Abstract
A gene of antimicrobial peptide cecropin P1 (CP1) was inserted into the vector plasmid pPCV91 under the control of promoter 35S RNA of cauliflower mosaic virus (CaMV 35S) containing four enhancer sequences CaMV 35S and nontranslated leader sequence Ω RNA of tobacco mosaic virus. The recombinant vector obtained was used for agrobacterial transformation of tobacco plants (Nicotiana tabacum L., variety Samsun) with the polymerase chain reaction (PCR)-based method. The presence of gene CP1 in the genome of plants was proven by western-blot analysis and testing the antibiotic activity of plant extracts. In different plant lines, the level of cecropin P1 synthesis amounted 0.02–0.2% of total soluble plant leaf protein. The transgenic plants, unlike the control ones, displayed enhanced tolerance to phytopathogenic microorganisms and oxidative stress. It was established that the ability of the transgenic plants to express cecropin P1 is transmitted to progeny.
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Abbreviations
- SOD:
-
superoxide dismutase
- Cf:
-
cefatoxime
- CaMV 35S:
-
promoter 35S RNA cauliflower mosaic virus (CaMV)
- CP1:
-
cecropin P1
- Hm:
-
hygromycin
- hpt :
-
hygromycin phosphotransferase gene
- pnos:
-
promoter of agrobacterial nopalin synthase
References
Ohshima, M., Mitruhara, I., Okamoto, M., Sawano, S., Nishiyama, K., Kaku, U., Natori, S., and Ohashi, Y.J., Enhanced resistance to bacterial diseases of transgenic tobacco plants overexpressing sarcotoxin IA, a bactericidal peptide of insect, J. Biochem., 1999, vol. 125, pp. 431–435.
Jan, P.S., Huang, H.Y., and Chen, H.M., Expression of a synthesized gene encoding cationic peptide cecropin B in transgenic tomato plants protects against bacterial diseases, Appl. Environ. Microbiol., 2010, vol. 76, pp. 769–775.
Campo, S., Manrique, S., Garcia-Martinez, J., and San Segundo, S., Production of cecropin A in transgenic rice plants has an impact on host gene expression, Plant Biotechnol. J., 2008, vol. 6, pp. 585–608.
Martemyanov, K.A., Shirokov, V.A., Kurnasov, O.V., Gudkov, A.T., and Spirin, A.S., Cell-free production of biologically active polypeptides: application to the synthesis of antibacterial peptide cecropin, Protein Expr. Purif., 2001, vol. 21, pp. 456–461.
Mitsuhara, L.H., Matsufuru, M., Ohshima, M., Kaku, H., Nakajima, Y., Murai, N., Natori, S., and Ohashi, Y., Induced expression of sarcotoxin IA enhanced host resistance against both bacterial and fungal pathogens in transgenic tobacco, Mol. Plant-Microbe Interact., 2000, vol. 13, pp. 860–868.
Jaynes, J.M., Nagpala, P., Destefano-Beltran, L., Huang, J.H., Kim, J.H., Denny, T., and Cetiner, S., Expression of a cecropin B lytic peptide analog in transgenic tobacco confers enhanced resistance to bacterial wilt caused by Pseudomonas solanacearum, Plant Sci., 1993, vol. 89, pp. 43–53.
Zakharchenko, N.S., Rukavtsova, E.B., Gudkov, A.T., and Buryanov, Ya.I., Enhanced resistance to phytopathogenic bacteria in transgenic tobacco plants with synthetic gene of antimicrobial peptide cecropin P1, Russ. J. Genet., 2005, vol. 41, pp. 1187–1193.
Zakharchenko, N.S., Rukavtsova, E.B., Shkol’naya, L.A., Yukhmanova, A.A., and Bur’yanov, Ya.I., Expression of the artificial gene encoding anti-microbial peptide cecropin P1 increases the resistance of transgenic potato plants to potato blight and white rot, Dokl. Biol. Sci., 2007, vol. 415, pp. 267–269.
Martemyanov, K.A., Spirin, A.S., and Gudkov, A.T., Synthesis, cloning and expression of genes for antibacterial peptides: cecropin, magainin, and bombinin, Biotechnol. Lett., 1996, vol. 18, pp. 1357–1362.
Strizhov, N.I., Keller, M., Mathur, J., Koncz-Kalman, Z., Bosch, D., Prudovsky, E., Schell, J., Sneh, B., Koncz, C., and Ziberstein, A., A synthetic crylC gene, encoding a Bacillus thuringiensis δ-endotoxin, confers Spodoptera resistance in alfalfa and tobacco, Proc. Natl. Acad. Sci. USA, 1996, vol. 93, pp. 15 012–15 017.
Murashige, T. and Skoog, F., A revised medium for rapid growth and bioassays with tobacco cultures, Physiol. Plant., 1962, vol. 15, pp. 473–497.
Dye, D.M., A taxonomic study of the genus Erwinia the “Carotovora” group, New Zealand J. Sci., 1969, vol. 12, pp. 81–97.
Haldimann, A. and Wanner, B.L., Conditional-replication, integration, excision, and retrieval plasmid-host systems for gene structure-function studies of bacteria, J. Bacteriol., 2001, vol. 183, pp. 6384–6393.
Maniatis, T., Frisch, E.F., and Sambrook, J., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor: Cold Spring Harbor Lab., 1982.
