• Korean Journal of Environmental Agriculture
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• v.40 no.3
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• pp.186-193
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• 2021

BACKGROUND: Before generating transgenic plant using the CRISPR-Cas9 system, the efficiency test of sgRNAs is recommended to reduce the time and effort for plant transformation and regeneration process. The efficiency of the sgRNA can be measured through the transient expression of sgRNA and Cas9 gene in tomato cotyledon; however, we found that the calculated efficiency showed a large variation. It is necessary to increase the precision of the experiment to obtain reliable sgRNA efficiency data from transient expression. METHODS AND RESULTS: The cotyledon of 11^th, 15^th, 19^th, and 23^rd-day-old tomato (Solanum lycopersicum cv. Micro-Tom) were used for expressing CRISPR-Cas9 transiently. The agrobacterium harboring sgRNA for targeting ALS2 gene of tomato was injected through the stomata of leaf adaxial side and the genomic DNA was extracted in 5 days after injection. The target gene edition was identified by amplifying DNA fragment of target region and analyzing with Illumina sequencing method. The target gene editing efficiency was calculated by counting base deletion and insertion events from total target sequence read. CONCLUSION: The CRISPR-Cas9 editing efficiency varied with tomato cotyledon age. The highest efficiency was observed at the 19-day-old cotyledons. Both the median and mean were the highest at this stage and the sample variability was also minimized. We found that the transgene of CRISPR-Cas9 system was strongly correlated with plant leaf development and suggested the optimum cotyledon leaf age for Agrobacterium-mediated transfection in tomato.

• The Plant Pathology Journal
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• v.38 no.6
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• pp.656-664
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• 2022

Pectobacterium odoriferum is the primary causative agent in Kimchi cabbage soft-rot diseases. The pathogenic bacteria Pectobacterium genera are responsible for significant yield losses in crops. However, P. odoriferum shares a vast range of hosts with P. carotovorum, P. versatile, and P. brasiliense, and has similar biochemical, phenotypic, and genetic characteristics to these species. Therefore, it is essential to develop a P. odoriferumspecific diagnostic method for soft-rot disease because of the complicated diagnostic process and management as described above. Therefore, in this study, to select P. odoriferum-specific genes, species-specific genes were selected using the data of the P. odoriferum JK2.1 whole genome and similar bacterial species registered with NCBI. Thereafter, the specificity of the selected gene was tested through blast analysis. We identified novel species-specific genes to detect and quantify targeted P. odoriferum and designed specific primer sets targeting HAD family hydrolases. It was confirmed that the selected primer set formed a specific amplicon of 360 bp only in the DNA of P. odoriferum using 29 Pectobacterium species and related species. Furthermore, the population density of P. odoriferum can be estimated without genomic DNA extraction through SYBR Green-based real-time quantitative PCR using a primer set in plants. As a result, the newly developed diagnostic method enables rapid and accurate diagnosis and continuous monitoring of soft-rot disease in Kimchi cabbage without additional procedures from the plant tissue.

• Korean Journal of Plant Tissue Culture
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• v.28 no.6
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• pp.297-304
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• 2001

Scrophularia buergeriana Misrule has been contaminated with various pathogens in condition of field and storage period. This study was carried out for production of multiple stress resistance plant containing disease resistance that CGST gene expressed in transgenic Scrophularia buergeriana Misrule genome. Glutathione S-Transferases (GSTs) detoxify endobiotic and xenobiotic compounds by covalent linking of tripeptide glutathione to hydrophobic substrate. GST enzymes have been identified and characterized in insects, bacteria, and many plant species. A cDNA clone of GST was introduced into Scrophularia buergeriana Miquel by transformation with Agrobacterium tumefaciences. In coporation of the CGST gene into S. buergeriana Misrule was confirmed by PCR analysis of genomic DNA. Influence of exposure to darkness on the regeneration potential and transformation frequence were assessed. The activity of GST in transgenic plants was two times higher than that of non-transgenic plants. As a result of anti-microbe assays, the crude extract protein of transgenic plants showed the antimicrobial effects higher than control plants.

