Abstract
Panax notoginseng is one of the most widely used traditional herbs for the treatment of various diseases, in which saponins were the main active components. At present, the research of P. notoginseng mainly focused on the discovery of new compounds and pharmacology. However, there were few studies on the molecular mechanism of the synthesis of secondary metabolites of P. notoginseng. In our study, four coding sequences (CDS) encoding the key enzymes involved in saponin biosynthesis were cloned, namely farnesyl diphosphate synthase (FPS), squalene synthase (SS), squalene epoxidase (SE), and dammarenediol-II synthase (DS), which contained open reading frame (ORF) of 1029 bp, 1248 bp, 1614 bp, and 2310 bp, and coded 342, 415, 537, and 769 amino acids, respectively. At the same time, their domains, secondary structures, three-dimensional structures, and phylogenetics trees were analyzed by kinds of bioinformatics tools. Their phylogenetics relationships were also analyzed. In addition, GFP (Green fluorescent protein) fusion genes were constructed by the plasmid transformation system to determine the subcellular localization. The results of subcellular localization showed that FPS, SE, and DS were mainly located in cytomembrane and its surrounding, while SS was located both in cytoplasm and cytomembrane. Our findings provided data demonstrating the expression patterns of genes involved in saponin biosynthesis and would facilitate efforts to further elucidate the biosynthesis of the bioactive components in P. notoginseng.
Highlights
P. notoginseng (Burk.) F.H.Chen, called ‘San-Qi’ in Chinese, is one of the most widely used traditional herbs for the treatment of various conditions, such as cardiovascular diseases, inflammation, various body pains, traumas, and internal and external bleeding due to injury [1]
Under the gene gun bombardment, the recombinant plasmids and the GFP alone were expressed in the onion epidermal cells, respectively
Isolation of Four Full-Length cDNA Sequences Involved in Saponin Biosynthesis
Summary
P. notoginseng (Burk.) F.H.Chen, called ‘San-Qi’ in Chinese, is one of the most widely used traditional herbs for the treatment of various conditions, such as cardiovascular diseases, inflammation, various body pains, traumas, and internal and external bleeding due to injury [1]. Extensive chemical studies on this plant proved the dammarane-type saponins to be the main bioactive principles. The ginsengsides Rg1, Rb1, and notoginsengside R1 are the main effective components responsible for blood circulation, nourishing blood, improving myocardial ischemia, anti-arrhythmia, antishock, sedative, increasing intelligence, anti-aging, anti-oxidation, and antitumor activities [3,4,5,6,7,8,9]. The biosynthetic pathway of the plant dammarane-type saponins could be divided into three stages (Figure 1). The first stage leads to the synthesis of the isopentenyl pyrophosphate (IPP) and dimethylallyl diphosphate (DMAPP) through the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway and/or the mevalonate (MVA) pathway. IPP and DMAPP catalyze the synthesis of 2,3-oxidosqualene by isopentenyl transferase and anthracycline cyclase. All MEP pathway enzymes are located in plastids, whereas the MVA pathway enzymes can be in the cytosol or peroxisomes [11,12,13]
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