摘要
目的:建立并优化多伞阿魏简单重复序列区间(ISSR)-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Mg2+、dNTPs、引物、Taq DNA聚合酶、DNA模板最佳水平范围。对Mg2+、dNTPs、引物、Taq DNA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SPSS 18.0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Mg2+、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 Buffer 5μl、Mg2+2.0 mmol/L、dNTPs 0.4 mmol/L、引物0.4μmol/L、Taq DNA聚合酶1.75 u/50μl、DNA模板0.25 ng/μl。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.
出处
《中国药房》
CAS
CSCD
2014年第15期1345-1349,共5页
China Pharmacy
基金
国家自然科学基金资助项目(No.81060364)
