• 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.

• Proceedings of the Korea Society of Environmental Toocicology Conference
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• 2001.05a
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• pp.129-129
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• 2001

1,2-Dibromo-3-chloropropane (DBCP), a soil fumigant against nematodes, is a genotoxic carcinogen and also is classified by World Wildlife Fund as endocrine disruptors. DBCP has been extensively studied on genotoxicity, carcinogenicity, and damage in male reproductive-related organs. However, information on precise mechanism of mutagenesis and carcinogenesis of DBCP is yet unknown. (omitted)

• Journal of Radiation Industry
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• v.5 no.4
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• pp.285-295
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• 2011

Mushrooms belonging to macrofungi have been consumed by humans for their nutritional and medicinal values for centuries throughout the world. Mushroom farming is practiced in more than 100 countries of the world, with production increasing at a rate of 7% per annum. High yield and good quality are always the principal goals for agriculturally important crops, including mushrooms. Several breeding methods are employed for strain improvement such as mass selection based on the natural chance mutation and induced mutation (mutation breeding), protoplast fusion technology, cross breeding and transgenic breeding. However, mutation breeding has shown prominent success in crop plant improvement. Though several-hundred mutant crop varieties have been developed around the world, the mutation breeding of mushrooms is limited. This review paper explores the potential application of radiation on the development of mutant varieties of mushrooms for breeding with desired traits such as better quality and productivity.

• Plant Biotechnology Reports
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• v.4 no.3
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• pp.201-211
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• 2010

We present a highly effective T-DNA inserted gene screening method as part of a reverse genetics model system using the Chinese cabbage (Brassica rapa L. spp. pekinensis). Three-step two-dimensional (2D) matrix strategies are potentially accurate and useful for the identification of specific T-DNA inserted mutants from a large population. To construct our Chinese cabbage model, we utilized a forward genetics screening approach for the abnormal phenotypes that were obtained from transgenic plants of Brassica rapa generated with Agrobacteria tumefaciens containing the pRCV2 vector. From one transgenic plant with an abnormal phenotype, we observed that the st1 gene (which is related to senescence-associated process proteins) contained a T-DNA fragment, and that its expression level was decreased. This T-DNA insert was then used as a control to construct an effective screening pool. As a result, the optimum template concentration was found to be 0.1-1 ng in our PCR strategy. For other conditions, positive changes to the Gibbs free energy prevented the formation of oligo dimers and hairpin loop structures, and autosegment extension gave better results for long fragment amplification. Using this effective reverse genetics screening method, only 23 PCR reactions were necessary to select a target gene from a pool of 100 individual DNAs. Finally, we also confirmed that the sequence we obtained from the above method was identical to the flanking sequence isolated by rescue cloning.

• Horticultural Science & Technology
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• v.33 no.2
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• pp.242-250
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• 2015

Insertional mutagenesis induced by T-DNA or transposon tagging offers possibilities for analysis of gene function. However, its potential remains limited unless good methods for detecting the target locus are developed. We describe a PCR technique for efficient identification of DNA sequences adjacent to the inserted T-DNA in a higher plant, Chinese cabbage (Brassica rapa ssp. pekinensis). This strategy, which we named variable argument thermal asymmetric interlaced PCR (VA-TAIL PCR), was designed by modifying a single-step annealing-extension PCR by including a touch-up PCR protocol and using long gene-specific primers. Amplification efficiency of this PCR program was significantly increased by employing an autosegment extension method and linked sequence strategy in nested long gene-specific primers. For this technique, arbitrary degenerate (AD) primers specific to B. rapa were designed by analyzing the Integr8 proteome database. These primers showed higher accuracy and utility in the identification of flanking DNA sequences from individual transgenic Chinese cabbages in a large T-DNA inserted population. The VA-TAIL PCR method described in this study allows the identification of DNA regions flanking known DNA fragments. This method has potential biotechnological applications, being highly suitable for identification of target genomic loci in insertional mutagenesis screens.

