大鼠肝tRNA^(Ile)基因的克隆与体外转录

Cloning and in vitro Transcription of Rat Liver tRNA^(Ile) Gene

采用PCR扩增出大鼠肝tRNAIle基因，构建了重组质粒pGWIW，并用T7RNA聚合酶／启动子系统对其进行了体外表达．经过对转录产物片段大小及运用Northernblot鉴定，证明获得了不含修饰核苷酸的大鼠肝tRNAIle生物学活性检测显示：合成基因体外转录产物氨基酰化活性是天然tRNA的40％。

Rat liver tRNAIle gene was amplified by PCR from recombinant plasmid rec-M13mp18. The PCR product was digested by Hind Ⅲ and Sfi Ⅰ, then cloned into pGEM-9zf(－), thusthe recombinant plasmid pGWIW was constructed and was used to transcript the rat liver tRNAIlegene in vitro by T7 system. The in vitro transcription products were detected by denaturingPAGE and Northern blot. The results show that the in vitro expression products of rat liver tRNAIle were obtained. The tRNAIle transcript can be aminoacylated by mixed rat liver tRNA synthetase and has an activity only forty percent of the wild type tRNAIle. The results indicate thatmodified nucleosides may play a crucial role in the recognition by mammal isoleucyl-tRNA synthetase.