• Genomics & Informatics
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• v.6 no.2
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• pp.64-67
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• 2008

The Korean Rice Ds-tagging lines Database (KRDD) is designed to provide information about Ac/Ds insertion lines and activation tagging lines using japonica rice. This database has provided information on 18,158 Ds lines, which includes the ID, description, photo image, sequence information, and gene characteristics. The KRDD is visualized using a web-based graphical view, and anonymous users can query and browse the data using the search function. It has four major menus of web pages: (i) a Blast Search menu of a mutant line; Blast from rice Ds-tagging mutant lines; (ii) a primer design tool to identify genotypes of Ds insertion lines; (iii) a Phenotype menu for Ds lines, searching by identification name and phenotype characteristics; and (iv) a Management menu for Ds lines.

• Journal of Plant Biotechnology
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• v.37 no.4
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• pp.483-493
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• 2010

Knock-out of a gene by insertional mutagenesis is a direct way to address its function through the mutant phenotype. Among ca. 15,000 gene-trapped Ds insertion lines of rice, we identified one line from selected sensitive lines in highly salt stress. We conducted gene tagging by TAIL-PCR, and DNA gel blot analysis from salt sensitive mutant. A gene encoding an oligopeptide transporter (OPT family) homologue was disrupted by the insertion of a Ds transposon into the OsOPT10 gene that was located shot arm of chromosome 8. The OsOPT10 gene (NP_001062118.) has 6 exons and encodes a protein (752 aa) containing the OPT family domain. RT-PCR analysis showed that the expression of OsOPT10 gene was rapidly and strongly induced by stresses such as high-salinity (250 mM), osmotic, drought, $100\;{\mu}M$ ABA. The subcellular localization assay indicated that OsOPT10 was localized specifically in the plasma membrane. Overexpression of OsOPT10 in Arabidopsis thaliana and rice conferred tolerance of transgenic plants to salt stress. Further we found expression levels of some stress related genes were inhibited in OsOPT10 transgenic plants. These results suggested that OsOPT10 might play crucial but differential roles in plant responses to various abiotic stresses.

• Journal of Plant Biotechnology
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• v.38 no.1
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• pp.94-104
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• 2011

Salt stress is a major environmental factor influencing plant growth and development. To identify salt tolerance determinants, we systematically screened salt sensitive rice mutants by use of the Activator/Dissociation (Ac/Ds) transposon tagging system. In this study, we focused on the salt sensitive mutant line, designated SSM-1. A gene encoding a NAC transcription factor homologue was disrupted by the insertion of a Ds transposon into SSM-1 line. The OsNAC075 gene (EU541472) has 7 exons and encodes a protein (486-aa) containing the NAC domain in its N-terminal region. Sequence comparison showed that the OsNAC075 protein had a strikingly conserved region at the N-terminus, which is considered as the characteristic of the NAC protein family. OsNAC075 protein was orthologous to Arabidopsis thaliana ANAC075. Phylogenetic analysis confirmed OsNAC075 belonged to the OsNAC3 subfamily, which plays an important role in response to stress stimuli. RT-PCR analysis showed that the expression of OsNAC075 gene was rapidly and strongly induced by stresses such as NaCl, ABA and low temperature ($4^{\circ}C$). Our data suggest that OsNAC075 holds promising utility in improving salt tolerance in rice.

• Journal of Crop Science and Biotechnology
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• v.11 no.4
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• pp.237-242
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• 2008

