WO2008135603A2 - Over-expression of maize cox viia subunit for enhanced yield - Google Patents

Over-expression of maize cox viia subunit for enhanced yield Download PDF

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Publication number
WO2008135603A2
WO2008135603A2 PCT/EP2008/055709 EP2008055709W WO2008135603A2 WO 2008135603 A2 WO2008135603 A2 WO 2008135603A2 EP 2008055709 W EP2008055709 W EP 2008055709W WO 2008135603 A2 WO2008135603 A2 WO 2008135603A2
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plant
cox
vila
nucleotide sequence
subunit
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PCT/EP2008/055709
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French (fr)
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WO2008135603A3 (en
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Wesley B Bruce
Rajeev Gupta
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Cropdesign N.V.
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    • 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
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0053—Oxidoreductases (1.) acting on a heme group of donors (1.9)
    • 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 invention relates to the field of molecular biology. More specifically, this invention pertains to increased yield in a plant conferred by expression of a COX Vila subunit polypeptide.
  • Cereal grain development can be divided into two main stages, grain development and grain filling.
  • the first stage grain enlargement, involves early, rapid division of the zygote and nucleus. Cell division is followed by the influx of water, which drives cell extension (Briarty, et al., (1979) Annals of Botany 44:641-658).
  • the second stage grain filling
  • cell division slows and then ceases and storage products are accumulated, until maturity when the endosperm serves its function as a starch store (Briarty, et al., (1979) Annals of Botany 44:641-658; and Bewley and Black (1994) Seeds: Physiology of Development and Germination (2d ed.
  • ADPG ADPglucose
  • the mitochondrial respiratory chain which consists of multiple transmembrane protein complexes, is responsible for ATP synthesis. Electron flow within these transmembrane complexes leads to the transport of protons across the inner mitochondrial membrane and the generation of a membrane potential. ATP is synthesized when protons flow back to the mitochondrial matrix through a channel in an ATP-synthesizing complex. Given the complexity of the ATP synthesis pathway, it is likely that synchronous improvement in different components of the whole pathway may allow increased ATP production, and lead to grain yield improvements.
  • compositions and methods for increasing yield of a plant include expression cassettes comprising operably linked coding sequences for a cytochrome c oxidase (COX) Vila subunit polypeptide (COX Vila subunit), or a biologically active fragment thereof having COX Vila subunit activity, and an operably linked promoter.
  • COX cytochrome c oxidase
  • COX Vila subunit cytochrome c oxidase
  • a biologically active fragment thereof having COX Vila subunit activity or an operably linked promoter.
  • the COX Vila subunit polypeptide is the Zea mays (Zm) COX Vila subunit polypeptide set forth in SEQ ID NO: 2
  • the expression cassette comprises the coding sequence for the Zm COX Vila subunit polypeptide as set forth in nucleotides 81-281 of SEQ ID NO: 1 or in SEQ ID NO: 3, or a nucleotide sequence encoding the COX Vila subunit polypeptide set forth in SEQ ID NO: 2.
  • the expression cassette comprises the coding sequence for a biologically active fragment of a COX Vila subunit polypeptide.
  • the operably linked promoter can be any promoter that drives expression in a plant cell, including, but not limited to, a constitutive promoter, an inducible promoter, or a tissue-preferred promoter. Further provided are plants, plant cells, and plant seeds comprising these expression cassettes. In some embodiments, the expression cassettes are stably incorporated into the genomes of the plants, plant cells, and plant seeds. These expression cassettes find use in the methods of the invention to increase plant yield, particularly seed yield of crop plants as described herein.
  • Methods of the invention comprise increasing the expression levels of a COX Vila subunit polypeptide within the cells of a plant, particularly expression of a Zm COX Vila subunit polypeptide, or biologically active fragment thereof having COX Vila subunit activity, thereby increasing yield in the plant.
  • the methods comprise introducing into a plant of interest at least one expression cassette of the invention, comprising a nucleotide sequence that includes a coding sequence for a COX Vila subunit polypeptide, or a biologically active fragment thereof, where the coding sequence is operably linked to a promoter that drives expression in a plant cell.
  • the methods of the invention find use in producing plants having a phenotype that contributes to increased yield, including, but not limited to, increased total seed number, increased total seed weight, increased number of seeds filled, increased harvest index, and combinations thereof.
  • the Zm COX Vila subunit promoter sequence is useful for driving expression of polynucleotides of interest in a plant.
  • Figure 1 shows an alignment of the maize COX VIIa subunit protein sequence (SEQ ID NO: 2) with the Triticum aestivum (SEQ ID NO: 5), Oryza sativa (SEQ ID NO: 6), Sorghum sp. (SEQ ID NO: 7), Arabidopsis thaliana (SEQ ID NO: 8), Cyamopsis tetragonolobus (SEQ ID NO: 9), Lilium longiflorum (SEQ ID NO: 10), and Glycine max (SEQ ID NO: 11 ) COX Vila subunit protein sequences.
  • the three underlined domains refer to the alpha-helix, turn and transmembrane domains, respectively.
  • a consensus sequence (SEQ ID NO: 12) is also shown.
  • the present invention provides methods and compositions for increasing the level of a COX Vila subunit in a plant, thereby increasing yield in a plant or plant part thereof, compared to a wild-type or control plant.
  • the COX Vila subunit is a member of the COX complex in mitochondrial membranes involved in oxidizing the reduced form of cytochrome c, which then contributes to the proton-motive gradient used to drive ATP synthesis in the cell.
  • Methods of the invention comprise genetically altering a plant to express or overexpress a COX Vila subunit or a biologically active fragment or variant thereof. Increasing expression of the COX Vila subunit or fragment or variant thereof within the cells of a plant, particularly the vegetative cells, results in a plant with increased yield.
  • Increasing expression means an increase in the amount of COX Vila subunit mRNA or polypeptide.
  • increased expression includes, but is not limited to, at least a 20% increase in the amount of COX Vila subunit mRNA or polypeptide within the cells of a plant as compared to the endogenous COX Vila subunit mRNA or polypeptide, such as, but not limited to, at least a 30%, 50%, 75%, 100% or 200% increase of COX Vila subunit mRNA or polypeptide.
  • increased yield is intended increased total seed number, increased total seed weight, increased number of seeds filled, increased harvest index (i.e., the ratio of grain weight to total plant weight), or any combination thereof.
  • a COX Vila subunit or a biologically active fragment or variant thereof is a polypeptide that has COX Vila subunit activity.
  • the COX Vila subunit is important in stabilizing the integrity of the COX complex, as well as playing a regulatory role in the oxidation of cytochrome c (Poyton and McEwen (1996) Annu. Rev. Biochem. 65:563-607).
  • the COX Vila subunit may be a target for greater selective pressure for specific isoforms due to high energy needs of certain tissues or organisms, suggesting that this Vila subunit may be one of the rate-limiting subunits for COX activity (Jobson, et al., (2004) Proc. Natl.
  • a COX Vila subunit or a biologically active fragment or variant thereof that has COX Vila subunit activity increases yield in a plant, at least for total seed number, total seed weight, number of seeds filled, harvest index, and combinations thereof. Other increases in plant yield are also possible.
  • a polypeptide that has COX Vila subunit activity is referred to as a "COX Vila subunit,” a “COX Vila subunit polypeptide,” or a “COX Vila subunit protein,” and a polynucleotide that encodes a polypeptide that has COX Vila subunit activity is referred to as a "COX Vila subunit polynucleotide.”
  • COX Vila subunit polynucleotides for use in the methods of the present invention include, for example, the coding sequence of the Zm COX Vila subunit ortholog as set forth in nucleotides 81-281 of SEQ ID NO: 1 or in SEQ ID NO: 3, sequences encoding the Zm COX Vila subunit protein set forth in SEQ ID NO: 2, and fragments and variants thereof as defined below.
  • COX Vila subunit polypeptides of the present invention include, for example, the Zm COX Vila subunit protein set forth in SEQ ID NO: 2 and biologically active fragments and variants thereof as defined herein below.
  • the Zm COX Vila genomic sequence is set forth in SEQ ID NO: 4.
  • the Zm COX Vila promoter sequence is set forth as nucleotides 1 through 1012 of SEQ ID NO: 4.
  • the Zm COX Vila subunit protein (SEQ ID NO: 2) shares a high degree of sequence identity with COX Vila subunit proteins isolated from other plant species, including, for example, COX Vila subunit proteins from Triticum aestivum (SEQ ID NO: 5), Oryza sativa (SEQ ID NO: 6), Sorghum sp. (SEQ ID NO: 7), Arabidopsis thaliana (SEQ ID NO: 8), Cyamopsis tetragonolobus (SEQ ID NO: 9), Lilium longiflorum (SEQ ID NO: 10), and Glycine max (SEQ ID NO: 11 ) (see the alignment in Figure 1 ).
  • the term "increased yield” also means any improvement in the yield of any measured plant product, such as total seed number, total seed weight, number of seeds filled, harvest index, and the like.
  • the increase in yield can comprise a 0.1%, 0.5%, 1%, 3%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or greater increase in measured plant product.
  • the increased plant yield can comprise about a 0.5 fold, 1 fold, 2 fold, 4 fold, 8 fold, 16 fold or 32 fold increase in measured plant products.
  • an increase in the total seed weight of rice or corn from a transgenic crop comprising the COX Vila subunit polypeptide, as compared to the total seed weight from rice or corn without the COX Vila subunit polypeptide, cultivated under the same conditions, would be considered an increased yield.
  • the nutritional value of a crop plant can be increased.
  • the methods of the present invention comprise increasing the expression of a COX Vila subunit in plants, particularly expression of the Zm COX Vila subunit or biologically active fragment or variant thereof having COX Vila subunit activity.
