Molecular Cloning And Sequence Analysis Of FPS In Panax Notoginseng (Burkill) F.H. Chen Ex C.Y. Wu & K.M. Feng

Object: A cDNA encoding farnesyl pyrophasphate synthase (FPS) that is one of key enzymes in the synthesis of triterpene saponins from Panax notoginseng (Burkill) F. H. Chen ex C. Y. Wu & K. M. Feng was cloned and sequenced.This works provided a foundation for exploring the mechanism of saponins biosynthesis at the molecular level and application in the other medical plants.Methods: The modificated single-step guanidinium isothiocyanate, guanidinium isothiocyanate with high concentration of salt, CTAB extraction method and Thomas'RNA extraction method were applied to isolate total RNA from root and stem of Panax notoginseng that is rich in polyphenolics, polysaccharides and secondary metabolites such as saponins. The total RNA samples obtained by the above methods were tested by ultraviolet spectrophotometry, agarose gel electrophoresis and RT-PCR. The cDNA, encoding FPS in Panax notoginseng was amplified by 3' RACE and 5' RACE strategy with the total RNA of root as the template that was obtained by the modificated single-step guanidinium isothiocyanate. The full-length cDNA of FPS was cloned into the pMD 18-T Vecter and sequenced.Result: The modificated single-step guanidinium isothiocyanate and guanidinium isothiocyanate with high concentration of salt are more effective.The integrality and high purity of total RNA can fit the demands of molecular biology experiment such as reverse-transcription by RT-PCR.The fragments of FPS was ligated and cloned into the pMD 18-T Vecter and sequenced.The analysis results revealed that the full-length cDNA had 1409 bp with an open reading frame encoding 343 amino acids of protein.The FPS sequence had 95%, 87%, 86%, 86% amino acid sequence homology and 81%, 66%, 68%, 66% DNA homology to the...