翻白草总DNA的提取与ISSR-PCR体系的建立与优化

Establishment and Optimization of ISSR-PCR Reaction System and Genome DNA of Discolor cinquefoil Herb

[目的]针对翻白草ISSR的反应特点，建立稳定可靠的ISSR分子指纹标记反应体系，为进一步研究翻白草的居群差异奠定基础。[方法]通过筛选引物并设定影响翻白草ISSR反应的诸因子的不同浓度，检测ISSR不同反应体系的扩增效果；通过分析非特异性条带的产生原因并进行条件优化，建立翻白草ISSR稳定可靠的反应体系。[结果]首次建立了可用于翻白草ISSR-PCR分析的最适宜的反应体系，确定了25μl PCR反应体系中各试剂终浓度：1×Buffer缓冲液，1U TaqDNA聚合酶，2．5mmol／LMg^2＋，0．25mmol／LdNTP，0．4μmol／L引物，DNA模板约20～30ng，退火温度在50～56℃；实验表明：Taq酶质量、DNA模板品质、退火温度、Mg^2＋浓度、dNTP浓度均对ISSR反应结果具有较大影响。[结论]所建立的翻白草ISSR反应体系具有标记位点清晰、反应系统稳定、检测多态性能力较强、重复性好等特点，可以较好地应用于翻白草的居群鉴别及居群分子生态的研究。

To establish and optimize ISSR-PCR system of Discolor cinquefoil Herb according to the ISSR-PCR characters of Discolor cinquefoil Herb the effects of ISSR-PCRs was examined with selected primers and different concentrations in the ISSR-PCRs,and the reliable ISSR-PCR systems for Discolor cinquefoil Herb populations was established through the analysis of the reasons for occurrence of differential bands and optimizing reaction conditions. The optimal ISSR-PCR system in Discolor cinquefoil Herb was reported for the first time,and 25μl ISSR-PCR system contained 1 ×Taq buffer, 1 U Taq DNA polymerase,2.5 mmol/L MgCl2,0.25 mmol/L dNTP,0.4 μmol/L primer and 20- 30 ng template DNA. The appropriate annealing temperature was among 50- 56 ℃. ISSR-PCRs were significantly influenced by Taq DNA polymerase, template DNA quantity and annealing temperature, concentrations of Mg^2＋ and dNTP, etc. The ISSR-PCR systems, which were established in this paper for studying Discolor Cinquefoil Herb, could provide clear reliable abundant polymorphisms molecular markers and were suitable for studying population authentication and population molecular ecology of Discolor Cinquefoil Herb.