正交设计优化翼梗五味子ISSR-PCR反应体系

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。

Optimization for ISSR-PCR reaction system on Schisandra henryi by orthogonal design

Objective To optimize the each factor affecting on ISSR-PCR reaction system of Schisandra henryi and establish the stable ISSR-PCR system. Methods Based on the analysis of orthogonal design test, an orthogonal design was used to optimize the ISSR-PCR amplification system on S. henryi by five factors (Tag polymerise, Mg²⁺, DNA template, dNTP, and primer) at four concentration levels, respectively. Results A suitable ISSR-PCR reaction system was constructed with the 20 μL reaction system containing 1.00 U Taq polymerise, Mg²⁺1.50 mmol/L, DNA template 40.00 ng, dNTP 0.25 mmol/L, and primer 0.50 μmol/L. Twelve effective ISSR primers were selected and the optional annealing temperature of every one primers was fixed. Conclusion ISSR-PCR is significantly influenced by the concentration of S. henryi. This ISSR-PCR system could provide clear bands, reliable reaction system, and abundant polymorphisms. It proves a reference for molecular research of S. henryi.