川楝内生放线菌多样性及抗菌活性筛选

Diversity and antimicrobial activity of endophytic actinobacteria isolated from Melia toosendan Sieb. et Zucc.

摘要:

【目的】探究药用植物川楝内生放线菌多样性，从中挖掘出新的放线菌菌株，发现新的潜在农业生防和医药先导化合物。【方法】从四川境内的资阳、遂宁以及重庆万州采集川楝的根、茎、叶、果、皮，采用纯培养方法，用4种培养基共分离获得148株放线菌。通过形态学观察筛选出60株放线菌进行RFLP分析，选出代表菌株进行16S rRNA基因序列分析。以3株细菌和6株病原真菌作为指示菌株，检测初筛出的60株菌株的抗菌活性以及聚酮合酶(PKSⅠ、PKSⅡ)基因、非核糖体多肽合成酶(NRPS)基因和卤化酶(Halo)基因。【结果】基于16S rRNA-RFLP分析，60株放线菌被分成10簇，筛选出25株代表菌株分别属于7个属，包括Streptomyces、Micromonospora、Planotetraspora、Streptosporangium、Nocardiopsis、Prauseria、Microbispora，其中链霉菌占73.3%。供试的川楝内生放线菌对细菌、真菌有不同程度的抗菌活性；其中含有4类化合物合成基因的菌株占10%?55%。【结论】药用植物川楝内生放线菌具有丰富的多样性，且不同地区不同部位川楝组织中放线菌的种群存在差异；分离菌株广谱的抗菌活性证明，川楝内生放线菌在次生代谢产物合成方面具有巨大潜力，这为进一步的药物开发提供了丰富的菌种资源。

Abstract:

[Objective] In order to explore new potential agricultural biocontrol actinobacteria strains and discover drug lead compounds, the diversity of endophytic actinobacteria isolated from medicinal Melia toosendan Sieb. et Zucc. were researched. [Methods] 148 endophytic actinobacteria were isolated from Melia toosendan Sieb. et Zucc. collected in Ziyang, Suining of Sichuan Province and Chongqing by four isolation medium. Based on morphological characteristic, 60 isolates were selected and analyzed by 16S rRNA-RFLP, the representative strains were further sequenced. Antibacterial activity of 60 strains were evaluated by three bacteria and six pathogenic fungi strains, respectively, and the genes coding for polyketide synthases (PKSI, PKSII), nonribosomal peptide synthetase (NRPS) and halogenated enzyme gene (Halo) were amplified. [Results] 60 strains of actinobacteria were divided into 10 clusters based on 16S rRNA-RFLP analysis, 25 strains of representative strains belonging to 7 genera: Streptomyces, Micromonospora, Planotetraspora, Streptosporangium, Nocardiopsis, Prauseria, Microbispora, the genus Streptomyces accounted for 73.3%. An evaluation of antimicrobial activity showed most isolates possessed activity towards pathogenic bacteria and fungi. The frequency of PCR amplification of 4 synthesis genes was 10%?55%. [Conclusion] This study indicated that endophytic actinobacteria associated Melia toosendan Sieb. et Zucc. are abundant and the distribution is influenced by sampling sites and plant organs, which possessed potential diverse bioactivities and can be further explored.