| Peer-Reviewed

Evaluation of the Diphtheria Toxin A Subunit Gene (DT-A) as a Non-conditional Negative Selectable Marker in Tobacco and Rice

Received: 21 July 2016    Accepted: 1 August 2016    Published: 29 August 2016
Views:       Downloads:
Abstract

The diphtheria toxin A subunit gene (DT-A) from Corynebacterium diphtheriae inhibits protein synthesis in eukaryotes. In this study, toxicity of the DT-A gene was evaluated by a transgenic approach in tobacco and rice. The DT-A gene was cloned under transcriptional control of the CaMV 35S promoter and transformed into tobacco. Similarly, CaMV 35S and the maize Ubi1 promoter-driven DT-A gene constructs were transformed into rice. The deployment of the DT-A gene in both tobacco and rice drastically reduced the recovery of transgenic plants in comparison to pCAMBIA1301 (without DT-A). Southern blot analyses of the transgenic plants were done using the hph- and DT-A gene-specific probes to check the presence of the hph and DT-A genes. All the tobacco and rice transgenic plants showed hybridization to junction fragments upon using the hph gene probe. Southern blotting with the DT-A probe revealed that all the transgenic plants either did not have the DT-A gene or harboured truncated DT-A gene in the integrated T-DNAs. None of the transgenic plants carried the complete DT-A gene. The results showed that the DT-A gene can be used as a good non-conditional negative selectable marker in both tobacco and rice. Both CaMV 35S promoter- and Ubi1 promoter-driven DT-A genes were effective as non-conditional negative selectable markers in rice.

Published in Journal of Plant Sciences (Volume 4, Issue 5)
DOI 10.11648/j.jps.20160405.13
Page(s) 106-112
Creative Commons

This is an Open Access article, distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution and reproduction in any medium or format, provided the original work is properly cited.

Copyright

Copyright © The Author(s), 2024. Published by Science Publishing Group

Keywords

Agrobacterium tumefaciens, Diphtheria Toxin, Negative Selectable Marker, Rice, Tobacco

