Abstract
Several strategies involving the use of antisense and ribozyme constructs in different expression vectors were investigated as methods of suppressing gene expressionin planta. We had previously identified an efficiently cleaving ribozyme (Rz), with two catalytic units and 60 nucleotide (nt) of complementary sequence, to the ligninforming peroxidase of tobacco (TPX). This Rz was cloned behind the 35S CaMV (35S) and nopaline synthase (NOS) promoters, and into a vector utilising the tobacco tyrosine tRNA for expression. For comparison with more traditional antisense strategies, full-length TPX antisense (AS) constructs were also constructed behind the NOS and 35S promoters. Populations of transgenic tobacco containing these constructs were produced and compared to control plants transformed with the vector only. Significant suppression of peroxidase expression in the range of 40–80% was seen in the T0 and T1 populations carrying 35S-AS, 35S-Rz and tRNA-Rz constructs. Co-segregation of the suppressed peroxidase phenotype and the tRNA-Rz transgenes was demonstrated. Northern blot analysis indicated that levels of TPX mRNA were lower in the Rz plants. No evidence of mRNA cleavage was observed and thus it was unclear if the Rz constructs were acting as Rzsin vivo. Transgenic plants containing the tRNA-Rz construct had significantly lower levels of peroxidase than the other transgenic plants. There was no significant difference in levels of suppression of TPX between the short Rz in the 35S vector and the full-length AS constructs. Although peroxidase levels were significantly reduced in transgenic plants carrying 35S-AS, 35S-Rz and tRNA-Rz constructs, no significant difference in lignin levels was observed.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
An, G., Ebert, P.R., Bitra, A. and H, S.B. (1988) Binary vectors. In Gelvin, S., Schilperoot, R.A. and Verma, D.P.S. (eds), Plant Molecular Biology Manual, pp. A3/1-A3/19. Dordrecht, Netherlands. Kluwer Academic Publishers.
Boudet, A.M., Lapierre, C. and Grima-Pettenati, J. (1995) Tansley Review No. 80. Biochemistry and molecular biology of lignification.New Phytol. 129, 203–36.
Bourque, J.E. and Folk, W.R. (1992) Suppression of gene expression in plant cells utilizing AS sequences transcribed by RNA polymerase III.Plant Mol. Biol. 19, 641–7.
Cannon, M., Platz, J., O'Leary, M., Sookdeo, C. and Cannon, F. (1990) Organ-specific modulation of gene expression in transgenic plants using AS RNA.Plant Mol. Biol. 15, 39–47.
Cantor, G.H., McElwain, T.F., Birkebak, T.A. and Palmer, G. (1993) Ribozyme cleavesrex/tax mRNA and inhibits bovine leukemic virus expression.Proc. Natl Acad. Sci. USA 90, 10932–6.
Clancy, M.J., Wilson, R.K. (1966) Development and application of a new chemical method for predicting the digestibility and intake of herbage samples. Proc. Xth International Grasslands Congress, Helsinki.
Cotten, M. and Birnstiel, M.L. (1989) Ribozyme mediated destruction of RNAin vivo.EMBO J 8: 3861–6.
Crisell, P., Thompson, S. and James, W. (1993) Inhibition of HIV-1 replication by ribozymes that show poor activityin vitro.Nucl. Acids Res. 21, 5251–5.
Diaz-de-Leon, F., Klotz, K.L. and Lagrimini, L.M. (1993) Nucleotide sequence of the tobacco (Nicotiana tabacum) anionic peroxidase gene.Plant Physiol. 101, 1117–8.
Erfat, S., Leiser, M., Wu, Y.J., Fusco-DeMane, D., Emran, O.A., Surana, M., Jetton, T.L., Magnuson, M.A., Weir, G. and Fleischer, N. (1994) Ribozyme-mediated attenuation of pancreatic B-dell glucokinase expression in transgenic mice results in impaired glucose-induced insulin secretion.Proc. Natl Acad. Sci. USA 91, 2051–5.
Gerlach, W.L. and Bedbrook, J.R. (1979) Cloning and characterisation of ribosomal RNA genes from wheat and barley.Nucl. Acids Res. 7, 1869–85.
Horsch, R.B., Fry, J.E., Hoffman, N.L., Eichholtz, D., Rogers, S.G. and Fraley, R.T. (1985) A simple and general method for transferring genes into plants.Science 27, 1229–31.
Krol, A.R. Van der, Mol, J.N.M. and Stuitje, A.R. (1988) Antisense genes in plants: an overview.Gene 72, 45–50.
Lagrimini, L.M., Burkhart, W., Moyer, M. and Rothstein, S. (1987) Molecular cloning of complementary DNA encoding the lignin-forming peroxidase from tobacco: molecular analysis and tissue-specific expression.Proc. Natl Acad. Sci. USA 84, 7542–6.
