Multicellular living organisms and unmodified parts thereof and – Method of introducing a polynucleotide molecule into or... – Nonplant protein is expressed from the polynucleotide
Reexamination Certificate
1997-09-26
2001-08-28
Fox, David T. (Department: 1638)
Multicellular living organisms and unmodified parts thereof and
Method of introducing a polynucleotide molecule into or...
Nonplant protein is expressed from the polynucleotide
C435S468000, C435S091500, C435S410000, C435S419000, C435S252100, C435S252300, C800S290000, C800S295000, C800S298000, C800S289000, C800S320200, C800S306000, C800S320000
Reexamination Certificate
active
06281412
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to a method for producing plants with novel properties, more specifically, a method for producing salt-tolerant and/or osmotolerant plants which are highly resistant to environmental stresses.
DESCRIPTION OF THE PRIOR ART
One effective way to prevent global warming is greening of uncultivated soils such as desert or salt-accumulated soil. As a means therefor, the development of plants which are resistant to environmental stresses in combination with an engineering solution such as irrigation plays an important role in controlling encroachment of desert, promoting greening and preventing global warming.
Salt accumulation causes the following damages: (1) accumulated salt lowers the water potential in soil to prevent plants from absorbing water; (2) the salt absorbed (penetrated) into plants disturbs their metabolism; (3) salt inhibits the absorption of other ions necessary for viability (Sato, F., Plant Cell Engineering, Supplement, “Environmental Problems and Phytobiotechnology”, pp. 33-39, 1994). Especially, the inhibition of water absorption causes plants to lose turgor pressure and close stoma. Thus, photosynthesis is deteriorated and growth is seriously inhibited.
Plants have evolved various mechanisms to adapt themselves to such environments. In a simple adaptation model, plant cells keep an osmotic difference between the inside and outside of the cells in some way, and restore turgor by water absorption. For example, halobacteria, which are not plants however, keep an osmotic balance between the inside and outside by accumulating salt in the cells. In this case, however, it is difficult to adapt them to environmental (osmotic) changes, because intracellular metabolic enzymes per se need to be salt-tolerant.
Therefore, a better adaptation mechanism is the synthesis of a specific compound called “compatible solute” for keeping an intracellular osmosis depending on extrinsic osmotic changes as many salt-tolerant plants do so.
As the compatible solute, bipolar compounds such as glycinebetaine or proline and polyols such as pinitol, sorbitol or mannitol are known. These compounds are characterized by low molecular weight, high water-solubility, low metabolizability, non-influence on metabolism, etc., and are suitable for osmoregulation.
Among others, glycinebetaine (hereinafter referred to as betaine) is noted as a compatible solute found in plants and bacteria which are adaptable to salt-accumulated and/or water-deficient environment. Betaine is thought as a compatible solute found in higher plants such as Chenopodeaceae, Gramineae, Solanaceae, as well as cyanobacteria,
Escherichia coli
, etc. (for example, see Rhodes, D. and Hanson, A. D., Annu. Rev. Plant Physiol. Plant Mol. Biol. 44:357-3584, 1993). Betaine is an osmoprotective substance which keeps an osmotic balance with environments (Robinson, S. P. and Jones, G. P., Aust. J. Plant Physiol. 13:659-668, 1986) and prevents the dissociation of soluble enzymes due to high salt concentration (Gabbay-Azaria et al., Arch. Biochem. Biophys. 264:333-339, 1988). In addition, betaine can protect photosystem II complex against high salt concentration by stabilization of neighboring proteins and manganese cluster within photosynthetic oxygen-evolving complex (for example, see Murata et al., FEBS Lett. 296:187-189, 1992).
In
Escherichia coli
and spinach (
Spinacia oleracea
), betaine is biosynthesized from choline via two steps of oxidation as shown in
FIG. 1.
E. coli
contains two dehydrogenases; one is a membrane-bound oxygen-dependent choline dehydrogenase which oxidizes choline to betainealdehyde (Landfald, B. and Strom, A., J. Bacteriol. 165:849-855, 1986), and the other is a soluble NAD-dependent betainealdehyde dehydrogenase which oxidizes betainealdehyde to betaine (Falkenberg, P. and Strom, A. R., Biochim. Biophys. Acta. 1034:253-259, 1990). In higher plants, it has been demonstrated that betaine is synthesized in the chloroplasts via a similar pathway to
E. coli
. In spinach (
Spinacia oleracea
), the first step of oxidation is catalysed by a ferredoxin-dependent choline monooxygenase (Brouquisse, R. et al., Plant Physiol. 90:322-329, 1989) and an NAD-dependent betainealdehyde dehydrogenase which catalyzes the second step of oxidation (Weretilnyk, E. A. et al., Planta. 178:342-352, 1989) has already been isolated. These plants were found to increase the activities of the both enzymes and thereby the amount of betaine under salt stress (for example, see Hanson, A. D. et al., Proc. Natl. Acad. Sci. U.S.A. 82:3678-3682, 1985).
