• Molecules and Cells
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• v.28 no.5
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• pp.431-439
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• 2009

Rice Oryza sativa accelerated cell death and resistance 1 (OsACDR1) encodes a putative Raf-like mitogen-activated protein kinase kinase kinase (MAPKKK). We had previously reported upregulation of the OsACDR1 transcript by a range of environmental stimuli involved in eliciting defense-related pathways. Here we apply biochemical, gain and loss-of-function approaches to characterize OsACDR1 function in rice. The OsACDR1 protein showed autophosphorylation and possessed kinase activity. Rice plants overexpressing OsACDR1 exhibited spontaneous hypersensitive response (HR)-like lesions on leaves, upregulation of defense-related marker genes and accumulation of phenolic compounds and secondary metabolites (phytoalexins). These transgenic plants also acquired enhanced resistance to a fungal pathogen (Magnaporthe grisea) and showed inhibition of appressorial penetration on the leaf surface. In contrast, loss-of-function and RNA silenced OsACDR1 rice mutant plants showed downregulation of defense-related marker genes expressions and susceptibility to M. grisea. Furthermore, transient expression of an OsACDR1:GFP fusion protein in rice protoplast and onion epidermal cells revealed its localization to the nucleus. These results indicate that OsACDR1 plays an important role in the positive regulation of disease resistance in rice.

• Journal of agriculture & life science
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• v.46 no.1
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• pp.163-176
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• 2012

TTo increase productivity of a strong extracellular alkaline protease (BCAP), stable strains of Bacillus clausii I-52 carrying another copy of BCAP gene in the chromosome were developed. Integrative vector, pHPS9-fuBCAP carrying BCAP promoter, ribosome binding site, signal sequence and active protease gene was constructed and transferred into B. clausii I-52, and integration of the constructed plasmid into chromosome was identified by PCR. An investigation was carried out on BCAP production by B. clausii I-52 and transformant C5 showing the highest relative activity of alkaline protease using submerged fermentation. Maximum enzyme activity was produced when cells were grown under the submerged fermentation conditions at $37^{\circ}C$ for 48 h with an aeration rate of 1 vvm and agitation rate of 650 rpm in a optimized medium (soybean meal 2%, wheat flour 1%, sodium citrate 0.5%, $K_2HPO_4$ 0.4%, $Na_2HPO_4$ 0.1%, NaCl 0.4%, $MgSO_47H_2O$ 0.01%, $FeSO_47H_2O$ 0.05%, liquid maltose 2.5%, $Na_2CO_3$ 0.6%). A protease yield of approximately 134,670U/ml was achieved using an optimized media, which show an increase of approximately 1.6-fold compared to that of non-transformant (83,960 U/ml). When the stability of transformant C5 was examined, the integrated plasmid pHPS9-fuBCAP was detected in the transformant after cultivation for 8 days, suggesting that it maintained stably in the chromosomal DNA of transformant C5.

• Journal of Plant Biotechnology
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• v.48 no.1
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• pp.34-43
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• 2021

Targeted genome editing using the CRISPR/Cas9 system is a ground-breaking technology that is being widely used to produce plants with useful traits. However, for woody plants, only a few successful attempts have been reported. These successes have used Agrobacterium-mediated transformation, which has been reported to be very efficient at producing genetically modified trees. Nonetheless, there are unresolved problems with plasmid sequences that remain in the plant genome. In this study, we demonstrated a DNA-free genome editing technique in which purified CRISPR/Cas9 ribonucleoproteins (RNPs) are delivered directly to the protoplasts of a hybrid poplar (Populus alba × Populus glandulosa). We designed three single-guide RNAs (sgRNAs) to target the stress-associated protein 1 gene (PagSAP1) in the hybrid poplar. Deep sequencing results showed that pre-assembled RNPs had a more efficient target mutagenesis insertion and deletion (indel) frequency than did non-assembled RNPs. Moreover, the RNP of sgRNA3 had a significantly higher editing efficacy than those of sgRNA1 and sgRNA2. Our results suggest that the CRISPR/Cas9 ribonucleoprotein-mediated transfection approach is useful for the production of transgene-free genome-edited tree plants.

• Journal of Mushroom
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• v.14 no.4
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• pp.142-154
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• 2016

