为了明确自主分离的天然抗真菌活性产物——纳他霉素产生菌A01和A02与已知纳他霉素产生菌的遗传相似性,采用RAPD技术对这2株菌和3个产纳他霉素的标准菌株进行了比较分析。利用筛选出的7个随机引物对5个菌株的PCR扩增共得到154条清晰稳定的DNA条带,其中同源性条带86条,多态性条带68条,分别占总条带数的55.8%和44.2%,平均每个引物扩增出9.7个多态性条带,得到了丰富的DNA指纹图谱。所得数据经NTSYS pc 2.10e软件聚类分析,表明5个菌株间的遗传距离为0.0991~0.4738,其中A02与其它菌株间的遗传距离均较远。结合前期的分类研究结果,确证了菌株A02为新的纳他霉素产生菌。
[Objective] The aim of this study was to investigate the preparation method and amplification system of antagonistic streptomyces DNA templates based on AFLP assays, and also provide a basis for the application of AFLP technology in the analysis of streptomyces or even actinomyces. [Method] The DNAs were extracted by the modified CTAB method and amplified by the Pst Ⅰ/Mse Ⅰ AFLP kit and its reaction system. The amplified products were analyzed by the denatured polyacrylamide gel electrophoresis. [Result] The genomic DNAs of ten antagonistic strains of Streptomyces were extracted and tested. The result of 0.8% agarose gel electrophoresis showed that the major DNA bands were clear without degradation and RNA residue, with the fragment sizes ranging from 37.64 to 40.86 Kb. By ultraviolet spectrophotometry, the OD260/OD280 values varying from 1.625 to 1.833 were obtained. Furthermore, the agarose gel electrophoresis of DNA products digested by Pst Ⅰ/Mse Ⅰ presented the dispersed fluorescent long band, which indicated that the enzymatic hydrolysis was fully carried out. The amplified bands of DNA templates by the screened three pairs of primers were clear with rich polymorphism. [Conclusion] The preparation method and amplification system of DNA template established in this study can be used in the AFLP analysis of Streptomyces.
