两种柑橘线粒体DNA的获得与AFLP分析

为了研究柑橘种属间线粒体基因组的遗传多样性以及温州蜜柑胞质雄性不育的分子基础，采用了两个柑橘品种‘国庆1号’温州蜜柑和无核‘冰糖橙’的胚性愈伤组织作为提取线粒体的材料。不连续蔗糖梯度超速离心法纯化线粒体。提取的线粒体完整，具很强的荧光活性。mtDNA片段在20kb左右，且无降解。叶绿体探针rubisco L、核探针45 s rRNA Southem blotting没有检测到叶绿体DNA与核DNA，而线粒体探针apt6杂交获得一条5kb大小的带，说明用此提取方法可获得无污染的mtDNA。4对AFLP引物共得到98条带，其中14条带有多态性。

Obtaining mtDNA of Two Types of Citrus and the AFLP Analysis

To study the genetic diversity of mitochondrial genome in Citrus and molecular mechanism of cytoplasmic male sterility in Satsuma mandarin( C.unshiu ),we present a method for isolation of mitochondrial DNA from embryogenic callus of Citrus. The results showed that discontinuous sucrose density gradient centrifugation method was effective for purifying mitochondria which possessed fluorescent activity.Southern blotting showed no contamination of cpDNA and nuclear DNA using chloroplast probe rubisco L,nuclear probe 45s rRNA respectively where as a 5kb size fragment was gained after being hybridized by mitochondrial probe apt 6. Consequently we acquired high pure,not contaminated big segment of mtDNA.The fragments of polymorphic mtDNA extracted from Guoqing 1 Satsuma mandarin and Bingtang Cheng sweet orange were found by AFLP assay.