贯叶连翘ISSR-PCR反应体系的建立与条件优化

摘要

[目的]建立贯叶连翘的ISSR-PCR反应体系,并对其条件进行优化.[方法]以贯叶连翘基因组DNA为模板,用L16(45)正交试验设计系统分析引物浓度、Taq DNA聚合酶浓度、Mg2浓度、dNTP浓度和模板DNA浓度5种因素对贯叶连翘ISSR-PCR反应扩增结果的影响.[结果]正交试验设计的方法可以用于贯叶连翘1SSR-PCR反应体系的建立,经过优化得到贯叶连翘ISSR-PCR反应体系的最佳条件为:20μISSR-PCR反应体系中含10×PCR buffer,Mg2+浓度1.2 mmol/L,Taq DNA聚合酶浓度50 U/ml,DNA浓度20 ng/μl,dNTP浓度250 μmol/L,引物浓度0.75 μmol/L.[结论]试验建立的贯叶连翘的ISSR-PCR反应体系重复性好、分辨率高,结果稳定可靠.%[ Objective ] To establish the ISSR-PCR Amplification System for Hypericum perforatum Linn. , and to optimize the condition. [Method] With genome DNA of H. Perforation as the template, L16(45) orthogonal test system was adopted; the effects of primer concentration , Taq DNA polymerase, Mg2+ concentration, dNTP concentration, and template DNA concentration on the Amplification results of ISSR-PCR were researched. [Result] Orthogonal test could be used to establish the ISSR-PCR reaction system of H. Perforatum. And the optimal condition of ISSR-PCR reaction system was as follows:20 μl ISSR-PCR reaction system had 10×PCR buffer, 1.2 mmol/L Mg2+ , 50 U/ml Taq DNA polymerase, 20 ng/μl DNA, 250 μmol/L dNTP, and 0.75 μmol/L primer. [Conclusion]The established ISSR-PCR reaction system for H. Perforatum had good repeatability, high resolution ratio, and stable and reliable results.