河南神仙洞放线菌资源勘探及抗菌活性筛选  

Study on diversity and bioactivity of actinomycetes isolated from the Shen xian dong in Henan Province

作　　者：戴素娟[1] 李静[1] 刘少伟[1] 李小俊[1] 蒋忠科[1] 姜蓉[1] 孙承航[1] 

机构地区：[1]中国医学科学院&北京协和医学院医药生物技术研究所,北京100050

出　　处：《中国抗生素杂志》2015年第9期641-648,共8页Chinese Journal of Antibiotics

基　　金：2014年度中央级公益性科研院所基本科研业务专项(IMBF201407);国家自然科学基金(81172963和81321004);十二五"重大新药创制"科技重大专项(2012ZX09301-002-001-018)

摘　　要：目的勘探河南神仙洞放线菌多样性,以期发现药用微生物资源,为新抗生素发现奠定基础。方法采用10种分离培养基,以稀释涂布法分离放线菌;采用16S r RNA基因序列比对分析开展初步鉴别;经液体发酵,发酵液乙酸乙酯萃取,菌丝体丙酮浸提,获得提取浓缩物样品,采用纸片扩散法对样品进行抗菌活性筛选,活性菌株进行次级代谢产物生物合成基因NRPS,PKS I和PKS II的筛选。结果从6份土壤样品中共纯化到179株放线菌;经16S r RNA基因序列比对分析,它们分布于11个科18个属,其中链霉菌属为优势菌属;菌株S6R2A4-9的16S r RNA基因序列与最近有效菌株Flindersiella endophytica EUM 378T的相似率为93.58%,为潜在新属;发酵74株放线菌,其中52株在至少一个抗菌活性筛选中为阳性,总阳性率为70.27%。52株有活性的放线菌中,48株菌存在至少一个次级代谢产物生物合成基因簇,阳性率为92.3%,其中17株同时具有3种生物合成基因簇。结论河南新密神仙洞土壤中存在较为丰富的药用放线菌资源,具有从中发现放线菌新物种及新抗生素的潜力。Objective To study the diversity and bioactivity of actinomycetes isolated from Shen Xian Dong in Henan Province, China. Methods 179 isolates were obtained by using standard serial dilution plating method, with ten different types of cultural media. Actinomycete strains were analyzed by comparison of 16S rRNA gene sequence. Fermentation broths and mycelia were extracted by ethyl acetate and acetone, respectively. The antifungal and antibacterial activities of the crude extracts were tested by disk diffusion method. Screening of PKS I, PKS II and NRPS genes of the bioactive strains were carried out by PCR and agarose gel electrophoresis. Results 179 strains distributed in 18 different genera affiliated with 11 families. The dominant genus was Streptomyces. The 16S rRNA sequence similarity between strain S6R2A4-9 and the nearest strain Flindersiella endophytica EUM 378r in EzBioCloud database （FJ805430） was 93.58%, which indicated that strain S6R2A4-9 is a potential new genus of Nocardioidaceae. Antimicrobial assay showed that 52 among 74 strains exhibited positive results in antifungal or antibacterial assay, and the total positive rate was 70.27%. PCR results showed 48 in 52 bioactive strains have at least one biosynthetic gene including NRPS, PKS I and PKS II, the total positive rate was 92.3%. 17 strains have the all three biosynthetic genes. Conclusion The study demonstrated that 18 genera of actinomycetes were isolated from soil samples collected from Shen Xian Dong, many of them had antifungal or antibacterial activities, which could be a potential source for the discovery of new antibiotics.

关 键 词：洞穴 放线菌 多样性 生物合成基因簇 生物活性 

分 类 号：R978.1[医药卫生—药品]