In order to confirm the presence of putative glucoamylase gene in Tricholoma matsutake genome, the genomic DNA was prepared from T. matsutake NBRC30773 strain and was used as template to clone the glucoamylases gene (TmGlu1). We obtained the nucleotide sequence of TmGlu1 and its franking region. The coding region (from ATG to stop codon) is 2,186 bp. The locations of exons and introns were determined from the nucleotide sequences of 3’- and 5’-RACE PCR and RT- PCR products. On the other hand, to investigate the relationship between composition of medium and glucoamylase expression, we checked the expression level of glucoamylase gene by realtime reverse transcription PCR and measurement of glucoamylase enzyme activity. It was found that enzyme activity of glucoamylase was very low in different medium. Expression of glucoamylases gene appeared to not be affected by different carbon source.

In the past studies of Lyophlium shimeji, it was reported that the quantity of sufficient starch used as a carbon source was able to supply the factor that allows successful fruit-body formation without raising osmotic pressure in the medium. Glucoamylase are exo Glucosyl hydrolase, which catalyze the release glucose from the nonreducing ends of amylose, amylopectin, and other polysaccharides. Glucoamylase genes are found in many prokaryotic and eukaryotic microbes that use starch as a carbon source. It was believed to be important in the utilization of starch by the basidiomycetous fungus. Glucoamylase activity in the medium increased markedly during fruit-body formation. So study of the characteristic of glucoamylase in Pholiota nameko will provide the basis for P .nameko fruit body formation. In this research, in order to confirm the presence of glucoamylase gene in P. nameko genome, the genomic DNA was prepared from P. nameko NGW19-6 strain and was used as template to amplify the glucoamylases gene (PnGlu1). To prepare genomic DNA from the P. nameko NGW19-6 strain, the mycelium was grown on 10 ml of PD liquid medium (potato 200 g/l ,Glucose 20 g/l) prepared with tap water in a 100 ml Erlenmeyer flask and at 25°C for 7 days. Genomic DNA fragment encoding the glucoamylase protein (PnGlu1) were amplified by PCR with degenerate primer F15-GP2-AF/F15-GP2-BR. The primer pair was designed based on the amino acid sequences GLGEPKF and FDLWEEI, respectively, which are conserved in the glucoamylase protein of Laccaria bicolor. This produce fragments of approximately 400 bp. Next, to amplify the whole genomic clone of PnGlu1, oligonucleotide primer PnGP2F/ PnGP2R were designed based on the nucleotide sequence of DNA fragments amplified by cassette PCR method. The produced fragment has significant homology with glucoamylase of L. bicolor. To investigate the relationship between different composition of medium and glucoamylase expression, we checked the expression level of glucoamylase gene by realtime RT-PCR and measurement of glucoamylase enzyme activity.