• Title/Summary/Keyword: Southern blot

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Environmental risk assessment of genetically modified Herbicide-Tolerant zoysiagrass (Event: Jeju Green21) (제초제저항성 들잔디(Zoysia japonica Steud.) 이벤트 Jeju Green21의 환경위해성평가)

  • Bae, Tae-Woong;Kang, Hong-Gyu;Song, In-Ja;Sun, Hyeon-Jin;Ko, Suk-Min;Song, Pill-Soon;Lee, Hyo-Yeon
    • Journal of Plant Biotechnology
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    • v.38 no.2
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    • pp.105-116
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    • 2011
  • Transgenic zoysiagrass (Zoysia japonica Steud.) expressing the bar gene inserted in the plant genome has been generated previously through Agrobacterium tumefaciens-mediated transformation. The GM zoysiagrass (event: JG21) permits efficient management of weed control of widely cultivated zoysiagrass fields, reducing the frequency and cost of using various herbicides for weed control. Now we have carried out the environmental risk assessment of JG21 prior to applying to the governmental regulatory agency for the commercial release of the GM turf grass outside of test plots. The morphological phenotypes, molecular analysis, weediness and gene flow from each test plot of JG21 and wild-type zoysiagrasses have been evaluated by selectively analyzing environmental effects. There were no marked differences in morphological phenotypes between JG21 and wild-type grasses. The JG21 retained its stable integration in the host plant in T1 generation, exhibiting a 3:1 segregation ratio according to the Mendelian genetics. We confirmed the copy number (1) of JG21 by using Southern blot analysis, as the transgenic plants were tolerant to ammonium glufosinate throughout the culture period. From cross-fertilization and gene flow studies, we found a 9% cross-pollination rate at the center of JG21 field and 0% at distances over 3 m from the field. The JG21 and wild-type zoysiagrass plants are not considered "weed" because zoysiagrasses generally are not dominant and do not spread into weedy areas easily. We assessed the horizontal gene transfer (HGT) of the transgene DNA to soil microorganisms from JG21 and wild-type plants. The bar gene was not detected from the total genomic DNA extracted from each rhizosphere soil of GM and non-GM Zoysia grass fields. Through the monitoring of JG21 transgene's unintentional release into the environment, we found no evidence for either pollen mediated gene flow of zoysiagrass or seed dispersal from the test field within a 3 km radius of the natural habitat.

Characterization of Transgenic Tall Fescue Plants Overexpressing NDP Kinase Gene in Response to Cold Stress (NDP Kinase 유전자를 과발현시킨 형질전환 톨 페스큐 식물체의 저온 스트레스에 대한 내성 특성)

  • Lee, Sang-Hoon;Lee, Ki-Won;Kim, Kyung-Hee;Yun, Dae-Jin;Kwak, Sang-Soo;Lee, Byung-Hyun
    • Journal of The Korean Society of Grassland and Forage Science
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    • v.29 no.4
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    • pp.299-306
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    • 2009
  • Oxidative stress is the main limiting factor in crop productivity. To solve global environmental problems using the plant biotechnology, we have developed on the oxidative stress-tolerant transgenic tall fescue plants via Agrobacterium-mediated genetic transformation method. In order to develop transgenic tall fescue (Festuca arundinacea Schreb.) plants with enhanced tolerance to multiple environmental stresses, nucleotide diphosphate kinase gene under the control of CaMV35S promoter were introduced into genome of tall fescue plants. Proteomic analysis revealed that transgenic tall fescue not only accumulated NDP kinase 2 protein in their cells, but also induced several other antioxindative enzyme-related proteins. When leaf discs of transgenic plants were subjected to cold stress, they showed approximately 30% less damage than wild-type plants. In addition, transgenic tall fescue plants showed normal growth when transgenic plants were subjected to $4^{\circ}C$ for 3 days treatments. These results suggest that transgene is important in ROS scavenging by induction of antioxidative proteins, and could improve abiotic stress tolerance in transgenic tall fescue plants.

