本文關(guān)鍵詞：龍灣火山湖放線菌的分離鑒定及活性代謝產(chǎn)物研究　出處：《東北農(nóng)業(yè)大學(xué)》2016年博士論文　論文類(lèi)型：學(xué)位論文

  更多相關(guān)文章： 火山湖放線菌 特殊生境 多相分類(lèi) 活性代謝產(chǎn)物 Streptomyces vulcanius Catellatospora vulcania Micromonospora vulcania

【摘要】：放線菌次級(jí)代謝產(chǎn)物豐富,作為微生物天然藥物的一項(xiàng)重要來(lái)源,其資源的開(kāi)發(fā)與利用一直以來(lái)備受科研人員們的廣泛關(guān)注。隨著人類(lèi)社會(huì)的發(fā)展,人們對(duì)高活性、新構(gòu)型天然藥物的需求愈加強(qiáng)烈,然而,伴隨常規(guī)生態(tài)系統(tǒng)中放線菌資源研究的深入,獲得新型天然藥物產(chǎn)生菌的難度愈來(lái)愈大,這極大地影響了新藥的開(kāi)發(fā)效率。因此,尋找新的分離源,從特殊生境中分離放線菌是提高新型天然藥物產(chǎn)生菌分離效率的有效途徑。三角龍灣火山湖,坐落于中國(guó)吉林省輝南縣,由20萬(wàn)年前火山噴發(fā)后地下水上升聚集而成的火山口湖,以其獨(dú)特的地貌特征形成了特異的生態(tài)系統(tǒng),為豐富放線菌物種資源,發(fā)掘新型天然化合物提供了新的分離來(lái)源。因此,本研究以龍灣火山湖土壤沉積物為分離對(duì)象,對(duì)其進(jìn)行放線菌的分離、多相鑒定、物種組成分析以及拮抗菌株活性產(chǎn)物研究。研究結(jié)果如下:(1)分離獲得火山湖放線菌158株。通過(guò)培養(yǎng)和形態(tài)特征觀察后對(duì)菌株進(jìn)行初步歸類(lèi)。選擇形態(tài)特征具有代表性的菌株進(jìn)行16S rRNA基因測(cè)序。經(jīng)過(guò)Ez Taxon服務(wù)器在線分析與典型菌株的序列進(jìn)行相似性比對(duì),確定菌株的歸屬。分析結(jié)果顯示,火山湖放線菌分布于放線菌目的8個(gè)屬內(nèi):鏈霉菌屬(Streptomyces,76%),小單孢菌屬(Micromonospora,14%),野野村氏菌屬,(Nonomuraea,4%),游動(dòng)放線菌屬(Actinoplanes,2%),小鏈孢菌屬(Catellatospora,1%),地嗜皮菌屬(Geodermatophilus,1%),擬諾卡氏屬(Nocardiopsis,1%),紅球菌屬(Rhodococcus,1%)。對(duì)候選新種放線菌進(jìn)行評(píng)估分析,通過(guò)多相分類(lèi)學(xué)鑒定了3株放線菌(NEAU-C3、NEAU-JM1和NEAU-JM2)為新種放線菌。(2)菌株NEAU-C3的多相分類(lèi)學(xué)鑒定:16S rRNA基因測(cè)序結(jié)果分析顯示,菌株NEAU-C3從屬于鏈霉菌菌屬,并與菌株Streptomyces hygroscopicus subsp.ossamyceticus JCM4965T(97.7%)和Streptomyces torulosus JCM 4872T(97.7%)相似性最高。系統(tǒng)發(fā)育分析結(jié)果顯示,菌株NEAU-C3在Neighbour-joining和Maximum-likelihood兩種建樹(shù)圖譜中形成了一獨(dú)立分支,并且與最高相似性菌株S.hygroscopicus subsp.ossamyceticus JCM 4965T和S.torulosus JCM 4872T的DNA同源性均小于新菌種鑒定閾值70%,結(jié)合表型特征及生理生化特征上的差別,確定菌株NEAU-C3為鏈霉菌菌屬的一個(gè)新種,根據(jù)分離源信息,將菌株NEAU-C3命名為Streptomyces vulcanius。(3)菌株NEAU-JM1的多相分類(lèi)學(xué)鑒定:16S r RNA基因測(cè)序結(jié)果分析顯示,菌株NEAU-JM1從屬于小鏈孢菌屬,并與菌株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。系統(tǒng)發(fā)育分析結(jié)果顯示,菌株NEAU-JM1在Neighbour-joining和Maximum-likelihood兩種建樹(shù)圖譜中與菌株C.parii NEAU-CL2T形成了一獨(dú)立分支,并與菌株C.coxensis DSM 44901T,C.aurea NEAU-SH16T,C.citrea DSM 44097T,C.methionotrophica JCM 7543T和C.chokoriensis JCM 12950T在系統(tǒng)發(fā)育圖譜中最相近。DNA同源性結(jié)果顯示菌株NEAU-JM1與所有相似菌的同源性均小于新菌種鑒定閾值70%,結(jié)合表型特征及生理生化特征上的差別,確定菌株NEAU-JM1為小鏈孢菌屬的一個(gè)新種,根據(jù)分離源信息,將菌株NEAU-JM1命名為Catellatospora vulcania。(4)菌株NEAU-JM2的多相分類(lèi)學(xué)鑒定:16S r RNA基因測(cè)序結(jié)果分析顯示,菌株NEAU-JM2從屬于小單孢菌屬,并與菌株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。系統(tǒng)發(fā)育分析結(jié)果顯示,菌株NEAU-JM2在Neighbour-joining和Maximum-likelihood兩種建樹(shù)圖中,均與菌株M.maoerensis NEAU-MES19T和M.matsumotoense JCM 9104T建在同一分支。菌株NEAU-JM2的gyrB基因相似性比對(duì)分析顯示,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%)為其相似性最高的菌株,進(jìn)一步系統(tǒng)發(fā)育分析顯示,菌株NEAU-JM2形成一獨(dú)立分支,不與其任何最高相似性菌株鄰近。通過(guò)系統(tǒng)發(fā)育分析、形態(tài)特征、生理生化特征比較及DNA同源性分析確定菌株NEAU-JM2為小單孢菌菌屬的一個(gè)新種,根據(jù)分離源信息,將菌株NEAU-JM2命名為Micromonospora vulcania。(5)抑菌活性測(cè)試實(shí)驗(yàn)結(jié)果顯示,菌株LW1-3對(duì)10種病原真菌均表現(xiàn)出了良好的抗菌活性,結(jié)合細(xì)菌抑制實(shí)驗(yàn)結(jié)果,對(duì)菌株LW1-3進(jìn)行后續(xù)的化合物分離,得到2個(gè)單體化合物(compound 1和compound 2),經(jīng)結(jié)構(gòu)鑒定分別為巴佛洛霉素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).

【學(xué)位授予單位】：東北農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類(lèi)號(hào)】：Q93

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