• 한국작물학회지
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• 제54권4호
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• pp.427-432
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• 2009

Seashore Paspalum (Paspalum vaginatum Swartz) is a warm season grass and indigenous to tropical and subtropical regions of coastal areas worldwide. The species is used as feed for cattle and horses and has been very successful for golf courses worldwide. One of the most outstanding characteristics of seashore paspalum is its tolerance to saline soils compared to other warm season turfgrasses. The development of new seashore paspalum cultivars with improved traits could be facilitated through the application of biotechnological strategies. The purpose of this study was to product for herbicide resistant seashore paspalum using Arobacterium-mediated transformation and this study is the first report on transformation and herbicideresistant transgenic plants in seashore paspalum. Embryogenic calli were induced from the seeded variety of pseashore paspalum. Embryogenic calli were transformed with Agrobacterium tumefaciens strain EHA105 carrying the binary vector pCAMBIA3301 with two genes encoding gusA and bar. Transformed calli and plants were selected on medium containing 3 mg/l PPT. PCR detected the presence of the gusA and bar gene, indicating both genes are integrated into the genome of seashore paspalum. A chlorophenol red assay was used to confirm that the bar gene was expressed. By application of herbicide BASTA, the herbicide resistance in the transgenic seashore paspalum plants was confirmed.

• 원예과학기술지
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• 제34권2호
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• pp.314-323
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• 2016

A transient ihpRNA-induced gene silencing system based on Agrobacterium-mediated injection infiltration has been established to evaluate candidate genes involved in proanthocyanidin (PAs) biosynthesis in persimmon (Diospyros kaki Thunb.). We chose DkPDS (phytoene desaturase) as a gene-silencing target to evaluate the newly developed transient gene silencing system. Our qRT-PCR analysis indicated that two ihpRNA constructs (pHG-PDS5' and pHG-PDS3') targeted DkPDS, which also led to significantly reduce expression of DkPDS in 'Mopanshi' persimmon leaves. To further confirm the reliability of the system, we successfully utilized it for DkLAR (leucoanthocyanidin reductase) gene silencing. The expression levels of DkLAR in 'Mopanshi' and 'Eshi 1' leaves were ca. 6-fold and ca. 5-fold lower than those in leaves harboring empty vector (pHG-GFP), respectively. DMACA (4-dimethylaminocinnamaldehyde) staining and the Folin-Ciocalteau assay showed that the accumulation of PAs was markedly inhibited in 'Mopanshi', 'Eshi 1' and 'Youhou' leaves. These results indicate that DkLAR plays an important role in the accumulation of PAs in persimmon. The transient ihpRNA-induced gene silencing method developed in this study is a highly efficient and useful tool for functional analysis of persimmon genes involved in PA biosynthesis.

• Journal of Plant Biotechnology
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• 제7권2호
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• pp.97-103
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• 2005

This study was carried out to obtain basic information for gene manipulation in potent edible tobacco (Nicotiana tabacum cv. TI 516). N. tabacum cv. TI 516 is a plant for a possible candidate to use as an edible vaccine, since it contains a low level of nicotine. The effective plant regeneration system through leaf disc culture was achieved using a MS basal medium supplemented with 0.1 mg $1^{-1}$ NAA and 0.5 mg $1^{-1}$ BA. In order to transform the N. tabacum cv. TI 516 with the green fluorescent protein (GFP) gene, Agrobacterium tumefaciens LBA 4404 containing the GFP gene was used. Genomic PCR confirmed the integration of the GFP gene into nuclear genome of transgenic plants. Expression of the GFP gene was identified in callus, apical meristem and root tissue of transgenic N. tabacum cv. TI 516 plants using fluorescence microscopy. Western blot analysis revealed the expression of GFP protein in the transgenic edible tobacco plants. The amount of GFP protein detected in the transgenic tobacco plants was approximately 0.16% of the total soluble plant protein (TSP), which was determined by ELISA.

• 식물조직배양학회지
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• 제27권1호
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• pp.39-45
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• 2000

전위유전자 표지를 이용한 2배체 야생근연종 감자의 유용 유전자 cloning 체계를 개발하기 위하여 옥수수의 진위유전자 Ac를 조작하여 전위효소는 생산하나 이동은 불가능하도록 제작된 immobilized Ac (iAc)와 자체 이동은 불가능하나 iAc의 작용에 의해 이동이 가능한 Ds를 Agrobacterium tumefaciens를 이용한 형질전환에 의해 2배체 근연종 감자 (Solanum tuberosum Group Phureja) 계통 1.22에 도입하였다. iAc및 Ds 삽입 binary vector들을 보유하는Agrobacterium 계통들로 형질전환 처리된 1.22 기내배양신초의 잎과 줄기 절편의 경우 50mg/L 의 kanamycin이 첨가된 배지에서도 캘러스를 형성하였으며, 잎 절편으로부터 재분화신초가 획득되었다. 형질전환 처리되지 않은 1.22 신초는 100mg/L의 kanamycin이 첨가된 배지에서 전혀 발근이 되지 않았으나, 형질전환 처리에 의해 획득된 재분화 신초는 동일 배지에서 발근이 되었다. iAc와 Ds의 염기배열에 대해 특이적인 oligonucleotide primer들을 이용하여 형질전환 처리 획득 식물들로부터 추출된 DNA에 대한 PCR분석을 실시한 결과, 사용된 primer들의 iAc 와 Ds의 염기배열에 있어서의 위치에 의해 예상되는 크기의 DNA들이 형성되어 iAc 와 Ds의 1.22 genome내 도입이 확인되었다.