Transformation of Gourd through Leaf Explant Regeneration (잎 절편의 재분화에 의한 참박 형질전환)

  • Cho, Song-Mi;Moon, Sun-Jin;Chung, Soo-Jin;Kim, Mi-Seong;Kim, Young-Cheol;Yang, Kwang-Yeol;Choi, Yong-Soo;Sapkota, Kumar;Cho, Baik-Ho;Kim, Kwang-Sang
    • Korean Journal of Plant Resources
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    • v.19 no.5
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    • pp.634-639
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    • 2006
  • In order to develop a disease-resistant root stock for the growth of watermelon, an efficient regeneration system of the gourd(Lagenaria leucantha Duch.) inbred line GO701-2 via organogenesis was established in this experiment. Using proximal parts of cotyledon explant excised from germinated seedling in vitro, maximum adventitious shoot formation (39%) was achieved on MS medium where cytokinin (BA) and auxin (IAA) were added at a concentration of 3mg/L and 0.1mg/L, respectively. Roots of the elongated shoots were successfully formed on MS medium without adding any plant growth regulators. The cucumber CsGolS1 gene known as a resistance gene against biotic and abiotic stresses, was constructed into the binary vector pBI121 under the control of CaMV 35S promoter. When the gene was introduced into the genome of gourd by Agrobacterium-mediated transformation, putative transgenic plants were obtained with the transformation efficiency of approximately 20 percent.

Characterization of the genomes of Aujeszky's disease virus isolated in Korea (국내분리 오제스키병 바이러스의 게놈 유전자 특성 분석)

  • Hyun, Bang-Hun;Kim, In-Joong;Pyo, Hyun-Mi;Cha, Sang-Ho;Park, Ji-Yeun;Song, Jae-Young;Cho, In-Soo;Yang, Chang-Bum;An, Soo-Hwan;Lee, Joong-Bok
    • Korean Journal of Veterinary Research
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    • v.49 no.1
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    • pp.45-57
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    • 2009
  • The molecular genetic characterization of Aujeszky's disease virus (ADV) Yangsan strain (ADVYS), a Korean isolate, was investigated by analyzing the electrophoresis patterns and the physical maps of the viral DNA digested with various endonucleases. To establish DNA library for ADV-YS, twelve major BamHI restricted segments were cloned. Each location of the segments in the ADV genome was determined by sequence comparison with the sequences reported in Genbank and those sequences of the both termini of the segments. Physical maps were constructed based on the electrophoresis patterns of the digested viral DNA by restriction endonuclease and the results of Southern blot analyses with various DIG labeled probes originated from those of enzyme restricted segments of virulent (Shope) and avirulent (Bartha) strain. Comparing ADV-YS with a standard strain of Kaplan in the maps of restriction enzymes, following major respects were identified: (i) disappearance of BamHI restriction site between the first and second BamHI segments, (ii) creation of the BamHI restriction site in the fifth segment, and (iii) generation of the BglII site in the unique short (US) region. The genome of ADV-YS also contains a type 2 herpesvirus DNA molecule (in which the US region only inverts itself relative to the unique longregion) like all other ADV strains except Norden strain(type3), analyzed up to date. The size of the ADV genome estimated from the sizes of the restriction enzyme fragments, was approximately 145.3 kb (BamHI) or 145.4 kb (BglII). BamHI enzyme cleavage patterns were compared among the five Korean ADV isolates: Yangsan, Yongin, Dangjin, Jincheon and Iksan strains. Difference either in the number or in the size of the DNA fragments, suspected regions of termini of IR and TR, could be detected among all five strains.

Factors Effecting Agrobacterium Mediated Transformation and Regeneration of Populus nigra × P. maximowiczii (Agrobacterium tumefaciens에 의한 양황철나무의 형질전환(形質轉換) 요인(要因))

  • Park, Young Goo;Shin, Dong Won;Kim, Joung Hee
    • Journal of Korean Society of Forest Science
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    • v.79 no.3
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    • pp.278-284
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    • 1990
  • We have demonstrated expression of bacterial genes transferred into cells of Populus nigra ${\times}$ P. maximowiczii by A. tumefaciens strain 6044 (pGA 472). We determined the optimum concentration of kanamycin sulfate for effective selection of punctured leaf transformed using Agrobacterium binary vector pGA 472 containing a neomycine phosphotransferase gene (NPT-II) which confers kanamycin resistance. The combination of cefotaxime (200mg/l) and carbenicillin (300mg/l) showed good performance of discarding Agrobacterium from inoculated punctured leaf. A relatively low concentration (10mg/l) of kanamycin sulfate inhibited callus and shoots induction from punctured leaf. Number of shoots regenerated from co-cultured punctured leaf was 3.0 on MS basal medium supplemented with 10 mg/l kanamycin sulfate, while that of not co-cultured punctured leaf was none. The regeneration rate was 10% from the punctured leaf co-cultured on MS medium with 10 mg/l kanamycin. Regenerated shoots are developing from micropropagation for Southern blot analysis and inheritance of the kanamycin resistance trait (NPT-II).

