EP1576163A2 - Genes de plantes associes a un phenotype proteagineux - Google Patents

Genes de plantes associes a un phenotype proteagineux

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Publication number
EP1576163A2
EP1576163A2 EP02773582A EP02773582A EP1576163A2 EP 1576163 A2 EP1576163 A2 EP 1576163A2 EP 02773582 A EP02773582 A EP 02773582A EP 02773582 A EP02773582 A EP 02773582A EP 1576163 A2 EP1576163 A2 EP 1576163A2
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EP
European Patent Office
Prior art keywords
plant
nucleic acid
ofthe
acid molecule
polypeptide
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP02773582A
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German (de)
English (en)
Other versions
EP1576163A4 (fr
Inventor
Wenpei Su
Nancy Andon
Paul Haynes
Steven P. Briggs
Bret Cooper
Tong Zhu
Stephen A. Goff
Fumiaki Katagiri
Joel Kreps
Todd Moughamer
Nicholas Provart
Darrell Ricke
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Syngenta Participations AG
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Syngenta Participations AG
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Publication of EP1576163A2 publication Critical patent/EP1576163A2/fr
Publication of EP1576163A4 publication Critical patent/EP1576163A4/fr
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
    • C12N15/8251—Amino acid content, e.g. synthetic storage proteins, altering amino acid biosynthesis
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
    • Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants

Definitions

  • the present invention generally relates to the field of plant molecular biology, and more specifically to plant genes useful to alter the protein content or level in plants and to develop molecular markers for plant breeding..
  • Maize is the world's most widely grown cereal crop and an essential food source for millions ofthe world's poor. More than half of the world's malnourished children live in countries where maize is an important food.
  • maize gruel is the main food mothers use to wean their babies, and maize is the single largest source of calories. But babies who subsist on maize can face a dangerous lack of protein during a critical stage of physical and mental development as diets high in maize lack two essential amino acids needed to prevent malnutrition.
  • the prolamine (zein) fraction of storage proteins comprises over 50% ofthe total protein in the mature seed, however, ⁇ -zein polypeptides which are especially abundant contain extremely low levels ofthe essential amino acids lysine and tryptophan.
  • zein prolamine
  • ⁇ -zein polypeptides which are especially abundant contain extremely low levels ofthe essential amino acids lysine and tryptophan.
  • maize seed protein is deficient in these amino acids because such a large proportion of the total seed storage protein is contributed by the ⁇ -zeins (Mertz et al., 1964).
  • the development of breeding steps to improve maize based on the manipulation of zein profile is hampered by the complexity ofthe zein proteins.
  • the term "zein" encompasses a family of some 100 related proteins.
  • C-zeins include proteins with molecular weights of 19,000 and 22,000 daltons; 3-zeins include proteins with a molecular weight of 14,000 daltons; ( ⁇ zeins include proteins with molecular weights of 27,000 and 26,000 daltons; and ⁇ -zeins include proteins having a molecular weight of 10,000 daltons.
  • the ⁇ -zeins are the major zein proteins found in the endosperm of maize kernels. However, the complexity of zein proteins goes beyond these size classes. Protein sequence analyses indicates that there is microheterogenicity in zein amino acid sequences. This is in accord with isoelectric focusing analyses which show charge differences in zein proteins. Over 70 genes encoding the zein proteins have been identified (Rubenstein, 1982), and the zein genes appear to be located on at least three chromosomes. Thus, the zein proteins are encoded by a multigene family.
  • Down regulation of gene expression in a plant may also occur through expression of a particular transgene.
  • This type of down regulation is referred to as co-suppression and involves coordinate silencing of a transgene and a second transgene or a homologous endogenous gene (Matzke and Matzke, 1995).
  • cosuppression of a herbicide resistance gene in tobacco (Brandle et al., 1995), polygalacturomdase in tomato (Flavell, 1994) and chalcone synthase in petunia (U. S. Patent No. 5,034,323) have been demonstrated.
  • Flavell (1994) suggested that multicopy genes, or gene families, must have evolved to avoid cosuppression in order for multiple copies of related genes to be expressed in a plant.
  • the new corn variety was prepared which contains nearly twice as much usable protein as other maize grown in the tropics and yields 10 percent more grain.
  • the new maize variety called "quality protein maize” (QPM)
  • QPM quality protein maize
  • the new maize variety was developed through traditional plant breeding and looks and tastes like normal maize, but the nutritive value of its protein is nearly equivalent to cow's milk.
  • the varieties produce 70-100 percent more of lysine and tryptophan.
  • a bumper crop ofthe maize is expected in the coming months from more than one million hectares (2.5 million acres) currently under cultivation in 11 countries. Economists expect that by 2003, the number of hectares sown to QPM will triple to approximately 3.5 million hectares (8.75 million acres).
  • Plants are increasingly used a "protein factories" for production of industrial or therapeutic polypeptides, such as antigens, antibodies (e.g., monoclonal), cytokines, vaccines. Methods for increased yield and/or quality or ease of downstream processing are needed.
  • Proteins and genes involved in a tropical high protein trait corn germplasm are disclosed, as well as their use to genetically modify cereals for higher protein yield and better protein quality.
  • a total 11 genes (and thir orthologs) are identified for use in protein trait modification in cereals, particularly corn. These genes belong to two groups: one group of proteins is associated with seed protein storage and the other group is generally related to seed stress response or proteins that are unregulated during seed maturation. Possibly the stress response mechanism has co-evolved with the high protein trait. Higher protein yield in com and other cereals can be achieved by manipulating the gene expression level of these genes and other regulatory genes regulating the stress mechanism.
  • the invention provides isolated nucleic acid molecules, e.g., DNA, comprising a plant nucleotide sequence encoding a polypeptide that is expressed in cells of a plant, e.g., embryos, mature embryos, endosperm, shoot, root, leaf and developing seed, from high protein varieties of plants, relative to cells of a plant from a corresponding lower protein variety.
  • the invention provides a nucleic acid molecule comprising a plant nucleotide sequence comprising an open reading frame encoding a polypeptide which is substantially similar to a polypeptide comprising any one of SEQ ID Nos. 1-36.
  • this sequence may be overexpressed individually, in the sense or antisense orientation, or in combination with other sequences, to confer altered nutritional properties to the plant relative to a plant that does not comprise and/or express the sequence(s).
  • the protein content may be enhanced, while in another embodiment it may be reduced, e.g., low protein products such as rice for individuals that are intolerant or sensitive to certain proteins.
  • Low protein content plants or seeds can be a superior form for production of heterologous industrially or therapeutically important proteins in plants, and plant seeds by, for example, reducing levels of abundant endogenous proteins. To avoid detrimental effects to the plants, such modulation can be controlled using inducible promoters.
  • One system employs hybrid two component systems such as Gal4/Cl, in which the controlled promoter(s) is on or off only in the hybrid, not the parental lines.
  • the overexpression may be constitutive, or it may be preferable to express the sequence from an inducible promoter including a promoter which is responsive to external stimuli, such as chemical application, or environmental stimuli, so as to avoid possible deleterious effects on plant growth.
  • High protein varieties of plants are those which have at least a 1%, preferably at least 5%, and more preferably at least 10%, increase in protein content or level relative to a corresponding control plant.
  • a high protein line or variety preferably may have a protein content in whole kernel that is at least 14.5%, more preferably at least 15.5%, in embyro at least 11%, more preferably 18.3%, and in endosperm at least 13.5%, more preferably at least 14.2%>.
  • High protein varieties of maize are well known to the art, see, for example, U.S. Patent Nos. 5,986,182, 5,936,143, 5,907,089, 5,900,528, 5,850,031, 5,824,855, 5,824,854, 5,763,756, and 5,675,065.
  • protein expression profiles from embryos of normal and high protein varieties of maize were compared using two-dimensional SDS-PAGE analysis in order to identify differentially expressed genes.
  • proteomic technology to the high protein corn germplasm has revealed more than 120 genes that are differentially expressed in high protein lines.
  • genes may encode structural or regulatory proteins, and hence are of potential use in manipulating protein content in maize (corn) and other cereals such as wheat and rice, e.g., for manipulating seed protein phenotype and for the development of molecular markers for plant breeding.
  • the results provide a novel function for unknown or previously uncharacterize ⁇ V mischaracterized genes, and may lead to useful regulatory genes for particular traits, structural genes or molecular markers. Further, by using a segregating population, the results also provide the necessary means to identify genes specifically related to the high protein phenotype rather than those that are merely causally associated.
  • the identified proteins (polypeptides) and their corresponding genes can be used to: 1) manipulate protein content or levels in corn and other cereal species, e.g., by using the genes as molecular markers in breeding or in transgenic plants; 2) isolate orthologs from other crop species such as rice and wheat; 3) generate antibodies and develop protein-based assays for breeding selection; and 4) identify common transcriptional regulatory elements and factors which bind those elements, i.e.; the upstream regions ofthe genes associated with the high protein trait.
  • Non-protein based methods may also be employed to identify the nucleic acid molecules ofthe invention.
  • an array of nucleic acid samples e.g., a plurality of oligonucleotides, each plurality corresponding to a different plant gene, on a solid substrate, e.g., a DNA chip, and probes corresponding to nucleic acid obtained from plant sources that express genes associated with protein content and probes to nucleic acid obtained from plant sources that do not express those genes or express the genes at a reduced level, can be used to systematically identify genes associated with increased protein levels.
  • the nucleotide sequence in the nucleic acid molecule ofthe invention is from plant DNA, either a dicot or a monocot, which encodes a polypeptide that is substantially similar to a polypeptide comprising any one of SEQ ID NOs: 1-36. More preferably, the nucleotide sequence is from plant DNA that is substantially similar to a nucleic acid segment encoding a polypeptide comprising any one of SEQ ID NOs: 1-36.
  • substantially similar when used herein with respect to a polypeptide means a polypeptide corresponding to a reference polypeptide, wherein the polypeptide has substantially the same structure and function as the reference polypeptide, e.g., where only changes in amino acid sequence are those which do not affect the polypeptide function.
  • the percentage of identity between the substantially similar and the reference polypeptide or amino acid sequence is at least 65%, 66%, 67%, 68%, 69%>, 70%, e.g., 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, and even 90% or more, e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, up to at least 99%o, where the reference polypeptide is a polypeptide comprising any one of SEQ ID NOs: 1-36.
  • an agent e.g., an antibody, which specifically binds to one ofthe polypeptides, specifically binds to the other.
  • the term “substantially similar”, when used herein with respect to a nucleotide sequence, means a nucleotide sequence corresponding to a reference nucleotide sequence, wherein the corresponding sequence encodes a polypeptide having substantially the same structure and function as the polypeptide encoded by the reference nucleotide sequence.
  • the term “substantially similar” is specifically intended to include nucleotide sequences wherein the sequence has been modified to optimize expression in particular cells.
  • the percentage of identity between the substantially similar nucleotide sequence and the reference nucleotide sequence is at least 65%, 66%, 67%, 68%, 69%, 70%, e.g., 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, ' 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, and even 90% or more, e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, up to at least 99%, wherein the reference sequence is one which encodes a polypeptide comprising any one of SEQ ID NOs: 1-36, or the complement thereof.
  • Sequence comparisons maybe carried out using a Smith- Waterman sequence alignment algorithm (see e.g. Waterman (1995) or http://www hto.usc.edu/software/seqahi index.html).
  • the locals program, version 1.16 is preferably used with following parameters: match: 1, mismatch penalty: 0.33, open-gap penalty: 2, extended-gap penalty: 2.
  • nucleotide sequence that is "substantially similar" to a reference nucleotide sequence hybridizes to the reference nucleotide sequence in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO 4 , 1 mM EDTA at 50°C with washing in 2X SSC, 0.1% SDS at 50°C, more desirably in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO 4 , 1 mM EDTA at 50°C with washing in IX SSC, 0.1% SDS at 50°C, more desirably still in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO 4 , 1 mM EDTA at 50°C with washing in 0.5X SSC, 0.1% SDS at 50°C, preferably in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO 4 , 1 mM EDTA at 50°C with washing
  • the isolated nucleic acid molecules ofthe invention also include orthologs of the sequences encoding the polypeptides comprising the amino acid sequences disclosed herein, including, but not limited to, dicots and monocots, preferably cereal plants, e.g., wheat or rice.
  • An ortholog is a gene from a different species that encodes a product having the same function as the product encoded by a gene from a reference organism. The encoded ortholog products likely have at least 70%> sequence identity to each other.
  • the invention includes an isolated nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide having at least 70%> identity to a polypeptide comprising one or more ofthe sequences disclosed herein.
  • GenBank GenBank or one found at http://bioserver.myongjiac.kr/rjce.html (for rice) may be employed to identify sequences related to the disclosed sequences, e.g., orthologs in cereal crops such as rice.
  • recombinant DNA techniques such as hybridization or PCR may be employed to identify sequences related to the disclosed sequences.
  • the polypeptide has substantial identity, i.e., at least 70%>, e.g., 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, and even 90% or more, e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and at least 99%o, amino acid sequence identity to a polypeptide comprising any one of SEQ ID NOs: 1-36.
  • the invention also provides anti-sense nucleic acid molecules corresponding to the genes identified herein.
  • expression cassettes e.g., recombinant vectors, and host cells, comprising the nucleic acid molecule ofthe invention.
  • the nucleic acid molecules ofthe invention are useful to provide plants with enhanced protein content, identify common transcriptional regulatory factors which bind upstream ofthe coding region of genes associated with high protein content and as markers for breeding selection.
  • the compositions ofthe invention include plant nucleic acid sequences and the amino acid sequences for the polypeptides or partial-length polypeptides encoded thereby which are useful to provide enhanced nutritional characteristics to a plant, preferably by enhancing protein content or levels.
  • Methods ofthe invention involve stably transforming a plant with one or more of at least a portion of these nucleotide sequences operably linked to a promoter capable of driving expression of that nucleotide sequence in a plant cell.
  • portion or fragment as it relates to a nucleic acid molecule, sequence or segment ofthe invention, when it is linked to other sequences for expression, is meant a sequence having at least 80 nucleotides, more preferably at least 150 nucleotides, and still more preferably at least 400 nucleotides. If not employed for expressing, a "portion” or “fragment” means at least 9, preferably 12, more preferably 15, even more preferably at least 20, consecutive nucleotides, e.g., probes and primers (oligonucleotides), corresponding to the nucleotide sequence ofthe nucleic acid molecules of the invention.
  • the method comprises introducing to a plant, plant cell, or plant tissue an expression cassette comprising at least one of nucleic acid molecules ofthe invention so as to yield a transformed differentiated plant, transformed cell or transformed tissue.
  • Transformed cells or tissue can be regenerated to provide a transformed differentiated plant.
  • the transformed differentiated plant preferably expresses the nucleic acid molecule in an amount that yields a transformed plant having enhanced protein content, e.g., in seed, to a corresponding nontransformed plant.
  • the present invention also provides a transformed plant prepared by the method, progeny and seed thereof.
  • a transformed (transgenic) plant ofthe invention includes plants, dicots or monocots, the genome of which is augmented by a nucleic acid molecule ofthe invention, or in which the corresponding gene has been disrupted, e.g., to result in a loss, a decrease or an alteration, in the function ofthe product encoded by the gene.
  • the nucleic acid molecules ofthe invention are thus useful for targeted gene disruption, as well as for markers and probes.
  • the invention also includes recombinant nucleic acid molecules which have been modified so as to comprise codons other than those present in the unmodified sequence.
  • the recombinant nucleic acid molecules ofthe invention include those in which the modified codons specify amino acids that are the same as those specified by the codons in the unmodified sequence, as well as those that specify different amino acids, i.e., they encode a variant polypeptide having one or more amino acid substitutions relative to the polypeptide encoded by the unmodified sequence.
  • the invention further includes a nucleotide sequence which is complementary to one (hereinafter "test" sequence) which hybridizes under stringent conditions with the nucleic acid molecules ofthe invention as well as RNA which is encoded by the nucleic acid molecule.
  • test sequence a nucleotide sequence which is complementary to one
  • RNA which is encoded by the nucleic acid molecule.
  • either a denatured test or nucleic acid molecule ofthe invention is preferably first bound to a support and hybridization is effected for a specified period of time at a temperature of, e.g., between 55 and 70°C, in double strength citrate buffered saline (SC) containing 0.1% SDS followed by rinsing ofthe support at the same temperature but with a buffer having a reduced SC concentration.
