华细辛AFLP反应体系的建立和优化

Establishment and optimization of AFLP reaction system in Asarum sieboldii

目的建立优化的华细辛Asarum sieboldiiAFLP反应体系,为华细辛遗传多样性的研究提供技术支持。方法以华细辛叶片为材料,对基因组DNA提取、酶切连接、PCR扩增等操作过程中各关键因素进行分析,筛选最适条件,建立完善的AFLP反应体系。结果建立了优化的华细辛AFLP分析体系:在基因组DNA提取时,提取液中添加巯基乙醇、样品温浴时间为30 min;Tru1Ⅰ和PstⅠ的酶切时间分别为3 h;在扩增反应中,Mg2+终浓度为1.5 mmol/L、TaqDNA聚合酶用量为0.2μL。结论本反应体系可获得良好的AFLP分析结果,可用于华细辛遗传多样性研究。

Objective To establish and optimize AFLP reaction system used in providing necessary technique basis for genetic diversity analysis in Asarurn sieboldii. Methods Leaves of A. sieboldii were used as experimental materials to analyze various essential elements of the whole process, such as quality of extracted DNA, time of restriction digest and ligation, concentration of Mg^2＋ , primers and Taq DNA polymerase during PCR amplification etc. , so that the most optimal AFLP reaction system could be built up. Results The optimal AFLP reaction system of A. sieboldii has been constructed, in the genomic DNA extraction, mercaptoethanol being utilized and samples being incubated about 30 min at 65 ℃. the genomic DNA being digested by Trul I and Pst I for 3 h respectively; in PCR amplification, the final concentration of Mg^2＋ being 1.5 mmol/L and the volume of Taq DNA polymerase being 0.2 μL. Conclusion The present reaction system for A. sieboldii is able to gain the favorable results of AFLP analysis and can be used in the genetic diversity research of the species.