  • the methods comprise introducing into a plant of interest at least one nucleotide construct comprising a nucleotide sequence encoding the COX Vila subunit protein or a biologically active fragment or variant thereof operably linked to a promoter that drives expression in a plant cell.
  • the COX Vila subunit is the coding sequence of the Zm COX Vila subunit ortholog as set forth in nucleotides 81-281 of SEQ ID NO: 1 or in SEQ ID NO: 3, or biologically active fragment or variant thereof and the plant is a monocot, such as maize or rice.
  • COX Vila subunit for example, the Zm COX Vila subunit protein or biologically active fragment or variant thereof, within a plant increases yield in the plant.
  • Zm COX Vila subunit coding sequence, and fragments and variants thereof, find particular use in increasing yield in a monocot plant, for example maize or rice, as this COX Vila subunit has evolved to function within the monocot cellular environment.
  • an "isolated” or “purified” polynucleotide or protein, or biologically active portion thereof, is substantially or essentially free from components that normally accompany or interact with the polynucleotide or protein as found in its naturally occurring environment.
  • an isolated or purified polynucleotide or protein 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" polynucleotide is free of sequences (optimally protein encoding sequences) that naturally flank the polynucleotide (i.e., sequences located at the 5' and 3' ends of the polynucleotide) in the genomic DNA of the organism from which the polynucleotide is derived.
  • the isolated polynucleotide can contain less than about 5 kb, 4 kb, 3 kb,
  • a protein that is substantially free of cellular material includes preparations of protein having less than about 30%, 20%, 10%, 5% or 1% (by dry weight) of contaminating protein.
  • optimally culture medium represents less than about 30%, 20%, 10%, 5% or 1% (by dry weight) of chemical precursors or non-protein-of-interest chemicals.
  • fragments and variants of COX Vila subunit polynucleotides and polypeptides encoded thereby are also encompassed by the present invention.
  • fragment refers to a portion of the polynucleotide or a portion of the amino acid sequence and hence protein encoded thereby.
  • Fragments of a polynucleotide may encode protein fragments that retain the biological activity of the original protein and hence confer COX Vila subunit activity.
  • fragments of a nucleotide sequence may range from at least about 20 nucleotides, about 50 nucleotides, about 100 nucleotides, and up to the full-length polynucleotide encoding a COX Vila subunit polypeptide.
  • a fragment of a COX Vila subunit polynucleotide that encodes a biologically active portion of a COX Vila subunit polypeptide will encode at least 15, 25, 30 or 50 contiguous amino acids, or up to the total number of amino acids present in a full-length COX Vila subunit polypeptide (for example, 67 amino acids for the Zm COX Vila subunit polypeptide of SEQ ID NO: 2).
  • a biologically active portion of a COX Vila subunit polypeptide can be prepared by isolating a portion of a COX Vila subunit polynucleotide, expressing the encoded portion of the COX Vila subunit polypeptide (e.g., by recombinant expression in vitro), and assessing the activity of the encoded portion of the COX Vila subunit polypeptide.
  • Polynucleotides that are fragments of a COX Vila subunit polynucleotide comprise at least 16, 20, 50, 75, 100, 150, 200, 250, 300, 350, 400 or 450 contiguous nucleotides, or up to the number of nucleotides present in a full- length COX Vila subunit polynucleotide (for example, 485 contiguous nucleotides for the Zm COX Vila subunit nucleotide sequence of SEQ ID NO: 1 or 201 contiguous nucleotides for the Zm COX Vila subunit coding sequence of SEQ ID NO: 3).
  • variants refers to substantially similar sequences.
  • a variant comprises a polynucleotide having deletions (i.e., truncations) at the 5' and/or 3' end; deletion and/or addition of one or more nucleotides at one or more internal sites in the native polynucleotide; and/or substitution of one or more nucleotides at one or more sites in the native polynucleotide.
  • a "native" polynucleotide or polypeptide comprises a naturally occurring nucleotide sequence or amino acid sequence, respectively.
  • conservative variants include those sequences that, because of the degeneracy of the genetic code, encode the amino acid sequence of a COX Vila subunit polypeptide, for example, the Zm COX Vila subunit of SEQ ID NO: 2.
  • 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 polynucleotides also include synthetically derived polynucleotides, such as those generated, for example, by using site-directed mutagenesis or "shuffling.”
  • variants of a particular polynucleotide for example, the Zm COX Vila subunit sequence set forth in SEQ ID NO: 1 , or the Zm COX Vila subunit coding sequence set forth in nucleotides 81-281 of SEQ ID NO: 1 or in SEQ ID NO: 3 have at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to that particular polynucleotide as determined by sequence alignment programs and parameters as described elsewhere herein.
  • Variants of a particular polynucleotide can also be evaluated by comparison of the percent sequence identity between the polypeptide encoded by a variant polynucleotide and the polypeptide encoded by the reference polynucleotide.
  • the variant of a COX Vila subunit polynucleotide is an isolated polynucleotide that encodes a COX Vila subunit polypeptide having a given percent identity to the Zm COX Vila subunit polypeptide of SEQ ID NO: 2. Percent sequence identity between any two polypeptides can be calculated using sequence alignment programs and parameters described elsewhere herein.
  • the percent sequence identity between the two encoded polypeptides is at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity.
  • Variant protein is intended to mean a protein derived from a native and/or original protein by deletion (so-called truncation) of one or more amino acids at the N-terminal and/or C-terminal end of the protein; deletion and/or addition of one or more amino acids at one or more internal sites in the protein; or substitution of one or more amino acids at one or more sites in the protein.
  • Variant proteins encompassed by the present invention are biologically active, that is they continue to possess the desired COX Vila subunit activity as described herein (i.e., increasing yield in a plant, at least for total seed number, total seed weight, number of seeds filled, harvest index, and combinations thereof).
  • Biologically active variants of a COX Vila subunit polypeptide will have at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the amino acid sequence for the native protein as determined by sequence alignment programs and parameters described elsewhere herein.
  • a biologically active variant of a COX Vila subunit polypeptide may differ from that polypeptide by as few as 1-15 amino acid residues, as few as 1-10, such as 6-10, as few as 5, as few as 4, 3, 2 or even 1 amino acid residue.
  • COX Vila subunit polypeptides for use in practicing the invention may be 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 and fragments of the Zm COX Vila subunit protein of SEQ ID NO: 2 can be prepared by mutations in the encoding polynucleotide, for example, the sequence set forth in SEQ ID NO: 1 , or the coding sequence set forth in nucleotides 81-281 of SEQ ID NO: 1 or in SEQ ID NO: 3.
  • Methods for mutagenesis and polynucleotide alterations are well known in the art. See, for example, Kunkel (1985) Proc. Natl.
  • COX Vila subunit polypeptide can be evaluated by routine screening assays (see, e.g., Aggeler and Capaldi (1990) J. Biol. Chem. 265:16389-93; Calder and McEwen (1991 ) MoI. Microbiol. 5:1769-77), including the assays disclosed herein below.
  • Variant COX Vila subunit polynucleotides and COX Vila subunits for use in the methods of the invention also encompass sequences and proteins derived from a mutagenic and recombinogenic procedure such as DNA shuffling. With such a procedure, one or more different COX Vila subunit polypeptide coding sequences can be manipulated to create a new COX Vila subunit polypeptide possessing the desired properties. In 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.
  • sequence motifs encoding a domain of interest may be shuffled between the Zm COX Vila subunit sequence of SEQ ID NO: 1 or SEQ ID NO: 3 and other known COX Vila subunit genes to obtain a new gene coding for a COX Vila subunit protein with an improved property of interest, such as a particular type of increased yield in a plant, for example, increased total seed number, increased total seed weight, increased number of seeds filled, increased harvest index, or any combination thereof.
  • Strategies for such DNA shuffling are known in the art. See, for example, Stemmer (1994) Proc. Natl. Acad. Sci.
  • compositions of the invention also include isolated nucleic acid molecules comprising the Zm COX Vila promoter nucleotide sequence set forth as nucleotides 1 through 1012 of SEQ ID NO:
  • promoter is intended a regulatory region of DNA usually comprising a TATA box capable of directing RNA polymerase Il to initiate RNA synthesis at the appropriate transcription initiation site for a particular coding sequence.
  • a promoter may additionally comprise other recognition sequences generally positioned upstream or 5' to the TATA box, referred to as upstream promoter elements, which influence the transcription initiation rate. It is recognized that having identified the nucleotide sequence for the promoter region disclosed herein, it is within the state of the art to isolate and identify additional regulatory elements in the 5'-untranslated region upstream from the particular promoter region defined herein. Thus for example, the promoter region disclosed herein may further comprise upstream regulatory elements that confer tissue-preferred expression of heterologous nucleotide sequences operably linked to the disclosed promoter sequence. See particularly, Australian Patent No.
  • Fragments and variants of the disclosed Zm COX Vila promoter nucleotide sequence are also encompassed by the present invention.
  • fragment is intended a portion of the nucleotide sequence. Fragments of a promoter nucleotide sequence may retain biological activity and hence retain their transcriptional regulatory activity. Thus, for example, less than the entire promoter sequence disclosed herein may be utilized to drive expression of an operably linked nucleotide sequence of interest, such as a nucleotide sequence encoding a heterologous protein. Alternatively, fragments of a promoter nucleotide sequence that are useful as hybridization probes generally do not retain biological activity. Thus, fragments of a promoter nucleotide sequence may range from at least about 20 nucleotides, about 50 nucleotides, about 100 nucleotides, and up to the full-length promoter nucleotide sequence of the invention.
  • a fragment of a Zm COX Vila promoter nucleotide sequence may encode a biologically active portion of the Zm COX Vila promoter, or it may be a fragment that can be used as a hybridization probe or PCR primer using methods disclosed herein.
