拮抗链霉菌AFLP分析技术体系的研究

[目的]探索基于AFLP的拮抗链霉菌DNA模板制备方法及其扩增体系，为AFLP技术在链霉菌乃至放线菌资源分析中的应用提供依据。[方法]以改进的CTAB法提取DNA，利用Pst I／Mse I型AFLP试剂盒及其反应体系进行扩增，采用5％变性聚丙烯酰胺凝胶电泳分析扩增结果。[结果]提取了10个拮抗链霉菌菌株的基因组DNA，0.8％琼脂糖凝胶电泳检测显示其主带清晰，片段大小为37.64-40.86Kb，无降解现象，亦无RNA残留；其OD260/OD280为1.625-1.833；Pst I／Mse I双酶切产物琼脂糖电泳呈弥散荧光长带，说明酶解充分；筛选出的3对引物对DNA模板的扩增谱带清晰，多态性丰富。[结论]该研究建立的DNA模板制备方法及其扩增反应体系可用于链霉菌的AFLP分析。

Studies on the Technological System for AFLP in Antagonistic Strains from Streptomyces

[Objective] This research was to study the preparation method of DNA template and amplification system for AFLP in antagonistic strains from Streptomyces,so as to provide the basic data for the application of AFLP in the resource analysis of Streptomyces and even actinomycete.[Method] The genomic DNAs were extracted by using the modified CTAB method.The amplifications were carried out with the reaction system of PstⅠ/MseⅠAFLP kit purchased from Beijing Dingguo Biotechnology Co.Ltd.The amplified products were analyzed via 5% denatured polyacrylamide gel electrophoresis.[Result] The genomic DNA samples extracted from 10 strains presented the clear main bands composed of 37.64-40.86 Kb fragments by 0.8% agarose gel electrophoresis,which shows no degradation and no RNA residue,and could be completely digested by Pst I/Mse I.The OD260/OD280 values of the DNA samples varied within the range of 1.625-1.833.The clear bands with high polymorphism were obtained by amplifying with 3 screened primer pairs.[Conclusion] The modified CTAB method for DNA extraction and the amplification system tested in the research are suitable for the AFLP analysis of Streptomyces.