悬铃木AFLP反应体系优化及引物筛选

摘要

以三球悬铃木为试验材料,通过对影响AFLP反应体系的各主要因素的研究,建立了悬铃木AFLP分析技术的体系.结果表明,悬铃木AFLP最佳酶切体系(20 μL)为模板DNA 1 000 ng,PstⅠ和MseⅠ各为3U,在37℃下双酶切3h;在20 μL最佳连接体系中,15 μL酶切产物为3U,T4连接酶,0.25 μmol·L-1Pst I接头,2.5μmol·L-1 MseⅠ接头,1 μL 10×T4 buffer,在22℃下连接12 h；在20 μL最佳预扩反应体系中稀释15倍的连接产物5 μL,2.0 mmol·L-1 Mg2+,2 UTaq酶,200 μmol·L-1 dNTP,0.5μmol·L-1 PstⅠ和Mse Ⅰ引物(P+AGA/M+ ATC)；在20 μL最佳选择性扩增反应体系中5μL稀释15倍的预扩增产物,2.0 mmol·L-1的Mg2+,2U Taq酶,200 μmol·L-1 dNTP,0.5μmol·L-1 Pst I和MseI引物(P+AGA/M+ ATC).最后,利用上面的体系筛选出了92对适宜于悬铃木AFLP分析的引物.%Optimization of AFLP reaction system and its primer selection were investudied with Platanus orientalis as experimental materials in this paper. The results showed that the optimal digestion system (20μL ) included 100 ng the template DNA,3 U both Pst I and Mse I each digesting at 37℃ for 3 h; The best connection system (20μL) was 15μL digestion products 3 U,T,ligase,0.25μmol·L-1 Pst I adaptor,2.5μmol·L-1 Mse I adaptor,1μL 10 x T4 buffer connecting at 22℃ for 10 h; The optimal pre-amplification system (20μL) included 5μL 1/15 of the connection products,2.0 mmol·L-1 Mg2+ ,2 U Taq enzyme,200μmol·L-1 dNTP,0.5μmol·L-1 Pst I and Mse I primer(P+AGA/M+ATC);The optimal selective amplification system (20μL) was 5μL 1/15 pre-amplification products,2.0 mmol·L-1of Mg2+ ,2 U Taq enzyme,200μmol·L-1 dNTP,0.5μmol·L-1 Pst I and Mse I primers (P + AGA/M + ATC). Moreover,screening of 92 pairs of primers was suitable for the AFLP analysis of Platanus orientalis with the optimized AFLP system.