珍稀濒危植物宝华玉兰ISSR-PCR反应体系建立及优化

Establishment and Optimization of ISSR-PCR Reaction System for Rare and Endangered Plant Magnolia zenii

为建立和优化宝华玉兰(Magnolia zenii Cheng)ISSR-PCR反应体系,采用正交试验和单因子试验方法对影响PCR扩增体系中的Mg^(2+)、d NTPs、DNA模板、引物和Taq聚合酶一定浓度的用量5个因子进行分析比较。结果显示,宝华玉兰ISSR-PCR 25μL反应体系中的5个因子最佳水平分别为DNA模板(20 ng/μL)3.5μL,引物(10μmol/L)1.25μL,Taq聚合酶(5 U/μL)0.2μL,Mg^(2+)(25 mmol/L)2.0μL,d NTPs(2.5 mmol/L)0.8μL。宝华玉兰ISSR-PCR反应的最优体系建立,为进一步利用ISSR对其进行分子标记辅助育种、分子身份证构建和遗传多样性分析等后续研究奠定了基础。

To establish and optimize the ISSR-PCR reaction system for Magnolia zenii Cheng, orthogonal experiment and single factor design were conducted to analyze the effect of 5 factors inluding DNA template, dose of Taq DNA polymerase,primers, Mg2 ＋and d NTPs in amplification system. The results showed that the optimal ISSR-PCR reaction system（25 μL）mixture contains 3.5 μL DNA template（20 ng / μL）,1.25 μL primers（10 μmol / L each）, 0.2 μL Taq DNA polymerase（5 U / μL）,2.0 μL Mg2＋（25 mmol / L）, 0.8 μL d NTPs（2.5 mmol / L）. The establishment of ISSR-PCR reaction system for M. zenii laid the foundation for further use of ISSR in molecular marker-assisted breeding, molecular identification and genetic diversity study.