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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
Volume 4, Issue 5, October 2016, Pages: 106-112
Received: Jul. 21, 2016; Accepted: Aug. 1, 2016; Published: Aug. 29, 2016
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Pachamuthu Kannan, Department of Plant Biotechnology, School of Biotechnology, Madurai Kamaraj University, Madurai, Tamil Nadu, India
Bharat Bhusan Majhi, Department of Plant Biotechnology, School of Biotechnology, Madurai Kamaraj University, Madurai, Tamil Nadu, India
Karuppannan Veluthambi, Department of Plant Biotechnology, School of Biotechnology, Madurai Kamaraj University, Madurai, Tamil Nadu, India
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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.
Agrobacterium tumefaciens, Diphtheria Toxin, Negative Selectable Marker, Rice, Tobacco
To cite this article
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, Journal of Plant Sciences. Vol. 4, No. 5, 2016, pp. 106-112. doi: 10.11648/j.jps.20160405.13
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This article is an open access article distributed under the Creative Commons Attribution License ( which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
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.
Czako, M., An, G (1991) Expression of DNA coding for diphtheria toxin chain A is toxic to plant cells. Plant Physiol 95, 687-692.
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.
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.
Xiang, C., Guerra, D. J (1993) The anti-nptII gene. A potential negative selectable marker for plants. Plant Physiol 102, 287-293.
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.
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.
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.
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.
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.
Terada, R., Urawa, H., Inagaki, Y., Tsugane, K., Iida, S (2002) Efficient gene targeting by homologous recombination in rice. Nat Biotechnol 20, 1030-1034.
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.
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.
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.
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.
Pappenheimer, A. M. J (1977) Diphtheria toxin. Annu Rev Biochem 46, 69–94.
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.
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.
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.
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.
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.
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.
Murashige, T., Skoog, F (1962) A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol Plant 15, 473-497.
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.
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.
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.
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.
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.
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.
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