摘要
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 study the biodiversity and bioactivity of actinobacteria isolated from mangrove plants collected from Zhanjiang Mangrove National Nature Reserve, Guangdong Province, China. Methods Samples were ground into powder after surface sterilization and cultured onto 10 different media for isolation. Strains were analyzed by comparison of 16S rRNA gene sequences. Fermentation broths were extracted by ethyl acetate to obtain crude extracts. The antifungal and antibacterial activities of the crude extracts were tested by disk diffusion method. The nematicidal activities were tested by 96 well plate fluid model. Preliminary screening of PKS I, PKS II and NRPS genes were carried out by PCR and agarose gel electrophoresis. Results Total 159 strains obtained were distributed in 19 genera affiliated with 12 families of 8 orders. The 16S rRNA gene sequence similarity between strain IP4SC6 and its nearest strain Phycicoccus jejuensis KSW2-15T（DQ345443） in EzBioCloud database was 96.81%. The N-J phylogenetic tree based on 16S rRNA gene sequences showed that the strain IP4SC6 was clustered with validly named species in the genus Phycicoccus and formed a distinct and independent branch, which indicated that strain IP4SC6 is a potentially new species of the genus Phycicoccus. Antimicrobial assay showed that 46 among 88 strains exhibited positive results in antifungal or antibacterial assay, and the total positive rate was 52.27%. 36 in 46 strains with antimicrobial activity might have at least one antibiotic biosynthetic gene including NRPS, PKS I and PKS II, and 8 strains might have all three kinds of biosynthetic genes of antibiotics. LC50 values of nematicidal strain NA6SC10, TW2L1 and NA2S2 were 83.2, 138 and 132 mg/mL, separatively. Conclusion Mangrove plants in Zhanjiang Mangrove National Nature Reserve are rich in bioactive endophytic actinobacteria, which could be a potential source for the discovery of new taxa and new antibiotics.
出处
《中国抗生素杂志》
CAS
CSCD
北大核心
2016年第1期26-34,共9页
Chinese Journal of Antibiotics
基金
国家自然科学基金(No.81172963
81321004)
国家高技术研究发展计划863计划课题(No.2011A A10A204)
十二五"重大新药创制"科技重大专项(No.2012ZX09301-002-001-018)
关键词
红树林
放线菌
耐药菌
秀丽隐杆线虫
生物多样性
Mangrove
Actinobacteria
Drug-resistant bacteria
Caenorhabditis elegans
Biodiversity
