海南雷公笋的SCoT-PCR体系优化及有效引物筛选

Optimization of SCoT-PCR System and Screening of Effective Primers for Costus speciosus of Hainan

[目的]分析DNA、引物,dNTPs、Taq DNA聚合酶4种因素对雷公笋SCoT-PCR扩增结果的影响,通过最优的SCoT-PCR反应体系筛选多态性引物,为雷公笋种质资源分子标记研究提供条件。[方法]以海南雷公笋为材料,采用单因素试验和正交试验方法。[结果]Taq酶对雷公笋SCoT-PCR扩增的影响最大,其次是模板DNA和dNTPs,最后是引物;最优反应体系为20μL体系中40 ng DNA,0.30μmol/L引物,0.25 mmol/L dNTPs,3.00 U Taq酶。经验证,该体系获得的扩增产物清晰稳定;应用该体系从40条SCoT引物筛选出8条多态性好,且适合雷公笋扩增的引物,多态性条带占62.64%。[结论]该研究为使用SCoT分子标记技术对雷公笋开展深入研究提供了重要的理论基础和技术支持。

[Objective]In order to analyze the effects of DNA,primers,dNTPs and Taq DNA polymerase on the SCoT-PCR amplification results,the polymorphic primers were selected through the optimal SCoT-PCR reaction system to provide conditions for the study of molecular markers of the Costus speciosus resources.[Method]Unifactor test and orthogonal test methods were used.[Result]Taq had the greatest effect on SCoT-PCR amplification,followed by template DNA and dNTPs,and the last was primer.The optimal reaction system was 40 ng DNA,0.30μmol/L primer,0.25 mmol/L dNTPs,3.00 U Taq enzyme in the 20 system.The amplification products obtained by this system were clear and stable.8 primers with good polymorphism were selected from 40 SCoT primers,and the polymorphism bands accounted for 62.64%.[Conclusion]This study provided an important theoretical basis and technical support for further study of Costus speciosus with SCoT molecular marker technology.