3种黄连属濒危植物内生放线菌多样性及其抗菌消炎活性初探

At present, the need for Coptidis Rhizoma is increasing due to clinical adverse reactions of anti-inflammatory drugs and the emergence of drug-resistant bacteria, which leads to the deficiency of wild Coptis spp.. Some endophytes could excrete the same or similar compounds that may be involved in a symbiotic association with the host plants. Therefore, the research of endophytic actinomyces reside in Coptis spp. provide a new way to develop novel antimicrobial and anti-inflammatory drugs. In this study, three endangered plants, which are included in Chinese Pharmacopoeia, Coptis chinensis Franch., Coptis deltoidea C. Y. Cheng et Hsiao and Coptis teeta Wall., are used as raw materials. Culture-independent analysis will be implemented through Miseq high-throughput sequencing technology on the endophytic actinomyces. Systematic research of the separation method is suitable for endophytes associated with Coptis spp. will be carried out, according to the results of culture-independent approach and the analysis of physical and chemical properties of Coptis spp., to increase the number of rare actinomycetes, and to get as much as possible pure cultures. Systematic identification of isolated strains will be executed by the polyphasic taxonomic analysis, to further establish relevant actinomycetes repository. Through comprehensive analyzing the results from culture-independent and culture-dependent approaches, the diversity and community structure of endophytic actinomycetes associated with the three endangered medicinal coptis species will be revealed. In order to get active actinomycetes, which can be used to develop new drugs in the future, the isolated strains are screened for antimicrobial and anti-inflammatory activities. This study will provide basis for later studies and the development of new drugs, and has significant meaning for rescuing and exploring endophytic actinomycetes resources isolated from endangered plants.

目前临床抗炎药物的不良反应和耐药菌的出现，使副作用小、不易耐药的传统抗菌消炎药材黄连的需求增加，导致其原植物野生资源匮乏。内生菌具有产生与其宿主植物相同或相近化合物的能力，研究黄连植物的内生放线菌可为新型抗菌消炎药物开发提供新途径。本研究以中国药典收录的“味连、雅连、云连”的3种濒危原植物为材料，运用Miseq高通量测序技术对其内生放线菌进行免培养分析；同时结合免培养及样品理化分析结果，系统研究适合黄连内生放线菌的分离方法，提高稀有放线菌出菌率，获得尽量多的纯培养物，运用多相分类技术对分离到的菌株进行系统鉴定，建立相关放线菌资源库；综合分析免培养与纯培养结果，初步揭示3种濒危黄连属药用植物内生放线菌的多样性及类群结构。并对分离到的纯化菌株进行抗菌、抗炎活性筛选，以期得到具有开发潜力的活性菌株，为后期相关药物开发及其他研究提供基础。本研究对抢救和研究我国濒危植物内生放线菌资源具有科学意义。

黄连为我国传统药用植物，近年来受到药用价值及经济价值的诱惑，黄连属药用植物遭到过度采挖，导致野生居群急剧减少。内生菌具有产生与其宿主植物相同或相近化合物的能力，研究黄连植物的内生菌可为新型活性物质发现提供思路。本项目以中国药典收录的“味连、雅连、云连”的3种濒危原植物为材料，运用16S rRNA基因扩增子高通量测序技术及纯培养两种方法对其内生细菌进行多样性及群落结构分析。研究结果表明：通过免培养分析发现，黄连根内所含细菌及放线菌的多样性高于茎和叶，且差异显著；而在三种黄连之间以雅连多样性最好，但无显著差异；人工黄连内生菌多样性稍高于野生黄连，但差异不显著。在门水平上，黄连以Cyanobacteria、Proteobacteria、Actinobacteria、Firmicutes、Bacteroidetes为主要类群，其中放线菌门占18.87%；属水平上，Mycobacterium、Rhizobium、Salmonella、Bradyrhizobium为主要类群，其中放线菌以Mycobacterium和Nocardioides两个属为主要类群。通过云连微生物群落组成与根际土壤主要养分因子及根茎小檗碱含量的相关性分析，发现根茎内小檗碱含量与根际土壤总氮、总磷、总钾及速效钾含量成正相关，且根际土壤总钾含量与Microbacterium正相关，而Microbacterium 又和根茎内小檗碱含量表现出强正相关性。这提示我们根际土壤钾含量可能会影响Microbacterium 的富集而影响小檗碱的生成，农业领域可通过针对性的施肥促进黄连有效成分的快速积累，有关机理及验证工作有待后续深入研究。通过完善并建立纯培养方法，共得到内生细菌及放线菌315株，分属于3门5纲15目24科47属，其中内生放线菌112株，分属于1纲7目11科21属，并通过16S rRNA基因测序分析，完成纯培养细菌及放线菌的系统鉴定，确定其分类地位。通过体外抗菌消炎活性筛选，得到抗菌菌株15株，抗炎菌株1株。本研究对抢救和研究我国濒危植物内生菌资源及指导其农业栽培具有一定意义。