宽叶缬草DNA的提取及正交优化RAPD反应体系  

DNA extraction from Valeriana officinalis L. var. latifolia Miq and orthogonal optimization of RAPD reaction system

作　　者：钱志瑶 周道堂 黄秀平[1] 周镁[1] 陈祖云[1] 覃容贵[1] 

机构地区：[1]贵阳医学院药学院,贵州贵阳550025

出　　处：《生物技术》2015年第2期143-146,132,共5页Biotechnology

基　　金：贵州省科技攻关项目("贵州缬草类药材的道地性研究";黔科合ZY[2011]3008号);贵州省高层次人才特助经费("宽叶缬草油的质量标准研究";黔科合LG字[2012]013号)资助

摘　　要：[目的]确定适合宽叶缬草RAPD分析的DNA提取方法以及建立最佳RAPD反应体系。[方法]比较宽叶缬草基因组DNA的两种提取方法(经典CTAB法、试剂盒法);采用正交设计L16(45),针对Taq DNA聚合酶浓度,d NTP浓度,Mg2+浓度,引物浓度,DNA模板浓度进行RAPD扩增,确立最佳RAPD反应体系。[结果]综合比较,试剂盒法较适合宽叶缬草基因组DNA提取;25μl最适宽叶缬草RAPD反应体系为:2.0 U Taq DNA聚合酶、0.4 mmol/L d NTP、4.0 mmol/L Mg2+、4.0μmol/L随机引物、60 ng模板DNA、2.5μl 10×buffer。[结论]试剂盒DNA提取法和正交优化的反应体系适用于宽叶缬草的RAPD分析,为进一步研究黔产宽叶缬草药材遗传多样性奠定了基础。[ Objective ] To select a suitable method of DNA extraction and RAPD reaction system for Valeriana officinalis L. var. latifolia Miq. [ Methods] To compare the genomic DNA extraction method from CTAB and extraction kit. The suitable RAPD reaction system was establish by orthogonal design L16 （45）. [Results] The extraction kit was suited to extract the genomic DNA of Valeriana officinalis L. var. latifolia Miq. The optimal conditions in 25 μl RAPD system were 2. 0 U Taq DNA Polymerase,0.4 mmol/L dNTP,4.0 mmol/L Mg2＋ ,4. 0 μmol/L Primer,60 ng DNA template,2. 5 μl 10 × buffer. [ Conclusion ] The extraction kit and the optimized reaction system by orthogonal design was suitable for RAPD analysis of Valeriana officinalis L. var. latifolia Miq, which provided the base to analyze genetic diversity of Valeriana officinalis L. var. latifolia Miq.

关 键 词：宽叶缬草 基因组DNA 随机扩增多态性DNA 正交设计 体系优化 

分 类 号：R931.2[医药卫生—生药学]