菠菜胆碱单加氧酶基因cDNA的克隆及重组植物表达载体的构建

Cloning cDNA of Choline Monooxygenase Gene from Spinach and Construction of Recombinant Plant Expression Vector

从菠菜叶片中提取总RNA,用RT-PCR方法扩增到菠菜胆碱单加氧酶(CMO)基因cDNA,然后将CMO cDNA克隆至pBluescript SK+载体上,序列分析结果表明,该CMO cDNA全长为1 424 bp,开放阅读框1 320 bp,编码440个氨基酸。核酸序列同源性分析表明,该cDNA与已发表的菠菜CMO基因具有99.8%同源性,氨基酸序列同源性达99.6%。在此基础上以pBI121和pBIrd为基础,构建了组成型启动子CaMV 35S和逆境诱导表达启动子rd29A驱动的CMO基因的植物表达载体。

Total RNA was extracted from leaf of spinach. The cDNA of choline monooxygenase （CMO） was obtained using the reverse transcription-polymerase chain reaction （RT-PCR） method, and then cloned into the vector pBluescript SK^＋. The sequence analysis demonstrated that the fulllength CMO cDNA was 1 424 bp, and the open reading frame was 1 320 bp which encoded a 440-amion acid polypeptide. Homology analysis of the nucleotide sequence and the deduced amino acid sequence showed that the spinach CMO gene shared 99.8% and 99.6% identity with the published sequence respectively. Two plant expression vectors of the CMO gene driven by the constitutive promoter CaMV 35S and the stress induced promoter rd29A were constructed respectively based on the vectors of pBI121 and pBIrd.