丝状真菌PCR模板DNA的快速制备方法  

A Rapid Method of Preparing DNA Template of Filamentous Fungi for PCR Amplification

作　　者：罗中钦[1] 程琳[2] 张茜[3] 陈国华[1] 

机构地区：[1]中国农业科学院蔬菜花卉研究所,北京100081 [2]山西师范大学生命科学学院,临汾041004 [3]北京师范大学生命科学学院,北京100875

出　　处：《生物技术通报》2015年第9期79-83,共5页Biotechnology Bulletin

基　　金：国家自然科学基金项目(31301618)

摘　　要：以尖孢镰刀菌(Fusarium oxysporum)为材料,旨为建立沸水浴获得PCR模板DNA的新方法。】取少量真菌菌丝置于一定体积50 mmol/L Na OH溶液中沸水浴10 min,再加入1/10体积1 mol/L Tris-HCl(p H8.0)缓冲液,12 000 r/min离心后,上清用作PCR的DNA模板。结果显示,该方法扩增效率高,扩增条带清晰,且Taq酶适应性强,适合快速制备丝状真菌的模板DNA。该方法经济、简单、快速、安全高效,可用于丝状真菌转化子的高通量筛选和菌株的快速鉴定。This work aims to establish a novel method of extracting DNA template of filamentous fungi for PCR amplification with boiling-water bath and Fusarium oxysporum as raw material. The procedures of this method are as the following：exposing the fungal mycelia in certain volume of 50 mmol/L NaOH solution under boiling water bath, adding 1/10 volume of 1mol/L Tris-HCl（pH8.0）buffer, centrifuging it at 12 000 r/min for 10 min, and using the supernatant as the DNA template for PCR amplification. Results demonstrated that the efficiency of PCR amplification with the DNA template prepared by the method was high, the amplified bands were clear, and adaptability to various Taq DNA polymerases was strong;This indicated that the method was suitable for the preparation of DNA template of filamentous fungi. Conclusively, this method is economic, simple, rapid, safe and high-efficient, and can be utilized for high-throughput screening of filamentous fungal transformants and rapid identification of isolated strains.

关 键 词：丝状真菌 尖孢镰刀菌 PCR扩增 TAQ DNA聚合酶 

分 类 号：S432.44[农业科学—植物病理学]