Methods for improving seed characteristics

TECHNICAL FIELD

[0002] The present invention relates to the field of crop improvement. Specifically, the present invention relates to the identification and use of genes encoding molecules involved in altering the number and characteristics of aleurone cells in plant seeds and the use of these genes and mutants thereof to improve nutritional, industrial, and agricultural traits in seed and grain.

BACKGROUND OF THE INVENTION

[0003] Most common cereal grains contain cells called aleurone cells that occur as the most external layer of the endosperm. Aleurone cells are generally oil and protein-rich and secrete enzymes allowing the mobilization of endosperm reserves during seed germination. Barley, oats, and some varieties of rice have been reported to have 2 to 4 layers of aleurone cells (Sawicki et al., 1952, and Hoshikawa et al, 1967). Maize commonly has only a single layer making up only 2% or less of the weight of the kernel (Hinton et al, 1953). A South American race of maize, Coroico, has been found to contain from 2 to 6 layers of aleurone cells (Wolf et al, 1972). This characteristic has been referred to as the Mal (multiple aleurone) phenotype by Welch et al, 1993.

[0004] When compared to grains having only a single layer of aleurone cells, varieties having multiple aleurone cell layers contain increased amounts of minerals (Welch et al., 1993) and protein (Wolf, ibid). However, the Mal phenotype is inherited as a partially dominant trait and is highly variable in the Coroico line. To date, a reliable, multiple aleurone trait has not been available in maize or other cereal crops that could be used in a breeding program to take advantage of the unique characteristics of aleurone cells.

[0005] Thus, a transgenic or breeding approach that alters the number of aleurone cells in a grain crop as well as the characteristics of the aleurone cells would be highly desirable.

SUMMARY OF THE INVENTION

[0006] Several genes have been isolated that are known to affect the differentiation of the aleurone layer in crop seeds. This invention foresees using these nucleic acids, or polypeptides, or variants thereof, to improve seed and grain quality by altering the number, type, and/or characteristics of aleurone cells.

DETAILED DESCRIPTION OF THE INVENTION

[0007] Units, prefixes, and symbols may be denoted in their SI accepted form. Unless otherwise indicated, nucleic acids are written left to right in 5′ to 3′ orientation; amino acid sequences are written left to right in amino to carboxy orientation, respectively. Numeric ranges recited within the specification are inclusive of the numbers defining the range and include each integer within the defined range. Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes. Unless otherwise provided for, software, electrical, and electronics terms as used herein are as defined in The New IEEE Standard Dictionary of Electrical and Electronics Terms (5th edition, 1993). The terms defined below are more fully defined by reference to the specification as a whole.

[0008] By “seed” or “kernel” is intended to include the grain or ripened ovule of a plant, or more broadly, a propagative plant structure. The terms “seed” and “kernel” are used interchangeably herein.

[0009] The term “grain” as used herein means the mature seed produced by commercial growers for purposes other than growing or reproducing the species.

[0010] As used herein the term “recombinant” means having parts combined in a configuration not found in nature.

[0011] The term “coding sequence” or “coding region” refers to a nucleotide sequence that codes for a specific amino acid sequence.

[0012] The term “expression” as used herein, refers to the transcription and accumulation of sense (mRNA) or antisense RNA derived from a nucleic acid fragment of the invention. Expression may also refer to translation of mRNA into a polypeptide.

[0013] The term “isolated” refers to material, such as a nucleic acid or a protein, which is: (1) substantially or essentially free from components which normally accompany or interact with the material as found in its naturally occurring environment or (2) if the material is in its natural environment, the material has been altered by deliberate human intervention to a composition and/or placed at a locus in the cell other than the locus native to the material.

[0014] As used herein, the term “nucleic acid” means a polynucleotide and includes single or multi-stranded polymers of deoxyribonucleotide or ribonucleotide bases. Nucleic acids may also include fragments and modified nucleotides. Therefore, as used herein, the terms “nucleic acid” and “polynucleotide” are used interchangably.

[0015] As used herein, “polypeptide” means proteins, protein fragments, modified proteins (e.g., glycosylated, phosphorylated, or other modifications), amino acid sequences and synthetic amino acid sequences. The polypeptide can be modified or not. Therefore, as used herein, “polypeptide” and “protein” are used interchangably.

[0016] As used herein, “plant” includes plants and plant parts including but not limited to plant cells and plant tissues such as leaves, stems, roots, flowers, pollen, and seeds.

[0017] As used herein, “promoter” includes reference to a region of DNA upstream from the start of transcription and involved in recognition and binding of RNA polymerase and other proteins to initiate transcription.

[0018] By “fragment” is intended a portion of the nucleotide sequence or a portion of the amino acid sequence and hence protein encoded thereby. Fragments of a nucleotide sequence may encode protein fragments that retain the biological activity of the native nucleic acid ie: “functional fragments”. Alternatively, fragments of a nucleotide sequence that can be useful as hybridization probes may not encode fragment proteins retaining biological activity. Thus, fragments of a nucleotide sequence are generally greater than 25, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, or 700 nucleotides and up to and including the entire nucleotide sequence encoding the proteins of the invention. Generally the probes are less than 1000 nucleotides and often less than 500 nucleotides. Fragments of the invention include antisense sequences used to decrease expression of the inventive polynucleotides. Such antisense fragments may vary in length ranging from greater than 25, 50, 100, 200, 300, 400, 500, 600, or 700 nucleotides and up to and including the entire coding sequence.

[0019] By “functional equivalent” as applied to a polynucleotide or a protein is intended a polynucleotide or a protein of sufficient length to modulate the level of protein activity in a plant cell. A polynucleotide functional equivalent can be in sense or antisense orientation.

[0020] By “variants” is intended substantially similar sequences. Generally, nucleic acid sequence variants of the invention will have at least 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 99% sequence identity to the native nucleotide sequence, wherein the % sequence identity is based on the entire sequence and is determined by GAP 10 analysis using default parameters. Generally, polypeptide sequence variants of the invention will have at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 98% sequence identity to the native protein, wherein the % sequence identity is based on the entire sequence and is determined by GAP 10 analysis using default parameters. GAP uses the algorithm of Needleman and Wunsch (J. Mol. Biol. 48:443-453, 1970) to find the alignment of two complete sequences that maximizes the number of matches and minimizes the number of gaps.

[0021] As used herein “transformation” includes stable transformation and transient transformation.

[0022] As used herein “stable transformation” refers to the transfer of a nucleic acid fragment into a genome of a host organism (this includes both nuclear and organelle genomes) resulting in genetically stable inheritance. In addition to traditional methods, stable transformation includes the alteration of gene expression by any means including chimeraplasty or transposon insertion.

[0023] “Non-ruminant animal” means an animal with a simple stomach divided into the esophageal, cardia, fundus and pylorus regions. A non-ruminant animal additionally implies a species of animal without a functional rumen. A rumen is a section of the digestive system where feedstuff/food is soaked and subjected to digestion by microorganisms before passing on through the digestive tract. This phenomenon does not occur in a non-ruminant animal. The term non-ruminant animal includes, but is not limited to: humans, swine, poultry, cats and dogs.

[0024] The term “aleurone cell” as used herein refers to an epidermis-like cell layer in the seeds of many crop species. This aleurone layer functions as “an important source of hydrolytic enzymes required for remobilization of stored starch and protein during germination” (Becraft et al., Science, 1996 273:1406-1409.)

[0025] Wild-type aleurone cells contain oil bodies, phytic acid inclusions and protein carbohydrate bodies. Storage proteins are in the form of globulins. Wild-type aleurone cells contain very little or no starch granules. As a seed matures, aleurone cells go through a maturation and desiccation program. After seed desiccation, mature aleurone cells respond to GA stimulation by initiation of transcription of genes encoding amylases, proteases and glucanases upon inhibition. A marker of aleurone cells is that they can activate transcription from the barley Ltp2 promoter (Kalla, R. et al., 1994). Sudan red can be used to visualize the high concentration of oil bodies in the aleurone layer, as well as the low amount of oil bodies in nearby starchy endosperm cells. Aleurone cells may also be transgenically manipulated to alter expression in—including but not limited to—type or amount of: oil bodies and their contents, storage proteins, protein-carbohydrate bodies, phytate deposition, and responsiveness to GA in the form of transcriptional activation of one or more of the genes activated in germinating seeds.

[0026] It is expected that modulating the expression of the nucleic acids of the present invention will modulate signaling in the aleurone cell developmental pathway providing methods to alter the number and characteristics of the aleurone cells.

[0027] Increasing the aleurone cell number is a mechanism for increasing the concentration of plant sterols (phytosterols) and their derivatives. Phytosterols have been shown to have important applications to human health by, for example, decreasing serum cholesterol concentrations. Sitostanol, a sterol derivative that is particularly effective in reducing cholesterol, is found at unusually high concentrations in maize aleurone cells.

[0028] As used herein, “Dek1” is a polynucleotide of the present invention that encodes a polypeptide which is a protein involved in aleurone cell differentiation, and refers to one or more amino acid sequences, in modified or unmodified form, that is necessary and sufficient for aleurone cell fate determination in a transformed plant. The term is also inclusive of fragments, variants, homologs, alleles or precursors (e.g., preproproteins or proproteins) that, when introduced into a plant or plant germ line by way of—but not limited to—transformation or breeding, result in changes in the aleurone cells.

[0029] The Dek1 gene has been identified as a calpain based on the presence of the cystein proteinase domain shared by all members of the calpain super gene family. The sequence of the single copy gene ZmDek1-calpain spans about 24 000 bp (SEQ ID NO: 25). A transcriptional start-site is predicted at bp1440 and the coding sequence is between bp 2682-23732, containing 31 exons. Arabidopsis thaliana also has a single copy Del1-calpain orthologue (SEQ ID NO: 28) that shares a remarkable conservation with the maize gene, including an identical exon structure.

[0030] The maize Dek1 coding region is 7110 bp, compared with 6453 bp for Arabidopsis. Based on MPSS transcript profiling technology (Lynx Therapeutics, Inc. 25861 Industrial Blvd. Hayward, Calif. 94545). Maize Dek1 is expressed at a low level in many tissues of the maize plant, including kernel, roots, tassel, stem sheaths, leaf, seedlings and roots. Using RT-PCR, the transcript is detected in unfertilized ovules and in developing grains up to 15 DAP, but very little at 22 DAP.

[0031] The isolated maize dek1 promoter region is characterized by a putative TATA box at positions 2283-2287 of SEQ ID NO:25 and a putative CAAT box at positions 2333-2337 of SEQ ID NO:25.

[0032] Kernels of the dek1 corn mutant lack aleurone cells and the endosperm of such mutants consists of starchy endosperm cells in the periphery normally occupied by aleurone cells. Positional cues specify and maintain aleurone cell fate in maize endosperm development. A reversion of a dek1 mutant allele to wild-type restores aleurone cell identity to peripheral starchy endosperm cells in which this event occurs (Becraft et al., Development, 127(18):4039-48, 2000).

[0033] The maize DEK1 protein is 2159 amino acid residues (239.0 kDa) and 2151 amino acids for Arabidopis: the two protein sharing 70% overall identity. The DEK1 proteins are identified as members of the calpain superfamily by their conserved cystein proteinase domain II (from aa position 1708-2013 of SEQ ID NO:24) shared by all members of this superfamily, plus similarity in domain III (from aa position 2014-2163) present in many calpains. The identity between maize and Arabidopsis in these two domains is 88 and 83%, respectively.

[0034] DEK1 proteins from maize and Arabidopsis differ from conventional calpains by their N-terminal extensions of 1435 amino acids. This part of the protein contains five distinguishable domains, including a predicted plasma membrane targeting signal in the N-terminus (positions 1-30 of SEQ ID NO:24). This region is followed by a predicted membrane spanning peptide sequence (positions 31-335 of SEQ ID NO:24). A similar domain, containing 15 predicted membrane-spanning peptide sequences, is found further downstream (positions 620-1108 of SEQ ID NO:24). The identity between maize and Arabidopsis in these two regions is 72% and 64%, respectively. The two membrane spanning domains are separated by a loop region (positions 336-628 of SEQ ID NO:24). This region is the least conserved part of the maize and Arabidopis DEK1 proteins, with 57% identity.

