狭边大叶藓(<em>Rhodobryum ontariense</em>)ISSR-PCR反应体系的建立与优化

狭边大叶藓(Rhodobryum ontariense)ISSR-PCR反应体系的建立与优化

摘要: 研究目的是获得适用于狭边大叶藓(Rhodobryum ontariense)遗传多样性研究的ISSR-PCR反应标准化程序。通过单因素试验设计,对Mg²⁺、dNTPs、TaqDNA聚合酶、模板、引物浓度,以及退火温度、循环次数等影响ISSR扩增的主要因素进行了研究和优化。结果表明,UBC808、UBC811、UBC812、UBC825、UBC826、UBC841、UBC888及UBC891引物适用于该研究;20μL ISSR-PCR最适反应体系包括:6 ng DNA模板、0.4μmol/L引物、2.25 mmol/L Mg²⁺、0.6 UTaqDNA聚合酶、0.4 mmol/L dNTPs。扩增程序为:94℃预变性4 m in;然后94℃变性1 m in,48~50℃(根据不同引物确定)复性2 m in,72℃延伸1 m in,共进行40个循环;最后,72℃延伸7 m in,4℃保存。

Abstract: To seek a standardizing program of ISSR technique for the genetic diversity analysis of a rare medicinal plant Rhodobryum ontariense,a single-factor experiment was designed to optimize ISSR-PCR amplification system.A suitable ISSR reaction system was obtained,that is:20 μL reaction system containing 6 ng DNA template,0.4 μmol/L primer,2.25 mmol/L Mg²⁺,0.6 U Taq DNA polymerase,0.4 mmol/L dNTPs.The profile of ISSR amplification was 4 min at 94℃,followed by 40 cycles of denaturation for 1 min at 94℃,annealing for 1 min at 48~50℃(according to specific primer),and extension for 1 min at 72℃,and ending with a final extension for 7 min at 72℃,stored at 4℃.