红豆杉内生真菌遗传多样性分析及生物转化研究
摘要
红豆杉又称紫杉、赤柏松,为红豆杉科红豆杉属植物,属珍稀植物濒危物种,有“活化石植物”之称。内生真菌是指在其生活史的一定阶段或全部阶段生活在植物的各种组织,对植物组织不会引起病害症状的真菌,已被作为活性次生代谢产物的重要来源。目前,红豆杉内生真菌的研究已成为医药领域的一个热点。
本试验首先利用传统的形态学鉴定方法对20株来自南方红豆杉(Taxus chinensis)和云南红豆杉(Taxus yunnanensis)的供试菌株进行形态学鉴定,结果显示：20株供试菌株被分为4类,分别为曲霉属,黑孢霉属,青霉属,木霉属。再选取7株代表内生真菌,以紫杉醇标准品作对照品,结合TLC、UV和HPLC技术,进一步研究内生真菌代谢产物的相关性,结果显示,EFTⅤ-01、EFTⅠ-03具有分泌紫杉醇或紫杉醇类似物的能力。研究发现同一种属的不同菌株间的代谢产物具有一定相关性。
为了筛选出更优良的紫杉醇产生菌,本文采用相关序列扩增多态性(SRAP)分子标记技术对20株红豆杉内生真菌菌株进行遗传多样性分析。结果显示：24对srap引物共扩增出584条带,其中多态性条带446条,多态性比率为76.4%,分子量范围为100bp～2000bp,在以相似系数0.601为阈值时,SRAP标记可将20株菌株分为4类,在以相似系数0.921为阈值时可将20株菌株进一步分为9类。研究表明红豆杉内生真菌具有丰富的遗传多样性,SRAP能较好地应用于红豆杉内生真菌种间和种内的遗传多样性分析,为产紫杉醇红豆杉内生真菌的进一步研究提供了依据。
本文结合超高效液相色谱(UPLC)和GC-MS技术,调查红豆杉内生真菌对葡萄干提取物和艾叶油的生物转化,以期获得更多萜类化合物。结果显示：以葡萄干醇提物为转化底物,目标菌株EFTV-01可能产新化合物：2,2-二甲基辛醇、2,4-二异丙基-1,1-二甲基-环己烷、甾体化合物；以艾叶油为转化底物,目标菌株EFTV-01能转化生产新的萜类化合物：丁醚脲、δ-榄香烯、δ-杜松烯和β-法呢烯。
Taxus, a genus of yews, known as the "living fossil plant", is a small coniferous tree or shrub in the yew family Taxaceae. Endophytic fungi are microorganisms living within the tissues of host plants without causing discernible manifestation of disease, and have proven to be one of the important sources of a variety of structurally novel active secondary metabolites with anticancer, antimicrobial and other biological activities. Endophytic fungi of Taxus have attracted great attention on the areas of medicine research.
20 endophytic fungi were classified by morphological identification method. The results showed that:20 endophytic fungi were classified four groups by traditional morphological identification, including Aspergillus, Nigrospora, Penicillium, Trichoderma. The relevance of secondary metabolites of endophytic fungi was studied combined with TLC, UV and HPLC techniques in this research. The results showed EFTⅤ-01, EFTⅠ-03 have the ability to yield paclitaxel or paclitaxel analogues. It suggested that metabolites of strains from the same genus have some relevance.
The genetic diversity of 20 endophytic fungi was analyzed by sequence-related amplified polymorphism (SRAP) technique. SRAP analysis was done with 24 pairs of primers. The data were analyzed by NTSYS-pc 2.1 software. All strains were uniquely distinguished by 24 pairs of SRAP primers with 584 bands and 446 polymorphism bands which generated 76.4% polymorphic ratio, with the size ranging from 100 to 2000 bp, and clustered into four major groups at GS=0.601 and further separated into nine sub-groups at GS=0.921 by UPGMA cluster. The results suggested that endophytic fungi from taxus possessed highly genetic diversity. It suggested that SRAP could really reveal the genetic diversity of endophytic fungi strains from Taxus, and also could be used as a method for genetic diversity analysis among endophytic fungi from Taxus. This study provides foundation for further study of the taxol-producing endophytic fungi based on our analysis.
The microbial transformation of raisins extract and absinthe oil by endophytic fungi isolated from the taxus was investigated to obtain some new pharmaceutical active ingredients. Screening test for the microbial transformation was carried out using TLC and UPLC and GC-MS techniques. The results showed that endophytic fungi EFTⅤ-01 can produce three new compounds, including 2,2-Dimethyloctanol, 2,4-diisopropyl-1,1-dimethyl-Cyclohexane and cyclic 1,2-ethanediyl acetal, (5.alpha.)-Cholestan-3-one, with the substrate raisins ethanol extract. It also can produce new terpenoids, including (-)-(Z)-Myrtanyl acetate, delta.-Elemene, delta.-Cadinene and beta.-Farnesene, with the substrate absinthe oil.
