论文部分内容阅读
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。
To explore various factors affecting the Amplified Fragment Length Polymor-phism (AFLP) in Codonopsis pilosula and to establish and optimize the AFLP reaction system. Taking the fresh leaves of Codonopsis pilosula as material, the genomic DNA was extracted by the improved CTAB method, the suitable reaction time was investigated, and the products of dilution and multiplication were set at different dilutions. Finally, polyacrylamide gel electrophoresis, silver Dye detection. In this study, an AFLP system for Codonopsis pilosula was established: 300 ng of genomic DNA was completely digested with restriction enzymes Mse I and EcoR I, the digestion condition was 37 ° C for 3 h, and the ligation conditions were 16 ° C for 12 h; Connect the product 1 times, pre-expansion products 150-fold dilution. After amplification, a total of 90 polymorphic bands were obtained from 2 pairs of E + 3 / M + 3 primer combinations. The bands of PAGE electrophoresis were clear and stable with high resolution. The reaction system established in this study is suitable for DNA-AFLP analysis of Codonopsis pilosula.