[0035] Over-expression or modification of this protein can increase the number of aleurone layer cells, or the production of aleurone cells in parts of the seed that do not normally possess these cells. Further, creation of a dominant version of the dek1 protein would render the signal transduction pathway responsible for aleurone cell fate specification constitutively active in the cells expressing the gene—inducing formation of aleurone layers independent of the positional cues proposed to be involved in aleurone cell formation.

[0036] The crinkly 4 (cr4)(Becraft, P. W., Science, 1996, 273:1406-1409) gene encodes a putative receptor kinase that is involved in the signaling pathway controlling aleurone layer differentiation. Over-expression or modification of this protein can increase the number of aleurone layer cells.

[0037] A dominant version of cr4 can be obtained by functional screening mutated cr4 gene. The cr4 kinase domain as described in Becraft can be fused to the extracellular domain of the clv1 (Clark S. E., et al. Cell. 1997 May 16;89(4):575-85) or bri1 (Wang Z. Y., et al, Nature. 2001 Mar 15;410(6826):380-3) gene to form a chimeric receptor that can be activated by CLV3 (Trotochaud A. E., et al, Science Jul. 28, 2000 289(5479):613-7) or brassinosteroid.

[0038] As used herein “Superal” is a polynucleotide of the present invention that encodes a polypeptide involved in or interacts with the signal transduction pathway that determines aleurone cell fate. The kernels of superal mutants are characterized by as many as eight layers of aleurone cells. Down regulation of this protein can increase the number of aleurone cells in the seed.

Nucleic Acids

[0039] The isolated nucleic acids of the present invention can be made using standard recombinant methods, synthetic techniques, or combinations thereof known in the art. In some embodiments, the polynucleotides of the present invention can be cloned, amplified, or otherwise constructed from monocots or dicots. Typical plants include maize, barley, wheat, rice, rye, oats, millet, soybeans, arabidopsis, tobacco, canola, sunflower, or sorghum.

[0040] Functional fragments included in the invention can be obtained using primers which selectively hybridize under stringent conditions. Primers are generally at least 12 bases in length and can be as high as 200 bases, but will generally be from 15 to 75, or more likely from 15 to 50 bases. Functional fragments can be identified using a variety of techniques such as restriction analysis, Southern analysis, primer extension analysis, and DNA sequence analysis.

[0041] The present invention includes a plurality of polynucleotides that encode for the DEK1 amino acid sequence. The degeneracy of the genetic code allows for such “silent variations” which can be used, for example, to selectively hybridize and detect allelic variants of polynucleotides of the present invention. Additionally, the present invention includes isolated nucleic acids comprising allelic variants. The term “allele” as used herein refers to a related nucleic acid of the same gene.

[0042] Variants of nucleic acids included in the invention can be obtained, for example, by oligonucleotide-directed mutagenesis, linker-scanning mutagenesis, mutagenesis using the polymerase chain reaction, and the like. See, for example, pages 8.0.3-8.5.9 Current Protocols in Molecular Biology, Ausubel et al., Eds., Greene Publishing and Wiley-Interscience, New York (1995). Also, see generally, McPherson (ed.), DIRECTED MUTAGENESIS: A Practical Approach, (IRL Press, 1991). Thus, the present invention also encompasses DNA molecules comprising nucleotide sequences that have substantial sequence similarity with the inventive sequences.

[0043] Variants included in the invention may contain individual substitutions, deletions or additions to the nucleic acid or polypeptide sequences which alter, add or delete a single amino acid or a small percentage of amino acids in the encoded sequence. A “conservatively modified variant” is an alteration which results in the substitution of an amino acid with a chemically similar amino acid. When the nucleic acid is prepared or altered synthetically, advantage can be taken of known codon preferences of the intended host.

[0044] The present invention also includes “shufflents” produced by sequence shuffling of the inventive polynucleotides to obtain a desired characteristic. Sequence shuffling is described in PCT publication No. 96/19256. See also, Zhang, J. H., et al., Proc. Natl. Acad. Sci. USA 94:4504-4509 (1997).

[0045] The present invention also includes a promoter region natively associated with the maize dek1 coding region. By “natively associated” is meant a promoter region as it exists prior to its isolation from its associated in vivo coding region.

[0046] The present invention also includes the use of 5′ and/or 3′ UTR regions for modulation of translation of heterologous coding sequences. Positive sequence motifs include translational initiation consensus sequences (Kozak, Nucleic Acids Res.15:8125 (1987)) and the 7-methylguanosine cap structure (Drummond et al., Nucleic Acids Res. 13:7375 (1985)). Negative elements include stable intramolecular 5′ UTR stem-loop structures (Muesing et al., Cell 48:691 (1987)) and AUG sequences or short open reading frames preceded by an appropriate AUG in the 5′ UTR (Kozak, supra, Rao et al., Mol. and Cell. Biol. 8:284 (1988)).

[0047] Further, the polypeptide-encoding segments of the polynucleotides of the present invention can be modified to alter codon usage. Altered codon usage can be employed to alter translational efficiency. Codon usage in the coding regions of the polynucleotides of the present invention can be analyzed statistically using commercially available software packages such as “Codon Preference” available from the University of Wisconsin Genetics Computer Group (see Devereaux et al., Nucleic Acids Res. 12:387-395 (1984)) or MacVector 4.1 (Eastman Kodak Co., New Haven, Conn.).

[0048] For example, the inventive nucleic acids can be optimized for enhanced expression in plants of interest. See, for example, EPA0359472; WO91/16432; Perlak et al. (1991) Proc. Natl. Acad. Sci. USA 88:3324-3328; and Murray et al. (1989) Nucleic Acids Res. 17:477-498. In this manner, the polynucleotides can be synthesized utilizing plant-preferred codons. See, for example, Murray et al. (1989) Nucleic Acids Res. 17:477-498, the disclosure of which is incorporated herein by reference.

[0049] The present invention provides subsequences comprising isolated nucleic acids containing at least 20 contiguous bases of the inventive sequences. For example the isolated nucleic acid includes those comprising at least 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700 or 800 contiguous nucleotides of the inventive sequences. Subsequences of the isolated nucleic acid can be used to modulate or detect gene expression by introducing into the subsequences compounds which bind, intercalate, cleave and/or crosslink to nucleic acids.

[0050] The nucleic acids of the invention may conveniently comprise a multi-cloning site comprising one or more endonuclease restriction sites inserted into the nucleic acid to aid in isolation of the polynucleotide. Also, translatable sequences may be inserted to aid in the isolation of the translated polynucleotide of the present invention. For example, a hexa-histidine marker sequence provides a convenient means to purify the proteins of the present invention.

[0051] A polynucleotide of the present invention can be attached to a vector, adapter, promoter, transit peptide or linker for cloning and/or expression of a polynucleotide of the present invention. Additional sequences may be added to such cloning and/or expression sequences to optimize their function in cloning and/or expression, to aid in isolation of the polynucleotide, or to improve the introduction of the polynucleotide into a cell. Use of cloning vectors, expression vectors, adapters, and linkers is well known and extensively described in the art. For a description of such nucleic acids see, for example, Stratagene Cloning Systems, Catalogs 1995, 1996, 1997 (La Jolla, Calif.); and, Amersham Life Sciences, Inc, Catalog '97 (Arlington Heights, Ill.).

[0052] The isolated nucleic acid compositions of this invention, such as RNA, cDNA, genomic DNA, or a hybrid thereof, can be obtained from plant biological sources using any number of cloning methodologies known to those of skill in the art. In some embodiments, oligonucleotide probes which selectively hybridize, under stringent conditions, to the polynucleotides of the present invention are used to identify the desired sequence in a cDNA or genomic DNA library.

[0053] Exemplary total RNA and mRNA isolation protocols are described in Plant Molecular Biology: A Laboratory Manual, Clark, Ed., Springer-Verlag, Berlin (1997); and, Current Protocols in Molecular Biology, Ausubel et al., Eds., Greene Publishing and Wiley-Interscience, New York (1995). Total RNA and mRNA isolation kits are commercially available from vendors such as Stratagene (La Jolla, Calif.), Clonetech (Palo Alto, Calif.), Pharmacia (Piscataway, N.J.), and 5′-3′ (Paoli, Pa.). See also, U.S. Pat. Nos. 5,614,391; and, 5,459,253.

[0054] Typical cDNA synthesis protocols are well known to the skilled artisan and are described in such standard references as: Plant Molecular Biology: A Laboratory Manual, Clark, Ed., Springer-Verlag, Berlin (1997); and, Current Protocols in Molecular Biology, Ausubel et al., Eds., Greene Publishing and Wiley-Interscience, New York (1995). cDNA synthesis kits are available from a variety of commercial vendors such as Stratagene or Pharmacia.

[0055] An exemplary method of constructing a greater than 95% pure full-length cDNA library is described by Carninci et al., Genomics 37:327-336 (1996). Other methods for producing full-length libraries are known in the art. See, e.g., Edery et al., Mol. Cell Biol. 15(6):3363-3371 (1995); and PCT Application WO 96/34981.

[0056] It is often convenient to normalize a cDNA library to create a library in which each clone is more equally represented. A number of approaches to normalize cDNA libraries are known in the art. Construction of normalized libraries is described in Ko, Nucl. Acids. Res. 18(19):5705-5711 (1990); Patanjali et al., Proc. Natl. Acad. U.S.A. 88:1943-1947 (1991); U.S. Pat. Nos. 5,482,685 and 5,637,685; and Soares et al., Proc. Natl. Acad. Sci. USA 91:9228-9232 (1994).

[0057] Subtracted cDNA libraries are another means to increase the proportion of less abundant cDNA species. See, Foote et al., in Plant Molecular Biology: A Laboratory Manual, Clark, Ed., Springer-Verlag, Berlin (1997); Kho and Zarbl, Technique 3(2):58-63 (1991); Sive and St. John, Nucl. Acids Res. 16(22):10937 (1988); Current Protocols in Molecular Biology, Ausubel et al., Eds., Greene Publishing and Wiley-Interscience, New York (1995); and, Swaroop et al., Nucl. Acids Res. 19(8):1954 (1991). cDNA subtraction kits are commercially available. See, e.g., PCR-Select (Clontech).

[0058] To construct genomic libraries, large segments of genomic DNA are generated by random fragmentation. Examples of appropriate molecular biological techniques and instructions are found in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory, Vols. 1-3 (1989), Methods in Enzymology, Vol. 152: Guide to Molecular Cloning Techniques, Berger and Kimmel, Eds., San Diego: Academic Press, Inc. (1987), Current Protocols in Molecular Biology, Ausubel et al., Eds., Greene Publishing and Wiley-Interscience, New York (1995); Plant Molecular Biology: A Laboratory Manual, Clark, Ed., Springer-Verlag, Berlin (1997). Kits for construction of genomic libraries are also commercially available.

[0059] The cDNA or genomic library can be screened using a probe based upon the sequence of a nucleic acid of the present invention such as those disclosed herein. Probes may be used to hybridize with genomic DNA or cDNA sequences to isolate homologous polynucleotides in the same or different plant species. Those of skill in the art will appreciate that various degrees of stringency of hybridization can be employed in the assay; and either the hybridization or the wash medium can be stringent. The degree of stringency can be controlled by temperature, ionic strength, pH and the presence of a partially denaturing solvent such as formamide.

[0060] Typically, stringent hybridization conditions will be those in which the salt concentration is less than about 1.5 M Na ion, typically about 0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30° C. for short probes (e.g., 10 to 50 nucleotides) and at least about 60° C. for long probes (e.g., greater than 50 nucleotides). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide.

[0061] Exemplary low stringency conditions include hybridization with a buffer solution of 30 to 35% formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulfate) at 37° C., and a wash in 1× to 2×SSC (20×SSC=3.0 M NaCl/0.3 M trisodium citrate) at 50° C. Exemplary moderate stringency conditions include hybridization in 40 to 45% formamide, 1 M NaCl, 1% SDS at 37° C., and a wash in 0.5× to 1×SSC at 55° C. Exemplary high stringency conditions include hybridization in 50% formamide, 1 M NaCl, 1% SDS at 37° C., and a wash in 0.1×SSC at 60° C. Typically the time of hybridization is from 4 to 16 hours.

