云南美登木内生真菌及其抗结核活性成分的研究
其他题名Studies on endophytic fungi from Maytenus hookeri and their antituberculosis constituents
倪志伟
学位类型硕士
导师曾英
2007-05-30
学位授予单位中国科学院昆明植物研究所
学位授予地点昆明植物研究所
学位专业植物学
关键词云南美登木 内生真菌 活性追踪分离 抗结核 Chaetoglobosin b
摘要植物内生真菌为具有生物活性天然产物的开发提供了丰富资源,其中不乏大量有药用价值的成分。本论文以本实验室分离自云南美登木(Maytenus hookeri)的内生真菌为研究对象,获取了对结核分支杆菌(Mycobacterium tuberculosis,MTB)具有有效抑制生长作用的成分。首先将内生真菌活化培养,通过形态学的特征,结合核糖体rRNA基因转录单元内间隔区(Internal Transcribed Spacer,ITS)序列分析对可培养真菌进行分类鉴定,同时以金黄色葡萄球菌、结核分支杆菌、白色念珠菌、酿酒酵母和橙色青霉UC-4376为指示菌株,通过纸片扩散法检测各真菌PDA发酵粗提物的抑菌活性。研究结果表明:鉴定出的24株云南美登木内生真菌,分别属于5目(丛梗孢目Moniliales、肉座菌目Hypocreales、鹿角菌目Xylariales、球壳孢目Sphaeropsidales、黑盘孢目Melanconiales)、9科(丛梗孢科Moniliaceae、暗梗孢科Dematiaceae、瘤座孢科Tuberculariaceae、麦角菌科Clavicipitaceae、毛壳菌科Chaetomiaceae、鹿角菌科Xylariaceae、腐皮壳菌科Diapothaceae、球壳孢科Sphaeropsidaceae、黑盘孢科Melanconiaceae)、14属(青霉属Penicillium、曲霉属Aspergillus、轮枝菌属Verticillium、木霉菌属Trichoderma、链格孢菌属Alternaria、节孢霉属Arthrinium、镰刀菌属Fusarium、被毛孢属Hirsutella、毛壳菌属Chaetomium、炭角菌属Xylaria、间座壳属Diaporthe、拟茎点霉属Phomopsis、葡萄座腔菌属Botryosphaeria、刺盘孢属Colletotrichum)。有抑菌活性的菌株大多分布于丛梗孢目(Moniliales);其中,Ly50'(毛壳菌属)、Lr82(木霉菌属)、Lr417(镰刀菌属)三株真菌的发酵产物具有显著抑制结核分支杆菌生长的活性。我们选择抑菌活性明显的Ly50'、Lr82分别进行10 L体积的PDA琼脂平板固体发酵培养,通过纸片扩散法进行活性追踪分离(Bioassay-guided fractionation,Bioassay-directed fractionation),从Ly50'的发酵产物中分离得到有效抗结核活性成分—chaetoglobosin B,这是首次发现chaetoglobosin B具有抗MTB活性;同时发现Lr82发酵产物的有效抗MTB组分为脂肽类化合物。 本论文通过活性追踪方法来进行分离,针对性地得到与靶标微生物拮抗的活性天然产物。对同一内生真菌若采用不同的靶标微生物做为指示菌株,则发现其中蕴含不同的活性组分。对内生真菌次生代谢产物的活性追踪研究将会开启一扇有应用价值的天然产物宝库的大门。
其他摘要Bioactive natural products from endophytic fungi have enormous potential as the source of new medicine. In this thesis, the endophytic fungi from Maytenus hookeri were studied and their antimicrobial constituents against Mycobacterium tuberculosis were identified. The endophytic fungi were cultivated on PDA medium after activation. They were phylogenetically identified through analyses of morphological characteristic traits combined with nucleotide sequence of internal transcribed spacers (ITS). Their antimicrobial activities were examined with crude extracts via disk diffusion bioassay against microbes of Staphylococus aureus, Mycobacterium tuberculosis, Candida albicans, Saccharomyces ceevisiae, Penicilium avellaneum UC-4376. Results showed that 24 strains of the endophytic fungi from M. hookeri could be classified to be 14 genera (Penicillium, Aspergillus, Verticillium, Trichoderma, Alternaria, Arthrinium, Fusarium, Hirsutella, Chaetomium, Xylaria, Diaporthe, Phomopsis, Botryosphaeria, Colletotrichum) of 9 families (Moniliaceae, Dematiaceae, Tuberculariaceae, Clavicipitaceae, Chaetomiaceae, Xylariaceae, Diapothaceae, Sphaeropsidaceae, Melanconiaceae) of 5 orders (Moniliales, Hypocreales, Xylariales, Sphaeropsidales, Melanconiales). In addition, most of the antimicrobial strains belonged to Moniliales. Crude extracts from Ly50' (Chaetomium), Lr82 (Trichoderma) and Lr417 (Fusarium) fermentation significantly inhibit the growth of M. tuberculosis. Ly50' and Lr82 were therefore incubated on 10L solid-state PDA medium for further study, respectively. Based on bioassay-guided fractionation, chaetoglobosin B from Ly50' was determined as the potent component against M. tuberculosis. The constituents against M. tuberculosis in Lr82 were analyzed as lipopeptides. Bioassay-guided fractionation mentioned in the thesis may give a explicit target towards the bioactive constituents. If more targets are given, more valuable active constituents can be separated efficiently through an appropriate high-throughput screening. It is believed that the application of bioassay-guided fractionation might enlighten the access to the natural products.
页数146
语种中文
文献类型学位论文
条目标识符http://ir.kib.ac.cn/handle/151853/208
专题昆明植物所硕博研究生毕业学位论文
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倪志伟. 云南美登木内生真菌及其抗结核活性成分的研究[D]. 昆明植物研究所. 中国科学院昆明植物研究所,2007.
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