Multicellular living organisms and unmodified parts thereof and – Plant – seedling – plant seed – or plant part – per se – Higher plant – seedling – plant seed – or plant part
Reexamination Certificate
1999-04-28
2003-12-30
Nelson, Amy J. (Department: 1638)
Multicellular living organisms and unmodified parts thereof and
Plant, seedling, plant seed, or plant part, per se
Higher plant, seedling, plant seed, or plant part
C800S289000, C536S023600
Reexamination Certificate
active
06670528
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a transgenic plant containing a gene in which a DNA encoding a protein that binds to dehydration responsive element (DRE) and regulates the transcription of genes located downstream of DRE is ligated downstream of a stress responsive promoter.
2. Prior Art
In the natural world, plants are living under various environmental stresses such as dehydration, high temperature, low temperature or salt. Unlike animals, plants cannot protect themselves from stresses by moving. Thus, plants have acquired various stress tolerance mechanisms during the courses of their evolution. For example, low temperature tolerant plants (
Arabidopsis thaliana
, spinach, lettuce, garden pea, barley, beet, etc.) have less unsaturated fatty acid content in their biomembrane lipid than low temperature sensitive plants (maize, rice, pumpkin, cucumber, banana, tomato, etc.). Therefore, even when the former plants are exposed to low temperatures, phase transition is hard to occur in their biomembrane lipid and, thus, low temperature injury does not occur easily.
To date, dehydration, low temperature or salt tolerant lines have been selected and crossed in attempts to artificially create environmental stress tolerant plants. However, a long time is needed for such selection, and the crossing method is only applicable between limited species. Thus, it has been difficult to create a plant with high environmental stress tolerance.
As biotechnology progressed recently, trials have been made to create dehydration, low temperature or salt tolerant plants by using transgenic technology which introduces into plants a specific, heterologous gene. Those genes which have been used for the creation of environmental stress tolerant plants include synthesis enzyme genes for osmoprotecting substances (mannitol, proline, glycine betaine, etc.) and modification enzyme genes for cell membrane lipid. Specifically, as the mannitol synthesis enzyme gene,
Escherichia coli
-derived mannitol 1-phosphate dehydrogenase gene [Science 259:508-510 (1993)] was used. As the proline synthesis enzyme gene, bean-derived &Dgr;
1
-proline-5-carboxylate synthetase gene [Plant Physiol. 108:1387-1394 (1995)] was used. As the glycine betaine synthesis enzyme gene, bacterium-derived choline dehydrogenase gene [Plant J. 12:1334-1342 (1997)] was used. As the cell membrane lipid modification enzyme gene,
Arabidopsis thaliana
-derived &ohgr;-3 fatty acid desaturase gene [Plant Physiol. 105:601-605 (1994)] and blue-green alga-derived &Dgr;9 desaturase gene [Nature Biotech. 14:1003-1006 (1996) were used. However, the resultant plants into which these genes were introduced were instable in stress tolerance or low in tolerance level; none of them have been put into practical use to date.
Further, it is reported that a plurality of genes are involved in the acquisition of dehydration, low temperature or salt tolerance in plants [Plant Physiol., 115:327-334 (1997)]. Therefore, a gene encoding a transcription factor capable of activating simultaneously the expression of a plurality of genes involved in the acquisition of stress tolerance has been introduced into plants, yielding plants with high stress tolerance. However, when a gene which induces the expression of a plurality of genes is introduced into a host plant, the genes are activated at the same time. As a result, the energy of the host plant is directed to production of the products of these genes and intracellular metabolism of such gene products, which often brings about delay in the growth of the host plant or dwarfing of the plant.
OBJECTS AND SUMMARY OF THE INVENTION
It is an object of the present invention to provide a transgenic plant containing a gene in which a DNA encoding a protein that binds to a stress responsive element and regulates the transcription of genes located downstream of the element is ligated downstream of a stress responsive promoter, the transgenic plant having improved tolerance to environmental stresses (such as dehydration, low temperature and salt) and being free from dwarfing.