  • SC citrate buffered saline
  • SC citrate buffered saline
  • a buffer having a reduced SC concentration buffers are typically single strength SC containing 0.1 % SDS, half strength SC containing 0.1% SDS and one-tenth strength SC containing 0.1% SDS.
  • the present invention also provides a method to identify a polypeptide which is associated with a high protein phenotype.
  • the method comprises separating a plurality of polypeptides from a sample comprising polypeptides, wherein the sample is from a plant having a high protein content. Then the separated sample of polypeptides from a plant having a high protein content is compared to a separated sample of polypeptides from a corresponding plant with lower protein content.
  • polypeptides are identified that are present in the sample from a plant having a high protein content that are not present in the sample from the plant with lower protein content.
  • nucleic acid molecule comprising a nucleotide sequence that directs transcription, e.g., a promoter, of a linked nucleic acid fragment in a host cell, such as a plant cell.
  • the nucleotide sequence is from plant genomic DNA which has at least 65%, 66%, 61%, 68%, 69%, 70%, e.g., 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, and even 90% or more, e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99%, nucleotide sequence identity to a sequence of a promoter from a plant gene encoding a polypeptide comprising any one of SEQ ID NOs: 1-36.
  • the isolated nucleic acid molecule comprises a plant nucleotide sequence which is the promoter region for a gene encoding any one of SEQ ID NOs: 1-36, or is structurally related to the promoter for a gene encoding SEQ ID NOs: 1-36, i.e., is an orthologous promoter, and is linked to a plant structural gene.
  • the present invention further provides an expression cassette or a recombinant vector containing the nucleic acid molecule, and the vector may be a plasmid.
  • cassettes or vectors when present in a plant, plant cell or plant tissue result in transcription ofthe linked nucleic acid fragment in the plant, plant tissue or plant cell.
  • the expression cassettes or vectors ofthe invention may optionally include other regulatory sequences, e.g., transcription terminator sequences, introns and/or enhancers, and may be contained in a host cell.
  • the expression cassette or vector may augment the genome of a transformed plant or may be maintained extrachromosomally.
  • the expression cassette or vector may further have a Ti plasmid and be contained in an Agrobacterium tumefaciens cell; it may be carried on a microparticle, wherein the microparticle is suitable for ballistic transformation of a plant cell; or it may be contained in a plant cell protoplast.
  • the expression cassette can be contained in a plant, plant cell or plant tissue from a dicot or a monocot.
  • the plant may be a cereal plant.
  • the present invention further provides a method of augmenting a plant genome by contacting plant cells with an expression cassette or vector ofthe invention, i.e., one having a nucleotide sequence that directs transcription ofa linked nucleic acid fragment in a plant cell, wherein the nucleotide sequence is from plant genomic DNA that has at least 65 > , and more preferably at least 70%>, identity to the sequence of a promoter from a gene encoding a polypeptide comprising any one of SEQ ID NOs: 1-36 so as to yield transformed plant cells; and regenerating the transformed plant cells to provide a differentiated transformed plant, wherein the differentiated transformed plant expresses the linked fragment in the cells ofthe plant.
  • the present invention also provides a plant prepared by the method, progeny and seed thereof.
  • Figure 1 shows the protein content in various sources from high protein and control maize lines.
  • Figures 2A and 2B illustrate a two dimensional gel with proteins from a control (#530; panel A) or high protein (#465; panel B) maize line.
  • Figures 2C and 2D illustrate another comparision of protein expression profile of high protein germplasm and normal corn line.
  • Figures 3A to 3H show the peptide and criteria (e.g., Xcro > 2 and Den > 0.01) employed to search databases for the corresponding full length protein for 18 ofthe proteins shown in the attached Sequence Listing which is incorporated herein.
  • criteria e.g., Xcro > 2 and Den > 0.01
  • Figures 4A and 4B are representative vectors for over- or under-expression of genes in seed.
  • Sequence Listing shows the amino acid sequence of proteins, high protein phenotype genes and proteins, or the orthologs thereof, which are preferentially expressed in high protein maize lines relative to lines with lower protein content.
  • Sequences 1 to 36 are the high protein involved proteins. Odd numbered SEQ ID Nos are are protein-encoding orfs and the even numbered SEQ ID NOs are amino acids sequences.
  • Sequences 37 to 45 are representative peptides identified by the MS as described in the Examples.
  • nucleic acid refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form, composed of monomers (nucleotides) containing a sugar, phosphate and a base which is either a purine or pyrimidine. Unless specifically limited, the term encompasses nucleic acids containing known analogs of natural nucleotides which have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides.
  • nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences as well as the sequence explicitly indicated.
  • degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et al., 1991; Ohtsuka et al, 1985; Rossolini et al., 1994).
  • a "nucleic acid fragment" is a fraction ofa given nucleic acid molecule.
  • nucleic acid sequence refers to a polymer of DNA or RNA which can be single- or double-stranded, optionally containing synthetic, non-natural or altered nucleotide bases capable of incorporation into DNA or RNA polymers.
  • nucleic acid refers to a polymer of DNA or RNA which can be single- or double-stranded, optionally containing synthetic, non-natural or altered nucleotide bases capable of incorporation into DNA or RNA polymers.
  • nucleic acid may also be used interchangeably with gene, cDNA, DNA and RNA encoded by a gene.
  • an "isolated” or “purified” DNA molecule or an “isolated” or “purified” polypeptide is a DNA molecule or polypeptide that, by the hand of man, exists apart from its native environment and is therefore not a product of nature.
  • An isolated DNA molecule or polypeptide may exist in a purified form or may exist in a non-native environment such as, for example, a transgenic host cell.
  • an "isolated” or “purified” nucleic acid molecule or protein, or biologically active portion thereof is substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized.
  • an "isolated" nucleic acid is free of sequences that naturally flank the nucleic acid (i.e., sequences located at the 5' and 3' ends ofthe nucleic acid) in the genomic DNA ofthe organism from which the nucleic acid is derived.
  • the isolated nucleic acid molecule can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kb of nucleotide sequences that naturally flank the nucleic acid molecule in genomic DNA ofthe cell from which the nucleic acid is derived.
  • a protein that is substantially free of cellular material includes preparations of protein or polypeptide having less than about 30%, 20%,, 10%, 5%, (by dry weight) of contaminating protein.
  • culture medium represents less than about 30%, 20%, 10%, or 5%, (by dry weight) of chemical precursors or non-protein-of- interest chemicals.
  • Fragments and variants ofthe disclosed nucleotide sequences and proteins or partial-length proteins encoded thereby are also encompassed by the present invention.
  • fragment or portion is meant a full length or less than full length ofthe nucleotide sequence encoding, or the amino acid sequence of, a polypeptide or protein.
  • fragments or portions ofa nucleotide sequence that are useful as hybridization probes generally do not encode fragment proteins retaining biological activity.
  • fragments or portions of a nucleotide sequence may range from at least about 9 nucleotides, about 12 nucleotides, about 20 nucleotides, about 50 nucleotides, about 100 nucleotides or more.
  • genes include coding sequences and/or the regulatory sequences required for their expression.
  • gene refers to a nucleic acid fragment that expresses mRNA, functional RNA, or specific protein, including regulatory sequences.
  • Genes also include nonexpressed DNA segments that, for example, form recognition sequences for other proteins.
  • Genes can be obtained from a variety of sources, including cloning from a source of interest or synthesizing from known or predicted sequence information, and may include sequences designed to have desired parameters.
  • Naturally occurring is used to describe an object that can be found in nature as distinct from being artificially produced by man.
  • a protein or nucleotide sequence present in an organism which can be isolated from a source in nature and which has not been intentionally modified by man in the laboratory, is naturally occurring.
  • a "marker gene” encodes a selectable or screenable trait.
  • Selectable marker is a gene whose expression in a cell gives the cell a selective advantage.
  • the selective advantage possessed by the cells transformed with the selectable marker gene may be due to their ability to grow in the presence of a negative selective agent, such as an antibiotic or a herbicide, compared to the growth of non-transformed cells.
  • the selective advantage possessed by the transformed cells, compared to non-transformed cells may also be due to their enhanced or novel capacity to utilize an added compound as a nutrient, growth factor or energy source.
  • Selectable marker gene also refers to a gene or a combination of genes whose expression in a cell gives the cell both a negative and/or a positive selective advantage.
  • chimeric refers to any gene or DNA that contains 1) DNA sequences, including regulatory and coding sequences, that are not found together in nature, or 2) sequences encoding parts of proteins not naturally adjoined, or 3) parts of promoters that are not naturally adjoined. Accordingly, a chimeric gene may comprise regulatory sequences and coding sequences that are derived from different sources, or comprise regulatory sequences and coding sequences derived from the same source, but arranged in a manner different from that found in nature.
  • transgene refers to a gene that has been introduced into the genome by transformation and is stably maintained.
  • Transgenes may include, for example, DNA that is either heterologous or homologous to the DNA of a particular plant to be transformed. Additionally, transgenes may comprise native genes inserted into a non-native organism, or chimeric genes.
  • endogenous gene refers to a native gene in its natural location in the genome of an organism.
  • a “foreign” gene refers to a gene not normally found in the host organism but that is introduced by gene transfer.
  • variants include those sequences that, because ofthe degeneracy ofthe genetic code, encode the identical amino acid sequence ofthe native protein.
  • Naturally occurring allelic variants such as these can be identified with the use of well-known molecular biology techniques, as, for example, with polymerase chain reaction (PCR) and hybridization techniques.
  • variant nucleotide sequences also include synthetically derived nucleotide sequences, such as those generated, for example, by using site-directed mutagenesis which encode the native protein, as well as those that encode a polypeptide having amino acid substitutions.
  • nucleotide sequence variants ofthe invention will have at least 40, 50, 60, to 70%», e.g., preferably 71%>, 72%, 73%, 74%, 75%, 76%, 77%, 78%, to 79%, generally at least 80%, e.g., 81%-84%, at least 85%, e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, to 98%, sequence identity to the native (endogenous) nucleotide sequence.
  • DNA shuffling is a method to introduce mutations or rearrangements, preferably randomly, in a DNA molecule or to generate exchanges of DNA sequences between two or more DNA molecules, preferably randomly.
  • the DNA molecule resulting from DNA shuffling is a shuffled DNA molecule that is a non-naturally occurring DNA molecule derived from at least one template DNA molecule.
  • the shuffled DNA preferably encodes a variant polypeptide modified with respect to the polypeptide encoded by the template DNA, and may have an altered biological activity with respect to the polypeptide encoded by the template DNA.
  • the nucleic acid molecules ofthe invention can be optimized for enhanced expression in plants of interest.
  • nucleotide sequences can be optimized for expression in any plant. It is recognized that all or any part ofthe gene sequence may be optimized or synthetic. That is, synthetic or partially optimized sequences may also be used. Variant nucleotide sequences and proteins also encompass sequences and protein derived from a mutagenic and recombinogenic procedure such as DNA shuffling.
  • one or more different coding sequences can be manipulated to create a new polypeptide possessing the desired properties, hi this manner, libraries of recombinant polynucleotides are generated from a population of related sequence polynucleotides comprising sequence regions that have substantial sequence identity and can be homologously recombined in vitro or in vivo.
  • Strategies for such DNA shuffling are known in the art. See, for example, Stemmer (1994); Stemmer (1994); Crameri et al. (1997); Moore et al. (1997); Zhang et al. (1997); Crameri et al. (1998); and U.S. Patent Nos. 5,605,793 and 5,837,458.
  • Constantly modified variations of aparticular nucleic acid sequence refers to those nucleic acid sequences that encode identical or essentially identical amino acid sequences, or where the nucleic acid sequence does not encode an amino acid sequence, to essentially identical sequences. Because ofthe degeneracy ofthe genetic code, a large number of functionally identical nucleic acids encode any given polypeptide. For instance the codons CGT, CGC, CGA, CGG, AGA, and AGG all encode the amino acid arginine. Thus, at every position where an arginine is specified by a codon, the codon can be altered to any ofthe corresponding codons described without altering the encoded protein.
  • nucleic acid variations are “silent variations” which are one species of “conservatively modified variations.” Every nucleic acid sequence described herein which encodes a polypeptide also describes every possible silent variation, except where otherwise noted.
  • each codon in a nucleic acid except ATG, which is ordinarily the only codon for methionine
  • each "silent variation" of a nucleic acid which encodes a polypeptide is implicit in each described sequence.
  • "Recombinant DNA molecule” is a combination of DNA sequences that are joined together using recombinant DNA technology and procedures used to join together DNA sequences as described, for example, in Sambrook et al.
  • heterologous DNA sequence each refer to a sequence that originates from a source foreign to the particular host cell or, if from the same source, is modified from its original form.
  • a heterologous gene in a host cell includes a gene that is endogenous to the particular host cell but has been modified through, for example, the use of DNA shuffling.
  • the terms also include non- naturally occurring multiple copies of a naturally occurring DNA sequence.
  • the terms refer to a DNA segment that is foreign or heterologous to the cell, or homologous to the cell but in a position within the host cell nucleic acid in which the element is not ordinarily found. Exogenous DNA segments are expressed to yield exogenous polypeptides.
  • a “homologous” DNA sequence is a DNA sequence that is naturally associated with a host cell into which it is introduced. "Wild-type” refers to the normal gene, or organism found in nature without any known mutation.
  • Gene refers to the complete genetic material of an organism.
  • Vector is defined to include, inter alia, any plasmid, cosmid, phage or Agrobacterium binary vector in double or single stranded linear or circular form which may or may not be self transmissible or mobilizable, and which can transform prokaryotic or eukaryotic host either by integration into the cellular genome or exist extrachromosomally (e.g., autonomous replicating plasmid with an origin of replication).
  • shuttle vectors by which is meant a DNA vehicle capable, naturally or by design, of replication in two different host organisms, which may be selected from actinomycetes and related species, bacteria and eukaryotic (e.g., higher plant, mammalian, yeast or fungal cells).
  • Codoning vectors typically contain one or a small number of restriction endonuclease recognition sites at which foreign DNA sequences can be inserted in a determinable fashion without loss of essential biological function ofthe vector, as well as a marker gene that is suitable for use in the identification and selection of cells transformed with the cloning vector. Marker genes typically include genes that provide tetracycline resistance, hygromycin resistance or ampicillin resistance.
  • Expression cassette as used herein means a DNA sequence capable of directing expression of a particular nucleotide sequence in an appropriate host cell, comprising a promoter operably linked to the nucleotide sequence of interest which is operably linked to termination signals. It also typically comprises sequences required for proper translation ofthe nucleotide sequence.
  • the coding region usually codes for a protein of interest but may also code for a functional RNA of interest, for example antisense RNA or a nontranslated RNA, in the sense or antisense direction.
  • the expression cassette comprising the nucleotide sequence of interest may be chimeric, meaning that at least one of its components is heterologous with respect to at least one of its other components.
  • the expression cassette may also be one which is naturally occurring but has been obtained in a recombinant form useful for heterologous expression.
  • the expression ofthe nucleotide sequence in the expression cassette may be under the control ofa constitutive promoter or of an inducible promoter which initiates transcription only when the host cell is exposed to some particular external stimulus. In the case ofa multicellular organism, the promoter can also be specific to a particular tissue or organ or stage of development.
  • Such expression cassettes will comprise the transcriptional initiation region ofthe invention linked to a nucleotide sequence of interest.
  • Such an expression cassette is provided with a plurality of restriction sites for insertion ofthe gene of interest to be under the transcriptional regulation ofthe regulatory regions.
  • the expression cassette may additionally contain selectable marker genes.
  • the transcriptional cassette will include in the 5'-3' direction of transcription, a transcriptional and translational initiation region, a DNA sequence of interest, and a transcriptional and translational termination region functional in plants.
  • the termination region may be native with the transcriptional initiation region, may be native with the DNA sequence of interest, or may be derived from another source.
  • Convenient termination regions are available from the Ti-plasmid of A. tumefaciens, such as the octopine synthase and nopaline synthase termination regions. See also, Guerineau et al. (1991); Proudfoot (1991); Sanfacon et al. (1991); Mogen et al. (1990); Munroe et al.
  • An oligonucleotide corresponding to a nucleic acid molecule ofthe invention may be about 30 or fewer nucleotides in length (e.g., 9, 12, 15, 18, 20, 21 or 24, or any number between 9 and 30).
  • primers are upwards of 14 nucleotides in length.
