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Ruirong Yi,Hiroyuki Mukaiyama,Takashi Tachikawa,Norihiro Shimomura,Tadanori Aimi 한국버섯학회 2011 한국버섯학회지 Vol.9 No.1
In the bipolar basidiomycete Pholiota nameko, a pair of homeodomain protein genes located at the A mating-type locus regulates mating compatibility. In the present study, we used a DNA-mediated transformation system in P. nameko to investigate the homeodomain proteins that control the clamp formation. When a single homeodomain protein gene (A3- hox1 or A3-hox2) from the A3 monokaryon strain was introduced into the A4 monokaryon strain, the transformants produced many pseudo-clamps but very few clamps. When two homeodomain protein genes (A3-hox1 and A3-hox2) were transformed either separately or together into the A4 monokaryon, the ratio of clamps to the clamp-like cells in the transformants was significantly increased to approximately 50%. We, therefore, concluded that the gene dosage of homeodomain protein genes is important for clamp formation. When the sip promoter was connected to the coding region of A3-hox1 and A3-hox2 and the fused fragments were introduced into NGW19-6 (A4), the transformants achieved more than 85% clamp formation and exhibited two nuclei per cell, similar to the dikaryon (NGW12 -163 × NGW19-6). The results of real-time RT-PCR confirmed that sip promoter activity is greater than that of the native promoter of homeodomain protein genes in P. nameko. So, we concluded that nearly 100% clamp formation requires high expression levels of homeodomain protein genes and that altered expression of the A mating-type genes alone is sufficient to drive true clamp formation.
Jianing Wan,Ruirong Yi,Yan Li,Yukiko Kinjo,Aki Sadashima,Takao Terashita,Katsuji Yamanaka,Tadanori Aimi 한국버섯학회 2011 한국버섯학회지 Vol.9 No.2
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.
Yan LI,Ruirong YI,Jia ning WAN,Yukiko KINJO,Norihiro Shimomura,Tadanori AIMI 한국버섯학회 2010 한국버섯학회지 Vol.8 No.4
Basidiomycetes can degrade lignocellulosic biomass, and some basidiomycetes produce alcohol dehydrogenase, so it is feasible to produce alcohol from basidiomycetes. Agaricus blazei, Flammulina velutipes and Tricholoma matsutake have been used for mushroom fermentation to produce alcohol. To investigate whether Pholilta nameko can be used for mushroom fermentation, and find out the relationship between mannitol-1-phosphate dehydrogenase and alcohol dehydrogenase, we cloned mannitol-1-phosphate dehydrogenase gene from P. nameko, which is a zinc-containing long- chain alcohol dehydrogenase. Mpd, the gene encoding mannitol-1-phosphate dehydrogenase (MPD), has been sequenced and characterized from basiodiomycete P. nameko. The length of the coding region is 1360bp. The gene encodes a putative protein of 359 amino acids; predicted protein molecular weight is 38.6 kDa and an isoelectric point is PI = 7.34. The locations of exons and introns in the gene were deduced on the basis of interruptions in the amino acid sequence that were homologous to those in the MPD of Laccaria bicolor, the coding region was split into 6 exons and 5 introns. The protein deduced from the gene MPD showed more than 46% sequence identity to 20 fungal MPDs or alcohol dehydrogenases documented in the Gene bank protein database, based on BLASTP analysis, and was phylogenetically close to the MPDs of L. bicolor and Coprinopsis cinerea. This protein shared the same conserved domain with the alcohol dehydrogenase.
Analysis of glcoamylase gene from Pholiota nameko
Yukiko Kinjo,Yan Li,Ruirong Yi,Jia ning Wan,Takeshi Yamaguchi,Norihiro Shimomura,Tadanori Aimi 한국버섯학회 2010 한국버섯학회지 Vol.8 No.4
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.