• Molecules and Cells
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• v.27 no.4
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• pp.467-473
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• 2009

Our previous study suggested that OsBWMK1, a gene which encodes a member of the rice MAP kinase family, generates transcript variants which show distinct expression patterns in response to environmental stresses. The transcript variants are generated by alternative splicing and by use of alternative promoters. To test whether the two alternative promoters, pOsBWMK1L (promoter for the OsBWMK1L splice variant) and pOsBWMK1S (promoter for the OsBWMK1S splice variant), are biologically functional, we analyzed transgenic plants expressing GUS fusion constructs for each promoter. Both pOsBWMK1L and pOsBWMK1S are biologically active, although the activity of pOsBWMK1S is lower than that of pOsBWMK1L. Histochemical analysis revealed that pOsBWMK1L is constitutively active in most tissues at various developmental stages in rice and Arabidopsis, whereas pOsBWMK1S activity is spatially and temporally restricted. Furthermore, the expression of pOsBWMK1S::GUS was upregulated in response to hydrogen peroxide, a plant defense signaling molecule, in both plant species. These results suggest that the differential expression of OsBWMK1 splice variants is the result of alternative promoter usage and, moreover, that the mechanisms controlling OsBWMK1 gene expression are conserved in both monocot and dicot plants.

• Journal of Plant Biotechnology
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• v.37 no.3
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• pp.250-261
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• 2010

The expression of vaccine antigens in transgenic plants has the potential to provide a convenient, stable, safe approach for oral vaccination alternative to traditional parenteral vaccines. Over the past two decades, many different vaccine antigens expressed via the plant nuclear genome have elicited appropriate immunoglobulin responses and have conferred protection upon oral delivery. Up to date, efforts to produce antigen proteins in plants have focused on potato, tobacco, tomato, banana, and seed (maize, rice, soybean, etc). The choice of promoters affects transgene transcription, resulting in changes not only in concentration, but also in the stage tissue and cell specificity of its expression. Inclusion of mucosal adjuvants during immunization with the vaccine antigen has been an important step towards the success of plant-derived vaccines. In animal and Phase I clinical trials several plant-derived vaccine antigens have been found to be safe and induce sufficiently high immune response. Future areas of research should further characterize the induction of the mucosal immune response and appropriate dosage for delivery system of animal and human vaccines. This article reviews the current status of development in the area of the use of plant for the development of oral vaccines.

• Proceedings of the Botanical Society of Korea Conference
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• 1996.07a
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• pp.10-18
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• 1996

Recently, we have reported a rice MADS-box gene, OsMADS1, as a molecular factor triggering flower formation; this has been well studied in a heterologous system (Chung et al., 1994). In order to study whether the OsMADS1 homolog exists in other plant species, the OsMADS1 cDNA was used as a probe to screen a tobacco cDNA library, and a potential homolog, NtMADS3, was isolated. Sequence analysis revealed that the gene shares 56.1% identity in whole amino acids with OsMADS1. Like OsMADS1, the NtMADS3 gene starts to express at a very early stage of flower development, and the expression continues up to flower maturation. In the tobacco flower, the gene is expressed in whorl 2,3 and 4, corresponding to the petal, stamen, and carpel, respectively. Upon ectopic expression in the homologous system, NtMADS3 caused a trasition from inflorescence shoot meristem into floral meristem, reducing flowering time dramatically. These phenotypes strongly suggest the NtMADS3 gene is the OsMADS1 homolog of tobacco. Hybrids between the OsMADS1 and the NtMADS3 plants were also generated. The hybrids flowered even earlier than these two transgenic plants. The detailed studies are discussed here.

• Biotechnology and Bioprocess Engineering:BBE
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• v.10 no.6
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• pp.482-493
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• 2005