  • primers of 16-24 nucleotides in length may be preferred.
  • probing can be done with entire restriction fragments ofthe gene disclosed herein which may be 100's or even 1000's of nucleotides in length.
  • Coding sequence refers to a DNA or RNA sequence that codes for a specific amino acid sequence and excludes the non-coding sequences. It may constitute an "uninterrupted coding sequence", i.e., lacking an intron, such as in a cDNA or it may include one or more introns bounded by appropriate splice junctions.
  • An "intron” is a sequence of RNA which is contained in the primary transcript but which is removed through cleavage and re-ligation of the RNA within the cell to create the mature mRNA that can be translated into a protein.
  • the terms “open reading frame” and “ORF” refer to the amino acid sequence encoded between translation initiation and termination codons of a coding sequence.
  • initiation codon and “termination codon” refer to a unit of three adjacent nucleotides ('codon') in a coding sequence that specifies initiation and chain termination, respectively, of protein synthesis (mRNA translation).
  • a "functional RNA” refers to an antisense RNA, ribozyme, or other RNA that is not translated.
  • RNA transcript refers to the product resulting from RNA polymerase catalyzed transcription of a DNA sequence.
  • the primary transcript When the RNA transcript is a perfect complementary copy ofthe DNA sequence, it is referred to as the primary transcript or it may be a RNA sequence derived from posttranscriptional processing ofthe primary transcript and is referred to as the mature RNA.
  • Messenger RNA (mRNA) refers to the RNA that is without introns and that can be translated into protein by the cell.
  • cDNA refers to a single- or a double-stranded DNA that is complementary to and derived from mRNA.
  • Regulatory sequences each refer to nucleotide sequences located upstream (5' non-coding sequences), within, or downstream (3' non-coding sequences) of a coding sequence, and which influence the transcription, RNA processing or stability, or translation ofthe associated coding sequence. Regulatory sequences include enhancers, promoters, translation leader sequences, introns, and polyadenylation signal sequences. They include natural and synthetic sequences as well as sequences which may be a combination of synthetic and natural sequences. As is noted above, the term “suitable regulatory sequences” is not limited to promoters. However, some suitable regulatory sequences useful in the present invention will include, but are not limited to constitutive plant promoters, plant tissue-specific promoters, plant development specific promoters, inducible plant promoters and viral promoters.
  • 5' non-coding sequence refers to a nucleotide sequence located 5' (upstream) to the coding sequence. It is present in the fully processed mRNA upstream ofthe initiation codon and may affect processing ofthe primary transcript to mRNA, mRNA stability or translation efficiency (Turner et al., 1995).
  • 3' non-coding sequence refers to nucleotide sequences located 3' (downstream) to a coding sequence and include polyadenylation signal sequences and other sequences encoding regulatory signals capable of affecting mRNA processing or gene expression.
  • the polyadenylation signal is usually characterized by affecting the addition of polyadenylic acid tracts to the 3' end ofthe mRNA precursor.
  • the use of different 3' non-coding sequences is exemplified by Ingelbrecht et al., 1989.
  • translation leader sequence refers to that DNA sequence portion of a gene between the promoter and coding sequence that is transcribed into RNA and is present in the fully processed mRNA upstream (5') ofthe translation start codon.
  • the translation leader sequence may affect processing ofthe primary transcript to mRNA, mRNA stability or translation efficiency.
  • mature protein refers to a post-translationally processed polypeptide without its signal peptide.
  • Precursor protein refers to the primary product of translation of an mRNA.
  • Signal peptide refers to the amino terminal extension of a polypeptide, which is translated in conjunction with the polypeptide forming a precursor peptide and which is required for its entrance into the secretory pathway.
  • signal sequence refers to a nucleotide sequence that encodes the signal peptide.
  • Intracellular localization sequence refers to a nucleotide sequence that encodes an intracellular targeting signal.
  • An “intracellular targeting signal” is an amino acid sequence that is translated in conjunction with a protein and directs it to a particular sub- cellular compartment.
  • Endoplasmic reticulum (ER) stop transit signal refers to a carboxy- terminal extension of a polypeptide, which is translated in conjunction with the polypeptide and causes a protein that enters the secretory pathway to be retained in the ER.
  • ER stop transit sequence refers to a nucleotide sequence that encodes the ER targeting signal.
  • Other intracellular targeting sequences encode targeting signals active in seeds and/or leaves and vacuolar targeting signals.
  • Promoter refers to a nucleotide sequence, usually upstream (5') to its coding sequence, which controls the expression ofthe coding sequence by providing the recognition for RNA polymerase and other factors required for proper transcription.
  • Promoter includes a minimal promoter that is a short DNA sequence comprised of a TATA- box and other sequences that serve to specify the site of transcription initiation, to which regulatory elements are added for control of expression.
  • Promoter also refers to a nucleotide sequence that includes a minimal promoter plus regulatory elements that is capable of controlling the expression of a coding sequence or functional RNA. This type of promoter sequence consists of proximal and more distal upstream elements, the latter elements often referred to as enhancers.
  • an “enhancer” is a DNA sequence which can stimulate promoter activity and may be an innate element ofthe promoter or a heterologous element inserted to enhance the level or tissue specificity of a promoter. It is capable of operating in both orientations (normal or flipped), and is capable of functioning even when moved either upstream or downstream from the promoter. Both enhancers and other upstream promoter elements bind sequence-specific DNA-binding proteins that mediate their effects. Promoters may be derived in their entirety from a native gene, or be composed of different elements derived from different promoters found in nature, or even be comprised of synthetic DNA segments. A promoter may also contain DNA sequences that are involved in the binding of protein factors which control the effectiveness of transcription initiation in response to physiological or developmental conditions.
  • the "initiation site” is the position surrounding the first nucleotide that is part ofthe transcribed sequence, which is also defined as position +1. With respect to this site all other sequences ofthe gene and its controlling regions are numbered. Downstream sequences (i.e., further protein encoding sequences in the 3' direction) are denominated positive, while upstream sequences (mostly ofthe controlling regions in the 5' direction) are denominated negative.
  • Promoter elements particularly a TATA element, that are inactive or that have greatly reduced promoter activity in the absence of upstream activation are referred to as "minimal or core promoters.”
  • the minimal promoter functions to permit transcription.
  • a “minimal or core promoter” thus consists only of all basal elements needed for transcription initiation, e.g., a TATA box and/or an initiator.
  • Constutive expression refers to expression using a constitutive or regulated promoter.
  • Consditional and “regulated expression” refer to expression controlled by a regulated promoter.
  • Constant promoter refers to a promoter that is able to express the gene that it controls in all or nearly all ofthe plant tissues during all or nearly all developmental stages of the plant.
  • Each ofthe transcription-activating elements do not exhibit an absolute tissue-specificity, but mediate transcriptional activation in most plant parts at a level of >1% ofthe level reached in the part ofthe plant in which transcription is most active.
  • Regular promoter refers to promoters that direct gene expression not constitutively, but in a temporally- and/or spatially-regulated manner, and include both tissue-specific and inducible promoters. It includes natural and synthetic sequences as well as sequences which may be a combination of synthetic and natural sequences. Different promoters may direct the expression of a gene in different tissues or cell types, or at different stages of development, or in response to different environmental conditions. New promoters of various types useful in plant cells are constantly being discovered, numerous examples may be found in the compilation by Okamuro et al. (1989). Since in most cases the exact boundaries of regulatory sequences have not been completely defined, DNA fragments of different lengths may have identical promoter activity.
  • Typical regulated promoters useful in plants include but are not limited to safener-inducible promoters, promoters derived from the tetracycline-inducible system, promoters derived from salicylate-inducible systems, promoters derived from alcohol- inducible systems, promoters derived from glucocorticoid-inducible system, promoters derived from pathogen-inducible systems, and promoters derived from ecdysome-inducible systems.
  • tissue-specific promoter refers to regulated promoters that are not expressed in all plant cells but only in one or more cell types in specific organs (such as leaves or seeds), specific tissues (such as embryo or cotyledon), or specific cell types (such as leaf parenchyma or seed storage cells). These also include promoters that are temporally regulated, such as in early or late embryogenesis, during fruit ripening in developing seeds or fruit, in fully differentiated leaf, or at the onset of senescence.
  • “Inducible promoter” refers to those regulated promoters that can be turned on in one or more cell types by an external stimulus, such as a chemical, light, hormone, stress, or a pathogen.
  • “Operably-linked” refers to the association of nucleic acid sequences on single nucleic acid fragment so that the function of one is affected by the other.
  • a regulatory DNA sequence is said to be “operably linked to” or “associated with” a DNA sequence that codes for an RNA or a polypeptide ifthe two sequences are situated such that the regulatory DNA sequence affects expression ofthe coding DNA sequence (i.e., that the coding sequence or functional RNA is under the transcriptional control ofthe promoter). Coding sequences can be operably-linked to regulatory sequences in sense or antisense orientation.
  • “Expression” refers to the transcription and/or translation of an endogenous gene or a transgene in plants.
  • expression may refer to the transcription ofthe antisense DNA only.
  • expression refers to the transcription and stable accumulation of sense (mRNA) or functional RNA. Expression may also refer to the production of protein.
  • altered levels refers to the level of expression in transgenic cells or organisms that differs from that of normal or untransformed cells or organisms.
  • “Overexpression” refers to the level of expression in transgenic cells or organisms that exceeds levels of expression in normal or untransformed cells or organisms.
  • Antisense inhibition refers to the production of antisense RNA transcripts capable of suppressing the expression of protein from an endogenous gene or a transgene.
  • Codon and transwitch each refer to the production of sense RNA transcripts capable of suppressing the expression of identical or substantially similar transgene or endogenous genes (U.S. Patent No. 5,231,020).
  • Gene silencing refers to homology-dependent suppression of viral genes, transgenes, or endogenous nuclear genes. Gene silencing may be transcriptional, when the suppression is due to decreased transcription ofthe affected genes, or post-transcriptional, when the suppression is due to increased turnover (degradation) of RNA species homologous to the affected genes. (English et al., 1996). Gene silencing includes virus-induced gene silencing (Ruiz et al., 1998).
  • Silencing suppressor gene refers to a gene whose expression leads to counteracting gene silencing and enhanced expression of silenced genes.
  • Silencing suppressor genes may be of plant, non-plant, or viral origin. Examples include, but are not limited to HC-Pro, Pl-HC- Pro, and 2b proteins. Other examples include one or more genes in TGMV-B genome.
  • Transcription stop fragment refers to nucleotide sequences that contain one or more regulatory signals, such as polyadenylation signal sequences, capable of terminating transcription. Examples include the 3' non-regulatory regions of genes encoding nopaline synthase and the small subunit of ribulose bisphosphate carboxylase.
  • Translation stop fragment refers to nucleotide sequences that contain one or more regulatory signals, such as one or more termination codons in all three frames, capable of terminating translation. Insertion of a translation stop fragment adjacent to or near the initiation codon at the 5' end ofthe coding sequence will result in no translation or improper translation. Excision ofthe translation stop fragment by site-specific recombination will leave a site-specific sequence in the coding sequence that does not interfere with proper translation using the initiation codon.
  • c ⁇ -acting sequence and "cts-acting element” refer to DNA or RNA sequences whose functions require them to be on the same molecule.
  • An example of a cis- acting sequence on the replicon is the viral replication origin.
  • trans-acting sequence and "trans-acting element” refer to DNA or RNA sequences whose function does not require them to be on the same molecule.
  • Chrosomally-integrated refers to the integration of a foreign gene or DNA construct into the host DNA by covalent bonds. Where genes are not “chromosomally integrated” they may be “transiently expressed.” Transient expression of a gene refers to the expression of a gene that is not integrated into the host chromosome but functions independently, either as part of an autonomously replicating plasmid or expression cassette, for example, or as part of another biological system such as a virus.
  • sequence relationships between two or more nucleic acids or polynucleotides are used to describe the sequence relationships between two or more nucleic acids or polynucleotides: (a) “reference sequence”, (b) “comparison window”, (c) “sequence identity”, (d) “percentage of sequence identity”, and (e) “substantial identity”.
  • reference sequence is a defined sequence used as a basis for sequence comparison.
  • a reference sequence may be a subset or the entirety of a specified sequence; for example, as a segment ofa full length cDNA or gene sequence, or the complete cDNA or gene sequence.
  • comparison window makes reference to a contiguous and specified segment of a polynucleotide sequence, wherein the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment ofthe two sequences.
  • the comparison window is at least 20 contiguous nucleotides in length, and optionally can be 30, 40, 50, 100, or longer.
  • Computer implementations of these mathematical algorithms can be utilized for comparison of sequences to determine sequence identity.
  • Such implementations include, but are not limited to: CLUSTAL in the PC/Gene program (available from Intelligenetics,
  • HSPs high scoring sequence pairs
  • Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always > 0) and N (penalty score for mismatching residues; always ⁇ 0).
  • M forward score for a pair of matching residues
  • N penalty score for mismatching residues; always ⁇ 0.
  • a scoring matrix is used to calculate the cumulative score. Extension ofthe word hits in each direction are halted when the cumulative alignment score falls off by the quantity X from its maximum achieved value, the cumulative score goes to zero or below due to the accumulation of one or more negative-scoring residue alignments, or the end of either sequence is reached.
  • the BLAST algorithm also performs a statistical analysis ofthe similarity between two sequences (see, e.g., Karlin & Altschul (1993).
  • BLAST smallest sum probability
  • P(N) the smallest sum probability
  • a test nucleic acid sequence is considered similar to a reference sequence if the smallest sum probability in a comparison ofthe test nucleic acid sequence to the reference nucleic acid sequence is less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.
  • Gapped BLAST in BLAST
  • BLAST 2.0 can be utilized as described in Altschul et al. (1997).
  • PSI-BLAST in BLAST 2.0
  • PSI-BLAST can be used to perform an iterated search that detects distant relationships between molecules. See Altschul et al., supra.
  • the default parameters ofthe respective programs e.g. BLASTN for nucleotide sequences, BLASTX for proteins
  • the BLASTP program uses as defaults a wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, 1989). See http://www.ncbi.nlm.nih.gov. Alignment may also be performed manually by inspection.
  • comparison of nucleotide sequences for determination of percent sequence identity to the promoter sequences disclosed herein is preferably made using the BlastN program (version 1.4.7 or later) with its default parameters or any equivalent program.
  • equivalent program is intended any sequence comparison program that, for any two sequences in question, generates an alignment having identical nucleotide or amino acid residue matches and an identical percent sequence identity when compared to the corresponding alignment generated by the preferred program.
  • sequence identity or “identity” in the context of two nucleic acid or polypeptide sequences makes reference to a specified percentage of residues in the two sequences that are the same when aligned for maximum correspondence over a specified comparison window, as measured by sequence comparison algorithms or by visual inspection.
  • percentage of sequence identity is used in reference to proteins it is recognized that residue positions which are not identical often differ by conservative amino acid substitutions, where amino acid residues are substituted for other amino acid residues with similar chemical properties (e.g., charge or hydrophobicity) and therefore do not change the functional properties ofthe molecule.
  • sequences differ in conservative substitutions the percent sequence identity maybe adjusted upwards to correct for the conservative nature ofthe substitution. Sequences that differ by such conservative substitutions are said to have
  • sequence similarity or “similarity.”
  • Means for making this adjustment are well known to those of skill in the art. Typically this involves scoring a conservative substitution as a partial rather than a full mismatch, thereby increasing the percentage sequence identity. Thus, for example, where an identical amino acid is given a score of 1 and a non-conservative substitution is given a score of zero, a conservative substitution is given a score between zero and 1. The scoring of conservative substitutions is calculated, e.g., as implemented in the program PC/GENE (Litelligenetics, Mountain View, California).
  • percentage of sequence identity means the value determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment ofthe two sequences. The percentage is calculated by determining the number of positions at which- the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison, and multiplying the result by 100 to yield the percentage of sequence identity.
  • polynucleotide sequences means that a polynucleotide comprises a sequence that has at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, or 79%, preferably at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%, more preferably at least 90%, 91%, 92%, 93%, or 94%, and most preferably at least
  • stringent conditions are selected to be about 5°C lower than the thermal melting point (T m ) for the specific sequence at a defined ionic strength and pH.
  • stringent conditions encompass temperatures in the range of about 1°C to about 20°C, depending upon the desired degree of stringency as otherwise qualified herein.