  • a biologically active portion of a Zm COX Vila promoter can be prepared by isolating a portion of the Zm COX Vila promoter nucleotide sequence of the invention, and assessing the activity of the portion of the Zm COX Vila promoter.
  • Nucleic acid molecules that are fragments of a Zm COX Vila promoter nucleotide sequence comprise at least 16, 20, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1012 nucleotides or up to the number of nucleotides present in a full-length Zm COX Vila promoter nucleotide sequence disclosed herein (i.e., 1012 nucleotides for the Zm COX Vila promoter set forth as nucleotides 1-1012 of SEQ ID NO: 4). Assays to determine the activity of a promoter sequence are well known in the art.
  • a Zm COX Vila promoter fragment or variant may be operably linked to the nucleotide sequence encoding any reporter protein, such as the ⁇ -glucuronidase protein (GUS reporter) or the luciferase protein.
  • GUS reporter ⁇ -glucuronidase protein
  • the DNA construct is inserted into the genome of a plant or plant cell, and the mRNA or protein level of the reporter sequence is determined. See, for example, Eulgem, et al., (1999) EMBO Journal 18:4689-4699.
  • the Zm COX Vila subunit polynucleotide and Zm COX Vila promoter sequence for use in the methods of the invention can be used to isolate corresponding COX Vila subunit sequences and promoter sequences from other plants, including other monocots. In this manner, methods such as PCR, hybridization, and the like can be used to identify such sequences based on their sequence homology to the Zm COX Vila subunit sequence set forth in SEQ ID NO: 1 , the Zm COX Vila subunit coding sequence set forth in nucleotides 81-281 of SEQ ID NO: 1 or in SEQ ID NO: 3, or the Zm COX Vila promoter sequence set forth as nucleotides 1 through 1012 of SEQ ID NO: 4.
  • Sequences isolated based on their sequence identity to the entire Zm COX Vila subunit nucleotide sequence or Zm COX Vila promoter sequence set forth herein, or to variants and fragments thereof, are encompassed by the present invention.
  • Such sequences include sequences that are orthologs of the disclosed sequences.
  • "Orthologs" is intended to mean genes derived from a common ancestral gene and which are found in different species as a result of speciation. Genes found in different species are considered orthologs when their nucleotide sequences and/or their encoded protein sequences share at least 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence identity.
  • 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 and are disclosed in Sambrook, et al., (1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, New York). See also, Innis, et ai, eds. (1990) PCR Protocols: A Guide to Methods and Applications (Academic Press, New York); Innis and Gelfand, eds.
  • PCR PCR Strategies
  • nested primers single specific primers
  • degenerate primers gene-specific primers
  • vector-specific primers partially mismatched primers
  • hybridization techniques all or part of a known polynucleotide is used as a probe that selectively hybridizes to other corresponding polynucleotides 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 Zm COX Vila subunit nucleotide sequence of SEQ ID NO: 1 , or the Zm COX Vila subunit coding sequence set forth in nucleotides 81-281 of SEQ ID NO: 1 or in SEQ ID NO: 3.
  • Methods for preparation of probes for hybridization and for construction of cDNA and genomic libraries are generally known in the art and are disclosed in Sambrook, et al., (1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, New York).
  • the entire Zm COX Vila subunit polynucleotide disclosed in SEQ ID NO: 1 , nucleotides 81-281 of SEQ ID NO: 1 , or SEQ ID NO: 3, or one or more portions thereof may be used as a probe capable of specifically hybridizing to corresponding COX Vila subunit polynucleotides and messenger RNAs.
  • probes include sequences that are unique among COX Vila subunit polynucleotide sequences and are optimally at least about 10 nucleotides in length, and most optimally at least about 20 nucleotides in length.
  • Such probes may be used to amplify corresponding COX Vila subunit polynucleotides from a chosen plant by PCR. This technique may be used to isolate additional COX Vila subunit coding sequences from a desired plant or as a diagnostic assay to determine the presence of COX Vila subunit coding sequences in a plant.
  • Hybridization techniques include hybridization screening of plated DNA libraries (either plaques or colonies; see, for example, Sambrook, et al., (1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, New York).
  • Hybridization of such sequences may be carried out under stringent conditions.
  • stringent conditions or “stringent hybridization conditions” is intended conditions under which a probe will hybridize to its target sequence to a detectably greater degree than to other sequences (e.g., at least 2-fold over background).
  • Stringent conditions are sequence-dependent and will be different in different circumstances.
  • target sequences that are 100% complementary to the probe can be identified (homologous probing).
  • stringency conditions can be adjusted to allow some mismatching in sequences so that lower degrees of similarity are detected (heterologous probing).
  • a probe is less than about 1000 nucleotides in length, optimally less than 500 nucleotides in length.
  • stringent conditions will be those in which the salt concentration is less than about 1.5 M Na ion, typically about 0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30 0 C for short probes (e.g., 10 to 50 nucleotides) and at least about 60 0 C for long probes (e.g., greater than 50 nucleotides).
  • Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide.
  • Exemplary moderate stringency conditions include hybridization in 40 to 45% formamide, 1.0 M NaCI, 1 % SDS at 37°C, and a wash in 0.5X to 1X SSC at 55 to 60 0 C.
  • Exemplary high stringency conditions include hybridization in 50% formamide, 1 M NaCI, 1% SDS at 37°C, and a wash in 0.1 X SSC at 60 to 65°C.
  • wash buffers may comprise about 0.1% to about 1% SDS. Duration of hybridization is generally less than about 24 hours, usually about 4 to about 12 hours. The duration of the wash time will be at least a length of time sufficient to reach equilibrium.
  • T m 81.5 0 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 of the hybrid in base pairs.
  • the T m is the temperature (under defined ionic strength and pH) at which 50% of a complementary target sequence hybridizes to a perfectly matched probe. T m is reduced by about 1 °C for each 1 % of mismatching; thus, T m , hybridization, and/or wash conditions can be adjusted to hybridize to sequences of the desired identity. For example, if sequences with >90% identity are sought, the T m can be decreased 10 0 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.
  • sequence relationships between two or more polynucleotides or polypeptides are used to describe the sequence relationships between two or more polynucleotides or polypeptides: (a) “reference sequence”, (b) “comparison window”, (c) “sequence identity”, and, (d) “percentage of sequence 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 of a 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 of the two polynucleotides.
  • 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, Mountain View, California); the ALIGN program (Version 2.0) and GAP, BESTFIT, BLAST, FASTA, and TFASTA in the GCG Wisconsin Genetics Software Package, Version 10 (available from Accelrys Inc., 9685 Scranton Road, San Diego, California, USA). Alignments using these programs can be performed using the default parameters.
  • the CLUSTAL program is well described by Higgins, et al., (1988) Gene 73:237-244 (1988); Higgins, et al., (1989) CABIOS 5:151-153; Corpet, et al., (1988) Nucleic Acids Res. 16:10881-90; Huang, et al., (1992) CABIOS 8:155-65; and Pearson, et al., (1994) Meth. MoI. Biol. 24:307-331.
  • the ALIGN program is based on the algorithm of Myers and Miller (1988) supra. A PAM 120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used with the ALIGN program when comparing amino acid sequences.
  • Gapped BLAST in BLAST 2.0
  • PSI-BLAST in BLAST 2.0
  • the default parameters of the respective programs e.g., BLASTN for nucleotide sequences, BLASTX for proteins
  • BLAST software is publicly available on the NCBI website. Alignment may also be performed manually by inspection.
  • sequence identity/similarity values provided herein refer to the value obtained using GAP Version 10 using the following parameters: % identity and % similarity for a nucleotide sequence using GAP Weight of 50 and Length Weight of 3, and the nwsgapdna.cmp scoring matrix; % identity and % similarity for an amino acid sequence using GAP Weight of 8 and Length Weight of 2, and the BLOSUM62 scoring matrix; or any equivalent program thereof.
  • 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 GAP Version 10.
  • GAP uses the algorithm of Needleman and Wunsch (1970) J. MoI. Biol. 48:443-453, to find the alignment of two complete sequences that maximizes the number of matches and minimizes the number of gaps. GAP considers all possible alignments and gap positions and creates the alignment with the largest number of matched bases and the fewest gaps. It allows for the provision of a gap creation penalty and a gap extension penalty in units of matched bases. GAP must make a profit of gap creation penalty number of matches for each gap it inserts. If a gap extension penalty greater than zero is chosen, GAP must, in addition, make a profit for each gap inserted of the length of the gap times the gap extension penalty.
  • gap creation penalty values and gap extension penalty values in Version 10 of the GCG Wisconsin Genetics Software Package for protein sequences are 8 and 2, respectively.
  • the default gap creation penalty is 50 while the default gap extension penalty is 3.
  • the gap creation and gap extension penalties can be expressed as an integer selected from the group of integers consisting of from 0 to 200.
  • the gap creation and gap extension penalties can be 0, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65 or greater.
  • GAP presents one member of the family of best alignments. There may be many members of this family, but no other member has a better quality. GAP displays four figures of merit for alignments: Quality, Ratio, Identity, and Similarity.
  • the Quality is the metric maximized in order to align the sequences. Ratio is the quality divided by the number of bases in the shorter segment.
  • Percent Identity is the percent of the symbols that actually match.
  • Percent Similarity is the percent of the symbols that are similar. Symbols that are across from gaps are ignored.
  • a similarity is scored when the scoring matrix value for a pair of symbols is greater than or equal to 0.50, the similarity threshold.
  • the scoring matrix used in Version 10 of the GCG Wisconsin Genetics Software Package is BLOSUM62 (see, Henikoff and Henikoff (1989) Proc. Natl. Acad. Sci. USA 89:10915).
  • sequence identity or “identity” in the context of two polynucleotides or polypeptide sequences makes reference to the residues in the two sequences that are the same when aligned for maximum correspondence over a specified comparison window.
