• Journal of Plant Biotechnology
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• v.30 no.1
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• pp.13-18
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• 2003

This study was carried out to investigate the expression patterns of foreign gene that controlled by tuber-specific patatin promoter in transgenic potatoes. Potato leaf disc cultured in vitro were transformed by the Agrobacterium strain LBA4404 containing pBl121 or pATGUS from potato cv. Desiree. In order to select the transgenic lines, gene-specific primers deduced from the NPTII were synthesized and used for polymerase chain reaction. The down part of the putative transgenic potatoes was transplanted weekly onto sucrose-enriched medium to accelerate the microtuber formation. RNA gel blot analysis was performed on the total RNAs obtained from tuber that had been harvested at a week interval. Also, histochemical assay was observed in the explants transformed with either pBI121 or pATGUS. Results showed that the transgenic plant containing pATGUS expressed GUS transcripts mainly at the tuber, not in stem, with the highest expression level in 5 weeks-grown microtubers. In contrast to pATGUS plants, the transformed plants with pBI121 showed an equal expression pattern throughout the whole developing stages. Consistent with RNA gel blot analysis, histochemical GUS staining and enzyme activity exhibited pATGUS transcripts were at the highest level in 5 weeks cultures. From these results, we suggest that the best stage to analyze the foreign gene introduced by patatin promoter into potato plants is at 5 weeks cultures after tuber formation.

• Journal of The Korean Society of Grassland and Forage Science
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• v.26 no.2
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• pp.83-90
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• 2006

To develop transgenic birdsfoot trefoil (Lotus corniculatus L.) plants tolerant to environmental stress, Arabidopsis NDPK gene (AtNDPK) was introduced into birdsfoot trefoil plants using Agrobacterium-mediated transformation and expressed powerfully under the control of the E35S promoter. The expression vector, pEN-K was used for introduction of AtNDPK gene into birdsfoot trefoil plaits. The transformed calli were selected on kanamycin containing medium and then regenerated. The transformed birdsfoot trefoil plants were cultivated for 4 months on BOi2Y medium. Genomic DNA PCR and Southern blot analysis confirmed the incorporation of AtNDPK into the birdsfoot trefoil genome.

• Journal of Plant Biotechnology
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• v.29 no.4
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• pp.235-240
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• 2002

We report the new method for the screening of genetically modified potato by competitive duplex-PCR using the potato specific single oligomer primer for the internal control and CaMV 35S promoter or NOS terminator specific primers. The single oligomer primer (rAGU4A) amplify the potato specific internal control band from the homozygous potato genomic DNA in the RAPD profiles of all analyzed potato varieties. The 530 bp internal control DNA was amplified independently to CaMV 35S promoter or NOS terminator DNA and identified as repetitive or microsatellite DNA of potato (AF541972). With this new technique, the transgenic potatoes which were transformed with vectors contained the different foreign genes are analyzed. In case of the commercialized transgenic potato varieties, 'Hew Leafs', those two genetic factors are used for promoter and terminator respectively So, this new PCR technique should be a promising method of cost effective and accurate screening for the commercialized GM potatoes on market.

• Journal of Plant Biotechnology
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• v.34 no.4
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• pp.375-380
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• 2007

This study was conducted to develop herbicide-resistance domestic maize plants by introducing the CP4 5-enol-pyruvylshikimate-3-phosphate synthase (CP4 EPSPS) gene using Agrobacterium tumefaciens-mediated immature embryo transformation. Immature embryos of five genotypes (HW1, KL103, HW3, HW4, HW7) were co-cultivated with strains Agrobacterium tumefaciens (strain C58C1) containing the binary vector (pCAMBIA2300) carrying Ubiquitin promoter-CP4 EPSPS gene and Cauliflower mosaic virus 35S (CaMV35S) promoter-nptll gene conferring resistance to paromomycin as a selective agent. The presence and expression of CP4 EPSPS transgene were confirmed by PCR, RT-PCR and Northern blot analysis, respectively. Also, the resistance to glyphosate in the transgenic maize ($T_1$) was analyzed by shikimate accumulation assay. The frequency (%) of paromomycin-resistance callus was 0.37, 0.03, 2.20, 2.37, and 0.81% in pure lines HW1, KL103, HW3, HW4 and HW7, respectively. EPSP transgene sequences were amplified in putative transgenic plants that regenerated from paromomycin-resistance calli of two inbred lines (HW3, HW4). Of them, RT-PCR and Northern blot analyses revealed that the transgene was only expressed in two transgenic events (M266, M104) of HW4 inbred line, and a mild glyphosate resistance of transgenic event (M266) was confirmed by the lower shikimate accumulation in leaf segments. These results demonstrate that transgenic maize with herbicide-resistance traits in Korean genotype can be genetically obtained.

• Journal of Microbiology and Biotechnology
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• v.25 no.8
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• pp.1241-1245
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• 2015

The aim of the present work was to examine the putative promoter region of the operon ansPAB and to determine the general elements required for the regulation of transcriptional activity. The transcriptional start site of the ansPAB promoter was determined by using highresolution S1-nuclease mapping. Sequence analysis of this region showed -10 and -35 elements, which were consistent with consensus sequences for R. etli promoters that are recognized by the major form of RNA polymerase containing the σ⁷⁰ transcription factor. Mutation studies affecting several regions located upstream of the transcriptional start site confirmed the importance of these elements on transcriptional expression.

