党参DNA-AFLP分析体系建立及条件优化

Establishment and Optimization of DNA-AFLP Analysis of Codonopsis Pilosula

探讨影响党参(Codonopsis pilosula)扩增片断长度多态性(Amplified Fragment Length Polymor-phism,AFLP)的各种因素,建立并优化党参AFLP反应体系。以党参鲜嫩叶片为材料,采用改进的CTAB法提取基因组DNA,考察适合的酶切反应时间、对连接产物和预扩产物设置不同稀释倍数进行扩增,最后聚丙烯酰胺凝胶电泳分离,银染检测。本研究建立了适用于党参的AFLP体系:300 ng的基因组DNA被限制性内切酶Mse I和EcoR I完全酶切,酶切条件为37°C 3 h,连接条件为16°C 12 h;连接产物1倍,预扩产物150倍稀释。扩增后,2对E+3/M+3引物组合共得到90条多态性条带,PAGE电泳条带清晰,稳定,分辨率高。本研究建立的反应体系适用于党参的DNA-AFLP分析研究。

The factors affecting DNA- AFLP （DNA amplified fragment length polymorphism） were investigated to develop and optimize a DNA - AFLP reaction system of Codonopsis pilosula. Total genomic DNA was extracted from fresh leaves using CTAB protocol with minor modifications. The reaction time of digestion was investigated. Then ligation products were provided for pre-amplification and selective amplification of various concentration gradients. Amplified fragments were separated electrophoretically by 6G denaturing PAGE （polyacrylamide gel electrophoresis） and silver-staining was performed. The DNA - AFLP reaction system of Codonopsis pilosula was as follows： 300 ng of genomic DNA was thoroughly digested at 37 ℃ for 3 h, and ligated at 16 ℃ for 12 h. Furthermore, the sample dilution multiplication was primitive multiple for pre-amplification and 150 - fold for selective amplification under the proper system concentration. According to the above reaction system, 90 polymorphic bands were obtained with 2 E＋3/M＋3 primier combinations. The polymorphous bands with high resolution in PAGE were clear and Stable. The DNA - AFLP reaction system established and optimized in this experiment is suitable for the analysis of Codonopsis pilosula.