8种贵州药用植物内生放线菌的分离及多样性研究

Diversity of endophytic actinobacteria isolated from medicinal plants collected from Guizhou Province

摘要:

【背景】药用植物内生菌能产生多种活性较好的次级代谢产物，是新抗生素发现的重要来源。目前，药用植物内生菌的研究主要集中在内生真菌，内生放线菌研究相对较少，是一个尚未充分开发的新领域。【目的】研究贵州药用植物内生放线菌多样性，以期发现新种放线菌或产新活性物质的放线菌，为微生物药物研究奠定基础。【方法】药用植物样品表面消毒后用粉碎机粉碎，采用9种不同分离培养基分离放线菌；通过提取放线菌基因组DNA，PCR扩增16S rRNA基因序列，测定序列并提交Ezbiocloud数据库进行序列比对，构建系统进化树，进行内生放线菌多样性分析。【结果】通过形态特征初步排重，从8种药用植物样品中共分离得到内生菌62株，测定其16S rRNA基因序列，序列比对结果表明其中的57株菌分布于放线菌域的5目8科16属，分别是短小杆菌属(Curtobacterium)、链霉菌属(Streptomyces)、微杆菌属(Microbacterium)、纤维单胞菌属(Cellulomonas)、植物杆菌属(Plantibacter)、原小单孢菌属(Promicromonospora)、动球菌属(Kineococcus)、壤球菌属(Agrococcus)、沼杆菌属(Patulibacter)、气微菌属(Aeromicrobium)、拉贝达氏菌属(Labedella)、叶居菌属(Frondihabitans)、冷杆菌属(Frigoribacterium)、节杆菌属(Arthrobacter)、动孢菌属(Kineosporia)、Amnibacterium属，其中短小杆菌属(Curtobacterium)为优势菌属，菌株M8JJ-5和M2KJ-4的16S rRNA基因序列分别与有效发表菌株Amnibacterium kyonggiense KSL51201-037T (FJ527819)和Aeromicrobium fastidiosum DSM10552T (Z78209)的相似性最高，相似性分别为97.29%和98.95%，可能为潜在新物种。【结论】贵州药用植物中蕴藏着多样性丰富的放线菌且具有从中发现新放线菌的潜力，能为新抗生素的发现提供菌种储备，也为今后对该地区放线菌资源的认识和开发利用提供理论依据。

Abstract:

[Background] Endophyte from medicinal plants were important resource for discovery of new antibiotics due to their ability of producing a wide variety of secondary metabolites with good activity. Concerning the endophytic fungi, many studies have been carried out, but few reported about endophytic actinobacteria. [Objective] This study is aimed at analyzing the diversity of actinobacteria isolated from medicinal plants in Guizhou Province. [Methods] Various tissues from 8 kinds of medicinal plants as samples were collected in Guizhou Province, China. Samples were grinded into powders after surface sterilization and cultured on 9 different media for strain isolation. To determine the phylogenetic position of the strains, 16S rRNA gene sequences were obtained by PCR amplification and direct sequencing, then compared with EzBioCloud database, finally carried out phylogenetic analysis based on 16S rRNA gene sequences. [Results] A total of 62 strains were obtained, 57 of which were actinobacteria and were affiliated to 16 genera 8 families 5 order, contained genus Curtobacterium, Streptomyces, Microbacterium, Cellulomonas, Plantibacter, Promicromonospora, Kineococcus, Agrococcus, Patulibacter, Aeromicrobium, Labedella, Frondihabitans, Frigoribacterium, Arthrobacter, Kineosporia and Amnibacterium. The dominant genus is Curtobacterium. The highest similarity of 16S rRNA gene sequence between strain M8JJ-5 and the validly described species Amnibacterium kyonggiense KSL51201-037T (FJ527819), between strain M2KJ-4 and the validly described species Aeromicrobium fastidiosum DSM10552T (Z78209) was 97.29% and 98.95%, respectively, which indicated that the strains M8JJ-5 and M2KJ-4 were potential new taxa. [Conclusion] The study demonstrates that diversity of endophytic actinobacteria from medicinal plants are rich and medicinal plants as a special resource for discovery of new pharmaceutical microorganisms deserved to be further explored.