塔里木盆地胀果甘草内生放线菌多样性及抗菌活性分析

Diversity and antimicrobial activity of endophytic actinobacteria from Glycyrrhizain flata Bat.

【目的】发掘具有开发前景的放线菌资源,对分离自新疆胀果甘草的内生放线菌的多样性、抗菌活性和次级代谢产物合成相关基因进行研究。【方法】采用5种培养基和3种前处理方法,从胀果甘草中分离获得80株放线菌。基于菌株形态学特征,对36株代表菌株进行抗菌活性检测,通过特异性引物扩增方法,检测了PKS I、PKS II、NPRS和卤化酶基因,探究其合成天然产物的潜在能力。结合筛选结果,选取其中20株代表菌,经16S r RNA基因测序,对其进行系统发育分析。【结果】培养基E2和E3结合热处理的分离效果较好;86.1%的代表菌株对供试的细菌、病原真菌表现出了不同程度的抗菌活性,PKS I、PKS II、NRPS基因和卤化酶基因阳性检出率分别为16.7%、72.2%、25.0%和11.1%。具有活性功能的代表菌株经16S r RNA基因测序分析,分别属于链霉菌属(Streptomyces)、小单胞菌属(Micromonospora)、红球菌属(Rhodococcus)和游动放线菌属(Actinoplanes)4个属,其中链霉菌属(Streptomyces)为优势菌属,占60%以上。【结论】胀果甘草是我国传统的药用植物,其植株内部蕴藏着丰富的放线菌资源,并在次生代谢产物合成方面拥有巨大潜力,具有进一步开发的价值。

[Objective] In order to explore actinobacteria resources, the diversity, antimicrobial activity and secondary metabolites biosynthesis genes of endophytic actinobacteria isolated from Glycyrrhizain flata Bat. were studied. [Methods] 80 endophytic actinobacteria strains were isolated from Glycyrrhizain flata Bat. by employing five selective media and three isolated procedures. 36 representative strains were selected for antimicrobial activity analysis and amplifying genes coding for polyketide synthases（PKS I, PKS II）, nonribosomal peptide synthetases（NRPS） and halogenated enzyme gene（Halo）. Based on the results of antimicrobial activity, 20 representatives were chosen for 16 S rRNA gene sequencing. [Results] The better separating conditions were observed with Medium E2 and E3 combined with dry-air treatment. 86.1% out of 36 representative strains showed antimicrobial activity against at least one of tested pathogenic organisms, and the number of strains with PKSI, PKSII, NRPS and halogenated enzyme gene fragments matching the anticipated length were 16.7%, 72.2%, 25.0%, 11.1%. According to the phylogenetic analysis, twenty isolates were belonging to Streptomyces, Micromonospora, Rhodococcus and Actinoplanes, respectively. Streptomyces was the most common endophytic actinobacteria of Glycyrrhizain flata Bat., accounting for above 60%. [Conclusion] Liquorice is traditional chinese medicinal plant, the strains isolated from Glycyrrhizain flata Bat. are potent source which produce multiple compounds and can be further explored.