title: Characterization and function of the 3-hydroxy-3-methylglutaryl-CoA reductase gene in Alisma orientale (Sam.) Juz. and its relationship with protostane triterpene production.
abstract: 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; 2252 bp; GenBank accession no. KP342318) with an open reading frame (ORF) of 1809 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 (Rosetta) 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.
authors.affiliation: Jiangsu Province Key Laboratory for Molecular and Medical Biotechnology, Life Sciences College, Nanjing Normal University, Nanjing, 210023, China; School of Pharmacy, Nanjing University of Chinese Medicine, Nanjing, 210023, China.
authors.forename: Wei
authors.initials: W
authors.lastname: Gu
authors.affiliation: School of Pharmacy, Nanjing University of Chinese Medicine, Nanjing, 210023, China.
authors.forename: Chao
authors.initials: C
authors.lastname: Geng
authors.affiliation: School of Pharmacy, Nanjing University of Chinese Medicine, Nanjing, 210023, China.
authors.forename: Wenda
authors.initials: W
authors.lastname: Xue
authors.affiliation: School of Pharmacy, Nanjing University of Chinese Medicine, Nanjing, 210023, China.
authors.forename: Qinan
authors.initials: Q
authors.lastname: Wu
authors.affiliation: School of Pharmacy, Nanjing University of Chinese Medicine, Nanjing, 210023, China.
authors.forename: Jianguo
authors.initials: J
authors.lastname: Chao
authors.affiliation: School of Pharmacy, Nanjing University of Chinese Medicine, Nanjing, 210023, China.
authors.forename: Fei
authors.initials: F
authors.lastname: Xu
authors.affiliation: School of Pharmacy, Nanjing University of Chinese Medicine, Nanjing, 210023, China.
authors.forename: Hongmei
authors.initials: H
authors.lastname: Sun
authors.affiliation: School of Pharmacy, Nanjing University of Chinese Medicine, Nanjing, 210023, China.
authors.forename: Ling
authors.initials: L
authors.lastname: Jiang
authors.affiliation: School of Pharmacy, Nanjing University of Chinese Medicine, Nanjing, 210023, China.
authors.forename: Yun
authors.initials: Y
authors.lastname: Han
authors.affiliation: Jiangsu Province Key Laboratory for Molecular and Medical Biotechnology, Life Sciences College, Nanjing Normal University, Nanjing, 210023, China. Electronic address: shuangquan_z@sina.cn.
authors.forename: Shuangquan
authors.initials: S
authors.lastname: Zhang
mesh_terms: D034902:Alisma / Q000201:enzymology* / Q000235:genetics* / Q000254:growth & development; D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000378:metabolism; D001483:Base Sequence; D002783:Cholestenones / Q000737:chemistry / Q000378:metabolism; D003001:Cloning, Molecular; D019295:Computational Biology; D017124:Conserved Sequence / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D004797:Enzyme-Linked Immunosorbent Assay; D008401:Gas Chromatography-Mass Spectrometry; D018506:Gene Expression Regulation, Plant; D017343:Genes, Plant; D006903:Hydroxymethylglutaryl CoA Reductases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D010802:Phylogeny; D017434:Protein Structure, Tertiary; D011817:Rabbits; D017386:Sequence Homology, Amino Acid; D014315:Triterpenes / Q000378:metabolism*
chemical_list: C000604763:29-norprotostane; D000906:Antibodies; D002783:Cholestenones; D018076:DNA, Complementary; D014315:Triterpenes; C526957:alisol B 23-acetate; D006903:Hydroxymethylglutaryl CoA Reductases
keywords: Alisma orientale (Sam.) Juz.; AoHMGR; Expression; Protostane triterpenes