中国水仙高分子量核DNA提取方法的建立

Development of a Method for Preparation of High Molecular Weight DNA from Narcissus tazetta L.var.chinensis Roem

以中国水仙(Narcissus tazetta L.var.Chinensis Roem)黄化叶片为材料,比较了2种提取方法对高分子量核DNA(HMW-DNA)制备效果的影响。结果表明,改良Zhang法与Peterson法相比,前者更适合于中国水仙叶片HMW-DNA的提取。将得到的细胞核用低熔点琼脂糖包埋,琼脂糖小块用蛋白酶K最适消化时间为24h,脉冲场凝胶电泳分析结果表明,所获得的HMW-DNA大于1 Mb,这些核DNA能够被限制性内切酶消化;用Southern blot检测,结果表明没有其它细胞器DNA的污染;适合于中国水仙BAC基因组文库的构建。

High molecular weight nuclear DNA （HMW-DNA）from the leaves of Narcissus tazetta L.var. Chinensis Roem was prepared by two methods（ZhangSs and Peterson^s method）and the extracting effects of the two preparing methods were compared. The results showed that Zhang＇s method was more suitable for preparation of HMW-DNA in Narcissus. The isolated nuclei were embedded with low-melting-point agarose. The optimal digestion time of plugs was 24 hours by Proteinase K. The pulse field gel electrophoresis（PFGE）results showed that the isolated HMW-I）NAs were more than 1 Mb in size and were digested completely or partially by restriction enzymes.The results showed that there were no others organelle DNA in plugs by Southern blot analysis. Therefore, Zhang＇s method was suitable for the construction of a BAC library in Narcissus.