  • sequence identity or “identity” in the context of two polynucleotides or polypeptide sequences makes reference to the residues in the two sequences that are the same when aligned for maximum correspondence over a specified comparison window.
  • 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 of the molecule.
  • sequences differ in conservative substitutions the percent sequence identity may be adjusted upwards to correct for the conservative nature of the 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, for example, as implemented in the program PC/GENE (Intelligenetics, 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 of the 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 is not intended to be limited to polynucleotides comprising DNA.
  • polynucleotides can comprise ribonucleotides and combinations of ribonucleotides and deoxyribonucleotides.
  • deoxyribonucleotides and ribonucleotides include both naturally occurring molecules and synthetic analogues.
  • polynucleotides also encompass all forms of sequences including, but not limited to, single-stranded forms, double-stranded forms, hairpins, stem-and-loop structures, and the like.
  • the COX Vila subunit polynucleotide for example, the Zm COX Vila subunit polynucleotide or fragment or variant thereof, can be provided in expression cassettes for expression in the plant of interest.
  • the cassette will include 5' and 3' regulatory sequences operably linked to the COX Vila subunit polynucleotide.
  • Operably linked is intended to mean a functional linkage between two or more elements.
  • an operable linkage between a polynucleotide of interest and a regulatory sequence i.e., a promoter
  • Operably linked elements may be contiguous or non-contiguous.
  • the cassette may additionally contain at least one additional gene to be cotransformed into the plant.
  • the additional gene(s) can be provided on multiple expression cassettes.
  • Such an expression cassette is provided with a plurality of restriction sites and/or recombination sites for insertion of the COX Vila subunit polynucleotide to be under the transcriptional regulation of the regulatory regions.
  • the expression cassette may additionally contain other genes, including other selectable marker genes.
  • the expression cassette will include in the 5'-3' direction of transcription a transcriptional and translational initiation region (i.e., a promoter), a COX Vila subunit polynucleotide of the invention, for example, SEQ ID NO: 1 , nucleotides 81-281 of SEQ ID NO: 1 , SEQ ID NO: 3, or fragment or variant thereof, and a transcriptional and translational termination region (i.e., termination region) functional in plants.
  • the regulatory regions (i.e., promoters, transcriptional regulatory regions, and translational termination regions) and/or the COX Vila subunit polynucleotide may be native/analogous to the host cell or to each other.
  • the regulatory regions and/or the COX Vila subunit polynucleotide may be heterologous to the host cell or to each other.
  • heterologous in reference to a sequence is a sequence that originates from a foreign species, or, if from the same species, is substantially modified from its native form in composition and/or genomic locus by deliberate human intervention.
  • a promoter operably linked to a heterologous polynucleotide is from a species different from the species from which the polynucleotide was derived, or, if from the same/analogous species, one or both are substantially modified from their original form and/or genomic locus, or the promoter is not the native promoter for the operably linked polynucleotide. While it may be optimal to express the COX Vila subunit polynucleotides using heterologous promoters, the native promoter sequences may be used. Such constructs can change expression levels of the encoded polypeptide in the plant or plant cell. Thus, the phenotype of the plant or cell can be altered.
  • the termination region may be native with the transcriptional initiation region, may be native with the operably linked COX Vila subunit polynucleotide of interest, may be native with the plant host, or may be derived from another source (i.e., foreign or heterologous) to the promoter, the COX Vila subunit polynucleotide of interest, the plant host, or any combination thereof.
  • Convenient termination regions for use in the present invention include those available from the Ti-plasmid of A. tumefaciens, such as the octopine synthase and nopaline synthase termination regions. See also, Guerineau, et al., (1991 ) MoI. Gen. Genet.
  • Methods are known in the art for increasing expression of a polypeptide of interest in a plant or plant cell, for example, by inserting into the polypeptide coding sequence one or two G/C-rich codons (such as GCG or GCT) immediately adjacent to and downstream of the initiating methionine ATG codon.
  • G/C-rich codons such as GCG or GCT
  • the COX Vila subunit polynucleotides may be optimized for increased expression in the transformed plant. See, for example, Campbell and Gowri (1990) Plant Physiol. 92:1-1 1 for a discussion of host-preferred codon usage. Methods are available in the art for synthesizing plant-preferred genes. See, for example, U.S. Patent Nos.
  • Additional sequence modifications are known to enhance gene expression in a particular plant host. These include elimination of sequences encoding spurious polyadenylation signals, exon-intron splice site signals, transposon-like repeats, and other such well-characterized sequences that may be deleterious to gene expression.
  • the G-C content of the sequence may be adjusted to levels average for a given plant host, as calculated by reference to known genes expressed in the host cell. When possible, the sequence is modified to avoid predicted hairpin secondary mRNA structures.
  • the expression cassettes may additionally contain 5' leader sequences. Such leader sequences can act to enhance translation.
  • Translation leaders are known in the art and include: picornavirus leaders, for example, EMCV leader (Encephalomyocarditis 5' noncoding region) (Elroy-Stein, et al., (1989) Proc. Natl. Acad. Sci.
  • potyvirus leaders for example, TEV leader (Tobacco Etch Virus) (GaIMe, et ah, (1995) Gene 165(2):233- 238), MDMV leader (Maize Dwarf Mosaic Virus) ⁇ Virology 154:9-20), and human immunoglobulin heavy-chain binding protein (BiP) (Macejak, et ai, (1991 ) Nature 353:90-94); untranslated leader from the coat protein mRNA of alfalfa mosaic virus (AMV RNA 4) (Jobling, et al., (1987) Nature 325:622-625); tobacco mosaic virus leader (TMV) (GaIMe, et al., (1989) in Molecular Biology of RNA, ed.
  • TEV leader tobacco Etch Virus
  • MDMV leader Mainze Dwarf Mosaic Virus
  • BiP human immunoglobulin heavy-chain binding protein
  • AMV RNA 4 untranslated leader from the coat protein mRNA of
  • Cech (Liss, New York), pp. 237-256); and maize chlorotic mottle virus leader (MCMV) (Lommel, et al., (1991 ) Virology 81 :382-385). See also, Della-Cioppa, et al., (1987) Plant Physiol. 84:965-968.
  • MCMV chlorotic mottle virus leader
  • the various polynucleotide fragments may be manipulated, so as to provide for sequences to be in the proper orientation and, as appropriate, in the proper reading frame.
  • adapters or linkers may be employed to join the fragments or other manipulations may be involved to provide for convenient restriction sites, removal of superfluous material such as the removal of restriction sites, or the like.
  • in vitro mutagenesis, primer repair, restriction, annealing, resubstitutions, for example, transitions and transversions may be involved.
  • a number of promoters can be used in the practice of the invention, including the native promoter of the COX Vila subunit polynucleotide sequence of interest.
  • the promoters can be selected for increased expression.
  • the COX Vila subunit polynucleotides of interest can be combined with constitutive, tissue-preferred, or other promoters for expression in plants.
  • Such constitutive promoters include, for example, the core promoter of the Rsyn7 promoter and other constitutive promoters disclosed in WO 99/43838 and U.S. Patent No. 6,072,050; the core CaMV 35S promoter (Odell, et al., (1985) Nature 313:810-812); rice actin (McElroy, et al., (1990) Plant Cell 2:163-171 ); ubiquitin (UBI) (Christensen, et al., (1989) Plant MoI. Biol. 12:619-632 and Christensen, et al., (1992) Plant MoI. Biol.
  • UBI ubiquitin
  • pEMU Last, et al., (1991 ) Theor. Appl. Genet. 81 :581-588); MAS (Velten, et al., (1984) EMBO J. 3:2723-2730); ALS promoter (U.S. Patent No. 5,659,026), and the like.
  • Other constitutive promoters include, for example, those described in U.S. Patent Nos. 5,608,149; 5,608,144; 5,604,121 ; 5,569,597; 5,466,785; 5,399,680; 5,268,463; 5,608,142; and 6,177,611.
  • Tissue-preferred promoters can be utilized to target enhanced COX Vila subunit polypeptide expression within a particular plant tissue.
  • Tissue-preferred promoters include those disclosed in Yamamoto, et al., (1997) Plant J. 12(2):255-265; Kawamata, et al., (1997) Plant Cell Physiol. 38(7):792-803; Hansen, et al., (1997) MoI. Gen Genet. 254(3):337-343; Russell, et al., (1997) Transgenic Res. 6(2):157-168; Rinehart, et al., (1996) Plant Physiol. 1 12(3):1331-1341 ; Van Camp, et al., (1996) Plant Physiol.
  • the expression cassette can also comprise a selectable marker gene for the selection of transformed cells.
  • Selectable marker genes are utilized for the selection of transformed cells or tissues.
  • Marker genes include genes encoding antibiotic resistance, such as those encoding neomycin phosphotransferase Il (NEO) and hygromycin phosphotransferase (HPT), as well as genes conferring resistance to herbicidal compounds, such as glufosinate ammonium, bromoxynil, imidazolinones, and 2,4-dichlorophenoxyacetate (2,4-D).
  • Additional selectable markers include phenotypic markers such as ⁇ -galactosidase and fluorescent proteins such as green fluorescent protein (GFP) (Su, et al., (2004) Biotechnol. Bioeng. 85:610-9 and Fetter, et al., (2004) Plant Cell 16:215-28), cyanofluorescent protein (CYP) (Bolte, et al., (2004) J. Cell Science 117:943-54 and Kato, et al., (2002) Plant Physiol 129:913-42), and yellow fluorescent protein (PhiYFPTM from Evrogen, see, Bolte, et al., (2004) J. Cell Science 117:943-54).
  • GFP green fluorescent protein
  • CYP cyanofluorescent protein
  • the present invention also provides a method for increasing the concentration and/or activity of a COX Vila subunit polypeptide, for example, the Zm COX Vila subunit protein of SEQ ID NO: 2 or biologically active fragment or variant thereof, in a plant.
