白术叶片DNA提取方法的优化

Optimization of DNA Extraction Method for Atractylodes macrocephala Leaves

[目的]优化白术叶片DNA提取方法,提取高质量DNA,用于分子生物学研究。[方法]以白术叶片为材料,比较常规CTAB法、改良CTAB法、SDS法、试剂盒法提取DNA的效果,并对改良CTAB法中的裂解温度、裂解时间、核分离液解离次数进行优化,用琼脂糖凝胶电泳和微量核酸测定仪检测DNA的浓度和纯度。最后通过ISSR-PCR验证优化的改良CTAB法提取DNA的效果。[结果]4种方法中,改良CTAB法提取白术叶片DNA效果最好。针对叶片的改良CTAB法的优化条件为裂解温度65℃、裂解时间20 min、解离次数2次;ISSR-PCR验证结果表明扩增条带清晰稳定,无拖尾。[结论]优化后的改良CTAB法提取白术叶片DNA质量高,可用于白术种质资源遗传多样性分析。

[Objective]To optimize the DNA extraction method of Atractylodes macrocephala leaves and extract high-quality DNA for molecular biology research.[Method]A.macrocephala leaves were used as material,the DNA extraction effects of four methods(conventional CTAB,modified CTAB,SDS and Kit method)were compared,and the cracking temperature,cracking time and times of nuclear separation liquid extraction in the modified CTAB method were optimized,the concentration and purity of the extracted DNA were detected by agarose gel electrophoresis and spectrophotometer.Finally,the effects of DNA extracted by the modified CTAB method after optimization was verified by ISSR analysis.[Result]Among the four extraction methods,the modified CTAB method had the best extraction effects of DNA.The optimal extraction conditions of the improved CTAB method of leaves were as follows:cracking temperature was 65℃,cracking time was twenty minutes,dissociation times was twice.The ISSR-PCR validation results showed that the amplified bands were clear and stable,without any trailing.[Conclusion]The optimized CTAB method was effective in extracting DNA of A.macrocephala leaves.And it could be used for genetic diversity analysis of A.macrocephala.