羌活和宽叶羌活简单重复序列区间扩增反应体系的正交优化

Optimization for ISSR-PCR Reaction System in Notopterygium incisum Ting and N.forbesii Boiss Using Orthogonal Design

目的通过探讨羌活和宽叶羌活ISSR实验中各种因素的影响，以建立羌活和宽叶羌活的简单重复序列区间扩增（ISSR—PCR）最佳反应体系。方法利用正交实验设计L9（3^4）对羌活和宽叶羌活ISSR-PCR反应体系的4因素（Mg^2＋，dNTP，引物，Taq酶）在3个水平上进行优化实验，PCR结果用SPSS13．0统计软件分析，并设置梯度PCR进行引物退火温度筛选。结果在20μl ISSR—PCR反应体系中，各组分的最适浓度为：2．0mmol／L Mg^2＋，250μmol／L dNTPs，0．50μmol／L引物，1U Taq酶，40ng DNA模板；模板DNA 40—90μg，不同引物有不同的最佳退火温度，范围在48—52℃。结论该优化体系的建立为进一步进行羌活和宽叶羌活种群间遗传多样性研究提供了参考。

Objective The optimal ISSR - PCR reaction system for Notopergium incisum Ting and N. forbesii Boissin was constructed by studying the influence of ISSR factors. Methods The orthogonal design with three levels of four factors （ Taq DNA polymerase, Mg^2＋, dNTPs and primer） was used to optimize ISSR-PCR amplification reaction system of N. incisum, and N. forbesii. The data of PCR results were analysed with SPSS software, and the different annealing temperatures were also studied by gradient PCR. Results The optimal ISSR - PCR reaction system contained 2.0 mmol/L Mg^2＋, 1U Taq DNA polymerase, 250μmol/L dNTPs, 0.50μmol/L primer and 40 ng DNA template in the total volume of 20 μl. The Annealing temperature ranged from 48 to 52℃. Conclusion The optimal ISSR - PCR reaction system provides the basis for further study on the genetic diversity among the populations of N. incisum, and N. forbesii.