Tritepenoids Constituents And Key Enzymes Involved In Triterpenoids Biosynthesis Pathway From The Roots Of Euphorbia Pekinensis Rupr

"Jing Daji", the root of Euphorbia pekinensis Rupr, is a common medicine in the folk, and widely used for treatment of edema and ascites caused by various reasons. Wild resources of Euphorbia pekinensis has become more scarce at present because of environmental disruption. At the same time Euphorbia pekinensis is a perennial plant with persistent roots and need many years cultivation, therefore it is a major challenge for researchers and is an important issue for the field of traditional Chinese medicine modernization to solve the decreasing resources. The main medicine composition of Euphorbia pekinensis are triterpenoids. The research on the metabolic pathway of triterpenoids is more clearly and key genes in metabolic pathway regulated triterpenoids biosynthesis have made some progress. Some works can be done from different directions to solve resource shortage of medicine plant, such as changing extracting method to improve the rate of extraction of active ingredients, or analyzing the active components and using biological engineering technology to improve the content of the active ingredient. The following aspects had been done in this study:1) The chemical components of non-polar factions exctracted from the roots of Euphorbia pekinensis were analyzed by GC/MS. Twenty-three compounds were separated and identified. Triterpenoids, steroids and esters were the major components, and triterpenoids were the more. The chemical components of non-polar factions could inhibit the growth of cell line NCI-H460 cell with an inhibiting rate of 99.51% at 20μg/mL and the DPPH radical scavenging rate was 96.68% when the concentration was 4 mg/mL.2) Response surface methodology was used to optimize the extraction technique for total triterpenoids from roots of Euphorbia pekinensis by supersonic wave. By regression analysis and verification experiment, the results showed that the optimum conditions of alcohol concentration, solid liquid ratio, extraction temperature, ultrasonic extraction time and ultrasonic power were 95%, 20 mL/g, 70℃, 32 min and 390 W respectively.3) A full-length cDNA encoding 3-hydroxy-3-methylglutoryl-Coenzyme A reductase (HMGR; EC1.1.1.34), which catalyzes the first committed step of isoprenoids biosynthesis in MVA pathway, was isolated from young leaves of Euphorbia Pekinensis Rupr. by rapid amplification of cDNA ends (RACE) for the first time. The full-length cDNA of HMGR (designated as EpHMGR, GenBank Accession NO. EF062569 ) was 2200 bp containing a 1752 bp ORF encoding 583 amino acids. Bioinformatic analyses revealed that the deduced EpHMGR had extensive homology with other plant HMGRs and contained two transmembrane domains and a catalytic domain. The predicted 3-D model of EpHMGR had a typical spatial structure of HMGRs. Southern blot analysis indicated that at most two copies of EpHMGR gene existed in Euphorbia Pekinensis genome. Tissue expression analysis revealed that EpHMGR expressed strongly in roots, weakly in stems and leaves. The functional colour complementation assay indicated that EpHMGR could accelerate the biosynthesis of carotenoids in the Escherichia coli transformant, demonstrating that EpHMGR plays an influential role in isoprenoid biosynthesis.4) A cDNA encoding farnesyl diphosphate synthase (FPS) from E.pekinensis was cloned and characterized. The full-length cDNA named EpFPS (Genbank Accession Number FJ755465) contained 1431 bp with an open reading frame of 1029 bp encoding a polypeptie of 342 amino acids. The deduced amino acid sequence of the EpFPS named EpFPS exhibited a high homology with other plant FPS, and contained five conserved domains. Phylogenetic analysis showed that EpFPS belonged to the plant FPS group. Southern blot analysis revealed a small FPS gene family in E.pekinensis. Tissue expression pattern analysis revealed that EpFPS expressed strongly in root, weak in leaf and stem. Functional complementation of EpFPS in an ERG20-disrupted yeast strain indicated that the cloned cDNA encoded a farnesyl diphosphate synthase.5) Salicylic acid (SA) and methyl jasmonate (MeJA) were used to study the response of two functional gene to two elicitors. To MeJA treated callus, the content of EpHMGR expression was the highest after 12 h, and then was slowly lower, while the content of EpFPS expression was the highest after 48 h, then slower lower. To SA treated callus, the content of EpHMGR expression was the highest after 12 h, and then was slowly lower, while the content of EpFPS expression was the highest after 48 h, then slower lower. The results provides a theoretical basis for the adoption of certain chemical means and provides candidate genes to use biological engineering technology to increase active ingredients in Euphorbia pekinensis.