蛋黄果SCoT-PCR反应体系的建立与优化

Establishment and optimization of SCoT-PCR reaction system for Lucuma nervosa A.DC

为建立和优化蛋黄果SCoT-PCR反应体系,以“仙桃1号”蛋黄果为试材,SCoT1为引物,采用L_(16)(4^(5))正交试验,对影响SCoT-PCR反应的DNA、Mg^(2+)、dNTPs、引物及Taq聚合酶等因素进行优化;并选用3份地理位置相距较远的蛋黄果种质为模板,对优化反应体系进行验证。结果表明,不同因素对蛋黄果SCoT-PCR反应体系的影响存在差异,但不显著,其中最大的影响因素是Taq聚合酶含量,其次是dNTPs浓度、模板DNA含量、引物浓度和Mg^(2+)浓度。蛋黄果SCoT-PCR最佳反应体系(20μL)为模板DNA 80 ng、3 mmol/L Mg^(2+)、0.25 mmol/L dNTPs、0.4μmol/L引物、Taq 2 U。在该体系下,3份蛋黄果种质均能稳定扩增出清晰明亮、数目丰富且稳定性高的条带,说明优化的SCoT-PCR反应体系适用于蛋黄果SCoT分子标记。

To establish and optimize the SCoT-PCR reaction system for Lucuma nervosa A.DC,factors affecting SCoT-PCR reaction such as template DNA,Mg^(2+),dNTPs,primers and Taq DNA polymerases were optimized using L.nervosa A.DC(Xiantao No.1)as research material and SCoT1 as primer by orthogonal experiment of L_(16)(4^(5)).DNA samples from three germplasms of L.nervosa A.DC located far away from each other were selected as templates to verify the optimized reaction system.The results showed that the effects of various factors on the SCoT-PCR reaction system of L.nervosa A.DC were different,but not significant.The most important factor was the concentration of Taq DNA polymerase,followed by the concentrations of dNTPs,template DNA,primers and Mg^(2+).The optimal SCoT-PCR reaction system(20μL)of L.nervosa A.DC was 80 ng template DNA,3 mmol/L Mg^(2+),0.25 mmol/L dNTPs,0.4μmol/L primer,2 U Taq DNA polymerases.With this system,the clear,bright,abundant and stable bands could be amplified from three germplasms,which indicated that the optimized SCoT-PCR reaction system was suitable for use with L.nervosa A.DC SCoT molecular markers.