C.aurea NEAU-SH16T, C.citrea DSM 44097T, C.methionotrophica JCM 7543T and C.chokoriensis JCM 12950T in the system development of the most similar.DNA homology showed that strain NEAU-JM1 and the homology of all similar bacteria were less than the threshold of 70% new species identification, combined with the characteristics and physiological and biochemical characteristics of phenotype differences, determine a new strain NEAU-JM1 as microstreptospora, according to the separation of the source of information, the strain of NEAU-JM1 named Catellatospora vulcania. (4) NEAU-JM2 strains identification of Polyphasic Taxonomy: 16S R RNA gene sequencing analysis showed that strain NEAU-JM2 belongs to the genus Micromonospora, and strain Micromonospora maoerensis NEAU-MES19T (99.5%) zeae (98.9% NEAU-gq9T, Micromonospora Micromonospora saelicesensis Lupac), 09T (98.8%) and Micromonospora matsumotoense JCM 9104T (98.8%) the highest similarity. Phylogenetic analysis showed that strain NEAU-JM2 in Neighbour-joining and Maximum-likelihood two kinds of construction graph, and M.maoerensis NEAU-MES19T and M.matsumotoense JCM 9104T was built in the same branch. The gyrB gene of strain NEAU-JM2 of sequence similarity analysis showed that M.maoerensis NEAU-MES19T (94.5%), M.zeae NEAU-gq9T (94%), M.saelicesensis Lupac 09T (94.8%) and M.matsumotoense JCM 9104T (92.9%) is the highest strain similar further phylogenetic analysis showed that NEAU-JM2 strain formed a separate branch, with the highest similarity of any strain proximity. Through phylogenetic analysis, morphological characteristics, physiological analysis of a new strain NEAU-JM2 was identified as Micromonospora sp. and DNA homology comparison according to the biochemical characteristics, separation of source information, the strain of NEAU-JM2 named Micromonospora vulcania. (5) antibacterial activity test The results showed that the strain LW1-3 of 10 kinds of pathogenic fungi showed good antibacterial activity, the combination of bacterial inhibition experiments, for subsequent separation of strain LW1-3 compounds, 2 compounds (compound 1 and compound 2), they are identified as bafilomycins A1 (bafilomycin A1) and buffy Los mycomycin K (bafilomycin K).

【学位授予单位】：东北农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：Q93

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