• Title/Summary/Keyword: Genomic Southern blot analysis

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Isolation of a Rice Genomic Clone Encoding Ribulose-1,5-bisphosphate Carboxylase (리블로스 1,5- 이인산 탄산화효소 유전자의 분리 및 특성규명)

  • Park, Sung-Soon;Kim, Hee-Jin;Kim, Chung-Ho;Kim, Han-Jip;Lee, Jong-Seob;Lee, Kwang-Woong;Choi, Yang-Do
    • Applied Biological Chemistry
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    • v.37 no.5
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    • pp.361-369
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    • 1994
  • To study the light-induced expression mechanism and protein transport into the chloroplast, a rice genomic clone (GrbcS) for the small subunit of ribulose 1,5-bisphosphate carboxylase (rbcS) was isolated and its nucleotide sequence was determined. Nucleotide sequence analysis of GrbcS revealed that the gene consists of two exons interrupted by an intron, encoding a protein of 175 amino acids including a transit peptide of 47 amino acids. These structural features of GrbcS are consistent with those of other rbcS genes from monocot species. Genomic Southern blot analysis suggested that the rbcS genes are present as a relatively small multigene family in the rice genome. Comparison of the nucleotide and deduced amino acid sequences to other rice rbcSs shows close sequence similaritiy. Conserved DNA sequences present in other light-responsive genes are also found in the 5’ upstream region of GrbcS such as G-box, 3AF1-binding site and GATA site. The possible function of these putative regulatory elements are discussed.

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The Structural Characterization of the Putative DNA-Binding Protein BldB from Streptomyces Lividans

  • Ochiriin, Tsogbadrakh-Mishig;Kang, Sa-Ouk
    • Proceedings of the Korean Biophysical Society Conference
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    • 2002.06b
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    • pp.49-49
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    • 2002
  • Mutants blocked at the earliest stages of morphological development in Streptomyces species are called bld mutants. We have cloned bldB gene ORF from Slividans. Genomic Southern blot analysis for main strains S.lividans, S.seoulensis, S.coelicolor A3(2), and S.griseus indicated that bldB gene is conserved in all main Streptomyces strains.(omitted)

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Transposon Tn5 Mutagenesis of Bradyrhizobium japonicum: A Histidine Auxotrophic Mutant of B. japonicum Shows Defective Nodulation Phenotype on Soybean

  • So, Jae-Seong
    • Journal of Microbiology and Biotechnology
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    • v.5 no.2
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    • pp.110-113
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    • 1995
  • Transposon Tn5 was used to induce random insertional mutations in Bradyrhizobium japonicum, a soybean endosymbiont. By genomic Southern blot analysis, transposition events were found to have occurred randomly throughout the B. japonicum genome. After screening 3, 626 mutants by auxotrophy test, a histidine auxotroph was isolated. Upon plant infection test, the His mutant showed a 3~4 day delay in nodule formation.

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Differential Expression of Three Catalase Genes in the Small Radish (Rhaphanus sativus L. var. sativus)

  • Kwon, Soon Il;Lee, Hyoungseok;An, Chung Sun
    • Molecules and Cells
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    • v.24 no.1
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    • pp.37-44
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    • 2007
  • Three catalase cDNA clones were isolated from the small radish (Raphanus sativus L.). Their nucleotide and deduced amino acid sequences showed the greatest homology to those of Arabidopsis. Genomic Southern blot analysis, using RsCat1 cDNA as a probe, showed that catalases are encoded by small multigene family in the small radish. Nondenaturing polyacrylamide gels revealed the presence of several catalase isozymes, the levels of which varied among the organs examined. The isozyme activities were assigned the individual catalase genes by Northern analysis using total RNA from different organs. The three catalase genes were differentially expressed in response to treatments such as white light, xenobiotics, osmoticum, and UV. Their expression in seedlings was controlled by the circadian clock under a light/dark cycle and/or in constant light. Interestingly, RsCat1 transcripts peaked in the morning, while those of RsCat2 and RsCat3 peaked in the early evening. Our results suggest that the RsCat enzymes are involved in defense against the oxidative stress induced by environmental changes.