  • concentration and/or activity is increased by at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% relative to a wild-type or control plant, plant part, or cell that did not have a COX Vila subunit sequence of the invention introduced.
  • Increasing the concentration and/or activity of a COX Vila subunit polypeptide in the present invention may occur during and/or subsequent to growth of the plant to the desired stage of development.
  • COX Vila subunit polypeptides such as the Zm COX Vila subunit protein or fragment or variant thereof are increased in monocots, including, but not limited to, maize and rice.
  • the expression level of the COX Vila subunit polypeptide can be measured directly, for example, by assaying for the level of the COX Vila subunit polypeptide in the plant, or indirectly, for example, by measuring the COX Vila subunit activity of the polypeptide in the plant. Methods for determining COX Vila subunit activity of a polypeptide of interest are described elsewhere herein. Additionally, the level of the COX Vila subunit mRNA can be measured by methods well known to one of skill in the art, including, for example, Northern analysis.
  • the COX Vila subunit polypeptide or polynucleotide is introduced into the plant cell.
  • many methods are known the art for providing a polypeptide to a plant including, but not limited to, direct introduction of the polypeptide into the plant and introducing into the plant (transiently or stably) a polynucleotide construct encoding a polypeptide having COX Vila subunit activity.
  • a plant cell having the introduced sequence of the invention is selected using methods known to those of skill in the art such as, but not limited to, Southern blot analysis, DNA sequencing, PCR analysis, or phenotypic analysis (for increased yield, such as increased total seed number, increased total seed weight, increased number of seeds filled, increased harvest index, or any combination thereof).
  • a plant or plant part modified by the foregoing embodiments is grown under plant forming conditions for a time sufficient to increase the concentration and/or activity of the COX Vila subunit polypeptide, for example, the Zm COX Vila subunit protein or fragment or variant thereof, in the plant. Plant forming conditions are well known in the art and discussed briefly elsewhere herein.
  • COX Vila subunit polypeptide may be increased by employing a polynucleotide that is not capable of directing, in a transformed plant, the expression of a protein or an RNA.
  • COX Vila subunit polynucleotides such as the Zm COX Vila subunit gene may be used to design polynucleotide constructs that can be employed in methods for altering or mutating a genomic nucleotide sequence in an organism.
  • Such polynucleotide constructs include, but are not limited to, RNA:DNA vectors, RNA:DNA mutational vectors, RNA:DNA repair vectors, mixed-duplex oligonucleotides, self- complementary RNA:DNA oligonucleotides, and recombinogenic oligonucleobases.
  • Such nucleotide constructs and methods of use are known in the art. See, U.S. Patent Nos. 5,565,350; 5,731 ,181 ; 5,756,325; 5,760,012; 5,795,972; and 5,871 ,984; all of which are herein incorporated by reference.
  • COX Vila subunit polypeptide for example, the Zm COX Vila subunit protein of SEQ ID NO: 2 or fragment or variant thereof, may be increased by altering the gene encoding the COX Vila subunit polypeptide or its promoter. See, for example, Kmiec, U.S. Patent 5,565,350; Zarling, et al., PCT/US93/03868.
  • the genome may be altered following the introduction of a COX Vila subunit polynucleotide, such as the Zm COX Vila subunit sequence of SEQ ID NO: 1 , or the Zm COX Vila subunit coding sequence set forth in nucleotides 81-281 of SEQ ID NO: 1 or in SEQ ID NO: 3, into a cell.
  • a COX Vila subunit polynucleotide such as the Zm COX Vila subunit sequence of SEQ ID NO: 1 , or the Zm COX Vila subunit coding sequence set forth in nucleotides 81-281 of SEQ ID NO: 1 or in SEQ ID NO: 3
  • the polynucleotide, or any part thereof may incorporate into the genome of the plant.
  • Alterations to the genome of the present invention include, but are not limited to, additions, deletions, and substitutions of nucleotides into the genome. While the methods of the present invention do not depend on additions, deletions, and substitutions of any particular number of nucleotides, it is recognized that such additions, deletions, or substitutions comprises at least one nucleotide. Accordingly, in some embodiments, the methods of the invention involve introducing a COX
  • Introducing is intended to mean presenting to the plant the COX Vila subunit polynucleotide or polypeptide in such a manner that the sequence gains access to the interior of a cell of the plant.
  • the methods of the invention do not depend on a particular method for introducing a sequence into a plant, only that the polynucleotide or polypeptides gains access to the interior of at least one cell of the plant.
  • Methods for introducing COX Vila subunit polynucleotide or polypeptides into plants are known in the art including, but not limited to, stable transformation methods, transient transformation methods, and virus-mediated methods.
  • “Stable transformation” is intended to mean that the nucleotide construct introduced into a plant integrates into the genome of the plant and is capable of being inherited by the progeny thereof.
  • “Transient transformation” is intended to mean that a polynucleotide is introduced into the plant and does not integrate into the genome of the plant or a polypeptide is introduced into a plant.
  • Transformation protocols as well as protocols for introducing COX Vila subunit polypeptides or polynucleotide sequences into plants may vary depending on the type of plant or plant cell targeted for transformation.
  • the methods of the present invention involve transformation protocols suitable for introducing COX Vila subunit polypeptides or polynucleotide sequences into monocots.
  • Suitable methods of introducing COX Vila subunit polypeptides and polynucleotides into plant cells include microinjection (Crossway, et al., (1986) Biotechniques 4320-334), electroporation
  • transient transformation methods include, but are not limited to, the introduction of the COX Vila subunit polypeptide, for example, the Zm COX Vila subunit protein of SEQ ID NO: 2 or biologically active fragment or variant thereof, directly into the plant or the introduction of a transcript into the plant.
  • transient transformation methods include, for example, microinjection or particle bombardment. See, for example, Crossway, et al., (1986) MoI Gen.
  • COX Vila subunit polynucleotide for example, the Zm COX Vila subunit sequence of SEQ ID NO: 1 , the Zm
  • ID NO: 3 or fragment or variant thereof encoding a COX Vila subunit polypeptide, can be transiently transformed into the plant using techniques known in the art.
  • Such techniques include viral vector systems and the precipitation of the polynucleotide in a manner that precludes subsequent release of the DNA.
  • the transcription from the particle-bound DNA can occur, but the frequency with which it is released to become integrated into the genome is greatly reduced.
  • Such methods include the use of particles coated with polyethylimine (PEI; Sigma #P3143).
  • COX Vila subunit polynucleotides may be introduced into plants by contacting plants with a virus or viral nucleic acids. Generally, such methods involve incorporating a nucleotide construct within a viral DNA or RNA molecule. It is recognized that a COX Vila subunit polypeptide of interest may be initially synthesized as part of a viral polyprotein, which later may be processed by proteolysis in vivo or in vitro to produce the desired recombinant protein. Further, it is recognized that useful promoters may include promoters utilized for transcription by viral RNA polymerases.
  • a polynucleotide can be contained in a transfer cassette flanked by two non-recombinogenic recombination sites.
  • the transfer cassette is introduced into a plant having stably incorporated into its genome a target site that is flanked by two non-recombinogenic recombination sites that correspond to the sites of the transfer cassette.
  • An appropriate recombinase is provided and the transfer cassette is integrated at the target site.
  • the polynucleotide of interest is thereby integrated at a specific chromosomal position in the plant genome.
  • the cells that have been transformed may be grown into plants in accordance with conventional ways. See, for example, McCormick, et al., (1986) Plant Cell Reports 5:81-84. These plants may then be grown, and either pollinated with the same transformed strain or different strains, and the resulting progeny having expression of the desired phenotypic characteristic, for example, increased total seed number, increased total seed weight, increased number of seeds filled, increased harvest index, or any combination thereof, identified. Two or more generations may be grown to ensure that expression of the desired phenotypic characteristic is stably maintained and inherited and then seeds harvested to ensure expression of the desired phenotypic characteristic has been achieved.
  • transformed seed also referred to as "transgenic seed” having a polynucleotide described herein, for example, an expression cassette comprising the Zm COX Vila subunit sequence of SEQ ID NO: 1 , the Zm COX Vila subunit coding sequence set forth in nucleotides 81-281 of SEQ ID NO: 1 or in SEQ ID NO: 3, or fragment or variant thereof encoding a COX VIIa subunit polypeptide, stably incorporated into their genome.
  • Plants of the invention may be produced by any suitable method, including breeding.
  • Plant breeding can be used to introduce desired characteristics (e.g., a stably incorporated transgene) into a particular plant line of interest, and can be performed in any of several different ways.
  • Pedigree breeding starts with the crossing of two genotypes, such as an elite line of interest and one other elite inbred line having one or more desirable characteristics (i.e., having stably incorporated a polynucleotide of interest, having a modulated activity and/or level of the polypeptide of interest, etc.) which complements the elite plant line of interest. If the two original parents do not provide all the desired characteristics, other sources can be included in the breeding population.
  • the inbred line comprises homozygous alleles at about 95% or more of its loci.
  • backcrossing can also be used in combination with pedigree breeding to modify an elite line of interest and a hybrid that is made using the modified elite line.
  • backcrossing can be used to transfer one or more specifically desirable traits from one line, the donor parent, to an inbred called the recurrent parent, which has overall good agronomic characteristics yet lacks that desirable trait or traits.
  • the same procedure can be used to move the progeny toward the genotype of the recurrent parent but at the same time retain many components of the nonrecurrent parent by stopping the backcrossing at an early stage and proceeding with selfing and selection. For example, an F1 , such as a commercial hybrid, is created.
  • This commercial hybrid may be backcrossed to one of its parent lines to create a BC1 or BC2.