本试验首先利用传统的形态学鉴定方法对20株来自南方红豆杉(Taxus chinensis)和云南红豆杉(Taxus yunnanensis)的供试菌株进行形态学鉴定,结果显示：20株供试菌株被分为4类,分别为曲霉属,黑孢霉属,青霉属,木霉属。再选取7株代表内生真菌,以紫杉醇标准品作对照品,结合TLC、UV和HPLC技术,进一步研究内生真菌代谢产物的相关性,结果显示,EFTⅤ-01、EFTⅠ-03具有分泌紫杉醇或紫杉醇类似物的能力。研究发现同一种属的不同菌株间的代谢产物具有一定相关性。
为了筛选出更优良的紫杉醇产生菌,本文采用相关序列扩增多态性(SRAP)分子标记技术对20株红豆杉内生真菌菌株进行遗传多样性分析。结果显示：24对srap引物共扩增出584条带,其中多态性条带446条,多态性比率为76.4%,分子量范围为100bp～2000bp,在以相似系数0.601为阈值时,SRAP标记可将20株菌株分为4类,在以相似系数0.921为阈值时可将20株菌株进一步分为9类。研究表明红豆杉内生真菌具有丰富的遗传多样性,SRAP能较好地应用于红豆杉内生真菌种间和种内的遗传多样性分析,为产紫杉醇红豆杉内生真菌的进一步研究提供了依据。
本文结合超高效液相色谱(UPLC)和GC-MS技术,调查红豆杉内生真菌对葡萄干提取物和艾叶油的生物转化,以期获得更多萜类化合物。结果显示：以葡萄干醇提物为转化底物,目标菌株EFTV-01可能产新化合物：2,2-二甲基辛醇、2,4-二异丙基-1,1-二甲基-环己烷、甾体化合物；以艾叶油为转化底物,目标菌株EFTV-01能转化生产新的萜类化合物：丁醚脲、δ-榄香烯、δ-杜松烯和β-法呢烯。
Taxus, a genus of yews, known as the "living fossil plant", is a small coniferous tree or shrub in the yew family Taxaceae. Endophytic fungi are microorganisms living within the tissues of host plants without causing discernible manifestation of disease, and have proven to be one of the important sources of a variety of structurally novel active secondary metabolites with anticancer, antimicrobial and other biological activities. Endophytic fungi of Taxus have attracted great attention on the areas of medicine research.
20 endophytic fungi were classified by morphological identification method. The results showed that:20 endophytic fungi were classified four groups by traditional morphological identification, including Aspergillus, Nigrospora, Penicillium, Trichoderma. The relevance of secondary metabolites of endophytic fungi was studied combined with TLC, UV and HPLC techniques in this research. The results showed EFTⅤ-01, EFTⅠ-03 have the ability to yield paclitaxel or paclitaxel analogues. It suggested that metabolites of strains from the same genus have some relevance.
The genetic diversity of 20 endophytic fungi was analyzed by sequence-related amplified polymorphism (SRAP) technique. SRAP analysis was done with 24 pairs of primers. The data were analyzed by NTSYS-pc 2.1 software. All strains were uniquely distinguished by 24 pairs of SRAP primers with 584 bands and 446 polymorphism bands which generated 76.4% polymorphic ratio, with the size ranging from 100 to 2000 bp, and clustered into four major groups at GS=0.601 and further separated into nine sub-groups at GS=0.921 by UPGMA cluster. The results suggested that endophytic fungi from taxus possessed highly genetic diversity. It suggested that SRAP could really reveal the genetic diversity of endophytic fungi strains from Taxus, and also could be used as a method for genetic diversity analysis among endophytic fungi from Taxus. This study provides foundation for further study of the taxol-producing endophytic fungi based on our analysis.
The microbial transformation of raisins extract and absinthe oil by endophytic fungi isolated from the taxus was investigated to obtain some new pharmaceutical active ingredients. Screening test for the microbial transformation was carried out using TLC and UPLC and GC-MS techniques. The results showed that endophytic fungi EFTⅤ-01 can produce three new compounds, including 2,2-Dimethyloctanol, 2,4-diisopropyl-1,1-dimethyl-Cyclohexane and cyclic 1,2-ethanediyl acetal, (5.alpha.)-Cholestan-3-one, with the substrate raisins ethanol extract. It also can produce new terpenoids, including (-)-(Z)-Myrtanyl acetate, delta.-Elemene, delta.-Cadinene and beta.-Farnesene, with the substrate absinthe oil.
引文
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