EMBRYONIC LETHALS AND T-DNA INSERTIONAL MUTAGENESIS IN ARABIDOPSIS
EMBRYONIC LETHALS AND T-DNA INSERTIONAL MUTAGENESIS IN ARABIDOPSIS
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DOI：
10.1105/tpc.3.2.149
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发表时间：
1991-02-01
期刊：
影响因子：
11.6
通讯作者：
MEINKE, D
中科院分区：
文献类型：
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作者：
ERRAMPALLI, D;PATTON, D;MEINKE, D
T-DNA 插入诱变代表了一种有前途的分子分离植物胚胎发育过程中具有基本功能的基因的方法。 我们在本报告中描述了用根癌农杆菌种子转化后胚胎发育缺陷的 18 个拟南芥突变体的分离和表征。 随机 T-DNA 插入预计会导致隐性胚胎死亡的高频率，因为胚胎发生需要许多靶基因。 这些实验中使用的共整合 Ti 质粒含有胭脂碱合酶和新霉素磷酸转移酶基因标记。 使用胭脂碱测定和卡那霉素抗性来估计分离家族中存在的功能插入片段的数量。 九个家族似乎在突变基因内部或附近含有 T-DNA 插入片段。 八个家族显然没有用功能性插入物标记，而是似乎包含在转化过程中诱导的突变。 DNA 凝胶印迹杂交与内部和右边界探针揭示了与 T-DNA 插入相关的各种重排。 提出了一种通用策略来简化标记胚胎突变体的鉴定并促进植物胚胎发生所需基因的分子分离。
T-DNA insertional mutagenesis represents a promising approach to the molecular isolation of genes with essential functions during plant embryo development. We describe in this report the isolation and characterization of 18 mutants of Arabidopsis thaliana defective in embryo development following seed transformation with Agrobacterium tumefaciens. Random T-DNA insertion was expected to result in a high frequency of recessive embryonic lethals because many target genes are required for embryogenesis. The cointegrate Ti plasmid used in these experiments contained the nopaline synthase and neomycin phosphotransferase gene markers. Nopaline assays and resistance to kanamycin were used to estimate the number of functional inserts present in segregating families. Nine families appeared to contain a T-DNA insert either within or adjacent to the mutant gene. Eight families were clearly not tagged with a functional insert and appeared instead to contain mutations induced during the transformation process. DNA gel blot hybridization with internal and right border probes revealed a variety of rearrangements associated with T-DNA insertion. A general strategy is presented to simplify the identification of tagged embryonic mutants and facilitate the molecular isolation of genes required for plant embryogenesis.