Multicellular living organisms and unmodified parts thereof and – Method of introducing a polynucleotide molecule into or... – The polynucleotide alters plant part growth
Reexamination Certificate
1999-01-08
2003-05-06
Fox, David T. (Department: 1638)
Multicellular living organisms and unmodified parts thereof and
Method of introducing a polynucleotide molecule into or...
The polynucleotide alters plant part growth
C800S271000, C800S274000, C800S278000, C800S285000, C800S286000, C800S287000, C800S303000, C536S023600, C435S468000
Reexamination Certificate
active
06559357
ABSTRACT:
FIELD OF THE INVENTION
The present invention is directed to plant genetic engineering. In particular, it relates to new methods for altering plant mass (for instance organ size/mass) and fertility in plants.
BACKGROUND OF THE INVENTION
Control of organ mass/size and fertility in plants is a significant goal in commercial agriculture. Plant shoot vegetative organs and/or structures (e.g. leaves, stems and tubers), roots, flowers and floral organs and/or structures (e.g. bracts, sepals, petals, stamens, carpels, anthers and ovules), seed (including embryo, endosperm, and seed coat) and fruit (the mature ovary) are the harvested product of numerous agronomically-important crop plants. Therefore the ability to manipulate the size/mass of these organs/structures through genetic control would be an important agricultural tool. Similarly, induction of sterility in plants is useful in limiting plant pollination and reproduction until it is economically desirable. For example, male sterile plants are often desirable in crops where hybrid vigor increases yield.
The intrinsic plant organ size is determined genetically, although it can be altered greatly by environment signals (e.g. growth conditions). In general, larger organs consist of larger numbers of cells. Since neither cell migration nor cell death plays a major role during plant development, the number of cells in plant organs depends on cell proliferation. Precise regulation of cell proliferation is also necessary for proper development of reproductive organs that make plants fertile. While some basic research has identified genes involved in plant organ development and fertility, little is known about genetic control of cell proliferation or its link to organogenesis including organ size/mass control and fertility in plants. Therefore an important goal is to understand the connection between genes that control organogenesis and genes that control cell proliferation. A great deal of basic research has shown that the components (e.g., cyclin dependent kinases, cyclins and their inhibitors) and mechanisms (e.g., regulation by phosphorylations, ubiquitin-mediated proteolysis) that control the cell cycle in yeast and animals are conserved in higher plants (Burssens, et al.
Plant Physiol Biochem.
36:9-19 (1998)).
In Arabidopsis, the developing flower includes the ovule. Wild-type ovule development in Arabidopsis has been extensively analyzed (Robinson-Beers et al.,
Plant Cell
4:1237-1249 (1992); Modrusan, et al.
Plant Cell.
6:333-349 (1994) and Schneitz et al.,
Plant J.
7:731-749 (1995)). A variety of mutations that affect ovule development have been identified (Klucher et al.,
Plant Cell
8:137-153 (1996); Elliott et al.,
Plant Cell.
8:155-168 (1996); Baker, et al.,
Genetics.
145:1109-1124 (1997); Robinson-Beers, et al.,
Plant Cell.
4:1237-1249 (1992); Modrusan et al.
Plant Cell.
6:333-349 (1994); Ray, A., et al.
Proc Natl Acad Sci USA.
91:5761-5765 (1994); Lang, et al.,
Genetics
137:1101-1110 (1994); Leon-Kloosterziel
Plant Cell.
6:385-392 (1994); Gaiser et al.,
Plant Cell
7:333-345 (1995)), and some of them have been found that specifically affect patterns of cell division (Schneitz, et al.
Development.
124:1367-1376 (1997)). Of those, several genes have been cloned; AINTEGUMENTA (ANT) (Klucher et al.
Plant Cell.
8:137-153 (1996); Elliott et al.,
Plant Cell.
8:155-168 (1996)), AGAMOUS, (Yanofsky et al.,
Nature.
346:35-39 (1990); Bowman et al.,
Plant Cell.
3:749-758 (1991)), SUPERMAN (Sakai et al.,
Nature.
378:199-203 (1995)). Because these genes are expressed and function not only in developing ovules but also in various developing organs, analysis of these mutations and genes has provided general information about the control of cell proliferation during plant development.
In spite of the recent progress in defining the genetic control of plant cell proliferation, little progress has been reported in the identification and analysis of genes effecting agronomically important traits such as organ mass/size, fertility and the like through regulating cell proliferation. Characterization of such genes would allow for the genetic engineering of plants with a variety of desirable traits. The present invention addresses these and other needs.
SUMMARY OF THE INVENTION
The present invention provides methods for modulating cell proliferation and thus cell number in plants by modulating ANT activity in plants. Typically, the methods comprise modulating the expression of ANT in plants and selecting for plants with altered size/mass, fertility, or both. In some preferred embodiments, the ANT activity is increased and plants with increased cell proliferation and thus increased cell number are selected. One method for modulating ANT expression is by introducing into a plant an expression cassette containing a heterologous ANT nucleic acid operably linked to a promoter. Examples of possible ANT nucleic acids that can be used include the nucleic acids shown in SEQ ID NO:1 and SEQ ID NO:4. Other examples include nucleic acids that encode the polypeptides shown in either SEQ ID NO:2 or SEQ ID NO:5.
The plant promoters used in the methods of the invention are not critical to the invention. The promoter can be constitutive, inducible or specific for an organ, tissue, or cell. In some embodiments a promoter from an ANT gene, e.g. SEQ ID NO: 3, is used. Expression of the ANT nucleic acids of the invention can be directed to any desired organ, tissue, or cell in the plant. In some preferred embodiments of the invention, the promoter directs expression of the ANT nucleic acid in shoot vegetative organs/structures, such as leaf, stem and tuber. In other preferred embodiments, the promoter directs expression of the ANT nucleic acid in roots. In other preferred embodiments, the promoter directs expression of the ANT nucleic acid in flowers or floral organs/structures, such as bracts, sepals, petals, stamens, carpels, anthers and ovules. In different embodiments, the promoter directs expression of the ANT nucleic acid in seeds (e.g. embryo, endosperm, and seed coat) or fruit.
In another embodiment of the invention, a heterologous gene is expressed in meristematic tissue of a plant by introducing into a plant an expression cassette containing an ANT promoter operably linked to a heterologous polynucleotide. In a preferred embodiment of this invention, the ANT promoter is shown in SEQ ID NO:3.
The invention also provides isolated nucleic acid molecules comprising an ANT nucleic acid that specifically hybridizes to SEQ ID NO: 4, which is isolated from
Brassica napus.
Definitions
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.
The term “promoter” refers to regions or sequence located upstream and/or downstream from the start of transcription and which are involved in recognition and binding of RNA polymerase and other proteins to initiate transcription. A “plant promoter” is a promoter capable of initiating transcription in plant cells.
The term “plant” includes whole plants, shoot vegetative organs/structures (e.g. leaves, stems and tubers), roots, flowers and floral organs/structures (e.g. bracts, sepals, petals, stamens, carpels, anthers and ovules), seed (including embryo, endosperm, and seed coat) and fruit (the mature ovary), plant tissue (e.g. vascular tissue, ground tissue, and the like) and cells (e.g. guard cells, egg cells, trichomes and the like), 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 and lower plants amenable to transformation techniques, including angiosperms (monocotyledonous and dicotyledonous plants), gymnosperms, ferns, and multicellular algae. It includes plants of a variety of ploidy levels, including aneuploid, polyploid, diploid, haploid an
Fischer Robert L.
Mizukami Yukiko
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