Biomass is originally photosynthesized from inorgainic compounds such as $CO_2$, minerals, water and solar energy. Recent studies have shown that anaerobic bacteria have the ability to convert recalcitrant biomass such as cellullosic or chitinoic materials to useful compounds. The biomass containing agricultural waste, unutilized wood and other garbage is expected to utilize as feed, food and fuel by microbial degradation and other metabolic functions. In this study we isolated several anaerobic, cellulolytic and chitinolytic bacteria from rumen fluid, compost and soil to study their related enzymes and genes. The anaerobic and cellulolytic bacteria, Clostridium thermocellum, Clostridium stercorarium, and Clostridium josui, were isolated from compost and the chitinolytic Clostridium paraputrificum from beach soil and Ruminococcus albus was isolated from cow rumen. After isolation, novel cellulase and xylanase genes from these anaerobes were cloned and expressed in Escherichia coli. The properties of the cloned enzymes showed that some of them were the components of the enzyme (cellulase) complex, i.e., cellulosome, which is known to form complexes by binding cohesin domains on the cellulase integrating protein (Cip: or core protein) and dockerin domains on the enzymes. Several dockerin and cohesin polypeptides were independently produced by E. coli and their binding properties were specified with BIAcore by measuring surface plasmon resonance. Three pairs of cohesin-dockerin with differing binding specificities were selected. Two of their genes encoding their respective cohesin polypeptides were combined to one gene and expressed in E. coli as a chimeric core protein, on which two dockerin-dehydrogenase chimeras, the dockerin-formaldehyde dehydrogenase and the dockerin-NADH dehydrogenase are planning to bind for catalyzing $CO_2$ reduction to formic acid by feeding NADH. This reaction may represent a novel strategy for the reduction of the green house gases. Enzymes from the anaerobes were also expressed in tobacco and rice plants. The activity of a xylanase from C. stercorarium was detected in leaves, stems, and rice grain under the control of CaMV35S promoter. The digestibility of transgenic rice leaves in goat rumen was slightly accelerated. C. paraputrificum was found to solubilize shrimp shells and chitin to generate hydrogen gas. Hydrogen productivity (1.7 mol $H_2/mol$ glucos) of the organism was improved up to 1.8 times by additional expression of the own hydrogenase gene in C. paraputrficum using a modified vector of Clostridiu, perfringens. The hydrygen producing microflora from soil, garbage and dried pelletted garbage, known as refuse derived fuel(RDF), were also found to be effective in converting biomass waste to hydrogen gas.

• Proceedings of the Korean Society of Crop Science Conference
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• 2017.06a
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• pp.167-167
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• 2017

Our previous study for developing seeds of Iksan 526 (I.526), an inbred line of resveratrol-producing transgenic rice line, showed that, in 20 days after heading (DAH) seeds, resveratrol was almost saturated and accumulation of piceid was highest though the expression of Arachis hypogaea resveratrol synthase 3 (AhRS3, GenBank DQ124938) was highest in 31 DAH seeds. In this study, it was investigated how the overexpression of AhRS3 affects phenylpropanoid pathway genes. p-Coumaroyl-CoA is derived from phenylpropanoid pathway and used as a substrate of AhRS3 reaction for resveratrol production. In 6, 13, 20, 31 and 41 (45 for Dongjin) DAH seeds of I526 and Dongjin, a wild type of I.526, respectively, the expression pattern of phenylpropanoid pathway genes, including phenylalanine ammonia-lyase (PAL: LOC_Os02g41630.2, LOC_Os04g43760.1), cinnamate 4-hydroxylase (C4H: LOC_Os05g25640.1), 4-coumarate-CoA ligase (4CL: LOC_Os02g08100.1), cinnamoyl-CoA reductase (CCR: LOC_ Os09g25150.1, LOC_Os08g34280.1), hydroxycinnamoyl-CoA shikimate/quinate hydroxycinnamoyl transferase (HCT: LOC_Os04g42250.2, LOC_Os02g39850.1) and cinnamyl alcohol dehydrogenase (CAD: LOC_Os02g09490.1), was examined using real time (RT)-PCR. Compared to developing seeds of Dongjin, RT-PCR results showed that the expression pattern of phenylpropanoid pathway genes was modified in developing seeds of I.526. In most genes, except for CAD, of I.526 developing seeds, the gene expression was highest in 20 DAH corresponding to biosynthesis of resveratrol and piceid, i.e. the expression of phenylpropanoid pathway genes was gradually increased by 20 DAH and decreased as seeds develop. Especially, in Dongjin, the highest expression of PALs and 4CL was in 6 DAH and their expression was gradually decreased as seeds develop. These genes expression data also exhibited that, in developing seeds of I.526, phenylpropanoid pathway genes were slightly or significantly (in some genes) upregulated compared to Dongjin. Therefore, the overexpression of AhRS3 changed the expression pattern of phenylpropanoid pathway genes in I.526 developing seeds and this modification for gene expression is closely related to biosynthesis of resveratrol and piceid.