References
[1] Koprek, T., McElroy, D., Louwerse, J., Williams-Carrier, R., Lemaux, P. G (1999) Negative selection systems for transgenic barley (Hordeum vulgare L.): comparison of bacterial cod A-and cytochrome P450 gene-mediated selection. Plant J 19, 719-726.
[2] Czako, M., An, G (1991) Expression of DNA coding for diphtheria toxin chain A is toxic to plant cells. Plant Physiol 95, 687-692.
[3] Koning, A., Jones, A., Fillatti, J. J., Comai, L., Lassner, M. W (1992) Arrest of embryo development in Brassica napus mediated by modified Pseudomonas aeruginosa exotoxin A. Plant Mol Biol 18, 247-258.
[4] Mariani, C., De Beuckeleer, M., Truettner, J., Leemans, J., Goldberg, R. B (1990) Induction of male sterility in plants by a chimaeric ribonuclease gene. Nature 347, 737-741.
[5] Xiang, C., Guerra, D. J (1993) The anti-nptII gene. A potential negative selectable marker for plants. Plant Physiol 102, 287-293.
[6] Stirpe, F., Williams, D. G., Onyon, L. J., Legg, R. F., Stevens, W. A (1981) Dianthins, ribosome-damage proteins with anti-viral properties from Dianthus caryophyllus L. (carnation). Biochem. J 195, 399-405.
[7] Shah, J. M., Veluthambi, K (2010) DIANTHIN, a negative selection marker in tobacco, is non-toxic in transgenic rice and confers sheath blight resistance. Biol Plant 54, 443-450.
[8] Majhi, B. B., Bhosale, R., Jawkar, S., Veluthambi, K (2014) Evaluation of codA, tms2 and ABRIN-A as negative selectable markers in transgenic tobacco and rice. In Vitro Cell Dev Biol-Plant 50, 541-551.
[9] Dai, S., Carcamo, R., Zhang, Z., Chen, S., Beachy, R. N (2001) The bacterial cytosine deaminase gene used as a conditional negative selection marker in transgenic rice plants. Plant Cell Rep 20, 738-743.
[10] Osakabe, K., Nishizawa-Yokoi, A., Ohtsuki, N., Osakabe, Y., Toki, S (2013) A mutated cytosine deaminase gene, codA (D314A), as an efficient negative selection marker for gene targeting in rice. Plant Cell Physiol 55, 658-665.
[11] Terada, R., Urawa, H., Inagaki, Y., Tsugane, K., Iida, S (2002) Efficient gene targeting by homologous recombination in rice. Nat Biotechnol 20, 1030-1034.
[12] Iida, S., Terada, R (2005) Modification of endogenous natural genes by gene targeting in rice and other higher plants. Plant Mol Biol 59, 205-219.
[13] RamanaRao, M. V., Veluthambi, K (2010) Selectable marker elimination in the T0 generation by Agrobacterium-mediated co-transformation involving Mungbean yellow mosaic virus TrAP as a non-conditional negative selectable marker and bar for transient positive selection. Plant cell Rep 29, 473-483.
[14] De Block, M., Debrouwer, D., Moens, T (1997) The development of a nuclear male sterility system in wheat. Expression of the barnase gene under the control of tapetum specific promoters. Theor Appl Genet 95, 125-131.
[15] Hanson, B., Engler, D., Moy, Y., Newman, B., Ralston, E., Gutterson, N (1999) A simple method to enrich an Agrobacterium-transformed population for plants containing only T-DNA sequences. Plant J 19, 727-734.
[16] Pappenheimer, A. M. J (1977) Diphtheria toxin. Annu Rev Biochem 46, 69–94.
[17] Terada, R., Asao, H., Iida, S (2004) A large-scale Agrobacterium-mediated transformation procedure with a strong positive-negative selection for gene targeting in rice (Oryza sativa L.). Plant Cell Rep 22, 653-659.
[18] Mattheakis, L. C., Shen, W. H., Collier, R. J (1992) DPH5, a methyltransferase gene required for diphthamide biosynthesis in Saccharomyces cerevisiae. Mol Cell Biol 12, 4026-4037.
[19] Palmiter, R. D., Behringer, R. R., Quaife, C. J., Maxwell, F., Maxwell, I. H., Brinster, R. L (1987) Cell lineage ablation in transgenic mice by cell-specific expression of a toxin gene. Cell 50, 435-443.
[20] Hellens, R. P., Edwards, E. A., Leyland, N. R., Bean, S., Mullineaux, P. M (2000) pGreen: a versatile and flexible binary Ti vector for Agrobacterium-mediated plant transformation. Plant Mol Biol 42, 819-832.
[21] Komari, T., Hiei, Y., Saito, Y., Murai, N., Kumashiro, T (1996) Vectors carrying two separate T-DNAs for co-transformation of higher plants mediated by Agrobacterium tumefaciens and segregation of transformants free from selection markers. Plant J 10,165-174.
[22] Sunilkumar, G., Vijayachandra, K., Veluthambi, K (1999) Preincubation of cut tobacco leaf explants promotes Agrobacterium-mediated transformation by increasing vir gene induction. Plant Sci 141, 51-58.
[23] Murashige, T., Skoog, F (1962) A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol Plant 15, 473-497.
[24] Sridevi, G., Sabapathi, N., Meena, P., Nandakumar, R., Samiyappan, R., Muthukrishnan, S., Veluthambi, K (2003) Transgenic indica rice variety Pusa Basmati 1 constitutively expressing a rice chitinase gene exhibits enhanced resistance to Rhizoctonia solani. J. Plant Biochem Biotechnol 12, 93-101.
[25] Rogers, S. O., Bendich, A. J (1988) Extraction of DNA from plant tissues. In: Gelvin S. B, Schilperoort R. A, Verma D. P. S (eds) Plant molecular biology manual, vol A6. Kluwer, Dordrecht, pp 1-10.
[26] Odell, J. T., Nagy, F., Chua, N. H (1985) Identification of DNA sequences required for activity of the cauliflower mosaic virus 35S promoter. Nature 313, 810-812.
[27] Gupta, P., Raghuvanshi, S., Tyagi, A. K (2001) Assessment of the efficiency of various gene promoters via biolistics in leaf and regenerating seed callus of millets, Eleusine coracana and Echinochloa crusgalli. Plant Biotechnol J 18, 275-282.
[28] Potenza, C., Aleman, L., Sengupta-Gopalan, C (2004) Targeting transgene expression in research, agricultural, and environmental applications: promoters used in plant transformation. In Vitro Cell Dev Biol-Plant 40, 1-22.
[29] Park, S. H., Yi, N., Kim Y. S., Jeong, M. H., Bang, S. W., Choi, Y. D., Kim, J. K (2010) Analysis of five novel putative constitutive gene promoters in transgenic rice plants. J Exp Bot 61, 2459-2467.
Cite This Article
  • APA Style

    Pachamuthu Kannan, Bharat Bhusan Majhi, Karuppannan Veluthambi. (2016). Evaluation of the Diphtheria Toxin A Subunit Gene (DT-A) as a Non-conditional Negative Selectable Marker in Tobacco and Rice. Journal of Plant Sciences, 4(5), 106-112. https://doi.org/10.11648/j.jps.20160405.13

    Copy | Download

    ACS Style

    Pachamuthu Kannan; Bharat Bhusan Majhi; Karuppannan Veluthambi. Evaluation of the Diphtheria Toxin A Subunit Gene (DT-A) as a Non-conditional Negative Selectable Marker in Tobacco and Rice. J. Plant Sci. 2016, 4(5), 106-112. doi: 10.11648/j.jps.20160405.13

