喜树种子内生放线菌的分离鉴定及抗菌活性物质初分离

Isolation and identification of endophytic actinomycetes from the seeds of Camptotheca acuminata Decne. and isolation of antimicrobial substances from those endophytic actinomycetes

摘要:

【目的】探究含有抗肿瘤活性喜树碱的喜树(Camptotheca acuminata Decne.)种子中内生放线菌的多样性，以及从内生放线菌中寻找新型活性化合物产生菌。【方法】利用放线菌富集培养基分离喜树种子内生放线菌，并根据菌落形态及16S rRNA基因序列同源性分析进行菌种鉴定；以及利用与病原微生物共培养方法检测分离菌株的抗菌活性。【结果】从上海交通大学校园的喜树种子中共分离到33株内生疑似放线菌，经基于16S rRNA基因序列分析的分子鉴定，确定其中21株属于链霉菌，1株属于拟诺卡氏菌，另有3株由于序列相似性低于95%而无法分类；8株因未能克隆16S rRNA基因序列而未确定分类。这11株疑似内生放线菌是否是新放线菌(种)类群，有待后续深入研究。初步的抗细菌、真菌活性检测发现，分离到的内生放线菌中42.42%以上对立枯丝核菌(Rhizoctonia solani Kühn)有很强的抑制菌核形成的作用，54.54%内生菌有较强抑制芽孢杆菌生长的作用。并且挑选其中具有代表性的菌株Streptomyces sp. CXSZ1进行代谢产物的分析，初步发现其代谢产物中含有抗细菌活性成分。【结论】喜树种子内生放线菌的分离、鉴定及生物活性物质分离鉴定的研究工作，一方面可以为揭示喜树等高等植物种子内生菌的起源、演化等提供有价值的信息，同时也为新结构、新活性化合物发掘提供实验材料。

Abstract:

[Objective] The seeds of Camptotheca acuminata Decne. containing camptothecin (an anticancer agent), was investigated to explore the endophytic actinomycetes diversity and screen for new active compounds producing strain. [Methods] The seeds of C. acuminata Decne. were sliced and plated on different selective media after surface sterilization. Colonies that looked like actinomycetes were selected, and identified according to the colonial morphology and 16S rRNA sequence analysis. Strains isolated were tested for antimicrobial activity using co-culture method. [Results] Thirty-three suspected endophytic actinomycetes strains were isolated from the C. acuminata Decne. seeds collected in campus of Shanghai Jiao Tong University. Among the suspected endophytic actinomycetes isolated, twenty-one were identified as Streptomyces, one was Nocardiopsis by 16S rRNA sequence analysis. Three endophytes were left to be determined due to low sequence similarity (less than 95%). The other eight strains were failed to be identified because of failure of their 16S rRNA sequence cloning. Further study was needed to determine whether or not these eleven strains were new actinomycetes species. Preliminary antibacterial and antifungal activity survey of strains isolated showed that approximately 42.42% of them can strongly inhibit the sclerotia formation of Rhizoctonia solani Kühn, and 54.54% of them can strongly inhibit the growth of Bacillus subtilis. Further, the metabolites of one representative strain—Streptomyces sp. CXSZ1 was studied and found to contain antibacterial compounds. [Conclusion] Study of isolation and identification of endophytic actinomycetes from the C. acuminata Decne. seeds and isolation of antimicrobial substances from the endophytic actinomycetes not only revealed the origination and evolution of endophytes in C. acuminata Decne. seeds, but also provided unique material for mining new structural and new bio-active compounds.