[0062] An extensive guide to the hybridization of nucleic acids is found in Tijssen, Laboratory Techniques in Biochemistry and Molecular Biology—Hybridization with Nucleic Acid Probes, Part I, Chapter 2 “Overview of principles of hybridization and the strategy of nucleic acid probe assays”, Elsevier, New York (1993); and Current Protocols in Molecular Biology, Chapter 2, Ausubel et al., Eds., Greene Publishing and Wiley-Interscience, New York (1995). Often, cDNA libraries will be normalized to increase the representation of relatively rare cDNAs.

[0063] The nucleic acids of the invention can be amplified from nucleic acid samples using amplification techniques. For instance, polymerase chain reaction (PCR) technology can be used to amplify the sequences of polynucleotides of the present invention and related polynucleotides directly from genomic DNA or cDNA libraries. PCR and other in vitro amplification methods may also be useful, for example, to clone nucleic acid sequences that code for proteins to be expressed, to make nucleic acids to use as probes for detecting the presence of the desired mRNA in samples, for nucleic acid sequencing, or for other purposes.

[0064] Examples of techniques useful for in vitro amplification methods are found in Berger, Sambrook, and Ausubel, as well as Mullis et al., U.S. Pat. No. 4,683,202 (1987); and, PCR Protocols A Guide to Methods and Applications, Innis et al., Eds., Academic Press Inc., San Diego, Calif. (1990). Commercially available kits for genomic PCR amplification are known in the art. See, e.g., Advantage-GC Genomic PCR Kit (Clontech). The T4 gene 32 protein (Boehringer Mannheim) can be used to improve yield of long PCR products. PCR-based screening methods have also been described. Wilfinger et al. describe a PCR-based method in which the longest cDNA is identified in the first step so that incomplete clones can be eliminated from study. BioTechniques, 22(3):481-486 (1997).

[0065] In one aspect of the invention, nucleic acids can be amplified from a plant nucleic acid library. The nucleic acid library may be a cDNA library, a genomic library, or a library generally constructed from nuclear transcripts at any stage of intron processing. Libraries can be made from a variety of plant tissues such as ears, seedlings, leaves, stalks, roots, pollen, or seeds. Good results have been obtained using tissues such as corn nucellus 5 days after silking, corn embryos 20 days after pollination, and dissected embryo sacs at 4, 5, and 6, days after pollination.

[0066] Alternatively, the sequences of the invention can be used to isolate corresponding sequences in other organisms, particularly other plants, more particularly, other monocots. In this manner, methods such as PCR, hybridization, and the like can be used to identify such sequences having substantial sequence similarity to the sequences of the invention. See, for example, Sambrook et al., (1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, N.Y.). and Innis et al. (1990), PCR Protocols: A Guide to Methods and Applications (Academic Press, New York). Coding sequences isolated based on their sequence identity to the entire inventive coding sequences set forth herein or to fragments thereof are encompassed by the present invention.

[0067] The isolated nucleic acids of the present invention can also be prepared by direct chemical synthesis by methods such as the phosphotriester method of Narang et al., Meth. Enzymol. 68:90-99 (1979); the phosphodiester method of Brown et al., Meth. Enzymol. 68:109-151 (1979); the diethylphosphoramidite method of Beaucage et al., Tetra. Lett. 22:1859-1862 (1981); the solid phase phosphoramidite triester method described by Beaucage and Caruthers, Tetra. Letts. 22(20):1859-1862 (1981), e.g., using an automated synthesizer, e.g., as described in Needham-VanDevanter et al., Nucleic Acids Res. 12:6159-6168 (1984); and, the solid support method of U.S. Pat. No. 4,458,066. Chemical synthesis generally produces a single stranded oligonucleotide. This may be converted into double stranded DNA by hybridization with a complementary sequence, or by polymerization with a DNA polymerase using the single strand as a template. One of skill will recognize that while chemical synthesis of DNA is limited to sequences of about 100 bases, longer sequences may be obtained by the ligation of shorter sequences.

Regulatory Sequences

[0068] Transformation of organisms with exogenous DNA sequences can provide commercially desirable strains. In the case of plants, transformation can lead to new varieties having heritable traits of insect resistance, herbicide resistance, and other agronomically important nutritional or morphological properties.

[0069] Diverse regulatory sequences are needed as undesirable biochemical interactions result from using the same regulatory sequence to control more than one gene. For example, transformation with multiple copies of a regulatory element may cause homologous recombination between two or more expression systems, formation of hairpin loops caused from two copies of the same promoter or enhancer in opposite orientation in close proximity, competition between identical expression systems for binding to common promoter-specific regulatory factors, and inappropriate expression levels of an exogenous gene due to trans effects of a second promoter or enhancer.

[0070] The limited number of known regulatory sequences does not cover the desired range of conditions for transgenic expression. Accordingly, any new sequence having a regulatory role in promoting, enhancing or otherwise regulating gene expression could be used in new combinations with known regulatory sequences and would be a valuable contribution to the art.

[0071] By “promoter” or “transcriptional initiation region” is intended a regulatory region of DNA usually comprising a TATA box capable of directing RNA polymerase II to initiate RNA synthesis at the appropriate transcription initiation site for a particular coding sequence. A promoter may additionally comprise other recognition sequences generally positioned upstream or 5′ to the TATA box, referred to as upstream promoter elements, which influence the transcription initiation rate. It is recognized that having identified the nucleotide sequences for the promoter regions disclosed herein, it is within the state of the art to isolate and identify further regulatory elements in the 5′ untranslated region upstream from the particular promoter regions identified herein. Thus the promoter regions disclosed herein are generally further defined by comprising upstream regulatory elements such as those responsible for tissue and temporal expression of the coding sequence, enhancers and the like. In the same manner, the promoter elements which enable expression in the desired tissue such as the seed can be identified, isolated, and used with other core promoters to confirm seed-preferred expression.

[0072] It is recognized that the promoters may be used with their native coding sequences to increase or decrease expression resulting in a change in phenotype in the transformed plant.

[0073] A regulatory sequence “variant” is a modified form of a regulatory sequence wherein one or more bases have been modified, removed or added. For example, a routine way to remove part of a DNA sequence is to use an exonuclease in combination with DNA amplification to produce unidirectional nested deletions of double stranded DNA clones. A commercial kit for this purpose is sold under the trade name Exo-Size™ (New England Biolabs, Beverly, Mass.). Briefly, this procedure entails incubating exonuclease III with DNA to remove progressively nucleotides in the 3′ to 5′ direction at 5′ overhangs, blunt ends or nicks in the DNA template. However, exonuclease III is unable to remove nucleotides at 3′, 4-base overhangs. Timed digests of a clone with this enzyme produces unidirectional nested deletions. After digestion, mung bean nuclease removes single stranded DNA overhangs and the blunt ends are ligated with T4 DNA ligase.

[0074] One example of a regulatory sequence variant is a promoter formed by one or more deletions from a larger promoter. The 5′ portion of a promoter up to the TATA box near the transcription start site can be deleted without abolishing promoter activity, as described by Zhu et al., The Plant Cell 7:1681-89 (1995).

[0075] Those fragments of promoter nucleotide sequences disclosed herein that operate to promote the seed-preferred expression of an operably linked heterologous nucleotide sequence, are referred to as “functional fragments”. These fragments will comprise at least about 20 contiguous nucleotides, preferably at least about 50 contiguous nucleotides, more preferably at least about 75 contiguous nucleotides, even more preferably at least about 100 contiguous nucleotides of the particular promoter nucleotide sequence disclosed herein. The nucleotides of such fragments will usually comprise the TATA recognition sequence of the particular promoter sequence. Such fragments may be obtained by use of restriction enzymes to cleave the naturally occurring promoter nucleotide sequences disclosed herein; by synthesizing a nucleotide sequence from the naturally occurring promoter DNA sequence; or may be obtained through the use of PCR technology. See particularly, Mullis et al. (1987) Methods Enzymol. 155:335-350, and Erlich, ed. (1989) PCR Technology (Stockton Press, New York). Again, variants of these promoter fragments, such as those resulting from site-directed mutagenesis, are encompassed by the compositions of the present invention.

[0076] An artisan can modify the regulatory sequences as summarized above and expect to obtain a functional product. Others have made these kinds of simple modifications resulting in functional fragments. For example, Rieping and Schoffl reported that “[c]omplete reconstruction of a native (heat shock) promoter region increased” transgenic expression “only very little, but deletion of CCAAT box sequences reduced CAT expression five-fold.” Mol. Gen. Genet. 231:226-32 (1992). The same authors also reported using a “series of deletion mutants of a soybean heat shock (hs) promoter” and that heat inducible “activities were detected except in plants containing a transcriptional fusion devoid of all but 18 nucleotides at the 5′ terminus of the hs gene transcript.” Mol. Gen. Genet. 217:246-53 (1989).

[0077] The coding sequence expressed by the promoters of the invention may be used for varying the phenotype of the seeds. Various changes in phenotype are of interest including modifying the fatty acid composition in seeds, altering the starch or carbohydrate profile, altering the amino acid content of the seed, and the like. These results can be achieved by providing expression of heterologous or increased expression of endogenous products in seeds. Alternatively, the results can be achieved by providing for a reduction of expression of one or more endogenous products, particularly enzymes or cofactors in the seed. These changes result in a change in phenotype of the transformed seed.

[0078] A wide variety of polypeptides or RNA, such as an antisense sequence, can be expressed transgenically under the control of an inventive regulatory sequence. In practice, DNA that codes for a desired polypeptide or RNA is fused to a promoter to achieve a high level of expression. In many cases, the controlled polypeptide or RNA may not be native to the organism in which it is expressed. DNA coding for the polypeptide may be modified to reflect preferred codon usage in the particular species that is the transformation target.

[0079] In cases where the controlled gene is to be expressed in a plant seed or tissue, especially desired are regulatory elements of a “seed tissue-preferred” or “seed tissue-specific” promoter, that is, a promoter that drives high expression of the heterologous DNA segment in seed tissue where control of genes involved in seed metabolism is desired, and little or no expression in other parts of the plant. Manufacture of the protein encoded by the heterologous DNA segment in other parts of the plant needlessly expends the plant's energy.

[0080] A regulatory sequence of the invention is advantageously combined with a selectable marker gene, in physical proximity to the introduced DNA segment. The marker gene allows recovery of transformed cells by positive genetic selection or screening. The selectable marker gene also provides continuing selection pressure on a transgenic plant population, to ensure that the introduced DNA segment is retained by the transgenic organism.

[0081] Many commonly used positive selectable marker genes for transformation have been isolated from bacteria and code for enzymes that metabolically detoxify a selective chemical agent which may be an antibiotic or a herbicide. Other positive selection marker genes encode an altered target which is insensitive to the inhibitor.

[0082] In order to create an expression vector containing the gene and a regulatory sequence to control the gene, an expression cassette first is made by inserting a cloned gene, or a DNA segment comprising the desired regulatory sequences fused to a DNA sequence encoding a desired high-value protein as described above, into a plasmid.

[0083] One or more copies of the expression cassette containing the introduced DNA is transferred to an expression vector. In a preferred embodiment, the vector also contains a gene encoding a selection marker which functionally is linked to a promoter regulatory sequence that controls transcription initiation.

[0084] Transformation protocols as well as protocols for introducing nucleotide sequences into plants may vary depending on the type of plant or plant cell, i.e., monocot or dicot, targeted for transformation as described further herein.

Expression Cassettes

[0085] Expression cassettes comprising isolated nucleic acids of the present invention are provided. An expression cassette will typically comprise a polynucleotide of the present invention operably linked to transcriptional initiation regulatory sequences which will direct the transcription of the polynucleotide in the intended host cell, such as tissues of a transformed plant.

[0086] The construction of such expression cassettes which can be employed in conjunction with the present invention is well known to those of skill in the art in light of the present disclosure. See, e.g., Sambrook et al.; Molecular Cloning: A Laboratory Manual; Cold Spring Harbor, N.Y.; (1989); Gelvin et al.; Plant Molecular Biology Manual (1990); Plant Biotechnology: Commercial Prospects and Problems, eds. Prakash et al.; Oxford & IBH Publishing Co.; New Delhi, India; (1993); and Heslot et al.; Molecular Biology and Genetic Engineering of Yeasts; CRC Press, Inc., USA; (1992); each incorporated herein in its entirety by reference.

[0087] For example, plant expression vectors may include one or more cloned plant genes under the transcriptional control of 5′ and 3′ regulatory sequences and a dominant selectable marker. Such plant expression vectors may also contain, if desired, a promoter regulatory region (e.g., one conferring inducible, constitutive, environmentally- or developmentally-regulated, or cell- or tissue-specific/selective expression), a transcription initiation start site, a ribosome binding site, an RNA processing signal, a transcription termination site, and/or a polyadenylation signal.