A cDNA Clone for the 5' Exon of Chloroplast ATP Synthase Subunit I Gene (atpF) from Broccoli (Brassica oleracea L. var. Italica) and Its Expression Pattern

  • Choo Bong Hong
    • Journal of Plant Biology
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    • v.38 no.2
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    • pp.137-141
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    • 1995
  • We isolated a cDNA clone, BLSC1, encoding 5' exon of ATP synthase CF0 subunit I from broccoli. BLSC1 is 285 nucleotides long which consists of a 5' noncoding region of 34 nucleotides, a 5' exon of 145 nucleotides and an intron of 106 nucleotides. The 5' exon codes for 48 amino acids which reveals mostly hydrophobic. The amino acid sequence deduced from BLSC1 shares 83%, 83% and 91% identities with the genes coding for atpF from wheat, rice and spinach, respectively. Genomic Southern blot analysis for BLSC1 showed a typically strong signal for a gene located in the chloroplast genome. Northern blot analysis identified three major classes of transcripts showing strong positive signals in the leaves, but only trace amounts of the transcripts were identified in the other organs like stems, flowr buds and roots.

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Transformation of Orchardgrass (Dactylis glomerata L.) with Glutathione Reductase Gene (Glutathione Reductase 유전자의 도입에 의한 오차드그래스의 형질전환)

  • 이효신;배은경;김기용;원성혜;정민섭;조진기
    • Journal of The Korean Society of Grassland and Forage Science
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    • v.21 no.1
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    • pp.21-26
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    • 2001
  • To develop transgenic orchardgrass resistant to reactive oxygen species produced from environmental stresses, a vector with the cytosolic glutathione reductase cDNA (BcGRl) from Chinese cabbage was constructed under the control of the cauliflower mosaic virus 35S promoter and was introduced into orchardgrass using Agrobacterium tumefaciens EHA101. Transgenic plants from hygromycin-selected calli of orchardgrass did not show any morphological difference from wild-type plants. The results of PCR amplification and genomic Southern blot analysis confirmed the integration of foreign gene into the chromosome of transgenic orchardgrass. Northern blot analysis with total RNA from leaves also confirmed the constitutive expression of BcGR1 in transgenic orchardgrass.

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Molecular Characterization of a Chinese cabbage cDNA, C-DH, Predominantly Induced by Water-Deficit Stress and Plant Hormone, ABA (수분부족 및 식물호르몬, ABA에 의하여 발현이 유도되는 배추의 C-DH cDNA에 대한 분자적 특성)

  • 정나은;이균오;홍창휘;정배교;박정동;이상열
    • Korean Journal Plant Pathology
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    • v.14 no.3
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    • pp.240-246
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    • 1998
  • A cDNA encoding desiccation-related protein was isolated from a flower bud cDNA library of Chinese cabbage (C-DH) and its nucleotide sequence was characterized. It contains 679 bp nucleotides with 501 bp open reading frame. The amino acid sequence of the putative protein showed the highest amino acid sequence homology (79 % identity) to dehydrin protein in Gossypium hirsutum. Also, the C-DH shares 48-52% amino acid sequence identity with the other typical dehydrin proteins in plant cells. When the amino acid sequence of their proteins were aligned, several peptide motifs were well conserved, of which function has to be solved. Particularly the C-DH contains 15 additional amino acids at its N-terminus. Genomic Southern blot analysis using the coding region of C-DH showed that the C-DH consists of a single copy gene in Chinese cabbage genome. The C-DH mRNA, whose transcript size is 0.7 kb, was expressed with a tissue-specific manner. It was highly expressed in seed, flower buds and low expression as detected in root, stem or leaf tissues of Chinese cabbage. And the transcript level of C-DH was significantly induced by the treatment of plant hormone, abscisic acid and water-deficit conditions.

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Cloning And Characterization of Pathogen-Inducible EREBP-Like Transcription Factor(CaNR19) From Hot Pepper (Capsicum annuum L.)