  • Nucleic acids that do not hybridize to each other under stringent conditions are still substantially identical ifthe polypeptides they encode are substantially identical. This may occur, e.g., when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code.
  • One indication that two nucleic acid sequences are substantially identical is when the polypeptide encoded by the first nucleic acid is immunologically cross reactive with the polypeptide encoded by the second nucleic acid.
  • substantially identical in the context of a peptide indicates that a peptide comprises a sequence with at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, or 79%, preferably 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%, more preferably at least 90%, 91%, 92%, 93%, or 94%, or even more preferably, 95%, 96%, 97%, 98% or 99%o, sequence identity to the reference sequence over a specified comparison window.
  • optimal alignment is conducted using the homology alignment algorithm of Needleman and Wunsch, 1970.
  • a peptide is substantially identical to a second peptide, for example, where the two peptides differ only by a conservative substitution.
  • sequence comparison typically one sequence acts as a reference sequence to which test sequences are compared.
  • test and reference sequences are input into a computer, subsequence coordinates are designated if necessary, and sequence algorithm program parameters are designated.
  • sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters.
  • hybridizing specifically to refers to the binding, duplexing, or hybridizing of a molecule only to a particular nucleotide sequence under stringent conditions when that sequence is present in a complex mixture (e.g., total cellular) DNA or RNA.
  • Bod(s) substantially refers to complementary hybridization between a probe nucleic acid and a target nucleic acid and embraces minor mismatches that can be accommodated by reducing the stringency ofthe hybridization media to achieve the desired detection ofthe target nucleic acid sequence.
  • Stringent hybridization conditions and “stringent hybridization wash conditions” in the context of nucleic acid hybridization experiments such as Southern and Northern hybridizations are sequence dependent, and are different under different environmental parameters. Longer sequences hybridize specifically at higher temperatures.
  • the T m is the temperature (under defined ionic strength and pH) at which 50% ofthe target sequence hybridizes to a perfectly matched probe. Specificity is typically the function of post- hybridization washes, the critical factors being the ionic strength and temperature ofthe final wash solution.
  • T m can be approximated from the equation of Meinkoth and Wahl, 1984; T m 81.5°C + 16.6 (log M) +0.41 (%GC) - 0.61 (% form) - 500/L; where M is the molarity of monovalent cations, %>GC is the percentage of guanosine and cytosine nucleotides in the DNA, %> form is the percentage of formamide in the hybridization solution, and L is the length ofthe hybrid in base pairs. T m is reduced by about 1°C for each 1% of mismatching; thus, T m , hybridization, and/or wash conditions canbe adjusted to hybridize to sequences ofthe desired identity.
  • the T m can be decreased 10°C.
  • stringent conditions are selected to be about 5°C lower than the thermal melting point (T m ) for the specific sequence and its complement at a defined ionic strength and pH.
  • a high stringency wash is preceded by a low stringency wash to remove background probe signal.
  • An example medium stringency wash for a duplex of, e.g., more than 100 nucleotides, is IX SSC at 45°C for 15 minutes.
  • An example low stringency wash for a duplex of, e.g., more than 100 nucleotides, is 4-6X SSC at 40°C for 15 minutes.
  • stringent conditions typically involve salt concentrations of less than about 1.5 M, more preferably about 0.01 to 1.0 M, Na ion concentration (or other salts) at pH 7.0 to 8.3, and the temperature is typically at least about 30°C and at least about 60°C for long robes (e.g., >50 nucleotides).
  • Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide.
  • a signal to noise ratio of 2X (or higher) than that observed for an unrelated probe in the particular hybridization assay indicates detection of a specific hybridization.
  • Nucleic acids that do not hybridize to each other under stringent conditions are still substantially identical ifthe proteins that they encode are substantially identical. This occurs, e.g., when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code.
  • Very stringent conditions are selected to be equal to the T m for a particular probe.
  • An example of stringent conditions for hybridization of complementary nucleic acids which have more than 100 complementary residues on a filter in a Southern or Northern blot is 50% formamide, e.g., hybridization in 50%> formamide, 1 M NaCl, 1% SDS at 37°C, and a wash in 0. IX SSC at 60 to 65°C.
  • Exemplary moderate stringency conditions include hybridization in 40 to 45% > formamide, 1.0 M NaCl, 1% SDS at 37°C, and a wash in 0.5X to IX SSC at 55 to 60°C.
  • a reference nucleotide sequence preferably hybridizes to the reference nucleotide sequence in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO 4 , 1 mM EDTA at 50°C with washing in 2X SSC, 0.1% SDS at 50°C, more desirably in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO 4 , 1 mM EDTA at 50°C with washing in IX SSC, 0.1%) SDS at 50°C, more desirably still in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO 4 , 1 mM EDTA at 50°C with washing in 0.5X SSC, 0.1% SDS at 50°C, preferably in 7% sodium dode
  • variant polypeptide is intended a polypeptide derived from the native protein by deletion (so-called truncation) or addition of one or more amino acids to the N-terminal and/or C-terminal end ofthe native protein; deletion or addition of one or more amino acids at one or more sites in the native protein; or substitution of one or more amino acids at one or more sites in the native protein.
  • variants may results form, for example, genetic polymorphism or from human manipulation. Methods for such manipulations are generally known in the art.
  • polypeptides ofthe invention maybe altered in various ways including amino acid substitutions, deletions, truncations, and insertions. Methods for such manipulations are generally known in the art.
  • amino acid sequence variants ofthe polypeptides can be prepared by mutations in the DNA. Methods for mutagenesis and nucleotide sequence alterations are well known in the art. See, for example, Kunkel (1985); Kunkel et al. (1987); U. S. Patent No. 4,873,192; Walker and Gaastra (1983), and the references cited therein.
  • Guidance as to appropriate amino acid substitutions that do not affect biological activity ofthe protein of interest may be found in the model of Dayhoff et al. (1978). Conservative substitutions, such as exchanging one amino acid with another having similar properties, are preferred.
  • the genes and nucleotide sequences ofthe invention include both the naturally occurring sequences as well as mutant forms.
  • the polypeptides ofthe invention encompass both naturally occurring proteins as well as variations and modified forms thereof. Such variants will continue to possess the desired activity.
  • the deletions, insertions, and substitutions ofthe polypeptide sequence encompassed herein are not expected to produce radical changes in the characteristics ofthe polypeptide. However, when it is difficult to predict the exact effect ofthe substitution, deletion, or insertion in advance of doing so, one skilled in the art will appreciate that the effect will be evaluated by routine screening assays.
  • Production tissue refers to mature, harvestable tissue consisting of non-dividing, [0 terminally-differentiated cells. It excludes young, growing tissue consisting of germline, meristematic, and not-fully-differentiated cells.
  • “Germline cells” refer to cells that are destined to be gametes and whose genetic material is heritable.
  • plant refers to any plant, particularly to seed plant
  • plant cell is a L5 structural and physiological unit ofthe plant, e.g., a cell which comprises a cell wall or a protoplast.
  • the plant cell may be in form of an isolated single cell or a cultured cell, or as a part of higher organized unit such as, for example, a plant tissue, or a plant organ.
  • Plant tissue includes differentiated and undifferentiated tissues or plants, including but not limited to roots, stems, shoots, leaves, pollen, seeds, tumor tissue and various forms of 20 cells and culture such as single cells, protoplast, embryos, and callus tissue.
  • the plant tissue may be in plants or in organ, tissue or cell culture.
  • altered plant trait means any phenotypic or genotypic change in a transgenic plant relative to the wild-type or non-transgenic plant host.
  • transgenic refers to the transfer of a nucleic acid fragment into the 5 genome of a host cell, resulting in genetically stable inheritance.
  • Host cells containing the transformed nucleic acid fragments are referred to as "transgenic” cells, and organisms comprising transgenic cells are referred to as "transgenic organisms".
  • transgenic organisms Examples of methods of transformation of plants and plant cells include Agrobacterium-ms ⁇ atQd transformation (De Blaere et al, 1987) and particle bombardment technology (Klein et al., 1987; U.S. Patent No. 0 4,945,050). Whole plants may be regenerated from transgenic cells by methods well known to the skilled artisan (see, for example, Fromm et al., 1990).
  • Transformed refers to a host cell or organism such as a bacterium or a plant into which a heterologous nucleic acid molecule has been introduced.
  • the nucleic acid molecule can be stably integrated into the genome generally known in the art and are disclosed in Sambrook et al. (1989). See also Innis et al. (1995); and Gelfand (1995); and Innis and Gelfand (1999).
  • Known methods of PCR include, but are not limited to, methods using paired primers, nested primers, single specific primers, degenerate primers, 5 gene-specific primers, vector-specific primers, partially mismatched primers, and the like.
  • “transformed,” “transformant,” and “transgenic” plants or calli have been through the transformation process and contain a foreign gene integrated into their chromosome.
  • the term “untransformed” refers to normal plants that have not been through the transformation process.
  • a “transgenic plant” is a plant having one or more plant cells that contain an expression [0 vector.
  • Transiently transformed refers to cells in which transgenes and foreign DNA have been introduced (for example, by such methods as Agrobacterium-mediated transformation or biolistic bombardment), but not selected for stable maintenance.
  • “Stably transformed” refers to cells that have been selected and regenerated on a L 5 selection media following transformation.
  • Transient expression refers to transgene expression in cells, e.g., after transformation with recombinant virus or by such methods as Agrobacterium-mediated transformation, electroporation, or biolistic bombardment, but not selected for its stable maintenance.
  • Genetically stable and “heritable” refer to chromosomally-integrated genetic elements 20 that are stably maintained in the plant and stably inherited by progeny through successive generations.
  • Primary transformant and “TO generation” refer to transgenic plants that are ofthe same genetic generation as the tissue which was initially transformed (i.e., not having gone through meiosis and fertilization since transformation). 25 “Secondary transformants” and the “Tl, T2, T3, etc. generations” refer to transgenic plants derived from primary transformants through one or more meiotic and fertilization cycles. They may be derived by self-fertilization of primary or secondary transformants or crosses of primary or secondary transformants with other transformed or untransformed plants.
  • “Significant increase” is an increase that is larger than the margin of error inherent in 0 the measurement technique, preferably an increase by about 2-fold or greater.
  • This invention relates to isolated plant nucleic acid molecules, sequences and segments (fragments), the expression of which is increased in plants with increased protein content or levels, as well as the endogenous plant promoters for those expressed molecules, sequences or segments.
  • Preferred sources for the nucleic acid molecules ofthe invention include, but are not limited to, corn (Zea mays), Brassica sp. (e.g., B. napus, B.
  • rapa, B.juncea particularly those Brassica species useful as sources of seed oil, alfalfa (Medicago sativa), rice (Ot ⁇ za sativa), rye (Secale cereale), sorghum (Sorghum bicolor, Sorghum vulgare), millet (e.g., pearl millet (Pennisetum glaucum), proso millet (Panicum miliaceum), foxtail millet (Setaria italica), finger millet (Eleusine coracana)), sunflower (Helianthus annuus), safflower
  • genus Lemna L. aequinoctialis, L. disperma, L. ecuadoriensis, L. gibba, L. japonica, L. minor, L. miniscula, L. obscura, L. perpusilla, L. tenera, L. trisulca, L. turionifera, L. valdiviana
  • genus Spirodela S. intermedia, S. polyrrhiza, S.punctata
  • genus Woffia Wa. angusta, Wa. arrhiza, Wa. australina, Wa. borealis, Wa. brasiliensis, Wa. columbiana, Wa.
  • Lemnaceae Any other genera or species of Lemnaceae, if they exist, are also aspects ofthe present invention.
  • Lemna gibba, Lemna minor, and Lemna miniscula are preferred, vtit . Lemna minor and Lemna miniscula being most preferred.
  • Lemna species can be classified using the taxonomic scheme described by Landolt, Biosystematic Investigation on the Family of Duckweeds: The family of Lemnaceae - A Monograph Study. Geobatanischen Institut ⁇ TH, founded Rubel, Zurich (1986)); vegetables including tomatoes (Lycopersicon esculentwn), lettuce (e.g., Lactuca sativa), green beans (Phaseolus vulgaris), lima beans (Phaseolus limensis), peas (Lathyrus spp.), and members ofthe genus Cucumis such as cucumber (C. sativus), cantaloupe (C. cantalupensis), and musk melon (C. melo).
  • tomatoes Loxicon esculentwn
  • lettuce e.g., Lactuca sativa
  • green beans Phaseeolus vulgaris
  • lima beans Phaseeolus limensis
  • peas Lathyrus spp.
  • Ornamentals include azalea (Rhododendron spp.), hydrangea (Macrophylla hydrangea), hibiscus (Hibiscus rosasanensis), roses (Rosa spp.), tulips (Tulipa spp.), daffodils (Narcissus spp.), petunias (Petunia hybrida), carnation
  • Conifers that may be employed in practicing the present invention include, for example, pines such as loblolly pine (Pinus taeda), slash pine (Pinus elliotii), ponderosa pine (Pinus ponderosa), lodgepole pine (Pinus contorta), and Monterey pine (Pinus radiata), Douglas-fir (Pseudotsuga menziesii); Western hemlock (Tsuga canadensis); Sitka spruce (Picea glauca); redwood (Sequoia sempervirens); true firs such as silver fir (Abies amabilis) and balsam fir (Abies balsamea); and cedars such as Western red cedar (Thuja plicata) and Alaska yellow-cedar (Chamaecypar
  • Leguminous plants include beans and peas.
  • Beans include guar, locust bean, fenugreek, soybean, garden beans, cowpea, mungbean, lima bean, fava bean, lentils, chickpea, etc.
  • Legumes include, but are not limited to, Arachis, e.g., peanuts, Vicia, e.g., crown vetch, hairy vetch, adzuki bean, mung bean, and chickpea, Lupinus, e.g., lupine, trifolium, Phaseolus, e.g., common bean and lima bean, Pisum, e.g., field bean, Melilotus, e.g., clover, Medicago, e.g., alfalfa, Lotus, e.g., trefoil, lens, e.g., lentil, and false indigo, Acacia, aneth, artichoke, arugula, blackberry, canola, cilantro, Clementines, escarole, eucalyptus, fennel, grapefruit, honey dew, jicama, kiwifruit, lemon, lime, mushroom, nut, okra, orange
  • Preferred forage and turf grass nucleic acid sources for use in the methods ofthe invention include alfalfa, orchard grass, tall fescue, perennial ryegrass, creeping bent grass, and redtop.
  • the nucleic acid sources are crop plants and in particular cereals (for example, corn, alfalfa, sunflower, rice, Brassica, canola, soybean, barley, soybean, sugarbeet, cotton, safflower, peanut, sorghum, wheat, millet, tobacco, etc.), and even more preferably corn and soybean.
  • the present invention is directed to a nucleic acid molecule comprising a nucleotide sequence isolated from any plant which encodes a polypeptide having at least 70% amino acid sequence identity to a polypeptide comprising SEQ ID NOs. 1-36 or a promoter for said nucleotide sequence.
  • a nucleic acid molecule comprising a nucleotide sequence isolated from any plant which encodes a polypeptide having at least 70% amino acid sequence identity to a polypeptide comprising SEQ ID NOs. 1-36 or a promoter for said nucleotide sequence.
  • orthologs of those sequences may be identified or isolated from the genome of any desired organism, preferably from another plant, according to well known techniques based on their sequence similarity to the coding sequences, e.g., hybridization, PCR or computer generated sequence comparisons.
  • genomic DNA fragments or cDNA fragments i.e., genomic or cDNA libraries
  • suitable genomic and cDNA libraries may be prepared from any cell or tissue of an organism.
  • Such techniques include hybridization screening of plated DNA libraries (either plaques or colonies; see, e.g., Sambrook et al, 1989) and amplification by PCR using oligonucleotide primers preferably corresponding to sequence domains conserved among related polypeptide or subsequences ofthe nucleotide sequences provided herein (see, e.g., Innis et al., 1990). These methods are particularly well suited to the isolation of gene sequences from organisms closely related to the organism from which the probe sequence is derived.
  • oligonucleotide primers can be designed for use in PCR reactions to amplify corresponding DNA sequences from cDNA or genomic DNA extracted from any plant of interest. Methods for designing PCR primers and PCR cloning are generally known in the art as discussed hereinabove.
  • hybridization techniques all or part of a known nucleotide sequence is used as a probe that selectively hybridizes to other corresponding nucleotide sequences present in a population of cloned genomic DNA fragments or cDNA fragments (i.e., genomic or cDNA libraries) from a chosen organism.
