兜兰属植物ISSR-PCR反应体系的优化

以兜兰为试材,用改进的CTAB法提取总DNA,采用正交试验设计,分析Mg2+浓度、dNTP浓度、引物浓度和Taq DNA聚合酶用量对ISSR-PCR扩增的影响,并通过梯度PCR确定最佳退火温度,最终建立兜兰ISSR扩增反应体系。最佳反应体系为:25μL体系中,含10×PCR buffer 2.5μL、1.5mmol/L Mg2+、0.15mmol/L dNTP、0.6μmol/L引物、1.0 UTaq酶和40 ng模板DNA。反应程序为:94℃5 min,94℃30 s,56.2℃45 s,72℃1 min,共40个循环;72℃延伸7 min。

Optimization of ISSR-PCR Reaction System for Paphiopedilum

The genomic DNA of Paphiopedilum was extracted with improved CTAB method, consequently, the effects of Mg2＋ , dNTP, and primer concentrations, as well as the usage of Taq DNA polymerase on ISSR- PCR amplification were optimized using an orthogonal experimental design. The optimal anneal temperature of primer were determined by gradient PCR. The 25μL ISSR-PCR cocktail contained 2.5 uL 10× PCR buffer, 1.5 mmol/L Mg2＋ , 0.15 mmol/L dNTP, 0.6 umol/L primer, 1.0 U Taq polymerase and 40 ng template DNA gave the best amplification result. PCR procedures were described as follows： 94℃ for 5 min, followed by 40 cycles of denaturing at 94℃ for 30 s, annealing at 56. 2℃for 45 s, and extending at 72℃for 1 min, finally an extension at 72℃for 7 min.