Molecular Cloning And Analysis Of Expression Profiles Of Mevalonate Diphosphate Decarboxylase Gene From Ganoderma Lucidum

Ganoderma lucidum belongs to the Basidiomycetes, is well-known for its role in increasing wisdom and longevity in the opinion of traditional Chinese medicine. Modern medical research shows that several compounds such as polysaccharides, nucleoside, furan derivatives, steroids, alkaloids with biomedicinal activity, which have been isolated from this species. Triterpenoids is the most important compound with biomedicinal activities in anti-tumor, inhibit the release of histamine, anti-HIV and inhibit cholesterol biosynthesis, and so on.As a secondary metabolite, triterpenes of G. lucidum are biosynthesized via the mevalonate pathway. This pathway leads to the synthesis of both sterols and isoprenoids in eukaryotic cells. Mevalonate diphosphate decarboxylase is a key enzyme in the biosynthesis of isoprenoid and sterol. It catalyses the decarboxylation of the six-carbon mevalonate diphosphate (MVA-PP) to the five-carbon isopentenyl diphosphate (IPP). Thus, the MVD expression level may directly affect the content of triterpenoids in G. lucidum. Besides, triterpenes is the main bio-active component, so studying the expression profiles of MVD is an highly significance to enhance the medicinal value of G. lucidum.Degenerate primers were designed according to conservative sites of protein sequences from related species and a353bp DNA fragment was obtained. Three specific primers were designed according to the353-bp fragment, for5’SEFA-PCR, to amplify the5’end of MVD gene and the promotor.3’end sequence of MVD gene was obtained using traditional method. By comparing and aligning the nucleotide sequences from the5’-SEFA and3’-RACE, the full length cDNA (1203bp), genomic DNA (1312bp) which include three exons and two introns and promotor (1193bp) were amplified. The phylogenetic tree reconstructed with MEGA4.0implied GIMVD was closely related to the MVD of Cryptococcus neoformans. MVD contains typical GHMP kinase domains according structural domain prediction. The cDNA of MVD was inserted into yeast expression vector pYES2to investigate the function of GIMVD in S. cerevisiae.A pET-24b vector with MVD insertion was constructed and transformed to E. coli Rosetta (DE3) to express the MVD. The protein was purified by affinity chromatography using a Ni column and immunized New Zealand rabbit to obtain polyclonal antibody. Protein levels of MVD were detected using polyclonal antibody of MVD. To examine different developmental stages and under the conditions of methyl jasmonate-induced the mRNA levels of the MVD, we monitored the gene transcript levels by real-time RT-PCR. The results showed that the expression level of primordia was higher than the mycelia both in the mRNA level and protein level. The highest mRNA level was observed at a concentration of100μM of MeJA while the highest protein level was200μM and the induced effects are not obvious. To further investigate the function of the gene, we constructed the gene overexpression vector in an attempt to over-expression MVD of the gene in G. lucidum.In order to investigate the gene expression profile of MVD, the promoter sequence of MVD was analyzed and seven CAAT boxes, three GC boxes and one TATA box with typical features eukaryotic promoter elements were found. The potential regulatory elements, such as fungal elicitor responsive element, MYB binding site and two MeJA responsive elements were also found in the promotor region. Additionally, we investigated the promotor activity in different concentrations of MeJA induced and promoter deletional analysis using beta-glucuronidase (GUS) as a reporter. The results showed that the promoter demonstrated the highest activity when the concentration of MeJA was150μM; Deletion analysis of the MVD promoter shows that deletions of-1193to-243nt and-1193to-143nt resulted in a strong expression of the GUS gene compared with other deletions.