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
2000-03-21
2004-06-08
Nelson, Amy (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...
C435S419000, C435S468000, C536S024100, C800S298000, C800S306000, C800S312000, C800S314000, C800S317200, C800S317300, C800S317400, C800S320000, C800S320100, C800S320200, C800S320300, C800S322000
Reexamination Certificate
active
06747189
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates generally to transgenic plants. More specifically, it relates to methods and compositions for transgene expression using a
Zea mays
glycine rich protein promoter.
2. Description of the Related Art
An important aspect in the production of genetically engineered crops is obtaining sufficient levels of transgene expression in the appropriate plant tissues. In this respect, the selection of promoters for directing expression of a given transgene is crucial. Promoters which are useful for plant transgene expression include those that are inducible, viral, synthetic, constitutive as described (Poszkowski et al., 1989; Odell et al., 1985), temporally regulated, spatially regulated, and spatio-temporally regulated (Chau et al., 1989).
A number of plant promoters have been described with various expression characteristics. Examples of some constitutive promoters which have been described include the rice actin 1 (Wang et al., 1992; U.S. Pat. No. 5,641,876), CaMV 35S (Odell et al., 1985), CaMV 19S (Lawton et al., 1987), nos (Ebert et al., 1987), Adh (Walker et al., 1987), and sucrose synthase (Yang & Russell, 1990).
Examples of tissue specific promoters which have been described include the lectin (Vodkin et al., 1983; Lindstrom et al., 1990), corn alcohol dehydrogenase 1 (Vogel et al., 1989; Dennis et al., 1984), corn light harvesting complex (Simpson, 1986; Bansal et al., 1992), corn heat shock protein (Odell et al., 1985; Rochester et al., 1986), pea small subunit RuBP carboxylase (Poulsen et al., 1986; Cashmore et al., 1983), Ti plasmid mannopine synthase (Langridge et al., 1989), Ti plasmid nopaline synthase (Langridge et al., 1989), petunia chalcone isomerase (Van Tunen et al., 1988), bean glycine rich protein 1 (Keller et al., 1989), truncated CaMV 35s (Odell et al., 1985), potato patatin (Wenzler et al., 1989), root cell (Conkling et al., 1990), maize zein (Reina et al., 1990; Kriz et al., 1987; Wandelt and Feix, 1989; Langridge and Feix, 1983; Reina et al., 1990), globulin-1 (Belanger and Kriz et al., 1991), (&agr;-tubulin, cab (Sullivan et al., 1989), PEPCase (Hudspeth & Grula, 1989), R gene complex-associated promoters (Chandler et al., 1989), and chalcone synthase promoters (Franken et al., 1991).
Inducible promoters which have been described include ABA- and turgor-inducible promoters, the promoter of the auxin-binding protein gene (Schwob et al., 1993), the UDP glucose flavonoid glycosyl-transferase gene promoter (Ralston et al., 1988); the MPI proteinase inhibitor promoter (Cordero et al., 1994), and the glyceraldehyde-3-phosphate dehydrogenase gene promoter (Kohler et al., 1995; Quigley et al., 1989; Martinez et al., 1989).
A class of genes which are expressed in an inducible manner are glycine-rich proteins (GRPs). GRPs are a class of proteins characterized by their high content of glycine residues, which often occur in repetitive blocks (Goddemeier et al., 1998). Many GRPs are thought to be structural wall proteins or RNA-binding proteins (Mar Alba et al., 1994). Genes encoding glycine rich proteins have been described, for example, from maize (Didierjean et al., 1992; Baysdorfer, Genbank Accession No. AF034945) sorghum (Cretin and Puigdomenech, 1990), and rice (Genbank Accession No. AF009411). One maize GRP coding sequence in particular, the expression of which was found to be water and wounding stress-inducible, was isolated by Gomez et al., (1988), the sequence of which is given by Genbank Accession No. GI:22312. However, this reference failed to provide the sequence of the promoter of the gene.
