食用向日葵SSR-PCR反应体系的优化

Optimization of SSR-PCR Reaction System in Confectionary Sunflower

为建立食用向日葵分子标记反应体系,以食用向日葵四叶期叶片为DNA模板提取材料,采用单因素试验和正交试验设计,对SSR-PCR反应体系中的6因素(10×PCR Buffer、Mg2+、d NTPs、引物、Taq DNA聚合酶和DNA模板)在5水平上进行正交优化试验,并比较了不同浓度Mg2+、Taq DNA聚合酶、模板DNA对扩增效果的影响,结果表明,各因素水平变化对反应体系的影响为Mg2+>Taq DNA聚合酶(引物)>DNA模板>10×PCR Buffer>d NTPs。最终建立食用向日葵SSR-PCR最佳反应体系为:在总体系为20μL的SSR-PCR反应体系中包括10×PCR Buffer 0.2mmol/L、Mg2+2.0 mmol/L、d NTPs 1.8 mmol/L、Taq DNA聚合酶0.2 U、DNA 50 ng、引物1.5 mmol/L。

In order to established confectionary sunflower molecular markers reaction system,the study assum-ing DNA template from four-leaf stage of confectionary sunflowers leaves for experimental materials,this study used the single factor test and orthogonal design for optimizing six SSR-PCR reaction system factors（ 10 × PCR Buffer, Mg2 ＋,dNTPs,primers,Taq DNA polymerase,DNA template and compared different concentrations of Mg2 ＋, dNTPs,Taq DNA polymerase and DNA template amplification effect of the impact in SSR-PCR reaction system. The result showed that the range of the influence of six factors in the reaction system was Mg2 ＋ 〉Taq DNA polymerase （ primer）〉DNA template〉10 × PCR Buffer〉dNTPs. And this study established confectionary sunflower SSR-PCR reaction system（20 μL）：10 × PCR Buffer 0. 2 mmol/L,Mg2 ＋ 2. 0 mmol/L,dNTPs 1. 8 mmol/L,Taq DNA polymer-ase 0. 2 U,DNA 50 ng,primer 1. 5 mmol/L.