  • the hybridization probes may be genomic DNA fragments, cDNA fragments, RNA fragments, or other oligonucleotides, and may be labeled with a detectable group such as 32 P, or any other detectable marker.
  • probes for hybridization can be made by labeling synthetic oligonucleotides based on the sequence ofthe invention.
  • sequences that hybridize to the sequences disclosed herein will have at least 40% to 50%, about 60% to 70% and even about 80% 85%, 90%, 95% to 98% or more identity with the disclosed sequences. That is, the sequence similarity of sequences may range, sharing at least about 40%, to 50%, about 60% to 70%, and even about 80%>, 85%, 90%, 95%> to 98%o sequence similarity.
  • nucleic acid molecules ofthe invention can also be identified by, for example, a search of known databases for genes encoding polypeptides having a specified amino acid sequence identity. Methods of alignment of sequences for comparison are well known in the art and are described hereinabove.
  • Globulin-1 s allele precursor and Globulin-2 precursor are embryo storage protein. Reference describing these two gene family include (1) Biochem Genet 1989 Apr;27(3-4):239-51 Characterization of embryo globulins encoded by the maize Gib genes. Kriz AL. and (2) Characterization ofthe maize Globulin-2 gene and analysis of two null alleles. Kriz AL, Wallace NH Biochem Genet 1991 Jun;29(5-6):241-54, which are incorporated by reference. Oleosin is a proteins associated with seed oil body.
  • Glucose and ribitol dehydrogenase homolog is an embryo-specifc protein, up-regulated during seed maturation, and is further described in Alexander R, Alamillo JM, Salamini F, Bartels D Planta 1994;192(4):519-25
  • a novel embryo-specific barley cDNA clone encodes a protein with homologies to bacterial glucose and ribitol dehydrogenase, which is inco ⁇ orated by reference.
  • ZMPK1 precursor is a putative receptor protein kinase related to stress response, and further described in Zhang R, Walker JC (1993) Structure and expression ofthe S locus-related genes of maize. Plant Mol Biol 21: 1171-1174, whichis inco ⁇ orated herein by reference.
  • Glutathione S-transferase is an enzyme for transferring glutathione to many substrates, including cytotoxic substances, and is further described in Marrs K.A. The function and regulation of glutathione S- transferases in plants, Annu. Rev. Plant Physiol. Plant. Mol. Biol. 1996 , Vol. 47: 127-158, which is inco ⁇ orated herein by reference.
  • Thioredoxin dependent peroxidase is an enzyme involved in Antioxidative Defense System, further described in RB Van Huystee, Some Molecular Aspects Of Plant Peroxidase Biosynthetic Studies, Annu. Rev. Plant Physiol. Plant. Mol. Biol. 1987 , Vol. 38 : 205-219, which is inco ⁇ orated herein by reference.
  • RAB28 protein is an ABA induced gene, in late embryogenesis in repsponse to water stress, further described in Pla M, Gomez J, Goday A, Pages M Regulation ofthe abscisic acid-responsive gene rab28 in maize viviparous mutants, Mol Gen Genet. 1991 Dec;230(3):394-400, which is incoproated herein by reference.
  • Dehydrin dHNl belongs to a group of proteins that are stress induced and involved in stress tolerance, further described in Zeevaart JAD, Creelman RA 1988 Metabolisms and physiology of abscisic acids. Annu Rev Plant Physiol Mol Biol 39:439-473, which is incoproated herein by reference.
  • Hydroxymethylglutaryl-CoA reductase which is a key enzyme involved in catalyzing an early reaction unique to isoprenoid biosynthesis., further described in (1) Kato-Emori S, Higashi K, Hosoya K, Kobayashi T, Ezura H. Cloning and characterization ofthe gene encoding 3- hydroxy-3-methylglutaryl coenzyme A reductase in melon (Cucumis melo L. reticulatus). Mol Genet Genomics. 2001 Mar;265(l): 135-42, and (2) Caelles C, Ferrer A, Balcells L, Hegardt FG, Boronat A.
  • proteomics approach was used to identify genes that were differentially expressed in high protein corn lines. Over 150 differentially expressed protein spots were identified and analyzed as described in the experimental conditions. Provded herein are genes, their proteins, as shown in the Sequence Listing, and their orthologs, applicable to the methods and compositons ofthe present invention.
  • the nature ofthe candidate genes and their potential roles in contributing to the high protein phenotype is presented, however, the inventor is by no means to be limited by any one proposed mechanism.
  • proteins positively annotated two groups of proteins are outstanding and are believed to be intimately related to the corn high protein phenotype: one group represents the seed storage proteins, including globulins and oleosin. These major seed protein storage components are believed to directly contribute to the high protein phenotype.
  • a second group of proteins can be roughly characterized as stress related proteins, such as the heat shock protein, dehydrin and a regulatory gene rab28 involved in ABA related stress response. These two groups of proteins or genes are part ofthe same mechanism that contributes to the high protein yield.
  • ABI3 which is a Arabidopsis gene that involved in seed storage protein biosynthesis, is reported as a key player in temperature stress.
  • One hypothesis for this relationship is that plants that are more stress resistant, such as more heat tolerant, will grow better, therefore have more grain yield including grain protein.
  • other global regulators that have a significant impact on stress related response can be used to manipulate seed protein content, such as the ABI3 from Arabidopsis and its homolog in rice, and in particular the genes ofthe present invention.
  • Seed storage proteins directly contribute to the high protein phenotype.
  • Antibodies developed against the two embryo specific globulin proteins, glbl and 2 were used to deterimine that their protein levels in the high protein inbred lines are significantly higher than in the control line (1.5-2 fold).
  • Gene expression patterns of selected genes were studied in rice gene expression profiling experiments.
  • Both seed protein content and protein quality can be changed by using these genes, hi one embodiment a transgenic approach to over express or down regulate these genes in the seeds can be employed to increase grain protein content. Meanwhile, as is evident from their amino acid sequences (see Sequence Listing), some of these genes and proteins are biased to a special amino acid profile, over expressing of these proteins in seed can change the seed protein property. In particular, nutrionally enhanced seed, more complete or elevated in one or more amino acids, can be obtained. For example, poultry, like swine, have a specific amino acid that, if deficient, will reduce the animal's performance on the feed. For poultry, the limiting amino acid is methionine, while for swine the limiting amino acid is lysine. Accordingly, the present invention can provide feed that contains, or can be formulated to contain, an increase in the amount of methionine (or lysine for swine) and general protein to keep the desired protein gross energy ratio in the diet.
  • genes disclosed herein are useful as genetic markers in marker assisted breeding programs to select high protein lines during plant breeding.
  • antibodies to the proteins ofthe invention for use in ELISA for example
  • DNA markers that are linked to these genes, or the genes themselves find use to predict genomic profile and thus trait outcome of siblings in breeding practices.
  • the genes and proteins find use in production of effective protein production factories. For example, down regulation of one or more ofthe proteins can provide a seed that is reduced in these proteins thus allowing increased cellular resources for expression of industrially or therapeutically important polypeptides. This can best be done by inducible regulation o the one or more genes. In one embodiment reduction of storage protein content is achieved by anti-sense, for example RNAi methods.
  • a two component Gal4/Cl system can be used to provide an inducible system. Two components in separate inbred lines are inactive, but create hybrids in which gene mosulation is activated to create plants with lower protein yield. This methd also finds use to create low protein lines that are less aller genie.
  • the present invention also encompasses expression cassettes, preferably in the form of a recombinant vectors comprising the nucleic acid sequences ofthe invention.
  • the expression cassette comprises regulatory elements for expression ofthe nucleotide sequences in a host cell capable of expressing the nucleotide sequences.
  • regulatory elements usually comprise promoter and termination signals and preferably also comprise elements allowing efficient translation of polypeptides encoded by the nucleic acid sequences ofthe present invention.
  • sequences adjacent to the initiating methionine may require modification. For example, they can be modified by the inclusion of sequences known to be effective in plants.
  • Vectors comprising the nucleic acid sequences are usually capable of replication in particular host cells, e.g., as extrachromosomal molecules, and are therefore used to amplify the nucleic acid sequences of this invention in the host cells.
  • host cells for such vectors are plant cells.
  • promoters shown to be functional in plants are driven by promoters shown to be functional in plants.
  • the choice of promoter will vary depending on the temporal and spatial requirements for expression, and also depending on the target species, hi many cases, expression in multiple tissues is desirable.
  • many promoters from dicotyledons have been shown to be operational in monocotyledons and vice versa, ideally dicotyledonous promoters are selected for expression in dicotyledons, and monocotyledonous promoters for expression in monocotyledons.
  • promoters include, but are not limited to, constitutive, inducible, temporally regulated, developmentally regulated, chemically regulated, stress-responsive, tissue-preferred and tissue-specific promoters.
  • Promoter sequences are known to be strong or weak.
  • a strong promoter provides for a high level of gene expression, whereas a weak promoter provides for a very low level of gene expression.
  • An inducible promoter is a promoter that provides for the turning on and off of gene expression in response to an exogenously added agent, or to an environmental or developmental stimulus.
  • a bacterial promoter such as the P tac promoter can be induced to varying levels of gene expression depending on the level of isothiopropylgalactoside added to the transformed bacterial cells.
  • An isolated promoter sequence that is a strong promoter for heterologous nucleic acid is advantageous because it provides for a sufficient level of gene expression to allow for easy detection and selection of transformed cells and provides for a high level of gene expression when desired.
  • Preferred promoters that are expressed constitutively include promoters from genes encoding actin or ubiquitin and the CaMN 35S and 19S promoters.
  • the nucleotide sequences of this invention can also be expressed under the regulation of promoters that are chemically regulated. This enables the nucleic acid sequence or encoded polypeptide to be synthesized only when the crop plants are treated with the inducing chemicals.
  • Preferred technology for chemical induction of gene expression is detailed in the published application EP 0 332 104 (to Ciba-Geigy) and U.S. Patent 5,614,395.
  • a preferred promoter for chemical induction is the tobacco PR- la promoter.
  • Tissue-specific or tissue-preferential promoters useful in the present invention are also useful.
  • promoters which confer seed-specific expression such as those disclosed by Schemthaner et al. (1988); anther (tapetal) specific promoter B6 (Huffman et al.); and pistil- specific promoters such as a modified S13 promoter (Dzelkalns et al., 1993).
  • Preferred tissue specific expression patterns include green tissue-specific, root-specific, stem-specific, and flower-specific. Promoters suitable for expression in green tissue include many which regulate genes involved in photosynthesis and many of these have been cloned from both monocotyledons and dicotyledons.
  • a preferred promoter is the maize PEPC promoter from the phosphoenol carboxylase gene (Hudspeth & Grula, 1989).
  • a preferred promoter for root-specific expression is that described by de Framond (1991; EP 0 452 269 to Ciba-Geigy).
  • a preferred stem specific promoter is that described in U.S. Patent No. 5,625,136 (to Ciba-Geigy) and which drives expression ofthe maize trpA gene. -
  • promoters which direct specific or enhanced expression in certain plant tissues will be known to those of skill in the art in light ofthe present disclosure. These include, for example, the rbcS promoter, specific for green tissue; the ocs, nos, and m ⁇ s promoters which have higher activity in roots or wounded leaf tissue; a truncated (-90 to +8) 35S promoter which directs enhanced expression in roots, an tubulin gene that directs expression in roots and promoters derived from zein storage protein genes which direct expression in endosperm.
  • ocs octopine synthase
  • Preferred plant promoters include, but are not limited to, a promoter such as the CaMV 35S promoter, an enhanced 35S promoter or others such as CaMV 19S, nos, Adhl, sucrose synthase, V-tubulin, ubiquitin, actin, cab, PEPCase or those associated with the R gene complex.
  • a promoter such as the CaMV 35S promoter, an enhanced 35S promoter or others such as CaMV 19S, nos, Adhl, sucrose synthase, V-tubulin, ubiquitin, actin, cab, PEPCase or those associated with the R gene complex.
  • promoters may include the U2 and U5 snRNA promoters from maize, the promoter from alcohol dehydrogenase, the Z4 promoter from a gene encoding the Z4 22 kD zein protein, the Z10 promoter from a gene encoding a 10 kD zein protein, a Z27 promoter from a gene encoding a 27 kD zein protein, the A20 promoter from the gene encoding a 19 kD -zein protein, inducible promoters, such as the light inducible promoter derived from the pea rbcS gene and the actin promoter from rice; seed specific promoters, such as the phaseolin promoter from beans, may also be used.
  • Other promoters useful in the practice ofthe invention are known to those of skill in the art.
  • tissue specific promoters which have been described include the lectin (Vodkin, 1983; Lindstrom et al., 1990,) corn alcohol dehydrogenase 1 (Vogel et al., 1992; Demiis et al, 1984), com light harvesting complex (Simpson, 1985; Bansal et al., 1992), corn heat shock protein (Odell et al., 1985; Rochester et al., 1986), pea small subunit RuBP carboxylase (Poulsen et al., 1986; Cashmore et al., 1983), Ti plasmid mannopine synthase (Langridge et al., 1989), Ti plasmid nopaline synthase (Langridge et al., 1989), petunia chalcone isomerase (vanTunen et al., 1988), bean glycine rich protein 1 (Keller et al., 1989), truncated CaMV 35s (Odell
  • Inducible promoters that have been described include the ABA- and turgor-inducible promoters, the promoter ofthe auxin-binding protein gene (Schwob et al., 1993), the UDP glucose flavonoid glycosyl-transferase gene promoter (Ralston et al., 1988), the MPI proteinase inhibitor promoter (Cordero et al., 1994), and the glyceraldehyde-3-phosphate dehydrogenase gene promoter (Kohler et al., 1995; Quigley et al., 1989; Martinez et al., 1989).
  • tissue-specific regulated genes and/or promoters have been reported in plants. These include genes encoding the seed storage proteins (such as napin, cruciferin, beta- conglycinin, and phaseolin) zein or oil body proteins (such as oleosin), or genes involved in fatty acid biosynthesis (including acyl carrier protein, stearoyl-ACP desaturase. and fatty acid desaturases (fad 2-1)), and other genes expressed during embryo development (such as Bce4, see, for example. EP 255378 and Kridl et al., 1991). Particularly useful for seed-specific expression is the pea vicilin promoter (Czako et al., 1992). (See also U.S. Pat. No.
  • cDNA clones that are preferentially expressed in cotton fiber have been isolated (John et al., 1992).
  • cDNA clones from tomato displaying differential expression during fruit development have been isolated and characterized (Mansson et al., 1985, Slater et al., 1985).
  • the promoter for polygalacturonase gene is active in fruit ripening.
  • the polygalacturonase gene is described in U.S. Patent No. 4,535,060, U.S. Patent No. 4,769,061, U.S. Patent No. 4,801,590, and U.S. Patent No. 5,107,065, which disclosures are inco ⁇ orated herein by reference.
  • tissue-specific promoters include those that direct expression in leaf cells following damage to the leaf (for example, from chewing insects), in tubers (for example, patatin gene promoter), and in fiber cells (an example of a developmentally-regulated fiber cell protein is E6 (John et al., 1992). The E6 gene is most active in fiber, although low levels of transcripts are found in leaf, ovule and flower.
  • tissue-specificity of some "tissue-specific" promoters may not be absolute and may be tested by one skilled in the art using the diphtheria toxin sequence.
  • tissue-specific expression with "leaky” expression by a combination of different tissue-specific promoters (Beals et al., 1997).
  • Other tissue-specific promoters can be isolated by one skilled in the art (see U.S. 5,589,379).
  • Several inducible promoters (“gene switches") have been reported. Many are described in the review by Gatz (1996 and 1997).
  • RNA transcript that interferes with translation ofthe mRNA ofthe native DNA sequence.
  • Other elements include those that can be regulated by endogenous or exogenous agents, e.g., by DNA binding proteins such as zinc finger proteins, including naturally occurring zinc finger proteins or chimeric zinc finger proteins (see, e.g., U.S. Patent No. 5,789,538, WO 99/48909; WO 99/45132; WO 98/53060; WO 98/53057; WO 98/53058; WO 00/23464; WO 95/19431 ; and WO 98/54311) or myb-like transcription factors.
