Effects Of Exogenous Factors On Farnesyl Diphosphate Synthase Gene For Synthesis Of Triterpene In Inonotus Obliquus

Inonotus obliquus (Fr.) Pilat always grew on birch trees. The main pharmacological active components of Ⅰ.obliquus were triterpenoids. It was an important class of secondary metabolites and an important indicator of its efficacy and medicinal value for I.obliquus.In triterpene biosynthetic pathway of Ⅰ.obliquus, farnesyl diphosphate synthase (FPS) was the key enzyme that located at the first multiple branch point in the isoprenoid biosynthetic pathway. It suggested that the flux of subsequent products was determinated by changing in activity and expression level of FPS. Therefore, based on regulation and expression of FPS gene, the molecular mechanism of triterpenoids synthesis pathway in Ⅰ.obliquus is further elucidated. The most conserved regions of FPS amino acid sequence from a high affinity species determinated by DNAMAN, and then the degenerate oligonucleotides primers were designed and synthesized. For total DNA as a template, a 582 bp FPS gene-specific DNA fragment of Ⅰ.obliquus was amplified by PCR. According SEFA-PCR method, specific primers were designed on the basis of the specific fragment of FPS gene and the 5’end of FPS gene was amplified. Total RNA was extracted and reverse-transcribed to cDNA. We employed 3’-RACE strategy to obtain the sequence of 3’end of FPS gene. According to 5’and 3’terminal sequence, the promoter and terminator regions were determinated. Specific primers were designed according to in the regions of the promoter and terminator sequence, respectively. Full-length DNA and cDNA for FPS gene were amplified by DNA and cDNA as a template. Analysising of the FPS DNA and cDNA sequence, it indicated that the full-length DNA sequence was 1186 bp and 1098 bp for cDNA contained an open reading frame (ORF), the ORF encoded a 365 amino acid polypeptide. Amino acid sequence comparison used the NCBI Blast Program indicated that FPS of Ⅰ.obliquus is closely related to that of Ganoderma lucidum. The amino acid sequence also contains four highly conserved regions. A phylogenies tree of amino acid sequence was constructed with the program of Clustal X and MEGA 6.0. The result demonstrated that FPS of I.obliquus belongs to the type I FPS (eukaryotic). The cDNA sequence of FPS gene was cloned in to yeast expression vector pYF1845. The recombinant plasmid was introduced in to a defective yeast strain CC25, whose growth requires ergosterol. The positive transformants could be selected on SC(-ura) medium at 30 ℃. The specific substrates were hydrolyzed by fermentation broth of positive transformant. A specific products were detected by HPLC to confirm the FPS gene expression.To study effects of some substances existed in some microbes or plants on metabolic pathways of Ⅰ.obliquus. Some substances may contribute to the accumulation of biomass and active compounds of Ⅰ.obliquus, so that the original role of I.obliquus could be supplemented or enhanced. Therefore, birch bark and rhizosphere soil, methyl jasmonate, Frankia sp and Rhizobium indigoferae were selected as inductive agents. Different concentrations of inductive agents induced mycelia growth. For biomass and triterpenoids content of Ⅰ.obliquus as an indicator, the most suitable inductive agent and the appropriate dose were selected to promote growth of mycelia. For biomass, acceleration effect of Frankia sp fermentation on mycelia biomass is maximum, followed by Rhizobium indigoferae fermentation, methyl jasmonate solution, birch bark and rhizosphere soil decoction. For triterpenoids content, acceleration effect of Rhizobium indigoferae fermentation on triterpenoids accumulation is maximum, followed by Frankia sp fermentation, birch bark decoction, methyl jasmonate solution, rhizosphere soil decoction have inhibition effect on triterpenoids accumulation. We monitored the levels of the FPS mRNA at different growth stages by altered concentrations of additives by Real-Time PCR (RT-PCR). Birch bark decoction, methyl jasmonate solution, Frankia sp and Rhizobium indigoferae fermentation have acceleration effect on expression value of FPS. however that of rhizosphere soil decoction is have inhibition effect on its expression value. It suggested that FPS expression levels were significantly positive correlation to triterpene contents, under the same conditions.