ABSTRACT
3-Hydroxy-3-methylglutaryl-CoA synthase (HMGS) is the first committed enzyme in the MVA pathway and involved in the biosynthesis of terpenes in Tripterygium wilfordii. The full-length cDNA and a 515 bp RNAi target fragment of TwHMGS were ligated into the pH7WG2D and pK7GWIWG2D vectors to respectively overexpress and silence, TwHMGS was overexpressed and silenced in T. wilfordii suspension cells using biolistic-gun mediated transformation, which resulted in 2-fold increase and a drop to 70% in the expression level compared to cells with empty vector controls. During TwHMGS overexpression, the expression of TwHMGR, TwDXR and TwTPS7v2 was significantly upregulated to the control. In the RNAi group, the expression of TwHMGR, TwDXS, TwDXR and TwMCT visibly displayed downregulation to the control. The cells with TwHMGS overexpressed produced twice higher than the control value. These results proved that differential expression of TwHMGS determined the production of triptolide in T. wilfordii and laterally caused different trends of relative gene expression in the terpene biosynthetic pathway. Finally, the substrate acetyl-CoA was docked into the active site of TwHMGS, suggesting the key residues including His247, Lys256 and Arg296 undergo electrostatic or H-bond interactions with acetyl-CoA.
ABSTRACT
Objective: To clone and characterize a 3-hydroxy-3-methylglutaryl-CoA synthase (HMGS) gene which involved in the triterpenoid biosynthesis pathway in Sanghuangporus baumii. Methods: The HMGS gene cDNA full-length sequence was cloned by RACE technology. Characteristics including the physicochemical properties and conserved domain of the deduced HMGS protein were determined by a series of bioinformatics tools. The entire protein-coding cDNA of HMGS was cloned into the expression vector pET-32a (+). Then the recombinant plasmid was transformed into E. coli BL21 (DE3) cells. With IPTG induction, SDS-PAGE was used to investigate the situation of expression. Additionally, qRT-PCR technology was performed to measure the transcript levels of HMGS gene in the triterpenoid pathway during different developmental stages of S. baumii. Results: The full-length nucleotide sequence of HMGS was 1 930 bp, containing a complete open reading frame of 1 458 bp which encoded a polypeptide of 485 amino acids. Bioinformatics analysis of the amino acid sequence showed that the molecular weight of encoded protein was 52 750, and theoretical isoelectric point was 5.60. This protein was a hydrophilic protein, without transmembrane and signal peptide sequence. The constructed phylogenetic tree showed that HMGS from S. baumii had the highest similarity with HMGS from Fomitiporia mediterranea. The prokaryotic expression vector pET-32a-HMGS was sucessfully obtained. SDS-PAGE results showed that a significant protein band was in consistent with molecular weight of the predicted protein. Moreover, the results showed that the transcript levels of HMGS gene were in dynamic change. The transcript levels in the mycelium stage were higher than that in the fruiting body stage. For instance, the highest transcript level of HMGS was at 14 d and was 2.33-fold higher than the 5 d. Conclusion: Molecular characterization of HMGS will be useful for further functional elucidation of the gene involving in triterpenoid biosynthesis pathway in S. baumii.
ABSTRACT
Objective To clone and analyze 3-hydroxy-3-methylglutaryl coenzyme-A synthase (HMGS) and 3-hydroxy-3-methylglutaryl coenzyme-A reductase (HMGR) genes from Panax notoginseng of four-year old during the flowering period, the key genes involved in the mevalonic acid pathway for saponin biosynthesis. Methods The cDNA sequences of PnHMGS1 and PnHMGR2 were obtained by reverse transcription PCR (RT-PCR) and rapid amplification of cDNA ends (RACE) methods and were analyzed in their secondary structures, subcellular localizations, domains, and the three-dimensional structures of putative proteins by the bioinformatics tools. Fusion genes were constructed by the prokaryotic expression system. Results The two genes were cloned, named as PnHMGS1 and PnHMGR2, respectively, and were both predicted to be located in the chloroplast. PnHMGS1 (1410 bp) encoded a predictive unstable protein with 469 amino acids and covered hydroxymethylglutaryl-CoA synthase domain. PnHMGR2 (1690 bp) also encoded an unstable protein with 589 amino acids and possessed a hydroxymethylglutaryl-coenzyme A reductase domain and two transmembrane regions. Both of the genes were expressed most in flowers followed by roots, stems, and least in leaves. Conclusion PnHMGS1 and PnHMGR2 are firstly cloned from P. notoginseng as the new member of the HMGR family, and they show the same expression profile as P. ginseng and P. quinquefolius.