摘要
[Objective] To investigate the impacts of ISSR-PCR amplification factors, for the establishment and optimization of ISSR-PCR reaction system for Ligusticum chuanxiong hort. [Method] Using genomic DNA of Chuanxiong leaf extracted via an improved CTAB method as template, single factor analysis was performed to investigate the impacts of DNA template concentration, Mg2＋ concentration, dNTPs concentration, primer concentration, Taq DNA polymerase concentration on ISSR-PCR amplification and to optimize this system for Ligusticum chuanxiong hort. [Result] The ISSR-PCR amplification（25 μl） suitable for Ligusticum chuanxiong hort. was determined to be composed of 2.5 μl of 10×reaction buffer, 2.1 mmol/L MgCl2, 300 μmol/L dNTPs, 0.4 μmol/L primer, 1.0 U Taq DNA polymerase and 20-40 ng genomic DNA. [Conclusion] Our study laid basis for analyzing the genetic diversity of Ligusticum chuanxiong hort. resources distributed in 17 different areas of China.
[目的]建立和优化适合川芎的ISSR-PCR反应体系。[方法]以川芎叶片为材料,利用改良CTAB法提取了叶片基因组DNA,利用单因素试验分析了DNA模板、Mg2＋、dNTP、引物和TaqDNA聚合酶的浓度对ISSR-PCR反应的影响,对适合川芎的ISSR-PCR反应条件进行了优化。[结果]确定了适合川芎的ISSR-PCR反应体系为25.0μl,其中含2.5μl10×TaqDNA聚合酶缓冲液、2.1mmol/LMgCl2、300μmol/LdNTP、0.4μmol/L引物、1.0UTaqDNA聚合酶和20~40ng模板DNA。[结论]为进一步对全国17个不同分布区的川芎资源进行遗传多样性研究奠定了基础。
基金
Supported by the10th Five Years Key Programs for Science and Technology Development of China(2004BA721A31)~~
