何首乌扩增长度多态性反应及银染体系的建立

Establishment of Polymorphism Reaction for Amplified Fragment Length and Silver- staining System for Polygonum multiflorum Thunb

目的通过对各主要影响因素的研究,建立适于何首乌扩增长度多态性(AFLP)反应体系及银染体系。方法采用改进的CTAB提取方法从何首乌的嫩叶中提取获得总DNA;从酶切DNA的量、时间等考察酶切体系,并设计正交实验进行预扩体系和选扩体系的优化。结果酶切体系DNA的适用量为400ng,37℃酶切5h;用较优的预扩增体系和选择性扩增得到的选择性扩增产物在6%变性聚丙烯酰胺凝胶上电泳,得到清晰的指纹图谱。结论所建立的反应体系可有效地应用于何首乌的分类鉴定和道地性鉴定。

Objective To establish the optimum polymorphism （AFLP） analysis system for amplified fragment length and silver staining system for Polygonum multiflorum Thunb. Methods The improved method of CTAB-DNA isolation was used to extract total DNA from Polygonum multiflorum Thunb. Several factors including the quantity and time for digested DNA were investigated and orthogonal test was used to optimize the system of pre-amplification and selective amplification. Results 400 ng DNA were digested with restriction enzymes at 37 ℃ about 5 h. The sharp fingerprint patterns were obtained by 6% denatured acrvlamide/bisacrvlimade gels electrophoresis with the AFLP silver-staining using the optimized system of preamplification and selective amplification. Conclusion The AFLP reaction system could be used to identify the species and local populations for Polygonum multiflorum Thunb. with efficiency, strong stability and reliability.