Summary
We have obtained transgenic tobacco plants overexpressing the enzyme glutamine synthetase (GS) by fusing an alfalfa GS gene to the cauliflower mosaic virus 35S promotor and integrating it intoNicotiana tabacum var. W38 plants byAgrobacterium tumefaciens mediated gene transfer. The amount of RNA specific to alfalfa GS was about 10 times higher in transgenic tobacco plants than in alfalfa. The alfalfa GS produced by these transgenic plants was identified by Western blotting and represented 5% of total soluble protein in the transformed plants, amounting to a 5-fold increase in specific GS activity and in a 20-fold increase in resistance to the GS inhibitorl-phosphinothricin in vitro. Tissue from GS overproducing plants showed a sevenfold lower amount of free NH3. The amino acid composition of the plant tissue was not altered significantly by GS overproduction. GS overproducing plants were fertile and grew normally. These data show that a high level of expression of a key metabolic enzyme such as glutamine synthetase does not interfere with growth and fertility of plants.
Similar content being viewed by others
References
Cullimore JV, Gebhardt C, Saarelainen R, Miflin BJ, Idler KB, Barker RF (1984) Glutamine Synthetase ofPhaseolus vulgaris L.: Organ-specific expression of a multigene family. J Mol Appl Genet 2:589–599
Deak M, Donn G, Feher A, Dudits D (1988) Dominant expression of a gene amplification related herbicide resistance in Medicago cell hybrids. Plant Cell Rep 7:158–161
Donn G, Tischer E, Smith JA, Goodman HM (1984) Herbicide-resistant alfalfa cells: an example of gene amplification in plants. J Mol Appl Genet 2:621–635
Eckes P (1985) Isolation, characterisation and functional analysis of a developmentally regulated gene inSolanum tuberosum. Thesis, University of Cologne, West Germany
Eckes P, Schell J, Willmitzer L (1985) Organ-specific expression of three leaf/stem specific cDNAs from potato is regulated by light and correlated with chloroplast development. Mol Gen Genet 199:216–224
Eckes P, Rosahl S, Schell J, Willmitzer L (1986) Isolation and characterisation of a light-inducible, organ-specific gene from potato and analysis of its expression after tagging and transfer into tobacco and potato shoots. Mol Gen Genet 205:14–22
Fluhr R, Kuhlemeier C, Nagy F, Chua N-H (1986) Organ-specific and light-induced expression of plant genes. Science 232:1106–1112
Horsch RB, Fry FE, Hoffmann NL, Eichholtz D, Rogers SG, Fraley RT (1985) A simple and general method for transferring genes into plants. Science 227:1229–1231
Jones J, Dunsmuir P, Bedbrook J (1985) High level expression of introduced chimaeric genes in regenerated transformed plants. EMBO J 4:2411–2418
Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685
Leason M, Cunliffe D, Parkin D, Lea PJ, Miflin BJ (1982) Inhibition of pea leaf glutamine synthetase by methionine sulphiximine, phosphinothricin and other glutamate analogues. Phytochemistry 21:855–857
Logemann J, Schell J, Willmitzer L (1987) Improved method for the isolation of RNA from plant tissues. Anal Biochem 163:16–20
Maniatis T, Fritsch EF, Sambrook J (1982) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, New York
McNally SF, Hirel B, Gadal P, Mann F, Stewart GR (1983) Glutamine synthetases of higher plants. Plant Physiol 72:22–25
Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue culture. Physiol Plant 15:473–497
Murray MG, Thompson WF (1980) Rapid isolation of high molecular weight plant DNA. Nucleic Acids Res 8:6323–6327
Nagy F, Morelli G, Rogers SG, Chua N-H (1985) Photoregulated expression of a pea rbcS gene in leaves of transgenic plants. EMBO J 4:3063–3068
Nato F, Hirel B, Nato A, Gadal P (1984) Chloroplastic glutamine synthetase from tobacco leaves: a glycosylated protein. FEBS Lett 175:443–446
Odell JT, Nagy F, Chua N-H (1985) Identification of DNA sequences required for activity of the cauliflower mosaic virus 35S promotor. Nature 313:810–812
Pietrzak M, Shillito RD, Hohn T, Potrykus I (1986) Expression in plants of two bacterial antibiotic resistance genes after protoplast transformation with a new plant expression vector. Nucleic Acids Res 14:5857–5868
Sanders PR, Winter JA, Barnason AR, Rogers SG, Fraley RT (1987) Comparison of cauliflower mosaic virus 35S and nopaline synthase promotors in transgenic plants. Nucleic Acids Res 15:1543–1558
Shah DM, Horsch RB, Klee HJ, Kishore GM, Winter JA, Tumer NE, Hironaka CM, Sanders PR, Gasser CS, Aykent S, Siegel NR, Rogers SG, Fraley RT (1986) Engineering herbicide tolerance in transgenic plants. Science 233:478–481
Stockhaus J, Eckes P, Rocha-Sosa M, Schell J, Willmitzer L (1987) Analysis of cis-active sequences involved in the leaf-specific expression of a potato gene in transgenic plants. Proc Natl Acad Sci USA 84:7943–7947
Tingey SV, Walker EI, Coruzzi GM (1987) Glutamine synthetase genes of pea encode distinct polypeptides which are differentially expressed in leaves, roots and nodules. EMBO J 6:1–9
Tischer E, DasSarma S, Goodman HM (1986) Nucleotide sequence of an alfalfa glutamine synthetase gene. Mol Gen Genet 203:221–229
Van Haute E, Joos H, Maes M, Warren G, Van Montagu M, Schell J (1983) Intergenic transfer and exchange recombination of restriction fragments cloned in pBR322: a novel strategy for the reversal genetics of the Ti-plasmid ofAgrobacterium tumefaciens. EMBO J 2:411–418
Vezina L-P, Hope HJ, Joy KW (1987) Isoenzymes of glutamine synthetase in roots of pea (Pisum sativum L. cv Little Marvel) and alfalfa (Medicago media Pers. cv Saranac). Plant Physiol 83:58–62
Author information
Authors and Affiliations
Additional information
Communicated by J. Schell
Rights and permissions
About this article
Cite this article
Eckes, P., Schmitt, P., Daub, W. et al. Overproduction of alfalfa glutamine synthetase in transgenic tobacco plants. Molec. Gen. Genet. 217, 263–268 (1989). https://doi.org/10.1007/BF02464891
Received:
Issue Date:
DOI: https://doi.org/10.1007/BF02464891