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Developing a Gene-trapping Approach for Gene Identification Using Nuclear Transfer in Zebrafish (지브라물고기 복제방법에 의한 유전자 동정 및 유전자트랩법 개발)

  • Lee, K.Y.
    • Journal of Animal Science and Technology
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    • v.46 no.2
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    • pp.155-164
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    • 2004
  • This involves identifying and cloning trapped genes from cultured cells carrying the gene-trap constructs and generating cloned zebrafish using these cells for functional study. Gene-trapping studies in gene-trapped cells were carried out in initial and cloned zebrafish carrying gene-trap events were successfully produced based on the nuclear transplantation technique. Two kind of retroviral gene-trap constructs were adopted. The first one(SA/GFP-TP), constructed in my laboratory, carries a GFP reporter gene containing a splicing acceptor and an internal neo gene. The second one(Neo-TP), obtained from Dr. Hicks (Hicks et al., 1997), contains a promoter-less neo gene located in the LTR sequence of a retroviral vector. The infected cells were subjected to drug selection(neomycin treatment) because the two constructs carry the neomycin resistant gene. All those cells survived the neomycin treatment should carry the proviral insertions. For Neo-TP, Isolated DNA from the neomycin-resistant fibroblast cells infected by Neo-TP, was digested with EcoR1 restriction enzyme and transformed into bacteria after ligation. This procedure led to the isolation of seven clones carrying flanking cellular DNA with a typical retroviral integration signature sequence. These clones contained genomic DNA ranging from 1kb to 7kb and sequences of 300-600 bp were obtained from each of the rescued plasmids. Database searching showed that all of them share high homology to zebrafish sequences. For fish cloning using tagged cells, initially, nucleus donors directly selected from a mixture of cells(Neo-TP cells) were used. A total of 44 embryos(3.7%) out of 1179 transplants were reached blastula stage; 8 of these embryos(0.8%) hatched and 3(0.3%) of them survived to adulthood. One out of three lived cloned zebrafish has an amplified fragment and was labeled with 32P.

BACTERIAL IDENTIFICATION WITH RANDOM-CLONED RESTRICTION FRAGMENT OF Porphyromonas endodontalis ATCC 35406 GENOMIC DNA (무작위로 클로닝한 Porphyromonas endodontalis ATCC 35406 지놈 DNA의 제한절편 hybridization법에 의한 세균동정)

  • Um, Won-Seok;Han, Yoon-Soo
    • Restorative Dentistry and Endodontics
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    • v.20 no.2
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    • pp.645-654
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    • 1995
  • Porphyromonas endodontalis is a black-pigmented anaerobic Gram negative rod which is associated with endodontal infections. It has been isolated from infected dental root canals and submucous abscesses of endodontal origin. DNA probe is an available alternative, offering the direct detection of a specific microorganism. Nucleic-acid probes can be off different types: whole different: whole-genomic, cloned or oligonucleotide probes. Wholegenomic probes are the most sensitive because the entire genome is used for possible hybridization sites. However, as genetically similar species of bacteria are likely to be present in specimences, cross-reactions need to be considered. Cloned probes are isolated sequences of DNA that do not show cross-reactivity and are produced in quantity by cloning in a plasmid vector. Cloned probes can approach the sensitivity found with whole-genomic probes while avoiding known cross-reacting species. Porphyromonas endodontalis ATCC 35406 (serotype $O_1K_1$) was selected in this experiment to develop specific cloned DNA probes. EcoR I-digested genomic DNA fragments of P. endodontalis ATCC 35406 were cloned into pUC18 plasmid vector. From the E. coli transformed with the recombinant plasmid 4 clones were selected to be tested as specific DNA probes. Restriction-digested whole-genomic DNAs prepared from P. gingivalis 38(serotype a), W50(serotype b), A7A1-28(serotype C), P. intermedia 9336(serotype b), G8-9K-3(serotype C), P. endodontalis ATCC 35406(serotype $O_1K_1$), A. a Y4(serotype b), 75(serotype a), 67(serotype c), were each seperated on agarose gel electrophoresis, blotted on nylon membranes, and were hybridized with digoxigenin-dUTP labeled probe. The results were as follows: 1. Three clones of 1.6kb(probe e), 1.6kb(probe f), and 0.9kb(probe h) in size, were obtained. These clones were identified to be a part of the genomic DNA of P. endodontalis ATCC 35406 judging from their specific hybridization to the genomic DNA fragments of their own size on Southern blot. 2. The clones of 4.9kb(probe i) was identified to be a part of the genomic DNA of P. endodontalis ATCC 35406. but not to specific for itself. It was hybridized to P. gingivalis A7A1-28, P. intermedia G89K-3.

