Molecular Cloning, Characterization, And Differential Expression Of A Farnesyl Diphosphate Synthase Gene From The Basidiomycetous Fungus Ganoderma Lucidum

Ganoderma lucidum,called "Lingzhi" in China,has a long history of use for the treatment of many different diseases.Ganoderic acid(GA) produced by this higher fungus has a number of important biological functions including cytotoxicity to hepatoma cells, inhibition of histamine release,inhibition of cholesterol synthesis and absorption, stimulation of platelet aggregation,and anti-HIV and anti-HIV-protease activities.The quality of G.lucidum products was determined by the content of GA.As a secondary metabolite,ganoderic acid is synthesized via the mevalonate pathway, where farnesyl diphosphate is converted to squalene,then to 2,3-oxidosqualene,and finally undergoes a series of cyclization,oxidation,and reduction reactions.Farnesyl-diphosphate(FPP) synthase(FPS;EC 2.5.1.1./EC 2.5.1.10) catalyzes the sequential 1'-4 condensation of two molecules of IPP with dimethylallyl diphosphate (DMAPP) and the resulting 10-carbon compound geranyl diphosphate(GPP) to produce the 15-carbon compound FPP.FPS is located at the first multiple branch point in the isoprenoid biosynthetic pathway,suggesting that FPP biosynthesis is tightly regulated. Therefore,changes in FPS activity might alter the flux of GA.Based on a sequence comparison between the FPS genes from other species, degenerate oligonucleotides from the most conserved regions were synthesized for PCR amplification of cDNA library.A 456 bp putative cDNA fragment was obtained and sequenced to confirm that this fragment shared high similarity with known fungal FPS sequences.Specific primers were designed on the basis of the specific 456-bp fragment of FPS gene of G.lucidum.Collaborated with the universal primers of the cDNA library vector,the 5' end of G.lucidum gene was amplified.Total RNA was prepared from G. lucidum primordium and reverse-transcribed to cDNA.We employed a 3' RACE strategy to obtain the sequence of 3' end of G.lucidum gene.Specific primers were designed according to the obtained sequence information and the full length of cDNA was amplified. Analysis of the GIFPS cDNA sequence indicated the presence of an open reading frame(ORF) of 1,083 bases.The ORF encoded a 360-amino acid polypeptide and a theoretical molecular mass of 41.27 kDa.The genomic sequence of G.lucidum FPS gene was amplified by the primes designed according to the 5' and 3' ends of FPS gene.The full-length genomic sequence was 1388 bp;the gene was consisted of 4 exons and 3 intronsProtein sequence comparison indicated that GlFPS is closely related(59%identity) to the FPS of Lactarius chrysorrheus,another basidiomycetous fungus.The alignment of GlFPS with orthologs available in the databases showed four highly conserved regions. GlFPS contains the typeⅠ(eukaryotic) FPS signature,which is consistent with traditional evolutionary affinities.The cDNA sequence of FPS gene was cloned into yeast expression vector pYF1845. The recombinant plasmid was introduced into a FPS-defective yeast mutant strain CC25, whose growth requires ergosterol.Transformants could grow on media lacking ergosterol at 36℃.The results indicate that GlFPS is a functional fungal gene encoding FPS in G. lucidum.The promoter sequence of FPS was amplified from G.lucidum genomic DNA by the SEFA-PCR method.Using Softberry software,predicted TATA boxes and CAAT boxes were found.The potential regulatory elements associated with sterol,hormone,and stress-related responses were also found in the GlFPS promoter region.We monitored the levels of the GlFPS mRNA at different developmental stages by competitive PCR.The GlFPS mRNA level was relatively low in mycelia.However,the fruiting process increased the GlFPS mRNA level.The higher GlFPS mRNA level was probably correlated with the increased triterpene synthesis as described by Hirotani et al..Treatment of mycelia with exogenous methyl jasmonate(MeJA) also caused a large accumulation of triterpene biosynthesis.Thus,we monitored the effect of MeJA on the GlFPS mRNA level in the mycelia.GlFPS transcripts accumulated following the addition of 50-150μm MeJA to the culture medium,whereas mycelia treated with 200μm MeJA showed reduced promotion of GlFPS mRNA.The highest mRNA level was observed at a concentration of 150μm.Interestingly,sequence analysis of the GlFPS promoter identified two potential MeJA responsive elements.Our results suggest that GlFPS is probably involved in the triterpene biosynthesis regulation pathways of MeJA signalling via the putative MeJA-response elements.