• Journal of Life Science
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• v.29 no.5
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• pp.524-529
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• 2019

Medicinal plants resources are becoming important assets since their usages have been expanded to the development of functional foods for human health, cosmetics and pharmaceutical industries. However, their phylogenetic origins and names are different from each country and quite often they are mixed each other resulting in the confusion for consumers. Particularly when they are very similar based on their morphological characteristics and distributed, it is extremely difficult to differentiate their origins even by specialists. Therefore, identification of each plant species is important for standardizing herbal medicine. Thistle is a medicinal and perennial plant. Obtaining information about the genetic diversity of plant populations is highly important for conservation and germplasm utilization. Although thistle is an important medicinal plant species registered in South Korea, no molecular markers are currently available to distinguish from other similar species from different countries. In this study, we developed single nucleotide polymorphism (SNP) markers derived from chloroplast genomic sequences to identify distinct Korean-specific thistle species via high resolution melting (HRM) curve analyses. We performed molecular authentication of four different kinds of thistle species from different regions using DNA sequences in the trnL-F and matK chloroplast intergenic region. The SNP markers developed in this study are useful for rapidly identifying specific thistle species from different country.

• Journal of Microbiology and Biotechnology
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• v.15 no.3
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• pp.502-509
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• 2005

Sexual reproduction plays an important role in the biology and epidemiology of oomycete plant pathogens such as the heterothallic species Phytophthora infestans. Recent worldwide dispersal of A2 mating type strains of P. infestans resulted in increased virulence, gene transfer, and genetic variation, creating new challenges for disease management. To develop a genetic assay for mating type identification in P. infestans, we used the Amplified Fragment Length Polymorphism (AFLP) technique. The primer combination E+AT/M+CTA detected a fragment specific to A1 mating type (Mat-A1) of P. infestans. This fragment was cloned and sequenced, and a pair of primers (INF-1, INF-2) were designed and used to differentiate P. infestans Mat-A1 from Mat-A2 strains. The Mat A1-specific fragment was detected using Southern blot analysis of PCR products amplified with primers INF-1 and INF-2 from genomic DNA of 14 P. infestans Mat-A1 strains, but not 13 P. infestans Mat-A2 strains or 8 other isolates representing several Phytophthora spp. Southern blot analysis of genomic DNAs of P. infestans isolates revealed a 1.6 kb restriction enzyme (EcoRI, BamHI, AvaI)-fragment only in Mat-A1 strains. The A1 mating type-specific primers amplified a unique band under stringent annealing temperatures of $63^{\circ}C-64^{\circ}C$, suggesting that this PCR assay could be developed into a useful method for mating type determination of P. infestans in field material.

• The Plant Pathology Journal
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• v.34 no.6
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• pp.506-513
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• 2018

Clubroot is one of the most economically important diseases of the Brassicaceae family. Clubroot disease is caused by the obligate parasite Plasmodiophora brassicae, which is difficult to study because it is nonculturable in the laboratory and its races are genetically variable worldwide. In Korea, there are at least five races that belongs to four pathotype groups. A recent study conducted in Korea attempted to develop molecular markers based on ribosomal DNA polymorphism to detect P. brassicae isolates, but none of those markers was either race-specific or pathotype-specific. Our current study aimed to develop race- and isolate-specific markers by exploiting genomic sequence variations. A total of 119 markers were developed based on unique variation exists in genomic sequences of each of the races. Only 12 markers were able to detect P. brassicae strains of each isolate or race. Ycheon14 markers was specific to isolates of race 2, Yeoncheon and Hoengseong. Ycheon9 and Ycheon10 markers were specific to Yeoncheon isolate (race 2, pathotype 3), ZJ1-3, ZJ1-4 and ZJ1-5 markers were specific to Haenam2 (race 4) isolate, ZJ1-35, ZJ1-40, ZJ1-41 and ZJ1-49 markers were specific to Hoengseong isolate and ZJ1-56 and ZJ1-64 markers were specific to Pyeongchang isolate (race 4, pathotype 3). The PCR-based sequence characterized amplified region (SCAR) markers developed in this study are able to detect five Korean isolates of P. brassicae. These markers can be utilized in identifying four Korean P. brassicae isolates from different regions. Additional effort is required to develop race- and isolate-specific markers for the remaining Korean isolates.

