灰毡毛忍冬ISSR扩增的反应体系和扩增程序研究  

Reaction System and Amplification Process of ISSR Amplification of Lonicera macranthoides

作　　者：周日宝[1] 王珊[1] 潘清平[1] 谭晓风[2] 陈玉秀[1] 吴佳[1] 

机构地区：[1]湖南中医药大学药学院,长沙市410208 [2]中南林业科技大学经济林育种与栽培国家林业局重点实验室,长沙市410004

出　　处：《中国药房》2009年第33期2626-2628,共3页China Pharmacy

基　　金：湖南省教育厅省属高校科研项目(01C236);湖南省卫生厅中医药科研基金课题(2006-062);长沙市科技计划项目(k0901078-21)

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。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 Mg^2＋ 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 Mg^2＋,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 ℃ 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. rnacranthoides

关 键 词：灰毡毛忍冬 微卫星分子标记技术 体系优化 扩增 

分 类 号：R282.7[医药卫生—中药学] R283.1[医药卫生—中医学]