• Title/Summary/Keyword: selectable marker

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One-Step Engineering of a Stable, Selectable Marker-Free Autoluminescent Acinetobacter baumannii for Rapid Continuous Assessment of Drug Activity

  • Jiang, Huofeng;Gao, Yamin;Zeng, Sheng;Wang, Shuai;Cao, Zhizhong;Tan, Yaoju;Yin, Huancai;Liu, Jianxiong;Zhang, Tianyu
    • Journal of Microbiology and Biotechnology
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    • v.29 no.9
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    • pp.1488-1493
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    • 2019
  • The rising cases of multidrug-resistant Acinetobacter baumannii (Ab) and the lack of effective drugs call for quick attention. Here, based on a Tn7 transposon and Xer/dif system, we constructed a stable, selectable marker-free autoluminescent Ab capable of producing visible light without extra substrates. Utilization of this autoluminescent reporter strain has the potential to reduce the time, effort and costs required for the evaluation of activities of anti-Ab drug candidates in vitro.

Tissue Culture Studies of Anthranilate Synthase the Tryptophan Biosynthetic Control Enzyme

  • Widholm, Jack.M.
    • Journal of Plant Biotechnology
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    • v.2 no.2
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    • pp.55-60
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    • 2000
  • Experiments initiated 30 years ago to obtain selectable markers have led to a series of studies of Trp biosynthesis and anthranilate synthase (AS) the control enzyme using largely plant tissue cultures since they have experimental properties that can be readily exploited. Enzymological and compound feeding studies provided evidence that AS is the control point in the Trp biosynthesis branch and that altering the AS feedback control by the selection of mutants resistant to the Trp analog 5-methyl-tryptophan (5MT) can lead to the overproduction of this important amino acid. Plants regenerated from these Trp overproducing lines of most species also had high free Trp levels but Nicotiana tabaum (tobacco) plants expressed the feedback altered AS only in cultured cells and not in the regenerated plants. further tests by transient and stable expression of the cloned promoter for the naturally occurring tobacco feedback-insensitive AS, denoted ASA2, confirmed the tissue culture specific nature of the expression control. The 5MT caused by the expression of a feedback-insensitive AS from tobacco has been used to select protoplast fusion hybrids with several species since the resistance is expressed dominantly. Recently the ASA2 gene has been used successfully as a selectable marker to select transformed Astragalus sinicus and Glycine max hairy roots induced by Agrobactetium rhizogenes. These results show that the ASA2y-subunit can interact with the y-subunit of another species to form active feedback-insensitive enzyme that may be useful for selecting transformed cells. Plastid DNA transformation of tobacco has also effectively expressed ASA2 in the compartment in which Trp biosynthesis is localized in the cell.

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Agrobacterium-mediated transformation produces transgenic oilseed rape with a high-yield trait (아그로박테리움 형질전환법을 이용한 수량증대 유채 식물체 개발)

  • Jong Bo Kim
    • Journal of Plant Biotechnology
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    • v.50
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    • pp.63-69
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    • 2023
  • This study includes the transformation of genes such as ORE7, the increase of gene expression, and the use of the bar gene as a selectable marker that shows herbicide resistance with Agrobacterium tumefaciens using hypocotyls from the oilseed rape "Youngsan" cultivar. To establish an Agrobacterium transformation system for the production of oilseed rape with a high-yield trait, infection time and co-cultivation period with Agrobacterium were tested. Therefore, when hypocotyls from the oilseed rape "Youngsan" cultivar were infected with Agrobacterium for 20 min and co-cultivated for 3 days, approximately 32-36 putatively transformed hypocotyls with shoots including roots survived from 100 inoculated hypocotyls after 4 weeks of transformation on a selection medium containing 20 mg/L of phosphinothricin (PPT) as a selectable agent. Additionally, a PCR assay was performed to confirm the insertion of target genes and showed the presence of the ORE7 gene as a high-yielding trait and the bar gene as a selectable marker. Treatment with 0.5% (v/v) Basta solution as a selectable agent for 6 days with leaves from transformed oilseed rape expressed the bar gene. Therefore, this study can contribute to the development of special oilseed rapes containing agriculturally useful traits such as herbicide resistance, drought tolerance, high yielding traits, and high oleic acid content.

