Cloning, Functional Expression, And Expressional Regulation Of The Ganoderma Lucidum Hydroxymethylglutaryl-Coenzyme A Reductase Gene

Ganoderma lucidum (Leyss.ex.Fr.) Karst (G. lucidum) is a species of Basidomycetes that belongs to Ganoderma of Hymenomycetes, which has been popular as a healthy food and medicine for more than 2000 years in China. 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.We have made some progress in G. lucidum HMGR geng cloning. The full-length genomic DNA sequence of HMGR gene has been amplified. Three cDNA fragments have been amplified using the cDNA of G. lucidum primordium as a template. And we predicted the cDNA full-length sequence based on the obtained three cDNA fragments and the full-length genomic DNA sequence. The promoter sequence of HMGR gene has been amplified by SEFA-PCR method.In my research, according to the HMGR gene cDNA specific sequence of G. lucidum, two primers were designed. The cDNA sequence of HMGR gene 3' UTR was obtained by the 3' RACE method with 3'-Full RACE Core Set Ver.2.0 (TaKaRa). Then two sets of primers were designed according to the known sequence to amplify the full-length cDNA. The cDNA sequence was 3681 bp, encoding 1226 amino acid.By HMGR gene structure prediction analysis, Three domains were found: an N-terminal hydrophobic domain (626 aa), a linker domain (147 aa), and a C-terminal catalytic domain (454 aa). The N-terminal hydrophobic domain of the G. lucidum protein was predicted to include eight transmembrance segments separated by hydrophilic loops. The cDNA sequence of HMGR gene only containing Linker and catalytic domain excluding the hydrophobic region of N-terminal was cloned into yeast expression vector pYF1845. The recombinant plasmid was introduced into a HMGR-defective yeast mutant strain JRY1130, whose growth requires mevalonate. Transformants could grow on media lacking mevalonate. It confirmed that the cDNA sequence of HMGR gene obtained could be funcional complemented. And it showed that N-terminal hydrophobic region is not necessary to the HMGR catalytic function.Ganoderma triterpenes is secondary metabolites, whose biosynthesis was regulated by developmental stage and inducible factor. The HMGR is the key enzyme in triterpene biosynthesis. We detected the HMGR gene expression of transcription levels under the different stages of development in G. lucidum by competitive RT-PCR. The result indicated that when the mycelium of G. lucidum growth to an appropriate stage (14 day), the HMGR gene expression was increased significantly with the elongation of incubation time.Methyl jasmonate (MJ) functions as part of a signal-transduction system involved in regulation of cell defense responses to a variety of adversity,and induce the biosynthesis of a variety of secondary metabolites. We studied the HMGR gene expression patterns and the contents of triterpene of G. lucidum mycelia treated with different concentrations and different induced time of MJ. The content of triterpene reached the highest when the concentration of MJ was 50μM, increasing by 58.6 % and 29.4% compared with negative control, and the difference is significant. Except MJ concentration of 10μM., when MJ with the other concentrations induced the mycelium, the HMGR gene expression was increased significantly. The HMGR gene expressional dosage was the largest with the MJ concentrations of 100μM, increasing by 10 times, compared with the negative control.The result indicated that there is a positive relationship between the induced time and the contents of triterpene of G. lucidum mycelia. The contents of triterpene of the mycelium increased with the elongation of the induced time of MJ. However, the HMGR gene expression is disproportionate with the induced time of MJ, and the results showed an approximately normal distribution. The HMGR gene expressional dosage is the largest when MJ induced the mycelium for 5 days which is 284 times comparing with the minimum when MJ induced the mycelium for 6 days.UV induced mutagenesis of Ganodema 's protoplast was carried out to obtain strains with high yield of triterpenes or polysaccharides. By crude screen (growth rates), careful screen (dry weights and contents of triterpene or polysaccharides) in their mycelium, and stability test (contents of triterpene or polysaccharides after successive generation) , two mutants UV-3 & UV-10 with steady properties and higher contents of triterpenes(P<0.01) and three mutants UV-25, UV-34 & UV-39 with steady properties and higher contents of polysaccharides (P<0.01) were obtained.As compared with the original strain the growth rate and the content of triterpenes in its fruiting body increased by 58.6 % and 29.4% ( P < 0.01), respectively. Sequence-related amplified polymorphism (SRAP) test showed that genetic differences were very obvious between the mutant UV-3 and the original strain.The RT-PCR was used to determine the mRNA expressional dosage of the HMGR gene among the original strain and two mutants with steady properties and higher contents of triterpenes. As a result, the mutants UV-3 and UV-10 were equal, and both of them were lower than the original strain on the mRNA expressional dosage of the HMGR gene...