锡林郭勒盟高原盐湖放线菌资源勘探及抗菌活性筛选

Exploration and antibacterial activity of actinomycetes isolated from saline lakes in Xilin Gol Plateau

目的探究锡林郭勒盟高原盐湖放线菌的多样性、新颖性及抗菌活性,为新型抗菌活性产物的发掘储备菌种资源。方法采用20种分离培养基,以平板涂布法分离放线菌;依据16S rRNA基因的同源性对菌株进行分子鉴定;PCR检测代表菌株的Ⅰ型聚酮合酶(PKS Ⅰ)、Ⅱ型聚酮合酶(PKS Ⅱ)和非核糖体多肽合成酶(NRPS)功能基因;菌株的发酵液上清和菌体分别经乙酸乙酯和丙酮提取,提取样品经纸片扩散法进行抗菌活性检测。目标菌株合成次级代谢产物的能力通过anti SMASH对基因组序列进行分析预测。结果从7份盐湖土壤样品中分离获得了365株放线菌,其分布于放线菌纲的8个目15个科28个属,优势菌属为链霉菌属和拟诺卡菌属。分离获得的链霉菌新颖性突出,13株链霉菌与近缘菌株16S rRNA基因的最高相似度≤98.0%,推测其归属于10个链霉菌属新种。受试放线菌对革兰阳性菌的抗菌活性显著,并从中筛选出3株抗菌作用强且抗菌谱较广的链霉菌;20株受试放线菌同时含有3种抗生素生物合成基因。菌株XMNu-295基因组含有24个潜在的次级代谢产物生物合成基因簇,以聚酮类、非核糖体多肽类和萜烯类等为主。结论锡林郭勒盟高原盐湖中可培养放线菌多样性较为丰富,新颖性突出,目标菌株值得进一步开展次级代谢产物的化学研究。

Objective The purpose of this study was to assess the diversity,novelty,and antibacterial activities of culturable actinomycetes from saline lakes in Xilin Gol Plateau.The study will provide strain resource for the discovery of novel natural products against pathogens.Methods Actinomycetes were isolated by using the spread plate method with 20 kinds of media.Isolates were identified by comparison of 16S rRNA genes.PKSⅠ,PKSⅡ,and NRPS antibiotic biosynthetic genes of representative isolates were screened by PCR.The fermented broth and mycelia of representative isolates were extracted with ethyl acetate and acetone,respectively.Antibacterial tests were carried out with the disk diffusion method.The genome sequence of target strain was analyzed by antiSMASH to evaluate the biosynthetic potential of secondary metabolites.Results A total of 365 actinomycete isolates were obtained from 7soil samples,which were affiliated with 28 genera in 15 families of 8 orders,and Streptomyces and Nocardiopsis were dominant genera.The novelty of Streptomyces isolates was notable,and the highest 16S rRNA gene similarities of the 13 isolates to relative strains were lower than or equal to 98.0%,which implied the 13 strains might belong to 10 novel Streptomyces species.Some test strains showed notable antibacterial activities against Gram-positive pathogenic bacteria,and 3 Streptomyces strains with strong antibacterial activity and broad antibacterial spectrum were obtained.A total of 20 test strains carried all the three kinds of antibiotic biosynthetic genes.A total of 24 secondary metabolite biosynthetic gene clusters were identified in the genome of strain XMNu-295,and the main clusters were PKS,NRPS,and Terpene.Conclusion Culturable actinomycetes from saline lakes in Xilin Gol Plateau have abundant diversity and notable novelty,and the secondary metabolites of the selected target strains are worthy of further study.