[0088] Constitutive, tissue-preferred or inducible promoters can be employed. Examples of constitutive promoters include the cauliflower mosaic virus (CaMV) 35S transcription initiation region, the 1′- or 2′- promoter derived from T-DNA of Agrobacterium tumefaciens, the actin promoter, the ubiquitin promoter, the histone H2B promoter (Nakayama et al., 1992, FEBS Lett 30:167-170), the Smas promoter, the cinnamyl alcohol dehydrogenase promoter (U.S. Pat. No. 5,683,439), the Nos promoter, the pEmu promoter, the rubisco promoter, the GRP1-8 promoter, and other transcription initiation regions from various plant genes known in the art.

[0089] Examples of inducible promoters are the Adh1 promoter which is inducible by hypoxia or cold stress, the Hsp70 promoter which is inducible by heat stress, the PPDK promoter which is inducible by light, the in2 promoter which is safener induced, the ERE promoter which is estrogen induced and the Pepcarboxylase promoter which is light induced.

[0090] Examples of promoters under developmental control include promoters that initiate transcription preferentially in certain tissues, such as leaves, roots, fruit, pollen, seeds, or flowers. An exemplary promoter is the anther specific promoter 5126 (U.S. Pat. Nos. 5,689,049 and 5,689,051). Examples of seed-preferred promoters include, but are not limited to, 22 kDa zein promoter, 27 kD gamma zein promoter and waxy promoter, (Boronat, A., et al., Plant Sci. 47:95-102 (1986); Reina, M., et al., Nucleic Acids Res. 18(21):6426 (1990); Kloesgen, R. B., et al., Mol. Gen. Genet. 203:237-244 (1986)), as well as the globulin 1, oleosin and the phaseolin promoters. A typical aleurone-preferred promoter is the Ltp2 promoter (Kalla, R., et al., Plant Journal, 4:849-860, 1994; see also SEQ ID NO:18). The barley nuc1 promoter (see SEQ ID NO:17), and cim1 promoter (U.S. Pat. No. 6,225,529 issued May 1, 2001) are examples of nucellus tissue preferred promoters. Developing endosperm promoters that may be used are the end1 and end2 promoters (see SEQ ID NOS:19 and 20). The disclosures each of these are incorporated herein by reference in their entirety.

[0091] Either heterologous or non-heterologous (i.e., endogenous) promoters can be employed to direct expression of the nucleic acids of the present invention. These promoters can also be used, for example, in expression cassettes to drive expression of antisense nucleic acids to reduce, increase, or alter concentration and/or composition of the proteins of the present invention in a desired tissue.

[0092] If polypeptide expression is desired, it is generally desirable to include a polyadenylation region at the 3′-end of a polynucleotide coding region. The polyadenylation region can be derived from the natural gene, from a variety of other plant genes, or from T-DNA. The 3′ end sequence to be added can be derived from, for example, the nopaline synthase or octopine synthase genes, or alternatively from another plant gene, or less preferably from any other eukaryotic gene.

[0093] An intron sequence can be added to the 5′ untranslated region or the coding sequence of the partial coding sequence to increase the amount of the mature message that accumulates. See for example Buchman and Berg, Mol. Cell Biol. 8:4395-4405 (1988); Callis et al., Genes Dev. 1:1183-1200 (1987). Use of maize introns Adh1-S intron 1, 2, and 6, the Bronze-1 intron are known in the art. See generally, The Maize Handbook, Chapter 116, Freeling and Walbot, Eds., Springer, N.Y. (1994).

[0094] The vector comprising the sequences from a polynucleotide of the present invention will typically comprise a marker gene which confers a selectable phenotype on plant cells. Usually, the selectable marker gene encodes antibiotic or herbicide resistance. Suitable genes include those coding for resistance to the antibiotics spectinomycin and streptomycin (e.g., the aada gene), the streptomycin phosphotransferase (SPT) gene coding for streptomycin resistance, the neomycin phosphotransferase (NPTII) gene encoding kanamycin or geneticin resistance, the hygromycin phosphotransferase (HPT) gene coding for hygromycin resistance.

[0095] Suitable genes coding for resistance to herbicides include those which act to inhibit the action of acetolactate synthase (ALS), in particular the sulfonylurea-type herbicides (e.g., the acetolactate synthase (ALS) gene containing mutations leading to such resistance in particular the S4 and/or Hra mutations), those which act to inhibit action of glutamine synthase, such as phosphinothricin or basta (e.g., the bar gene), or other such genes known in the art. The bar gene encodes resistance to the herbicide basta and the ALS gene encodes resistance to the herbicide chlorsulfuron.

[0096] Typical vectors useful for expression of genes in higher plants are well known in the art and include vectors derived from the tumor-inducing (Ti) plasmid of Agrobacterium tumefaciens described by Rogers et al., Meth. In Enzymol. 153:253-277 (1987). Exemplary A. tumefaciens vectors useful herein are plasmids pKYLX6 and pKYLX7 of Schardl et al., Gene 61:1-11 (1987) and Berger et al., Proc. Natl. Acad. Sci. USA 86:8402-8406 (1989). Another useful vector herein is plasmid pBl101.2 that is available from Clontech Laboratories, Inc. (Palo Alto, Calif.).

[0097] A variety of plant viruses that can be employed as vectors are known in the art and include cauliflower mosaic virus (CaMV), geminivirus, brome mosaic virus, and tobacco mosaic virus.

[0098] A polynucleotide of the present invention can be expressed in either sense or anti-sense orientation as desired. In plant cells, it has been shown that antisense RNA inhibits gene expression by preventing the accumulation of mRNA which encodes the enzyme of interest, see, e.g., Sheehy et al., Proc. Natl. Acad. Sci. USA 85:8805-8809 (1988); and Hiatt et al., U.S. Pat. No. 4,801,340.

[0099] Another method of suppression is sense suppression. Introduction of nucleic acid configured in the sense orientation has been shown to be an effective means by which to block the transcription of target genes. For an example of the use of this method to modulate expression of endogenous genes see, Napoli et al., The Plant Cell 2:279-289 (1990) and U.S. Pat. No. 5,034,323.

[0100] Recent work has shown suppression with the use of double stranded RNA. Such work is described in Tabara et al., Science 282:5388:430-431 (1998), WO 99/53050 and WO 98/53083.

[0101] Catalytic RNA molecules or ribozymes can also be used to inhibit expression of plant genes. The inclusion of ribozyme sequences within antisense RNAs confers RNA-cleaving activity upon them, thereby increasing the activity of the constructs. The design and use of target RNA-specific ribozymes is described in Haseloff et al., Nature 334:585-591 (1988).

[0102] A variety of cross-linking agents, alkylating agents and radical generating species as pendant groups on polynucleotides of the present invention can be used to bind, label, detect, and/or cleave nucleic acids. For example, Vlassov, V. V., et al., Nucleic Acids Res (1986) 14:4065-4076, describe covalent bonding of a single-stranded DNA fragment with alkylating derivatives of nucleotides complementary to target sequences. A report of similar work by the same group is that by Knorre, D. G., et al., Biochimie (1985) 67:785-789. Iverson and Dervan also showed sequence-specific cleavage of single-stranded DNA mediated by incorporation of a modified nucleotide which was capable of activating cleavage (J. Am. Chem. Soc. (1987) 109:1241-1243). Meyer, R. B., et al., J. Am. Chem. Soc. (1989) 111:8517-8519, effect covalent crosslinking to a target nucleotide using an alkylating agent complementary to the single-stranded target nucleotide sequence. A photoactivated crosslinking to single-stranded oligonucleotides mediated by psoralen was disclosed by Lee, B. L., et al., Biochemistry (1988) 27:3197-3203. Use of crosslinking in triple-helix forming probes was also disclosed by Home et al., J. Am. Chem. Soc. (1990) 112:2435-2437. Use of N4, N4-ethanocytosine as an alkylating agent to crosslink to single-stranded oligonucleotides has also been described by Webb and Matteucci, J. Am. Chem. Soc. (1986) 108:2764-2765; Nucleic Acids Res (1986) 14:7661-7674; Feteritz et al., J. Am. Chem. Soc. 113:4000 (1991). Various compounds to bind, detect, label, and/or cleave nucleic acids are known in the art. See, for example, U.S. Pat. Nos. 5,543,507; 5,672,593; 5,484,908; 5,256,648; and, 5,681,941.

Proteins

[0103] Proteins of the present invention include proteins having the disclosed sequences as well proteins coded by the disclosed polynucleotides. In addition, proteins of the present invention include proteins derived from the native protein by deletion, addition or substitution of one or more amino acids at one or more sites in the native protein. Such variants may result from, for example, genetic polymorphism or from human manipulation. Methods for such manipulations are generally known in the art.

[0104] For example, amino acid sequence variants of the polypeptide can be prepared by mutations in the cloned DNA sequence encoding the native protein of interest. Methods for mutagenesis and nucleotide sequence alterations are well known in the art. See, for example, Walker and Gaastra, eds. (1983) Techniques in Molecular Biology (MacMillan Publishing Company, New York); Kunkel (1985) Proc. Natl. Acad. Sci. USA 82:488-492; Kunkel et al. (1987) Methods Enzymol. 154:367-382; Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (Cold Spring Harbor, N.Y.); U.S. Pat. No. 4,873,192; and the references cited therein; herein incorporated by reference. Guidance as to appropriate amino acid substitutions that do not affect biological activity of the protein of interest may be found in the model of Dayhoff et al. (1978) Atlas of Protein Sequence and Structure (Natl. Biomed. Res. Found., Washington, D.C.), herein incorporated by reference. Conservative substitutions, such as exchanging one amino acid with another having similar properties, may be preferred.

[0105] In constructing variants of the proteins of interest, modifications to the nucleotide sequences encoding the variants can generally be made such that variants continue to possess the desired activity.

[0106] The isolated proteins of the present invention include a polypeptide comprising at least 25 contiguous amino acids encoded by any one of the nucleic acids of the present invention, or polypeptides that are conservatively modified variants thereof. The proteins of the present invention or variants thereof can comprise any number of contiguous amino acid residues from a polypeptide of the present invention, wherein that number is selected from the group of integers consisting of from 25 to the number of residues in a full-length polypeptide of the present invention. Optionally, this subsequence of contiguous amino acids is at least 25, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, or 500 amino acids in length.

[0107] The present invention includes catalytically active polypeptides (i.e., enzymes). Catalytically active polypeptides will generally have a specific activity of at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% that of the native (non-synthetic), endogenous polypeptide. Further, the substrate specificity (kcat/Km) is optionally substantially similar to the native (non-synthetic), endogenous polypeptide. Typically, the Km will be at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% that of the native (non-synthetic), endogenous polypeptide. Methods of assaying and quantifying measures of enzymatic activity and substrate specificity (kcat/Km), are well known to those of skill in the art. See, e.g., Segel, Biochemical Calculations, 2nd ed., John Wiley and Sons, New York (1976).

[0108] The present invention includes modifications that can be made to an inventive protein. In particular, it may be desirable to diminish the activity of the gene. Other modifications may be made to facilitate the cloning, expression, or incorporation of the targeting molecule into a fusion protein. Such modifications are well known to those of skill in the art and include, for example, a methionine added at the amino terminus to provide an initiation site, or additional amino acids (e.g., poly His) placed on either terminus to create conveniently located restriction sites or termination codons or purification sequences.

[0109] Using the nucleic acids of the present invention, one may express a protein of the present invention in recombinantly engineered cells such as bacteria, yeast, insect, mammalian, or plant cells. The cells produce the protein in a non-natural condition (e.g., in quantity, composition, location, and/or time), because they have been genetically altered through human intervention to do so.

[0110] Typically, an intermediate host cell may be used in the practice of this invention to increase the copy number of the cloning vector. With an increased copy number, the vector containing the gene of interest can be isolated in significant quantities for introduction into the desired plant cells.

[0111] Host cells that can be used in the practice of this invention include prokaryotes and eukaryotes. Prokaryotes include bacterial hosts such as Eschericia coli, Salmonella typhimurium, and Serratia marcescens. Eukaryotic hosts such as yeast, insect cells or filamentous fungi may also be used in this invention.