Alternatively, choline oxidase from the gram-positive soil bacterium
Arthrobacter globiformis
is able to oxidize choline to betaine in one-step oxidation reaction (Ikuta, S. et al., J. Biochem. 82:1741-1749, 1977).
Attempts have been made to confer salt tolerance by integrating such two genes as found in
E. coli
and higher plants or choline oxidase gene into a plant to allow it constantly express these genes (for example, see Nomura M. et al., Plant Physiol. 107:703-708, 1995). However, no success has been achieved so far in obtaining a salt-tolerant and/or osmotolerant plant by integrating such genes into a plant, especially a higher plant expressing them stably.
Choline oxidase is commercially available, but its amino acid sequence has not been determined. Therefore, it would be highly desirable to determine a genetic sequence encoding choline oxidase which can efficiently convert choline into betaine and to integrate it into a plant to allow it to stably express said sequence, whereby producing a plant which is tolerant to environmental (osmotic) changes such as high salt concentration.
SUMMARY OF THE INVENTION
After profound study to solve the above problems, the present inventors succeeded in isolating a novel gene encoding choline oxidase (The Japanese Society of Plant Physiologist, Annual meeting of 1994, the 34th Symposium held Mar. 28-30, 1994) and integrating it into cyanobacteria, brassicaceous and gramineous plants to obtain salt-tolerant and/or osmotolerant plants.
Accordingly, this invention provides a method for producing salt-tolerant and/or osmotolerant plants, which comprises the step of transforming a plant with a recombinant vector carrying a gene encoding choline oxidase, as well as the salt-tolerant and/or osmotolerant plants obtained by said method.
REFERENCES:
patent: 97/24026 (1997-07-01), None
Rathinasabapathi, B. et al., Metabolic Engineering of Glycine Betaine Synthesis: Plant Betaine Aldehyde Dehydrogenases Lacking Typical Transit Peptides are Argeted to Tobacco Chloroplasts where they confer Betaine Aldehyde Resistance. vol. 193, 1994, pp. 155-162.
P. Deshnium et al., A. globiformis codA gene. Feb. 27, 1995 (EMBL Accession No. X84895).
Mark A. Mackay et al., Organic osmoregulatory solutes in cyanobacteria. Chemical Abstracs, vol. 101, No. 25, Dec. 17, 1984.
Holmstoem, K. -O et al., Production of the 1-3, 5-10Escherichia ColiBetaine-Aldehyde Dehydrogenase, An Enzyme required for the Synthesis of the Osmoprotectant Glycine Betaine, In Transgenic Plants. vol. 6, No. 5, 1994, pp. 749-758.
Reed, R.H. et al., The Responses of Cyanobacteria to Salt Stress, vol. 28, Apr. 1987.
Rathinasabapathi, B. et al., Cultivated And Wild Rices Do Not Accumulate Glycinebetaine Due to Deficiencies in Two Biosynthetic Steps. vol. 33, May 1993, pp. 534-538.
Saneoka, H. et al., Salt Tolerance of Glycinebetaine-Deficient and -Containing Maize Lines, vol. 107, Feb. 1995, pp. 631-638.
McCue, K. et al., Drought and Salt Tolerance: Towards Understanding and Application. vol. 8, Dec. 1990, pp. 358-362.
Pilon-Smits, E.A.H., et al., Improved performancepf trnasgenice fructan-accumulating tobacco under drought stress, vol. 107, Jan. 1995, pp. 125-130.
Tarczynki, M. C. et al., Stress Protection of Transgenic Tobacco by Production of the Osmolyte Mannitol. vol. 259, Jan. 22, 1993, pp. 508-510.
Deshnium et al. Plant Molecular Biology. 1995. vol. 29: 897-907.*
Roche et al. Plant Molecular Biology. 1993. vol. 22: 971-983.*
Wan and Lemaux. Plant Physiol. 1
Birch & Stewart Kolasch & Birch, LLP
Fox David T.
Suntory Limited
Zaghmout Ousama M-Faiz
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