产PK和NRP类抗生素苦豆子内生放线菌分子筛选及抗生素类型鉴定

Screening and identification of polyketide and non-ribosomal peptide antibiotics from endophytic actinomycetes of Sophora alopecuroides L.

摘要:

【目的】从19株苦豆子内生拮抗放线菌中筛选PKSⅠ、PKSⅡ和NRPS基因阳性菌株，并对其产抗生素种类进行初步鉴定，为苦豆子内生放线菌资源的合理开发和利用提供理论依据。【方法】分别以PKSⅠ、PKSⅡ和NRPS基因引物对19株拮抗菌株进行特异性扩增，筛选阳性菌株；以7种抗生素标准样品为对照，采用TLC和HPLC法对阳性菌株所产抗生素类型进行鉴定。【结果】PKSⅠ、PKSⅡ和NRPS基因阳性菌株率分别为47.4%、10.5%和21.1%；9株内生放线菌发酵液中各有1个峰的洗脱时间与麦迪霉素的洗脱时间一致，菌株NDZKDS69的发酵液中有4个峰的洗脱时间分别与麦迪霉素、乙酰螺旋霉素、替考拉宁和土霉素标准样品的洗脱时间一致。【结论】苦豆子内生放线菌中链霉菌属(Strepomyces)菌株是大环内脂类、芳香环类和非核糖体多肽类抗生素的丰富菌源；分子指纹图谱和化学指纹图谱检测结果一致，且建立的TLC和HPLC法检测抗生素的方法简便、快捷、灵敏、重复性良好。

Abstract:

[Objective] Screened positive strains which had PKSⅠ, PKSⅡ and NRPS genes from 19 endophytic antagonism actinomycetes strains of Sophora alopecuroides L. and preliminary identified types of antibiotics produced by them, which could also provide research methods and theory on rational development and use of endophytic actinomycetes from Sophora alopecuroides L. [Methods] Specific amplified 19 endophytic antagonism actinomycetes by primers of PKSⅠ,PKSⅡ and NRPS genes for screening positive strains. Then 7 kinds of antibiotics were used as contrast and preliminary identified antibiotics produced by positive strains which had PKSⅠ, PKSⅡ and NRPS genes by TLC and HPLC methods. [Results] The rates of positive strains of PKSⅠ, PKSⅡ and NRPS genes were 47.4%, 10.5% and 21.1% respectively. The fermentation broth of 9 endophytic actinomycetes had a peak respectively which had the same elution time with medemycin, the fermentation broth of strain NDZKDS69 had four peaks which had the same elution time with medemycin, acetylspiramycin, teicoplanin and oxytetracycline respectively. [Conclusion] Strepomyces of endophytic actinomycetes from Sophora alopecuroides L. were rich sources of macrolide antibiotic, aromatic polyketide antibiotics and non ribosomal peptide antibiotics. The testing results of molecular fingerprinting and chemical fingerprint chromatography were consistent, and the methods of TLC and HPLC were founded in this paper were convenient, rapid, sensitive and had better repeatability.