正交实验设计优化白木香ISSR-PCR反应体系的研究

Optimization for ISSR-PCR System of Aquilaria Sinensis (Lour.) Spreng Using Orthogonal Design

利用正交实验设计的方法,对白木香ISSR-PCR(简单重复序列区间-多聚酶链式反应)反应体系中的5种主要因素(dNTP、Mg2+、模板DNA、引物和Taq酶)在4个水平上进行优化筛选,建立了白木香ISSR-PCR反应的最佳体系(25μl)为:dNTP 0.2 mmo1/L、Mg2+2.5 mmol/L、引物0.5μmol/L、模板DNA 100 ng和Taq酶1.0 U。通过梯度PCR反应得到相应引物的最佳退火温度为49.2℃。这一体系的建立为今后利用ISSR标记技术研究白木香的遗传多样性提供了标准化程序。

The orthogonal design was used to optimize inter simple sequence repeat PCR （ISSR-PCR） amplification system on Aquilaria sinensis （Lour.） Spreng in five factors （dNTP,Mg^2＋ ,DNA template, primer and Taq DNA polyrnerase） at four levels respectively. The most suitable ISSR-PCR system for A. sinensis was established,namely 25 μl reaction system containing 0. 2 mmol/L dNTP,2.5 mmol/L Mg^2＋ ,100 ng DNA template,0. 5 vmol/L primer, and 1.0 U Taq DNA polymerase. The optimal annealing temperature for ISSR-PCR reaction was proposed by gradient PCR and it was 49.2℃. The result provided a standardizing ISSR-PCR program for the analysis of genetic diversity of A. sinensis.