琯溪蜜柚高分量核DNA提取方法的建立

Development of Method for Preparation of High Molecular Weight DNA from Citrus grandis (L.) Osbeck‘Guanximiyou’

以琯溪蜜柚[Citrus grandis(L.)Osbeck'Guanximiyou']叶片为材料,比较了两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,前者更适合于琯溪蜜柚HMW-DNA的提取。改良Zhang法用温和的物理方法破碎细胞壁,在一定的渗透压下保证细胞核的完整性,运用miracloth和低速离心去除大部分的多糖和细胞壁碎片,经显微观察,所提取的细胞核完整、纯净、质量高;将这些细胞核用低熔点琼脂糖包埋,以琼脂糖小块的方式保存细胞核,以保持核的稳定;琼脂糖小块的最适消化时间为48h,脉冲场凝胶电泳分析结果表明所获得的HMW-DNA大于1Mb,这些核DNA能够被限制性内切酶消化,适合于琯溪蜜柚BAC基因组文库的构建。

High molecular weight nuclear DNA（HMW-DNA）from leaves of Guanximiyou [Citrus grandis （L.） Osbeck ‘Guanximiyou’] was prepared by two methods（Zhang’s 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 Guanximiyou.The cell walls were broken down by gentle physical homogenization to keep integrity of nuclei under suitable osmolarity.Debris of cell walls and most of polysaccharides were removed by filtering with miracloth and centrifugation under slow speed.The isolated nuclei were proved to be intact,pure,and high-quality by observation under microscope and were embedded with low-melting-point agarose to preserve the isolated nuclei in plugs for keeping stability of nuclei.The optimum digestion time of plugs was 48 hours,the pulse field gel electrophoresis （PFGE）results showed that the isolated HMW-DNAs were more than 1 Mb in size and were digested completely or partially by restriction enzymes,therefore,Zhang’s method was suitable for construction of BAC library in Guanximiyou.