海菜花ISSR-PCR反应体系的优化

Establishment of ISSR-PCR Reaction System in the Ottelia acuminata(Gagnep.) Dand

以渐危植物海菜花基因组DNA为研究对象,筛选引物UBC-818[序列为(CA)_8G]研究并确定了该引物适宜的退火温度,对影响ISSR扩增效果的主要参数进行筛选和优化,确定了海菜花的ISSR分析的最佳反应体系:15μL反应体系中含25 ng模板DNA,0.2 mmol·L^(-1)引物,0.2μmol·L^(-1)dNTPs,2.0mmol·L^(-1)Mg^(2+),0.5 UTaq酶,1.5μL 10×PCR Buffer Mg^(2+)(-)[100 mmol·L^(-1)Tris-HCl(pH 8.3),500 mmol·L^(-1)KCl].

The ISSR reaction system of endangered plant Ottelia acuminata （Gagnep.） Dand was optimized. The annealing temperature of primers was affirmed with the primer UBC-818 [primer sequence： （CA）8G]. We optimized the main reaction condition of ISSR experiment such as concentrations of template DNA, Mg^2＋, dNTPS, Taq DNA Polymerase and Primer. The result showed that optimal concentrations of ISSR reaction system was 25 ng template DNA. 0.2 μmol.L^-1 Primer, 0.2 mmol.L^-1dNTPs, 2.0 mmol.L^-1 Mg^2＋, 0.5 U Taq DNA Polymerase, 1.5 μL 10×PCR Buffer Mg^2＋ （-） [100 mmol.L^-1 Tris-HCl （pH 8.3） and 500 mmol-L^-1 KCI] per 15 μL reaction volume.