title: Cloning and characterization of an elicitor-responsive gene encoding 3-hydroxy-3-methylglutaryl coenzyme A reductase involved in 20-hydroxyecdysone production in cell cultures of Cyanotis arachnoidea.
abstract: Cyanotis arachnoidea contains a rich source of bioactive phytoecdysteroids (i.e. analogues of insect steroid hormones). 3-Hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) supplies mevalonate for the synthesis of many secondary metabolites including 20-hydroxyecdysone (20E), one of metabolism-enhancing phytoecdysteroids. In this study, in order to develop a sustainable source of 20E, cell suspension cultures were established from shoot cultures of C. arachnoidea, and a full length cDNA encoding HMGR (designated as CaHMGR) was cloned and characterized. The cDNA contained 2037 nucleotides with a complete open reading frame (ORF) of 1800 nucleotides, which was predicted to encode a peptide of 599 amino acids. Expression analysis by real-time PCR revealed that CaHMGR mRNA was abundant in C. arachnoidea stems, roots and leaves. When cultivated in Murashige & Skoog medium supplemented with 0.2 mg L(-1) 1-naphthlcetic acid (NAA) and 3.0 mg L(-1) 6-benzyladenine (6-BA), C. arachnoidea cells in suspension culture grew rapidly, yielding 20E (124.14 μg L(-1)) after 12 days. The content of 20E in cell cultures elicited by 0.2 mM methyl jasmonate (MeJA), 100 mg L(-1) yeast elicitor (YE) or 25 μM AgNO3 was increased 8-, 2-, and 6-fold over the control, respectively. Quantitative real-time PCR analysis showed that CaHMGR was expressed at a higher level under the treatment of MeJA or Ag(+) elicitor. Our results suggested that 20E accumulation may be the result of the expression up-regulation of CaHMGR involved in the biosynthesis under the treatment of various elicitors.
authors.affiliation: College of Pharmaceutical Sciences, Soochow University, Suzhou 215123, China.
authors.forename: Qiu Jun
authors.initials: QJ
authors.lastname: Wang
authors.affiliation: School of Architecture and Urban Environment, Soochow University, Suzhou 215123, China.
authors.forename: Li Ping
authors.initials: LP
authors.lastname: Zheng
authors.affiliation: Institute of Horticultural Crops, Yunnan Academy of Agricultural Sciences, Kunming 650205, China.
authors.forename: Pei Fei
authors.initials: PF
authors.lastname: Zhao
authors.affiliation: College of Pharmaceutical Sciences, Soochow University, Suzhou 215123, China.
authors.forename: Yi Lu
authors.initials: YL
authors.lastname: Zhao
authors.affiliation: College of Pharmaceutical Sciences, Soochow University, Suzhou 215123, China. Electronic address: jwwang@suda.edu.cn.
authors.forename: Jian Wen
authors.initials: JW
authors.lastname: Wang
mesh_terms: D000085:Acetates / Q000494:pharmacology; D000214:Acyl Coenzyme A / Q000378:metabolism; D002264:Carboxylic Acids / Q000494:pharmacology; D003001:Cloning, Molecular; D034821:Commelinaceae / Q000737:chemistry / Q000235:genetics*; D003517:Cyclopentanes / Q000494:pharmacology; D004441:Ecdysterone / Q000378:metabolism; D018506:Gene Expression Regulation, Plant / Q000187:drug effects; D009281:Naphthalenes / Q000494:pharmacology; D016366:Open Reading Frames; D054883:Oxylipins / Q000494:pharmacology; D060888:Real-Time Polymerase Chain Reaction
chemical_list: D000085:Acetates; D000214:Acyl Coenzyme A; D002264:Carboxylic Acids; D003517:Cyclopentanes; D009281:Naphthalenes; D054883:Oxylipins; C008047:3-hydroxy-3-methylglutaryl-coenzyme A; C085312:1-naphthoic acid; D004441:Ecdysterone; C072239:methyl jasmonate
keywords: 20-Hydroxyecdysterone; 3-Hydroxy-3-methylglutaryl coenzyme A reductase; Cyanotis arachnoidea; Elicitation; Gene expression