Cloning And Sequence Analysis Of A Glyceraldehyde-3-Phosphate Dehydrogenase Gene From Ganoderma Lucidum And Establishment Of A Genetic Transformation System For Ganoderma Lucidum

Ganoderma lucidum(Leyss.:Fr.)Karst(G lucidum)is a species of basidomycetes that belongs to Ganodermaceae of Aphyllophorales,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.The quality of G.lucidum products was determined by the content of GA.Attempts to enhance and extend the medicinal applications of G.lucidum through strain improvement have been hampered by the lack of an efficient transformation system. In other fungi,one approach to alleviating this shortfall has been the construction of transformation vectors in appropriate hosts using a strong endogenous promoter,however, to our knowledge,no homologous gene promoters have been reported in G.lucidum. Previous observations in the yeast,Saccharomyces cerevisiae,and in higher eukaryotes have suggested that the glyceraldehyde-3-phosphate dehydrogenase(gpd,EC 1.2.1.12) gene(gpd)is under the control of a highly active promoter.In such cases,the GPD protein is a tetramer composed of identical subunits and comprises up to 5%of the soluble cellular protein.Furthermore,2-5%of the poly(A)+ RNA in yeast is gpd mRNA.The flanking regulatory regions of these gpd genes have also been used successfully for the construction of transformation vectors in different fungi.Amino acid sequences conserved among a number of known gpd gene were used to design two degenerate oligonucleotide primers for PCR amplification of G.lucidum genomic DNA.Sequencing of the amplification product revealed a fragment of 624 bp encoding an amino acid sequence corresponding to gpd.Specific primers were designed according to the genomic DNA sequence.Collaborated with the universal primers of the cDNA library vector,the 5′and 3′end of G.lucidum gene was amplified.According to the sequence of the 5′and 3′end of G.lucidum gpd gene,two specific primers were designed, and the full length of cDNA was amplified from the cDNA library.The cDNA sequence was 1014bp,encoding 337 amino acid.Blast x program was used to search NCBI database and analyze the homology of the deduced G.lucidum GPD amino acid sequence.The sequence showed a relatively high homology with the sequence of Pycnoporus coccineus. Two specific primers were designed and synthesized based on the sequence of the gpd cDNA sequence,the full length genomic sequence(1401 bp)of G.lucidum gpd gene was amplified from G.Iucidum genomic DNA.Comparing the genomic DNA sequence with the cDNA sequence of gpd gene,7 exons and 6 introns,with a typical intron's border "GT—AG" can be found.One conserved catalysis site were found using sequence alignment with some fungi gpd genes.A phylogentic tree of GPD was constructed with the program of Clustal X and PAUP 4.0.The results demonstrated that G.lucidum gpd gene is most closely related to basidiomycotina.The 5'-flanking region of G.lucidum was cloned by means of TaKaRa LA PCR^TMin vitro Cloning Kit according to the manufacturer's instructions(TaKaRa).Gene-specific primer(GSP)was designed from G.lucidum gpd sequence.There was one typical TATA box(TATAAAA)and two CAAT boxes(CCAAT)located in the 5'-flanking region.By the use of the promoter prediction software,two possible core promoter regions were found at the 5'-flanking regionThe gpd,pgk,qut,and qa boxes featured in the promoter of A. nidulans and A.niger gpd gene were not found in in the gpd promoter of G.lucidum.The hygromycin B phosphotransferase(hph)gene with cauliflower mosaic virus (CaMV)35Sterminator was amplified from pCAMBIA1300,then the amplified fragment was cloned into a pCAMBIA1300 mothboard and a 1.2kb promoter region of G.lucidum gpd gene was introduced to drive hph gene expression.The resulting plasmid,GLP-1300, was used for the transformation experiment.The transformation efficiency of this PEG-mediated method reached to 20 transformants/μg plasmid+10⁷protoplast.Transformants could still show HmB resistance after cultivating over 5 subcultures on media without HmB.An analysis of Ganoderma lucidum transformants using PCR revealed that foreign gene had integrated into Ganoderma lucidum's genome.