Toward the solution of the above problem, the present inventors have cloned a novel transcription factor gene that regulates the expression of genes involved in the acquisition of dehydration, low temperature or salt stress tolerance, and introduced into a plant this novel gene ligated downstream of a stress responsive promoter. As a result, the inventors have succeeded in creating a plant which has remarkably improved tolerance to dehydration, low temperature or salt and which is free from dwarfing. Thus, the present invention has been achieved.
The present invention relates to a transgenic plant containing a gene in which a DNA encoding the following protein (a) or (b) is ligated downstream of a stress responsive promoter:
(a) a protein consisting of the amino acid sequence as shown in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8 or SEQ ID NO: 10;
(b) a protein which consists of the amino acid sequence having deletion, substitution or addition of at least one amino acid in the amino acid sequence as shown in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8 or SEQ ID NO: 10 and which regulates the transcription of genes located downstream of a stress responsive element.
Further, the present invention relates to a transgenic plant containing a gene in which the following DNA (c) or (d) is ligated downstream of a stress responsive promoter:
(c) a DNA consisting of the nucleotide sequence as shown in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7 or SEQ ID NO: 9;
(d) a DNA which hybridizes with the DNA consisting of the nucleotide sequence as shown in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7 or SEQ ID NO: 9 under stringent conditions and which codes for a protein that regulates the transcription of genes located downstream of a stress responsive element.
Specific examples of the stress include dehydration stress, low temperature stress and salt stress.
As the stress responsive promoter, at least one selected from the group consisting of rd29A gene promoter, rd29B gene promoter, rd17 gene promoter, rd22 gene promoter, DREB1A gene promoter, cor6.6 gene promoter, cor15a gene promoter, erd1 gene promoter and kin1 gene promoter may be given.
This specification includes part or all of the contents as described in the specification and/or drawings of Japanese Patent Application No. 10-292348, which is a priority document of the present application.
REFERENCES:
patent: 5891859 (1999-04-01), Thomashow et al.
patent: 6495742 (2002-12-01), Shinozaki et al.
Liu et al, Two Transcription Factors, DREB1 and DREB2, with an EREBP/AP2 DNA Binding Domain Sep. TwoCell. Signal Transduction Pathways in Drought-and Low-Temp-Respon. Gene Express., Respectively, in Arabidopsis, Aug. 1998, ThePlant Cell, V. 10, p. 1391-1406.*
ShinWari et al. An Arabidopsis Gene Family Encoding DRE/CRT Binding Proteins Involved in Low-Temperature-Responsive Gene Expression. Biochemical And Biophysical Research Communicatior 250, 161-170 (1998) Article No. RC 989267.*
Abe et al. Role of Arabidopsis MYC and MYB Homologs In Droughtand Abscisic Acid_Regulated Gene Expression. The Plant Cell, vol. 9, 1859-1868, Oct. 1997.*
Riechmann et al. Arabidopsis transcription factors: genome-wide comparative analysis among eukaryotes. Science 15 Dec. 2000, vol. 290, pp. 2105-2110.*
Busk, et al.; “Regulatory elements in vivo in the promoter of the abscisic acid responsive gene reb17 from maize”; The Plant Journal, vol. 11, No. 6, 1997, pp 1285-1295.
Jiang, et al.; “Requirement of a CCGAC cis-acting element for cold induction of the BN115 gene from winter Brassica napus”; Plant Molecular Biology; vol. 30, 1996, pp 679-684.
Ouellet, et al.; “The wheat wcs120 promoter is cold-inducible in both monocotyledonous and dicotyledonous species”; Federation of European Biochemical Societies Letters, vol. 423, 1998, pp. 324-328.
Yamaguchi-Shinozaki, Kazuko; Shinozaki, Kazuo; “A Novel cis-A
Kasuga Mie
Shinozaki Kazuko
Davidson Davidson & Kappel LLC
Independent Administrative Institute, Japan International Resear
Nelson Amy J.
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