Lazo, G.R., Stein, P.A. and Ludwig, R.A. (1991) A DNA transformation-competentArabidopsis genomic library inAgrobacterium.Bio/Technology 9, 963–7.
Liu, T.T.Y., Lagrimini, L.M., Chabbort, B. and Monties, B. (1993) Lignin content and composition in tobacco plants with over-and under-expressed peroxidase.Plant Physiol. 102, 103.
McIntyre, C.L. and Manners, J.M. (1996) Strategies for the suppression & peroxidase gene expression in tobacco. I. Designing efficient ribozymes.Transgenic Res. 5, 257–262.
McIntyre, C.L., Abrahams, S.L., Bettenay, H.M., Sandeman, R.A., Hayes, C., Sharp, K., Elliot, A., Manners, J.M. and Watson, J.M. (1993) Improving pastures digestibility: low lignin forages.Proc. XVIIth Int. Grassland Congr. 1081–4.
McIntyre, C.L., Rae, A.L., Curtis, M.D. and Manners, J.M. (1995) Sequence and expression of a caffeic acidO-methyl transferase cDNA homologue in the tropical forage legumeStylosanthes humilis.Aust. J. Plant Physiol. 22, 471–8.
Manners, J.M. and Scott, K.J. (1985) Reduced translatable messenger RNA activities in leaves of barley infected withErisyphe graminis f.sp.hordei.Physiol. Plant Path. 26, 297–306.
Mazzolini, L., Axelos, M., Lescure N. and Yot, P. (1992) Assaying synthetic ribozymes in plants: high-level expression of a functional hammerhead structure fails to inhibit target gene activity in transiently transformed protoplasts.Plant Mol. Biol. 20, 715–31.
Murashige, T. and Skoog, F. (1962) A revised medium for rapid growth and bioassays with tobaco tissue cultures.Physiol. Plant. 15, 473–97.
Rothstein, S.J. and Lagrimini, L.M. (1989) Silencing gene expression in plants.Oxford Surveys Plant Molecular Cell Biol. 6, 221–45.
Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning: a Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.
Schuch, W. (1991) The manipulation of plant gene expression using antisense RNA. In: Dennis, E.S., Llewellyn, D.J. (eds) Plant Gene Research—Molecular Approaches to Crop Improvement. New York; USA: Springer-Verlag.
Sherf, B.A., Bajar, A.M. and Kolarrukudy, P.E. (1993) Abolition of an inducible highly anionic peroxidase activity in transgenic tomato.Plant Physiol. 101, 201–8.
Shore, S.K., Nabissa, P.M., Reddy, E.P. (1993) Ribozyme-mediated cleavage of theBCRABL oncogene transcript:in vitro cleavage of RNA andin vivo loss of P210 protein-kinase activity.Oncogene 8, 3183–8.
Stange, N. and Beier, H. (1986) A gene for the major cytoplasmic tRNATyr fromNicotiana rustical contains a 13 nucleotide long intron.Nucl. Acids Res. 14, 8691.
Steinecke, P., Herget, T., Schreier, P.H. (1992) Expression of a chimeric ribozyme gene results in eudonucleolytic cleavage of target mRNA and a concomitant reduction of gene expressionin vivo.EMBO J 11, 1525–30.
Watson, C.F., Grierson, D. (1992) Antisense RNA in plants. In Hiatt, A. (ed),Transgenic Plants: Fundamentals and Applications, pp. 256–281. New York: Marcel Dekker, Inc.
Wegener, D., Steinecke, P., Herget, T., Petereit, I., Philipp, C. and Schreier, P.H. (1994) Expression of a reporter gene is reduced by a ribozyme in transgenic plants.Mol. Gen. Genet. 245, 465–70.
Yu, M., Ojwang, J., Yamada, O., Hampel, A., Rapapport, J., Looney, D. and Wong-Staal, F. (1993) A hairpin Rz inhibits expression of diverse strains of human immunodeficiency virus type I.Proc. Natl Acad. Sci., USA 90, 6340–4.
Zhao, J.J. and Pick, L. (1993) Generating loss-of-function phenotypes of thefushi tarazu gene with a targeted Rz inDrosophila.Nature 365, 448–51.
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
McIntyre, C.L., Bettenay, H.M. & Manners, J.M. Strategies for the suppression of peroxidase gene expression in tobacco. II.In vivo suppression of peroxidase activity in transgenic tobacco using ribozyme and antisense constructs. Transgenic Research 5, 263–270 (1996). https://doi.org/10.1007/BF01972880
Received:
Revised:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF01972880