海南东寨港真红树植物内生放线菌多样性及其抗菌活性

Diversity and antimicrobial activity of endophytic actinobacteria isolated from eumangroves collected in Dongzhaigang of Hainan Province

【目的】勘探海南东寨港真红树植物内生放线菌多样性,为发现放线菌新物种和新抗生素奠定基础。【方法】样品经表面消毒后粉碎,用10种不同培养基分离放线菌;通过PCR扩增、测定并比对16S r RNA基因序列,开展放线菌多样性分析;通过发酵、萃取等处理方法得到四类样品,包括发酵原液、乙酸乙酯提取液及水层和菌体的丙酮浸泡提取液;采用纸片扩散法对样品进行抗菌活性筛选;基于PCR的基因筛选技术探测活性菌株可能存在的NRPS、PKS I、PKS II抗生素生物合成基因。【结果】经形态特征排重,从14种真红树植物样品中共得到放线菌146株,16S r RNA基因序列比对表明它们分布于13个科18个属,其中链霉菌属为优势菌属,菌株S3Cf-2和S3Af-1的16S r RNA基因序列分别与有效发表菌株Couchioplanes caeruleus DSM44103T(X93202)和Microlunatus terrae BS6T(JF806519)的相似率最高,分别为97.45%和97.43%,可能为新物种。对其中46株放线菌发酵样品的抗菌活性检测表明,40株具有抗菌活性,总阳性率为86.96%;活性菌株中,38株菌存在至少一种所探测的生物合成基因簇,阳性率为95%,其中14株同时具有所探测的3种抗生素生物合成基因簇。【结论】海南东寨港真红树植物中存在多样性丰富的药用放线菌资源,具有从中发现放线菌新物种及新抗生素的潜力。

[Objective] To study the diversity and bioactivity of actinobacteria isolated from Mangroves in Dongzhaigang, Hainan. [Methods] Various tissues from 14 kinds of mangrove plants as samples were collected from Dongzhaigang National Nature Reserve in Hainan, China. Samples were grinded into powders after surface sterilization and cultured on 10 different media for strain isolation. To determine the phylogenetic position of the strains, 16S rRNA gene sequences were obtained by PCR amplification and direct sequencing, then compared with gene database. The antifungal and antibacterial activities of the crude extracts were tested by disk diffusion method. Screening of PKS I, PKS II and NRPS genes was carried out by PCR and agarose electrophoresis. [Results] 146 strains affiliated to 18 genera were obtained. The dominant genus is Streptomyces. The highest similarity of 16S rRNA gene sequence between strain S3Cf-2 and the validly described species Couchioplanes caeruleus DSM44103T, between strain S3Af-1 and the validly described species Microlunatus terrae BS6T was 97.45% and 97.43%, respectively, which indicated that the strains S3Cf-2 and S3Af-1 were potential new taxa. The antimicrobial activity of crude extracts from the fermentation broths were tested against different pathogenic microorganisms by paper-disc diffusion method. Antimicrobial test showed that 40 out of 46 strains exhibited positive results in at least one antifungal or antibacterial assay, and the total positive rate was 86.96%. Thirty eight in 40 strains with antimicrobial activity, probably, had at least one antibiotic biosynthetic gene including NRPS, PKS I and PKS II, and 14 strains have all the three kinds of biosynthetic genes of antibiotics. [Conclusion] The study demonstrates that diversity of endophytic actinobacteria from mangrove are rich and mangrove tissues as a special niche for discovery of new pharmaceutical microorganisms are waiting to be explored.