天麻扩增酶切片段长度多态性反应体系的优化

Optimation of amplification length polymorphism (AFLP) reaction system with Gastrodia elata Blume

以新鲜天麻为材料,优化天麻扩增长度多态性(AFLP)反应体系中的几个关键参数。结果表明,用100～200ng/μL的天麻DNA2.5μL,反应体系25μL,37℃酶切连接过夜,酶切连接效果最佳;预扩产物稀释20倍,TaqDNA聚合酶为1 U,Mg2+(20 mM)1.5μL、dNTP(2 mM)2μL时选扩条带最清晰。体系优化后,扩增产物稳定,条带十分清晰,无背景干扰,这为构建天麻的AFLP指纹图谱和分子标记辅助育种提供了有力支撑。

In this study, the main factors influencing amplification length polymorphism （AFLP） reaction system were optimized on Gastrodin. The results showed that the effect of digestion connection was the best in 2.5μL of the 100-200 ng/μL of gastrodin DNA, 25μL of the reaction system, 37℃ overnight digestion connection; selective amplification was clear-cut strip in pre-expanded 20-fold diluted product, TaqDNA polymerase 1 U, Mg^2＋ （20 mM ）1.5 μL ,dNTP （2 mM） 2 μL. With the optimized system, amplified bands were clear and stable without disturbed background, which would provide a powerful tool for building the AFLP fingerprint of Gastrodin and molecular marker-assisted breeding.