For plants collected in the wild, both the mRNA and protein levels of the AoSEs increased from October to November and decreased slightly in December. the most important perennial medicinal plants in traditional Chinese medicine, where its rhizomes have been used for nearly 2000 years to eliminate dampness, reduce edema, and promote urinary excretion1. Protostane triterpenes from A.?orientalehave unique structural features that include a -CH3 on C10 and C14, an -CH3 on C8, and an can restore the sensitivity of multi-drug resistant cells to anti-tumor drugs, because it may take part in the transport of P-glycoprotein11. However, these compounds are found only in a few plant groups such as protostane triterpenes, of which the formation of the protostane triterpene skeleton is the core biosynthetic step. SE catalyzes the conversion of squalene to 2,3-oxidosqualene, which is the precursor of the triterpene skeleton. This enzyme is a non-cytochrome P450-type monooxygenase that participates in triterpene biosynthesis and functions as a rate-limiting step in the pathway18. At present, genes have been cloned from pharmacological plants such as in roots can promote the biosynthesis of triterpenoid saponins in expression causes the accumulation of ginsenosides in (GenBank accession numbers “type”:”entrez-nucleotide”,”attrs”:”text”:”KP342318″,”term_id”:”902556457″,”term_text”:”KP342318″KP342318, “type”:”entrez-nucleotide”,”attrs”:”text”:”HQ724508″,”term_id”:”317140568″,”term_text”:”HQ724508″HQ724508, and “type”:”entrez-nucleotide”,”attrs”:”text”:”JX866770″,”term_id”:”427542511″,”term_text”:”JX866770″JX866770, respectively)23C25, while characterization and functional analysis of SE in has not yet been reported. In 1971, jasmonic acid (JA) was first isolated as a plant growth hormone26. Jasmonates [JA, methyl jasmonate (MeJA), and related compounds] are lipid-derived signal molecules that have been shown to play important roles in the regulation of plant growth and development27,28. MeJA can regulate metabolic pathways and reaction rates through a series of signal transduction processes in the cells29. MeJA acts through a receptor in the plant cell membrane to regulate the expression of the key enzyme genes and transcription factors in biosynthetic pathways, and it can promote the production of secondary metabolites in plants30. The effect of MeJA on triterpene saponin biosynthesis has been reported in and the ginsenoside content are both increased in ginseng hairy or adventitious root cultures after MeJA treatment. The expression levels of and genes and then performed prokaryotic expression to identify the function of the AoSE proteins. We then prepared Vadadustat polyclonal antibodies to the AoSEs and determined their expression levels using immunodetection. We also analyzed the levels of the AoSE proteins and the alisol B 23-acetate contents at different growth stages in by Professor Gu Wei (College of Pharmacy, Nanjing University of Chinese Medicine). Beginning on October 15th, leaves, tubers, and roots of were collected every 15 days. Seedlings of were divided into the control and sample groups. MeJA dissolved in distilled water was applied to the leaves at a final concentration of 300?M. Leaves of the control group were treated with an equal volume of distilled water. The water Vadadustat control and MeJA solution were sprayed until the leaf surfaces were saturated. All plants were sampled at 0, 1, 2, 3, 4, and 5 days after treatment. Plants were rinsed with distilled water and dried using tissue paper. Subsequently, plant biomass (fresh weight) was determined for 10 complete plants in each group (each plant was an individual sample). Vadadustat All treatments were performed in five replicates. One-half of each sample was frozen in liquid nitrogen and stored at ?80?C to be used for RNA and protein extraction, and the other half was oven-dried at 60?C to a constant weight for extraction and HPLC analysis. The dry samples (0.5?g) were extracted with 20?ml of acetonitrile in an ultrasonic bath for 30?min, filtered through a 0.45?m membrane, Vadadustat and assayed by HPLC. HPLC analysis Samples were analyzed using a Waters 2695 series HPLC system (Waters Corporation, Milford, MA USA), equipped with a quaternary pump and a variable wavelength ultraviolet (UV) detector. The samples (20?L) were applied to a C18 analytical column (5 m, 4.6??250?mm; Phenomnex, Torrance, CA, USA) at a flow rate of 1 1?mL/min. The Rabbit Polyclonal to C1S mobile phase consisted of acetonitrile (A) and distilled water (B) and the gradient of the mobile phase was as follows: 0C10?min, 30% to 50% solvent A; 10C50?min, 50% to 90% solvent A. The column temperature was maintained at 25?C,.