【摘 要】
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Protostane triterpenes from Alisma orientale (Sam.) Juz.have exhibited distinct pharmacological properties that are currently in high demand.3-Hydroxy-3-methylglutaryl-CoA reductase (HMGR) is consider
【机 构】
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School of Pharmacy, Nanjing University of Chinese Medicine, Nanjing, 210023, China
【出 处】
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2016首届中国中药资源大会暨CSNR中药及天然药物资源研究专业委员会第十二届学术年会
论文部分内容阅读
Protostane triterpenes from Alisma orientale (Sam.) Juz.have exhibited distinct pharmacological properties that are currently in high demand.3-Hydroxy-3-methylglutaryl-CoA reductase (HMGR) is considered the first rate-limiting enzyme in isoprenoid biosynthesis via the mevalonic acid (MVA) pathway.In this study, we cloned a full-length cDNA of A.orientale (Sam.) Juz.HMGR (AoHMGR;2, 252 bp;GenBank accession no.KP342318) with an open reading frame (ORF) of 1,809 bp.The deduced protein sequence contained four conserved motifs and exhibited homology with HMGR proteins from other plants.We next expressed the cloned gene in Escherichia coli BL21 cells,collected the expressed products, and incubated those with 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA)to determine enzymatic activity.GC/MS analysis revealed that the products were able to catalyze HMG-CoA and NADPH to form MVA.The purified protein was used to immunize New Zealand rabbits and prepare an antibody against AoHMGR.Western blot results demonstrated that the antibodies specifically recognized AoHMGR protein in A.orientale (Sam.) Juz.We then established a rapid test to detect AoHMGR protein in the plant, and found the tuber to be the most AoHMGR protein-abundant organ in A.orientale (Sam.) Juz.Furthermore, we detected the expression level of AoHMGR and contents of the main active component, Alisol B 23-acetate, at different growth phases of A.orientale (Sam.) Juz.A significant positive correlation was identified, indicating that AoHMGR represents a key enzyme in the synthetic pathway of protostane triterpenes.
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