大花君子兰ISSR-PCR反应体系的建立与优化

Establishment and Optimization of ISSR-PCR Reaction System in Clivia miniata

以大花君子兰为材料研究了PCR反应体系的主要成分及退火温度对君子兰ISSR扩增结果的影响,并对影响PCR反应体系的主要成分及退火温度进行筛选和优化,确立了适合君子兰ISSR-PCR分析的最佳反应体系：在20μL的反应体系中,Mg^2＋为2.0 mmol/L,模板DNA用量为50 ng/μL,Taq酶0.4 U,dNTPs浓度为0.15 mmol/L,引物浓度为0.4 mmol/L。筛选出了13个适宜君子兰遗传分析的ISSR引物,确定最适宜退火温度为52-56℃。

The factors affecting the ISSR（inter-simple sequence repeat） result of Clivia（Clivia miniata） were studied.By adjusting Mg^2＋,dNTPs, primer,Taq DNA polymerase and annealing temperature were optimized.The optimal ISSR-PCR reaction conditions for Clivia miniata were determined as follows,in 20 μL PCR reaction system,2.0 mmol/L Mg2＋,50 ng/μL template DNA,0.4 U Taq DNA polymerase,0.15 mmol/L dNTPs,0.4 μmol/L primer.Thirteen ISSR primers that are suitable for Clivia were screened,and the optimal annealing temperature for the ISSR reaction was 52—56℃.