• Korean Journal of Plant Tissue Culture
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• v.25 no.3
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• pp.201-206
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• 1998

To develop the fruits of cucumber (Cucumis sativus L.) producing high yields of superoxide dismutase (SOD), the MnSOD cDNA from pea (Pisum sativum) under the control of the cauliflower mosaic virus 35S promoter was introduced into cucumber using Agrobacterium tumefaciens (strain LBA 4404)-mediated transformation. The kanamycin-resistant shoots were selected on the selection medium containing MS basal salt, 1.0 mg/L zeatin, 0.1 mg/L IAA, 300 mg/L claforan, and 100 mg/L kanamycin. After 6 weeks of culture on the selection medium, the shoots were transferred to MS medium containing 0.2 mg/L NAA to induce roots. PCR analysis using the primers for neomycin phosphotransferase (NPTII) gene revealed that three plantlets were transformed. The fruits of one transgenic plant had approximately 3.2-fold higher SOD activity than those of non-transgenic plants. MnSOD isoenzyme band was strongly detected on native gel in fruits of transgenic plants.

• Proceedings of the Korean Society of Plant Pathology Conference
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• 1994.06a
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• pp.86-102
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• 1994

To understand the molecular structure and pathogenesis mechanism of Korean garlic viruses, we have isolate cDNA clones for garlic viruses. The partial nucleotide sequences of 24 cDNA clones were determined and that of six clones containing poly (A) tail were compared with those of other plant viruses. One of those clones, V9 has 81.8% similarity in nucleotide sequence and 93.0% in deduced amino acid sequence, respectively, to the coat protein gene for garlic mosaic virus (GMV). Northern blot analysis with the clone V9 demonstrated that the genome of GMV is 7.8 kb long and has poly (A) tail. The anti-coat protein antibody for GMV recognizes 35 kDa polypeptide which could be the coat protein of GMV from infected garlic leaf extract or virus preparation. Clone G7 has about 62% of deduced amino acid sequence identity with the members of potyvirus group. Northern blot analysis with the clone G7 demonstrated that the genome of the potyvirus I garlic is 9.0 kb long and has poly (A) tail. The third clone, S81, shows 42% amino acid identity to the potexvirus. The other clones are under the characterization. To test the possibility of producing garlic virus resistant plant, we have designed a hairpin type ribozyme to cleave V9 RNA at the middle of the coat protein gene. From the cleavage reactions in vitro with two different sizes of RNA substrates, V9SUB (144 nucleotides) and V9 RNA (1,361 nucleotides), the ribozyme can cleave V9 sequence effectively at the predicted site. To study the activity of the ribozyme in vivo, plant transformation is in progress. Further possibilities to produce garlic virus resistant plant will be discussed.

• 한국균학회소식:학술대회논문집
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• 2015.05a
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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.

• Microbiology and Biotechnology Letters
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• v.33 no.2
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• pp.96-105
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• 2005

For screening of autoregulator receptor gene from Streptomyces longwoodensis, PCR was performed with primers of receptor gene designed on the basis of amino acid sequences of autoregulator receptor proteins with known function. PCR products were subcloned into the BamHIsite of pUC19 and transformed into the E. coli $DH5{\alpha}$. The isolated plasmid from transformant contained the fragment of 100 bp, which was detected on $2\%$ gel after BamHI treatment. The insert, 100 bp PCR product, was confirmed as the expected internal segment of gene encoding autoregulator receptor protein by sequencing. Southern and colony hybridizations with the 100 bp fragment as a probe allowed to select a genomic clone of S. longwoodensis, pSLT harboring a 4.4 kb SphI fragment. Nucleotide sequencing analyses revealed a 651 bp open reading frame(ORF) were isolated protein showing moderate homology ($35{\sim}46\%$) with the ${\Gamma}$-butyrolactone autoregulator receptors from Streptomyces sp., and this ORF was named sltR The sltR/pET-17b plasmid was constructed to overexpress the recombinant SltR protein (rSltR) in E. coli BL21 (DE3)/pLysS, and the rSltR protein was purified to homogeneity by DEAE-Sephacel column chromatography, and DEAE-5PW chromatography (HPLC). The molecular mass of the purified rSltR protein was 55 kDa by HPLC gel-filtration chromatography and 28 kDa by SDS-PAGE, indicating that the rSltR protein is present as a dimer. A binding assay with tritium-labeled autoregulators revealed that the rSltR has clear binding activity with a A-factor type autoregulator as the most effective ligand.

• Korean Journal of Plant Resources
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• v.16 no.1
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• pp.74-81
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• 2003

To obtain multi-shoot using zygotic embryos dissected from ginseng seed, the embryos were cultured on MS medium supplemented with CPA and BA. Effective multi-shoot induction was achieved on 0.5mg/ t CPA and 1.0mg/ t BA treatment. Among the various plant growth regulator treatment, MS basal medium with 1mg/ t 2,4-D and 0.5mg/ t kinetin was more competent and could be induced 4∼6 shoots per one embryo. Also, the best condition for pre-embryoid induction from ginseng cotyledon so as to ginseng transformation appeared to 1mg/ t 2,4-D and 0.5mg/ t kinetin treatment. The kanamycin level to select transformants varied greatly by different explant tyues. The petiole explants with leaf and embryo could survived up to 100$\mu\textrm{g}$ / ml kanamycin concentration where as petiole explants without leaf died all at the level. Conclusionally, our results suggest that optimum kanamycin concentration for ginseng transformation using somatic embryos is about 75∼100$\mu\textrm{g}$ / ml concentration.

