西藏仲巴五彩沙漠放线菌资源勘探及生物活性筛选

Bioprospecting and bioactivity screening of culturable actinobacteria from color desert in Dengpa, Tibet

摘要:

【背景】细菌耐药性问题日益严峻，新抗生素的研发速度远远落后于临床需要，从特殊生境中挖掘微生物药物资源有望解决以上问题。【目的】勘探西藏仲巴五彩沙漠土壤放线菌多样性并进行生物活性筛选，为发现药用放线菌资源、开发新型抗生素奠定基础。【方法】采用8种分离培养基，通过平板稀释涂布法分离放线菌；根据分离菌株的16S rRNA基因序列同源性分析放线菌多样性；采用PCR技术对分离的放线菌菌株进行II型聚酮合酶(PKS-II)酮缩酶结构域KS、非核糖体多肽合成酶(NRPS)腺苷酸化结构域A、安莎类抗生素生物合成前体3-氨基-5-羟基-苯甲酸合酶(AHBA)保守区、黄素腺嘌呤二核苷酸卤化酶(Halo)保守区抗生素生物合成基因检测；对生物合成基因检测阳性的菌株进行液体发酵，发酵液经乙酸乙酯萃取、菌体经丙酮浸提，获得提取浓缩物样品进行抑菌活性和抗氧化活性筛选。【结果】从4份土样中分离纯化到231株放线菌，分布于7个属，其中链霉菌为优势菌属。68株放线菌的生物合成基因分析显示至少具有1种生物合成基因簇，其中6株同时具有4种生物合成基因簇；进一步的抑菌活性检测显示所有检测的菌株至少表现为对1株检定菌具有抑菌活性，其中8株具有广谱抗菌活性；抗氧化活性筛选结果为13株显示总抗氧化能力阳性，10株具有较好的羟自由基清除能力，3株显示较强的氧自由基清除能力。【结论】西藏仲巴五彩沙漠土壤中含有较丰富的放线菌药用资源，具有从中发现放线菌新菌种和开发新抗生素的潜力。

Abstract:

[Background] The problem of bacterial drug resistance is becoming more and more serious. The development of new antibiotics far lags behind the clinical needs. The discovery of microbial drug resources from special habitats is expected to solve this problem. [Objective] Biodiversity and bioactivity screening of actinomycetes from color desert in Dengpa, Tibet were performed, which would lay a good foundation for discovery of the resources of medicinal actinomycetes and new antibiotics. [Methods] The actinobacterial strains were cultured on 8 different media. Biodiversity of actinobacterial strains were analyzed according to 16S rRNA gene sequences. Detection of PKS-II (ketosynthase, KS), NRPS (adenylation A domain), AHBA and Halo genes was performed by PCR and agarose gel electrophoresis. Screening of antimicrobial activity was carried out by filter paper method. Antioxidant ability including total antioxidant activity, hydroxyl and oxygen free radical scavenging rate was tested by the chemical method. [Results] a total of 231 strains were isolated from four soil samples from color desert in Dengpa, Tibet. 16S rRNA gene analysis revealed these strains were affiliated with seven actinobacterial genera. Streptomyces was the dominant genus. Screening of biosynthetic gene clusters showed that 68 strains had at least one of four biosynthetic gene clusters and 6 strains had all the tested biosynthetic gene clusters. Furthermore, secondary metabolites produced by these strains showed antimicrobial activity against certain pathogens and antioxidant capacity towards hydroxyl and oxygen free radical scavenging. Therein, eight strains displayed broad-spectrum antimicrobial activity. Thirteen strains showed the positive total antioxidant activity. Ten strains had good hydroxyl free radical scavenging and three strains showed positive oxygen free radical scavenging. [Conclusion] These results exhibited that there are abundant actinomycetes medicinal resources in color desert in Dengpa, Tibet, which was promising for discovering new strains of actinomycetes and developing new antibiotics.