【摘要】 本研究以芦笋基因组总DNA为模板,通过对芦笋SRAP反应体系的重要参数进行优化,建立了一套适用于芦笋的SRAP反应体系:25μL的反应体系中,模板DNA量80ng、Mg2+浓度3.0mmol/L、上下游引物各0.2μmol/L、dNTPs0.3mmol/L、Taq DNA聚合酶1U以及1×Buffer。扩增程序为:94℃预变性3min;94℃30s、35℃30s、72℃1.5min,5个循环;然后退火温度提高到50℃,35个循环;最后72℃延伸10min。比较琼脂糖凝胶和变性聚丙烯酰胺凝胶电泳检测SRAP扩增产物的多态性,结果发现:6%变性聚丙烯酰胺凝胶电泳检测扩增产物比琼脂糖的效果好。利用该优化体系筛选引物,从256个SRAP引物组合中筛选出239个,它们具有扩增条带清晰、丰富、重复性好的优点,证明了此优化体系稳定可靠。更多还原

【Abstract】 In this research,we utilized the total DNA of genome from asparagus to be as template for establishing a SRAP reaction system suitable for asparagus through optimization of significant parameters of SRAP reaction system. The results showed that the optimal reaction system was 25 μL total volume including 80 ng of template DNA,3.0 mmol/L of Mg2+,0.3 mmol/L of dNTPs,0.2 μmol/L of forward and reverse primers each and 1 U Taq DNA polymerase. The reaction program was 3 min of pre-denaturation at 94℃,five cycles of three steps:30 s of denaturation at 94℃,30 s of annealing at 35℃ and 1.5 min of elongation at 72℃. In the following 35 cycles,the annealing temperature was increased up to 50℃,with a final elongation step for 10 min at 72℃. Compared on polymorphism of SRAP-PCR products through agarose gels and denaturing polyacylamide gels,6% denaturing polyacylamide gels was better than agarose gels. Finally,239 primer combinations,which had clear,abundant and stable bands,were selected with the optimized system among the total 256 primer combinations. At the same time,it implied this optimized system was stable and reliable.更多还原