大葱叶绿体DNA的分离纯化及分子杂交鉴定

ISOLATION AND IDENTIFICATION OF ALLIUM FISTULOSUM CHLOROPLAST DNA

对大葱的心叶除去其管状叶内的粘液。经组织捣碎机粉碎、差速离心获叶绿体粗制品;再用蔗糖不连续梯度离心的方法纯化叶绿体。对纯化的叶绿体经蛋白酶及SDS处理后,用酚抽提、乙醇沉淀的方法获得叶绿体DNA。对本法获得的叶绿体DNA用限制性酶解,获得清晰的酶切电泳条带;与烟草rbc L基因探针杂交,确证本法获得的DNA是叶绿体DNA,可供分子克隆及基因分析等进一步研究。

The heart leaves of welsh onion (Allium fistoiosum var. giganteum Makino) were pretreated by removing the sticky liquid in tubular leaves. The chloroplast was obtained from the resulting tissues through liquid homogenizing, differential centrifugation and a stepwise sucrose gradient centrifugation. The chloroplast DNA was isolated from the purified chloroplast and digested by restriction enzyme. The probe of rbc L gene was hybridized with the restriction patterns and the gene was located on a fragment cleaved by Barn HI.