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目的:研究灰毡毛忍冬ISSR扩增的反应体系和扩增程序。方法:对影响灰毡毛忍冬ISSR扩增的反应体系的Mg2+浓度、dNTP浓度、引物浓度、Taq酶浓度、模板DNA浓度进行优化,考察退火温度对ISSR扩增的影响。结果:优化的反应体系为25μL中,Mg2+浓度为2.0mmol.L-1,dNTP浓度为0.2mmol.L-1,引物浓度为0.3μmol.L-1,Taq酶浓度为1.0U,模板DNA浓度为20ng。扩增程序为94℃预变性5min,1个循环;94℃30s,52℃45s,72℃90s,35个循环;72℃延伸5min,可扩增出清晰稳定的条带。结论:该条件适合于灰毡毛忍冬的ISSR扩增。
Objective: To study the reaction system and amplification procedure of ISSR amplification of Lonicera macranthoides. METHODS: The Mg2+ concentration, dNTP concentration, primer concentration, Taq enzyme concentration, and template DNA concentration in the reaction system affecting the ISSR amplification of Lonicera macranthoides were optimized. The effect of annealing temperature on the ISSR amplification was investigated. Results: The optimized reaction system was 25μL, Mg2+ concentration was 2.0mmol.L-1, dNTP concentration was 0.2mmol.L-1, primer concentration was 0.3μmol.L-1, Taq enzyme concentration was 1.0U, template DNA concentration. It is 20ng. The amplification procedure was pre-denatured at 94°C for 5 min, 1 cycle; 94°C for 30s, 52°C for 45s, 72°C for 90s, 35 cycles; 72°C for 5 min, a clear and stable band was amplified. Conclusion: This condition is suitable for ISSR amplification of Lonicera macranthoides.