[0112] Commonly used prokaryotic control sequences include such commonly used promoters as the beta lactamase (penicillinase) and lactose (lac) promoter systems (Chang et al., Nature 198:1056 (1977)), the tryptophan (trp) promoter system (Goeddel et al., Nucleic Acids Res. 8:4057 (1980)) and the lambda derived P L promoter and N-gene ribosome binding site (Shimatake et al., Nature 292:128 (1981)). The inclusion of selection markers in DNA vectors transfected in E. coli is also useful. Examples of such markers include genes specifying resistance to ampicillin, tetracycline, or chloramphenicol.

[0113] The vector is selected to allow introduction into the appropriate host cell. Bacterial vectors are typically of plasmid or phage origin. Expression systems for expressing a protein of the present invention are available using Bacillus sp. and Salmonella (Palva et al., Gene 22:229-235 (1983); Mosbach et al., Nature 302:543-545 (1983)).

[0114] Synthesis of heterologous proteins in yeast is well known. See Sherman, F., et al., Methods in Yeast Genetics, Cold Spring Harbor Laboratory (1982). Two widely utilized yeast for production of eukaryotic proteins are Saccharomyces cerevisiae and Pichia pastoris. Vectors, strains, and protocols for expression in Saccharomyces and Pichia are known in the art and available from commercial suppliers (e.g., Invitrogen). Suitable vectors usually have expression control sequences, such as promoters, including 3-phosphoglycerate kinase or alcohol oxidase, and an origin of replication, termination sequences and the like as desired.

[0115] The baculovirus expression system (BES) is a eukaryotic, helper-independent expression system which has been used to express hundreds of foreign genes (Luckow, V., Ch. 4 “Cloning and Expression of Heterologous Genes in Insect Cells with Baculovirus Vectors” in Recombinant DNA Technology and Applications, A. Prokop et al., Eds. McGraw-Hill, Inc. (1991); Luckow, V., Ch. 10 “Insect Expression Technology” in Principles & Practice of Protein Engineering, J. L. Cleland and C. S. Craig, Eds. John Wiley & Sons, (1994)).

[0116] Recombinant baculoviruses are generated by inserting the particular gene- or genes-of-interest into the baculovirus genome using established protocols with vectors and reagents from commercial suppliers (e.g., Invitrogen, Life Technologies Incorporated). Commercial vectors are readily available with various promoters, such as polyhedrin and p10, optional signal sequences for protein secretion, or affinity tags, such as 6×histidine. These recombinant viruses are grown, maintained and propagated in commercially available cell lines derived from several insect species including Spodoptera frugiperda and Trichoplusia ni. The insect cells can be cultured using well-established protocols in a variety of different media, for example, with and without bovine serum supplementation. The cultured cells are infected with the recombinant viruses and the gene-of-interest polypeptide is expressed. Proteins expressed with the baculovirus system have been extensively characterized and, in many cases, their post-translational modifications such as phosphorylation, acylation, etc., are identical to the natively expressed protein.

[0117] A protein of the present invention, once expressed, can be isolated from cells by lysing the cells and applying standard protein isolation techniques to the lysates. The monitoring of the purification process can be accomplished by using Western blot techniques or radioimmunoassay or other standard immunoassay techniques. Expression cassettes are also available which direct the expressed protein to be secreted from the cell into the media. In these cases, the expressed protein can be purified from the cell growth media using standard protein purification techniques.

[0118] The proteins of the present invention can also be constructed using non-cellular synthetic methods. Solid phase synthesis of proteins of less than about 50 amino acids in length may be accomplished by attaching the C-terminal amino acid of the sequence to an insoluble support followed by sequential addition of the remaining amino acids in the sequence. Techniques for solid phase synthesis are described by Barany and Merrifield, Solid-Phase Peptide Synthesis, pp. 3-284 in The Peptides: Analysis, Synthesis, Biology. Vol. 2 Special Methods in Peptide Synthesis, Part A.; Merrifield et al., J. Am. Chem. Soc. 85:2149-2156 (1963), and Stewart et al., Solid Phase Peptide Synthesis, 2nd ed., Pierce Chem. Co., Rockford, Ill. (1984). Proteins of greater length may be synthesized by condensation of the amino and carboxy termini of shorter fragments. Methods of forming peptide bonds by activation of a carboxy terminal end (e.g., by the use of the coupling reagent N,N′-dicyclohexylcarbodiimide)) are known to those of skill.

[0119] The proteins of this invention, recombinant or synthetic, may be purified to substantial purity by standard techniques well known in the art, including detergent solubilization, selective precipitation with such substances as ammonium sulfate, column chromatography, immunopurification methods, and others. See, for instance, R. Scopes, Protein Purification: Principles and Practice, Springer-Verlag: New York (1982); Deutscher, Guide to Protein Purification, Academic Press (1990). For example, antibodies may be raised to the proteins as described herein. Purification from E. coli can be achieved following procedures described in U.S. Pat. No. 4,511,503. Detection of the expressed protein is achieved by methods known in the art and include, for example, radioimmunoassays, Western blotting techniques or immunoprecipitation.

[0120] The present invention further provides a method for modulating (i.e., increasing or decreasing) the concentration or composition of the polypeptides of the present invention in a plant or part thereof. Modulation can be effected by increasing or decreasing the concentration and/or the composition (i.e., the ratio of the polypeptides of the present invention) in a plant.

[0121] The method comprises transforming a plant cell with an expression cassette comprising a polynucleotide of the present invention to obtain a transformed plant cell, growing the transformed plant cell under conditions allowing expression of the polynucleotide in the plant cell in an amount sufficient to modulate concentration and/or composition in the plant cell.

[0122] In some embodiments, the content and/or composition of polypeptides of the present invention in a plant may be modulated by altering, in vivo or in vitro, the promoter of a non-isolated gene of the present invention to up- or down-regulate gene expression. In some embodiments, the coding regions of native genes of the present invention can be altered via substitution, addition, insertion, or deletion to decrease activity of the encoded enzyme. See, e.g., Kmiec, U.S. Pat. No. 5,565,350; Zarling et al., PCT/US93/03868. One method of down-regulation of the protein involves using PEST sequences that provide a target for degradation of the protein.

[0123] In some embodiments, an isolated nucleic acid (e.g., a vector) comprising a promoter sequence is transfected into a plant cell. Subsequently, a plant cell comprising the promoter operably linked to a polynucleotide of the present invention is selected for by means known to those of skill in the art such as, but not limited to, Southern blot, DNA sequencing, or PCR analysis using primers specific to the promoter and to the gene and detecting amplicons produced therefrom. A plant or plant part altered or modified by the foregoing embodiments is grown under plant growing conditions for a time sufficient to: alter the aleurone cells in the seeds of the plant, to increase the level of oil in the seeds of the plant, or to otherwise improve the grain characteristics of the seed of the plant. Plant growing conditions are well known in the art.

[0124] In general, content of the polypeptide is increased or decreased by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% relative to a native control plant, plant part, or cell lacking the aforementioned expression cassette. Modulation in the present invention may occur during and/or subsequent to growth of the plant to the desired stage of development. Modulating nucleic acid expression temporally and/or in particular tissues can be controlled by employing the appropriate promoter operably linked to a polynucleotide of the present invention in, for example, sense or antisense orientation as discussed in greater detail, supra. Induction of expression of a polynucteotide of the present invention can also be controlled by exogenous administration of an effective amount of inducing compound. Inducible promoters and inducing compounds which activate expression from these promoters are well known in the art. In certain embodiments, the polypeptides of the present invention are modulated in monocots or dicots, for example: maize, wheat, rice, barley, soybean, arabidopsis, oats, sorghum, millet, rye, sunflower, safflower, alfalfa, canola, or cotton.

[0125] Means of detecting the proteins of the present invention are not critical aspects of the present invention. The proteins can be detected and/or quantified using any of a number of well-recognized immunological binding assays (see, e.g., U.S. Pat. Nos. 4,366,241; 4,376,110; 4,517,288; and 4,837,168). For a review of the general immunoassays, see also Methods in Cell Biology, Vol. 37: Antibodies in Cell Biology, Asai, Ed., Academic Press, Inc. New York (1993); Basic and Clinical Immunology 7th Edition, Stites & Terr, Eds. (1991). Moreover, the immunoassays of the present invention can be performed in any of several configurations, e.g., those reviewed in Enzyme Immunoassay, Maggio, Ed., CRC Press, Boca Raton, Fla. (1980); Tijan, Practice and Theory of Enzyme Immunoassays, Laboratory Techniques in Biochemistry and Molecular Biology, Elsevier Science Publishers B. V., Amsterdam (1985); Harlow and Lane, supra; Immunoassay: A Practical Guide, Chan, Ed., Academic Press, Orlando, Fla. (1987); Principles and Practice of Immunoassays, Price and Newman Eds., Stockton Press, NY (1991); and Non-isotopic Immunoassays, Ngo, Ed., Plenum Press, NY (1988).

[0126] Typical methods include Western blot (immunoblot) analysis, analytic biochemical methods such as electrophoresis, capillary electrophoresis, high performance liquid chromatography (HPLC), thin layer chromatography (TLC), hyperdiffusion chromatography, and the like, and various immunological methods such as fluid or gel precipitin reactions, immunodiffusion (single or double), immunoelectrophoresis, radioimmunoassays (RIAs), enzyme-linked immunosorbent assays (ELISAs), immunofluorescent assays, and the like.

[0127] Non-radioactive labels are often attached by indirect means. Generally, a ligand molecule (e.g., biotin) is covalently bound to the molecule. The ligand then binds to an anti-ligand (e.g., streptavidin) molecule which is either inherently detectable or covalently bound to a signal system, such as a detectable enzyme, a fluorescent compound, or a chemiluminescent compound. A number of ligands and anti-ligands can be used. Where a ligand has a natural anti-ligand, for example, biotin, thyroxine, and cortisol, it can be used in conjunction with the labeled, naturally occurring anti-ligands. Alternatively, any haptenic or antigenic compound can be used in combination with an antibody.

[0128] The molecules can also be conjugated directly to signal generating compounds, e.g., by conjugation with an enzyme or fluorophore. Enzymes of interest as labels will primarily be hydrolases, particularly phosphatases, esterases and glycosidases, or oxidoreductases, particularly peroxidases. Fluorescent compounds include fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, etc. Chemiluminescent compounds include luciferin, and 2,3-dihydrophthalazinediones, e.g., luminol. For a review of various labeling or signal producing systems which may be used, see, U.S. Pat. No. 4,391,904, which is incorporated herein by reference.

[0129] Some assay formats do not require the use of labeled components. For instance, agglutination assays can be used to detect the presence of the target antibodies. In this case, antigen-coated particles are agglutinated by samples comprising the target antibodies. In this format, none of the components need be labeled and the presence of the target antibody is detected by simple visual inspection.

[0130] The proteins of the present invention can be used for identifying compounds that bind to (e.g., substrates), and/or increase or decrease (i.e., modulate) the enzymatic activity of catalytically active polypeptides of the present invention. The method comprises contacting a polypeptide of the present invention with a compound whose ability to bind to or modulate enzyme activity is to be determined. The polypeptide employed will have at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% of the specific activity of the native, full-length polypeptide of the present invention (e.g., enzyme). Methods of measuring enzyme kinetics are well known in the art. See, e.g., Segel, Biochemical Calculations, 2nd ed., John Wiley and Sons, New York (1976).

[0131] Antibodies can be raised to a protein of the present invention, including individual, allelic, strain, or species variants, and fragments thereof, both in their naturally occurring (full-length) forms and in recombinant forms. Additionally, antibodies are raised to these proteins in either their native configurations or in non-native configurations. Anti-idiotypic antibodies can also be generated. Many methods of making antibodies are known to persons of skill.

[0132] In some instances, it is desirable to prepare monoclonal antibodies from various mammalian hosts, such as mice, rodents, primates, humans, etc. Description of techniques for preparing such monoclonal antibodies are found in, e.g., Basic and Clinical Immunology, 4th ed., Stites et al., Eds., Lange Medical Publications, Los Altos, Calif., and references cited therein; Harlow and Lane, Supra; Goding, Monoclonal Antibodies: Principles and Practice, 2nd ed., Academic Press, New York, N.Y. (1986); and Kohler and Milstein, Nature 256:495-497 (1975).

