采用正交设计方法优化观赏桃ISSR-PCR反应体系

摘要

In order to obtain the optimal ISSR-PCR reaction system of Prunus persica,and lay a foundation for studying genetic diversity of ornamental peach,concentrations of five factors (Taq DNA polymerase,Mg2 +,DNA template,dNTPs and primer) in P.persica ISSR-PCR amplification system were researched by using the method of orthogonal design,and data were analyzed by using software.The results showed that the optimal ISSR-PCR system was 0.4 U Taq DNA polymerase,1 mmol/L Mg2 +,10-80 ng DNA template,0.20 mmol/L dNPs and 0.3 μmol/L primer in 25 μL reaction system.The results of radient PCR showed the optimal annealing temperature for [SSR-PCR reaction was 49.3 ℃.The system was stable and reproducible,and could be used in the future researches on P.persica.%为了获得观赏桃ISSR-PCR最佳反应体系,并为观赏桃遗传多样性研究打下基础,采用正交设计的方法对观赏桃ISSR-PCR反应体系的5因素(Taq DNA聚合酶、Mg2+、模板DNA、dNTPs、引物)的浓度进行研究,用SPSS软件处理分析数据.试验结果表明,观赏桃的最佳反应体系(25 μL)为Taq DNA聚合酶0.4 U,1 mmol/L Mg2+,模板DNA10～80 ng,dNTPs 0.20 mmol/L,引物浓度0.3 μmol/L,并且在梯度退火试验后发现49.3℃为最佳退火温度.这一体系反应稳定、重复性强,可以用于观赏桃基因研究.