    Copy | Download

    AMA Style

    Pachamuthu Kannan, Bharat Bhusan Majhi, Karuppannan Veluthambi. Evaluation of the Diphtheria Toxin A Subunit Gene (DT-A) as a Non-conditional Negative Selectable Marker in Tobacco and Rice. J Plant Sci. 2016;4(5):106-112. doi: 10.11648/j.jps.20160405.13

    Copy | Download

  • @article{10.11648/j.jps.20160405.13,
      author = {Pachamuthu Kannan and Bharat Bhusan Majhi and Karuppannan Veluthambi},
      title = {Evaluation of the Diphtheria Toxin A Subunit Gene (DT-A) as a Non-conditional Negative Selectable Marker in Tobacco and Rice},
      journal = {Journal of Plant Sciences},
      volume = {4},
      number = {5},
      pages = {106-112},
      doi = {10.11648/j.jps.20160405.13},
      url = {https://doi.org/10.11648/j.jps.20160405.13},
      eprint = {https://article.sciencepublishinggroup.com/pdf/10.11648.j.jps.20160405.13},
      abstract = {The diphtheria toxin A subunit gene (DT-A) from Corynebacterium diphtheriae inhibits protein synthesis in eukaryotes. In this study, toxicity of the DT-A gene was evaluated by a transgenic approach in tobacco and rice. The DT-A gene was cloned under transcriptional control of the CaMV 35S promoter and transformed into tobacco. Similarly, CaMV 35S and the maize Ubi1 promoter-driven DT-A gene constructs were transformed into rice. The deployment of the DT-A gene in both tobacco and rice drastically reduced the recovery of transgenic plants in comparison to pCAMBIA1301 (without DT-A). Southern blot analyses of the transgenic plants were done using the hph- and DT-A gene-specific probes to check the presence of the hph and DT-A genes. All the tobacco and rice transgenic plants showed hybridization to junction fragments upon using the hph gene probe. Southern blotting with the DT-A probe revealed that all the transgenic plants either did not have the DT-A gene or harboured truncated DT-A gene in the integrated T-DNAs. None of the transgenic plants carried the complete DT-A gene. The results showed that the DT-A gene can be used as a good non-conditional negative selectable marker in both tobacco and rice. Both CaMV 35S promoter- and Ubi1 promoter-driven DT-A genes were effective as non-conditional negative selectable markers in rice.},
     year = {2016}
    }
    

    Copy | Download

  • TY  - JOUR
    T1  - Evaluation of the Diphtheria Toxin A Subunit Gene (DT-A) as a Non-conditional Negative Selectable Marker in Tobacco and Rice
    AU  - Pachamuthu Kannan
    AU  - Bharat Bhusan Majhi
    AU  - Karuppannan Veluthambi
    Y1  - 2016/08/29
    PY  - 2016
    N1  - https://doi.org/10.11648/j.jps.20160405.13
    DO  - 10.11648/j.jps.20160405.13
    T2  - Journal of Plant Sciences
    JF  - Journal of Plant Sciences
    JO  - Journal of Plant Sciences
    SP  - 106
    EP  - 112
    PB  - Science Publishing Group
    SN  - 2331-0731
    UR  - https://doi.org/10.11648/j.jps.20160405.13
    AB  - The diphtheria toxin A subunit gene (DT-A) from Corynebacterium diphtheriae inhibits protein synthesis in eukaryotes. In this study, toxicity of the DT-A gene was evaluated by a transgenic approach in tobacco and rice. The DT-A gene was cloned under transcriptional control of the CaMV 35S promoter and transformed into tobacco. Similarly, CaMV 35S and the maize Ubi1 promoter-driven DT-A gene constructs were transformed into rice. The deployment of the DT-A gene in both tobacco and rice drastically reduced the recovery of transgenic plants in comparison to pCAMBIA1301 (without DT-A). Southern blot analyses of the transgenic plants were done using the hph- and DT-A gene-specific probes to check the presence of the hph and DT-A genes. All the tobacco and rice transgenic plants showed hybridization to junction fragments upon using the hph gene probe. Southern blotting with the DT-A probe revealed that all the transgenic plants either did not have the DT-A gene or harboured truncated DT-A gene in the integrated T-DNAs. None of the transgenic plants carried the complete DT-A gene. The results showed that the DT-A gene can be used as a good non-conditional negative selectable marker in both tobacco and rice. Both CaMV 35S promoter- and Ubi1 promoter-driven DT-A genes were effective as non-conditional negative selectable markers in rice.
    VL  - 4
    IS  - 5
    ER  - 

    Copy | Download

Author Information
  • Department of Plant Biotechnology, School of Biotechnology, Madurai Kamaraj University, Madurai, Tamil Nadu, India

  • Department of Plant Biotechnology, School of Biotechnology, Madurai Kamaraj University, Madurai, Tamil Nadu, India

  • Department of Plant Biotechnology, School of Biotechnology, Madurai Kamaraj University, Madurai, Tamil Nadu, India

  • Sections