杨树基因组SRAP扩增体系的建立与优化

Establishment and Optimization of SRAP-PCR Reaction System of Populus

以响叶杨和银白杨及F1代为实验材料,利用正交设计直观分析法和单因素随机试验对杨树SRAP反应体系中各组分(TaqDNA聚合酶、dNTP、引物和Mg2+)的浓度进行优化;通过实验确定10μL反应体系为:引物0.35μmol/L,TaqDNA聚合酶0.5U,dNTP0.2mmol/L,Mg2+2.0mmol/L,10×PCRbuffer1μL,模板DNA1μl(20ng/μL)。同时利用最佳反应体系以及正交设计直观分析法对PCR扩增程序进行筛选,并通过梯度PCR试验,确定引物最佳退火温度。结果表明最佳反应程序为:开始94℃变性5min,反应前5个循环在94℃1min,37℃30s,72℃1min条件下运行;之后35个循环在94℃1min,54℃1min,72℃1min条件下运行,最后延伸7min;12℃保存。并利用两树种的杂种F1代对优化的反应体系和程序的可行性进行了验证。

Selecting Populus sadenopoda, P. alba and F1 （ P. sadenopoda × P. alba ） as the experimental materials, the SRAP-PCR reacting system of Populus was optimized by the orthogonal design along with single factor experiments. The results showed that the 10 μL volume of SRAP-PCR system contained 0.35μmol/L primer, 0. 5U Taq DNA polymerase , 0. 2 mmol/L dNTP, Mg^2＋ 2.0 mmol/L, 1μL 10 × PCR buffer, 1μL template DNA（20 ng/μL）. At the same time, using the best response system and the orthogonal design, the PCR amplification process was filtered. And the best primer annealing temperature was determined through gradient PCR test. The optimum SRAP-PCR reaction procedure was as follows： 94℃ 5 rain; 94℃ 1 min, 37℃ 30 s, 72℃ 1 min , 5 cycles; 94℃ 1min, 54℃ 1min, 72℃ lmin, 35cycles; 72℃ 7min; 12℃ forever. Then, the optimized reaction system and procedure was tested with F1 hybrid.