Multicellular living organisms and unmodified parts thereof and – Method of introducing a polynucleotide molecule into or... – The polynucleotide contains a tissue – organ – or cell...
Reexamination Certificate
1999-08-19
2001-05-01
Fox, David T. (Department: 1638)
Multicellular living organisms and unmodified parts thereof and
Method of introducing a polynucleotide molecule into or...
The polynucleotide contains a tissue, organ, or cell...
C800S278000, C800S281000, C800S320000, C800S320100, C800S320200, C800S320300, C435S069100, C435S320100, C435S419000, C435S468000, C435S412000, C536S023100, C536S023200, C536S024100, C536S023600
Reexamination Certificate
active
06225529
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to the field of plant molecular biology, more particularly to regulation of gene expression in plants.
BACKGROUND OF THE INVENTION
Expression of heterologous DNA sequences in a plant host is dependent upon the presence of an operably linked promoter that is functional within the plant host. Choice of the promoter sequence will determine when and where within the organism the heterologous DNA sequence is expressed. Where continuous expression is desired throughout the cells of a plant, constitutive promoters are utilized. In contrast, where gene expression in response to a stimulus is desired, inducible promoters are the regulatory element of choice. Where expression in specific tissues or organs are desired, tissue-specific promoters may be used. That is, they may drive expression in specific tissues or organs. Such tissue-specific promoters may be constitutive or inducible. In either case, additional regulatory sequences upstream and/or downstream from the core promoter sequence may be included in expression constructs of transformation vectors to bring about varying levels of expression of heterologous nucleotide sequences in a transgenic plant.
Frequently it is desirable to have constitutive or inducible expression of a DNA sequence in particular tissues or organs of a plant. For example, increased nutritional value of a plant might be accomplished by genetic manipulation of the plant's genome to comprise a seed-preferred promoter operably linked to a heterologous gene such that proteins with enhanced amino acid content are produced in the seed of the plant.
Alternatively, it might be desirable to inhibit expression of a native DNA sequence within a plant's tissues to achieve a desired phenotype. In this case, such inhibition might be accomplished with transformation of the plant to comprise a tissue-specific promoter operably linked to an antisense nucleotide sequence, such that expression of the antisense sequence produces an RNA transcript that interferes with translation of the mRNA of the native DNA sequence.
Seed development involves embryogenesis and maturation events as well as physiological adaptation processes that occur within the seed to insure progeny survival. Developing plant seeds accumulate and store carbohydrate, lipid, and protein that are subsequently used during germination. Expression of storage protein genes in seeds occurs primarily in the embryonic axis and cotyledons and in the endosperm of developing seeds but never in mature vegetative tissues. Generally, the expression patterns of seed proteins are highly regulated. This regulation includes spatial and temporal regulation during seed development. A variety of proteins accumulate and decay during embryogenesis and seed development and provide an excellent system for investigating different aspects of gene regulation as well as for providing regulatory sequences for use in genetic manipulation of plants.
Thus, isolation and characterization of seed-preferred promoters that can serve as regulatory regions for expression of heterologous nucleotide sequences of interest in a seed-preferred manner are needed for genetic manipulation of plants.
SUMMARY OF THE INVENTION
It is an object of the present invention to provide a novel nucleotide sequence for modulating gene expression in a plant.
It is a further object of the present invention to provide an isolated promoter capable of driving transcription in a seed-preferred manner.
It is a further object of the present invention to provide a method of improved control of an endogenous or exogenous product in the seed of a transformed plant.
It is a further object of the present invention to provide a method for providing useful changes in the phenotype of a seed of a transformed plant.
It is a further object of the present invention to provide a method for producing a novel product in the seed of a transformed plant.
It is a further object of the present invention to provide a method for producing a novel function in the seed of a transformed plant.
Therefore, in one aspect, the present invention relates to an isolated nucleic acid comprising a member selected from the group consisting of:
a) sequences capable of driving expression of coding regions selected from the group consisting of coding regions for Cim 1 (cytokinin-induced message); cZ19B1 (maize 19 KDa zein); or mi1ps (myo-inositol-1-phosphate synthase);
b) a sequence comprising at least 20 contiguous nucleotides of the sequence set forth in either of SEQ ID NOS 1, 4, or 7;
c) a sequence comprising a variant or fragment of the nucleotide sequence set forth in either of SEQ ID NOS 1, 4, or 7;
d) the nucleotide sequences set forth in SEQ ID NOS 1, 4, or 7;
e) nucleic acids having at least 60% sequence identity to SEQ ID NO 1, at least 81% sequence identity to SEQ ID NO 4, or at least 60% sequence identity to SEQ ID NO 7, wherein the % sequence identity is based on the entire sequence and is determined by GAP version 10 analysis using default parameters;
f) a sequence that hybridizes to any one of SEQ ID NOS: 1, 4, or 7, under low stringency conditions; and
g) a nucleic acid complementary to a nucleic acid of (a) through (f).
In other aspects, the present invention relates to expression cassettes comprising the promoter operably linked to a nucleotide sequence, vectors containing the expression cassette, and plants stably transformed with at least one expression cassette.
In a further aspect, the present invention relates to a method for modulating expression in the seed of a stably transformed plant comprising the steps of (a) transforming a plant cell with an expression cassette comprising the promoter of the present invention operably linked to at least one nucleotide sequence; (b) growing the plant cell under plant growing conditions and (c) regenerating a stably transformed plant from the plant cell wherein expression of the nucleotide sequence alters the phenotype of the seed.
Compositions and methods for regulating expression of heterologous nucleotide sequences in a plant are provided. Compositions are novel nucleotide sequences for seed-preferred plant promoters, more particularly transcriptional initiation regions isolated from the plant genes Cim1 (cytokinin-induced message); cZ19B1 (maize 19 KDa zein); and mi1ps (myo-inositol-1-phosphate synthase). A method for expressing a heterologous nucleotide sequence in a plant using the transcriptional initiation sequences disclosed herein is provided. The method comprises transforming a plant cell with a transformation vector that comprises a heterologous nucleotide sequence operably linked to one of the plant promoters of the present invention and regenerating a stably transformed plant from the transformed plant cell. In this manner, the promoter sequences are useful for controlling the expression of endogenous as well as exogenous products in a seed-preferred manner.
Downstream from and under the transcriptional initiation regulation of the seed-specific region will be a sequence of interest which will provide for modification of the phenotype of the seed. Such modification includes modulating the production of an endogenous product, as to amount, relative distribution, or the like, or production of an exogenous expression product to provide for a novel function or product in the seed.
DETAILED DESCRIPTION OF THE INVENTION
In accordance with the invention nucleotide constructs are provided that allow initiation of transcription in seed. Constructs of the invention comprise regulated transcriptional initiation regions associated with seed formation and seed tissues. Thus, the compositions of the present invention comprise novel nucleotide sequences for plant promoters, more particularly seed-preferred promoters for the genes Cim 1 (cytokinin-induced message); cZ19B1 (maize 19 KDa zein); and mi1ps (myo-inositol-1-phosphate synthase).
The promoters for these genes may be isolated from the 5′ untranslated region flanking their respective transcription initiation sites. Methods for
Lappegard Kathryn K.
Martino-Catt Susan J.
Fox David T.
Ibrahim Medina A.
Pioneer Hi-Bred International , Inc.
Pioneer Hi-Bred International Inc.
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