建泽泻法呢基焦磷酸合酶分子克隆、分布表达及生物信息学研究

Molecular cloning of farnesyl pyrophosphate synthase from Alisma orientale(Sam.) Juzep. and its distribution pattern and bioinformatics analysis

三萜类化合物是道地药材建泽泻中的主要药效成分,在癌症治疗方面有很大的应用潜力。法呢基焦磷酸合酶(FPPS)为三萜类化合物合成途径中的重要限速酶之一,本文运用同源克隆和快速cDNA末端扩增(RACE)技术相结合的方法,克隆了建泽泻FPPS基因的全长cDNA(accession no.HQ724508),FPPS cDNA全长为1 531 bp,含有1个1 032 bp的开放阅读框,编码343个氨基酸的蛋白,包含5个保守的功能域,其中两个富含天冬氨酸(DDXXD)。实时荧光定量PCR(QRT-PCR)结果显示建泽泻FPPS基因在各器官中均有表达,10月至12月上旬相对表达量呈上升趋势,后下降,其中叶片表达量较高,块茎、根、叶柄表达量相对较低。高效液相(HPLC)分析结果表明不同生长期建泽泻主要药效成分23-乙酰泽泻醇B变化趋势与FPPS基因表达量变化一致,初步证明FPPS是泽泻醇类化合物合成途径中的重要调控位点。本研究为利用植物基因工程改善中药材品质提供科学依据。

Triterpenes, which have large application potential in the treatment of cancer, are the main active components of genuine medicinal material Alisma orientale （Sam.） Juzep. Farnesyl pyrophosphate synthase （FPPS） is one of the important rate-limiting enzymes in the synthetic pathway of triterpenes. In this study the FPPS full length cDNA of the A. orientale, was cloned via homology-based cloning approach and rapid amplification of cDNA ends （RACE）. The full length of the FPPS cDNA was 1 531 bp （accession no. HQ724508）, which contained a full 1 032 bp ORF that encoded 343 amino acids. The deduced protein sequence exhibited five conserved motifs, two of which is riched of Asp （DDXXD）. The result of real-time quantitative PCR （QRT-PCR） showed that FPPS gene was expressed in different organs ofA. orientale. The expression increased from October to the first ten-day period of December, and then decreased. The FPPS gene expression was higher in leaves but lower in leafstalk, tuber and root. HPLC analysis of active components 23-acetyl-alismol B of A. orientale, during different periods indicated that its change trend should be consistent with FPPS gene expression. It can be primarily deduced that FPPS gene should be an important control point in the synthetic pathway of Alisma terpenes. This study may facilitate the quality of medicinal plants through gene engineeringin the future.