长春花法呢基焦磷酸合酶基因的克隆与组织表达分析

Expression Analysis of Farnesyl Pyrophosphate Synthase Gene in Catharanthus roseus

萜类吲哚生物碱是重要的植物次级代谢产物,是多种天然药物的药效基础,具有显著的消炎、抗病毒、抑制肿瘤等作用。而长春花(Catharanthus roseus)作为重要的药用植物可以通过次级代谢合成150多种萜类吲哚生物碱,但是长春花萜类化合物的代谢途径尚未完全解析,究其原因则是受到代谢通路中关键酶的限制。法呢基焦磷酸合成酶(FPPS)是植物萜类物质合成途径中的关键酶之一,但仅发现在拟南芥等植物的萜类化合物合成过程中发挥重要的作用。因此,为了深入探究FPPS对长春花次级代谢途径是否也发挥重要作用,本研究通过生物信息学、分子生物学等研究手段,基于拟南芥法呢基焦磷酸合成酶序列,对长春花转录组数据库进行序列比对分析,首次发现并成功克隆两个FPPS同源基因,并将其命名为CrFPPS1与CrFPPS2。进一步通过系统进化树分析表明,CrFPPS1和CrFPPS2与拟南芥、缬草等植物FPPS的氨基酸序列相似性均在80%以上。RT-qPCR结果显示CrFPPS在长春花不同组织表达特征不同,因此推测CrFPPS1与CrFPPS2的功能并不完全相同。初步表明CrFPPS是长春花次级代谢通路上具有生物学功能的蛋白质,这为研究长春花FPPS的功能及长春花次级代谢产物的生物合成奠定了理论基础。

Terpenoid indole alkaloids, as important secondary metabolites of plants, are the basis of many natural medicines, and they have significant effects on anti-inflammatory, anti-virus and anti-tumor. As an important medicinal plant, Catharanthus roseus can synthesize more than 150 terpenoid indole alkaloids through secondary metabolism, but the metabolic pathways of terpenoids have not been resolved altogether in Catharanthus roseus.The reason is that it is restricted by key enzymes in the metabolic pathway. Farnesyl pyrophosphate synthase(FPPS) is one of the key enzymes in the synthesis of terpenoids in plants, but it only plays an important role in the synthesis of terpenoids in plants such as Arabidopsis. Therefore, in order to explore whether FPPS also plays an important role in the secondary metabolic pathways of Catharanthus roseus, bioinformatics, molecular biology and other research methods were used in this study. Based on Arabidopsis thaliana pyrophosphate synthase sequence,two FPPS homologous genes were successfully cloned from Catharanthus roseus transcriptomic database, and named as CrFPPS1 and CrFPPS2 for the first time. Further phylogenetic tree analysis showed that CrFPPS1 and CrFPPS2 shared more than 80% similarity with FPPS of Arabidopsis thaliana and Valeriana officinalis. The RT-qPCR results show that CrFPPS has different expression characteristics in different tissues of Catharanthus roseus, so it is speculated that the functions of CrFPPS1 and CrFPPS2 are not exactly the same. It preliminarily reveals that CrFPPS are proteins with biological functions in the secondary metabolic pathway of Catharanthus roseus, which lays a theoretical foundation for studying the function of Catharanthus roseus FPPS and the biosynthesis of the secondary metabolites of Catharanthus roseus.