西洋参β-香树脂合成酶基因的克隆和生物信息学分析

Cloning of β-amyrin synthase gene from Panax quinquefolius and its bioinformatics analysis

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 obtain the full-length cDNA of β-amyrin synthase CoAS） involved in triterpene saponin biosynthesis in Panax quinquefolius and provide the reference for saponin biosynthesis and the regulation of secondary metabolism in P quinquefolius. Methods Based on large-scale ESTs sequencing and RACE technology, the full-length cDNA of P quinquefolius bAS （PqbAS） was obtained. Results The full-length cDNA of PqbAS （GenBank No. JX185490） was 2 309 bp which included an open reading frame （ORF） code of 631 amino acid peptide. Common conserved domains of oxidosqualene cyclases （OSCs） were found in PqbAS including active sites and conserved sequence. Singal P4.0 analysis showed that PqbAS was a non-secreted protein. Trnhmm 2.0 analysis showed that PqbAS was a non-transmembrane protein. Real-time fluorescence quantitative PCR analysis showed PqbAS gene expressed in various organs, higher in flowers and stems while relatively lower in roots and leaves. Conclusion The full-length ofbAS is first cloned, which could provide the foundation for the investigation on expression characteristics and its role in synthesis of saponin.