野生小果油茶ISSR-PCR反应体系的建立与优化

摘要

为建立一个具有良好稳定性和可重复性的野生小果油茶ISSR-PCR反应体系，以提取的野生小果油茶总DNA为供试材料，利用单因素试验，正交试验设计L16（45）和方差分析对DNA模板的量,引物浓度，dNTPs浓度， Mg2＋浓度，Taq DNA聚合酶的量5个影响ISSR-PCR反应体系的因素进行优化研究。确定野生小果油茶ISSR-PCR最优反应体系为：在20μL的反应体系中，DNA模板为30 ng，引物浓度为0.7μmol/L，dNTPs浓度为0.25 mmol/L，Mg2+浓度为2.0 mmol/L，Taq DNA聚合酶的量为1.75 U。方差分析结果表明，5个因素对体系的影响均未达到显著水平，其中引物浓度对反应体系影响最大。应用建立的体系对5份野生小果油茶样品进行扩增，证明了该体系具有稳定性和可重复性。%In order to establish a stability and repeatability reaction system of wild Camellia meiocarpa, five factors, the concentration of DNA template, primers, dNTPs, Mg2+and Taq DNA polymerase, were studied by the single factor tests, orthogonal experimental design L16(45)and variance analysis, with the total DNA of Camellia meiocarpa as an experimental material. The PCR amplification reaction process was optimized. The optimal system of ISSR-PCR reaction system was established as follows:30 ng DNA temple, 0.7μmol/L primers, 0.25 mmol/L dNTPs, 2.0 mmol/L Mg2+, 1.75U Taq DNA polymerase were contained in 20μL reaction system. The variance analysis shows that the effects of 5 factors on the system did not reach a significant level, and of them, the primer concentration had the greatest impact on the reaction system. The established system was applied in the amplifications of 5 varieties of wild Camellia meiocarpa Hu, and the results indicated the system had stability and repeatability.