Optimization of DNA isolation, ISSR-PCR system and primers screening of genuine species of rhubarb, an important herbal medicine in China
Optimization of DNA isolation, ISSR-PCR system and primers screening of genuine species of rhubarb, an important herbal medicine in China
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DOI：
10.5897/jmpr10.077
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发表时间：
2010-05
影响因子：
--
通讯作者：
Xu-Mei Wang
中科院分区：
文献类型：
--
作者：
Xu-Mei Wang
为了评价大黄属药用植物药用大黄、掌叶大黄和唐古特大黄的遗传多样性，并对这3种濒危药用植物进行鉴定，对DNA提取方法进行了优化，包括改良的CTAB法和试剂盒、简单重复序列（ISSRs）PCR体系和引物筛选。结果表明，改良CTAB法优于分离试剂盒法，在MgCl_2浓度为1.5mM、引物浓度为0.4 μM、dNTPs浓度为0.25mM、Taq DNA聚合酶浓度为1.0U、模板DNA浓度为50 ng的条件下，获得了最佳扩增条件。在所有PCR反应中观察到可再现的可扩增产物。根据该PCR体系，从100条引物中筛选出16条清晰度高、重复性好的引物。结果表明，优化后的DNA提取和PCR体系适用于3种大黄的遗传多样性分析。此外，本研究还建立了适合中国大黄遗传变异研究的DNA提取方法。关键词：核糖核酸，DNA提取，ISSR-PCR条件，引物筛选。
To estimate genetic diversity and to authenticate the three endangered and official genuine species, Rheum officinale, Rheum palmatum and Rheum tanguticum, of herbal medicine of rhubarb, the optimization of DNA isolation methods including modified CTAB and isolation kit, PCR system of inter-simple sequence repeats (ISSRs) and primers screening were investigated in the present work. Modified CTAB was a preferable choice compared to isolation kit, ISSR protocol was optimized based on the use of the concentration of MgCl2(1.5 mM), lower concentrations of primer (0.4 μM), dNTPs (0.25 mM), Taq DNA polymerase (1.0 U) and 50 ng of template DNA, resulted optimal amplification. Reproducible amplifiable products were observed in all PCR reactions. According to this PCR system, sixteen out of one hundred primers were chosen for their high clarity and repetition. Thus, the results indicated that the optimized protocol for DNA isolation and PCR system was amenable to three genuine species of rhubarb which is suitable for further work on genetic diversity analysis. Furthermore, here the suitable DNA isolation protocol for ISSR-PCR analysis can be used to study the genetic variation in the future in Rheum grown in China. Key words: Rhubarb, DNA isolation, ISSR-PCR condition, primer screening.