Transformation of Potato using the Phosphinothricin Acetyltransferase Gene as the Selectable Marker Gene (감자의 형질전환을 위한 표지유전자로서 Phosphinothricin Acetyltransferase 유전자의 이용)

  • Jeong, J.H.;Yang, D.C.;Bang, K.S.;Han, S.S.
    • Korean Journal of Weed Science
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    • v.18 no.3
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    • pp.205-213
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    • 1998
  • This experiment was carried out to produce herbicide resistant potatoes hawing only chimeric phosphinothricin acetyltransferase (PAT) genes without using antibiotic selectable marker. The pDY502 vector having only PAT gene was reconstructed for transformation of potato. The reconstructed vector was introduced to Agrobacterium tumefaciens MP90 disarmed, and they were used for potato transformation. Hormonal requirement for plant regeneration from leaves and stem explants of potato was investigated. From this experiment, MS medium treated with IBA 0.1 mg/L + BA 0.5 mg/L was the best for potato regeneration, and the ratio of shoot regeneration was 54% for leaf and 46% for stem in that condition. For transformation, explants of potato leaves and stems were cocultured with A. tumefaciens MP90 containing reconstructed vector harvoring only PAT gene. When the potato explants were placed on various concentrations of bialaphos and all the potato explants were dead on medium with over 5.0mg/L bialaphos. By this selection methods, the explants cocultured with Agrobacterium produced the putative transgenic shoots on medium with 5mg/L bialaphos treatment after 3-4 weeks. Second selection was performed by transferring the shoot tips of putative transgenic to medium containing 20mg/L of bialaphos. The shoot tips grew well on the second selection medium, indicating the production of successful transgenic plants. But normal shoots were dead in same cytotoxic medium. Incorporation of the PAT gene into transgenic potatos were confirmed by PCR analysis of DNA and Southern hybridization. These results show that the PAT gene can serve as a selectable marker and herbicide resistant genes for transformation of potato.

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The Function of ArgE Gene in Transgenic Rice Plants

  • Guo, Jia;Seong, Eun-Soo;Cho, Joon-Hyeong;Wang, Myeong-Hyeon
    • Korean Journal of Plant Resources
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    • v.20 no.6
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    • pp.524-529
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    • 2007
  • We carried out to study the function of ArgE in transgenic rice plants, which were confirmed by PCR analysis and hygromycin selection. Transgenic rice plants were with selectable marker gene(HPT) inserted in genome of the rice. Southern analysis with hpt probe confirmed by two restriction enzymes that copy numbers of the selectable gene was introduced into the plant genome. We displayed that the relationship between drought stress and ArgE gene with the overexpressing rice plants. From this result, we observed that the degree of leaves damage has no difference in control and transgenic lines. The total RNAs were extracted from 6 weeks-seedling in normal condition in order to examine their expression levels with ArgE-overexpressed transgenic rice. In particular, expression patterns of genes encoding enzymes involved in abiotic stress, including drought and salt stresses. OsGF14a and OsSalt were investigated by reverse transcription-PCR(RT-PCR). Expression levels of the OsSalt gene decreased significantly in transgenic rice plants compared to control plant. However, ion leakage measurement did not demonstrate any leaves damage change between control and ArgE transgenic plants exposure to mannitol treatment. These results suggest that expression of the ArgE is not involved in tolerance for drought stress in rice but may playa role of signaling networks for salt-induced genes.

Asymmetry of Cotransduction Frequency Occured When the Transposon Is Used as a Marker (전위인자가 표식인자로 사용될때 발생하는 동시형질도입의 비대칭성)

  • 김창훈;박찬규
    • Korean Journal of Microbiology
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    • v.30 no.2
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    • pp.83-87
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    • 1992
  • When the transposon is used as a selectable marker. the cotransduction frequency depends on the selection order of the markers. In this study. we adopted a mathematical approach to explain this phenomenon. At first, the formation of transducing particles were considered in five different configurations. Then the probability functions indicating the possibilities for a marker to be fixed were mathematically formulated on the basis of probability density concept. After actual values and useful assumptions were integrated into the Formula. resulting frequencies from theoretical calculations were compared with actual data. Such analysis let us conclude that the asymmetry in the frequency arose from the lack of homology required for homologous recombination due to the transposon insertion and from the suppression of recombination around the region where the transposon is inserted.

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