[0133] Other suitable techniques involve selection of libraries of recombinant antibodies in phage or similar vectors (see, e.g., Huse et al., Science 246:1275-1281 (1989); and Ward et al., Nature 341:544-546 (1989); and Vaughan et al., Nature Biotechnology 14:309-314 (1996)). Alternatively, high avidity human monoclonal antibodies can be obtained from transgenic mice comprising fragments of the unrearranged human heavy and light chain Ig loci (i.e., minilocus transgenic mice). Fishwild et al., Nature Biotech. 14:845-851 (1996). Also, recombinant immunoglobulins may be produced. See, Cabilly, U.S. Pat. No. 4,816,567; and Queen et al., Proc. Natl. Acad. Sci. U.S.A. 86:10029-10033 (1989).

[0134] The antibodies of this invention can be used for affinity chromatography in isolating proteins of the present invention, for screening expression libraries for particular expression products such as normal or abnormal protein or for raising anti-idiotypic antibodies which are useful for detecting or diagnosing various pathological conditions related to the presence of the respective antigens.

[0135] Frequently, the proteins and antibodies of the present invention may be labeled by joining, either covalently or non-covalently, a substance which provides for a detectable signal. A wide variety of labels and conjugation techniques are known and are reported extensively in both the scientific and patent literature. Suitable labels include radionucleotides, enzymes, substrates, cofactors, inhibitors, fluorescent moieties, chemiluminescent moieties, magnetic particles, and the like.

Transformation of Cells

[0136] The method of transformation is not critical to the present invention; various methods of transformation are currently available. As newer methods are available to transform crops or other host cells they may be directly applied. Accordingly, a wide variety of methods have been developed to insert a DNA sequence into the genome of a host cell to obtain the transcription and/or translation of the sequence to effect phenotypic changes in the organism. Thus, any method which provides for efficient transformation/transfection may be employed.

[0137] A DNA sequence coding for the desired polynucleotide of the present invention, for example a cDNA or a genomic sequence encoding a full length protein or functional portion thereof, can be used to construct an expression cassette which can be introduced into the desired plant. Isolated nucleic acid acids of the present invention can be introduced into plants according to techniques known in the art. Generally, expression cassettes as described above and suitable for transformation of plant cells are prepared.

[0138] Techniques for transforming a wide variety of higher plant species are well known and described in the technical, scientific, and patent literature. See, for example, Weising et al., Ann. Rev. Genet 22:421-477 (1988). For example, the DNA construct may be introduced directly into the genomic DNA of the plant cell using techniques such as electroporation, PEG poration, particle bombardment, silicon fiber delivery, or microinjection of plant cell protoplasts or embryogenic callus. See, e.g., Tomes et al., Direct DNA Transfer into Intact Plant Cells Via Microprojectile Bombardment. pp. 197-213 in Plant Cell, Tissue and Organ Culture, Fundamental Methods, Eds. O. L. Gamborg and G. C. Phillips, Springer-Verlag Berlin Heidelberg New York, 1995. Alternatively, the DNA constructs may be combined with suitable T-DNA flanking regions and introduced into a conventional Agrobacterium tumefaciens host vector. The virulence functions of the Agrobacterium tumefaciens host will direct the insertion of the construct and adjacent marker into the plant cell DNA when the cell is infected by the bacteria. See, U.S. Pat. No. 5,591,616.

[0139] The introduction of DNA constructs using polyethylene glycol precipitation is described in Paszkowski et al., Embo J. 3:2717-2722 (1984). Electroporation techniques are described in Fromm et al., Proc. Natl. Acad. Sci. U.S.A. 82:5824 (1985). Ballistic transformation techniques are described in Klein et al., Nature 327:70-73 (1987).

[0140]

Agrobacterium tumefaciens
-meditated transformation techniques are well described in the scientific literature. See, for example Horsch et al., Science 233:496-498 (1984), and Fraley et al., Proc. Natl. Acad. Sci. 80:4803 (1983). For instance, Agrobacterium transformation of maize is described in U.S. Pat. No. 5,981,840; 5,591,616 and 5,731,179 herein incorporated in their entirety by reference. Agrobacterium transformation of soybean is described in U.S. Pat. No. 5,563,055.

[0141] Other methods of transformation include (1) Agrobacterium rhizogenes-mediated transformation (see, e.g., Lichtenstein and Fuller In: Genetic Engineering, Vol. 6, P. W. J. Rigby, Ed., London, Academic Press, 1987; and Lichtenstein, C. P. and Draper, J. In: DNA Cloning, Vol. II, D. M. Glover, Ed., Oxford, IRI Press, 1985), Application PCT/U.S.87/02512 (WO 88/02405 published Apr. 7, 1988) describes the use of A. rhizogenes strain A4 and its Ri plasmid along with A. tumefaciens vectors pARC8 or pARC16, (2) liposome-mediated DNA uptake (see, e.g., Freeman et al., Plant Cell Physiol. 25:1353 (1984)), and (3) the vortexing method (see, e.g., Kindle, Proc. Natl. Acad. Sci. USA 87:1228 (1990)).

[0142] DNA can also be introduced into plants by direct DNA transfer into pollen as described by Zhou et al., Methods in Enzymology, 101:433 (1983); D. Hess, Intern Rev. Cytol., 107:367 (1987); Luo et al., Plant Mol. Biol. Reporter, 6:165 (1988). Expression of polypeptide coding polynucleotides can be obtained by injection of the DNA into reproductive organs of a plant as described by Pena et al., Nature, 325:274 (1987). DNA can also be injected directly into the cells of immature embryos and the rehydration of desiccated embryos as described by Neuhaus et al., Theor. Appl. Genet. 75:30 (1987); and Benbrook et al., in Proceedings Bio Expo 1986, Butterworth, Stoneham, Mass., pp. 27-54 (1986).

[0143] Animal and lower eukaryotic (e.g., yeast) host cells are competent or rendered competent for transformation by various means. There are several well-known methods of introducing DNA into animal cells. These include: calcium phosphate precipitation, fusion of the recipient cells with bacterial protoplasts containing the DNA, treatment of the recipient cells with liposomes containing the DNA, DEAE dextran, electroporation, biolistics, and micro-injection of the DNA directly into the cells. The transfected cells are cultured by means well known in the art. Kuchler, R. J., Biochemical Methods in Cell Culture and Virology, Dowden, Hutchinson and Ross, Inc. (1977).

Transgenic Plant Regeneration

[0144] Transformed plant cells which are derived by any of the above transformation techniques can be cultured to regenerate a whole plant which possesses the transformed genotype. Such regeneration techniques often rely on manipulation of certain phytohormones in a tissue culture growth medium, typically relying on a biocide and/or herbicide marker that has been introduced together with a polynucleotide of the present invention. For transformation and regeneration of maize see, Gordon-Kamm et al., The Plant Cell 2:603-618 (1990).

[0145] Plants cells transformed with a plant expression vector can be regenerated, e.g., from single cells, callus tissue or leaf discs according to standard plant tissue culture techniques. It is well known in the art that various cells, tissues, and organs from almost any plant can be successfully cultured to regenerate an entire plant. Plant regeneration from cultured protoplasts is described in Evans et al., Protoplasts Isolation and Culture, Handbook of Plant Cell Culture, Macmillan Publishing Company, New York, pp.124-176 (1983); and Binding, Regeneration of Plants, Plant Protoplasts, CRC Press, Boca Raton, pp. 21-73 (1985).

[0146] The regeneration of plants containing the foreign gene introduced by Agrobacterium can be achieved as described by Horsch et al., Science 227:1229-1231 (1985) and Fraley et al., Proc. Natl. Acad. Sci. U.S.A. 80:4803 (1983). This procedure typically produces shoots within two to four weeks and these transformant shoots are then transferred to an appropriate root-inducing medium containing the selective agent and an antibiotic to prevent bacterial growth. Transgenic plants of the present invention may be fertile or sterile.

[0147] Regeneration can also be obtained from plant callus, explants, organs, or parts thereof. Such regeneration techniques are described generally in Klee et al., Ann. Rev. of Plant Phys. 38:467-486 (1987). The regeneration of plants from either single plant protoplasts or various explants is well known in the art. See, for example, Methods for Plant Molecular Biology, A. Weissbach and H. Weissbach, eds., Academic Press, Inc., San Diego, Calif. (1988). For maize cell culture and regeneration see generally, The Maize Handbook, Freeling and Walbot, Eds., Springer, N.Y. (1994); Corn and Corn Improvement, 3rd edition, Sprague and Dudley Eds., American Society of Agronomy, Madison, Wis. (1988).

[0148] One of skill will recognize that after the expression cassette is stably incorporated in transgenic plants and confirmed to be operable, it can be introduced into other plants by sexual crossing. Any of a number of standard breeding techniques can be used, depending upon the species to be crossed.

[0149] In vegetatively propagated crops, mature transgenic plants can be propagated by the taking of cuttings, via production of apomictic seed, or by tissue culture techniques to produce multiple identical plants. Selection of desirable transgenics is made and new varieties are obtained and propagated vegetatively for commercial use. In seed propagated crops, mature transgenic plants can be self crossed to produce a homozygous inbred plant. The inbred plant produces seed containing the newly introduced heterologous nucleic acid. These seeds can be grown to produce plants that would produce the selected phenotype.

[0150] Parts obtained from the regenerated plant, such as flowers, seeds, leaves, branches, fruit, and the like are included in the invention, provided that these parts comprise cells comprising the isolated nucleic acid of the present invention. Progeny and variants, and mutants of the regenerated plants are also included within the scope of the invention, provided that these parts comprise the introduced nucleic acid sequences.

[0151] Transgenic plants expressing a selectable marker can be screened for transmission of the nucleic acid of the present invention by, for example, standard immunoblot and DNA detection techniques. Transgenic lines are also typically evaluated on levels of expression of the heterologous nucleic acid. Expression at the RNA level can be determined initially to identify and quantitate expression-positive plants. Standard techniques for RNA analysis can be employed and include PCR amplification assays using oligonucleotide primers designed to amplify only the heterologous RNA templates and solution hybridization assays using heterologous nucleic acid-specific probes. The RNA-positive plants can then be analyzed for protein expression by Western immunoblot analysis using the specifically reactive antibodies of the present invention. In addition, in situ hybridization and immunocytochemistry according to standard protocols can be done using heterologous nucleic acid specific polynucleotide probes and antibodies, respectively, to localize sites of expression within transgenic tissue. Generally, a number of transgenic lines are usually screened for the incorporated nucleic acid to identify and select plants with the most appropriate expression profiles.

[0152] Transgenic plants of the present invention can be homozygous for the added heterologous nucleic acid; i.e., a transgenic plant that contains two added nucleic acid sequences, one gene at the same locus on each chromosome of a chromosome pair. A homozygous transgenic plant can be obtained by sexually mating (selfing) a heterozygous transgenic plant that contains a single added heterologous nucleic acid, germinating some of the seed produced and analyzing the resulting plants produced for altered expression of a polynucleotide of the present invention relative to a control plant (i.e., native, non-transgenic). Back-crossing to a parental plant and out-crossing with a non-transgenic plant (such as Coroico) are also contemplated. Alternatively, propagation of heterozygous transgenic plants could be accomplished through apomixis.

[0153] The present invention provides a method of genotyping a plant comprising a polynucleotide of the present invention. Genotyping provides a means of distinguishing homologs of a chromosome pair and can be used to differentiate segregants in a plant population. Molecular marker methods can be used for phylogenetic studies, characterizing genetic relationships among crop varieties, identifying crosses or somatic hybrids, localizing chromosomal segments affecting monogenic traits, map based cloning, and the study of quantitative inheritance. See, e.g., Plant Molecular Biology: A Laboratory Manual, Chapter 7, Clark, Ed., Springer-Verlag, Berlin (1997). For molecular marker methods, see generally, The DNA Revolution by Andrew H. Paterson 1996 (Chapter 2) in: Genome Mapping in Plants (ed. Andrew H. Paterson) by Academic Press/R. G. Landis Company, Austin, Tex., pp.7-21.

[0154] The particular method of genotyping in the present invention may employ any number of molecular marker analytic techniques such as, but not limited to, restriction fragment length polymorphisms (RFLPs). RFLPs are the product of allelic differences between DNA restriction fragments caused by nucleotide sequence variability. Thus, the present invention further provides a means to follow segregation of a gene or nucleic acid of the present invention as well as chromosomal sequences genetically linked to these genes or nucleic acids using such techniques as RFLP analysis.

