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Gene cloning of a Pleurotus ostreatus lipoxygenase involved in the biosynthesis of mushroom aroma
Yuji Tasaki,Shungo Toyama,Toshio Joh 한국버섯학회 2010 한국버섯학회지 Vol.8 No.4
Lipoxygenase (LOX) is considered to be a key enzyme in the biosynthetic pathways of the most important mushroom aroma, 1-octen-3-ol. In previous work, we purified and characterized a LOX from Pleurotus ostreatus (probably H1 strain) fruit bodies [1] and also determined its partial amino acid sequence. In this study, to clarify the biosynthetic mechanism of 1-octen-3-ol, we isolated cDNA and genomic DNA corresponding to a LOX (Polox1) gene of P. ostreatus H1, and analyzed the expression of the gene in the fruit bodies. A commercial P. ostreatus H1 strain (Onuki kinjin, Utsunomiya, Japan) was used in this study. To isolate the Polox1 cDNA, RT-PCR was done using degenerate primers designed from the partial amino acid sequence. This approach generated a single DNA band of approximately 1.1 kbp, which was cloned and sequenced. The deduced amino acid sequence showed high similarity to LOXs of some ascomycetes fungi. To obtain the full-length cDNA of Polox1, clones corresponding to the Polox1 gene were isolated by plaque hybridization from a cDNA library of the P. ostreatus H1 fruit body. DNA sequences of all clones were determined. The 5’ end of the Polox1 cDNA was amplified by the 5’ RACE method and cloned. The full-length cDNA of Polox1 is 2,031 bp long and contains 640 amino acid residues. The deduced amino acid sequence contains LOX iron-binding catalytic domain signature sequences. Next, to determine the genomic DNA sequence of the Polox1 gene, inverse PCR and PCR was done with P. ostreatus H1 genomic DNA. After inverse PCR and PCR, 3.3 and 1.9 kbp DNA fragments, respectively, were amplified and sequenced. Sequence comparison between cDNA and genomic DNA showed that Polox1 gene contained one intron. To investigate expression of the Polox1 gene, northern blot analysis and measurement of LOX activity were performed. P. ostreatus fruit bodies were produced in a sawdust medium containing beech sawdust and rice bran and separated into pileus and stipe. Two transcripts were detected by northern blot analysis in both pileus and stipe. The band intensities were relatively higher in the stipe than in the pileus. The level of LOX activity in the stipe was 3.8 times higher than that in the pileus. By Southern blot analysis, several major bands were detected after the digestion of 4 restriction enzymes. These blot analyses suggest that the Polox1 gene is probably a member of a small gene family. [1] T. Kuribayashi et al., J. Agric. Food Chem., 50, 1247 (2002).
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Yasuhiro Sadanaga,Tadashi Kobashi,Akie Yuba,Shungo Kato,Yoshizumi Kajii,Akinori Takami,Hiroshi Bandow 한국대기환경학회 2015 Asian Journal of Atmospheric Environment (AJAE) Vol.9 No.4
We conducted intensive observations of ozone, CO, NOx (=NO and NO2), NOy (total odd nitrogen species including particulate nitrate) and total nitrate (the sum of gaseous HNO3 and particulate nitrate) at Cape Hedo, Okinawa, Japan, from 19 March to 3 April, 2009, to investigate ozone production during long-range transport from the Asian continent. Ozone production efficiency (OPE) was used to evaluate photochemical ozone production. OPE is defined as the number of molecules of ozone produced photochemically during the lifetime of a NOx molecule. OPE is calculated by the ratio of the concentration increase of ozone to that of NOz (= NOy -NOx). Average OPE during observation was estimated to be 12.6±0.5, but concentrations of ozone increased nonlinearly with those of NOz. This non-linearity suggests that OPE depends on air mass origin and NOz concentrations. There were very different values of OPE for the same air mass origin, so that only the air mass origin alone does not control OPE. OPE was low when NOz concentration was high. We examined the correlation between NOz and CO/NOy ratios, which we used instead of the ratio of nonmethane hydrocarbons (NMHCs) to NOx. The CO/NOy ratios decreased with increasing NOz concentrations. These results indicate that competition reactions of OH with NMHCs and NO2 are the rate determining steps of photochemical ozone production during longrange transport from the Asian continent to Cape Hedo, for high concentrations of nitrogen oxides.
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