  • Progeny are selfed and selected so that the newly developed inbred has many of the attributes of the recurrent parent and yet several of the desired attributes of the non-recurrent parent. This approach leverages the value and strengths of the recurrent parent for use in new hybrids and breeding.
  • the COX Vila subunit polynucleotides of the present invention can be stacked with any combination of polynucleotide sequences of interest in order to create plants with a desired trait.
  • a "trait,” as used herein, refers to the phenotype derived from a particular sequence or groups of sequences.
  • the Zm COX Vila subunit polynucleotides of the present invention may be stacked with any other polynucleotides encoding polypeptides having COX Vila subunit activity, such as COX Vila subunit polynucleotides from Pinguicula (GenBank Accession No. AY601869), Utricularia (GenBank Accession No.
  • AY1 1 1015 Malus domestica (GenBank Accession No. CN994265), Picea glauca (GenBank Accession No. CO237069), Rosa chinensis (GenBank Accession No. BI978872), Callinectes sapidus (GenBank Accession No. CV086499), Glossina morsitans morsitans (GenBank Accession No. BX567633), Ambystoma mexicanum (GenBank Accession No. CN046134), Oryzias latipes (GenBank Accession No. BJ514640), and Oncorhynchus mykiss (GenBank Accession No. BX308258).
  • the combinations generated can also include multiple copies of any one of the polynucleotides of interest.
  • the polynucleotides of the present invention can also be stacked with any other gene or combination of genes to produce plants with a variety of desired trait combinations including, but not limited to, traits desirable for animal feed such as high oil genes (e.g., U.S. Patent No. 6,232,529); balanced amino acids (e.g., hordothionins (U.S. Patent Nos. 5,990,389; 5,885,801 ; 5,885,802; and 5,703,409); barley high lysine (Williamson, et ah, (1987) Eur. J. Biochem.
  • traits desirable for animal feed such as high oil genes (e.g., U.S. Patent No. 6,232,529); balanced amino acids (e.g., hordothionins (U.S. Patent Nos. 5,990,389; 5,885,801 ; 5,885,802; and 5,703,409); barley high lysine (Williamson
  • the polynucleotides of the present invention can also be stacked with traits desirable for disease or herbicide resistance (e.g., fumonisin detoxification genes (U.S. Patent No. 5,792,931 ); avirulence and disease resistance genes (Jones, et al., (1994) Science 266:789; Martin, et al., (1993) Science 262:1432; Mindrinos, et al., (1994) Cell 78:1089); acetolactate synthase (ALS) mutants that lead to herbicide resistance such as the S4 and/or Hra mutations; inhibitors of glutamine synthase such as phosphinothricin or basta (e.g., bar gene); and glyphosate resistance (e.g., the EPSPS gene and the GAT gene; see, for example, U.S.
  • traits desirable for disease or herbicide resistance e.g., fumonisin detoxification genes (U.S. Patent No. 5,79
  • stacked combinations can be created by any method including, but not limited to, crossbreeding plants by any conventional or TopCross methodology, or genetic transformation. If the sequences are stacked by genetically transforming the plants, the polynucleotide sequences of interest can be combined at any time and in any order. For example, a transgenic plant comprising one or more desired traits can be used as the target to introduce further traits by subsequent transformation. The traits can be introduced simultaneously in a co-transformation protocol with the polynucleotides of interest provided by any combination of transformation cassettes. For example, if two sequences will be introduced, the two sequences can be contained in separate transformation cassettes ⁇ trans) or contained on the same transformation cassette (cis).
  • sequences can be driven by the same promoter or by different promoters.
  • polynucleotide sequences can be stacked at a desired genomic location using a site-specific recombination system. See, for example, WO 99/25821 , WO 99/25854, WO 99/25840, WO 99/25855, and WO 99/25853, all of which are herein incorporated by reference.
  • the term "plant” includes plant cells, plant protoplasts, plant cell tissue cultures from which plants can be regenerated, plant calli, plant clumps, and plant cells that are intact in plants or parts of plants such as embryos, pollen, ovules, seeds, leaves, flowers, branches, fruit, kernels, ears, cobs, husks, stalks, roots, root tips, anthers, and the like. Grain is intended to mean the mature seed produced by commercial growers for purposes other than growing or reproducing the species. Progeny, variants, and mutants of the regenerated plants are also included within the scope of the invention, provided that these parts comprise the introduced polynucleotides. Thus, the invention provides transgenic seeds produced by the plants of the invention.
  • a “subject plant or plant cell” is one in which a genetic alteration, such as transformation, has been effected as to a COX Vila subunit gene of interest, or is a plant or plant cell that is descended from a plant or cell so altered and which comprises the alteration.
  • a “control” or “control plant” or “control plant cell” provides a reference point for measuring changes in phenotype of the subject plant or plant cell.
  • a control plant or plant cell may comprise, for example: (a) a wild-type plant or cell, that is, of the same genotype as the starting material for the genetic alteration which resulted in the subject plant or cell; (b) a plant or plant cell of the same genotype as the starting material but which has been transformed with a null construct (that is, with a construct that has no known effect on the trait of interest, such as a construct comprising a marker gene); (c) a plant or plant cell that is a non-transformed segregant among progeny of a subject plant or plant cell; (d) a plant or plant cell genetically identical to the subject plant or plant cell but which is not exposed to conditions or stimuli that would induce expression of the gene of interest; or (e) the subject plant or plant cell itself, under conditions in which the COX Vila subunit gene of interest is not expressed.
  • the present invention may be used for transformation of any plant species, including, but not limited to, monocots and dicots.
  • plant species of interest 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 ⁇ 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 (Solarium tuberosum), peanuts (Arachis hypogaea), cotton (Gossypium barbadense, Gossypium hirsutum), sweet potato (Ipomoea batatus), cassava (Manihot
  • Vegetables include tomatoes (Lycopersicon esculentum), lettuce (e.g., Lactuca sativa), green beans (Phaseolus vulgaris), lima beans (Phaseolus limensis), peas (Lathyrus spp.), and members of the genus Cucumis such as cucumber (C. sativus), cantaloupe (C. cantalupensis), and musk melon (C. melo).
  • tomatoes Locopersicon esculentum
  • lettuce e.g., Lactuca sativa
  • green beans Phaseolus vulgaris
  • lima beans Phaseolus limensis
  • peas Lathyrus spp.
  • members of the genus Cucumis such as cucumber (C. sativus), cantaloupe (C. cantalupensis), and musk melon (C. melo).
  • Ornamentals include azalea (Rhododendron spp.), hydrangea (Macrophylla 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 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).
  • plants of the present invention are crop plants (for example, corn, alfalfa, sunflower, Brass
  • plants of interest are monocots, for example, corn (Zea mays), 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)), wheat (Triticum aestivum), sugarcane (Saccharum spp.), oats, and barley.
  • Other plants of interest include grain plants that provide seeds of interest, oil-seed plants, and leguminous plants.
  • Seeds of interest include grain seeds, such as corn, wheat, barley, rice, sorghum, rye, etc.
  • Oil-seed plants include cotton, soybean, safflower, sunflower, Brassica, maize, alfalfa, palm, coconut, etc.
  • Leguminous plants include beans and peas. Beans include guar, locust bean, fenugreek, soybean, garden beans, cowpea, mungbean, lima bean, fava bean, lentils, chickpea, etc.
  • nucleotide sequence for the Zm COX Vila promoter disclosed in the present invention as well as variants and fragments thereof are useful in the genetic manipulation of any plant when assembled within a DNA construct such that the promoter sequence is operably linked to a nucleotide sequence encoding a heterologous protein of interest.
  • the nucleotide sequence of the Zm COX Vila promoter of the invention, or fragment or variant thereof is provided in expression cassettes along with heterologous nucleotide sequences for expression in the plant of interest.
  • Synthetic hybrid promoter regions are known in the art. Such regions comprise upstream promoter elements of one nucleotide sequence operably linked to the promoter element of another nucleotide sequence.
  • heterologous gene expression is controlled by a synthetic hybrid promoter comprising the Zm COX Vila promoter sequence of the invention, or a variant or fragment thereof, operably linked to upstream promoter element(s) from a heterologous promoter.
  • Upstream promoter elements have been identified and may be used to generate a synthetic promoter. See, for example, Rushton, et al., (1998) Curr. Opin. Plant Biol. 1 :31 1-315.
  • a synthetic Zm COX VIIa promoter sequence may comprise duplications of upstream promoter elements found within the Zm COX Vila promoter sequence. It is recognized that the promoter sequence of the invention may be used with its native Zm COX Vila subunit coding sequence.
  • a DNA construct comprising the Zm COX Vila promoter operably linked with its native Zm COX Vila subunit coding sequence may be used to transform any plant of interest to bring about a desired phenotypic change, such as increased yield, for example, reflected in increased total seed number, increased total seed weight, increased number of seeds filled, increased harvest index, or any combination thereof.
  • expression cassettes will comprise a transcriptional initiation region comprising the Zm COX Vila promoter nucleotide sequence disclosed herein, or variant or fragment thereof, operably linked to the heterologous nucleotide sequence whose expression is to be controlled by the Zm COX Vila promoter of the invention.
  • the promoter nucleotide sequence and methods disclosed herein are useful in regulating expression of any heterologous nucleotide sequence in a host plant in order to vary the phenotype of a plant.
  • Various changes in phenotype are of interest, including, but not limited to, modifying the fatty acid composition in a plant, altering the amino acid content of a plant, altering a plant's pathogen defense mechanism, and the like. These results can be achieved by providing expression of heterologous products or increased expression of endogenous products in plants. Alternatively, the results can be achieved by providing for a reduction of expression of one or more endogenous products, particularly enzymes or cofactors in the plant. These changes result in a change in phenotype of the transformed plant.