[0155] Plants which can be used in the method of the invention include monocotyledonous and dicotyledonous plants. Typical plants include: maize, wheat, rice, barley, soybean, arabidopsis, oats, sorghum, millet, rye, sunflower, safflower, alfalfa, canola, or cotton.

[0156] Seeds derived from plants regenerated from transformed plant cells, plant parts or plant tissues, or progeny derived from the regenerated transformed plants, may be used directly as feed or food, or further processing may occur. Plants grown from these seeds can be effectively used in breeding programs to further incorporate and select for desirable traits.

[0157] All publications cited in this application are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

[0158] The present invention will be further described by reference to the following detailed examples. It is understood, however, that there are many extensions, variations, and modifications on the basic theme of the present invention beyond that shown in the examples and description, which are within the spirit and scope of the present invention.

[0159] Other objects, features, advantages and aspects of the present invention will become apparent to those of skill from the following description. It should be understood, however, that the following description and the specific examples, while indicating certain embodiments of the invention, are given by way of illustration only. Various changes and modifications within the spirit and scope of the disclosed invention will become readily apparent to those skilled in the art from reading the following description and from reading the other parts of the present disclosure.

EXAMPLES

Example 1

cDNA Library Construction

[0160] A. Total RNA Isolation

[0161] Total RNA was isolated from maize tissues with TRIzol Reagent (Life Technology Inc. Gaithersburg, Md.) using a modification of the guanidine isothiocyanate/acid-phenol procedure described by Chomczynski and Sacchi (Anal. Biochem. 162, 156 (1987)). In brief, plant tissue samples were pulverized in liquid nitrogen before the addition of the TRIzol Reagent, and then were further homogenized with a mortar and pestle. Addition of chloroform followed by centrifugation was conducted for separation of an aqueous phase and an organic phase. The total RNA was recovered by precipitation with isopropyl alcohol from the aqueous phase.

[0162] B. Poly(A)+RNA Isolation

[0163] The selection of poly(A)+RNA from total RNA was performed using PolyATract system (Promega Corporation. Madison, Wis.). In brief, biotinylated oligo(dT) primers were used to hybridize to the 3′ poly(A) tails on mRNA. The hybrids were captured using streptavidin coupled to paramagnetic particles and a magnetic separation stand. The mRNA was washed at high stringent condition and eluted by RNase-free deionized water.

[0164] C. cDNA Library Construction

[0165] cDNA synthesis was performed and unidirectional cDNA libraries were constructed using the SuperScript Plasmid System (Life Technology Inc. Gaithersburg, Md.). The first stand of cDNA was synthesized by priming an oligo(dT) primer containing a Not I site. The reaction was catalyzed by SuperScript Reverse Transcriptase II at 45° C. The second strand of cDNA was labeled with alpha-32P-dCTP and a portion of the reaction was analyzed by agarose gel electrophoresis to determine cDNA sizes. cDNA molecules smaller than 500 base pairs and unligated adapters were removed by Sephacryl-S400 chromatography. The selected cDNA molecules were ligated into pSPORT1 vector in between Not I and Sal I sites.

[0166] D. dek1 Isolation and Sequencing

[0167] dek1 maize cDNA fragments were used as probes to screen a maize Mo17 genomic bacterial artificial chromosome (BAC) library. The HindIII and EcoRI subfragments homologous to calcium-requiring cysteine proteinases (calpain) cDNA were identified in selected BAC clones by gel blot hybridization, cloned into a plasmid vector pBKSKS (Stratagene), and sequenced with the help of the ES::TN transposon insertion system (Epicentre Technologies, Madison, Wis.).

Example 2

Sequencing and cDNA Subtraction Procedures Used for Maize EST's

[0168] A. Sequencing Template Preparation

[0169] Individual colonies were picked and DNA was prepared either by PCR with M13 forward primers and M13 reverse primers, or by plasmid isolation. All the cDNA clones were sequenced using M13 reverse primers.

[0170] B. Q-bot Subtraction Procedure

[0171] cDNA libraries subjected to the subtraction procedure were plated out on 22×22 cm2 agar plate at density of about 3,000 colonies per plate. The plates were incubated in a 37° C. incubator for 12-24 hours. Colonies were picked into 384-well plates by a robot colony picker, Q-bot (GENETIX Limited). These plates were incubated overnight at 37° C.

[0172] Once sufficient colonies were picked, they were pinned onto 22×22 cm2 nylon membranes using Q-bot. Each membrane contained 9,216 colonies or 36,864 colonies. These membranes were placed onto individual agar plates with appropriate antibiotic. The plates were incubated at 37° C. for overnight.

[0173] After colonies were recovered on the second day, these filters were placed on filter paper prewetted with denaturing solution for four minutes, then were incubated on top of a boiling water bath for additional four minutes. The filters were then placed on filter paper prewetted with neutralizing solution for four minutes. After excess solution was removed by placing the filters on dry filter papers for one minute, the colony side of the filters were place into Proteinase K solution, incubated at 37° C. for 40-50 minutes. The filters were placed on dry filter papers to dry overnight. DNA was then cross-linked to nylon membrane by UV light treatment.

[0174] Colony hybridization was conducted as described by Sambrook, J., Fritsch, E. F. and Maniatis, T., (in Molecular Cloning: A Laboratory Manual, 2nd Edition). The following probes were used in colony hybridization:

[0175] 1. First strand cDNA from the same tissue from which the library was made to remove the most redundant clones.

[0176] 2. 48-192 most redundant cDNA clones from the same library based on previous sequencing data.

[0177] 3. 192 most redundant cDNA clones in the entire corn sequence database.

[0178] 4. A Sal-A20 oligonucleotide removes clones containing a poly A tail but no cDNA. See SEQ ID NO:22.

[0179] 5. cDNA clones derived from rRNA.

[0180] The image of the autoradiography was scanned into computer and the signal intensity and cold colony addresses of each colony was analyzed. Re-arraying of cold-colonies from 384 well plates to 96 well plates was conducted using Q-bot.

Example 3

Co-segregation Analysis of Superal Mutation

[0181] The maize superal mutation is characterized by multiple aleurone layers. Unlike wild-type maize, superal maize can have up to 8 layers of aleurone. It was isolated from the Pioneer TUSC population by phenotypic analysis; aleurone layers showed a red color when stained with the oil-specific stain red-fat7B. In the TUSC collection, a separate mutant with multiple aleurone layers was isolated by a microscopy screen, which when crossed to superal, demonstrated that this represents a second and independent allele.

[0182] The superal mutation is recessive and showed a simple 3:1 segregation in selfed ears.

[0183] Southern blots of the backcross population showed that a 1.6 kb BgIII fragment of mu1 (Del Giudice,L., et al, Mol. Gen. Genet. 222 (1), 71-76 (1990)) was co-segregated with the superal phenotype. When the band was cloned, the sequences flanking the mu1 insertion showed identity to SEQ ID NO:5. SEQ ID NO:5 showed complete co-segregation with the superal phenotype, consistent with this gene as the causal agent for the superal phenotype.

[0184] Mu1 was inserted in the 5′-UTR of the superal gene. A second independent allele with a mu8 (Fleenor, D., et al, Nucleic Acids Res. 18 (22), 6725 (1990)) insertion at 3′-UTR of superal gene showed the similar multiple aleurone layers, demonstrating that mutations resulting in down-regulation of SUPERAL was responsible for the multi-aleurone layer phenotype.

Example 4

Co-segregation Analysis of dek1 Mutation

[0185] The maize Dek1 mutation is characterized by a lack of aleurone layers.

[0186] Southern blots of a segregating population showed that plants with the Dek1 mutant phenotype did not contain a 3.0 kb Kpnl fragment representing the wild-type band. The plants were probed with the sequence flanking the co-segregating Mu1 insertion, indicating that maize Dek1 is responsible for the dek1 phenotype.

[0187] A second dek1 allele is being characterized.

Example 5

To Assay Gene Functionality

[0188] Dek1

[0189] Grain of the genotype dek1/dek1 (lacking aleurone cells) from Dek1/dek1 plants cultured in vitro had pericarp removed and were bombarded with constructs comprising maize Dek1 behind the beps promoter (Thorbjornsen, T. et al., 1996, Biochem J., 313:149-154, pp 152) driving gene transcription in peripheral starchy endosperm cells, and were co-bombarded with constructs comprising anthocyanin activators c1 (Paz-Ares et al., Embo J. 6(12):3553-8, 1987) and Ic (Ludwig, S. R. et al., Natl Acad Sci US 86(18):7092-6, 1989) behind the 35S promoter. Control constructs comprised only the activators with the 35S promoter.

[0190] Positive results were represented by kernels with red anthocyanine spots due to the presence of aleurone cells. Controls give no red spots due to lack of aleurone cells, as red anthocyanin coloration is expressed in aleurone cells but not starchy endosperm cells.

[0191] Alternatively, cyncytial stage dek1/dek1 endosperm of in vitro nucellus slab culture is micro injected with Dek1. Sectors of endosperm with aleurone cells, stemming from single nuclei or groups of nuclei in which Dek1 was incorporated, proves the function of Dek1 in aleurone cell signaling. The presence of aleurone cells was facilitated by using dek1/Dek1 plants that contained Ltp2::Gus gene constructs. In this genetic background, aleurone cells were detectable by the presence of the blue Gus stain. In these endosperms, the dek1/dek1 genotype of the endosperm was shown by the dek1/dek1 phenotype of the embryo, which lacks axis polarity. In these nucellus slab cultures, the embryos were never or extremely rarely transformed by injected DNA.

[0192] Superal

[0193] The function of the Superal gene as a negative regulator of aleurone cell fate in endosperm cells other than the peripheral layer of the endosperm was shown in co-bombardment experiments of in vitro cultivated colorless wild type grains with an Ltp2::superal cDNA construct and constructs comprising anthocyanin activators c1 (Paz-Ares, et al., Embo J. 6(12):3553-8, 1987) and Ic (Ludwig, S. R. et al., Natl Acad Sci US 86(18):7092-6, 1989) behind the 35S promoter. Control constructs comprised only the activators with the 35S promoter. Positive results were represented by kernels in which red anthocyanine spots did not appear or disappeared shortly after bombardment due to the de-differentiation of aleurone cells to starchy endosperm cells caused by ectopic expression of Superal in aleurone cells.

[0194] Alternatively, bombardment of in vitro cultivated grains with a genotype that gives red aleurone cells with the beps promoter::superal cDNA, demonstrate the wild-type function of SUPERAL as a suppressor of aleurone cell fate by the presence of white spots due to the de-differentiation of aleurone cells to starchy endosperm cells. Control bombardment with other genes under the control of the 35S promoter had no effect on aleurone cell coloration.

[0195] Alternatively, the function of Superal was demonstrated by the injection of Superal in homozygous superal syncytial endosperm of nucellus slab cultures. Endosperm with normal aleurone layers or sectors with normal aleurone layers demonstrated the wild-type function of Superal as a negative regulator of aleurone cell fate in deeper layers of the endosperm. Identification of the injected seeds as homozygous superal seeds was done by the embryo phenotype: homozygous superal embryos being arrested at an early developmental stage. The embryo was never or very rarely transformed by the injected DNA in nucellus slab cultures.

[0196] Alternatively, the function of SUPERAL is demonstrated by co-suppressing superal expression in transgenic seed. The superal gene introduced into plants under a seed specific promoter to silence endogenous superal gene expression, results in multi-aleurone layers.

Example 6

Vector Construction

[0197] All vectors are constructed using standard molecular biology techniques used by those of skill in the art (Sambrook et al., supra). Vectors are constructed for plant transformation using both particle bombardment and Agrobacterium transformation protocols. Plasmids are constructed by inserting the gene of interest into an expression cassette. For example, the Dek1 coding region, including the 5′ UTR and 3′ UTR is isolated from a full length EST clone. The fragment is ligated into an expression cassette comprising the beps promoter and nos terminator. Orientation is confirmed using a restriction enzyme digest. The expression cassette is linked to the selectable marker between the right and left borders of the T-DNA borders of an appropriate vector for Agrobacterium transformation. This vector is used for insert preparation for particle gun transformation as well as for generating Agrobacterium transformation vectors as described below. In this case, insert DNA for particle gun transformation is generated by isolating the insert from the vector.

