金钗石斛相关序列扩增多态性反应体系的正交优化研究

Optimization of sequence-related amplified polymorphism system in Dendrobium nobile based on orthogonal design

目的建立金钗石斛Dendrobiumnobile相关序列扩增多态性(SRAP)反应体系,为金钗石斛分子生物学研究提供技术支持。方法运用L25(56)正交设计对影响金钗石斛SRAP反应的5个因素:模板DNA、Mg2+、dNTPs、Taq酶、引物在5个水平上进行优化试验,对PCR结果进行极差分析。结果金钗石斛SRAP反应最佳体系为:25μL PCR体系中含有20 ng模板DNA,2.0 mmol/L Mg2+,0.2 mmol/L dNTPs,1 UTaq酶,0.6μmol/L引物。各因素对SRAP反应的影响依次为:Taq酶>Mg2+>dNTPs>模板DNA>引物。结论所建立的金钗石斛SRAP反应体系具有标记位点清晰、多态性丰富、稳定性好等特点,可用于金钗石斛分子生物学的研究。

Objective To establish the optimal sequence-related amplified polymorphism（SRAP） reaction system for molecular biology research in Dendrobium nobile.Methods The L25（56） orthogonal design was used to optimize SRAP-PCR amplification system of D.nobile on five levels,the experiment was performed on five factors： template DNA,Mg2＋,dNTPs,Taq DNA polymerase,and primer.The data generated by SRAP-PCR were analyzed.Results The optimal condition of the five important factors, template DNA,Mg2＋,dNTPs,Taq DNA polymerase,and primer in the 25 μL SRAP-PCR reaction system were 20 ng,2.0 mmol/L,0.2 mmol/L,1 U,and 0.6 μmol/L,respectively.The effective degree of the factors on the SRAP reaction was in the order： Taq DNA polymerase Mg2＋ dNTPs template DNA primer.Conclusion The present reaction system could provide clear bands,abundant polymorphisms,and reliable reaction.It is proved to be suitable for molecular biology research of D.nobile.