• The Plant Pathology Journal
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• 제19권3호
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• pp.162-165
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• 2003

Transformation of Fuji apple (Malus domestica 'Fuji') was performed using Agrobacterium tumefaciens harboring a coat protein (CP) gene of Cucumber mosaic virus (CMV). A plasmid DNA containing the virus CP and NPT II genes was introduced into the loaves of apple by th e Agrobacterium - mediated transformation procedure. Regenerated transformants of the apple were obtained by kanamycin resistance conferred by the introduced NPT II gene. PCR analysis showed that 3 out of 20 putatively selected R0 plant lines contain the CMV-CP gene. Nine putative transgenic lines out of 20 lines were investigated with the PCR analysis; 5 regenerants produced a 450 bp DNA band and 3 regenerants showed a 671 bp DNA band for the NPT II and CMV-CP genes, respectively. Southern hybyidization results demonstrate the successful integration of the CMV-CP gene into the genome of the apple. This is the first report on the generation of useful vius resistance source of transgenic apple for molecular breeding program.

• Plant Biotechnology Reports
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• 제5권1호
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• pp.71-77
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• 2011

Drought and salinity are the most important abiotic stresses that affect the normal growth and development of plants. Glycine betaine is one of the most important osmolytes present in higher plants that enable them to cope with environmental stresses through osmotic adjustment. In this study, a betaine aldehyde dehydrogenase (BADH) gene from spinach under the control of the stress-induced promoter rd29A from Arabidopsis thaliana was introduced into potato cultivar Gannongshu 2 by the Agrobacterium tumefaciens system. Putative transgenic plants were confirmed by Southern blot analysis. Northern hybridization analysis demonstrated that expression of BADH gene was induced by drought and NaCl stress in the transgenic potato plants. The BADH activity in the transgenic potato plants was between 10.8 and 11.7 U. There was a negative relationship (y = -2.2083x + 43.329, r = 0.9495) between BADH activity and the relative electrical conductivity of the transgenic potato plant leaves. Plant height increased by 0.4-0.9 cm and fresh weight per plant increased by 17-29% for the transgenic potato plants under NaCl and polyethylene glycol stresses compared with the control potato plants. These results indicated that the ability of transgenic plants to tolerate drought and salt was increased when their BADH activity was increased.

• Journal of Microbiology and Biotechnology
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• 제18권12호
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• pp.1874-1883
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• 2008

Bacterial diversity and the composition of individual communities during the composting process of swine and mushroom cultural wastes in a field-scale composter (Hazaka system) were examined using a PCR-based approach. The composting process was divided into six stages based on recorded temperature changes. Phylogenetic analysis of eighty 16S rRNA sequences from uncultured composting bacterial groups revealed the presence of representatives from three divisions, including plant pathogenic bacteria, high-molecule-degrading bacteria and spore-forming bacteria. The plant pathogen A. tumefaciens gradually decreased in abundance during the composting process and eventually disappeared during the thermophilic and cooling stage. A bacterium homologous to Bacillus humi first appeared at the early thermophilic stage and was established at the intermediate thermophilic, post-thermophilic, and cooling stages. It was not possible to isolate the B. humi during any of the stages using general culture techniques.

• Journal of Ginseng Research
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• 제41권3호
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• pp.403-410
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• 2017

Background: Prenyltransferases catalyze the sequential addition of isopentenyl diphosphate units to allylic prenyl diphosphate acceptors and are classified as either trans-prenyltransferases (TPTs) or cis-prenyltransferases (CPTs). The functions of CPTs have been well characterized in bacteria, yeast, and mammals compared to plants. The characterization of CPTs also has been less studied than TPTs. In the present study, molecular cloning and functional characterization of a CPT from a medicinal plant, Panax ginseng Mayer were addressed. Methods: Gene expression patterns of PgCPT1 were analyzed by quantitative reverse transcription polymerase chain reaction. In planta transformation was generated by floral dipping using Agrobacterium tumefaciens. Yeast transformation was performed by lithium acetate and heat-shock for $rer2{\Delta}$ complementation and yeast-two-hybrid assay. Results: The ginseng genome contains at least one family of three putative CPT genes. PgCPT1 is expressed in all organs, but more predominantly in the leaves. Overexpression of PgCPT1 did not show any plant growth defect, and its protein can complement yeast mutant $rer2{\Delta}$ via possible protein-protein interaction with PgCPTL2. Conclusion: Partial complementation of the yeast dolichol biosynthesis mutant $rer2{\Delta}$ suggested that PgCPT1 is involved in dolichol biosynthesis. Direct protein interaction between PgCPT1 and a human Nogo-B receptor homolog suggests that PgCPT1 requires an accessory component for proper function.

