Carneiro V T, Pelletier G, Small I
Laboratoire de Biologie Cellulaire, INRA, Versailles, France.
Plant Mol Biol. 1993 Jul;22(4):681-90. doi: 10.1007/BF00047408.
We have developed a simple, rapid and sensitive assay for tRNA gene expression in plant cells. A plant tRNA(Leu) gene was site-specifically mutated to encode each of the three anticodon sequences (CUA, UUA and UCA) that recognize, respectively, the amber, ochre and opal stop codons. The suppression activity of these genes was detected by their ability to restore transient beta-glucuronidase (GUS) expression in tobacco protoplasts electroporated with GUS genes containing premature stop codons. Protoplasts co-electroporated with the amber suppressor tRNA gene and a GUS gene containing a premature amber stop codon showed up to 20-25% of the activity found in protoplasts transfected with the functional control GUS gene. Ochre and opal suppressors presented maximum efficiencies of less than 1%. This system could be adapted to examine transcription, processing or aminoacylation of tRNAs in plant cells. In addition, phenotypically normal, fertile tobacco plants expressing a stably incorporated amber suppressor tRNA gene have been obtained. This suppressor tRNA can be used to transactivate a target gene containing a premature amber stop codon by a factor of at least several hundred-fold.
我们开发了一种用于检测植物细胞中tRNA基因表达的简单、快速且灵敏的测定方法。将一个植物tRNA（Leu）基因进行位点特异性突变，以编码分别识别琥珀色、赭石色和乳白型终止密码子的三种反密码子序列（CUA、UUA和UCA）。通过这些基因恢复在含有提前终止密码子的GUS基因电穿孔处理的烟草原生质体中瞬时β-葡萄糖醛酸酶（GUS）表达的能力，来检测这些基因的抑制活性。与琥珀色抑制tRNA基因和含有提前琥珀色终止密码子的GUS基因共电穿孔的原生质体，其活性高达用功能性对照GUS基因转染的原生质体中所发现活性的20% - 25%。赭石色和乳白型抑制子的最大效率低于1%。该系统可用于检测植物细胞中tRNA的转录、加工或氨酰化作用。此外，已经获得了表达稳定整合的琥珀色抑制tRNA基因的表型正常、可育的烟草植株。这种抑制tRNA可用于将含有提前琥珀色终止密码子的靶基因激活至少几百倍。
