滑叶藤ISSR-PCR反应体系建立及优化

Establishment and Optimization of ISSR-PCR Reaction System for Clematis fasciculifloora Franch.

为建立和优化滑叶藤ISSR-PCR反应体系,运用正交试验和单因子试验分析模板DNA,TagDNA聚合酶,Primer,Mg2+和dNTPs 5个因子对滑叶藤ISSR-PCR反应影响。结果表明了滑叶藤ISSR-PCR 25μL反应体系中5个因子最佳水平:DNA模板为8.4 ng,TagDNA聚合酶为2.0 u,Primer浓度为0.8 mmol/L,dNTPs浓度为0.68 mmol/L,Mg2+浓度为2.4 mmol/L。该反应体系为利用ISSR分子标记技术研究铁线莲属植物提供了可靠方法。

Effect for ISSR-PCR reaction of Clematis fasciculifloora Franch. on five factors （DNA tern-plate, TagDNA, primer, Mg^2＋ and dNTPs） were analyzed by orthogonal design and respective factor design for establishing and optimizing ISSR-PCR reaction system of C. fasciculifloora Franch.. As aresult, the optimal ISSR-PCR reaction system （25 μL） mixture contained 8.4 ng DNA template, 2.0 u TagDNA, 0. 8 mmol/L primer, 2.4 mmol/L Mg^2＋ and 0. 68 mmol/L dNTPs. Theory sustaining forstudying Clematis L. plant by ISSR molecular marker were afforded from this experiment.