烟草DNA的提取与SRAP反应体系的建立

以10个烟草栽培品种为试验材料,研究了烟草DNA的提取方法以及对建立烟草SRAP-PCR反应体系的影响因子设置梯度实验,筛选和建立可扩增多态性高、重复性好、带型清晰的最佳SRAP-PCR反应条件:在25μL的反应体系中,MgCl22.0mmol、dNTP200μmol,上下引物各30ng、DNA模板40ng、DNA聚合酶1.5U,扩增程序为:在94℃预变性5min,反应前5个循环在94℃1min,33℃1min,72℃1min条件下运行;随后的30个循环复性温度提高到53℃,最后72℃延伸5min。本文还讨论了不同分子标记在烟草中应用的优缺点以及SRAP分子标记在烟草上的应用前景,提出了应用SRAP分子标记构建烟草遗传图谱的可行性。 

DNA extraction and the establishment of SRAP reaction system in tobacco

Method of DNA extraction and factors influencing SRAP analysis were studied using ten tobacco cultivars. A reliable,effective and reproductive PCR reaction system for detecting SRAP was developed.Each 25μL PCR reaction mixture consisted of 40ng of genomic DNA,200μmol of dNTPs,2.0mmol of MgCl2,30ng of primer and 1.5 unit of Taq polymerase.Samples were subjected to thermal profile for amplification in an oven thermocycler:5 min of denaturing at 94℃,five cycles of three steps:1min of denaturing at 94℃,1min of annealing at 33℃ and 1min of elongation at 72℃. In the following 30 cycles the annealing temprerature was increased to 53℃,with a final elongation step of 5min at 72℃.Different molecular marks used in tobacco were also analyzed. It was shown that SRAP technology is a useful molecular marker system for mapping and gene tagging in tobacco.