【摘要】 目的:建立一个适于华中五味子研究用的AFLP反应体系。方法:以华中五味子硅胶干燥嫩叶为试材,采用改良CTAB法提取到高质量DNA。通过琼脂糖凝胶电泳和聚丙烯酰胺凝胶电泳对MseⅠ/EcoRⅠ双酶切、连接、预扩增和选择性扩增过程中的关键因素进行分析。结果:双酶切6h,片段主要集中在250～2000bp;连接产物和预扩增产物最适稀释倍数均为10倍;预扩增产物经选择性引物E-ACT/M-CAT和E-ACA/M-CAG扩增,琼脂糖电泳检测其主带分别集中在250～375bp和500～750bp,6%聚丙烯酰胺凝胶电泳检测及银染,条带清晰可辨。结论:该体系具有稳定性高、重复性好等优点,可用于华中五味子AFLP分析。更多还原

【Abstract】 Objective:To establish an appropriate AFLP(Amplified Fragment Length Polymorphism) analysis system for Schisandra sphenanthera Rehd. et Wils.(S. sphenanthera). Method: High quality genomic DNA from dry tender leaves of S. sphenanthera was isolated by improved CTAB method. The influencing factors of several reactions, including Mse I/EcoR I digest, adapter ligation, pre-amplification, selective amplification, were analyzed by the methods of agarose gel electrophoresis and polyacrylamide gel electrophoresis. Result: Restriction fragments were mainly concentrated between 250 bp and 2 000 bp after 6 hours digest. Ligation product and pre-amplification were diluted to the optimum 10 times. After 6 %polyacrylamide gel electrophoresis and silver staining, the clear AFLP fingerprintings have been obtained with primer pairs E-ACT/M-CAT and E-ACA/M-CAG amplication which were centralized 250～375 bp and 500～750 bp respectively by 0.8% agarose gel electrophoresis. Conclusion: With the virtue of high stability and good repeatability, AFLP system is very fit for S. sphenanthera analysis.更多还原