Cloning and tissue expression of geranylgeranyl diphosphate synthase gene from Andrographis paniculata / 中草药

ABSTRACT

Objective: Andrographolide is the main bioactive substance in Andrographis paniculata, and popularly used as active pharmaceutical ingredient (API). We have cloned the gene encoding geranylgeranyl diphosphate synthase from A. paniculata and characterized its tissue expression pattern. Methods: Total RNA was extracted with CTAB-LiCl extraction method; Conserved fragment was amplified and cloned with degenerated primers, and a full length ORF encoding geranylgeranyl diphosphate synthase was obtained with RACE method and analyzed by bioinformatic softwares, e.g. ProtParam. Tissue expression pattern was predicted with real time PCR. Results: We have cloned a 1 047 bp GGPS gene encoding a sequence with 348 amino acids. This amino acid sequence contained a plastid targeted N-terminal signal peptide and has high similarities with the GGPS protein from Catharanthus roseus. The GGPS gene has expressed in a dynamic state in stems and leaves of A. paniculata. The expression reached a high level at bud stage, then decreased at early flowering stage, increased at flowering and early seed setting stage again, and finally decreased at seed setting stage. Considering the above expression characteristics, biosynthesis of metabolites regulated by GGPS was deduced more active at bud stage and flowering and early seed setting stage. Conclusion: GGPS is a key enzyme in biosynthesis of andrographolide. We have cloned the GGPS gene from A. paniculata, and provide a sharp tool in genetic engineering of andrographolide biosynthsis pathway.