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Factors Affecting the Agrobacterium Mediated Transformation of 'Gala' Apple (사과 갈라 품종의 Agrobacterium이용 형질전환에 영향하는 요인)

  • 송관정;성은수;황정환;제갈성;차지은;김정희;신용억
    • Korean Journal of Plant Tissue Culture
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    • v.28 no.4
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    • pp.221-225
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    • 2001
  • Some factors of wounding methods, solidifying agents, origin of leaf explants, cone. of acetosyring-one, and MES affecting regeneration and transformation by Agrobacterium tumefaciens were investigated to establish an efficient transformation system of apple. Wounding by cutting the leaves merely showed the tendency of regeneration and transformation with higher efficiency compared with that of wounding by non-traumatic forcep when carrying out co-cultivation for three days after bacterial inoculation. While examining the solidifying agents of medium with the combination of agar (A)+Gelrit $e^{ }$ (G) in g. $L^{-1}$ , the higher concentration of Gelrit $e^{ }$ increased the efficiency of regeneration. However, there was no difference in the efficiency of transformation from the treatments of 2.5 G, 3.5 A+1.2 G, and 7.0 G. The origin of leaf explants showed no difference statistically in the efficiency of regeneration and transformation, but that from the shoots of proliferation medium showed the tendency with higher efficiency. The concentration of above 0.1 mM acetosyringon had an increase in the efficiency of regeneration and transformation and the concentration of 0.15 mM had the highest efficiency of transformation in the treatment of acetosyringone with different concentration. There was no effect of MES on regeneration and transformation.ion.

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Isolation and characterization of a monodehydroascorbate reductase gene in poplar (Populus alba × P. glandulosa) (현사시나무 monodehydroascorbate reductase (MDHAR) 유전자의 분리 및 발현특성)

  • Yoon, Seo-Kyung;Park, Eung-Jun;Bae, Eun-Kyung;Choi, Young-Im;Kim, Joon-Hyeok;Lee, Hyoshin
    • Journal of Plant Biotechnology
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    • v.41 no.4
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    • pp.194-200
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    • 2014
  • Monodehydroascorbate reductase (MDHAR) is an important enzyme that plays a role in the detoxification of reactive oxygen species (ROS) by maintaining reduced pool of ascorbate through recycling the oxidized form of ascorbate. In this study, we isolated a PagMDHAR1 gene from Populus alba ${\times}$ P. glandulosa, and investigated its expression characteristics. The PagMDHAR1 cDNA encodes a putative 434 amino acids containing FAD- and NAD(P)H-binding domains. Southern blot analysis indicated that a single nuclear gene encodes this enzyme. Northern hybridization analysis revealed that PagMDHAR1 is highly expressed in both suspension cells and flower tissues, while its expression levels were enhanced by drought, salt, cold, wounding and ABA. Therefore, PagMDHAR1 might be expressed in response to abiotic stress through the ABA-mediated signaling pathway in this poplar species, suggesting that the PagMDHAR1 plays an important role in the defense mechanisms against oxidative stress.

Efficient Transformation Method of Soybean Using Meristematic Tissues of Germinating Seeds (발아종자의 분열조직을 이용한 효율적인 콩 형질전환 방법)

  • Kim, Yul-Ho;Park, Hyang-Mi;Choi, Man-Soo;Sohn, Soo-In;Shin, Dong-Bum;Lee, Jang-Yong
    • Korean Journal of Breeding Science
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    • v.40 no.3
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    • pp.278-285
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    • 2008
  • An efficient transformation method for soybean [Glycine max (L.) Merr.] using meristematic tissues of germinating seeds has been established. The embryonic axes were excised from germinating seeds of Korean soybean cultivar, Iksannamulkong and 0.5-2 cm long segment containing meristematic tissues were prepared by cutting hypocotyl region. The explants were inoculated with Agrobacterium tumefaciens strain LBA4404 harboring a binary vector with the bar gene as a selectable marker gene and a ${\beta}-glucuronidase$ (GUSINT) reporter gene, and then co-cultured for 7 days on co-cultivation medium (CCM). The meristematic tissues were cultured on shoot induction medium (SIMP6) supplemented with 0.4 mg/l $N_6-benzylaminopurine$ (BAP) and 0.1 mg/l indolebutyric acid (IBA) in the presence of 6 mg/l L-phosphinotricin (PPT) for 2 weeks and the surviving explants were transferred to shoot elongation medium (SEMP6). Transformation was confirmed by Southern blot analysis and the transformation efficiencies ranged from 1.48 to 2.07%. The new modified transformation method was successfully implemented for obtaining several transgenic lines with SMV-CP gene. It is expected that this method could efficiently be used for the transformation of recalcitrant soybean cultivars.