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目的克隆西洋参三萜皂苷生物合成途径关键酶β-香树脂合成酶(bAS)全长cDNA,为研究西洋参皂苷生物合成与次生代谢调控奠定基础。方法采用大规模ESTs测序和cDNA末端扩增(RACE)技术克隆西洋参bAS基因。结果获得了西洋参bAS基因全长cDNA,命名为PqbAS(GenBank注册号:JX185490),其核苷酸序列长度为2 309 bp,含有1个开放阅读框,编码631个氨基酸多肽。保守结构域搜索显示PqbAS含有环氧角鲨烯环化酶(OSCs)共有的活性位点和保守基序。Singal P4.0分析表明PqbAS属于非分泌型蛋白,Tmhmm 2.0分析表明其为非跨膜蛋白。实时荧光定量PCR显示PqbAS基因在各个器官中均有表达,在花和幼茎中高表达,根和叶中表达量相对较低。结论首次克隆了PqbAS基因全长序列,为研究其表达特性以及在三萜皂苷生物合成中的功能奠定了基础。
Objective To clone the full-length cDNA of bAS, a key enzyme in the biosynthesis pathway of American tonic (Panax quinquefolium L.), and lay the foundation for the study on biosynthesis and secondary metabolism of ginsenosides. Methods Large scale ESTs sequencing and cDNA end amplification (RACE) were used to clone the bAS gene of American ginseng. Results The full-length cDNA of bAS gene was obtained and named as PqbAS (GenBank accession number: JX185490). The nucleotide sequence of the gene was 2 309 bp and contained an open reading frame encoding 631 amino acids. Conserved domain searches revealed that PqbAS contains the active site and conserved motifs common to epoxy squalene cyclase (OSCs). Singal P4.0 analysis showed that PqbAS belongs to a non-secreted protein and Tmhmm 2.0 analysis indicates that it is a non-transmembrane protein. Real-time PCR showed that PqbAS gene was expressed in all organs, highly expressed in flower and young stem, and relatively low in root and leaf. Conclusion The full-length PqbAS gene was cloned for the first time, which laid the foundation for the study of its expression characteristics and function in triterpenoid saponin biosynthesis.