A liguleless mutant, which showed complete loss of lamina joint region at the junction between leaf blade and leaf sheath, was isolated from a Ds insertional mutants derived from the source cultivar, Dongjin. This mutant could not affect other developmental patterns like phyllotaxis. Southern blot analysis, using GUS as a probe, revealed that the liguleless mutant contained three Ds copies transposed in the rice genome. Among the four genomic sequences flanking the Ds, one was mapped in the intergenic region (31661640 - 31661759), and the other two predicted a protein kinase domain (12098980 - 12098667) as an original insertion site within a starter line used for massive production of Ds insertional mutant lines. Another predicted and inserted in first exon of liguleless 1 protein (OsLG1) that was mapped in coding region (LOC_Os04g56170) of chromosome 4. RT-PCR revealed that the OsLG1 gene was not expressed liguleless mutants. Structure analysis of OsLG1 protein revealed that it predicted transcription factor with a highly conserved SBP domain consisting of 79 amino acids that overlapped a nuclear localization signal (NLS). RT-PCR revealed that OsLG1 is mainly expressed in vegetative organs.

• Proceedings of the Korean Society of Crop Science Conference
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• 2017.06a
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• pp.90-90
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• 2017

Regulation of seed dormancy is important in many grains to prevent pre-harvest sprouting. To identify and understand the gene related to seed dormancy regulation, we have screened for viviparous phenotypes of rice mutant lines generated by insertion of Ds transposon in a Korean Japonica cultivar (Dongjin) background. One of the mutants, which represented viviparous phenotype, was selected for further seed dormancy regulation studies and designated dor1. The dor1 mutant has single Ds insertion in the second exon of OsDor1 gene encoding glycine-rich protein. The seeds of dor1 mutant showed a higher germination potential and reduced abscisic acid (ABA) sensitivity compared to wild type Dongjin. Over-expression of Dor1 complements the viviparous phenotype of dor1 mutant, indicating that Dor1 function in seed dormancy regulation. Subcellular localization assay of Dor1-GFP fusion protein revealed that the OsDor1 protein mainly localized to membrane and the localization of OsDOR1 was influenced by presence of a giberelin (GA) receptor OsGID1. Further bimolecular fluorescence complementation (BiFC) analysis indicated that OsDOR1 interact with OsGID1. The combined results suggested that OsDOR1 regulates seed dormancy by interacting with OsGID1 in GA response. Additionally, expression of OsDOR1 partially complemented the cold sensitivity of Escherichia coli BX04 mutant lacking four cold shock proteins, indicating that OsDOR1 possessed RNA chaperone activity.

• Korean Journal of Breeding Science
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• v.40 no.1
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• pp.39-47
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• 2008

The 1,874 rice lines were selected from 3,000 Ds insertional mutant pool by Basta herbicide treatment and were surveyed for trait variation and molecular characteristics of genes knocked out by Ds insertion. Compared with "Donjin", an original japonica cultivar used for transformation, Ds insertion mutant pool showed large variation in major agronomic traits including tiller, panicle, and heading etc. Southern blot analysis demonstrated that these lines on the average had two Ds copies in Donjin genome, resulting in 38.4% of one copy, 32.5% of two copies, 16.7% of three copies, and 11.3% of over four copies. GUS analysis showed that 3.9% of lines (73/1,860) had tissue-specific expression in leaves, nodal parts, floral organs such as stigma and pollen, and roots. Data set obtained from agricultural trait variation and molecular characteristics for individual Ds insertional lines would provide researchers with more information for understanding the function of unknown rice genes controlling economically important traits.

• Plant Breeding and Biotechnology
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• v.6 no.4
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• pp.313-320
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• 2018

Rice is the staple food of more than 50% of the world population. Cultivated rice has the AA genome (diploid, 2n = 24) and small genome size of only 430 megabase (haploid genome). As the sequencing of rice genome was completed by the International Rice Genome Sequencing Project (IRGSP), many researchers in the world have been working to explore the gene function on rice genome. Insertional mutagenesis has been a powerful strategy for assessing gene function. In maize, well characterized transposable elements have traditionally been used to clone genes for which only phenotypic information is available. In rice endogenous mobile elements such as MITE and Tos have been used to generate gene-tagged populations. To date T-DNA and maize transposable element systems have been utilized as main insertional mutagens in rice. The Ac/Ds system offers the advantage of generating new mutants by secondary transposition from a single tagged gene. To enhance the efficiency of gene detection, advanced gene-tagging systems (i.e. activation, gene or enhancer trap) have been employed for functional genomic studies in rice. Internationally, there have been many projects to develop large scales of insertional mutagenized populations and databases of insertion sites has been established. Ultimate goals of these projects are to supply genetic materials and informations essential for functional analysis of rice genes and for breeding using agronomically important genes. In this report, we summarize the current status of Ac/Ds-mediated gene tagging systems that has been conducted by collaborative works in Korea.