Although the above studies have provided a number of useful tools for the generation of transgenic plants, there is still a great need in the art for novel promoter sequences with beneficial expression characteristics. The number of effective promoters available for use with transgenes in maize is not abundant. New promoters, especially promoters that will express differentially in maize tissues, are spatially and/or temporally expressed, or are induced to express by different environmental signals, would be useful. Such expression specific promoters could be useful in minimizing yield drag and other potential adverse physiological effects on maize growth and development that might be encountered by high-level, non-inducible, constitutive expression of a transgenic protein in a plant. A wider range of effective promoters also would make it possible to introduce multiple transgenes into a plant, each fused to a different promoter, thereby minimizing the risk of DNA sequence homology dependent transgene inactivation (co-suppression). Therefore, there is a great need in the art for the identification of novel inducible promoters which can be used for the high-level expression of selected transgenes in economically important crop plants.
SUMMARY OF THE INVENTION
In one aspect, the invention provides an isolated nucleic acid comprising a maize GRP promoter. Still further provided by the invention is a maize GRP promoter isolatable from the nucleic acid sequence of SEQ ID NO:1. In particular embodiments, a GRP promoter in accordance with the invention may comprise from about 95 to about 3536, about 110 to about 3536, about 125 to about 3536, about 250 to about 3536, about 400 to about 3536, about 750 to about 3536, about 1000 to about 3536, about 1500 to about 3536, about 2000 to about 3536, about 2500 to about 3536, or about 3000 to about 3536 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO:1, as well as all lengths of contiguous nucleotides within such sizes. In further embodiments, the GRP promoter may comprise the nucleic acid sequence of SEQ ID NO:1.
An isolated nucleic acid comprising a maize GRP promoter in accordance with the invention may further comprising an enhancer, for example, an intron. In one embodiment, the intron is a rice actin 1 intron or rice actin 2 intron. The isolated nucleic acid may further comprise a terminator, such as a rbcS terminator.
In another aspect, the invention provides a transgenic plant stably transformed with a selected DNA comprising a maize GRP promoter. In particular embodiments of the invention, the maize GRP promoter may comprise from about 95 to about 3536, about 110 to about 3536, about 125 to about 3536, about 250 to about 3536, about 400 to about 3536, about 750 to about 536, about 1000 to about 3536, about 1500 to about 3536, about 2000 to about 3536, about 2500 to about 3536, or about 3000 to about 3536 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO:1. In one embodiment of the invention, the GRP promoter comprises the nucleic acid sequence of SEQ ID NO:1.
The selected DNA may further comprise any additional desired sequences. In one embodiment of the invention, the selected DNA further comprises a selected heterologous coding region operably linked to the maize GRP promoter. Potentially any coding sequence could be employed with the maize GRP promoter, including a selected coding region which encodes a protein imparting insect resistance, bacterial disease resistance, fungal disease resistance, viral disease resistance, nematode disease resistance, herbicide resistance, enhanced grain composition or quality, enhanced nutrient utilization, enhanced environment or stress resistance, reduced mycotoxin contamination, male sterility, a selectable marker phenotype, a screenable marker phenotype, a negative selectable marker phenotype, or altered plant agronomic characteristics. Where the selected coding region encodes a protein imparting a selectable marker phenotype, the protein may be selected from, for example, the group consisting of phosphinothricin acetyltransferase, glyphosate resistant EPSPS, aminoglycoside phosphotransferase, hygromycin phosphotransferase, neomycin phosphotransferase, dalapon dehalogenase, bromoxynil resistant nitrilase, anthranilate synthase and glyphosate oxidoreductase. The selected coding region may be operably linked to a terminator, for example, an rbcS terminator, including a rice rb
Laccetti Lucille B.
McElroy David
Orozco, Jr. Emil M.
Collins Cynthia
DeKalb Genetics Corporation
Fulbright & Jaworski L.L.P.
Nelson Amy
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