• Proceedings of the Korean Society of Crop Science Conference
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• 2017.06a
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• pp.105-105
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• 2017

Our previous study for developing seeds of Iksan 526 (I.526), an inbred line of resveratrol-producing transgenic rice line, showed that, in 20 days after heading (DAH) seeds, resveratrol was almost saturated and accumulation of piceid was highest though the expression of Arachis hypogaea resveratrol synthase 3 (AhRS3, GenBank DQ124938) was highest in 31 DAH seeds. In this study, it was investigated how the overexpression of AhRS3 affects phenylpropanoid pathway genes. p-Coumaroyl-CoA is derived from phenylpropanoid pathway and used as a substrate of AhRS3 reaction for resveratrol production. In 6, 13, 20, 31 and 41 (45 for Dongjin) DAH seeds of I526 and Dongjin, a wild type of I.526, respectively, the expression pattern of phenylpropanoid pathway genes, including phenylalanine ammonia-lyase (PAL: LOC_Os02g41630.2, LOC_Os04g43760.1), cinnamate 4-hydroxylase (C4H: LOC_Os05g25640.1), 4-coumarate-CoA ligase (4CL: LOC_Os02g08100.1), cinnamoyl-CoA reductase (CCR: LOC_Os09g25150.1, LOC_Os08g34280.1), hydroxycinnamoyl-CoA shikimate/quinate hydroxycinnamoyl transferase (HCT: LOC_Os04g42250.2, LOC_Os02g39850.1) and cinnamyl alcohol dehydrogenase (CAD: LOC_Os02g09490.1), was examined using real time (RT)-PCR. Compared to developing seeds of Dongjin, RT-PCR results showed that the expression pattern of phenylpropanoid pathway genes was modified in developing seeds of I.526. In most genes, except for CAD, of I.526 developing seeds, the gene expression was highest in 20 DAH corresponding to biosynthesis of resveratrol and piceid, i.e. the expression of phenylpropanoid pathway genes was gradually increased by 20 DAH and decreased as seeds develop. Especially, in Dongjin, the highest expression of PALs and 4CL was in 6 DAH and their expression was gradually decreased as seeds develop. These genes expression data also exhibited that, in developing seeds of I.526, phenylpropanoid pathway genes were slightly or significantly (in some genes) upregulated compared to Dongjin. Therefore, the overexpression of AhRS3 changed the expression pattern of phenylpropanoid pathway genes in I.526 developing seeds and this modification for gene expression is closely related to biosynthesis of resveratrol and piceid.

• Journal of Plant Biotechnology
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• v.40 no.1
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• pp.37-42
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• 2013

Rice is one of the most important cereal crops as a model plant for functional genomics of monocotyledons and usually transformed using Agrobacterium tumefaciens. However, the transformation's process using previous method is still time consuming and uneconomical, low efficiency. In this study, we established a new method by modifying the general Agrobacterium protocol especially in the infection and co-cultivation, Agrobacterium elimination, infected calli's selection steps using liquid media. We directly inoculated Agrobacterium containing a ZjLsL gene under the control of constitutive promoter into the 1- to 3-week-old rice calli derived from mature seeds. After 3 days of co-cultivation, the infected calli were transferred onto liquid media of Agrobacterium elimination and calli's selection for 3 days. The calli were transferred to calli's growth solid media for 14 days and then the calli transferred to shoot induction and root induction media. Putative transformants were initially selected on the medium containing phosphinothricin, and the PAT protein verified by PAT strip test. This method in this study would lead to reduction of substantial labor and time to generate transgenic plants.

• Journal of Practical Agriculture & Fisheries Research
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• v.25 no.4
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• pp.138-147
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• 2024