[0198] The plasmid pSB11 is obtained from Japan Tobacco Inc. (Tokyo, Japan). The construction of pSB11 from pSB21 and the construction of pSB21 from starting vectors is described by Komari et al., 1996, Plant J. 10:165-174. The T-DNA of the plasmid is integrated in to the superbinary plasmid pSB1 (Saito et al., EP 672 752 A1) by homologous recombination between the two plasmids. The plasmid pSB1 is also obtained from Japan Tobacco Inc. These plasmids are either used for particle bombardment transformation, or for Agrobacterium-mediated transformation, after making a cointegrate in an appropriate Agrobacterium strain.

[0199] Competent cells of the Agrobacterium strain LBA4404 harboring pSB1 are created using the protocol as described by Lin (1995) in Methods in Molecular Biology, ed. Nickoloff, J. A. (Humana Press, Totowa, N.J.). The plasmid containing the expression cassettes is electroporated into competent cells of the Agrobacterium strain LBA4404 harboring pSB1 to create the cointegrate plasmid in Agrobacterium. Cells and DNA are prepared for electroporation by mixing 1 ul of plasmid DNA (˜100 ng) with 20 ul of competent Agrobacterium cells in a 0.2 cm electrode gap cuvette (Bio-Rad Cat# 165-2086, Hercules, Calif.). Electroporation is performed in a Bio-Rad Micropulser (Cat# 165-2100, Hercules, Calif.) using the EC2 setting, which delivers 2.5 kV to the cells. Successful recombination is verified by restriction analysis of the plasmid after transformation of the cointegrate plasmid back into E. coil DH5α cells.

Example 7

Plant Transformation

[0200] A. Particle Bombardment Transformation and Regeneration of Maize Callus

[0201] Immature maize embryos from greenhouse or field grown High type II donor plants are bombarded with a plasmid containing a calpain polynucleotide of the invention operably linked to an appropriate promoter. If the polynucleotide does not include a selectable marker, another plasmid containing a selectable marker gene can be co-precipitated on the particles used for bombardment. For example, a plasmid containing the PAT gene (Wohlleben et al., 1988, Gene 70:25-37) which confers resistance to the herbicide Bialaphos can be used. Transformation is performed as follows.

[0202] The ears are surface sterilized in 50% Chlorox bleach plus 0.5% Micro detergent for 20 minutes, and rinsed two times with sterile water. The immature embryos are excised and placed embryo axis side down (scutellum side up), 25 embryos per plate. These are cultured on 560L agar medium 4 days prior to bombardment in the dark. Medium 560L is an N6-based medium containing Eriksson's vitamins, thiamine, sucrose, 2,4-D, and silver nitrate. The day of bombardment, the embryos are transferred to 560Y medium for 4 hours and are arranged within the 2.5-cm target zone. Medium 560Y is a high osmoticum medium (560L with high sucrose concentration).

[0203] A plasmid vector comprising a polynucleotide of the invention operably linked to the selected promoter is constructed. This plasmid DNA, plus plasmid DNA containing a PAT selectable marker if needed, is precipitated onto 1.1 μm (average diameter) tungsten pellets using a CaCl2 precipitation procedure as follows: 100 μl prepared tungsten particles (0.6 mg) in water, 20 μl (2 μg) DNA in TrisEDTA buffer (1 μg total), 100 μl 2.5 M CaCl2, 40 μl 0.1 M spermidine.

[0204] Each reagent is added sequentially to the tungsten particle suspension. The final mixture is sonicated briefly. After the precipitation period, the tubes are centrifuged briefly, liquid removed, washed with 500 ml 100% ethanol, and centrifuged again for 30 seconds. Again the liquid is removed, and 60 μl 100% ethanol is added to the final tungsten particle pellet. For particle gun bombardment, the tungsten/DNA particles are briefly sonicated and 5 μl spotted onto the center of each macrocarrier and allowed to dry about 2 minutes before bombardment.

[0205] The sample plates are bombarded at a distance of 8 cm from the stopping screen to the tissue, using a DuPont biolistics helium particle gun. All samples receive a single shot at 650 PSI, with a total of ten aliquots taken from each tube of prepared particles/DNA.

[0206] Four to 12 hours post bombardment, the embryos are moved to 560P (a low osmoticum callus initiation medium similar to 560L but with lower silver nitrate), for 3-7 days, then transferred to 560R selection medium, an N6 based medium similar to 560P containing 3 mg/liter Bialaphos, and subcultured every 2 weeks. After approximately 10 weeks of selection, callus clones are sampled for PCR and activity of the polynucleotide of interest. Positive lines are transferred to 288J medium, an MS-based medium with lower sucrose and hormone levels, to initiate plant regeneration. Following somatic embryo maturation (2-4 weeks), well-developed somatic embryos are transferred to medium for germination and transferred to the lighted culture room. Approximately 7-10 days later, developing plantlets are transferred to medium in tubes for 7-10 days until plantlets are well established. Plants are then transferred to inserts in flats (equivalent to 2.5″ pot) containing potting soil and grown for 1 week in a growth chamber, subsequently grown an additional 1-2 weeks in the greenhouse, then transferred to Classic™ 600 pots (1.6 gallon) and grown to maturity. Plants are monitored for expression of the polynucleotide of interest.

[0207] B. Agrobacterium-mediated Transformation and Regeneration of Maize Callus

[0208] For Agrobacterum-mediated transformation of maize of a nucleotide sequence encoding a protein of the present invention, the method of Zhao was employed (U.S. Pat. No. 5,981,840, and PCT patent publication WO98/32326; the contents of which are hereby incorporated by reference).

[0209] Briefly, immature embryos were isolated from maize and the embryos contacted with a suspension of Agrobacterium containing a polynucleotide of the present invention, where the bacteria are capable of transferring the nucleotide sequence of interest to at least one cell of at least one of the immature embryos (step 1: the infection step). In this step the immature embryos were immersed in an Agrobacterium suspension for the initiation of inoculation. The embryos were co-cultured for a time with the Agrobacterium (step 2: the co-cultivation step). The immature embryos were cultured on solid medium following the infection step. Following this co-cultivation period an optional “resting” step is contemplated. In this resting step, the embryos were incubated in the presence of at least one antibiotic known to inhibit the growth of Agrobacterium without the addition of a selective agent for plant transformants (step 3: resting step). The immature embryos were cultured on solid medium with antibiotic, but without a selecting agent, for elimination of Agrobacterium and for a resting phase for the infected cells. Next, inoculated embryos were cultured on medium containing a selective agent and growing transformed callus was recovered (step 4: the selection step). The immature embryos were cultured on solid medium with a selective agent resulting in the selective growth of transformed cells. The callus was then regenerated into plants (step 5: the regeneration step), and calli grown on selective medium were cultured on solid medium to regenerate the plants.

[0210] C. DNA Isolation from Callus and Leaf Tissues

[0211] In order to screen putative transformation events for the presence of the transgene, genomic DNA is extracted from calluses or leaves using a modification of the CTAB (cetyltriethylammonium bromide, Sigma H5882) method described by Stacey and Isaac (1994). Approximately 100-200 mg of frozen tissues is ground into powder in liquid nitrogen and homogenised in 1 ml of CTAB extraction buffer (2% CTAB, 0.02 M EDTA, 0.1 M Tris-Cl pH 8, 1.4 M NaCl, 25 mM DTT) for 30 min at 65° C. Homogenised samples are allowed to cool at room temperature for 15 min before a single protein extraction with approximately 1 ml 24:1 v/v chloroform:octanol is done. Samples are centrifuged for 7 min at 13,000 rpm and the upper layer of supernatant collected using wide-mouthed pipefte tips. DNA is precipitated from the supernatant by incubation in 95% ethanol on ice for 1 h. DNA threads are spooled onto a glass hook, washed in 75% ethanol containing 0.2 M sodium acetate for 10 min, air-dried for 5 min and resuspended in TE buffer. Five μl RNAse A is added to the samples and incubated at 37° C. for 1 h.

[0212] For quantification of genomic DNA, gel electrophoresis is performed using a 0.8% agarose gel in 1×TBE buffer. One microliter of the samples are fractionated alongside 200, 400, 600 and 800 ng μl−1 λ uncut DNA markers.

Example 8

Overexpression of CR4 in Transgenic Corn

[0213] The maize cr4 gene (SEQ ID NO:15) was overexpressed in endosperm under the control of the Itp2 promoter (Kalla, R., et al., 1994) (Itp2::cr4::pinII) and 22 kDa zein promoter (Boronat, A., et al.,1986) (22 kDa zein::cr4::22 kDa zein) constructed as generally described in Example 6. A single vector containing both constructs was introduced into maize by agrobacterial transformation (see Example 7B). Transgenic plants showed an extra layer of aleurone cells in the endosperm. This additional layer of cells stained red by red fat-7B, indicating oil accumulation in the extra aleurone cells.

Example 9

Expression of Functional Domains in Bacteria and Plants

[0214] Functional domains that were used in bacterial and plant expression constructs were as follows:

[0215] External loop region: positions 1063-1938 of SEQ ID NO: 23 corresponding to positions 337-628 of SEQ ID NO: 24

[0216] Cytoplasmic domain: positions 3679-6531 of SEQ ID NO: 23 corresponding to positions 1209-2159 of SEQ ID NO:24

[0217] calpain proteinase Domain II: positions 5164-6069 of SEQ ID NO:23 corresponding to positions 1704-2005 of SEQ ID NO:24

[0218] calpain proteinase Domain III: positions 6082-6531 of SEQ ID NO:23 corresponding to positions 2010-2159 of SEQ ID NO:24

[0219] A. Bacterial expression

[0220] Calpain proteinase Domains II+III, and the external loop region were cloned into the pGEX-4T-3 vector (Amersham Biosciences) for expression in E. coli. After induction with IPTG, the bacterial lysates were analyzed for the presence of expressed protein. Proteins are expressed as fusion proteins with the 26 kDa glutathione S-transferase. Both constucts were shown to have GST activity.

[0221] Sequence analysis has indicated that dek1 has domain features similar to calpain. Domain II & III of calpain are highly conserved in dek1. In addition, it is reported that domain II of m-calpain has cysteine protease activity. Therefore, bacteria-expressed domain II+III of dek1 was used to determine if dek1 has calpain activity.

[0222] The so called caseinolytic assay was used for this determination. In this assay, degradation of casein was evaluated by electrophoresis on SDS-PAGE with gel staining. With partially purified Domain II+III, casein was clearly shown to be degraded. Caseinolytic activity of partially purified Domain II+III was higher than that of domain II of human m-calpain as reported.

[0223] B. Plant Expression

[0224] The external loop region, cytoplasmic domain, calpain proteinase domain II, and calpain proteinase domain III are each operably linked to the Itp2 and/or 22 kDa zein promoter to express altered aleurone phenotypes in plants.

[0225] Over-expression of the external loop region is expected to phenocopy the dek1 mutation, ie: lack of aleurone cells.

[0226] Expression of the cytoplasmic domain, domain II, and domain III are expected to increase the number of aleurone layers.

1Sequence Listing TableNucleotide (NT) orSEQ ID NO.DescriptionAmino Acid (AA)1maize dek1 cDNA incomplete se-NTquence2maize dek1 incomplete sequenceAA3Arabidopsis calpain cDNANT4Arabidopsis calpainAA5maize superal cDNANT6maize SUPERALAA7Arabidopsis superal cDNA homo-NTlog 18Arabidopsis SUPERAL homo-AAlog 19Arabidopsis superal cDNA homo-NTlog 210Arabidopsis SUPERAL homolog 2AA11Rice superal homologNT12Wheat superal homologNT13Soybean superal homologNT14Barley superal homologNT15maize crinkly4 (cr4) cDNANT16maize CRINKLY 4AA17barley nuc1 promoterNT18barley Ltp2 promoterNT19maize end1 promoterNT20maize end2 promoterNT21barley beps promoterNT22Sal A20 primerNT23maize dek1 cDNA from B73NT24maize DEK1 from B73AA25maize Mo17 dek1 (genomic)NT26Arabidopsis dek1 cDNANT27Arabidopsis DEK1AA28Arabidopsis dek1 (genomic)NT

[0227]

	Number	Date	Country
	60309719	Aug 2001	US
	60337444	Oct 2001	US

Methods for improving seed characteristics

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