Revenkova, E.V., Kraev, A.S., and Skryabin, K.G., Construction of disarmed derivative of the supervirulent Ti plasmid oTiBo542, Plant Biotechnology and Molecular Biology, Skryabin, K.G., Ed., Moscow: Pushchino Research Center, 1993, pp. 67–76.
An, G., Ebert, P., Mitra, A., and Ha, S.B., Binary vectors, Plant Molecular Biology Manual, Gelvin, S.B., Schilperoort, R.A., and Verma, D.P.S, Eds., Dordrecht: Kluwer Academic, 1988, A3, pp. 1–19.
Draper, J., Scott, R., and Hamil, J., Plant genetic transformation and gene expression, A Laboratory Manual, Draper, J., Scott, R., and Armitage, P., Walden, R., Eds., Oxford: Blackwell Science, 1988, pp. 69–160.
Edwards, K., Johnstone, C., and Thompson, C., A simple and rapid method for the preparation of plant genomic DNA for PCR analysis, Nucleic Acids Res., 1991, vol. 19, p. 1349.
Schagger, H. and Von Jagow, G., Tricine-sodium dodecyl sulfate-polyacrilamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa, Anal. Biochem., 1987, vol. 166, pp. 368–379.
Lehrer, R.I., Rosenman, M., Harwig, S.S.S.L., Jackson, R., and Eisenhauer, P., Ultrasensitive assay for endogenous antimicrobial polypeptides, J. Immunol. Methods, 1991, vol. 137, pp. 167–173.
Bellincampi, D., Dipierro, N., Salvi, G., Cervone, F., and de Lorenzo, G., Extracellular H2O2 induced by oligogalacturonides is not involved in the inhibition of the auxin-regulated rolB gene expression in tobacco leaf explants, Plant Physiol., 2000, vol. 122, pp. 1379–1385.
Beauchamp, C.O. and Fridovich, I., Superoxide dismutase: improved assays and an assay applicable to acrylamide gels, Anal. Biochem., 1971, vol. 44, pp. 276–287.
Uchiyama, M. and Mihara, M., Determination of malonaldehyde precursor in tissue by thiobarbituric acid test, Anal. Biochem., 1978, vol. 86, pp. 287–297.
Wintermans, I.F. and Mots, A., Spectrophotometric characteristics of chlorophylls a and b and their pheophytins in ethanol, Biochim. Biophys. Acta, 1965, vol. 109, pp. 448–453.
Nagy, F., Kay, S.A., and Chua, N.H., Analysis of gene expression in transgenic plants, Plant Molecular Biology Manual, Gelvin, S.B., Schilperoort, R.A., and Verma, D.P.S., Eds., Dordrecht: Kluwer Academic, 1988, B4, pp. 1–29.
Epple, P., Apel, K., and Bohlmann, H., Overexpression of an endogenous thionin enhances resistance of Arabidopsis against Fusarium oxysporum, Plant Cell, 1997, vol. 9, pp. 509–520.
Fang, R.X., Nagy, F., Sivasubramaniam, S., and Chua, N.H., Multiple cis regulatory elements for maximal expression of the cauliflower mosaic virus 35S promoter in transgenic plants, Plant Cell, 1989, vol. 1, pp. 141–150.
Gallie, D.R. and Kado, C.I., A translational enhancer derived from tobacco mosaic virus is functionally equivalent to a Shine-Dalgarno sequence, Proc. Natl. Acad. Sci. USA, 1989, vol. 86, pp. 129–132.
Mittova, V., Tal, M., Volokita, M., and Guy, M., Upregulation of the leaf mitochondrial and peroxisomal antioxidative systems in response to salt-induced oxidative stress in the wild salt-tolerant tomato species Lycopersicon pennellii, Plant Cell Environ., 2003, vol. 26, pp. 845–856.
Kreslavski, V.D., Los, D.A., Allakhverdiev, S.I., and Kuznetsov, Vl.V., Signaling role of reactive oxygen species in plants under stress, Russ. J. Plant Physiol., 2012, vol. 59, pp. 141–154.
Zakharchenko, N.S., Buryanov, Ya.I., Lebedeva, A.A., Pigoleva, S.V., Vetoshkina, D.V., Loktyushov, E.V., Chepurnova, M.A., Kreslavski, V.D., and Kosobryukhov, A.A., Physiological features of rapeseed plants expressing the gene for an antimicrobial peptide cecropin P1, Russ. J. Plant Physiol., 2013, vol. 60, pp. 411–419.
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Original Russian Text © N.S. Zakharchenko, N.I. Strizhov, L.A. Shkolnaya, S.V. Pigoleva, A.A. Lebedeva, E.B. Rukavtsova, O.V. Furs, T.V. Shevchuk, O.V. Dyachenko, Ya.I. Buryanov, 2015, published in Fiziologiya Rastenii, 2015, Vol. 62, No. 4, pp. 571–578.
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Zakharchenko, N.S., Strizhov, N.I., Shkolnaya, L.A. et al. Novel expression system for enhanced synthesis of antimicrobial peptide cecropin P1 in plants. Russ J Plant Physiol 62, 534–541 (2015). https://doi.org/10.1134/S1021443715040202
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DOI: https://doi.org/10.1134/S1021443715040202