Garcia Eric L, Mourad George S
Department of Biology, Indiana-Purdue University, 2101 East Coliseum Blvd, IN, USA.
Plant Mol Biol. 2004 May;55(1):121-34. doi: 10.1007/s11103-004-0500-z.
We fused four mutant omr1 alleles, encoding feedback-insensitive forms of Arabidopsis thaliana biosynthetic threonine dehydratase/deaminase (TD), to the CaMV 35S promoter and transformed these constructs into A. thaliana Columbia wild type plants. The mutant TD forms consisted of our previously isolated double mutant, omr1-1 , and three new site-directed mutants, omr1-5 , omr1-7 , and omr1-8 with single point mutations. We employed site-directed mutagenesis to assay the effects of amino acid substitutions in separate regulatory regions within the carboxy-terminal (C-term) allosteric end. TD assays and growth resistance to the isoleucine (Ile) toxic analog -O-methylthreonine (OMT) confirmed the desensitization to feedback inhibition and the viability of these mutant omr1 alleles as selectable markers, respectively. Two of the site-directed mutants, omr1-5 and omr1-7 , appeared to influence one of the two separate Ile-binding sites and had a notable 13-fold and 15-fold increase in free Ile, respectively. The omr1-8 appeared to influence the other Ile-binding site and resulted in a 2-fold increase in free Ile. The transgenic omr1-1 double mutant affecting both Ile-binding sites, however, displayed a 106-fold increase in free Ile revealing a profound synergistic interplay between these separate Ile-binding sites. While all of the four omr1 alleles conferred resistance to elevated concentrations of OMT, the progeny of omr1-1 initial transformants exhibited a bushy phenotype at the rosette stage. On the other hand, progeny of transformants omr1-5 , omr1-7 , and omr1-8 had a normal phenotype, undistinguishable from wild type. Therefore, alleles omr1-5 , omr1-7 , and omr1-8 , proved to be ideal as environmentally-friendly, dominant, selectable markers for plant transformation.
我们将四个突变的omr1等位基因（编码对反馈不敏感的拟南芥生物合成苏氨酸脱水酶/脱氨酶（TD）形式）与CaMV 35S启动子融合，并将这些构建体转化到拟南芥哥伦比亚野生型植株中。突变的TD形式包括我们之前分离的双突变体omr1-1，以及三个新的单点突变的定点突变体omr1-5、omr1-7和omr1-8。我们采用定点诱变来测定羧基末端（C端）变构末端内各个调节区域中氨基酸取代的影响。TD测定以及对异亮氨酸（Ile）毒性类似物-O-甲基苏氨酸（OMT）的生长抗性分别证实了对反馈抑制的脱敏作用以及这些突变的omr1等位基因作为选择标记的活力。两个定点突变体omr1-5和omr1-7似乎影响两个独立的Ile结合位点之一，并且游离Ile分别显著增加了13倍和15倍。omr1-8似乎影响另一个Ile结合位点，并导致游离Ile增加了2倍。然而，影响两个Ile结合位点的转基因omr1-1双突变体显示游离Ile增加了106倍，揭示了这些独立的Ile结合位点之间深刻的协同相互作用。虽然所有四个omr1等位基因都赋予了对高浓度OMT的抗性，但omr1-1初始转化体的后代在莲座期表现出丛生表型。另一方面，omr1-5、omr1-7和omr1-8转化体的后代具有正常表型，与野生型无法区分。因此，omr1-5、omr1-7和omr1-8等位基因被证明是用于植物转化的理想的环境友好型显性选择标记。
