Koning A, Jones A, Fillatti J J, Comai L, Lassner M W
Calgene Inc., Davis, CA 95616.
Plant Mol Biol. 1992 Jan;18(2):247-58. doi: 10.1007/BF00034953.
Intracellularly expressed cytotoxins are useful tools both to study the action of plant regulatory sequences in transgenic plants and to modify plant phenotype. We have engineered a low mammalian toxicity derivative of Pseudomonas aeruginosa exotoxin A for intracellular expression in plant cells by fusing the ADP ribosylating domain of the exotoxin gene to plant regulatory sequences. The efficacy of exotoxin A on plant cells was demonstrated by transient expression of the modified exotoxin gene in tobacco protoplasts: the exotoxin gene inhibited the expression of a co-electroporated beta-glucuronidase gene. An exotoxin with an introduced frameshift mutation was also effective at inhibiting beta-glucuronidase expression in the transient assay; the activity of the frameshifted gene was presumably a result of frameshifting during translation or initiation of translation at a codon other than AUG. When fused to napin regulatory sequences, the exotoxin gene specifically arrested embryo development in the seeds of transgenic Brassica napus plants concomitant with the onset of napin expression. The napin/exotoxin chimeric gene did not have the same pattern of expression in tobacco as in B. napus; in addition to exhibiting an inhibition of seed development, the transgenic tobacco plants were male-sterile.
细胞内表达的细胞毒素是研究植物调控序列在转基因植物中的作用以及改变植物表型的有用工具。我们通过将外毒素基因的ADP核糖基化结构域与植物调控序列融合，构建了一种低哺乳动物毒性的铜绿假单胞菌外毒素A衍生物，用于在植物细胞中进行细胞内表达。通过在烟草原生质体中瞬时表达修饰后的外毒素基因，证明了外毒素A对植物细胞的作用：外毒素基因抑制了共电穿孔的β-葡萄糖醛酸酶基因的表达。在瞬时试验中，带有引入移码突变的外毒素也能有效抑制β-葡萄糖醛酸酶的表达；移码基因的活性可能是翻译过程中移码或在除AUG以外的密码子处起始翻译的结果。当与napin调控序列融合时，外毒素基因在转基因甘蓝型油菜种子中伴随着napin表达的开始特异性地阻止了胚胎发育。napin/外毒素嵌合基因在烟草中的表达模式与在甘蓝型油菜中不同；除了表现出对种子发育的抑制外，转基因烟草植株还是雄性不育的。