  • DNA binding proteins such as zinc finger proteins, including naturally occurring zinc finger proteins or chimeric zinc finger proteins (see, e.g., U.S. Patent No. 5,789,538, WO 99/48909; WO 99/45132; WO 98/53060; WO 98/53057; WO 98/53058; WO 00/23464; WO 95/19431 ; and WO 98/54311) or myb-
  • a chimeric zinc finger protein may include amino acid sequences which bind to a specific DNA sequence (the zinc finger) and amino acid sequences that activate (e.g., GAL 4 sequences) or repress the transcription ofthe sequences linked to the specific DNA sequence.
  • amino acid sequences that bind to a specific DNA sequence the zinc finger
  • amino acid sequences that activate e.g., GAL 4 sequences
  • repress the transcription ofthe sequences linked to the specific DNA sequence e.g., GAL 4 sequences
  • Transcriptional terminators are responsible for the termination of transcription and correct mRNA polyadenylation.
  • the 3N nontranslated regulatory DNA sequence preferably includes from about 50 to about 1,000, more preferably about 100 to about 1,000, nucleotide base pairs and contains plant transcriptional and translational termination sequences. Appropriate transcriptional terminators and those which are known to function in
  • 3 plants include the CaMV 35S terminator, the tml terminator, the nopaline synthase terminator, the pea rbcS E9 terminator, the terminator for the T7 transcript from the octopine synthase gene of Agrobacterium tumefaciens, and the 3N end ofthe protease inhibitor I or II genes from potato or tomato, although other 3N elements known to those of skill in the art can also be employed.
  • the 5N regulatory region ofthe expression cassette may also include other enhancing sequences. Numerous sequences have been found to enhance gene expression in transgenic plants. These include sequences which have been shown to enhance expression such as intron sequences (e.g., from Adhl, bronzel or the sucrose synthase intron) and viral leader sequences (e.g., from TMV, MCMV and AMV). For example, a number of non-translated leader
  • leader sequences from Tobacco Mosaic Virus TMV
  • MCMV Maize Chlorotic Mottle Virus
  • AMV Alfalfa Mosaic Virus
  • Other leaders known in the art include but are not limited to: Picornavirus leaders, for example, EMCV leader (Encephalomyocarditis 5
  • TEV leader Tobacco Etch Virus
  • MDMV leader Maize Dwarf Mosaic Virus
  • Human immunoglobulin heavy-chain binding protein (BiP) leader Macejak et al., 1991
  • C. Targeting Sequences for example, TEV leader (Tobacco Etch Virus) (Allison et al., 1986); MDMV leader (Maize Dwarf Mosaic Virus); Human immunoglobulin heavy-chain binding protein (BiP) leader, (Macejak et al., 1991); Untranslated leader from the coat protein
  • the nucleotide sequences o the present invention may be target expression of different cellular localizations in the plant. In some cases, localization in the cytosol may be desirable, whereas in other cases, localization in some subcellular organelle, e.g., the nucleus, may be preferred. Subcellular localization of transgene encoded enzymes is undertaken using techniques well known in the art. Typically, the DNA encoding the target peptide from a known organelle-targeted gene product is manipulated and fused upstream of the nucleotide sequence. Many such target sequences are known for the chloroplast and their functioning in heterologous constructions has been shown. The expression ofthe nucleotide sequences ofthe present invention is also targeted to the endoplasmic reticulum or to the vacuoles ofthe host cells. Techniques to achieve this are well-known in the art.
  • Marker genes are genes that impart a distinct phenotype to cells expressing the marker gene and thus allow such transformed cells to be distinguished from cells that do not have the marker.
  • Such genes may encode either a selectable or screenable marker, depending on whether the marker confers a trait which one can 'select' for by chemical means, i.e., through the use of a selective agent (e.g., a herbicide, antibiotic, or the like), or whether it is simply a trait that one can identify through observation or testing, i.e., by 'screening' (e.g., the R-locus trait).
  • a selective agent e.g., a herbicide, antibiotic, or the like
  • 'screening' e.g., the R-locus trait
  • selectable or screenable marker genes are also genes which encode a "secretable marker” whose secretion can be detected as a means of identifying or selecting for transformed cells. Examples include markers which encode a secretable antigen that can be identified by antibody interaction, or even secretable enzymes which can be detected by their catalytic activity.
  • Secretable proteins fall into a number of classes, including small, diffusible proteins detectable, e.g., by ELISA; small active enzymes detectable in extracellular solution (e.g., V-amylase, ⁇ -lactamase, phosphinothricin acetyltransferase); and proteins that are inserted or trapped in the cell wall (e.g., proteins that include a leader sequence such as that found in the expression unit of extensin or tobacco PR-S).
  • small, diffusible proteins detectable e.g., by ELISA
  • small active enzymes detectable in extracellular solution e.g., V-amylase, ⁇ -lactamase, phosphinothricin acetyltransferase
  • proteins that are inserted or trapped in the cell wall e.g., proteins that include a leader sequence such as that found in the expression unit of extensin or tobacco PR-S.
  • a gene that encodes a polypeptide that becomes sequestered in the cell wall, and which polypeptide includes a unique epitope is considered to be particularly advantageous.
  • a secreted antigen marker would ideally employ an epitope sequence that would provide low background in plant tissue, a promoter-leader sequence that would impart efficient expression and targeting across the plasma membrane, and would produce protein that is bound in the cell wall and yet accessible to antibodies.
  • a normally secreted wall protein modified to include a unique epitope would satisfy all such requirements.
  • Possible selectable markers for use in connection with the present invention include, but are not limited to, a neo gene, which codes for kanamycin resistance and can be selected for using kanamycin, G418, a gene encoding resistance to bleomycin, and the like; a bar gene which codes for bialaphos resistance; a gene which encodes an altered EPSP synthase protein thus conferring glyphosate resistance; a nitrilase gene such as bxn from Klebsiella ozaenae which confers resistance to bromoxynil; a mutant acetolactate synthase gene (ALS) which confers resistance to imidazolinone, sulfonylurea or other ALS -inhibiting chemicals (European Patent Application 154,204, 1985); a methotrexate-resistant DHFR gene; a dalapon dehalogenase gene that confers resistance to the herbicide dalapon; or a mutated anthranilate synthase
  • a mutant EPSP synthase gene is employed, additional benefit may be realized through the inco ⁇ oration of a suitable chloroplast transit peptide, CTP (European Patent Application 0 218 571*, 1987).
  • CTP chloroplast transit peptide
  • An illustrative embodiment of a selectable marker gene capable of being used in systems to select transformants is the genes that encode the enzyme phosphinothricin acetyltransferase, such as the bar gene from Streptomyces hygroscopicus or the pat gene from Streptomyces viridochromogenes (U.S. Patent No. 5,550,318).
  • PPT phosphinothricin acetyltransferase
  • Screenable markers that may be employed include, but are not limited to, a 5- glucuronidase or uidA gene (GUS) which encodes an enzyme for which various chromogenic substrates are known; an R-locus gene, which encodes a product that regulates the production of anthocyanin pigments (red color) in plant tissues; a -lactamase gene, which encodes an enzyme for which various chromogenic substrates are known (e.g., PAD AC, a chromogenic cephalosporin); xylE gene which encodes a catechol dioxygenase that can convert chromogenic catechols; an -amylase gene; a tyrosinase gene which encodes an enzyme capable of oxidizing tyrosine to DOPA and dopaquinone which in turn condenses to form the easily detectable compound melanin; a -galactosidase gene, which encodes an enzyme for which there are chromogenic substrates;
  • the R gene complex in maize encodes a protein that acts to regulate the production of anthocyanin pigments in most seed and plant tissue.
  • Maize strains can have one, or as many as four, R alleles which combine to regulate pigmentation in a developmental and tissue specific manner.
  • a gene from the R gene complex was applied to maize transformation, because the expression of this gene in transformed cells does not harm the cells. Thus, an R gene introduced into such cells will cause the expression of a red pigment and, if stably inco ⁇ orated, can be visually scored as a red sector.
  • a maize line carries dominant alleles for genes encoding the enzymatic intermediates in the anthocyanin biosynthetic pathway (C2, Al, A2, Bzl and Bz2), but carries a recessive allele at the R locus, transformation of any cell from that line with R will result in red pigment formation.
  • Exemplary lines include Wisconsin 22 which contains the rg-Stadler allele and TR112, a K55 derivative which is r-g, b, PL
  • any genotype of maize can be utilized ifthe CI and R alleles are introduced together.
  • a further screenable marker contemplated for use in the present invention is firefly luciferase, encoded by the lux gene.
  • the presence ofthe lux gene in transformed cells may be detected using, for example, X-ray film, scintillation counting, fluorescent spectrophotometry, low-light video cameras, photon counting cameras or multiwell luminometry. It is also envisioned that this system may be developed for populational screening for bioluminescence, such as on tissue culture plates, or even for whole plant screening.
  • a vector ofthe invention can also further comprise plasmid DNA.
  • Plasmid vectors include additional DNA sequences that provide for easy selection, amplification, and transformation ofthe expression cassette in prokaryotic and eukaryotic cells, e.g., pUC-deriVed vectors such as pUC8, pUC9, pUCl 8, pUC19, pUC23, pUCl 19, and pUC120, pSK-derived vectors, pGEM-derived vectors, pSP-derived vectors, or pBS-derived vectors.
  • the additional DNA sequences include origins of replication to provide for autonomous replication ofthe vector, additional selectable marker genes, preferably encoding antibiotic or herbicide resistance, unique multiple cloning sites providing for multiple sites to insert DNA sequences or genes encoded in the expression cassette, and sequences that enhance transformation of prokaryotic and eukaryotic cells.
  • Another vector that is useful for expression in both plant and prokaryotic cells is the binary Ti plasmid (as disclosed in Schilperoort et al., U.S. Patent No. 4,940,838) as exemplified by vector pGA582.
  • This binary Ti plasmid vector has been previously characterized by An, cited supra.
  • This binary Ti vector can be replicated in prokaryotic bacteria such as E. coli and Agrobacterium.
  • the Agrobacterium plasmid vectors can be used to transfer the expression cassette to dicot plant cells, and under certain conditions to monocot cells, such as rice cells.
  • the binary Ti vectors preferably include the nopaline T DNA right and left borders to provide for efficient plant cell transformation, a selectable marker gene, unique multiple cloning sites in the T border regions, the colEl replication of origin and a wide host range replicon.
  • the binary Ti vectors carrying an expression cassette ofthe invention can be used to transform both prokaryotic and eukaryotic cells, but is preferably used to transform dicot plant cells.
  • Virtually any DNA may be used for delivery to recipient cells to ultimately produce fertile transgenic plants in accordance with the present invention.
  • DNA segments in the form of vectors and plasmids, or linear DNA fragments, in some instance containing only the DNA element to be expressed in the plant, and the like, may be employed.
  • Vectors, plasmids, cosmids, YACs (yeast artificial chromosomes) and DNA segments for use in transforming such cells will, of course, generally comprise the cDNA, gene or genes which one desires to introduce into the cells. These DNA constructs can further include structures such as promoters, enhancers, polylinkers, or even regulatory genes as desired.
  • the DNA segment or gene chosen for cellular introduction will often encode a protein which will be expressed in the resultant recombinant cells, such as will result in a screenable or selectable trait and/or which will impart an improved phenotype to the regenerated plant. However, this may not always be the case, and the present invention also encompasses transgenic plants inco ⁇ orating non-expressed transgenes.
  • the expression cassettes ofthe present invention can be introduced into a host cell, e.g., a plant cell, in a number of art-recognized ways. Those skilled in the art will appreciate that the choice of method might depend on the type of cell, e.g., monocotyledonous or dicotyledonous, targeted for transformation.
  • Vectors which may be used to transform plant tissue with the expression cassettes ofthe present invention include both Agrobacterium vectors and ballistic vectors, as well as vectors suitable for DNA-mediated transformation, e.g., direct uptake or via electroporation. However, cells other than plant cells may be transformed with the expression cassettes ofthe invention.
  • Suitable methods of transforming plant cells include, but are not limited to, microinjection (Crossway et al., 1986), direct DNA transfer to plant cells by PEG precipitation; liposomes; electroporation (Riggs et al., 1986, Agrobacteriwn-mediated transformation (Hinchee et al., 1988), direct gene transfer (Paszkowski et al., 1984), and ballistic particle acceleration using devices available from Agracetus, Inc., Madison, Wis. and BioRad, Hercules, Calif, (see, for example, Sanford et al., U.S. Pat. No. 4,945,050; and McCabe et al., 1988).
  • a nucleotide sequence ofthe present invention is directly transformed into the plastid genome.
  • Plastid transformation technology is extensively described in U.S. Patent Nos. 5,451,513, 5,545,817, and 5,545,818, in PCT application no. WO 95/16783, and in McBride et al., 1994.
  • the basic technique for chloroplast transformation involves introducing regions of cloned plastid DNA flanking a selectable marker together with the gene of interest into a suitable target tissue, e.g., using biolistics or protoplast transformation (e.g., calcium chloride or PEG mediated transformation).
  • the 1 to 1.5 kb flanking regions facilitate orthologous recombination with the plastid genome and thus allow the replacement or modification of specific regions ofthe plastome.
  • point mutations in the chloroplast 16S rRNA and ⁇ sl2 genes conferring resistance to spectinomycin and/or streptomycin are utilized as selectable markers for transfonnation (Svab et al., 1990; Staub et al., 1992). This resulted in stable homoplasmic transformants at a frequency of approximately one per 100 bombardments of target leaves. The presence of cloning sites between these markers allowed creation of a plastid targeting vector for introduction of foreign genes (Staub et al., 1993).
  • Plastid expression in which genes are inserted by orthologous recombination into all ofthe several thousand copies of the circular plastid genome present in each plant cell, takes advantage of the enormous copy number advantage over nuclear-expressed genes to permit expression levels that can readily exceed 10%o ofthe total soluble plant protein, hi a preferred embodiment, a nucleotide sequence ofthe present invention is inserted into a plastid targeting vector and transformed into the plastid genome of a desired plant host. Plants homoplastic for plastid genomes containing a nucleotide sequence ofthe present invention are obtained, and are preferentially capable of high expression ofthe nucleotide sequence.
  • Agrobacterium tumefaciens cells containing a vector comprising an expression cassette ofthe present invention, wherein the vector comprises a Ti plasmid are useful in methods of making transformed plants. Plant cells are infected with an Agrobacterium tumefaciens as described above to produce a transformed plant cell, and then a plant is regenerated from the transformed plant cell. Numerous Agrobacterium vector systems useful in carrying out the present invention are known. For example, U.S. Pat. No. 4,459,355 discloses a method for transforming susceptible plants, including dicots, with an Agrobacterium strain containing the Ti plasmid. The transformation of woody plants with an Agrobacterium vector is disclosed in U.S. Patent No. 4,795,855.
  • U.S. Patent No. 4,940,838 to Schilperoort et al. discloses a binary Agrobacterium vector (i.e., one in which the Agrobacterium contains one plasmid having the vir region of a Ti plasmid but no T region, and a second plasmid having a T region but no vir region) useful in carrying out the present invention. It is particularly preferred to use the binary type vectors of Ti and Ri plasmids of Agrobacterium spp.
  • Ti-derived vectors transform a wide variety of higher plants, including monocotyledonous and dicotyledonous plants, such as soybean, cotton, rape, tobacco, and rice (Pacciotti et al., 1985: Byrne et al., 1987; Sukhapinda et al., 1987; Lorz et al., 1985; Potrykus, 1985; Park et al., 1985 : Hiei et al., 1994.
  • the use of T-DNA to transform plant cells has received extensive study and is amply described (EP 120516; Hoekema, 1985; Knauf, et al., 1983; and An. et al., 1985.
  • the nucleotide sequences ofthe invention can be inserted into binary vectors as described in the examples.
  • Transformation of plants can be undertaken with a single DNA molecule or multiple DNA molecules (i.e., co-transformation), and both these techniques are suitable for use with the expression cassettes ofthe present invention.
  • Numerous transformation vectors are available for plant transformation, and the expression cassettes of this invention can be used in conjunction with any such vectors. The selection of vector will depend upon the preferred transformation technique and the target species for transformation.
  • Preferred plant cells for transformation include, but are not limited to, cells from plant such as com (Zea mays), Brassica sp. (e.g., B. napus, B. rapa, B.
