百部内生放线菌的分离、分类及次级代谢潜力

Isolation, classification and biosynthetic potential of endophytic actinomycetes from Stemona

摘要:

摘要:【目的】以对叶百部块根为材料分离内生放线菌，并对分离菌株进行分类、抗菌活性和次级代谢产物合成基因研究。【方法】样品经过严格的表面消毒，选用4 种培养基分离百部内生放线菌;分离菌株通过形态观察和16S rRNA 序列分析进行分类鉴定;采用琼脂移块法测试分离菌株的抗菌活性;通过PCR 检测分离菌株的PKS/NPRS 和卤化酶基因;使用HPLC-UV/VIS-ESI-MS/MS 分析发酵产物。【结果】从6 个样品中获得18 株内生放线菌，分属链霉菌属(Streptomyces)、小单孢菌属(Micromonospora)、假诺卡氏菌属(Pseudonocardia)和甲基杆菌属(Methylobacterium)。分离菌株绝大部分具有抗菌活性和次级代谢产物合成基因，其中13 株对耐药金黄色葡萄球菌和/或绿脓杆菌有拮抗活性，17 株具有PKS/NRPS 基因，8 株菌具有卤化酶基因，且卤化酶阳性代表菌株的发酵产物具有抗细菌活性和卤代化合物特征。【结论】百部作为一种传统中药，其内生放线菌以链霉菌和小单孢菌为主，在次级代谢产物合成方面具有很好的潜力，可作为一类重要微生物资源进行活性产物开发。

关键词:关键词:百部，内生放线菌，分类，抗菌活性，次级代谢基因

Abstract:

Abstract:［Objective］To isolate endophytic actinomycetes from earthnut samples of Stemona tuberosa Lour. collected from Xishuangbanna，Yunnan Province，and to study the classification，antimicrobial activity and secondary metabolic potential of the isolates． ［Methods］Samples were treated by a well-established surface-sterilized procedure，and plated on four selective media． The isolates were identified by morphology and 16S rRNA gene sequence analysis． Antimicrobial activity of the isolates was tested using agar diffusion method． Secondary metabolite genes of PKS /NRPS and halogense were detected by PCR amplification． HPLC-UV and VIS-ESI-MS /MS were used to analyze the fermentation product． ［Results］We obtained 18 actinomycete isolates from 6 Stemona earthnut samples． The isolates belonged to 4 genera，Streptomyces，Pseudonocardia，Micromonospora and Methylobacterium，and 10 of them were streptomycetes． The isolates also showed distinguish positive rates of antibacterial activity and secondary metabolite genes． Of them 13 strains showed antimicrobial activity against Staphlococcus aureus and/or Pseudomonas aeruginosa． Of them 17 isolates were positive for PKS/NRPS genes，and 8 strains were positive for halogenase gene. Moreover，the representative halogenase gene-positive strain produced metabolites that had typical MS peaks of halogen absorption． ［Conclusion］Based on the results of this study，the endophytic actinomycetes from Stemona，dominant by Streptomyces and Micromonospora，have good secondary metabolic potential and could serve as a promising resource for bioactive metabolite discovery in the future．

Key words:Keywords: Stemona，endophytic actinomycetes，classification，antimicrobial activity，secondary metabolite gene