• Journal of Life Science
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• v.16 no.3 s.76
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• pp.540-545
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• 2006

T-DNA-mediated transformation is a common method for generating transgenic plants with insertional mutagenesis. In order to identify important genes involved in shoot development, a system of promoter trap insertional mutagenesis was employed in Arabidopsis thaliana. For this system, an efficient promoter trap vector, pFGL561 was developed. The pFGL561 includes a basta-resistant gene, an intron with multiple splicing donor and acceptor sites, and a promoter-less GFP reporter gene. Using floral-dipping method, we made total 300 $T_1$ promoter-trap lines which were screened for GFP expression. GFP signals in the $T_1$ plants were detected with high frequency, 26.7%, and the signals were reconfirmed in $T_2$ plants. To isolate the genes that are involved in shoot development, phenotypes were analyzed in $T_2$ plants of the 19 $T_1$ lines that had GFP signals in shoot apex, and 6 $T_1$ lines were selected that had abnormal shoot development. These lines will be very useful for the investigation of shoot development.

• Asian-Australasian Journal of Animal Sciences
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• v.29 no.10
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• pp.1383-1391
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• 2016

Bovine embryonic stem cells have potential for use in research, such as transgenic cattle generation and the study of developmental gene regulation. The Nanog may play a critical role in maintenance of the undifferentiated state of embryonic stem cells in the bovine, as in murine and human. Nevertheless, efforts to study the bovine Nanog for pluripotency-maintaining factors have been insufficient. In this study, in order to understand the mechanisms of transcriptional regulation of the bovine Nanog, the 5'-flanking region of the Nanog was isolated from ear cells of Hanwoo. Results of transient transfection using a luciferase reporter gene under the control of serially deleted 5'-flanking sequences revealed that the -134 to -19 region contained the positive regulatory sequences for the transcription of the bovine Nanog. Results from mutagenesis studies demonstrated that the Sp1-binding site that is located in the proximal promoter region plays an important role in transcriptional activity of the bovine Nanog promoter. The electrophoretic mobility shift assay with the Sp1 specific antibody confirmed the specific binding of Sp1 transcription factor to this site. In addition, significant inhibition of Nanog promoter activity by the Sp1 mutant was observed in murine embryonic stem cells. Furthermore, chromatin-immunoprecipitation assay with the Sp1 specific antibody confirmed the specific binding of Sp1 transcription factor to this site. These results suggest that Sp1 is an essential regulatory factor for bovine Nanog transcriptional activity.

• Proceedings of the Korean Society of Crop Science Conference
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• 2017.06a
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• pp.163-163
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• 2017

A deletion analysis of the Oryza sativa dihydroflavonol reductase (DFR) promoter defined a 25 bp region (-386 to -362) sufficient to confer pericarp-specific expression of ${\beta}$ -glucuronidase(GUS) reporter gene in transgenic rice. Site-specific mutagenesis of these conserved sequences and subsequent expression analysis in calli which transiently expressed the mutated promoter::GUS gene showed that both bHLH (-386 to -381) and Myb (-368 to -362) binding sites in the DEL3 (-440 to 70) promoter were necessary for complete expression of the GUS gene including the tissue-specific expression of DFR::GUS gene. The GUS gene was expressed well in the mutated Myb (-368 to -362) binding site, but not as strong as in normal condition, implying that the Myb is also necessary to express GUS gene fully. Also, we found the non-epistatic relation between Rc and DFR. There were no changes of expression patterns GUS under the Rc and rc genotypes. Thus, DFR expression might be independent of the presence of functional Rc gene and suggested that Rc and Rd (DFR) share the same pathway controlling the regulation of flavonoid synthesis but not a direct positive transcriptional regulator of DFR gene.