  • juncea particularly those Brassica species useful as sources of seed oil, alfalfa (Medicago sativa), rice (Oryza sativa), rye (Secale cereale), sorghum (Sorghum bicolor, Sorghum vulgare), millet (e.g., pearl millet (Pennisetum glaucum), proso millet (Panicum miliaceum), foxtail millet (Setaria italica), finger millet (Eleusine coracana)), sunflower (Helianthus annuus), safflower (Carthamus tinctorius), wheat (Triticum aestivum), soybean (Glycine max), tobacco (Nicotiana tabacum), potato (Solanum tuberosum), peanuts (Arachis hypogaea), cotton (Gossypium barbadense, Gossypium hirsutum), sweet potato (Ipomoea batatus), cassava (Matiihot esculent
  • genus Lemna L. aequinoctialis, L. disperma, L. ecuadoriensis, L. gibba, L. japonica, L. minor, L. miniscida, L. obscura, L. perpusilla, L. tenera, L. trisulca, L. turionifera, L. valdiviana
  • genus Spirodela S. intermedia, S. polyrrhiza, S.punctata
  • genus Woffia Wa. angusta, Wa. arrhiza, Wa. australina, Wa. borealis, Wa. brasiliensis, Wa. columbiana, Wa.
  • Lemnaceae Any other genera or species of Lemnaceae, if they exist, are also aspects ofthe present invention. Lemna gibba, Lemna minor, and Lemna miniscula are preferred, with Lemna minor and Lemna miniscula being most preferred.
  • Lemna species can be classified using the taxonomic scheme described by Landolt, Biosystematic Investigation on the Family of Duckweeds: The family of Lemnaceae - A Monograph Study. Geobatanischen Institut ETH, founded Rubel, Zurich (1986)); vegetables including tomatoes (Lycopersicon esculentum), lettuce (e.g., Lactuca sativa), green beans (Phaseolus vulgaris), lima beans (Phaseolus limensis), peas (Lathyrus spp.), and members ofthe genus Cucumis such as cucumber (C. sativus), cantaloupe (C. cantalupensis), and musk melon (C. melo).
  • tomatoes Loxicon esculentum
  • lettuce e.g., Lactuca sativa
  • green beans Phaseeolus vulgaris
  • lima beans Phaseeolus limensis
  • peas Lathyrus spp.
  • Ornamentals include azalea (Rhododendron spp.), hydrangea (M ⁇ crophyll ⁇ hydrangea), hibiscus (Hibiscus rosasaj ensis), roses (Rosa spp.), tulips (Tulipa spp.), daffodils (Narcissus spp.), petunias (Petunia hybrida), carnation (Dianthus caryophyllus), poinsettia (Euphorbia pulcherrima), and chrysanthemum.
  • Conifers that may be employed in practicing the present invention include, for example, pines such as loblolly pine (Pinus taeda), slash pine (Pinus elliotii), ponderosa pine (Pinus ponderosa), lodgepole pine (Pinus contorta), and Monterey pine (Pinus radiata), Douglas-fir (Pseudotsuga menziesii); Western hemlock (Tsuga canadensis); Sitka spruce (Picea glauca); redwood (Sequoia sempervirens); true firs such as silver fir (Abies amabilis) and balsam fir (Abies balsamea); and cedars such as Western red cedar (Thuja plicata) and Alaska yellow-cedar (Chamaecyparis nootkatensis).
  • pines such as loblolly pine (Pinus taeda), slash pine (P
  • Leguminous plants include beans and peas.
  • Beans include guar, locust bean, fenugreek, soybean, garden beans, cowpea, mungbean, lima bean, fava bean, lentils, chickpea, etc.
  • Legumes include, but are not limited to, Arachis, e.g., peanuts, Vicia, e.g., crown vetch, hairy vetch, adzuki bean, mung bean, and chickpea, Lupinus, e.g., lupine, trifolium, Phaseolus, e.g., common bean and lima bean, Pisum, e.g., field bean, Melilotus, e.g., clover, Medicago, e.g., alfalfa, Lotus, e.g., trefoil, lens, e.g., lentil, and false indigo, Acacia, aneth, artichoke, arugula, blackberry, canola, cilantro, Clementines, escarole, eucalyptus, fennel, grapefruit, honey dew, jicama, kiwifruit, lemon, lime, mushroom, nut, okra, orange
  • Preferred forage and turf grass for use in the methods ofthe invention include alfalfa, orchard grass, tall fescue, perennial ryegrass, creeping bent grass, and redtop.
  • plants or cells to be transformed are crop plants and in particular cereals (for example, corn, alfalfa, sunflower, rice, Brassica, canola, soybean, barley, soybean, sugarbeet, cotton, safflower, peanut, sorghum, wheat, millet, tobacco, and the like), and even more preferably rice, corn and soybean.
  • cereals for example, corn, alfalfa, sunflower, rice, Brassica, canola, soybean, barley, soybean, sugarbeet, cotton, safflower, peanut, sorghum, wheat, millet, tobacco, and the like
  • rice, corn and soybean for example, corn, alfalfa, sunflower, rice, Brassica, canola, soybean, barley, soybean, sugarbeet, cotton, safflower, peanut, sorghum, wheat, millet, tobacco, and the like
  • the transformed host cells are monocot or dicot cells, including, but not limited to, wheat, corn (maize), rice, oat, barley, millet, rye, rape and alfalfa, as well as asparagus, tomato, egg plant, apple, pear, quince, cherry, apricot, pepper, melon, lettuce, cauliflower, Brassica, e.g., broccoli, cabbage, brussels sprout, sugar beet, sugar cane, sweetcorn, onion, carrot, leek, cucumber, tobacco, aubergine, beet, broad bean, carrot, celery, chicory, cotton, radish, pumpkin, hemp, buckwheat, orchardgrass, creeping bent top, redtop, ryegrass, tobacco, turfgrass, tall fescue, cow pea, endive, gourd, grape, raspberry, chenopodium, blueberry, pineapple, avocado, mango, banana, groundnut, nectarine, papaya, garlic, pea, peach,
  • the transformed host cells are monocot cells such as maize, rice, wheat, barley, oats, and sorghum, which can be regenerated into a transgenic plant.
  • Any plant tissue capable of subsequent clonal propagation, whether by organogenesis or embryogenesis, may be transformed with a vector ofthe present invention.
  • organogenesis means a process by which shoots and roots are developed sequentially from meristematic centers;
  • embryogenesis means a process by which shoots and roots develop together in a concerted fashion (not sequentially), whether from somatic cells or gametes.
  • tissue targets include leaf disks, pollen, embryos, cotyledons, hypocotyls, megagametophytes, callus tissue, existing meristematic tissue (e.g., apical meristems, axillary buds, and root meristems), and induced meristem tissue (e.g., cotyledon meristem and hypocotyl meristem).
  • tissue source for transformation will depend on the nature ofthe host plant and the transformation protocol.
  • Useful tissue sources include callus, suspension culture cells, protoplasts, leaf segments, stem segments, tassels, pollen, embryos, hypocotyls, tuber segments, meristematic regions, and the like.
  • the tissue source is selected and transformed so that it retains the ability to regenerate whole, fertile plants following transformation, i.e., contains totipotent cells.
  • Type I or Type II embryonic maize callus and immature embryos are preferred Zea mays tissue sources. Selection of tissue sources for transformation of monocots is described in PCT publication WO 95/06128. For certain plant species, different antibiotic or herbicide selection markers may be preferred.
  • Selection markers used routinely in transformation include the nptll gene which confers resistance to kanamycin and related antibiotics (Messing & Vierra, 1982); Bevan et al., 1983), the bar gene which confers resistance to the herbicide phosphinothricin (White et al., 1990, Spencer et al., 1990), the hph gene which confers resistance to the antibiotic hygromycin (Blochinger & Diggelmann), and the dhfr gene, which confers resistance to methotrexate (Bourouis et al., 1983).
  • the present invention also provides a transformed (transgenic) plant cell, in planta or explanta, including, but not limited to, a transformed plant cell from plants such as corn (Zea mays), Brassica sp. (e.g., B. napus, B. rapa, B.
  • a transformed plant cell from plants such as corn (Zea mays), Brassica sp. (e.g., B. napus, B. rapa, B.
  • juncea particularly those Brassica species useful as sources of seed oil, alfalfa (Medicago sativa), rice (Oryza sativa), rye (Secale cereale), sorghum (Sorghum bicolor, Sorghum vulgare), millet (e.g., pearl millet (Pennisetum glaucum), proso millet (Panicum miliaceum), foxtail millet (Setaria italica), finger millet (Eleusine coracana)), sunflower (Helianthus annuus), safflower (Carthamus tinctorius), wheat (Triticum aestivum), soybean (Glycine max), tobacco (Nicotiana tabacum), potato (Solanum tuberosum), peanuts (Arachis hypogaea), cotton (Gossypium barbadense, Gossypium hirsutum), sweet potato (Ipomoea batatas), cassava (Manihot esc
  • genus Lemna L. aequinoctialis, L. disperma, L. ecuadoriensis, L. gibba, L. japonica, L. minor, L. miniscula, L. obscura, L. perpusilla, L. tenera, L. trisulca, L. turionifera, L. valdiviana
  • genus Spirodela S. intermedia, S. polyrrhiza, S.punctata
  • genus Woffia Wa. angusta, Wa. arrhiza, Wa. australina, Wa. borealis, Wa. brasiliensis, Wa. columbiana, Wa.
  • Lemnaceae Any other genera or species of Lemnaceae, if they exist, are also aspects ofthe present invention. Lemna gibba, Lemna minor, and Lemna miniscula are preferred, with Lemna minor and Lemna miniscula being most preferred.
  • Lemna species can be classified using the taxonomic scheme described by Landolt, Biosystematic Investigation on the Family of Duckweeds: The family of Lemnaceae - A Monograph Study. Geobatanischen Institut ETH, founded Rubel, Zurich (1986)); vegetables including tomatoes (Lycopersicon esculentum), lettuce (e.g., Lactuca sativa), green beans (Phaseolus vulgaris), lima beans (Phaseolus limensis), peas (Lathyrus spp.), and members ofthe genus Cucumis such as cucumber (C. sativus), cantaloupe (C. cantalupensis), and musk melon (C. melo).
  • tomatoes Loxicon esculentum
  • lettuce e.g., Lactuca sativa
  • green beans Phaseeolus vulgaris
  • lima beans Phaseeolus limensis
  • peas Lathyrus spp.
  • Ornamentals include azalea (Rhododendron spp.), hydrangea (M ⁇ crophyll ⁇ hydrangea), hibiscus (Hibiscus rosasanensis), roses (Rosa spp.), tulips (Tulipa spp.), daffodils (Narcissus spp.), petunias (Petunia hybrida), carnation (Dianthus caryophyllus), poinsettia (Euphorbia pulcherrima), and chrysanthemum.
  • Conifers that may be employed in practicing the present invention include, for example, pines such as loblolly pine (Pinus t ⁇ ed ⁇ ), slash pine (Pinus elliotii), ponderosa pine (Pinus ponderosa), lodgepole pine (Pinus contorta), and Monterey pine (Pinus radiata), Douglas-fir (Pseudotsuga menziesii); Western hemlock (Tsuga canadensis); Sitka spruce (Picea glauca); redwood (Sequoia sempervirens); true firs such as silver fir (Abies amabilis) and balsam fir (Abies balsamea); and cedars such as Western red cedar (Thuja plicata) and Alaska yellow-cedar (Chamaecyparis nootkatensis).
  • pines such as loblolly pine (Pinus t ⁇ ed ⁇ ), slash pine (P
  • Leguminous plants include beans and peas.
  • Beans include guar, locust bean, fenugreek, soybean, garden beans, cowpea, mungbean, lima bean, fava bean, lentils, chickpea, etc.
  • Legumes include, but are not limited to, Arachis, e.g., peanuts, Vicia, e.g., crown vetch, hairy vetch, adzuki bean, mung bean,, and chickpea, Lupinus, e.g., lupine, trifolium, Phaseolus, e.g., common bean and lima bean, Pisum, e.g., field bean, Melilotus, e.g., clover, Medicago, e.g., alfalfa, Lotus, e.g., trefoil, lens, e.g., lentil, and false indigo, Acacia, aneth, artichoke, arugula, blackberry, canola, cilantro, Clementines, escarole, eucalyptus, fennel, grapefruit, honey dew, jicama, kiwifruit, lemon, lime, mushroom, nut, okra,
  • the transformed plants include, but are not limited to, transformed wheat, com (maize), rice, oat, barley, millet, rye, rape and alfalfa, as well as asparagus, tomato, egg plant, apple, pear, quince, cherry, apricot, pepper, melon, lettuce, cauliflower, Brassica, e.g., broccoli, cabbage, brussels sprout, sugar beet, sugar cane, sweetcorn, onion, carrot, leek, cucumber, tobacco, aubergine, beet, broad bean, carrot, celery, chicory, cotton, radish, pumpkin, hemp, buckwheat, orchardgrass, creeping bent top, redtop, ryegrass, tobacco, turfgrass, tall fescue, cow pea, endive, gourd, grape, raspberry, chenopodium, blueberry, pineapple, avocado, mango, banana, groundnut, nectarine, papaya, garlic, pea, peach, peanut, pepper, pineapple
  • assays include, for example, “molecular biological” assays well known to those of skill in the art, such as Southern and Northern blotting, in situ hybridization and nucleic acid-based amplification methods such as PCR or RT-PCR; "biochemical” assays, such as detecting the presence ofa protein product, e.g., by immunological means (ELISAs and Western blots) or by enzymatic function; plant part assays, such as leaf or root assays; and also, by analyzing the phenotype of the whole regenerated plant, e.g., for disease or pest resistance.
  • DNA may be isolated from cell lines or any plant parts to determine the presence ofthe preselected nucleic acid segment through the use of techniques well known to those skilled in the art. Note that intact sequences will not always be present, presumably due to rearrangement or deletion of sequences in the cell.
  • nucleic acid elements introduced through the methods of this invention may be determined by polymerase chain reaction (PCR). Using this technique discreet fragments of nucleic acid are amplified and detected by gel electrophoresis. This type of analysis permits one to determine whether a preselected nucleic acid segment is present in a stable transformant, but does not prove integration ofthe introduced preselected nucleic acid segment into the host cell genome. In addition, it is not possible using PCR techniques to determine whether transformants have exogenous genes introduced into different sites in the genome, i.e., whether transformants are of independent origin. It is contemplated that using PCR techniques it would be possible to clone fragments ofthe host genomic DNA adjacent to an introduced preselected DNA segment.
  • PCR polymerase chain reaction
  • Positive proof of DNA integration into the host genome and the independent identities of transformants maybe determined using the technique of Southern hybridization. Using this technique specific DNA sequences that were introduced into the host genome and flanking host DNA sequences can be identified. Hence the Southern hybridization pattern of a given transformant serves as an identifying characteristic of that transformant. In addition it is possible through Southern hybridization to demonstrate the presence of introduced preselected DNA segments in high molecular weight DNA, i.e., confirm that the introduced preselected DNA segment has been integrated into the host cell genome.
  • the technique of Southern hybridization provides information that is obtained using PCR, e.g., the presence ofa preselected DNA segment, but also demonstrates integration into the genome and characterizes each individual transformant.
  • Both PCR and Southern hybridization techniques can be used to demonstrate transmission of a preselected DNA segment to progeny.
  • the characteristic Southern hybridization pattern for a given transformant will segregate in progeny as one or more Mendelian genes (Spencer et al., 1992); Laursen et al., 1994) indicating stable inheritance ofthe gene.
  • the nonchimeric nature ofthe callus and the parental transformants (R 0 ) was suggested by germline transmission and the identical Southern blot hybridization patterns and intensities ofthe transforming DNA in callus, R 0 plants and Ri progeny that segregated for the transformed gene.
  • RNA may only be expressed in particular cells or tissue types and hence it will be necessary to prepare RNA for analysis from these tissues.
  • PCR techniques may also be used for detection and quantitation of RNA produced from introduced preselected DNA segments. In this application of PCR it is first necessary to reverse transcribe RNA into DNA, using enzymes such as reverse transcriptase, and then through the use of conventional PCR techniques amplify the DNA. h most instances PCR techniques, while useful, will not demonstrate integrity ofthe RNA product. Further information about the nature ofthe RNA product may be obtained by Northern blotting. This technique will demonstrate the presence of an RNA species and give information about the integrity of that RNA.
