Analysis Of The Influence Of Environmental Conditions On Ganoderic Acid Content Of Ganoderma Lucidum By The Orthogonal Design And Cloning Of The Hydroxymethylglutaryl-Coenzyme A Reductase Gene

Ganoderma lucidum (Leyss.: Fr.) Karst (G. lucidum) is a species of basidomycetes thatbelongs to Ganodermaceae of Aphyllophorales, which has been popular as a healthy foodand medicine for more than 2000 years in China. Ganoderic acid (GA) produced by thishigher fungus has a number of important biological functions including cytotoxicity tohepatoma cells, inhibition of histamine release, inhibition of cholesterol synthesis andabsorption, stimulation of platelet aggregation, and anti-HIV and anti-HIV-proteaseactivities. 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 finallyundergoes a series of cyclization, oxidation, and reduction reactions. Hydroxymethylglutar-yl-coenzyme A reductase (HMGR) is the key enzyme in triterpene biosynthesis in whichHMG-CoA was reduced to mevalonate using two molecules of NADP(H). There is apositive correlation between the expression level of HMGR and the amount of triterpenesproduced.The influence of environmental conditions on GA content of G. lucidum was investigatedby one-factor-at-a-time design and orthogonal design. In the one-factor-at-a-time design,the effects of different carbon sources, nitrogen sources, mineral ions and pH on the GAcontent was investigated. The results shown that sucrose, soybean power or peptone,ferrous sulfate and pH 6.0 were the most suitable carbon source (A), nitrogen source (B),mineral source (C) and initial pH (D) for GA content, respectively. Three optimal levels ofthe four factors were chosen for the orthogonal design. The result shown that the order ofeffects of the four factors on GA content was A＞C＞D＞B. The best level of factor A wasA₂ (sucrose) and the value was+0.34 mg/100mg DW. The optimal treatment combinationwas A₂B₁C₃D₁ and the GA content reached 2.63±0.011 mg/100 mg DW. The interactionsbetween mineral ion and nitrogen source, mineral ion and pH were extremely significant (P＜0.01). The highest interaction effect was (B₂×D₂) and the value was+0.19mg/100mgDW, which is higher than the value of the level effect of B₂ (peptone) and D₂(pH 5.0). Theresults proved that interactions between factors could not be ignored. With the confirmationof the variety of GA content under different culture conditions, the mechanisms of theexpression of the key enzymes HMGR will be studied in the research.Amino acid sequences conserved among a number of known HMGRs gene were used todesign two degenerate oligonucleotide primers for PCR amplification of G. lucidumgenomic DNA. Sequencing of the amplification product revealed a fragment of 511 bpencoding an amino acid sequence corresponding to HMGR.Two specific primers were designed and synthesized based on the sequence of theamplified 511-bp HMGR gene specific fragment and the full length genomic sequence(4262 bp) of G. lucidum HMGR gene was amplified from G. lucidum genomic DNA bythe SEFA-PCR method.Total RNA was prepared from G. lucidum primordium and used to synthesize cDNA.Specific primers were designed according to the genomic DNA sequence and the full lengthof cDNA was amplified. The cDNA sequence was 3681bp, encoding 1226 amino acid.Comparing the genomic DNA sequence with the cDNA sequence of HMGR gene, 7exons and 6 introns, with a typical intron's border "GT—AG" can be founded.Blast x program was used to search NCBI database and analyze the homology of thededuced G. lucidum HMGR amino acid sequence. The sequence showed a relatively highhomology with the sequence of C. neoformans. The NH₂-terminal amino acid sequenceshowed 46% homology with the sequence of C. neoformans and the COOH-terminal aminoacid sequence showed 83% homology with the sequence of C. neoformans.Two putative conserved HMG-CoA binding sites and two putative conserved NADP(H)binding sites were founded using sequence alignments with some fungi HMGRs. Twoputative PEST sequences, three transmembrane regions and a signal peptide were founded.Two-dimensional structural prediction of G. lucidum HMGR amino acid sequence wasperformed. A cAMP dependent protein kinase phosphorylation site and twelve proteinkinase C phosphorylations sites were founded and the HMGR protein was composed of41.5% a-helix, 11.3% extended strand and 47.1% random coil. A phylogentic tree ofHMGR was constructed with the program of Clustal X and MEGA 3.0. The resultdemonstrated that G. lucidum HMGR gene are more closely related to Basidiomycotina,such as C. neoformans and U.mayis. Competitive RT-PCR was performed to determine HMGR gene expression patternsunder different carbon source cultural conditions. The result demonstrated that there is apositive relationship between the expression level of HMGR gene and the amount oftriterpene produced using different carbon sources.The promoter sequence of HMGR was amplified from G. lucidum genomic DNA by theSEFA-PCR method. The promoter was analyzed using the Softberry software. The resultshowed that it contained several regulatory elements, such as TFII-I, GATA-1, GC-box,NF-Y, but the TATA box and the TTATT box could not found. Similar to human andhamster, the promoter also contained some important sterol regulated elements, such asSRE-1, SREBP and CREB.