Knowledge Resource Center for Ecological Environment in Arid Area
| 两株新疆来源稀有放线菌的多相分类和天然产物研究 | |
| 其他题名 | Investigation of polyphasic taxonomy and natural products of two rare actinomycetes isolated from Xinjiang region |
| 张丽 | |
| 出版年 | 2016 |
| 学位类型 | 硕士 |
| 导师 | 张立新 ; 刘梅 ; 刘雪婷 |
| 学位授予单位 | 中国科学院大学 |
| 中文摘要 | 天然产物是药物开发的主要来源，其中微生物天然产物是其重要组成部分。近几十年来，传统方法指导下的活性天然产物发现逐渐显示出瓶颈，新活性化合物的发现逐渐减少，因此亟需我们开发快速、高效获得新颖的活性天然产物的新方法。极端环境由于其特殊的生态环境因素给微生物带来特殊的生存压力，因此蕴藏着新颖的放线菌资源。越来越多报道表明，来自极端环境的稀有放线菌表现出能够产生活性新颖天然产物的潜力。相比较于链霉菌，稀有放线菌这一相对空白的研究领域，已逐渐成为天然产物挖掘的新方向。新疆地区生态环境复杂多样，包括高原湖泊，盐碱地，沙漠等极端生境，蕴藏着丰富新颖的微生物资源。本论文选择新疆极端环境作为研究对象，进行放线菌生物多样性的研究和天然产物活性评价。通过放线菌选择性分离，从18份新疆土壤样品中共获得排重放线菌260株，通过表观形态筛选，选择61株稀有放线菌形态菌株进行深入系统分类分析。16S rRNA基因序列系统进化分析显示，61株放线菌来源于8个科，11个属，其中包括5个稀有放线菌科和8个稀有放线菌属，显示出较高的稀有放线菌多样性。同时，基于16S rRNA基因序列比对，获得两株潜在新种Nocardia sp. XJ31和Prauserella sp. XJ46，并进行了深入研究。菌株XJ46分离自新疆沙漠环境，16S rRNA基因序列比对显示其与菌株Prauserella marina MS498T相似度最高（95.9%），进一步16S rRNA基因序列系统进化分析显示菌株XJ46在普氏菌属内形成以独立稳定的分支。本论文按照多相分类标准，对菌株XJ46进行了培养特征描述、生理生化特征分析、细胞化学组分分析和分子遗传分析，鉴定菌株XJ46为一株普氏菌属新种，并命名为Prauserella shujinwangii sp. nov.菌株XJ31分离自新疆环境，经过16S rRNA基因序列比对和系统进化分析，鉴定其属于诺卡氏菌属。通过21种不同成分培养基OSMAC发酵，发现菌株XJ31能够产生丰富的次级代谢产物。进一步结合抗BCG活性测试，选择了代谢产物丰富、有抗BCG活性的Noc05培养基进行放大发酵和天然产物分离。通过系统的化学分离工作，共得到14个化合物，采用HRESIMS、MALDI-TOF MS/MS、一维和二维NMR等技术，鉴定了以上化合物的结构，包含12个寡肽类化合物（铁螯合剂，siderophore），为nocardimicin B（1），nocardimicin D（2），nocardimicin F（3），nocardimicin J（4），nocardimicin K（5），nocardimicin L（6），nocardimicin M（7），nocardimicin N（8），nocardimicin O（9），nocardimicin P（10），nocardimicin Q（11），nocardimicin R（12），以及2个蒽醌类化合物为brasiliquinone D（13）和brasiliquinone E（14）。其中，10个是新化合物（4-12，14）。本论文还对分离得到的化合物进行抗BCG活性评价，结果显示两个蒽醌类化合物13和14有较好活性，MIC值均为25 μM。 |
| 英文摘要 | Natural products, especially those originated from actinomycetes, are the main source for drug development. During the last decades, few natural products were discovered via traditional bioassay-guided isolation strategy due to its limitations, so new method and strategy need to be investigated for natural products discovery. Untapped extreme environment reserves unique actinomycetes source due to the special living conditions and pressure. With more and more novel compounds isolated from rare actinomycetes from extreme environment, rare actinomycetes are showing great significances for natural product discovery. Comparing with Streptomyces, rare actinomycete has much more space remaining to be mined and it has been becoming a new direction for natural product development.Xinjiang region harbors various ecological systems and complicated extreme environment, including plateau lake, salty soil, alkali soil and desert. This thesis studied the biodiversity of actinomycetes from Xinjiang environment along with their acitve natural products. Eighteen samples were collected in different environment in Xinjiang region. Through actinomycetes selective isolation and dereplication, 260 strains were isolated and 61 strains with special morphology were subjected to systematic taxonomy analysis. Phylogenetic analysis based on 16S rRNA gene indicated that 61 strains belong to 8 families and 11 genera, including 5 rare actinomycete families and 8 rare actinomycete genera. According to 16S rRNA gene squence analysis, strain XJ31 and strain XJ46 were identified as new species.Strain XJ46 was isolated from Xinjiang desert. 16S rRNA gene sequence phylogenetic analysis showed that strain XJ46 belongs to genus Prauserella with the highest sequence similarity with Prauserella marina MS498T. Based on polyphasic taxonomy study, including phenotypic characteristics description, physiological and biochemical property analysis, chemosystematic analysis and 16S rRNA gene sequence analysis, strain XJ46 was proposed to represent a novel species of genus Prauserella, named Prauserella shujinwangii sp. nov.Strain XJ31 was isolated from Xinjiang region and identified to be a member of genus Nocardia based on 16S rRNA gene analysis. An OSMAC stratege was applied on strain XJ31 and revealed the highly diversified secondary metabolism profile based on fermentation on twenty-one media. Among these 21 media, medium Noc05 was chosen for large-scale fermentation and subsequently isolation of natural product due to its significant potential of metabolites production as well as good anti-BCG activity. Through systematic natural product isolation, 14 compounds were purified and their structures were elucidated using HRESIMS, MALDI-TOF MS/MS, 1D NMR and 2D NMR techniquies. Twelve compounds are oligopeptides (siderophores), including nocardimicin B (1), nocardimicin D (2), nocardimicin F (3), nocardimicin J (4), nocardimicin K (5), nocardimicin L (6), nocardimicin M (7), nocardimicin N (8), nocardimicin O (9), nocardimicin P (10), nocardimicin Q (11), nocardimicin R (12)；two compounds are identified as benz[α]anthraquinones, brasiliquinones D (13) and E (14). Ten out of 14 compounds are new (4-12，14). Two benz[α]anthraquinones 13 and 14 showed good bioactivity against BCG (MIC: 25 μM). |
| 中文关键词 | 新疆地区 ; 极端环境 ; 稀有放线菌 ; 多相分类 ; 天然产物 |
| 英文关键词 | Xinjiang region extreme environment rare actinomycetes polyphasic taxonomy natural product |
| 语种 | 中文 |
| 国家 | 中国 |
| 来源学科分类 | 生物工程 |
| 来源机构 | 中国科学院微生物研究所 |
| 资源类型 | 学位论文 |
| 条目标识符 | http://119.78.100.177/qdio/handle/2XILL650/287756 |
| 推荐引用方式 GB/T 7714 | 张丽. 两株新疆来源稀有放线菌的多相分类和天然产物研究[D]. 中国科学院大学,2016. |
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