蜡梅AFLP-银染体系的建立与优化

Establishment of Suitable Silver-stained AFLP Reactions for Chimonanthus praecox

为了建立蜡梅AFLP-银染的优化体系,采用EcoRⅠ和MseⅠ 2种限制性内切酶酶切组合,对该2种酶的用量、酶切时间、预扩增与选择扩增中的Mg^2＋浓度、Taq DNA聚合酶的用量、引物的浓度以及预扩增产物的稀释倍数等因素进行了多水平的筛选,同时探讨了温度及聚丙烯酰胺凝胶电泳（PAGE）的电流强度等因素对胶图的影响。结果表明：用5 U限制性内切酶酶切4 h便能在40μL的反应体系中完全切割500 ng的总DNA,酶切连接产物稀释10倍后在含有1.5 mmol/L Mg^2＋1、U Taq、预扩增引物E＋A和M＋C分别为40 ng的20μL反应体系中进行预扩增,将预扩增产物稀释30倍后在含有1.5 mmol/L Mg^2＋、0.6 U Taq、选择扩增引物E＋3为30 ng、M＋3为60 ng的20μL的体系中进行选择性扩增,能够得到质量比较好的AFLP胶图。此外,在进行PAGE电泳和硝酸银染色,将电泳的温度控制在50℃左右,电流控制在45～60 mA左右能提高胶图的质量。利用这种优化的反应体系,成功地评价了40个蜡梅基因型的遗传多样性,6对选择扩增引物组合表现出较高的稳定性、清晰度和多态性,它们分别是：E-AAC/M-CCC,E-ATC/M-CCC,E-AGA/M-CAG,E-AAA/M-CAC,E-ATA/M-CCA,E-ATT/M-CCA。

In this study,silver-stained amplified fragment length polymorphism （AFLP） was applied to study on the germplasm of Chimonanthus praecox. Based on screening several key reaction conditions, an optimal AFLP reaction system for C. praecox was established as follows, it was appropriate for 0. 5 g genomic DNA to be digested in a total volume of 40 μL for 4 h at 37 ℃ with 5 U EcoR Ⅰ and 5 U Mse Ⅰ ; 1.5 mmol/L Mg^2＋ was best for two amplifications （both performed in 20μL） ; in preamplification, 1 U Taq and 40 ng for each primer yielded the best image, while in selective amplification, 0. 6 U Taq and 60 ng of Mse Ⅰ primers which was twice than EcoR Ⅰ primers proved to be optimal; and the products obtained from preamplification should be reduced to 1/30 before being used as the template for selective amplification. In addition,the influence of other factors such as temperature and electrophoretic parameters was also discussed. As a result, there would be more efficient if temperature was about 50℃ and the current was controlled in the range of 45～60 mA while polyacrylamide gels electrophoresis （PAGE） was running. Using this suitable AFLP reaction system, six polymorphic primer combinations were screened out by evaluating genetic diversity among 76 C. praecox genotypes, which were E-AAC/M-CCC, E-ATC/M-CCC, E-AGA/M-CAG, E-AAA/M-CAC, E-ATA/M-CCA, E-ATT/M-CCA.