黔产苦参ISSR-PCR反应体系正交优化研究

Orthogonal Optimization of ISSR-PCR Reaction System of Suphora flavercens Ait.

[目的]确定适合苦参稳定的ISSR-PCR反应体系。[方法]采用经典的CTAB法提取苦参新鲜幼嫩叶片DNA;针对Mg2+、d NTPs、引物、模板DNA和Taq DNA聚合酶进行正交设计L_(16)(4~5),优化黔产苦参ISSR-PCR反应体系。[结果]最适ISSR-PCR反应体系为:模板DNA 90 ng、0.4μmol/L引物、2.0 mmol/L Mg^(2+)、TaqDNA聚合酶0.5 U、0.5 mmol/L d NTPs。[结论]建立的苦参ISSR-PCR反应体系,经过19份苦参样品检验,证明该体系稳定可靠,可用于苦参遗传多样性分析。

[Objective]To select a suitable method of ISSR-PCR reaction system for Suphora flavercens Ait. [Method] Modified CTAB method was used to extract the genomic DNA from fresh young leaves of S. flavercens. The suitable ISSR-PCR reaction system was established by orthogo-nal design L16（45） based on the Mg^2＋, dNTPs, primer,template DNA and Taq DNA. [Result]The optimal conditions in 25μL ISSR-PCR system were 90 ng DNA template, 0. 4μmol/L primer, 2. 0 mmol/L Mg^2＋, 0. 5 U TaqDNA Polymerase, and 0. 5 mmol/L dNTPs. [Conclusion]The es-tablished ISSR-PCR reaction system is verified by 19 samples of S. flavercens, which verifies that this system is stable and reliable, and can be used for the analysis of genetic diversity of S. flavercens.