小麦雄性不育材料RAPD扩增体系研究

Studies on the System of RAPD Reaction for Male-sterile Wheat

以光敏雄性不育系A31及其恢复系1376为材料,使用美国MGREACHE公司生产的PTC-200型PCR,对影响RAPD扩增的因素进行了比较研究,确定了模板、Mg2+、dNTPs、引物和TapDNA聚合酶的适宜浓度和最佳的循环次数,实验结果表明在25μL反应体系中使用20～40ng的模板DNA,1.5～2.0mmol/L的Mg2+,0.3～0.4mmol/L的dNTPs,0.15～0.20μmol/L随机引物,1.0～1.5单位TaqDNA聚合酶;反应条件为94℃预变性5min,然后进行94℃45s,36°45s,72°90s,45个循环后,再在72℃延伸10min,小麦雄性不育材料RAPD扩增效果较好。

Using wheat photoperiod-sensitive male sterile line (A31) and its restoring line (1376) as experimental materials, the factors that affected the RAPD pattern were studied .A stable RAPD reaction system for wheat was set up .This system was 25 μL total volume contained 20～60 ng template DNA,(1.5~2.0) mmol/L Mg^(2+),0.3～0.4 mmol/L dNTPs, 0.15～0.20 μmol/L random primer, 1.0~1.5 unit Taq polymerase. After pre-denaturing at 94℃ for 5 min, under the condition of denaturing at 94℃ for 45 seconds ,annealing at 36℃ for 45 seconds,extension at 72℃ for 90 seconds,amplifying for 45 cycles, and extension at 72℃for 5 min at last ,the results of amplification was good for RAPD research in male-sterile wheat.