正交设计优化胡萝卜ISSR-PCR反应体系

Optimization of ISSR-PCR Reaction System of Carrot by Orthogonal Design

为获得适于胡萝卜ISSR-PCR的反应体系,本研究以胡萝卜为试验材料,采用新型植物基因组DNA提取试剂盒提取胡萝卜嫩叶DNA,利用正交试验设计方法,对ISSR-PCR的5个影响因素:Mg2+、dNTP、Taq DNA聚合酶、引物和模板DNA进行了研究。结果得到胡萝卜ISSR-PCR的最佳反应体系:25μL的反应体系中含有1.5 mmol/L Mg2+、0.15 mmol/L dNTP、1.5 U Taq DNA聚合酶、0.2μmol/L引物、50 ng模板DNA。利用优化的反应体系,对最佳反应次数进行了筛选,结果表明优化的反应体系适宜的循环次数是35次。该体系可以为胡萝卜遗传多样性分析、基因定位等提供理论依据。

To obtain the optimum ISSR-PCR reaction system of carrot, the carrot genomic DNA was extracted from young leaf using new plant genomic DNA extraction kit. And five factors, including Mg2＋, dNTP, Taq DNA polymerase, primers and template DNA were researched to establish the optimum reaction system by orthogonal design, that is 25 p,L of PCR reaction mixture containing 1.5 mmol/L Mg2＋, 0.15 mmol/L dNTP, 1.5 U Taq DNA polymerase, 0.2 μmol/L primers and 50 ng template DNA. Then the best reaction times were screened by the optimum reaction system. The results showed that the best cycle number was 35. This system could provide theoretical foundation for carrot research on genetic diversity analysis, gene location, etc..