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
1999-08-10
2003-04-15
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
C800S278000, C800S287000, C800S290000, C800S298000, C435S419000, C435S468000, C435S194000
Reexamination Certificate
active
06548743
ABSTRACT:
BACKGROUND OF THE INVENTION
This invention relates to the manipulation of plant gene expression and the production of transgenic plants.
Plant growth and development relies on the integration of developmental and environmental signals. In response to these signals, undifferentiated cells at the meristems are able to develop into either vegetative or reproductive structures. The signaling and regulatory mechanisms underlying the maintenance and the differentiation of meristems are mostly unknown. Two plant hormones, auxin and cytokinin, are known to affect meristematic cell division activities. A common phenomenon termed apical dominance results from the suppression of secondary meristems by inhibitory levels of auxin produced at the actively growing apex. Application of cytokinin at the secondary meristems can, however, release this suppression. It is therefore likely that hormones such as auxin and cytokinin are also involved in the maintenance and the regulation of meristem differentiation.
Mitogen-activated protein kinase (MAPK) pathways have been implicated in transmitting hormonal and environmental signals to the cell nucleus in organisms ranging from yeast to humans. For example, in mammals, the primary responses to hormone, growth, and stress signals are mediated by a conserved signaling cascade consisting of three protein kinases, the mitogen-activated protein kinase (MAPK), mitogen-activated protein kinase kinase (MAPKK), and mitogen-activated protein kinase kinase kinase (MAPKKK). MAPKKK phosphorylates and activates MAPKK that, in turn, phosphorylates and activates MAPK. The activated MAPK can be translocated into the nucleus where it phosphorylates transcription factors that control gene expression (Herskowitz,
Cell
80: 187-197, 1995; Kyriakis et al.,
J. Biol. Chem.
271: 24313-24316, 1996). Additionally, in some mammalian cells, the activation of the same MAPK pathway can lead to either cell proliferation or differentiation depending on the duration of the activation. Moreover, dual-specificity MAPK phosphatases have recently been identified as specific regulators which act to turn off and attenuate MAPK signal transduction pathways.
SUMMARY OF THE INVENTION
In one aspect, the invention features a method for modifying a plant phenotype. The method, in general, includes the steps of: (a) introducing into a plant cell a transgene including DNA encoding a phosphatase domain of a dual-specificity mitogen-activating protein kinase (MAPK) phosphatase (or a phosphatase domain thereof) operably linked to a promoter functional in the plant cell to yield a transformed plant cell; and (b) regenerating a transgenic plant from the transformed plant cell, wherein the phosphatase domain of the dual-specificity MAPK phosphatase is expressed in the cells of the transgenic plant, thereby modifying the phenotype of the transgenic plant. In preferred embodiments, the dual-specificity MAPK phosphatase (or a phosphatase domain thereof) hydrolyzes phosphoserine/threonine and phosphotyrosine residues on a protein substrate. In other preferred embodiments, the dual-specificity MAPK phosphatase (or a phosphatase domain thereof) is a eukaryotic dual-specificity MAPK phosphatase (e.g., an approximately full-length MKP-1 or a polypeptide including approximately amino acids 1-314 of MKP-1). In particular applications, the method is useful for modifying a plant's phenotype for the production of plants having increased yield; increased flower production; early flowering; increased reproductive capacity; decreased vegetative growth; delayed senescence; decreased sensitivity to auxin; increased seed production; or increased regeneration capacity in vitro.
In related aspects, the invention features a plant (or plant cell, plant tissue, plant organ, or plant part) including a transgene capable of expressing a phosphatase domain of a dual-specificity MAPK phosphatase, wherein the transgene is expressed in the transgenic plant under the control of a promoter that is functional in a plant cell. In preferred embodiments, the dual-specificity MAPK phosphatase hydrolyzes phosphoserine/threonine and phosphotyrosine residues on a protein substrate. In other preferred embodiments, the dual-specificity MAPK phosphatase is a eukaryotic dual-specificity MAPK phosphatase (e.g., an approximately full-length MKP-1 or a polypeptide including approximately amino acids 1-314 of MKP-1).