• Korean Journal of Medicinal Crop Science
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• v.15 no.4
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• pp.285-290
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• 2007

This study was conducted to introduce phosphinothricin acetyl transferase (PAT) gene, resistant to basta which was non-selective herbicide, into balloon flower (Platycodon grandiflorum A. De. candolle). Seeds were germinated on MS medium, and 10-day-old immature cotyledon explants and 30-day-old leaf explants were cocultured with Agrobacterium tumefaciens strain MP 90 (pBinSyn) on 1/10 MS medium for 48 hours in the dark at $25^{\circ}C$. The cultures were transferred for selection of kanamycin-resistant shoots to the MS medium supplemented with 0.2 $mg/{\ell}$ NAA, 1.0 $mg/{\ell}$ BA, 3% sucrose, 100 $mg/{\ell}$ kanamycin, 500 $mg/{\ell}$ carbenicillin. Shoots were obtained from 10-day-old immature cotyledon explants after 4 weeks of culture. The shoots were subcultured twice every 4 weeks on the same medium for growth of transgenic shoots. Successful transformation was confirmed by histochemical GUS assay, PCR analysis, RT-PCR analysis, 10 $mg/{\ell}$ phosphinothricin treatment and 0.3% basta spray. The basta-resistant transgenic plants flowered normally.

• Korean Journal of Microbiology
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• v.34 no.3
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• pp.120-125
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• 1998

A plasmid pNPG contains a genomic DNA complementing npgA1 which is located on the left arm of linkage group I. It transformed Aspergillus nidulans at a high frequency. No abortive transformants were observed and the $Trp^+$ transformants were all $Npg^+$. The 10.4 kb Psti fragment of the genomic DNA was subcloned into pILJ16, which increased the transformation efficiency by more than 200-folds. The transformants were mitotically unstable and yielded $Arg^-$ conidia at the frequency of more than 80%. An additional gene cloned into the plasmid containing the fragment was always lost with $argB^+$ marker. These characteristics strongly indicate the possibility that the plasmids autonomously replicate. The full activity of enhanced transformation was retained on the 4.9 kb EcoRI-HaeIII fragment. The DNA segment was similar to AMA1 rather than ANS1 in function and designated AMA2.

• Plant Biotechnology Reports
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• v.4 no.4
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• pp.253-260
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• 2010

Plant secondary metabolites have always been a focus of study due to their important roles in human medicine and nutrition. We transferred the isoflavone synthase (IFS) gene into soybean [Glycine max (L.) Merr.] using the Agrobacterium-mediated transformation method in an attempt to produce transformed soybean plants which produced increased levels of the secondary metabolite, isoflavone. Although the trial to produce transgenic plant failed due to unestablished hygromycin selection, transformed callus cell lines were obtained. The induction rate and degree of callus were similar among the three cultivars tested, but light illumination positively influenced the frequency of callus formation, resulting in a callus induction rate of 74% for Kwangan, 67% for Sojin, and 73% for Duyou. Following seven to eight subcultures on selection media, the isoflavone content of the transformed callus lines were analyzed by high-performance liquid chromatography. The total amount of isoflavone in the transformed callus cell lines was three- to sixfold higher than that in control callus or seeds. Given the many positive effects of isoflavone on human health, it may be possible to adapt our transformed callus lines for industrialization through an alternative cell culture system to produce high concentrations of isoflavones.

• Korean Journal of Microbiology
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• v.51 no.3
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• pp.256-262
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• 2015

In order to study the effect of the receptor protein (SeaR), which is isolated from Saccharopolyspora erythraea, we introduced the SeaR gene to Streptomyces virginiae as host strains. An effective transformation procedure for S. virginiae was established based on transconjugation by Escherichia coli ET12567/pUZ8002 with a ${\varphi}C31$-derived integration vector, pSET152, which contained int, oriT, attP, and $ermEp^{\ast}$ (erythromycin promotor). Therefore, the pEV615 was introduced into S. virginiae by conjugation and integrated at the attB locus in the chromosome of the recipients by the ${\varphi}C31$ integrase (int) function. Transformants of S. virginiae containing the SeaR gene were confirmed by PCR and transcriptional expression of the SeaR gene in the transformants was analyzed by RT-PCR, respectively. And, we examined the production time of virginiamycins in the culture media of both the transformants and the wild type. The production time of virginiamycins in the wild type and transformants was the same. When 100 ng/ml of synthetic $VB-C_6$ was added to the state of 6 or 8 hour cultivation of wild type and transformants, respectively, the virginiamycins production was induced, meaning that the virginiamycins production in the wild type was detected 2 h early than transformants. From these results, SeaR expression was also affected to virginiamycins production in transformants derived from S. virginiae. In this study, we showed that the SeaR protein worked as a repressor in transformants.

• Journal of Plant Biotechnology
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• v.7 no.4
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• pp.233-240
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• 2005

Slices of embryonic axis of mature pea (Pisum sativum L. cv. Green Arrow) seeds were used as explant. Transformation of explants was done via Agrobacterium tumefaciens bearing vector pBI-P5CS construct. The best results for inoculation of explants were obtained when they were immersed for 90 s at a concentration of $6{\times}10^8$ cell $ml^(-1)$ of bacterial suspension. Transformed pea plants were selected on $50\;mg\;l^(-1)$ kanamycin and successful transformants were confirmed by PCR and blotting. Transgenic plants were further analyzed with RT-PCR to confirm the expression of P5CS. Transgenic plants and non-transgenic plants were treated with different concentrations of NaCl 0 (control), 100, 150 and 200 mM in culture medium. Measurement of proline content indicated that transgenic plants produced more amino acid proline in response to salt in comparison with non-transgenic plants. Photosynthetic efficiency in transgenic plants under salt-stress was more than that of non-transgenic plants.