Worldwide production of mushrooms has been increasing by 10-20% every year. Recently, Pleurotus eryngii and P. nebrodensis have become popular mushroom species for cultivation. In particular, China exceeded 8.7 million tons in 2002, which accounted for 71.5% of total world output. A similar trend was also observed in Korea. Two kinds of mushrooms-Gumji (金芝; Ganoderma) and Seoji-are described in the ancient book 'Samguksagi' (History of the three kingdoms; B.C 57~A.D 668; written by Bu Sik Kim in 1145) during the Korea-dynasty. Many kinds of mushrooms are also described in more than 17 ancient books during the Chosun-dynasty (1392~1910) in Korea. Approximately 200 commercial strains of 38 species of mushrooms were developed and distributed to cultivators. The somatic hybrid variety of oyster mushroom, 'Wonhyeong-neutari,' was developed by protoplast fusion, and distributed to growers in 1989. Further, the production of mushrooms as food was 199,829 metric tons, valued at 850 billion Korean Won (one trillion won if mushroom factory products are included) in 2015. In Korea, the major cultivated species are P. ostreatus, P. eryngii, Flammulina velutipes, Lentinula edodes, Agaricus bisporus, and Ganoderma lucidum, which account for 90% of the total production. Since mushroom export was initiated in 1960, the export and import of mushrooms have increased in Korea. Technology was developed for liquid spawn production, and automatic cultivation systems led to the reduction of production cost, resulting in the increase in mushroom export. However, some species were imported owing to high production costs for effective cultivation methods. In academia, RDA scientists have conducted mushroom genome projects since 1997. One of the main outcomes is the whole genome sequencing of Flammulina velutipes for molecular breeding. With regard to medicinal mushrooms, we have been conducting genome research on Cordyceps and its related species for developing functional foods. There are various kinds of beneficial substances in mushrooms; mushroom products, including pharmaceuticals, tonics, healthy beverages, functional biotransformants, and processed foods have also became available on the market. In addition, compost and feed can likewise be made from mushroom substrates after harvest.

• KSBB Journal
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• v.25 no.2
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• pp.155-166
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• 2010

Studies on the production of cell-wall bound innerpolysaccharides (IPS) (soluble ${\beta}$-D-glucan) have been performed by use of suspended myelial cultures of Inonotus obliquus. This product has promising potentials as an effective antidiabetic as well as an immunostimulating agents. As a first step to enhanced production of IPS, Intensive strain improvement programs were carried out by obtaining a large amounts of protoplasts for the isolation of single cell colonies. Rapid and large screening of high-yielding producers was possible because about fivefold higher amount of protoplasts ($2.3{\times}10^6$ protoplasts/mL) could be recovered with relatively high regeneration rates of $10^{-2}{\sim}10^{-3}$ by applying a modified filtration method, as compared to the previously used trapping method. A basic protocol necessary for UV-mutation of the protoplasts was also developed, resulting in several overproducing variants with good fermentation properties. Since the amount of IPS extracted from the mycelial cell walls of I. obliquus turned out to be almost constant per g DCW, increase in cell mass was considered the most important factor for the enhancement in IPS production. Therefore, attempts were made to screen mutant cells showing rapid mycelial growth rate in the final suspended cultures. Notably, the mutant strains showing an active cellgrowth in the preceding solid growth cultures were observed to produce higher amount of IPS in the suspended fermentations as well. A striking mutant, OBLQ756-15-5 strain, obtained from the survivors of a harsh UV-treated condition (97% death rate) was found to stably produce as high cell mass as 22 g DCW/L in the final fermentations. Currently, this strain is being tested for development of a scaled-up fermentation process for mass production of IPS.

• Korean Journal of Weed Science
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• v.10 no.4
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• pp.328-337
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• 1990

The incividual and combined effects of the chloroacetanilide herbicide pretilachlor and of the safener fenclorim on the growth and selected physiological processes of rice (Oryza sativa L., var 'Lemont')were evaluated under greenhouse and laboratory conditions. Fenclorim applied at rates ranging from 50 to 300 g a.i./ha antagonized the injurious effects caused by 150 to 900 g a.i./ha of pretilachlor on 15-day old wet-sown rice grown under greenhouse conditions. When used rates of 150 g/ha or higher, fenclorim reversed completely the effects of all doses of pretilachlor on rice. When the two compounds were given simultaneously, fenclorim enhanced the uptake of $^{14}C$pretilachlor into rice leaf mesophyll protoplasts measured for 1 hr, indicating that competition for uptake at the protoplast level is not involved in the protective action of this safener. The safener-induced stimulation of pretilachlor uptake was particularly evident when fenclorim was used at concentrations of 10, 20 and $40{\mu}M$. Following 4 hr of incubation, individual treatments with pretilachlor inhibited the in vitro incorporation of radiolabeled precursors into proteins, DNA, and lipids of rice leaf protoplasts only when used at the high concentration of $100{\mu}M$M. Individual treatments with high concentrations (10 or $100{\mu}M$) of the safener fenclorim inhibited the incorporation of radiolabeled precursors into proteins and lipids of rice protoplasts, but had no DNA synthesis. The combined effects of pretilachlor and fenclorim on the incorporation of radiolabeled precursors into these macromolecules of isolated rice mesophyll protoplasts appeared to be additive or slightly synergistic rather than antagonistic. Fenclorim at $1{\mu}M$ antagonized the effects of pretilachlor on total lipids of rice leaf protoplasts. In addition, individual and combined treat-menu with pretilachlor and fenclorim influenced the incoroporation of$^{14}C$acetate into polar lipids, triglycerides and steryl esters of rice leaf protoplas causing a redistribution of carbon in these lipid fractions. However, these effects were not large enough to explain the herbicidal activity of pretilachlor or to account for the protective action of the safener fenclorim. Overall, the uesults of the present study idnicate that the safener fenclorim does not seem to protect rice against pretilachlor injury by antagonizing its effects on protein, DNA, or lipid syntheses.