目的:建立并优化多伞阿魏简单重复序列区间(ISSR)一聚合酶链反应(PCR)体系。方法:通过单因素试验确定影响多伞阿魏ISSR—PCR扩增结果的Mg^2+、dNTPs、引物、TaqDNA聚合酶、DNA模板最佳水平范围。对Mg^2+、dNTPs、引物、TaqDNA聚合酶、模板DNA进行5因素4水平的正交试验,优选最佳ISSR—PCR体系条件。ISSR—PCR扩增结果用SPSS18.0统计软件分析。结果:不同水平的因素对PCR均有明显影响.其中Mg^2+、dNTPs、引物影响最大。多伞阿魏ISSR.PCR最佳反应体系为:在50μl的反应体系中含有10×Taq Buffer5lal、Mg^2+2.0mmol/L、dNTPs0.4mmol/L、引物0.4gmol/L、TaqDNA聚合酶1.75u/50μl、DNA模板0.25ng/gl。在此基础上.从100条引物中筛选出15条扩增稳定、多态性丰富的ISSR引物。结论:建立的多伞阿魏ISSR—PCR体系具有较高的稳定性和重现性,可为进一步利用ISSR技术进行多伞阿魏遗传多样性分析、不同种源鉴定及亲缘关系分析提供基础。
OBJECTIVE: To establish and optimize the ISSR-PCR system for Ferula ferulaeoides. METHODS: The ranges of Mg^2+, dNTPs, primers, Taq DNA, polymerase and template DNA, which influenced ISSR-PCR system ore ferulaeoides, were de- termined by single factor test; using them as factors and levels, the ISSR-PCR system was optimized by orthogonal experiment. ISSR-PCR result was analyzed by SPSS statistical software. RESULTS: Most of the factors at different levels had the significant effects on the result of PCR, and the most remarkable factors were the concentration of Mg^2+, dNTPs and primers. The optimized ISSR-PCR system of 50 p.1 Eferulaeoides as follows: 10×Taq Buffer 5 pl, Mg^2+ 2.0 mmol/L, dNTPs 0.4 mmol/L,primer 0.4 ~tmol/L, Taq DNA polymerase 1.75 u/50 pl , DNA template 0.25 ng/td. 15 ISSR primers with stable amplification and abundant polymor- phism were selected from 100 ISSR primers. CONCLUSIONS: Established ISSR-PCR system is stable and reproducible, and pro- vides the basis for further study on genetic diversity analysis, source identification and genetic relationship analysis of E ferulae- oides by 1SSR molecular marker technique.