  • An expression cassette comprising a nucleotide sequence selected from the group consisting of:
  • nucleotide sequence encodes a polypeptide having COX Vila subunit activity
  • nucleotide sequence encoding an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 2, wherein said nucleotide sequence encodes a polypeptide having COX Vila subunit activity; wherein said nucleotide sequence is operably linked to a promoter that drives expression in a plant.
  • a plant comprising the expression cassette of any one of items 1 to 3.
  • a method for increasing the level of a COX Vila subunit polypeptide in a plant comprising introducing into said plant the expression cassette of any one of items 1 to
  • a method for increasing yield in a plant comprising increasing expression of a COX Vila subunit polypeptide in said plant, wherein said COX Vila subunit polypeptide has COX Vila subunit activity and is selected from the group consisting of: (a) a polypeptide comprising an amino acid sequence having at least 80% sequence identity to the sequence set forth in SEQ ID NO: 2; and (b) a polypeptide comprising a functional fragment of the amino acid sequence set forth in SEQ ID NO: 2. 17.
  • said polypeptide comprises an amino acid sequence having at least 90% sequence identity to the sequence set forth in SEQ ID NO: 2.
  • nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 3, wherein said nucleotide sequence encodes a polypeptide having COX Vila subunit activity;
  • nucleotide sequence comprising at least 147 consecutive nucleotides of SEQ ID NO: 3, wherein said nucleotide sequence encodes a polypeptide having COX
  • nucleotide sequence encoding an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 2, wherein said nucleotide sequence encodes a polypeptide having COX Vila subunit activity; wherein said nucleotide sequence is operably linked to a promoter that drives expression in a plant.
  • nucleotide sequence selected from the group consisting of: (a) the nucleotide sequence set forth as nucleotides 1 through 1012 of SEQ ID NO:
  • nucleotide sequence with at least 98% sequence identity to the nucleotide sequence set forth as nucleotides 1 through 1012 of SEQ ID NO: 4, wherein said sequence drives expression in a plant cell.
  • An expression cassette comprising the polynucleotide of item 27 operably linked to a heterologous polynucleotide of interest.
  • the plant cell of item 30 wherein said plant cell is from a dicot.
  • said dicot is soybean, Brassica, sunflower, cotton, or alfalfa.
  • the cDNA that encoded the COX Vila polypeptide from maize was identified by sequence homology from a collection of ESTs generated from a maize cDNA library using BLAST 2.0 (Altschul, et al., (1990) J. MoI. Biol. 215:403) against the NCBI DNA sequence database. From the EST plasmid, the maize COX Vila cDNA fragment was amplified by PCR using Hifi Taq DNA polymerase in standard conditions with maize COX W/a-specific primers that included the AttB site for Gateway recombination cloning.
  • PCR fragment of the expected length was amplified and purified using standard methods as described by Sambrook, et al., (1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, New York).
  • the first step of the Gateway procedure the BP reaction, was then performed, during which the PCR fragment recombined in vivo with the pDONR201 plasmid to produce the "entry clone.”
  • Plasmid pDONR201 was purchased from Invitrogen, as part of the Gateway® technology (Invitrogen, Carlsbad, CA).
  • the entry clone was subsequently used in an LR reaction with a destination vector used for Oryza sativa transformation.
  • This vector contains as functional elements within the T-DNA borders, a plant selectable marker, a screenable marker, and a Gateway cassette intended for LR in vivo recombination with the sequence of interest already cloned in the entry clone.
  • Upstream of this Gateway cassette is the rice GOS2 promoter (Hensgens, et al., (1993) Plant MoI. Biol. 23:643-669) that confers moderate constitutive expression on the gene of interest.
  • the resulting expression vector pGOS2::ZmCOXVIIa was transformed into Agrobacterium tumefaciens strain LBA4044 and subsequently into Oryza sativa var. Nipponbare plants as described herein in Example 3. Transformed rice plants were grown and examined for various growth characteristics as described herein in Example 4.
  • High-velocity ballistic bombardment using metal particles coated with the nucleic acid constructs was used to transform wild-type rice and Cox Vila mutants (Klein, et al., (1987) Nature 327:70-73; U.S. Patent No. 4,945,050, incorporated by reference herein).
  • a Biolistic PDS-1000/He BioRAD Laboratories, Hercules, CA was used for these complementation experiments.
  • the particle bombardment technique was used to transform wild-type rice and Cox Vila mutants with two genomic DNA fragments: a 10.0 kb Muni fragment from wild-type rice that includes the 4.5 kb upstream and 3.8 kb downstream region of the COX VIIA gene, and a 5.1 kb EcoRI fragment from wild-type rice that includes the 1.7 kb upstream and 1.7 kb downstream region of the COX VIIA gene.
  • the bacterial hygromycin B phosphotransferase (Hpt II) gene from Streptomyces hygroscopicus (which confers resistance to the antibiotic) was used as the selectable marker for rice transformation.
  • the Hpt Il gene was engineered with the 35S promoter from Cauliflower Mosaic Virus and the termination and polyadenylation signals from the octopine synthase gene of Agrobacterium tumefaciens.
  • pML18 is described in WO 97/47731 , the disclosure of which is hereby incorporated by reference.
  • Embryogenic callus cultures derived from the scutellum of germinating rice seeds served as source material for transformation experiments. This material is generated by germinating sterile rice seeds on a callus initiation media (MS salts, Nitsch and Nitsch vitamins, 1.0 mg/l 2,4-D and 10 ⁇ M AgNO 3 ) in the dark at 27-28°C. Embryogenic callus proliferating from the scutellum of the embryos is then transferred to CM media (N6 salts, Nitsch and Nitsch vitamins, 1 mg/l 2,4-D; Chu, et al., (1985) Sci. Sinica 18:659-668).
  • CM media N6 salts, Nitsch and Nitsch vitamins, 1 mg/l 2,4-D; Chu, et al., (1985) Sci. Sinica 18:659-668.
  • Callus cultures are maintained on CM by routine sub-culture at two week intervals and used for transformation within 10 weeks of initiation.
  • Callus is prepared for transformation by subculturing 0.5-1.0 mm pieces approximately 1 mm apart, arranged in a circular area of about 4 cm in diameter, in the center of a circle of Whatman #541 paper placed on CM media. The plates with callus are incubated in the dark at 27-28°C for 3-5 days. Prior to bombardment, the filters with callus are transferred to CM supplemented with 0.25 M mannitol and 0.25 M sorbitol for 3 hr in the dark. The petri dish lids are then left ajar for 20-45 minutes in a sterile hood to allow moisture on tissue to dissipate.
  • Each of the genomic DNA fragment was co-precipitated with pML18 containing the selectable marker for rice transformation onto the surface of gold particles.
  • pML18 containing the selectable marker for rice transformation onto the surface of gold particles.
  • a total of 10 ⁇ g of DNA at a 2:1 ratio of trait:selectable marker DNAs were added to a 50 ⁇ l aliquot of gold particles that had been resuspended at a concentration of 60 mg ml "1 .
  • Calcium chloride 50 ⁇ l of a 2.5 M solution
  • spermidine (20 ⁇ l of a 0.1 M solution
  • the gold particles were then washed twice with 1 ml of absolute ethanol and resuspended in 50 ⁇ l of absolute ethanol and sonicated (bath sonicator) for one second to disperse the gold particles.
  • the gold suspension was incubated at -70 0 C for five minutes and sonicated (bath sonicator) to disperse the particles.
  • Six ⁇ l of the DNA-coated gold particles was then loaded onto mylar macrocarrier disks and the ethanol was allowed to evaporate.
  • a petri dish containing the tissue was placed in the chamber of the PDS-1000/He.
  • the air in the chamber was then evacuated to a vacuum of 28-29 inches Hg.
  • the macrocarrier was accelerated with a helium shock wave using a rupture membrane that bursts when the He pressure in the shock tube reaches 1080-1100 psi.
  • the tissue was placed approximately 8 cm from the stopping screen and the callus was bombarded two times. Two to four plates of tissue were bombarded in this way with the DNA-coated gold particles. Following bombardment, the callus tissue was transferred to CM media without supplemental sorbitol or mannitol.
  • SM media CM medium containing 50 mg/l hygromycin.
  • callus tissue was transferred from plates to sterile 50 ml conical tubes and weighed. Molten top-agar at 40 0 C was added using 2.5 ml of top agar/100 mg of callus. Callus clumps were broken into fragments of less than 2 mm diameter by repeated dispensing through a 10 ml pipette. Three ml aliquots of the callus suspension were plated onto fresh SM media and the plates were incubated in the dark for 4 weeks at 27-28°C. After 4 weeks, transgenic callus events were identified, transferred to fresh SM plates and grown for an additional 2 weeks in the dark at 27-28°C.
  • RM 1 media MS salts, Nitsch and Nitsch vitamins, 2% sucrose, 3% sorbitol, 0.4% gelrite +50 ppm hyg B
  • RM2 media MS salts, Nitsch and Nitsch vitamins, 3% sucrose, 0.4% gelrite + 50 ppm hyg B
  • Example 4 Overexpression of a COX Vila Sequence to Increase Yield in Rice Evaluation of TO, T1 , and T2 Rice Plants Transformed with pG0S2::ZmC0XVIIa Approximately 15 to 20 independent TO transformants were generated. The primary transformants were transferred from tissue culture chambers to a greenhouse for growing and harvest of T1 seed. Six events of which the T1 progeny segregated 3/1 for presence/absence of the transgene were retained. "Null plants” or “Null segregants” or “Nullizygotes” are the plants treated in the same way as a transgenic plant, but from which the transgene has segregated. Null plants can also be described as the homozygous negative transformants. For each of these events, approximately 10 T1 seedlings containing the transgene (hetero- and homozygotes), and approximately 10 T1 seedlings lacking the transgene (nullizygotes), were selected by PCR.
