摘要
转座子标签法是一种利用转座因子插入高等植物基因组中造成基因突变 ,然后通过分离转座因子插入的旁邻顺序 ,进而克隆出突变基因的策略。这种策略在高等植物功能基因组学的研究中是十分有用的。为此目的 ,将玉米的Ds因子及bar基因连接至载体pCAMBIA130 0的T DNA区域中 ,构建成重组Ti质粒pDsBar130 0。pDsBar130 0中T DNA区域中的潮霉素抗性基因可在转化过程中用作水稻转化植株的选择标记。插入在Ds因子中的bar基因可追踪转化后代的Ds因子。pDsBar130 0通过根瘤农杆菌介导引入水稻品种中花 11号的幼胚组织。从各转化愈伤组织中获得了 140 0株独立的Ds水稻转化植株。通过PPT抗性检测和PCR分析证明了水稻转化植株中Ds因子的整合。Southernblot分析了转化植株基因组中Ds因子的插入拷贝数 ,其中单拷贝插入比率约占 70 %。这些插有Ds因子的水稻转化植株 ,当引入自主型的Ac因子反式活化Ds因子后 ,可使Ds因子跳跃到不同位点上 。
Transposon tagging is a strategy used to generate insertional mutation in higher plants by transposon and isolate the mutated gene by cloning the DNA sequences flanking the inserted transposon. Therefore,this strategy may be useful in the study of the functional genomics in higher plants. For this purpose, we have constructed a recombinant Ti plasmid, in which the maize Ds sequence and a bar gene were ligated into the T DNA of pCAMBIA1300 to form a Ti plasmid pDsBar1300 (Fig.1). A bar gene inside the Ds can be used to trace the Ds element. pDsBar1300 was introduced into rice ( Oryza sativa L. subsp. japonica cv. Zhonghua No.11)immature embryos by Agrobacterium tumefaciens mediated transformation. Hygromycin resistant gene located in T DNA was used as selection marker for transgenic rice plants in the transforming process. A total number of 1 400 independent transgenic rice plants were generated from individually transformed calli. Ds integrity in transformed plants was assayed by PPT resistance and PCR amplification(Fig.2). Ds copy number in rice genome was estimated by Southern blot hybridization (Fig.3) and the results showed that about 70% were single copy number (Table 1). We suggest that these Ds insertion lines of transgenic rice plants can be used as materials to obtain more Ds insertion plants when Ds was transactivated and moved to elsewhere by introducing an autonomous Ac element into these plants.
基金
国家 8 63项目
国家自然科学基金 (批准号3 97890 2 0 )
中国科学院重点项目
国家重点基础研究规划项目 (批准号G19990 116)资助
关键词
水稻
Ds因子
农杆菌
转座因子标签
突变体
rice, Ds element, Agrobacterium tumefaciens, transposon tagging, mutant