• Journal of Ginseng Research
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• v.46 no.4
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• pp.505-514
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• 2022

Background: The roots of Panax ginseng contain two types of tetracyclic triterpenoid saponins, namely, protopanaxadiol (PPD)-type saponins and protopanaxatiol (PPT)-type saponins. In P. ginseng, the protopanaxadiol 6-hydroxylase (PPT synthase) enzyme catalyses protopanaxatriol (PPT) production from protopanaxadiol (PPD). In this study, we constructed homozygous mutant lines of ginseng by CRISPR/Cas9-mediated mutagenesis of the PPT synthase gene and obtained the mutant ginseng root lines having complete depletion of the PPT-type ginsenosides. Methods: Two sgRNAs (single guide RNAs) were designed for target mutations in the exon sequences of the two PPT synthase genes (both PPTa and PPTg sequences) with the CRISPR/Cas9 system. Transgenic ginseng roots were generated through Agrobacterium-mediated transformation. The mutant lines were screened by ginsenoside analysis and DNA sequencing. Result: Ginsenoside analysis revealed the complete depletion of PPT-type ginsenosides in three putative mutant lines (Cr4, Cr7, and Cr14). The reduction of PPT-type ginsenosides in mutant lines led to increased accumulation of PPD-type ginsenosides. The gene editing in the selected mutant lines was confirmed by targeted deep sequencing. Conclusion: We have established the genome editing protocol by CRISPR/Cas9 system in P. ginseng and demonstrated the mutated roots producing only PPD-type ginsenosides by depleting PPT-type ginsenosides. Because the pharmacological activity of PPD-group ginsenosides is significantly different from that of PPT-group ginsenosides, the new type of ginseng mutant producing only PPD-group ginsenosides may have new pharmacological characteristics compared to wild-type ginseng. This is the first report to generate target-induced mutations for the modification of saponin biosynthesis in Panax species using CRISPR-Cas9 system.

• Proceedings of the Korean Society of Plant Biotechnology Conference
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• 2005.11a
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• pp.95-105
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• 2005

Transposon-mediated insertional mutagenesis provides one of the most powerful tools for functional studies of genes in higher plants. This project has been performed to develop a large population of insertional mutations, and to construct databases of molecular information on Ds insertion sites in rice. Ultimate goals are to supply genetic materials and information to analyze gene function and to identify and utilize agronomically important genes for breeding purpose. Two strategies have been employed to generate the large scale of transposon population in a Japonica type rice, Dongjin Byeo; 1) genetic crosses between Ac and Ds lines and 2) plant regeneration from seeds carrying Ac and Ds. Our study showed that over 70% of regenerated plants generally carried independent Ds elements and high activity of transposition was detected only during regeneration period. Ds-flanking DNA amplified from leaf tissues of F2 and T1 (or T2) plants have been amplified via TAIL-PCR and directly sequenced. So far, over 65,000 Ds lines have been generated and over 9,500 Ds loci have been mapped on chromosomes by sequence analysis. Database of molecular information on Ds insertion sites has been constructed, and has been opened to the public and will be updated soon at http://www.niab.go.kr. Detailed functional analysis of more than 30 rice mutants has been performed. Several Ds-tagged rice genes that have been selected for functional analysis will be briefly introduced. We expect that a great deal of information and genetic resources of Ds lines would be obtained during the course of this project, which will be shared with domestic and international rice researchers. In addition to the Japonica rice, we have established the tagging system in an rice line of indica genetic background, MGRI079. MGRI079 (Indica/Japonica) was transformed with Agrobacteria carrying Ac and Ds T-DNA vectors. Among transgenic lines, we successfully identified single-copy Ds and Ac lines in MGR1079. These lines were served as ‘starter lines’ to mutagenize Indica genetic background. To achieve rapid, large scale generation of Ds transposant lines, MGR1079 transformants carrying homozygous Ac were crossed with ones with homozygous Ds, and $F_2$seeds were used for plant regeneration. In this year, over 2,000 regeneration plants were grown in the field. We are able to evaluate the tagging efficiency in the Indica genetic background in the fall.