灰枣成熟叶片DNA提取及ISSR反应体系构建

针对枣DNA提取须用幼嫩叶片或组织培养幼苗这一限制相关实验顺利进行的问题,对常用的DNA提取方法CTAB法进行了改良,在破碎细胞之后,先加入2%β巯基乙醇,3%PVP,100mmol/LEDTA以去除成熟叶片细胞质中的多酚等次生物质,再加核裂解液CTAB以释放DNA。改良的CTAB法提取灰枣成熟叶片基因组DNA,分光光度法检测该法提取的样品DNA得率为55.1～79.1μg/g,D260nm/280nm为1.78～1.87;琼脂糖凝胶电泳检测的结果表明,该法提取的样品DNA分子量为23kb左右;该法提取的样品DNA可以被EcoRI和HindⅢ限制性内切酶进行切割。在此基础上构建了方便快捷的灰枣ISSR反应体系。

A method for extracting DNA from mature leaves of Ziziphus jujuba cv. Huizao and ISSR reacting system construction

CTAB method was widely used in isolating plant genomic DNA.Up to now,extracting jujube DNA must use the young tender leaves or the in vitro cultured seedlings,so it is not convenient to do it.We have adopted the CTAB method in this article.Before breaking nuclei to free DNA by adding CTAB solution,β-mercaptoethanol,PVP and EDTA were used to remove secondary metabolites,such as polyphenols,and others that were the main parts of the cytoplasm and cell wall.The isolated genomic DNA was detected by spectrophotometry,agarose gel electrophoresis and enzymatic digestion.By improved CTAB,the yields were between 55.1 to 79.1 μg/g.D260nm/280nm values were between 1.78 to 1.87;molecular weight of isolated DNA was around 23 kb and could be cut by EcoRI and HindIII restriction enzyme.Based on these a jujube ISSR reacting system was set up.