正交设计优化金发藓科植物ISSR-PCR反应体系

Study on Optimization for ISSR-PCR Reaction System of Polytrichaceae using Orthogonal Design

目的:建立金发藓科植物ISSR-PCR反应的最佳体系。方法:利用正交设计的方法,对金发藓科植物(Polytrichaceae)IS-SR-PCR反应的5因素(Mg2+,dNTP,primer,DNA template,Taq DNA polymerase)4水平进行试验。结果:在20μl反应体系中,模板DNA50ng,1.6μmol/L的引物,1×反应缓冲液,3.2mmol/L的Mg2+,dNTP为1.2mmol/L,2U的TaqDNA聚合酶,反应程序为94℃预变性6min;94℃变性45s,57℃退火45s,72℃延伸2min,循环40次;72℃延伸10min。利用此结论,对20种金发藓科植物进行ISSR-PCR扩增,扩增产物的多态性为69.52%。利用引物841构建的指纹图谱,可区分20种金发藓科植物中的18种,分辨率达90%。金发藓科植物ISSR-PCR反应体系的建立,为今后利用ISSR标记技术开展金发藓科植物种间遗传多样性分析提供一个标准化程序。

Objective： The orthogonal design was used to optimize ISSR- PCR amplification system of Polytrichaceae in five factors （Mg^2＋ , dNTP, primer, DNA template, Taq DNA polymerase） at four levels respectively. A suitable ISSR reaction system was established , namely 20μl reaction system containing 50ng DNA template, 1.6μmoL/L primer, 1 × PCR buffer, 3.2mmol/L Mg^2＋ , 1.2mmol/L dNTP, 2U Taq DNA polymerase. The program of heating：pre- denaturation 6min at 94℃, then undergo a proceeding of reaction cycles as denaturation 45s at 94℃, anneal 45s at 57℃ ,extension 2min at 72℃ ,40 cycles,finally extension 10 min at 72℃. ISSR polymorphism among twenty Polytrichaceae was 69.52% ,and 18 out of 20 Polytriehaceae could be identified by 1SSR fingerprinter established with primer 841 .The result provided a standardizing program for the analysis of interspecies genetic diversity of Polytfichaceae.