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沙特阿拉伯沙漠可培养放线菌多样性及抗菌活性的研究

Study on the Diversity of Culturable Actinobacteria in Saudi Arabian Desert and Their Antibacterial Activities

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【Author in Chinese】 杨子文

【Supervisor】 李文均

【Author's Information】 中山大学, 生态学, 2018, 硕士

【Abstract in Chinese】 沙漠生态系统是地球上最大的陆地生态系统,然而到目前为止仍然没有系统且完整的对沙漠生态系统的微生物资源进行研究和利用。为此,本论文的研究着眼于沙漠生态系统的微生物尤其是放线菌资源,希望此研究能够对以后沙漠环境微生物资源的挖掘利用做出一定贡献。研究采用了经典的纯培养方法,挖掘了沙特阿拉伯沙漠环境的可培养放线菌资源,并对分离所获得的放线菌进行初步抗菌活性检测,期望从中挖掘到结构新颖的活性分子。最后选取4株潜在的放线菌新种进行多相分类鉴定,明确了它们的分类地位。选取了沙特阿拉伯沙漠生态系统中的5个不同样品作为研究对象,采取了有利于放线菌生长的预处理方法和不同的选择性分离培养基。总共分离并纯化了521株放线菌。根据菌落的形态、颜色等特征进行去重复,对合并后的401株菌进行了16S rRNA基因序列比对鉴定。结果表明:这些菌株分别属于放线菌门中的11个目22个科和37个属。采用16S rRNA基因序列比对的方法,发现了16S rRNA基因序列相似性在98.7%以下的潜在新分类单元菌株65株,占全部分离获得菌株的16.2%。在所有获得的放线菌菌株中,链霉菌属(Streptomyces)所占比例最高,其他例如植物内生放线菌属(Actinophytocola)、芽球菌属(Blastococcus)、白蚁菌属(Isoptericola)、解木聚糖微菌属(Xylanimicrobium)、游动双孢菌属(Planobispora)、假棒形杆菌属(Pseudoclavibacter)、纤维素菌属(Cellulosimicrobium)和类谷氨酸杆菌属(Paeniglutamicibacter)等稀有菌属也在这次分离结果中有所发现。选取200株前期分离得到的放线菌进行了抗菌活性检测。检测结果发现存在46株放线菌至少对3种及以上的指示菌有抑制作用,占测试菌株总数的23%,同时有10株对5种以上的指示菌都有抑制作用,表现出光谱的抗菌活性。同时在所有活性菌株中,链霉菌属展现出了较强的抗菌活性,是所有受测菌株所占比例最大的类群。除此之外还有5株菌对于青枯病致病菌茄科雷尔氏菌(Ralstonia solanacearum)有着明显的拮抗作用。结果表明,沙漠环境中的放线菌具有巨大的活性天然产物开发与应用的潜力,是寻找新型抗生素的一个宝库。实验选取四株放线菌菌株SYSU D8007~T,SYSU D8014~T,SYSU D8010~T和SYSU D8004~T进行了潜在新种的多相分类的研究工作,通过不同指标的检测及对比以及进化树的构建,明确了这4个菌株的分类地位,并分别命名为Microbacterium album sp.nov.,Microbacterium deserti sp.nov.,Saccharopolyspora deserti sp.nov.和Georgenia deserti sp.nov.。本课题通过纯培养的研究方法对沙特阿拉伯沙漠环境放线菌多样性进行了初步探索,为沙漠环境放线菌群落与资源的更进一步的系统性研究打下了基础。同时,分离菌株的抗菌活性筛选结果展现了沙漠环境中生存有大量具有强烈次级代谢能力的菌株,说明沙漠环境是一个筛选新的活性产物的宝库。

【Abstract】 Desert environment is the largest terrestrial ecosystem in the world,but scanty works have been done to explore their microbial resources and diversity.In the present study,we try to analyse the diversity and distribution of actinobacteria in the Saudi Arabian Desert environment by utilizing a culture-based laboratory method and screen these microbial resources for their antibacterial activities.521 actinobacterial strains were isolated in this study.Unique pretreatment method and a series of isolation media had been used to culture more actinobacteria.Initial characterization of these isolates on the basis of its morphology,there were grouped into 37 genera in 22 families and 11 order of the class Actinobacteria.65 of these isolates were further characterized by 16 S rRNA gene sequence analysis to be potential novel species of the families Streptomycetaceae,Pseudonocardiaceae,Actinophytocola,Blastococcus,Isoptericola,Xylanimicrobium,Planobispora,Pseudoclavibacter,Cellulosimicrobium and Paeniglutamicibacter.A total of 200 strains were further screened for antibacterial activities against indicator strains.The results indicated that 46 strains were inhibitory towards at least one of the three indicator bacteria.Of these 46 potential strains,sixteen showed strong inhibitory effect against at least 5 indicator strains.Majority of these potent strains were affiliated to the genus Streptomyces,while few belongs to other genera,thereby indicating that the desert actinobacterial resources have great potential for screening and development of active natural products.Through a polyphasic characterization method involving polyphase sort method,four new species were identified and named as Microbacterium album sp.nov.,Microbacterium deserti sp.nov.,Saccharopolyspora deserti sp.nov.and Georgenia deserti sp.nov..In conclusion,this study present the vast actinobacterial diversity of the Saudi Arabian desert environment by a culture dependent method and lays the root for the further systematic study of actinomycetes community and resources in desert environment.In addition,the screening results of the antibacterial activities indicated us that the desert environment lives a large number of strains which has a strong secondary metabolism ability and Illustrate that the desert environment is the treasure house of the new natural products.

【Keywords in Chinese】 沙特阿拉伯沙漠放线菌多样性抗菌活性
【Key words】 Saudi Arabiadesert Actinobacteriadiversityantibacteria
  • 【Contributor】 中山大学
  • 【Year of Internet Publish】201905
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