• Journal of Microbiology and Biotechnology
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• 제13권2호
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• pp.292-300
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• 2003

Pseudomonas fluorescens B16 is a plant glowth-prornoting rhizobacterium, which produces an antibacterial compound that is effective against plant root pathogens, such as Agrobacrerium tumefaciens and Raistonia solanacearum. We mutagenized the strain B16 with Omegon-Km and isolated six antibacterial-activity-deficient mutants. Two cosmid clones that hybridized with the mutant clones also were isolated from a genomic library of tile parent strain. Using deletion and complementation analyses, it was found that the biosynthesis genes resided in a 4.3-kb SalI-NarI fragment. When a plasmid clone carrying the fragment was introduced into P. fluorescens strain 1855.344, which does not exhibit any antibacterial activity, the transconjugants exhibited antibacterial activity, indicating that the plasmid clone carried all the genes essential for production of the antibacterial compound. DNA sequence analysis of the fragment identified four putative open reading frames (ORFs): orf1 through orf4 The deduced amino acid sequences of ORF1, ORF2, and ORF4 were similar to cystathionine gamma lyase, pyruvate formate-lyase activating enzyme, and transcriptional regulator, respectively, yet the amino acid sequence of ORF3 showed no similarities to any known proteins. It was also demonstrated that the antibacterial activity was responsible for biological control of the bacterial wilt caused by R. solanacearum.

• 한국균학회소식:학술대회논문집
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• 한국균학회 2015년도 춘계학술대회 및 임시총회
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• pp.53-53
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• 2015

Agrobacterium tumefaciens-mediated transformation(ATMT) of Flammulina velutipes was used to produce a diverse number of transformants to discover the functions of gene that is vital for its variation color, spore pattern and cellulolytic activity. Futhermore, the transformant pool will be used as a good genetic resource for studying gene functions. Agrobacterium-mediated transformation was conducted in order to generate intentional mutants of F. velutipes strain KACC42777. Then Agrobacterium tumefaciens AGL-1 harboring pBGgHg was transformed into F. velutipes. This method is use to determine the functional gene of F. velutipes. Inverse PCR was used to insert T-DNA into the tagged chromosomal DNA segments and conducting sequence analysis of the F. velutipes. But this experiment had trouble in diverse morphological mutants because of dikaryotic nature of mushroom. It needed to make monokaryotic fruiting varients which introduced genes of compatible mating types. In this study, next generation sequencing data was generated from 28 strains of Flammulina velutipes with different phenotypes using Illumina Hiseq platform. Filtered short reads were initially aligned to the reference genome (KACC42780) to construct a SNP matrix. And then we built a phylogenetic tree based on the validated SNPs. The inferred tree represented that white- and brown- fruitbody forming strains were generally separated although three brown strains, 4103, 4028, and 4195, were grouped with white ones. This topological relationship was consistently reappeared even when we used randomly selected SNPs. Group I containing 4062, 4148, and 4195 strains and group II containing 4188, 4190, and 4194 strains formed early-divergent lineages with robust nodal supports, suggesting that they are independent groups from the members in main clades. To elucidate the distinction between white-fruitbody forming strains isolated from Korea and Japan, phylogenetic analysis was performed using their SNP data with group I members as outgroup. However, no significant genetic variation was noticed in this study. A total of 28 strains of Flammulina velutipes were analyzed to identify the genomic regions responsible for producing white-fruiting body. NGS data was yielded by using Illumina Hiseq platform. Short reads were filtered by quality score and read length were mapped on the reference genome (KACC42780). Between the white- and brown fruitbody forming strains. There is a high possibility that SNPs can be detected among the white strains as homozygous because white phenotype is recessive in F. velutipes. Thus, we constructed SNP matrix within 8 white strains. SNPs discovered between mono3 and mono19, the parental monokaryotic strains of 4210 strain (white), were excluded from the candidate. If the genotypes of SNPs detected between white and brown strains were identical with those in mono3 and mono19 strains, they were included in candidate as a priority. As a result, if more than 5 candidates SNPs were localized in single gene, we regarded as they are possibly related to the white color. In F. velutipes genome, chr01, chr04, chr07,chr11 regions were identified to be associated with white fruitbody forming. White and Brown Fruitbody strains can be used as an identification marker for F. veluipes. We can develop some molecular markers to identify colored strains and discriminate national white varieties against Japanese ones.