Agrobacterium-mediated transformation (AMT) is a method that allows for the stable integration of DNA fragments into the plant genome. Transgenic plants generated through AMT typically exhibit a lower copy number of the transgene compared to those induced by particle bombardment. Furthermore, AMT offers a straightforward and efficient approach for generating transgenic plants. While the transformation efficiency of wheat is comparatively lower than that of other monocot plants such as Rice (Oryza sativa L.) and Maize (Zea mays L.), the cultivars 'Bobwhites' and 'Fielder' are commonly employed for wheat transformation. To date, there have been no reported instances of successful development of transgenic plants using Korean wheat varieties through AMT. This study aims to assess the transformation efficiency of 43 Korean wheat cultivars using the GUS assay, with the goal of identifying suitable Korean wheat cultivars for AMT. The pCAMBIA1301 vector, carrying the β-glucuronidase (GUS) gene, was incorporated into Agrobacterium strain EH105. Following the inoculation of Agrobacterium into immature embryos, GUS assays were conducted 'Saeol', 'Jopum', and 'Jonong' showed 100% (the number of embryos showing GUS spots/the number of embryos used for AMT) among 43 cultivars. In addition, cultivars with more than 70% were 'Saekeumgang', 'Jojung', 'Tapdong', 'Anbaek', 'Dabun', 'Sugang', 'Keumgang', 'Jeokjung', 'Seodun', 'Joeun', 'Dajung', and 'Baekjung'. It seems that the 15 cultivars above showed the possibility of using AMT. On the other hand, 'Yeonbaek', 'Goso', 'Baekgang', and 'Johan' showed less than 20% and GUS spots were not observed in 'Gru', 'Gobun', 'Milseong', and 'Shinmichal-1'. This study explores transient GUS expression in Korean wheat cultivars seven days after AMT. The observed initial high efficiency of transient transformation suggests the potential for subsequent stable transformation efficiency. Korean wheat cultivars demonstrating elevated transient transformation efficiency could serve as promising candidates for the development of stable transgenic wheat.

• 한국생물공학회:학술대회논문집
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• 2001.11a
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• pp.193-196
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• 2001

IL-18. formerly known as IGIF(interferon -gamma inducing factor), is structurally IL-l related but functionally IL-12 related pro-inflammatory cytokine. The human IL -18(hIL-lS), like IL-$1{\beta}$, is synthesized as a biologically inactive precursor of 24kDa lacking a signal peptide, and then cleaved into an active mature form by cystein protease IL-$1{\beta}$ converting enzyme (ICE: caspase- 1), We tested if the mature hIL -18 can be expressed and secreted into culture medium by transforming the forming gene construct consisting of a mature hIL-18 gene fused to signal peptide of rice amylase lA. Secondly, we were tested if the pro- IL-18 could be processed into a biologically active form by caspase-l like protease in plant. Cell suspension culture was established from the leaf-derived calli of transgenic tobacco plant. Southern and Northern blot analysis indicated the expression of both pro-hIL-18 and mature hIL-18 plant cells. Western blot analysis introduced the protein products of pro- hIL -18 and mhIL -18 were observed in transigenic cell lines. In addition, the molecular size of recombinant pro-hILl-18 and mhIL-18 were estimated to be 24kDa and 18kDa, respectively. ELISA revealed that the amount of pro- hIL -18 was 1.3ug per gram of fresh weight calli. Moreover, the presence of mhIL-18 was detected in the culture medium and it appeared to be 25ug/L.

• Journal of Microbiology and Biotechnology
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• v.22 no.11
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• pp.1557-1567
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• 2012

Glutathione reductase (GR, E.C. 1.6.4.2) is an important enzyme that reduces glutathione disulfide (GSSG) to a sulfydryl form (GSH) in the presence of an NADPH-dependent system. This is a critical antioxidant mechanism. Owing to the significance of GR, this enzyme has been examined in a number of animals, plants, and microbes. We performed a study to evaluate the molecular properties of GR (OsGR) from rice (Oryza sativa). To determine whether heterologous expression of OsGR can reduce the deleterious effects of unfavorable abiotic conditions, we constructed a transgenic Saccharomyces cerevisiae strain expressing the GR gene cloned into the yeast expression vector p426GPD. OsGR expression was confirmed by a semiquantitative reverse transcriptase polymerase chain reaction (semiquantitative RT-PCR) assay, Western-blotting, and a test for enzyme activity. OsGR expression increased the ability of the yeast cells to adapt and recover from $H_2O_2$-induced oxidative stress and various stimuli including heat shock and exposure to menadione, heavy metals (iron, zinc, copper, and cadmium), sodium dodecyl sulfate (SDS), ethanol, and sulfuric acid. However, augmented OsGR expression did not affect the yeast fermentation capacity owing to reduction of OsGR by multiple factors produced during the fermentation process. These results suggest that ectopic OsGR expression conferred acquired tolerance by improving cellular homeostasis and resistance against different stresses in the genetically modified yeast strain, but did not affect fermentation ability.