Multicellular living organisms and unmodified parts thereof and – Method of introducing a polynucleotide molecule into or... – The polynucleotide alters plant part growth
Reexamination Certificate
2001-03-19
2004-12-07
Mehta, Ashwin (Department: 1638)
Multicellular living organisms and unmodified parts thereof and
Method of introducing a polynucleotide molecule into or...
The polynucleotide alters plant part growth
C260S66500B, C260S66500B, C260S66500B
Reexamination Certificate
active
06828477
ABSTRACT:
FIELD OF THE INVENTION
The present invention is directed to plant genetic engineering. In particular, it relates to modulation of expression of genes controlling endosperm development in plants.
BACKGROUND OF THE INVENTION
A fundamental problem in biology is to understand how fertilization initiates reproductive development. In higher plants, the ovule generates the female gametophyte which is composed of egg, central, synergid and antipodal cells (Reiser, et al.,
Plant Cell,
1291-1301 (1993)). All are haploid except the central cell which contains two daughter nuclei that fuse prior to fertilization. One sperm nucleus fertilizes the egg to form the zygote, whereas another sperm nucleus fuses with the diploid central cell nucleus to form the triploid endosperm nucleus (van Went, et al.,
Embryology of Angiosperms
, pp. 273-318 (1984)). The two fertilization products undergo distinct patterns of development. In
Arabidopsis
, the embryo passes through a series of stages that have been defined morphologically as preglobular, globular, heart, cotyledon and maturation (Goldberg, R. B., et al.,
Science
(1994) 266: 605-614; Mansfield, S. G., et al.,
Arabidopsis: An Atlas of Morphology and Development
, pp. 367-383 (1994)). The primary endosperm nucleus undergoes a series of mitotic divisions to produce nuclei that migrate into the expanding central cell (Mansfield, S. G., et al.,
Arab Inf Serv
27: 53-64 (1990); Webb, M. C., et al.,
Planta
184: 187-195 (1991)). Cytokinesis sequesters endosperm cytoplasm and nuclei into discrete cells (Mansfield, S. G., et al.,
Arab Inf Serv
27: 65-72 (1990)) that produce storage proteins, starch, and lipids which support embryo growth (opes, M. A. et al.,
Plant Cell
5: 1383-1399 (1993)). Fertilization also activates development of the integument cell layers of the ovule that become the seed coat, and induces the ovary to grow and form the fruit, or silique, in
Arabidopsis.
Control of the expression of genes that control egg and central cell differentiation, or those that activate reproductive development in response to fertilization is useful in the production of plants with a range of desired traits. These and other advantages are provided by the present application.
SUMMARY OF THE INVENTION
The present invention provides methods of modulating fruit and seed development and other traits in plants. The methods involve providing a plant comprising a recombinant expression cassette containing an FIE nucleic acid linked to a plant promoter.
In some embodiments, transcription of the FIE nucleic acid inhibits expression of an endogenous FIE gene or activity the encoded protein. This embodiment is particularly useful, for instance, making embryo-less seed and parthenocarpic fruit. Alternatively, expression of the FIE nucleic acid may enhance expression of an endogenous FIE gene or FIE activity
In the expression cassettes, the plant promoter may be a constitutive promoter, for example, the CaMV 35S promoter. Alternatively, the promoter may be a tissue-specific promoter. Examples of tissue specific expression useful in the invention include ovule-specific or embryo-specific expression. For instance, the promoter sequence from the FIE genes disclosed here can be used to direct expression in relevant plant tissues.
The invention also provides seed or fruit produced by the methods described above. The seed or fruit of the invention comprise a recombinant expression cassette containing an FIE nucleic acid.
Definition
The phrase “nucleic acid sequence” refers to a single or double-stranded polymer of deoxyribonucleotide or ribonucleotide bases read from the 5′ to the 3′ end. It includes chromosomal DNA, self-replicating plasmids, infectious polymers of DNA or RNA and DNA or RNA that performs a primarily structural role.
A “promoter” is defined as an array of nucleic acid control sequences that direct transcription of an operably linked nucleic acid. As used herein, a “plant promoter” is a promoter that functions in plants. Promoters include necessary nucleic acid sequences near the start site of transcription, such as, in the case of a polymerase II type promoter, a TATA element. A promoter also optionally includes distal enhancer or repressor elements, which can be located as much as several thousand base pairs from the start site of transcription. A “constitutive” promoter is a promoter that is active under most environmental and developmental conditions. An “inducible” promoter is a promoter that is active under environmental or developmental regulation. The term “operably linked” refers to a functional linkage between a nucleic acid expression control sequence (such as a promoter, or array of transcription factor binding sites) and a second nucleic acid sequence, wherein the expression control sequence directs transcription of the nucleic acid corresponding to the second sequence.
The term “plant”, includes whole plants, plant organs (e.g., leaves, stems, flowers, roots, etc.), seeds and plant cells and progeny of same. The class of plants which can be used in the method of the invention is generally as broad as the class of higher plants amenable to transformation techniques, including angiosperms (monocotyledonous and dicotyledonous plants), as well as gymnosperms. It includes plants of a variety of ploidy levels, including polyploid, diploid, haploid and hemizygous.
A polynucleotide sequence is “heterologous to” an organism or a second polynucleotide sequence if it originates from a foreign species, or, if from the same species, is modified from its original form. For example, a promoter operably linked to a heterologous coding sequence refers to a coding sequence from a species different from that from which the promoter was derived, or, if from the same species, a coding sequence which is different from any naturally occurring allelic variants.
A polynucleotide “exogenous to” an individual plant is a polynucleotide which is introduced into the plant by any means other than by a sexual cross. Examples of means by which this can be accomplished are described below, and include
Agrobacterium
-mediated transformation, biolistic methods, electroporation, and the like. Such a plant containing the exogenous nucleic acid is referred to here as an R, generation transgenic plant. Transgenic plants which arise from sexual cross or by selfing are descendants of such a plant.
A “FIE nucleic acid” or “FIE polynucleotide sequence” of the invention is a subsequence or full length polynucleotide sequence of a gene which encodes a polypeptide involved in control of reproductive development and which, when mutated, allows for aspects of fertilization independent reproductive development. In some embodiments, the polypeptides of the invention have substantial sequence identity (as defined below) to a polycomb group gene of Drosophila. An exemplary nucleic acid of the invention is the
Arabidopsis
FIE1 and FIE3 sequences disclosed below. FIE polynucleotides are defined by their ability to hybridize under defined conditions to the exemplified nucleic acids or PCR products derived from them. An FIE polynucleotide is typically at least about 30-40 nucleotides to about 3000, usually less than about 5000 nucleotides in length. The nucleic acids contain coding sequence of from about 100 to about 2000 nucleotides, often from about 500 to about 1700 nucleotides in length.
FIE nucleic acids are a new class of plant regulatory genes that encode polypeptides with sequence identity to members of the polycomb group genes first identified in
Drosophila
. Polycomb group gene products and their homologues in other species are responsible for repression of homeotic genes. The proteins are a heterogenous group that interact with each other to form large complexes that bind DNA and thereby control gene expression. For a review of the current understanding of polycomb complex genes see, Pirrotta
Cur. Op. Genet. Dev.
7:249-258 (1997). Nine groups of polycomb genes have been identified. FIE1 (SEQ ID NO:1) is related to the group of polycomb genes encoding protein
Fischer Robert L.
Goldberg Robert B.
Harada John
Kiyosue Tomohiro
Margossian Linda
Mehta Ashwin
Regents of the University of California
Townsend and Townsend / and Crew LLP
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