• Korean Journal of Plant Tissue Culture
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• v.27 no.2
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• pp.95-100
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• 2000

The cytosolic glutathione reductase (GR) gene of Brassica campestris L. was introduced into several Japonica cultivars of rice by Agrobacterium tumefaciens and a large number of transgenic plants were produced. Three-week old calli were co-cultivated with A. tumefaciens strain EHA101 carrying the plasmid pIGR1. The efficiency of transformation was differed from rice cultivars. A Japonica cultivar, 'Daeribbyeo' appeared the highest efficiency (42.5%) of transformation among the four cultivars tested. The addition of acetosyringone (50 $\mu$M) during co-cultivation was a key to successful transformation. Transgene fragments were identified by PCR amplification and further confirmed by Southern blot analysis. Mendelian inheritance of the transgenes was confirmed in T$_1$ progeny.

• Environmental Analysis Health and Toxicology
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• v.20 no.1
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• pp.67-73
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• 2005

오염물질의 노출에 의해 발현이 변화되는 유전자의 발굴은 외부환경 자극에 대한 적응이나 반응의 메커니즘을 알아내는뎨 중요한 정보를 제공하며, 오염물질에 반응하는 유전자는 환경오염을 감지하는 분자 마커로 개발될 수 있다. DD-PCR 기법은 차등 발현 유전자들을 발굴해내기 위한 유용한 방법으로 사용되어 왔고, 본 연구는 이 방법을 이용하여 벤조피렌에 반응하는 조피볼락 유전자들의 발굴을 목적으로 진행되었다. 간조직에서 추출한 RNA로부터 벤조피렌의 노출에 의해 발현 양이 달라진 12개의 클론을 발굴하였고, 그 염기서 열을 분석하였다. 또한 벤조피렌의 노출시간을 각각 6, 12, 24시간으로 달리한 조피볼락에서 12개의 클론 중 4개의 클론에 대해 northern blot 분석이 실시되었으며, 이들 모두 노출시간에 따 라 발현양이 증가 또는 감소하는 것이 확인되었다. 본 연구결과는 오염물질의 영향에 의한 유전자들의 발현에 관한 전반적인 지식을 제공하였고, 나아가 환경오염이나 외부 스트레스를 감지해 낼 수 있는 바이오마커의 개발을 위한 첫 단계로서의 정보를 제공할 수 있을 것으로 생각된다.

• Korean Journal of Plant Tissue Culture
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• v.25 no.5
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• pp.341-346
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• 1998

In this paper we describe the cloning and expression of the genes encoding the flavonoid-biosynthetic enzyme dihydroflavonol 4-reductase (DFR) in Matthiola incana R. Br. A heterologous cDNA probe from Zea mays was used to isolate full-size DFR cDNA clone from a corolla-specific cDNA library. Comparison of the coding region of this DFR cDNA sequence including the sequences of Zea mays, Anthirrinum majus, Petunia hybrida, Callistephus chinensis, Dianthus caryophyllus and Rosa hybrida reveals a identity higher than 61% at the nucleotide level. The DFR transcript is G/C rich in monocotyledonous plants show a strong codon bias preferring codons with a G or C in the third position. The function of this nucleotide sequences were verified by comparison with amino acid sequences of the amino-terminus and tryptic peptides from purified plant enzyme, by northern blotting with mRNA from wild type and mutant plants and by in vitro expression yielding an enzymatically active reductase. Genomic southern blot analysis showed the presence of one gene for DFR in Matthiola incana. Northern blot analysis of the DFR wild type and mutant lines showed that the lack of DFR activity in the stable acyanic mutant k17b is clearly by a transcriptional block of the DFR gene.

• Applied Biological Chemistry
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• v.40 no.6
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• pp.495-500
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• 1997

To isolate a gene for cyclodextrin glycosyltransferase (CGTase) from alkalophilic Bacillus sp. E1, polymerase chain reaction (PCR) amplification was carried out. Direct molecular cloning of 1.2 kbp fragment and partial nucleotide sequence analysis of the PCR amplified clone, pH12, showed close homology with CGTases from Bacillus species. To investigate the genomic structure of the gene, Southern blot analysis of genomic DNA was carried out with the clone pH12 as a molecular probe. It showed that 5.3 kbp XbaI fragment was hybridized with the probe pH12. To isolate a genomic clone, genomic DNA library was constructed and a genomic clone for CGTase, pCGTE1, was isolated. Nucleotide sequence analysis of the clone pCGTE1 revealed that BCGTE1 contained 2,109 bp open reading frame encoding a polypeptide of 703 amino acids and showed over 94.3% amino acid sequence homology with CGTase of ${\beta}-cyclodextrin$ producer, Bacillus sp. KC201.(Received October 7, 1997; accepted October 20, 1997)

• Korean Journal of Plant Tissue Culture
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• v.25 no.2
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• pp.109-113
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• 1998

In this study we tried to transform an antimicrobial peptide gene (Shiva) under the promoter of tomato phenylalanine ammonia-lyase (tPAL5) into tobacco and potato plants. Antimicrobial peptide gene was isolated originally from giant silk moth (Hyalophora cecropia) and modified ie nucleotide sequence to increase antimicrobial activity. Transgenic tobacco plants were regenerated and their seeds were tested on the media containing kanamycin (500 mg/L). The results of PCR amplification and genomic Southern blot hybridization confirmed the integration of construct (tPAL5 promoter-Shiva-NOS-GUS-NOS) into chromosome. We observed that one of the transgenic tobacco plants showed chromosome rearrangement when integrated. In case of potato transformation, the efficiency of regeneration was maximized at the medium containing Zeatin 2mg/L, NAA 0.01mg/L, GA$_3$ 0.1mg/L. We also observed the high expression of GUS (${\beta}$-glucuronidase) enzyme which was located next to the terminator sequence of nopaline synthase gene (NOS) in the vascular tissue of stem, leaves of transgenic potatoes. This result suggested that a short sequence of Shiva gene (120 bp) and NOS terminator sequence might be served as a leader sequence of transcript when translated.

