<Review Article>Development of Transformation and Recombinant Gene Expression Systems in Pleurotus ostreatus

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A calmodulin inhibitor, W-7 influences the effect of cyclic adenosine 3', 5'-monophosphate signaling on ligninolytic enzyme gene expression in Phanerochaete chrysosporium

The capacity of white-rot fungi to degrade wood lignin may be highly applicable to the development of novel bioreactor systems, but the mechanisms underlying this function are not yet fully understood. Lignin peroxidase (LiP) and manganese peroxidase (MnP), which are thought to be very important for the ligninolytic property, demonstrated increased activity in Phanerochaete chrysosporium RP-78 (FGSC #9002, ATCC MYA-4764™) cultures following exposure to 5 mM cyclic adenosine 3', 5'-monophosphate (cAMP) and 500 μM 3'-isobutyl-1-methylxanthine (IBMX), a phosphodiesterase inhibitor. Real-time reverse transcription polymerase chain reaction (RT-PCR) analysis revealed that transcription of most LiP and MnP isozyme genes was statistically significantly upregulated in the presence of the cAMP and IBMX compared to the untreated condition. However, 100 μM calmodulin (CaM) inhibitor N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), which had insignificant effects on fungal growth and intracellular cAMP concentration, not only offset the increased activity and transcription induced by the drugs, but also decreased them to below basal levels. Like the isozyme genes, transcription of the CaM gene (cam) was also upregulated by cAMP and IBMX. These results suggest that cAMP signaling functions to increase the transcription of LiP and MnP through the induction of cam transcription

Marker-free genome editing in the edible mushroom, Pleurotus ostreatus, using transient expression of genes required for CRISPR/Cas9 and for selection

In a previous study, we reported a transient transformation system using repeated screening for hygromycin B (Hyg) resistance in the basidiomycete Ceriporiopsis subvermispora. In the present study, by combining this technique with CRISPR/Cas9, we demonstrated successful marker-free genome editing in Pleurotus ostreatus, which is one of the most economically important cultivated mushrooms as well as a model white-rot fungus. At first, transformant selection mediated by the transient expression of marker genes was demonstrated using a plasmid harboring the Hyg resistance gene (hph) in P. ostreatus. Then, genome editing of fcy1, which confers 5-fluorocytosine (5-FC) resistance to the host cell, was performed by the transient expression of Cas9, gRNA, and hph and strains with 5-FC resistance and Hyg sensitivity were isolated. Additionally, genome editing of fcy1 in these strains was confirmed by Sanger sequencing. To our knowledge, this is the first report of marker-free genome editing through the transient expression of Cas9, gRNA, and hph in agaricomycetes, which opens the door for repeated genome editing in these fungi

Evidence of means–end behavior in Asian elephants (Elephas maximus)

The present study explores to what extent Asian elephants show “means–end” behavior. We used captive Asian elephants (N = 2) to conduct four variations of the Piagetian “support” problem, which involves a goal object that is out of reach, but rests on a support within reach. In the first condition, elephants were simultaneously presented with two identical trays serving as the “support”, with the bait on one tray and the other tray left empty. In the next two conditions, the bait was placed on one tray, while additional bait was placed beside the other tray. In the last condition, both trays contained bait, but one of the trays had a small gap which prevented the elephants from reaching the reward. Subjects were required to choose and pull either tray with their trunk and to obtain the bait (i.e. goal). Results showed that one elephant performed all of the support problems significantly above chance after several sessions, suggesting that the elephant was capable of understanding that pulling the tray was the “means” for achieving the “end” of obtaining the bait. This study showed that elephants show means–end behavior when subjected to a Piagetian “support” task, and indicates that such goal-directed behavior occurs in species other than primates

ヒラタケにおける形質転換系と組換え遺伝子発現系の開発

京都大学0048新制・課程博士博士(農学)甲第8567号農博第1150号新制||農||809(附属図書館)学位論文||H12||N3446(農学部図書室)UT51-2000-M31京都大学大学院農学研究科林産工学専攻(主査)教授 桒原 正章, 教授 酒井 富久美, 教授 島田 幹夫学位規則第4条第1項該当Doctor of Agricultural ScienceKyoto UniversityDA

Summation by Asian Elephants (Elephas maximus)

Recent empirical evidence for complex cognition in elephants suggests that greater attention to comparative studies between non-human primates and other animals is warranted. We have previously shown that elephants possess the ability to judge the difference between two discrete quantities, and unlike other animals, their choice does not appear to be affected by distance or overall quantity. In this study, we investigated Asian elephants’ ability to perform summation, as exemplified by the ability to combine four quantities into two sums and subsequently compare them. We presented two discrete sums (3–7) to the elephants by baiting two buckets; they were loaded sequentially with two discrete quantities (1–5 pieces) of food per bucket. All three elephants selected the larger grand sum significantly more often than the smaller grand sum. Moreover, their performance was not affected by either distance to the bait or the overall quantity evaluated. Studies report that the performance of other animal species on similar tasks declines as distance to the bait decreases and as the overall quantities evaluated increase. These results suggest that the numerical cognition of Asian elephants may be different from that of other animals, but further study is required to elucidate the differences precisely