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-Co A 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.
We cloned and analyzed the two genes of the 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate reductase(HDR) gene family from Huperzia serrate.The two transcripts coding HDR,named Hs HDR1 and Hs HDR2,were discovered in the transcriptome dataset of H.serrate and were cloned by reverse transcription-polymerase chain reaction(RT-PCR).The physicochemical properties,protein domains,protein secondary structure,and 3D structure of the putative Hs HDR1 and Hs HDR2 proteins were analyzed.The full-length c DNA of the Hs HDR1 gene contained 1431 bp encoding a putative protein with 476 amino acids,whereas the Hs HDR2 gene contained 1428 bp encoding a putative protein of 475 amino acids.These two proteins contained the conserved domain of 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate reductase(PF02401),but without the transmembrane region and signal peptide.The most abundant expression of Hs HDR1 and Hs HDR2 was detected in H.serrate roots,followed by the stems and leaves.Our results provide a foundation for exploring the function of Hs HDR1 and Hs HDR2 in terpenoid and sterol biosynthesis in Huperziaceae plants.
