西沙石珊瑚共附生放线菌的分离培养及抗菌活性评价

Isolation and antibacterial activity of Xisha stone coral-associated actinomycetes

摘要:

【背景】珊瑚礁生态系统是海洋中一类极其重要的生态系统，健康珊瑚礁中丰富的共附生放线菌群体是珊瑚抵御各种致病菌的重要防线，因此，这类放线菌是寻找抗菌活性分子的重要资源，其药用潜力巨大。【目的】从西沙石珊瑚样品中分离共附生放线菌，并从中筛选具有良好抗菌活性的菌株。【方法】通过稀释涂布法分离珊瑚共附生放线菌，并根据16S rRNA基因序列构建系统发育树进行菌种鉴定；通过平板对峙法对放线菌进行抗菌活性筛选并确定目标菌株；将目标菌株涂布于不同氯化钠浓度的ISP2固体培养基上培养，测试其盐度耐受能力；通过平板对峙法对该菌株发酵产物的热稳定性和光稳定性进行测试；采用Nanopore和Illumina方法完成目标活性放线菌全基因组测序，并通过antiSMASH在线分析预测其次级代谢产物生物合成基因簇及其结构类型。【结果】从6份西沙石珊瑚样品中分离得到104株可培养放线菌，根据菌落形态和分离来源去重后对其中27株放线菌进行16S rRNA基因序列测序，通过序列比对和系统发育树分析将菌株初步鉴定为盐孢菌属(Salinispora) (25株)、链霉菌属(Streptomyces) (1株)和戈登菌属(Gordonia) (1株)。活性筛选结果表明，盐孢菌株SH098抑菌活性最好，该菌株在无盐的培养基中不生长，盐耐受范围为2%-5%，其发酵产物的抗菌活性具有良好的热稳定性和光稳定性。全基因组序列分析显示，盐孢菌株SH098基因组中21.93%的基因序列与次级代谢产物合成相关，预测的次级代谢产物结构类型多样，有待深入发掘。【结论】首次从我国西沙石珊瑚中分离得到具有嗜盐特性和抗农业病原细菌活性的盐孢菌，丰富了我国西沙海域来源的专性海洋放线菌资源，为海水养殖业的新型微生态制剂的研发提供新菌种资源，也为后期挖掘新型抗菌活性物质奠定了研究基础。

关键词:石珊瑚;共附生放线菌;分离培养;沙栖盐孢菌;抗菌活性

Abstract:

[Background] Coral reef ecosystem is extremely important in the marine ecosystem, in which the actinomycetes are the important line of defense against various pathogens. Therefore, coral-associated actinomycetes are important resources for molecules with antibacterial activity, indicating tremendous medicinal potential.[Objective] To isolate the culturable actinomycetes from samples of Xisha stone corals and screen out the strains with good antibacterial activity. [Methods] Coral-associated actinomycetes were isolated by dilution separation method and identified by phylogenetic analysis of 16S rRNA gene sequence. The antibacterial activity test was conducted by the plate confrontation method, and the target strain was identified. The salinity tolerance test was carried out by plating the strain on ISP2 solid medium with different concentrations of NaCl, and the thermostability and photostability of the fermentation products were tested by the plate confrontation method. The genome of the target active actinomycete was sequenced by Nanopore and Illumina sequencing, and the types of biosynthetic gene clusters of secondary metabolites were analyzed online by antiSMASH. [Results] A total of 104 culturable actinomycetes were isolated from 6 samples of Xisha stone corals, and 27 actinomycetes were obtained after removing duplicates according to colony morphology and sample source. The 16S rRNA gene sequencing of the 27 strains was performed, and they were identified as Salinispora (25 strains), Streptomyces (1 strain), and Gordonia (1 strain) according to sequence alignment and phylogenetic tree analysis. The results of activity screening showed that Salinispora sp. SH098 had the optimal antibacterial activity during the antibacterial activity test. Salinispora sp. SH098 did not grow in the salt-free medium and its salt-tolerant range was 2%-5%. Its fermentation products showed good thermostability and photostability. The whole genome sequence analysis showed that 21.93% of the gene sequences of strain SH098 were related to the biosynthesis of secondary metabolites. The structure types of predicted metabolites were diverse, which needed to be further explored. [Conclusion] This is the first report on Salinispora spp. with anti-agricultural pathogenic bacteria activity from Xisha stone corals in China, which enriches the obligate marine actinomycetes resources from the Xisha islands of China. It provides new resources for the development of new microecological agents applied to mariculture and also lays a foundation for mining antibacterial active substances.

Key words:stone coral;associated actinomycetes;isolation culture;Salinispora arenicola;antibacterial activity