用限制性酶切和Southern杂交对葡萄无核基因分子标记的分析  

Analysis of the DNA Sequences of Molecular Markers Linked to Grape Seedlessness Gene by Restriction Enzyme Cleavage and Southern Blot

作　　者：杨克强[1] 王跃进[1] 张今今[1] 王西平[1] 万怡震[1] 张剑侠[1] 

机构地区：[1]西北农林科技大学农业部西北园艺植物种质资源与遗传改良重点开放实验室

出　　处：《农业生物技术学报》2005年第3期294-298,共5页Journal of Agricultural Biotechnology

基　　金：国家自然科学基金项目(No.39970524);国家高技术研究与发展计划(863)项目(No.2004AA241170);教育部"跨世纪优秀人才培养计划"基金资助。

摘　　要：对葡萄(Vitis vinifera)无核基因特异标记39970524-5-564和39970524-6-1538的DNA序列进行了克隆和测序,其序列全长分别为564和1538bp,GenBank登录号为AY327513和AY327514。用Wingene231DNA分析软件对39970524-5-564和39970524-6-1538序列的限制性内切酶酶切位点进行了分析,可识别6个碱基及其以上序列的酶切位点分别有30和131个;EcoRⅠ和HindⅢ在39970524-5-564特异标记上没有酶切位点,而EcoRⅠ在39970524-6-1538第135个碱基处有一个酶切位点,HindⅢ在39970524-6-1538上没有酶切位点。用39970524-5-564DNA作探针对葡萄基因组DNA作Southern杂交,结果在无核亲本红光无核及无核基因供给者无核白和无核杂种上出现杂交带,而且呈单拷贝,而在有核亲本红地球及有核杂种上未出现杂交带,进一步证明了39970524-5-564特异片段来源于无核葡萄基因组,为检测和克隆葡萄无核基因提供了证据。The cloning and sequencing of two specific molecular markers linked to grape（Vitis vinifera） seedlessness gene indicatedthe full lengths of 564 and 1538 bp for marker 39970524-5-564 （GenBank accession number：AY327513） and marker 39970524-6-1538 （GenBank accession number： AY327514）, respectively. The restriction-sites of the sequences of the 39970524-5-564 and 39970524-6-1538 were analyzed by software ‘Wingene231’. The results showed that 30 restriction enzymes with recognition sites of equal or greater than 6 bases had cleavage sites on the sequences of 39970524-5-564 and 131 restriction enzymes had cleavage sites on the sequences of 39970524-6-1538. No cleavage sites on the sequence of 39970524-5-564 were found by EcoR Ⅰ and Hind Ⅲ ;but there was one cleavage site at 135 bp of the sequence of 39970524-6-1538 by EcoR Ⅰ and no cleavage site on this sequence by Hind Ⅲ. The DNA labeled marker 39970524-5-564 was used as a probe for Southern blotting with genomic DNA from Red Globe, Flame Seedless, Thompson Seedless, 4 seedless hybrids and 3 seeded hybrids. One band revealed a unique fragment presented in all seedless individuals but absented in all seeded individuals. It further covinced that specific marker 39970524-5-564 was derived from genome of seedless grape varieties, which provided scientific evidence of identifying and cloning seedlessness genes of grapes.

关 键 词：葡萄 无核基因 限制性酶切分析 SOUTHERN杂交 基因分子标记 无核葡萄 限制性酶切 DNA序列 基因组DNA GENBANK 

分 类 号：Q959.482[生物学—动物学] Q524