油葵种子DNA提取和RAPD反应体系的优化

目的]为利用RAPD技术进行油葵的亲缘关系分析及杂种鉴定奠定基础。方法]以6种杂交油葵种子为试材,采用改良的CTAB法提取油葵基因组DNA,并对提取DNA的浓度和纯度进行测定。通过单因子梯度试验,筛选出优化、稳定的RAPD-PCR反应体系。结果]采用改良CATB法提取的油葵DNA质量好、得率高,且无蛋白质和酚类的污染。油葵的最适RAPD-PCR反应体系为:2.0μl 10×Buffer、2.0 mmol/L Mg2+、150μmol/LdNTPs、1.5UTaq酶、2.0 ng/μl DNA模板、0.25μmol/L引物,总体积为20.0μl。利用该体系对不同含油量的6个油葵杂交种进行扩增,均表现为带形清晰稳定、带数适宜。结论]该RAPD技术体系适用于各种油葵杂交种的DNA分析。

DNA Extraction from Oil Sunflower Seeds and the Optimization of Its RAPD Reaction System

Objective] The research aimed to lay the foundation for the genetic relationship analysis of oil sunflower and hybrid identification by RAPD technology.Method] With 6 varieties of hybrid oil sunflower seeds as tested materials,genomic DNA were extracted from oil sunflower by the improved CATB method and the concentration and purity of the extracted DNA were determined.Through single-factor gradient test,the optimized and stable RAPD-PCR raction system was screened out.Result] DNA extracted from oil sunflower by the improved CATB method had no pollution of protein and phenols,with good quality and high yield.The optimum RAPD-PCR raction system was as follows: 2.0 μl 10×Buffer,2.0 mmol/L Mg2+,150 μmol/L dNTPs,1.5 U Taq enzyme,2.0 ng/μl DNA template and 0.25 μmol/L primers with total volume of 20.0 μl.6 oil sunflower hybrids with different oil content were amplified by this system and they all showed clear and stable bands type with suitable band number.Conclusion] This RAPD reaction system was suitable for DNA analysis of all kinds of oil sunflower hybrids.