摘要
为了探索建立烟草的ISSR-PCR最适反应体系,用单因素设计方法对影响ISSR-PCR反应体系的主要影响因素进行优化筛选。结果表明,烟草的最适反应体系为:反应总体积为20μL:2.00μL10×buffer,50ng(1μL)模板DNA,Taq聚合酶的用量为4U(0.8μL),0.6μmol/L(1.2μL)引物,0.75 mmol/L(0.6μL)Mg2+,0.3 mmol/L(2.4μL)d NTP,dd H2O12μL,退火温度为52℃。用筛选的引物对36份烟草种质分别进行PCR扩增,扩增条带清晰,清晰度和多态性都较好,可以认为该反应体系对烟草种质具有较好的可靠性和稳定性。
To establish the optimal ISSR-PCR reaction system for tobacco,the experiment used single factor design to optimize and screen the major factors influencing ISSR-PCR reaction system. Results showed that the optimal reaction system was as follows： 20 μL of total reaction capacity：2.00 μL10×buffer,50 ng（1 μL） DNA template,4 U（0.8 μL） Taq DNA polymerase, 0.6 μmol/L（1.2 μL） primers, 0.75 mmol/L（0.6 μL） Mg^2＋ and 0.3（2.4μL） mmol/L d NTP, dd H_2O 12 μL, with annealing temperature of 52 ℃. 36 tobacco germplasm were treated with PCR amplification using the selected primers. The acquired amplification bands were clear, with relatively good clearness and polymorphism. The results indicated that this reaction system showed better reliability and stability for tobacco germplasm.
作者
覃剑峰
何江
王新钧
王东贤
王卫峰
陈涛
覃旭
崔明勇
黄显雅
杨祥燕
彭欣怡
QIN Jianfeng;HE Jiang;WANG Xinjun;WANG Dongxian;WANG Weifeng;CHEN Tao;QIN Xu;CUI Mingyong;HUANG Xianya;YANG Xiangyan;PENG Xinyi(Guangxi Subtropical Crops Research Institute,Nanning 530001,China;Guangxi Tobacco Department,China National Tobacco Corporation,Nanning 530001,China)
出处
《农业研究与应用》
2018年第3期19-22,共4页
Agricultural Research and Application
基金
中国烟草总公司广西壮族自治区公司科技创新项目(桂烟科[2014]3号)
