阿克苏高盐咸水滩放线菌分离新策略及系统发育多样性

New isolation methods and phylogenetic diversity of Actinobacteria from hypersaline beach in Aksu

【目的】探讨高盐咸水滩放线菌的分离新策略,为高盐地区放线菌资源的分离提供理论依据。【方法】以甘油-精氨酸培养基、海藻糖-肌酸培养基、甘油-天冬氨酸培养基、甘露醇-酸水解酪蛋白培养基、干酪素-甘露醇、甘露醇-丙氨酸培养基、壳聚糖-天冬酰胺培养基和高氏一号琼脂培养基8种培养基为基础培养基,采用营养成分十倍稀释法、据土样理化性质模拟原始生态环境、免培养分子技术检测菌株的分离培养基及培养条件指导放线菌可培养以及借鉴半咸水海洋环境放线菌分离培养基等4种策略来优化设计培养基,进行阿克苏高盐咸水滩土样放线菌的分离,并采用细菌通用引物进行16S rRNA基因扩增和序列测定,并构建系统发育树。【结果】共分离到403株,分属于放线菌的8个亚目10个科,Streptomyces、Streptomonospora、Saccharomonospora、Plantactinospora、Nocardia、Amycolatopsis、Glycomyces、Micromonospora、Nocardiopsis、Isoptericola、Nonomuraea、Thermobifida、Actinopolyspora和Actinomadura等14个属。69.96%菌株属于链霉菌亚目(Streptomycineae),9.68%菌株属于链孢囊菌亚目(Streptosporangineae),9株为潜在新种。【结论】4种分离新策略显著地提高了高盐地区放线菌的可培养性,还发现了许多新物种,为放线菌的分离提供了新思路与途径。

[ Objective] We explored 4 new methods to improve the isolation of actinobacterial resources from high salt areas. [ Methods] Optimized media based on 4 new strategies were used for isolating actinobacteria from hypersaline beaches. Glycerin-arginine, trehalose-creatine, glycerol-asparticacid, mannitol-casein, casein-mannitol, mannitol-alanine, chitosan-asparagineand GAUZE＇ No. l were used as basic media. New isolation strategy includes 4 methods： ten-fold dilution culture, simulation of the original environment, actinobacterial culture guided by uncultured molecular technology detected, and reference of actinobacterial media for brackish marine environment. The 16S rRNA genes of the isolates were amplified with bacterial universal primers. The results of 16S rRNA gene sequences were compared with sequences obtained from GenBank databases. We constructed phylogenetic tree with the neighbor-joining method. [ Results ] No actinobacterial strains were isolated by 8 media of control group, while 403 strains were isolated by new strategies. The isolates by new methods were members of 14 genera （ Streptomyces, Streptomonospora, Saccharomonospora, Plantactinospora , Nocardia , Amycolatopsis , Glycomyces , Micromonospora , Nocardiopsis , Isoptericola , Nonomuraea , Thermobifida, Actinopolyspora, Actinomadura） of 10 families in 8 suborders. The most abundant and diverse isolates were the two suborders of Streptomycineae （ 69.96% ） and Streptosporangineaesuborder （ 9.68% ） within the phylum Actinobacteria, including 9 potential novel species. [ Conclusion ] New isolation methods significantly improved the actinobacterial culturability of hypersaline areas, and obtained many potential novel species, which provided a new and more effective way to isolate actinobacteria resources in hypersaline environments.