Cloning Of Gynostemma Farnesyl Pyrophosphate Synthase (FPS) Gene And Its Prokaryotic Expression

The race method was applied to identify the farnesyl pyrophosphatesynthase from Gynostemma pentaphyllum, and on this basis the enzyme wasexpressed in prokaryotic cells. The main results of this project were presentedbelow：First, the full lenth cDNA of the farnesyl pyrophosphate synthase fromGynostemma pentaphyllum was isolated. TaKaRa3’RACE and5’-Full RACEKit were used to cloning the full length cDNA of Gynostemma FPPS gene. Thefull length cDNA of Gynostemma FPPS composed of1288nucleotides wasobtained, The open reading frame (ORF) of Gynostemma FPPS gene is1029bpin length, corresponding to a predicted polypeptide of342amino acid residueswith a molecular mass of3.94×104. The results of homologous analysis inGenBank demonstrated that the sequences had88%~78%similarity on thenucleotide sequence and91%~74%similarity on the deduced amino acidsequence. Phylogenetic analysis on the amino acid sequence of GynostemmaFPPS with those of other plants showed that Gynostemma FPPS was closely related to Siraitia grosvenorii and Cucumis sativus which was consistent withthe expected results. It was also found kinship of Gynostemma, fructusmomordicae, cucumber and apple FPS is also close, but according to planttaxonomy adopted by NCBI table, Gynostemma belongs to Cucurbitaceaefamily, apple is Rosaceae family in Rosales. The evolution of plants of theCucurbitaceae and Rosaceae in different branches, there may be a branch pointof a FPS gene evolution.Secondly, Prokaryotic expression of Gynostemma FPS, re-designedprimers after obtaining Pentaphyllum FPS full-length cDNA sequence, the FPScDNA sequences and expression vector pET32a (+) to connect successfullyconstructed pET32a (+)/FPS recombinant plasmid, and in Escherichia coliBL21(DE3) we successfully expressed about54KD protein.For this study, the RACE method was used to isolate the full lenthcdna of Gynostemma FPS and the Enzyme was successfully expressed inProkaryotic Cells,which has important significance for the research of theTriterpenoids biosynthetic pathway and its molecular mechanisms inGynostemma, as well as the separation and purification of the enzyme activityand molecular evolution of the farnesyl pyrophosphate synthase.