从植物细胞核分离大分子量核DNA

研究了从植物中分离百万碱基对级大分子量核DNA的方法。该方法利用差速离心分离植物细胞核，经低熔点琼脂糖块或低熔点琼脂糖微珠包埋，蛋白酶K原位裂解后制备大分子量核DNA。结果表明，选择不同生长时期的材料和不同的包埋细胞核方式对大分子量核DNA的制备有很大的影响，由黄化苗或幼嫩的绿叶为材料分离细胞核，进行胶块包埋是制备大分子量核DNA的最佳条件。利用该法获得的DNA分子量在200kb－5．7Mb之间，主要集中在2．2～5．7Mb之间；每一胶块DNAE量为18～20μg。与包埋原生质体制备大分子量核DNA的方法相比，该方法获得的DNA纯度较高，去除了大部分细胞器DNA的污染；易于被限制性内切酶部分和完全消化，其消化结果具可重复性。该方法操作简单、适用植物种类广泛，用该方法从水稻（OryzasativaL．）、苹果（MaluspumilaMill．）、大豆（Glycinemax（L．）Merr．）、玉米（ZeamaysL．）等多种植物材料中成功地制备了大分子量核DNA。该方法制备的核DNA适用于植物的脉冲交变电泳基因组分析和构建人工细菌染色体文库和人工酵母染色体文库。

Isolation of High Molecular Weight DNA from Plant Nuclei

A method of isolating megabase DNA from plant tissue was studied. Generally it includes isolation of purified nuclei by differential centrifugation, embedment of the nuclei in either low-melting-point agarose plugs or microbeads, and digestion with proteinase K in situ to release high molecular weight (HMW) DNA. The results indicated that the size of the prepared HMW nuclear DNA were influenced by both growth period of plant materials and the ways of embedment. The optimal condition for preparing HMW nuclear DNA was to isolate nuclei from the etiolated seedlings or young green leaves and to embedment the nuclei in agarose plugs.The DNA in one plug prepared in this best condition was between 200 kb and 5. 7 Mb in size, mostly in the size of 2. 2 - 5. 7 Mb, and approximately 18 - 20 ug in amount. Since most of the organellar DNA was removed, the quality of HMW nuclear DNA prePared by this method was greatly improved comparing to those prepared by embedment of protoplast in agarose. The prepared DNA by this method was ready for partial and complete digestion with restriction enzymes and the digestion result was reproducible. The method is very simple and suitable for a wide range of plant taxa. The authors have used this method in isolating HMW nucIear DNA from rice (Oryza sativa L. ), soybean (Glycine max (L. ) Merr. ), apple (Malus pumila Mill. ), and corn (Zea may L. ) successfully. The HMW nuclear DNA thus prepared facilitates plant genome analysis by pulsed field gel electrophoresis (PFGE ) and construction of yeast and bacterial artificial chromsomal libralles.