观光木cpDNA非编码序列PCR反应体系优化及引物筛选

摘要

With single factor orthogonal design method, the experiments were conducted to optimize the main elements affecting cpDNA-PCR ampliifcation system forTsoongiodendron odorum. An optimal cpDNA-PCR system for T. odorum was set up as follows: 50 ng DNA template, 1×PCR buffer, 0.3μmol•L-1 primer, 2.5 mmol•L-1 MgCl2, 0.3 mmol•L-1 dNTP and 1 UTaqDNA polymerase in a total volume of 20μL. Five pairs of cpDNA non-coding regions primers which are appropriate for the study of molecular genealogy of T. odorum were screened out, they are:rpl32-trnL,psbJ-petA,3′rps16-5′trnK,atpI-atpH andpetL-psbE.%利用单因素与正交设计试验，对影响观光木cpDNA-PCR扩增的主要因子进行优化，建立了观光木cpDNA-PCR最适反应体系（20μL)，为：50 ng模板DNA、1×PCR buffer、0.2μmol·L-1引物、2 mmol·L-1 MgCl2、0.3 mmol·L-1dNTP以及1 UTaq DNA聚合酶；筛选出了适合观光木分子谱系地理学研究的非编码区序列引物，为 rpl32-trnL、psbJ-petA、3′rps16-5′trnK、atpI-atpH、petL-psbE。