• Journal of Microbiology and Biotechnology
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• v.29 no.5
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• pp.785-793
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• 2019

Black rot caused by Xanthomonas campestris pv. campestris (Xcc) is the most damaging disease in Brassica crops around the world. In this study, we developed a molecular marker specific to Xcc race 5. To do this, the available whole genome sequences of Xcc races/strains and Xc subspecies were aligned and identified a highly variable genomic region (XccR5-89.2). Subsequently, a primer set covering the 'XccR5-89.2' region was designed and tested against the genomic DNA of Xcc races/strains, Xc subspecies and other plant-infecting bacterial strains (Pseudomonas syringae pv. maculicola and Erwinia carotovora subsp. carotovora). The results showed that the 'XccR5-89.2' primer pair amplified a 2,172-bp fragment specific to Xcc race 5. Moreover, they also amplified a 1,515-bp fragment for Xcc race 1 and an over 3,000-bp fragment for Xcc race 3. However, they did not amplify any fragments from the remaining Xcc races/strains, subspecies or other bacterial strains. The 'XccR5-89.2' primer pair was further PCR amplified from race-unknown Xcc strains and ICMP8 was identified as race 5 among nine race-unknown Xcc strains. Further cloning and sequencing of the bands amplified from race 5 and ICMP8 with 'XccR5-89.2' primers revealed both carrying identical sequences. The results showed that the 'XccR5-89.2' marker can effectively and proficiently detect, and identify Xcc race 5 from Xcc races/strains, subspecies and other plant-infecting bacteria. To our knowledge, this is the first report for an Xcc race 5-specific molecular marker.

• BMB Reports
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• v.40 no.5
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• pp.625-635
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• 2007

The enzyme squalene synthase (EC 2.5.1.21) catalyzes a reductive dimerization of two farnesyl diphosphate (FPP) molecules into squalene, a key precursor for the sterol and triterpene biosynthesis. A full-length cDNA encoding squalene synthase (designated as TcSqS) was isolated from Taxus cuspidata, a kind of important medicinal plants producing potent anti-cancer drug, taxol. The full-length cDNA of TcSqS was 1765 bp and contained a 1230 bp open reading frame (ORF) encoding a polypeptide of 409 amino acids. Bioinformatic analysis revealed that the deduced TcSqS protein had high similarity with other plant squalene synthases and a predicted crystal structure similar to other class I isoprenoid biosynthetic enzymes. Southern blot analysis revealed that there was one copy of TcSqS gene in the genome of T. cuspidata. Semi-quantitative RT-PCR analysis and northern blotting analysis showed that TcSqS expressed constitutively in all tested tissues, with the highest expression in roots. The promoter region of TcSqS was also isolated by genomic walking and analysis showed that several cis-acting elements were present in the promoter region. The results of treatment experiments by different signaling components including methyl-jasmonate, salicylic acid and gibberellin revealed that the TcSqS expression level of treated cells had a prominent diversity to that of control, which was consistent with the prediction results of TcSqS promoter region in the PlantCARE database.

• Korean Journal of Medicinal Crop Science
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• v.12 no.2
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• pp.154-162
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• 2004

Hairy roots were induced from Korean ginseng (Panax ginseng C. A. Meyer) root explants and studied for their gene expression. A total of 3,000 ESTs (expressed sequence tags) from ginseng hairy root were determined and about 2,700 ESTs have a length of readable sequence, which result in 1,352 unique ESTs sequences. The 879 ESTs showed significant similarities to known nucleotide or amino acid sequences in other plant species, which were divided into eleven categories depending upon gene function. The remaining 473 sequences showed no significant matches, which are likely to be transcripts or to be matched to other organisms. The results indicated that the analysis of the ginseng hairy root ESTs by partial sequencing of random cDNA clones may be an efficient approach to isolate genes that are functional in ginseng root in a large scale. Our extensive EST analysis of genes expressed in ginseng hairy root not only contributes to the understanding of the dynamics of genome expression patterns in root organ but also adds data to the repertoire of all genomic genes.

• BMB Reports
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• v.39 no.5
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• pp.607-617
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• 2006

A novel calcium-dependent protein kinase gene (designated as IiCPK2) was cloned from tetraploid Isatis indigotica. The full-length cDNA of IiCPK2 was 2585 bp long with an open reading frame (ORF) of 1878 bp encoding a polypeptide of 625 amino acid residues. The predicted IiCPK2 polypeptide included three domains: a kinase domain, a junction domain (or autoinhibitory region), and a C-terminal calmodulin-like domain (or calcium-binding domain), which presented a typical structure of plant CDPKs. Further analysis of IiCPK2 genomic DNA revealed that it contained 7 exons, 6 introns and the length of most exons was highly conserved. Semi-quantitative RT-PCR revealed that the expression of IiCPK2 in root, stem and leaf were much higher in tetraploid sample than that in diploid progenitor. Further expression analysis revealed that gibberellin ($GA_3$), NaCl and cold treatments could up-regulate the IiCPK2 transcription. All our findings suggest that IiCPK2 might participate in the cold, high salinity and GA3 responsive pathways.