• Journal of Plant Biotechnology
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• v.35 no.4
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• pp.307-316
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• 2008

Transposon-mediated insertional mutagenesis is one of powerful strategy for assessing functions of genes in higher plants. In this report, we have selected highly susceptible and tolerance plant by screening about high salt (3% NaCl) and cold stresses ($4^{\circ}C$) from F2 seeds of 30,000 Ac/Ds insertional mutagenesis lines in rice (Oryza sativa L. cv. Dongjin). In order to identify the gene tagging, insertion of Ds element was analyzed by Southern blot and these results revealed that 19 lines were matched genotype of selected lines with phenotype from the first selected 212 lines, and 13 lines have one copy of Ds elements. The Franking Sequence Tags (FSTs) of selected mutant lines showed high similarities with the following known function genes: signal transduction and regulation of gene expression (transpoter, protease family protein and apical meristem family protein), osmotic stress response (heat shock protein, O-methyltransferase, glyceraldehyde-3-phosphate dehydrogenase and drought stress induce protein), vesicle trafficking (SYP 5 family protein) and senescence associated protein. The expression pattern of 19 genes were analyzed using RT-PCR under the abiotic stresses of 9 class; 250mM NaCl, osmotic, drought, 3% $H_2O_2$, $100{\mu}M$ ABA, $100{\mu}M$ IAA, 0.1 ppm 2,4-D, $4^{\circ}C$ cold and $38^{\circ}C$ high temperature. Isolated knock-out genes showed the positive response about 250 mM NaCl, drought, $H_2O_2$, PEG, IAA, 2,4-D, ABA treatment and low ($4^{\circ}C$) and high temperature ($38^{\circ}C$). The results from this study indicate that function of selected knock-out genes could be useful in improving of tolerance to abiotic stresses as an important transcriptional activators in rice.

• Journal of Plant Biotechnology
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• v.35 no.3
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• pp.177-183
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• 2008

Ac/Ds mutant lines of this study were transgenic rice plants, each of which harbored the maize transposable element Ds together with a GUS coding sequence under the control of a promoterless(Ds-GUS). We selected the mutants that were GUS expressed lines, because the GUS positive lines will be useful for identifying gene function in rice. One of these mutants was identified knock-out at Oszinc626(NP_001049991) gene, encoding a RING-H2 zinc-finger protein, by Ds insertion. In this mutant, while primary root development is normal, secondary root development from lateral root was very poor and seed development was incomplete compare with normal plant. RING zinc-finger proteins play important roles in the regulation of development in a variety of organisms. In the plant kingdom, a few genes encoding RING zinc-finger proteins have been documented with visible effects on plant growth and development. The consensus of the RING-H2(C3-H2-C3 type) domain for this group of protein is $Cys-X_2-Cys-X_{28}-Cys-X-His-X_2-His-X_2-Cys-X_{14}-Cys-X_2-Cys$. Oszinc626 encodes a predicted protein product of 445 amino acids residues with a molecular mass of 49 kDa, with a RING-zinc-finger motif located at the extreme end of the C-terminus. RT-PCR analysis indicated that the expression of Oszinc626 gene was induced by IAA, cold, dehydration, high-salinity and abscisic acid, but not by 2,4-D, and the transcription of Oszinc626 gene accumulated primarily in rice immature seeds, root meristem and shoots. The gene accumulation patterns were corresponded with GUS expression.