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目的对影响翼梗五味子ISSR-PCR扩增反应的各因素进行优化,建立稳定的反应体系。方法基于正交极差分析方法,以Taq酶、Mg2+、模板DNA、dNTP和引物5因素4水平的正交试验对翼梗五味子ISSR-PCR反应体系进行优化。结果翼梗五味子ISSR-PCR的最佳反应体系(20μL)为:Taq酶1.00 U、Mg2+1.50 mmol/L、模板DNA 40.00 ng、dNTP 0.25mmol/L、引物0.50μmol/L。筛选出12条扩增稳定、多态性丰富的ISSR引物,并确定了每个引物的最佳退火温度。结论所建立的翼梗五味子ISSR反应体系具有标记位点清晰、反应系统稳定、重复性好等优点,为翼梗五味子的分子水平研究提供实验依据。
Objective To optimize the factors affecting the ISSR-PCR amplification reaction of Schisandra chinensis and establish a stable reaction system. Methods The orthogonal design was used to optimize the ISSR-PCR reaction system of Schisandra chinensis with orthogonal design of Taq enzyme, Mg2 +, template DNA, dNTP and primer 5 factors. Results The optimal reaction system (20μL) for ISSR-PCR was as follows: 1.00 U of Taq DNA polymerase, 1.50 mmol / L of Mg2 +, 40.00 ng of template DNA, 0.25 mmol / L of dNTP and 0.50 μmol / L of primer. Twelve ISSR primers with stable amplification and rich polymorphism were screened and the optimal annealing temperature was determined for each primer. Conclusion The ISSR reaction system of Schisandra chinensis has the advantages of clear marker site, stable reaction system and good repeatability. It provides an experimental basis for the molecular level study of Schisandra chinensis.