山生柳SSR-PCR反应体系优化

Establishment and optimization of SSR-PCR system of Salix oritrepha

本研究以祁连山4个海拔梯度的山生柳(Salix oritrepha)为材料,采用L9(34)正交试验设计和单因子试验,分析山生柳SSR技术中PCR体系的主要成分对扩增结果的影响,并对引物SHUK123的适宜退火温度进行优化。结果表明,PCR反应体系的最佳条件:20μL体系中2.0mmol·L-1 Mg2+1μL,0.10mmol·L-1 dNTPs1.5μL,0.5UTaq酶用量为1μL,20ng·μL-1 DNA模板1μL,0.5μmol·L-1的上下游引物各2μL,10×TaqBuffer 2μL,ddH2O加至20μL。扩增反应程序:94℃高温预变性时间3min,94℃变性45s,Tm(不同退火温度)退火时间45s,72℃延伸30s,循环数30个,最后72℃后延伸时间5min,4℃保温。适宜退火温度为56℃。以上结果表明,此反应体系在山生柳PCR扩增中的稳定性和可重复性较好。

In this study, the SSR-PCR system of Salix oritrepha from four altitudes was optimized. The main factors of the system were studied using orthogonal design and single factor experiment. Meanwhile, the suitable annealing temperature of primer SHUK123 was optimized. The results of this study showed the optimized system was： 1μL Mg2＋ （2. 50 mmol·L-1 ）, 1. 5 μL dNTPs concentration （0. 10 mmol·L-1） , 1 μL Taq polymerase （0.5 U）, 1μL DNA template （20 ng·L-1）, 2 μL each primer concentration （0.5 mmol·L-1 ） , 2 μL 10 ×Taq Buffer in a mixture of 20μL. The PCR procedure included an initial step of 94℃ for 3 min, followed by 30 cycles of 94 ℃ for 45 s, annealing 45 s （annealing temperature varies with the primers）, 72℃ extension 30 s, and a final extension at 72 ℃ for 5 min. The products of PCR were saved at 4 ℃. The optimized annealing temperature of primer SHUK123 was 56 ℃. The optimized SSR-PCR system was stable and repeatable, which will be a good choice in the study of S. oritrepha.