Chen Z, Ulmasov B, Folk W R
Department of Biochemistry, University of Missouri-Columbia 65211, USA.
Plant Mol Biol. 1998 Jan;36(1):163-70. doi: 10.1023/a:1005996125011.
A nuclear tRNA(Lys) gene from Arabidopsis thaliana was cloned and mutated so as to express tRNAs with altered anticodons which bind to a UAG nonsense (amber) codon and to the Arg (AGG), Asn (AAC,AAT), Gln (CAG) or Glu (GAG) codons. Concomitantly, a codon in the firefly luciferase gene for a functionally important Lys was altered to an amber codon, or to Arg, Asn, Gln, Glu, Thr and Trp codons, so as to construct reporter genes reliant upon incorporation of Lys. The altered tRNA(Lys) and luciferase genes were introduced into Nicotiana benthamiana protoplasts and expression of the mutated tRNAs was verified by translational suppression of the mutant firefly luciferase genes. Expression of the amber suppressor tRNA(LysCUA) from non-replicative vectors promoted 10-40% suppression of the luciferase nonsense reporters while expression of the amber and missense tRNA(Lys) suppressor genes from a geminivirus vector capable of replication promoted 30-80% suppression of the luciferase nonsense reporter and up to 10% suppression of the luciferase missense reporters with Arg, Asn, Gln and Glu codons.
克隆并突变了拟南芥的一个核tRNA（Lys）基因，以表达具有改变的反密码子的tRNA，这些反密码子可与UAG无义（琥珀）密码子以及Arg（AGG）、Asn（AAC、AAT）、Gln（CAG）或Glu（GAG）密码子结合。同时，将萤火虫荧光素酶基因中一个对功能重要的Lys密码子改变为琥珀密码子，或改变为Arg、Asn、Gln、Glu、Thr和Trp密码子，从而构建依赖于Lys掺入的报告基因。将改变后的tRNA（Lys）和荧光素酶基因导入本氏烟草原生质体，并通过对突变萤火虫荧光素酶基因的翻译抑制来验证突变tRNA的表达。来自非复制型载体的琥珀抑制tRNA（LysCUA）的表达促进了荧光素酶无义报告基因10%-40%的抑制，而来自能够复制的双生病毒载体的琥珀和错义tRNA（Lys）抑制基因的表达促进了荧光素酶无义报告基因30%-80%的抑制，以及对带有Arg、Asn、Gln和Glu密码子的荧光素酶错义报告基因高达10%的抑制。
