麻疯树SRAP-PCR反应体系的优化

Optimization of SRAP-PCR Amplification System of Jatropha curcas

采用L9(45)正交设计对影响麻疯树SRAP反应的5个因子(DNA模板浓度、Mg2+浓度、dNTPs浓度、引物浓度、Taq酶)在4个水平上进行优化试验,PCR结果采用SPSS13.0进行分析。结果表明,各因素对SRAP反应的影响依次为:Mg2+>dNTPs>引物>Taq酶>DNA模板。对SRAP反应结果影响较大的3个因子(Mg2+浓度、dNTPs浓度、引物浓度)进行单因素试验,确立麻疯树SRAP反应最佳体系为:20μL的PCR体系中含有Mg2+2.5 mmol/L、引物0.4μmol/L、dNTPs150μmol/L、DNA模板60 ng、Taq酶0.5 U。将该体系用于麻疯树的SRAP扩增能获得稳定、清晰的多态性条带。

The orthogonal design was used to optimize 5 factors to influence SRAP-PCR amplification of Jatropha curcas at four levels and the result was analyzed by SPSS13.0 software. The results showed that the effect of different factors on SRAP amplification was ：Mg^2＋ 〉dNTPs〉primer〉 DNA template〉 Taq DNA polymerase. The optimum SRAP amplification system including 20~L PCR, 2. 5mmol/L, Mg^2＋ , 0.4 μmol/L primer, 150μmol/L dNTPs, 60 ng DNA template and 0.5 U Taq DNA polymerase was established based on the single-factor tests of Mg^2＋, dNTPs and primer. The optimum SRAP amplificatinn system cnuld be used in SRAP amplification of Jatropha curcas.