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目的勘探广东湛江红树林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,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;经发酵,离心,乙酸乙酯萃取上清液,旋转蒸发获得浓缩物,甲醇溶解制成样品;采用纸片扩散法对样品进行抗菌活性筛选;采用96孔板法,以秀丽隐杆线虫为对象,对样品进行杀线虫活性筛选;基于PCR的基因筛选技术初步探测活性菌株NRPS、PKS I、PKS II抗生素生物合成基因簇。结果经形态特征排重,从11份植物样品中共得到放线菌159株,16S r RNA基因序列比对分析表明,它们分布于8个目12个科19个属,其中链霉菌属为优势菌属,菌株IP4SC6为海藻球菌属潜在新种;发酵88株放线菌,发酵液乙酸乙酯萃取浓缩物测活结果表明,至少对一株检定菌表现为阳性的菌株有46株,总阳性率为52.27%,其中36株菌可能存在抗生素生物合成基因簇,8株可能同时具有3种抗生素生物合成基因簇;对秀丽隐杆线虫有较强杀虫活性的放线菌有3株,LC50值分别为83.2,132.0和138.0mg/m L。结论广东湛江红树林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 explore the diversity of endophytic actinomycetes in mangrove plantation of Zhanjiang, Guangdong and lay a foundation for the discovery of new actinomycetes and new antibiotics. Methods The samples were ground and disinfected, then the actinomycetes were isolated from 10 different media. The 16S rRNA gene sequences were determined by PCR, and the diversity of actinomycetes was analyzed. After fermentation, centrifugation and extraction with ethyl acetate The supernatant was concentrated by rotary evaporation and dissolved in methanol to prepare a sample. The antibacterial activity of the sample was screened by disk diffusion method. The nematicidal activity of the sample was tested by 96-well plate method. PCR-based gene screening technology to detect active strains NRPS, PKS I, PKS II antibiotics biosynthetic gene cluster. Results A total of 159 actinomycetes strains were obtained from 11 plant species through the morphological characteristics. The sequence alignment of 16S rRNA genes showed that they belonged to 19 genera and 12 genera in 8 orders, of which Streptomyces was dominant The strain IP4SC6 was a potential new species of the genus Klebsiella. The results of 88 strains of actinomycetes and ethyl acetate extract of fermentation broth showed that there were 46 strains positive for at least one strain. The total positive rate Of them were 52.27%, of which 36 strains might exist antibiotic biosynthetic gene cluster, 8 strains may have three antibiotic biosynthetic gene cluster at the same time, 3 strains actinomycetes which have stronger insecticidal activity against C. elegans, LC50 value 83.2, 132.0 and 138.0 mg / m L, respectively. Conclusion There are abundant resources of medicinal actinomycetes in mangrove plants of Zhanjiang, Guangdong Province, which have the potential of discovering new actinomycetes and new antibiotics.