• Korean Journal of Breeding Science
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• v.42 no.5
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• pp.534-539
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• 2010

'Hongro' is early-mid maturing cultivar with good quality like 'Tsugaru' and it has not preharvest drop. The CAX1 gene was introduced into Korean apple cultivar 'Hongro' by Agrobacterium tumefaciens LBA4404 harboring pBI121 to obtain transgenic apple with enhanced Ca level. The CAX1 gene playing the role of $H^+/Ca^{2+}$ transporter from Arabidopsis thaliana increases Ca concentration in several plants. Regenerated transgenic lines were confirmed by polymerase chain reaction (PCR) analysis and Southern blot analysis of genomic DNA for the existence of CAX1 gene. Southern blot analysis of 'Hongro' transformants showed that two putative transgenic lines were integrated with CAX1 gene in genomic DNA. The CAX1 comparative expression levels of two transgenic lines were higher than that of non-transformant evaluated by comparative quantification analysis using a real-time PCR. These two lines were multiplied in vitro, and micro-grafted on apple rootstocks 'M.9' in the isolated greenhouse. Since two years after micro-grafting, the fruits came into bearing. Compared to Ca level of the non-transgenic 'Hongro', that of the CAX1 transgenic 'Hongro' in the flesh and leaves was higher.

• Journal of Plant Biotechnology
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• v.38 no.3
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• pp.209-214
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• 2011

Transgenic lettuce plants were successfully obtained from hypocotyl explants inoculated with Agrobacterium tumefaciens, which harbored a binary vector plasmid with Bcl-2 gene, related to apoptosis. After culture and selection on MS medium a number of kanamycin-resistant plantlets were regenerated. Polymerase chain reaction, Southern blot analysis and Northern blot analysis were used to identify and characterize the transgenic plants with the integrated Bcl-2 gene. Over 100 transgenic plants have been established in soil and flowered in the greenhouse. T1 progeny of 100 transgenic lettuce inbred lines were inoculated with Sclerotinia sclerotiorum. Expression of the Bcl-2 peptide in transgenic lettuce plants provides high levels of field resistance against Sclerotinia sclerotiorum, causal agent of the agronomically important fungal disease of lettuce.

• The Korean Journal of Mycology
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• v.30 no.1
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• pp.66-72
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• 2002

To develop DNA markers for analysis of genetic characteristics of Phytophthora capsici population, randomly selected clones from HindIII-digested genomic DNA library of P. capsici 95CY3119 were surveyed by hybridizing to Southern blots of HindIII-digested total genomic DNA of P. capsici. Probe DNAs inserted into selected individual clones strongly hybridized with HindIII digests of P. capsici. Among probes examined, PC9 revealed the repetitive and highly polymorphic bands to HindIII digests of inter-and intra-field P. capsici isolates. Genetic diversity of individual isolates was also clearly revealed in cluster analysis based on its band patterns. The other probe, PC22, was hybridized only to DNA from P. capsici and this was highly repetitive. However, there was no response to other Phytophthora species and Pythium sp. These DNA probes could be used as very useful markers in analysing genetic diversity and identification for P. capsici population throughout the world.

• Microbiology and Biotechnology Letters
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• v.22 no.1
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• pp.37-44
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• 1994

The homology among the genes coding for degradation of bipheny(BP) and 4-chlorobiphenyl(4CB) was comparatively analyzed by Southern hybridization in several BP/4CB degrading bacterial strains. As the hybridization results of their genomic DNAs with pcbABCD as the DNA probe, the group of Pseudomonas sp. DJ-12. P08 and P27 strain was separated by the group of P20 and P1242 strains. The P. pseudoalcaligenes KF707 showed the hybidization signal which was homologous to the group of DJ-12, but they had different restriction endonuclease sites. The pcbAB genes in pCUl recombinant plasmid from Pseudomonas sp. DJ-12 appeared to be homologous to pchAB genes in pKTF20 cloned from P. pseudoalcaligenes KF707, but the C genes in both strains were not homologous. The bphABC in pKTF20 showed the signals homologous to the cbp ACB in pAW6194 cloned from P. putida OU83, but homologous signal was not found botween the pcbABCD genes in pCUl and the cbpADCB genes in pAW6194 recombbinant plasmid.

• Applied Biological Chemistry
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• v.36 no.3
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• pp.208-214
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• 1993

To investigate the gene rearrangement via short repeated sequences in chloroplast DNA, the pattern of heterologous gene clusters containing the 151 bp repeated sequence with the development of plastid was compared in rice and the homologous gene clusters from various plant sources were searched for comparative analysis. Southern blot analysis of rice DNA using rp12 gene containing 151 bp repeated sequence as a probe showed the presence of heterologous gene clusters. Such heterologous gene clusters varied with the development of plastid. Also it was observed that the heterologous gene clusters were observed in all of the rice cultivars used in this work. Finally the comparative analysis of DNA sequence of the homologous gene clusters from various plants showed the evolutionary gene rearragngement via short repeated sequence among plants. These results suggest the possible relationship between the plastid development and gene rearrangement through short repeated sequences.