  • RNA species can also be determined using dot or slot blot Northern hybridizations. These techniques are modifications of Northern blotting and will only demonstrate the presence or absence of an RNA species. While Southern blotting and PCR may be used to detect the preselected DNA segment in question, they do not provide information as to whether the preselected DNA segment is being expressed. Expression may be evaluated by specifically identifying the protein products ofthe introduced preselected DNA segments or evaluating the phenotypic changes brought about by their expression.
  • Assays for the production and identification of specific proteins may make use of physical-chemical, structural, functional, or other properties ofthe proteins.
  • Unique physical- chemical or structural properties allow the proteins to be separated and identified by electrophoretic procedures, such as native or denaturing gel electrophoresis or isoelectric focussing, or by chromatographic techniques such as ion exchange or gel exclusion chromatography.
  • the unique structures of individual proteins offer opportunities for use of specific antibodies to detect their presence in formats such as an ELISA assay. Combinations of approaches may be employed with even greater specificity such as Western blotting in which antibodies are used to locate individual gene products that have been separated by electrophoretic techniques. Additional techniques may be employed to absolutely confirm the identity ofthe product of interest such as evaluation by amino acid sequencing following purification. Although these are among the most commonly employed, other procedures may be additionally used.
  • Assay procedures may also be used to identify the expression of proteins by their functionality, especially the ability of enzymes to catalyze specific chemical reactions involving specific substrates and products. These reactions may be followed by providing and quantifying the loss of substrates or the generation of products ofthe reactions by physical or chemical procedures. Examples are as varied as the enzyme to be analyzed.
  • bioassays Very frequently the expression of a gene product is determined by evaluating the phenotypic results of its expression. These assays also may take many forms including but not limited to analyzing changes in the chemical composition, mo ⁇ hology, or physiological properties ofthe plant. Mo ⁇ hological changes may include greater stature or thicker stalks. Most often changes in response of plants or plant parts to imposed treatments are evaluated under carefully controlled conditions termed bioassays.
  • an expression cassette ofthe invention may be propagated in that species or moved into other varieties ofthe same species, particularly including commercial varieties, using traditional breeding techniques.
  • Particularly preferred plants ofthe invention include the agronomically important crops listed above.
  • the genetic properties engineered into the transgenic seeds and plants described above are passed on by sexual reproduction and can thus be maintained and propagated in progeny plants.
  • the present invention also relates to a transgenic plant cell, tissue, organ, seed or plant part obtained from the transgenic plant. Also included within the invention are transgenic descendants ofthe plant as well as transgenic plant cells, tissues, organs, seeds and plant parts obtained from the descendants.
  • the expression cassette in the transgenic plant is sexually transmitted.
  • the coding sequence is sexually transmitted through a complete normal sexual cycle ofthe R0 plant to the Rl generation.
  • the expression cassette is expressed in the cells, tissues, seeds or plant of a transgenic plant in an amount that is different than the amount in the cells, tissues, seeds or plant of a plant which only differs in that the expression cassette is absent.
  • the transgenic plants produced herein are thus expected to be useful for a variety of commercial and research pu ⁇ oses.
  • Transgenic plants can be created for use in traditional agriculture to possess traits beneficial to the grower (e.g., agronomic traits such as resistance to water deficit, pest resistance, herbicide resistance or increased yield), beneficial to the consumer ofthe grain harvested from the plant (e.g., improved nutritive content in human food or animal feed), or beneficial to the food processor (e.g., improved processing traits), h such uses, the plants are generally grown for the use of their grain in human or animal foods.
  • other parts ofthe plants including stalks, husks, vegetative parts, and the like, may also have utility, including use as part of animal silage or for ornamental proposes.
  • chemical constituents e.g., oils or starches
  • transgenic plants may be created which have enhanced or modified levels of such components.
  • Transgenic plants may also find use in the commercial manufacture of proteins or other molecules, where the molecule of interest is extracted or purified from plant parts, seeds, and the like.
  • Cells or tissue from the plants may also be cultured, grown in vitro, or fermented to manufacture such molecules.
  • the transgenic plants may also be used in commercial breeding programs, or may be crossed or bred to plants of related crop species. Improvements encoded by the expression cassette may be transferred, e.g., from maize cells to cells of other species, e.g., by protoplast fusion.
  • the transgenic plants may have many uses in research or breeding, including creation of new mutant plants through insertional mutagenesis, in order to identify beneficial mutants that might later be created by traditional mutation and selection.
  • An example would be the introduction of a recombinant DNA sequence encoding a transposable element that may be used for generating genetic variation.
  • the methods ofthe invention may also be used to create plants having unique "signature sequences" or other marker sequences which can be used to identify proprietary lines or varieties.
  • the transgenic plants and seeds according to the invention can be used in plant breeding which aims at the development of plants with improved properties conferred by the expression cassette, such as tolerance of viruses or other pests, or other stresses.
  • the various breeding steps are characterized by well-defined human intervention such as selecting the lines to be crossed, directing pollination ofthe parental lines, or selecting appropriate descendant plants. Depending on the desired properties different breeding measures are taken.
  • the relevant techniques are well known in the art and include but are not limited to hybridization, inbreeding, backcross breeding, multiline breeding, variety blend, interspecific hybridization, aneuploid techniques, etc.
  • Hybridization techniques also include the sterilization of plants to yield male or female sterile plants by mechanical, chemical or biochemical means. Cross pollination of a male sterile plant with pollen of a different line assures that the genome ofthe male sterile but female fertile plant will unifonnly obtain properties of both parental lines.
  • transgenic seeds and plants according to the invention can be used for the breeding of improved plant lines which for example increase the effectiveness of conventional methods such as herbicide or pesticide treatment or allow to dispense with said methods due to their modified genetic properties.
  • new crops with improved stress tolerance can be obtained which, due to their optimized genetic "equipment", yield harvested product of better quality than products which were not able to tolerate comparable adverse developmental conditions.
  • High protein com lines Wil500, WU578, WIO4605, control lines (WICY530 and LH59) and a segregating population derived from cross LH59XWIL578 (total of 53 lines) were obtained from Wilson Genetics.
  • High protein maize refers to germplasm having elevated levels of protein in the seed, typically above 14.5 % in the whole kernel, above 17%> in the embryo and above 13.5% in the endosperm (see Figure 1).
  • proteomic approaches 1) extraction of proteins from tissue including total kernels, mature/developing embyros, root and leaf from 2 week old seedlings, optionally exposed to fertilizer; 2) two-dimensional (2-D) separation of proteins by size and charge using gels using isoelectric focusing (IEF) and SDS-PAGE at three pH ranges (e.g., pH 3-10, pH 4-7 and pH 7-10); 3) image analysis of silver stained gels to identify differentially expressed proteins (by visual inspection and PDQUEST software); 4) gel excision and trypsin digestion of selected protein spots;
  • the IEF strips were re-equilibrated with a solution (2% SDS, 50 mM Tris, pH 6.9, 10% glycerol and 7 mM urea), and directly applied to a BioRad 8- 16%) gradient SDS-PAGE gel for electrophoresis.
  • the resultant gels were stained with silver using a BioRad silver staining kit according to the manufacturer's recommendations. 2D PAGE profiles were laser scanned and comparative analyses were performed using PDQuest software package (BioRad) . Only spots that were present/completely absent between normal and high protein lines were selected for further analysis. Protein spots were cut out ofthe gel either manually or using the BioRad spot cutter.
  • FIG. 2 is an example of two gels, one with proteins from control maize embryos (pH 5-8, spots 13-18, panel A) and another with proteins from a high protein line (pH 5-8, spots 1-12, panel B).
  • Figures 2C and 2D are further examples, in which the arrow points to a readily identifiable difference area that contains the various forms of globulins proteins in embryo as described in the invention.
  • Figures 3 A to 3H show a subset ofthe 38 proteins which were identified using different criteria (Xcorr and dCN). This genetic information is useful for marker development for breeding pu ⁇ oses in maize and for seed protein content manipulation in cereals in general.
  • Relative gene expression levels were determined and are presented in Table 2. All three genes were up regulated in the time course of rice seed development. The gene expression levels were determined by hybridizing the rice mRNA isolated at various developmental stages to an Affymetrix gene chip containing rice gene sequences. The rice genechip covered about 20,000 rice genes. A similar pattern of gene experession during corn seed maturation si expected.
  • Taqman analysis for key candidate gene expression was performed as follows. For one step RT-PCR amplification, total RNA was used in a 50 ml reaction using the master mixture of a Taq-Man One-Step RT-PCR Mix Reagnets (cat # 4309169, lot# 0006014) (PE Biosystems, Foster City, CA), following the manufacturer's protocol. The one step RT-PCR was conducted with an ABI Prism* 7900 HT Sequence Detection System (AB Applied Biosystems, Foster City, CA). The reactions were incubated for 30 min at 48° C for reverse transcription, and for 40 cycles of 15 s at 95° C, 60 s at 60° C for amplification. The ramp rate was set at 100% between two different temperature set points.
  • 50 ml Reaction was composed of 6.25 ml of 2 mM forward primer, AtTRX3-F (gtgtggaaatgacacagattgtga), 6.25 ml of 2 mM reverse primer, AtTRX3-R (agacgggtgcaatgaaacg), 6.25 ml of 2 mM TaqMan probe (6FAM- agacttcactgcaacatggtgcccac-TAMRA), AtTRX3_TaqMan, 1.25 ml of 40x MultiScribe & Rnase inhibitor Mix, 5 ml of template RNA (50 ng total RNA), and 25 ml of Master Mix w/o UNG (Taq-Man One-Step RT-PCR Mix Reagnets: cat # 4309169, lot# 0006014) (PE Biosystems, Foster City, CA).
  • Modulation of high protein trait by genes ofthe invention is readily determined using plant transformation sytems as described herein and as known in the art.
  • the Gateway cloning system was used to introduce genes ofthe invention into agrotransformation vectors for cereals, with seed specific promoters. See Figures 4A and 4B.
  • the embryo specific promoter is a globulin promoter
  • the ADPGPP gene promoter is used as the endosperm specific promoter. Use of these promoter constructs allows ease of cloning various genes under the control of these promoter to overexpress and/or downregulate the expression of these genes.
  • Gateway System cloning of ⁇ OPT003 & pOPT004 was as follows. Two oligos (NJ001 for & NJ002rev) were designed to amplify the Gateway Cassette A. These oligos contain restriction enzymes (Bel I and Spe I) to clone into Xba I and BamH I sites ofthe pNON4000 and p ⁇ OV4002 vector (note that Xba I is compatible with Spe I site and Bel I is compatible with BamH I site). The sGFP-M5 gene ofthe pNOV4000 and pNOV4002 plasmid is replaced with the Gateway cassette A in which we generated pOPTOOl and pOPT002 vector.
  • pOPTOOl, pOPT002 and pNOV2117 (agro) vector were digested and ligated with Kpn I and Hind in sites. The final products were transformed into DB3.1 E.Coli cells, and the pOPT003 and pOPT004 vectors were generated, as shown in the figures.
  • Protein determination was done as follows. For green house generated materials, seed protein were determined by elemental analysis, nitrogen to caculate the total protein yield, using conversion factor 6.25. A N/protein analyzer, FLASH EA 1112 Series , from CE Instruments were used in our experiment. Protein content for field generated seed materials were determined by NTR (Near Infrared) analysis.
  • Example 6 5 Vector construction for overexpression and gene "knockout” experiments.
  • Nectors used for expression of full-length genes ofthe embodiments ofthe invention of interest in plants are designed to overexpress the protein of interest and are of two general types, biolistic and binary, depending on the plant transformation method to be ) used.
  • biolistic transformation For biolistic vectors, the requirements are as follows:
  • a gene expression cassette consisting of a promoter (eg. ZmUBIint MOD), the gene of interest (typically, a full-length cD ⁇ A) and a transcriptional terminator (eg. Agrobacterium tumefaciens nos terminator);
  • a plant selectable marker cassette consisting of a promoter (eg. rice ActlD-BN MOD), selectable marker gene (eg. phosphomannose isomerase, PMI) and
  • transcriptional terminator eg. CaMV terminator
  • Vectors designed for transformation by Agrobacterium tumefaciens consist of:
  • Vectors designed for reducing or abolishing expression ofa single gene or of a family or related genes are also of two general types corresponding to the methodology used to downregulate gene expression: antisense or double-stranded RNA interference (dsRNAi).
  • dsRNAi double-stranded RNA interference
  • a full-length or partial gene fragment (typically, a portion ofthe cDNA) can be used in the same vectors described for full-length expression, as part ofthe gene expression cassette.
  • the coding region ofthe gene or gene fragment will be in the opposite orientation relative to the promoter; thus, mRNA will be made from the non-coding (antisense) strand inplanta.
  • dsRNAi vectors For dsRNAi vectors, a partial gene fragment (typically, 300 to 500 basepairs long) is used in the gene expression cassette, and is expressed in both the sense and antisense orientations, separated by a spacer region (typically, a plant intron, eg. the OsSHl intron 1, or a selectable marker, eg. conferring kanamycin resistance).
  • a spacer region typically, a plant intron, eg. the OsSHl intron 1, or a selectable marker, eg. conferring kanamycin resistance.
  • Nectors of this type are designed to form a double-stranded mR ⁇ A stem, resulting from the basepairing ofthe two complementary gene fragments inplanta.
  • Biolistic or binary vectors designed for overexpression or knockout can vary in a number of different ways, including eg. the selectable markers used in plant and bacteria, the transcriptional terminators used in the gene expression and plant selectable marker cassettes, and the methodologies used for cloning in gene or gene fragments of interest (typically, conventional restriction enzyme-mediated or GatewayTM recombinase-based cloning).
  • An important variant is the nature ofthe gene expression cassette promoter driving expression of the gene or gene fragment of interest in most tissues ofthe plants (constitutive, eg. ZmUBIint MOD), in specific plant tissues (eg. maize ADP-gpp for endosperm-specific expression), or in an inducible fashion (eg. GAL4bsBzl for estradiol-inducible expression in lines constitutively expressing the cognate transcriptional activator for this promoter).
  • a validated rice cD ⁇ A clone such as the OsPTll cD ⁇ A prepared in Example 14 above, in pCR2.1-TOPO is subcloned using conventional restriction enzyme-based cloning into a vector, downstream ofthe maize ubiquitin promoter and intron, and upstream ofthe Agrobacterium tumefaciens nos 3' end transcriptional terminator.
  • the resultant gene expression cassette (promoter, gene ofthe embodiments ofthe invention and terminator) is further subcloned, using conventional restriction enzyme-based cloning, into the pNON2117 binary vector, generating p ⁇ OVCA ⁇ D.
  • the p ⁇ OVCA ⁇ D binary vector is designed for transformation and over-expression of the gene ofthe embodiments ofthe invention in monocots. It consists of a binary backbone containing the sequences necessary for selection and growth in Escherichia coli DH-5 ⁇ (Invitrogen) and Agrobacterium tumefaciens LBA4404, including the bacterial spectinomycin antibiotic resistance aadA gene from E. coli transposon Tn7, origins of replication for E. coli (ColEl) and A. tumefaciens (VS1), and the A. tumefaciens virG gene.
  • p ⁇ OV2117 contains the T-DNA portion flanked by the right and left border sequences, and including the PositechTM (Syngenta) plant selectable marker and the gene ofthe embodiments ofthe invention gene expression cassette.
  • the PositechTM plant selectable marker confers resistance to mannose and in this instance consists ofthe maize ubiquitin promoter driving expression ofthe PMI (phosphomannose isomerase) gene, followed by the cauliflower mosaic virus transcriptional terminator.
  • pNONCAND is transformed into a rice cultivar (Kaybonnet) using Agrobacterium-mediated transformation, and mannose- resistant calli are selected and regenerated.
  • Agrobacterium is grown on YPC solid plates for 2-3 days prior to experiment initiation. Agrobacterial colonies are suspended in liquid MS media to an OD of 0.2 at ⁇ 600nm. Acetosyringone is added to the agrobacterial suspension to a concentration of 200 ⁇ M and agro is induced for 30min.
  • Co-Cultivation is continued for 2 days in the dark at 22°C.

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Abstract

L'invention porte sur des procédés visant à identifier des gènes de plantes associés à un phénotype protéagineux, ainsi que sur les gènes identifiés par ces procédés et sur leur utilisation.
EP02773582A 2001-09-26 2002-09-26 Genes de plantes associes a un phenotype proteagineux Withdrawn EP1576163A4 (fr)

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See also references of WO03027249A2 *

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