  • Yi, So-Young;Kim, Jee-Hyub;Yu, Seung-Hun;Park, Doil
    • Proceedings of the Korean Society of Plant Pathology Conference
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    • 2003.10a
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    • pp.77.2-78
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    • 2003
  • An EREBP/AP2-type transcription factor (CaPFl) was isolated by DDRT-PCR following inoculation of soybean pustule pathogen Xanthomonas axonopodis pv. glycines Bra which induces HR on pepper leaves. Genomic Southern blot analysis revealed that the CaPFl gene is present as a single copy within the hot pepper genome. The deduced amino acid sequence of CaPFl has two potential nuclear localization signals, a possible acidic activation domain, and an EREBP/AP2 motif that could bind to a conserved cis- element present in promoter region of many stress-induced genes. The mRNA level of CaPFl was induced by both biotic and abiotic stresses. We observed higher-level transcripts in resistance-induced pepper tissues than diseased tissues. Expression of CaPFl is also induced upon various abiotic stresses including ethephon, MeJA, cold stress, drought stress and salt stress treatments. To study the role of CPFI in plant, transgenic Arabidopsis and tobacco plants which express higher level of pepper CaPFl were generated. Global gene expression analysis of transgenic Arabidopsis by cDNA microarray indicated that expression of CaPFl in transgenic plants affect the expression of quite a few GCC box and DRE/CRT box-containing genes. Furthermore, the transgenic Arabidopsis and tobacco plant, expressing CaPFl showed tolerance against freezing temperature and enhanced resistance to Pseudomonas syrnigae pv. tabaci. Taken together, these results indicated that CaPFl is a novel EREBP/AP2 transcription factor in hot pepper plant and it may has a significant role(s) in regulation of biotic and abiotic stresses in plant.

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Silencing of CaCDPK4 ( Capsicum annuum Calcium Dependent Protein Kinase) and ItsOrtholog, NbCDPK5 Induces Cell Death in Nicotiana benthamiana

  • Eunsook Chung;Kim, Young-Cheol;Oh, Sang-Keun;Younghee Jung;Kim, Soo-Yong;Park, Doil
    • Proceedings of the Korean Society of Plant Pathology Conference
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    • 2003.10a
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    • pp.77.1-77
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    • 2003
  • We have isolated a full-length cDNA clone, CaCDPK4 encoding a typical calcium-dependent protein kinase (CDPK) from hot pepper cDNA library. Genomic southern blot analysis showed that it belongs to a multigene family, but represents a single copy gone in hot pepper genome. RNA expression pattern of this gene revealed that it is induced by infiltration of Xanthomonas axonopodis pv. glycines Bra into hot pepper leaves but not by water deficit stress. However, high salt treatment of NaCl (0.4 M) solution to hot pepper plants strongly induced CaCDPK4 gene. In addition, this gene is weakly responsive to the exogenous application of salicylic acid or ethephon. Biochemical study of the GST-CaCDPK4 recominant protein showed that it autophosphorylates in vitro and the presence of EGTA, a calcium chelater, eliminates the kinase activity of the recombinant protein. As a way to identify the in vivo function of CaCDPK4 in plants, VIGS (Virus-Induced Gene Silencing) was employed. Agrobacterium-mediated TRV silencing construct containing the kinase and calmodulin domain of CaCDPK4 resulted in cell death of Nicotiana benthamiana plants. A highly homologous H benthamiana CDPK gene, NbCDPK5, to CaCDPK4 was cloned from N. benthamiana cDNA library. VIGS of NbCDPK5 also resulted in cell death. The molecular characterization of this cell death phenotype is being under investigation.

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Selection of Transgenic Potato Plants Expressing Both CuZnSOD and APX in Chloroplasts with Enhanced Tolerance to Oxidative Stress (CuZnSOD와 APX를 엽록체에 발현시킨 산화스트레스 내성 형질전환 감자의 선발)

  • Tang, Li;Kwon, Suk-Yoon;Sung, Chang-K;Kwak, Sang-Soo;Lee, Haeng-Seoon
    • Journal of Plant Biotechnology
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    • v.31 no.2
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    • pp.109-113
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    • 2004
  • In order to develop transgenic potato plants with enhanced tolerance to multiple stress, we constructed the transformation vector expressing both superoxide dismutase and ascorbate peroxidase genes in chloroplasts under the control of a stress-inducible SWPA2 promoter. Transgenic potato plants (cv. Superior and Atlantic) were generated using an Agrobacterium-mediated transformation system. Transgenic potato plants were regenerated on MS medium containing 100mg/L kanamycin. Genomic Southern blot analysis confirmed the incorporation of foreign genes into the potato genome. When potato leaf discs were subjected to methyl viologen (MV) at 10 $\mu$M, transgenic plants showed higher tolerance than non-transgenic or vector-transformed plants. To further study we selected the transgenic plant lines with enhanced tolerance against MV. These plants will be used for further analysis of stress-tolerance to multiple environmental stresses.