一株抗真菌海洋放线菌的筛选、分离和多相分类鉴定

Screening,Isolation and Polyphasic Taxonomic Identification of an Anti-Fungal Marine Actinomycete

为开发利用宁波近海潮间带药用微生物资源,本研究自宁波慈溪海塘采集7份海泥样品,从中筛选、分离抗真菌放线菌,并对目标菌株进行多相分类鉴定,包括16S rRNA、gyrB基因序列分析、形态观察、生理生化鉴定及细胞化学组成分析。通过筛选培养获得了25个菌株,其中2P-4对白色念珠菌、黑曲霉表现出抑制生长的作用。细胞化学组成鉴定分析了2P-4全细胞糖、氨基酸、极性脂质、甲基萘醌和脂肪酸组成,结果表明,2P-4属于链霉菌属。2P-4的16S rRNA、gyrB基因序列分析也表明2P-4属于链霉菌属,并且与毒三链霉菌的关系最密切,其16S rRNA基因序列与Streptomyces toxytricini NBRC 13194~T的相似度为99.56%,gyrB基因序列与Streptomyces toxytricini NRRL B-5426~T的相似度为99.04%,但16S rRNA、gyrB基因序列在系统进化树上均形成独立分枝,支持2P-4与毒三链霉菌分属不同的种;此外,2P-4与其近缘种Streptomyces toxytricini NBRC 13194~T相比,两者在形态和生理生化特征方面存在明显差异。基于以上分析,初步鉴定2P-4为链霉菌属的一个新种。本研究为进一步探究2P-4抗真菌代谢产物提供了理论依据。

To develop and utilize medicinal microorganism resources of intertidal sediments,7 soil samples were collected from Cixi Haitang,Ningbo,to screen and isolate antifungal Actinomycetes,then polyphasic taxonomic identification of target strains were conducted,including 16 S rRNA and gyrB gene sequence analysis,morphological observation,physiological and biochemical identification and cytochemical composition analysis.25 strains were obtained by screening and culture,among which 2 P-4 showed inhibitory effects on the growth of Candida albicans and Aspergillus niger.The whole cell sugars,amino acids,polar lipids,methyl naphthoquinones and fatty acids composition of 2 P-4 were analyzed,indicating that 2 P-4 belonged to Streptomyces;sequence analysis of 16 S rRNA and gyrB gene also showed that 2 P-4 belonged to Streptomyces and was most closely related to Streptomyces toxytricini,and its 16 S rRNA gene sequence has 99.56%similarity to Streptomyces toxytricini NBRC 13194,its gyrB gene sequence has 99.04%similarity to Streptomyces toxytricini NRRL B-5426,however,16 S rRNA and gyrB gene sequences were classed into independent clades on the phylogenetic trees,supporting that 2 P-4 was not a member of Streptomyces toxytricini.In addition,there were significant differences in morphology,physiological and biochemical characteristics between 2 P-4 and its relative species Streptomyces toxytricini NBRC 13194.Based on these analyses,2 P-4 was preliminarily identified as a novel species of Streptomyces.This study provided experimental basis for further study of anti-fungal metabolites of 2 P-4.