• Korean Journal of Microbiology
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• v.35 no.4
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• pp.266-269
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• 1999

Bacteriophage E3 grows very rapidly and forms a large size plaque with a diameter of 1 cm. The promoter controlling the expression of the gene encoding the major capsid protein is thought to be most efficient. To find out this promoter, this gene was mapped in the genome according to the following procedure. The major capsid protein was purified from phage particle and the N-terminal amino acid sequence was revealed. Based on this sequence,a degernerate oligonucleotide probe was designed and used for screening of the genomic DNA fragments. From the DNA sequence of the selected clone, the gene encoding the major capsid protein was mapped at 70% of E3 genome. The expression of this gene was not sensitive to rifampicin which indicated the presence of E3's own RNA polymerase.

• Journal of Plant Biotechnology
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• v.32 no.4
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• pp.263-268
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• 2005

Porcine epidemic diarrhea virus (PEDV) causes acute enteritis in pigs of all ages and is often fatal for neonates. In order to develop sweetpotato plants expressing PEDV antigen, we constructed the vector expressing spike gene of PEDV under the control of sweetpotato sporamin promoter or constitutive CaMV 35S promoter. The spike protein region of PEDV was synthesized by PCR and linked to each promoter, Transgenic sweetpotato [Ipomoea batatas (L.) Lam. cv. Yulmi] plants were developed from embryogenic calli following Agrobacterium tumefaciens-mediated transformation. The co-cultured embryogenic calli transferred to selective MS medium containing 1 mg/L 2,4-D, 100 mg/L kanamycin, and 400 mg/L claforan. These embryogenic calli were subcultured to the same selection medium at 3 weeks interval. Kanamycin-resistant calli transferred to hormone-free MS medium with kanamycin gave rise to somatic embryos and then converted into plantlets in the same medium. Southern blot analysis confirmed that the spike gene of PEDV was inserted into the genome of the sweetpotato plants. RT-PCR revealed that the spike gene of PEDV was highly expressed in transgenic sweetpotato plants.

• Biotechnology and Bioprocess Engineering:BBE
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• v.11 no.2
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• pp.154-159
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• 2006

Interleukin-18 (IL-18), otherwise known as interferon-gamma-inducing factor (IGIF), is one of several well characterized and important cytokines that contribute to host defenses. The complementary DNA (cDNA) of mature human interleukin-18 gene (hIL-18) was fused with the signal peptide of the rice amylase 1A gene (Ramy1A) and introduced into the plant expression vector under the control of a duplicated CaMV 35S promoter. The recombinant plasmid was transformed into tobacco (Nicotiana tabacum L. cv Havana) using the Agrobacterium-mediated transformation method. The integration of the hlL-18 gene into the genome of transgenic tobacco plants was confirmed by polymerase chain reaction (PCR) amplification and its expression was observed in the suspension cells that were derived from the transgenic plant callus by using Northern blot analysis. The hlL-18 protein was detected in the extracts of the transgenic callus and in the medium of the transgenic tobacco suspension culture by using immunoblot analysis. Based upon enzyme-linked immunosorbant assay (ELISA) results, the expression level of the hlL-18 protein approximated $166{\mu}g/L$ in the suspension culture medium. Bioassay results from the induction of $interferon-{\gamma}$ from a KG-1 cell line indicated that the hlL-18 secreted into the suspension culture medium was bioactive.

• KSBB Journal
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• v.17 no.6
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• pp.577-581
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• 2002

In an attempt to produce recombinant proteins using the pollen enriched in some plant species, a 1.7 kb DNA encoding urease subunit B (UreB) amplified by PCR from Helicobacter pylori urease gene cluster in pH808 plasmid was cloned to be expressed under CaMV35S promoter in lily (Lilium longiflorum) pollen tubes elongated in vitro. Lily pollen at early germinating stage was transformed with the ureB DNA using Agrobacterium via vacuum infiltration and, incubated for a full pollen tube growth 16 - 24 h in the dark in the presence of kanamycin. DNA integration and expression in the transgenic pollen were analyzed by the standard molecular techniques and the results suggest that the pollen in vitro may be employed as a protein factory in a disposable fashion.

• Molecules and Cells
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• v.19 no.1
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• pp.81-87
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• 2005

Phytochelatins play an important role in heavy metal detoxification in plants as well as in other organisms. The Arabidopsis thaliana mutant cad1-3 does not produce detectable levels of phytochelatins in response to cadmium stress. The hypersensitivity of cad1-3 to cadmium stress is attributed to a mutation in the phytochelatin synthase 1 (AtPCS1) gene. However, A. thaliana also contains a functional phytochelatin synthase 2 (AtPCS2). In this study, we investigated why the cad1-3 mutant is hypersensitive to cadmium stress despite the presence of AtPCS2. Northern and Western blot analyses showed that expression of AtPCS2 is weak compared to AtPCS1 in both roots and shoots of transgenic Arabidopsis. The lower level of AtPCS2 expression was confirmed by RT-PCR analysis of wild type Arabidopsis. Moreover, no tissue-specific expression of AtPCS2 was observed. Even when AtPCS2 was under the control of the AtPCS1 promoter or of the cauliflower mosaic virus 35S promoter (CaMV 35S) it was not capable of fully complementing the cad1-3 mutant for cadmium resistance.