小桐子基因组DNA的提取及ISSR-PCR反应体系的优化

Optimization of Extracting Total DNA and ISSR-PCR Reaction of Jstropha curcas Linn

为从小桐子嫩叶中提取高质量的基因组DNA,采用改良CTAB法提取的基因组DNA通过OD260/OD280比值测定,琼脂糖凝胶电泳和ISSR-PCR扩增检测,结果表明改良CTAB法提取的DNA纯度高,可作为ISSR分子标记的模板。同时,采用正交设计的方法,对影响小桐子ISSR-PCR反应体系中的4个因素(引物、Taq DNA Polymerase、10×Buffer(含Mg2+)、dNTPs)在3个水平上进行优化试验。建立了适合小桐子的既稳定又能扩出最多数量谱带的ISSR最优反应体系,即20μl的反应体系中含有30ng模板DNA、0.2mmol/L dNTP、0.3 U Taq酶Polymerase、0.8μmol/L Primer、3.0mmol/L10×Buffer(含Mg2+)。

In order to get high-quality DNA from leaves of Jstropha curacs, an efficient procedure was developed based on the conventional CTAB methods. The resuhs from ratios of OD260/OD280, electrophoresis and ISSR-PCR amplification suggested that quality DNA extracted by using CTAB method produces was higher than that extracted with conventional CTAB method. Meanwhile, Orthogonal design was applied for optimizing four factors （Taq DNA polymerase, dNTP, Buffer （contain Mg^2＋, and primer） in the ISSR PCR amplification system on Jstropha curacs respectively. As a result, a satisfactory ISSR reaction system for Jstropha curacs with desirable repeatability and polymorphic bands was established. Each 20μl amplification reaction consisted of 30ng template DNA,0.2mmol/L dNTP, 0.3U Taq DNA Polymerase, 0.81μmol/L Primer, 3.0mmol/L 10× Buffer （contain Mg^2＋）.