蕺菜rDNA ITS序列分析的正交试验条件的初步研究

A Preliminary Study of Optimal Condition on PCR Amplification of rDNA ITS Sequence from Houttuynia Thunb

以蕺菜DNA为模板,采用正交试验设计,对影响蕺菜rDNA ITS区扩增的重要参数进行了优化试验,以期建立蕺菜rDNA ITS扩增反应的优化体系。结果表明,最优蕺菜ITS-PCR的反应体系为25μl反应体系中含Taq酶0.75 U、Mg^(2+)+(1 mmol/L)1.0μl、dNTP(0.15 mmol/L)1.5μl、引物对(0.6μmol/L)1.2μl、模板DNA(10 ng/μl)2.0μl、10×buffer(不含Mg^(2+))2.5μl。这一体系的建立为利用蕺菜rDNA ITS区研究蕺菜种质资源的系统进化提供了标准化程序。

Using DNA of Houttuynia cordata Thunb as template, the ITS was amplified, and the parameters affecting the amplification were optimized by orthogonal design. PCR conditions for amplifying rDNA ITS from Houttuynia Thunb were optimized. The results indicated that the best ITS- PCR reaction system should follow the conditions as follows： Taq DNA polymorase 0.75 U, Mg^2＋ （1 mmol/L） 1.0 μl, dNTP（0. 15 mmol/L）1. 5 μl, primers（0. 6 μmol/L）1. 2 μl, DNA （10 ng/μl） 2.0 μl, 10× buffer （Mg^2＋free）2.5 μl. The results provided a standard ITS- PCR program for the analysis of phylogenic and evolution of Houttuynia Thunb.