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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。
Triterpenoids are the main pharmacological components of Alzheimer’s disease. They have great potential in cancer treatment. Farnesyl pyrophosphate synthase (FPPS) is one of the important rate-limiting enzymes in the synthesis pathway of triterpenes. In this paper, we used the method of homologous cloning and rapid cDNA end amplification (RACE) The full-length cDNA of FPPS gene (accession no. HQ724508) contains an open reading frame of 1 032 bp with a total length of 1 531 bp and encodes a protein of 343 amino acids with 5 conserved domains, of which Two are rich in aspartic acid (DDXXD). Real-time quantitative PCR (QRT-PCR) showed that FPPS gene was expressed in various organs of Alzheimer’s disease. The relative expression increased from October to early December and then decreased. The expression of FPPS gene was higher in tuber and root , Petiole expression is relatively low. The results of HPLC showed that the change trend of 23-acetyl alisol B at the different growth stages was consistent with the change of FPPS gene expression. It was initially proved that FPPS is important in the synthesis of alisol Regulatory sites. This study provides a scientific basis for using plant genetic engineering to improve the quality of Chinese herbal medicines.