【摘要】 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。更多还原

【Abstract】 OBJECTIVE: To study the reaction system and amplification process of ISSR(inter-simple sequence repeat) amplification of L.macranthoides.METHODS: The influencing factors of ISSR such as Mg2+ concentration,dNTP concentration,primer concentration,Taq polymerase concentration,template DNA concentration were optimized and the effect of the annealing temperature on the ISSR amplification was investigated.RESULTS: The optimized reaction system was obtained as follows: each 25 μL reaction mixture contained 2.0 mmol·L-1 Mg2+,0.2 mmol·L-1 dNTP,0.3 μmol·L-1 primer,1.0 U Taq polymerase,20 ng template DNA.The amplification cycling was as follows: after an initial cycle of denaturation at 94 °C for 5 minutes,the reaction mixture was amplified for 35 cycles at 94 ℃ for 30 seconds,52 ℃ for 45 seconds and 72 ℃ for 90 seconds,then extension was performed at 72 ℃ for 5 minutes.Under these conditions clear and stable bands were obtained.CONCLUSION: These experiment conditions are suitable for ISSR amplification of L.macranthoides更多还原