In related aspects, the invention also features seeds and cells from a plant which includes a transgene capable of expressing a phosphatase domain of a dual-specificity MAPK phosphatase (or a phosphatase domain thereof).
In general, the phosphatase domain used in the methods or transgenic plants of the invention is generally expressed by itself, as a dual-specificity MAPK phosphatase polypeptide or phosphatase domain-containing fragment thereof, or as part of a genetically engineered chimeric polypeptide. Useful dual-specificity MAPK phosphatases include those that inactivate a MAPK pathway cascade; improve yields; increase flower production; promote early flowering; increase reproductive season; decrease vegetative growth; delay senescence; decrease sensitivity to auxin; increase seed production; or increase plant regeneration in vitro. Exemplary phosphatase domains include, without limitation, those that are substantially identical or identical to the phosphatase domains of MKP-1, MKP-2, MKP-3, MKP-4, PAC-1, MSG5, Pmp1, IphP, AtMKP1, AtMKP2, AtMKP3, or AtMKP3. Preferably, the methods and plants of the invention specifically utilize the phosphatase domain of MKP-1 or amino acids 1-314 of MKP-1. In other preferred embodiments, a full-length dual-specificity MAPK phosphatase polypeptide or a phosphatase domain containing fragment thereof that is substantially identical or identical to MKP-1, MKP-2, MKP-3, MKP-4, PAC-1, MSG5, Pmp1, IphP, AtMKP-1, AtMKP-2, AtMKP-3, or AtMKP-4 is utilized.
The DNA encoding the dual-specificity MAPK phosphatase polypeptide or phosphatase domain-containing fragment thereof is, in general, constitutively expressed in the transgenic plant. However, if desired, the domain may be inducibly expressed, or such a domain may be expressed in a cell-specific, tissue-specific, or organ-specific manner.
In other preferred embodiments, the invention features an isolated nucleic acid molecule including a sequence encoding a dual-specificity MAPK phosphatase having at least 40% identity with the amino acid sequence shown in
FIG. 7
(SEQ ID NO: 2). In preferred embodiments, the sequence that encodes a dual-specificity MAPK phosphatase includes the amino acid sequence shown in
FIG. 7
(SEQ ID NO: 2). Preferred nucleic acid molecules are obtained from cruciferous plants, for example,
Arabidopsis thaliana.
An exemplary nucleic acid molecule of a cruciferous dual-specificity MAPK phosphatase is shown in
FIG. 7
(SEQ ID NO: 1).
In still another aspect, the invention features a plant including an isolated nucleic acid molecule including a sequence (encoding a dual-specificity MAPK phosphatase having at least 40% identity with the amino acid sequence shown in
FIG. 7
(SEQ ID NO: 2). In addition, the invention features seeds and cells from such plants, as well as parts of such plants. These plants may be produced according to conventional methods of molecular biology using any crop or ornamental plant, e.g., those plants described herein.
In yet another aspect, the invention features a substantially pure dual-specificity MAPK phosphatase including an amino acid sequence that has at least 40% (and preferably, 50%, 60%, 70%, 80%, or 90%) identity to the amino acid sequence of
FIG. 7
(SEQ ID NO: 2) In preferred embodiments, the polypeptide is obtained from a cruciferous species, for example,
Arabidopsis thaliana.
Exemplary plants which are useful in the methods of the invention, as well as for generating the transgenic plants (or plant cells, plant tissues, plant organs, or plant parts) of the invention, include, without limitation, dicots and monocots, such as sugar cane, wheat, rice, maize, sugar beet, barley, mani
Chiu Wan-Ling
Sheen Jen
Clark & Elbing LLP
Fox David T.
Kubelik Anne
The General Hospital Corporation
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