  • T1 evaluation Based on the results of the T1 evaluation (described herein), four events that showed improved growth and yield characteristics at the T1 level were chosen for further characterization in the T2 generation. To this extent, seed batches from the positive T1 plants (both hetero- and homozygotes), were screened by monitoring marker expression. For each chosen event, the heterozygote seed batches were then selected for T2 evaluation. An equal number of positive and negative plants within each seed batch were transplanted for evaluation in the greenhouse (i.e., for each event 40 plants, of which 20 were positives for the transgene and 20 were negative for the transgene). For the four events, a total of 160 plants were evaluated in the T2 generation. Both T1 and T2 plants were transferred to a greenhouse and evaluated for vegetative growth parameters, as described herein.
  • the t-test was carried out to check for an effect of the gene within one transformation event, also described herein as "line-specific effect.”
  • the threshold for a significant line-specific effect is set at 10% probability level. Therefore, data with a p-value of the t-test under 10% means that the phenotype observed in the transgenic plants of that line was caused by the presence of the transgene.
  • some events may be under or below this threshold. This difference may be due to the difference in the position of the transgene within the rice genome (i.e., a gene might only have an effect in certain positions of the genome). Therefore, the "line- specific effect" is sometimes referred to as the "position-dependent effect.”
  • the selected plants were grown in a greenhouse. Each plant received a unique barcode label to link the phenotyping data unambiguously to the corresponding plant.
  • Transgenic plants and the corresponding nullizygotes were grown side-by-side at random positions. From the stage of sowing until the stage of maturity (i.e., the stage were there is no more increase in biomass), the plants were passed weekly through a digital imaging cabinet. At each time point digital images (2048x1536 pixels, 16 million colors) were taken of each plant from at least 6 different angles. The parameters described herein were derived in an automated way from the digital images using image analysis software.
  • Plants were also passed through a root-imaging system that digitally photographs the root morphology and mass from the base of the clear-bottom pots. Plant above-ground area and root mass were determined by counting the total number of pixels from plant parts discriminated from the background. The above-ground value was averaged for the pictures taken on the same time point from the different angles and was converted to a physical surface value expressed in square mm by calibration. Experiments have shown that the above-ground plant area, which corresponds to the total maximum area, measured this way correlates with the biomass of plant parts above-ground.
  • Immature maize embryos from greenhouse donor plants are bombarded with a plasmid containing a COX Vila subunit polynucleotide (such as the coding sequence of the Zm COX Vila subunit polynucleotide as set forth in nucleotides 81-281 of SEQ ID NO: 1 or in SEQ ID NO: 3) operably linked to a UBI promoter and the selectable marker gene PAT (Wohlleben, et al., (1988) Gene 70:25-37), which confers resistance to the herbicide Bialaphos.
  • the selectable marker gene is provided on a separate plasmid. Transformation is performed as follows. Media recipes follow below. Preparation of Target Tissue
  • the ears are husked and surface sterilized in 30% Clorox bleach plus 0.5% Micro detergent for 20 minutes, and rinsed two times with sterile water.
  • the immature embryos are excised and placed embryo axis side down (scutellum side up), 25 embryos per plate, on 560Y medium for 4 hours and then aligned within the 2.5 cm target zone in preparation for bombardment.
  • a plasmid vector comprising the COX Vila subunit sequence operably linked to a UBI promoter is made.
  • This plasmid DNA plus plasmid DNA containing a PAT selectable marker is precipitated onto 1.1 ⁇ m (average diameter) tungsten pellets using a CaCI 2 precipitation procedure as follows: 100 ⁇ l prepared tungsten particles in water; 10 ⁇ l (1 ⁇ g) DNA in Tris EDTA buffer (1 ⁇ g total DNA); 100 ⁇ l 2.5 M CaCI 2 ; and 10 ⁇ l 0.1 M spermidine.
  • Each reagent is added sequentially to the tungsten particle suspension, while maintained on the multitube vortexer.
  • the final mixture is sonicated briefly and allowed to incubate under constant vortexing for 10 minutes.
  • the tubes are centrifuged briefly, liquid removed, washed with 500 ml 100% ethanol, and centrifuged for 30 seconds. Again the liquid is removed, and 105 ⁇ l 100% ethanol is added to the final tungsten particle pellet.
  • the tungsten/DNA particles are briefly sonicated and 10 ⁇ l spotted onto the center of each macrocarrier and allowed to dry about 2 minutes before bombardment.
  • sample plates are bombarded at level #4 in a particle gun. All samples receive a single shot at 650 PSI, with a total of ten aliquots taken from each tube of prepared particles/DNA.
  • the embryos are kept on 560Y medium for 2 days, then transferred to 560R selection medium containing 3 mg/liter Bialaphos, and subcultured every 2 weeks. After approximately 10 weeks of selection, selection-resistant callus clones are transferred to 288J medium to initiate plant regeneration. Following somatic embryo maturation (2-4 weeks), well- developed somatic embryos are transferred to medium for germination and transferred to the lighted culture room. Approximately 7-10 days later, developing plantlets are transferred to 272V hormone-free medium in tubes for 7-10 days until plantlets are well established.
  • Plants are then transferred to inserts in flats (equivalent to 2.5" pot) containing potting soil and grown for 1 week in a growth chamber, subsequently grown an additional 1-2 weeks in the greenhouse, then transferred to classic 600 pots (1.6 gallon) and grown to maturity. Plants are monitored and scored for increased yield, such as increased total seed number, increased total seed weight, increased number of seeds filled, increased harvest index, or any combination thereof.
  • Bombardment medium comprises 4.0 g/l N6 basal salts (SIGMA C-1416), 1.0 ml/l Eriksson's Vitamin Mix (1000X SIGMA-151 1 ), 0.5 mg/l thiamine HCI, 120.0 g/l sucrose, 1.0 mg/l 2,4-D, and 2.88 g/l L-proline (brought to volume with D-I H 2 O following adjustment to pH 5.8 with KOH); 2.0 g/l Gelrite (added after bringing to volume with D-I H 2 O); and 8.5 mg/l silver nitrate (added after sterilizing the medium and cooling to room temperature).
  • Selection medium comprises 4.0 g/l N6 basal salts (SIGMA C-1416), 1.0 ml/l Eriksson's Vitamin Mix (1000X SIGMA-151 1 ), 0.5 mg/l thiamine HCI, 30.0 g/l sucrose, and 2.0 mg/l 2,4-D (brought to volume with D-I H 2 O following adjustment to pH 5.8 with KOH); 3.0 g/l Gelrite (added after bringing to volume with D-I H 2 O); and 0.85 mg/l silver nitrate and 3.0 mg/l Bialaphos(both added after sterilizing the medium and cooling to room temperature).
  • Plant regeneration medium (288J) comprises 4.3 g/l MS salts (GIBCO 1 1117-074), 5.0 ml/l MS vitamins stock solution (0.100 g nicotinic acid, 0.02 g/l thiamine HCL, 0.10 g/l pyridoxine HCL, and 0.40 g/l glycine brought to volume with polished D-I H 2 O) (Murashige and Skoog (1962) Physiol. Plant.
  • Hormone-free medium comprises 4.3 g/l MS salts (GIBCO 1 11 17-074), 5.0 ml/l MS vitamins stock solution (0.100 g/l nicotinic acid, 0.02 g/l thiamine HCL, 0.10 g/l pyridoxine HCL, and 0.40 g/l glycine brought to volume with polished D-I H 2 O), 0.1 g/l myo-inositol, and 40.0 g/l sucrose (brought to volume with polished D-I H 2 O after adjusting pH to 5.6); and 6 g/l bacto-agar (added after bringing to volume with polished D-I H 2 O), sterilized and cooled to 60 0 C.
  • immature embryos are isolated from maize and the embryos contacted with a suspension of Agrobacterium, where the bacteria are capable of transferring the COX Vila subunit polynucleotide to at least one cell of at least one of the immature embryos (step 1 : the infection step).
  • the immature embryos are immersed in an Agrobacterium suspension for the initiation of inoculation.
  • the embryos are co-cultured for a time with the Agrobacterium (step 2: the co-cultivation step).
  • the immature embryos are cultured on solid medium following the infection step. Following this co-cultivation period an optional "resting" step is contemplated.
  • step 3 resting step
  • the immature embryos are cultured on solid medium with antibiotic, but without a selecting agent, for elimination of Agrobacterium and for a resting phase for the infected cells.
  • step 4 inoculated embryos are cultured on medium containing a selective agent and growing transformed callus is recovered (step 4: the selection step).
  • the immature embryos are cultured on solid medium with a selective agent resulting in the selective growth of transformed cells.
  • the callus is then regenerated into plants (step 5: the regeneration step), and calli grown on selective medium are cultured on solid medium to regenerate the plants.
  • Soybean embryogenic suspension cultures (cv. Jack) are maintained in 35 ml liquid medium SB196 (see recipes below) on rotary shaker, 150 rpm, 26°C with cool white fluorescent lights on 16:8 hr day/night photoperiod at light intensity of 60-85 ⁇ E/m2/s. Cultures are subcultured every 7 days to two weeks by inoculating approximately 35 mg of tissue into 35 ml of fresh liquid SB196 (the preferred subculture interval is every 7 days).
  • Soybean embryogenic suspension cultures are transformed with the plasmids and DNA fragments described in the following examples by the method of particle gun bombardment (Klein, et al., (1987) Nature 327:70).
  • Soybean Embrvogenic Suspension Culture Initiation Soybean cultures are initiated twice each month with 5-7 days between each initiation.
  • soybeans with immature seeds from available soybean plants 45-55 days after planting are picked, removed from their shells and placed into a sterilized magenta box.
  • the soybean seeds are sterilized by shaking them for 15 minutes in a 5% Clorox solution with 1 drop of ivory soap (95 ml of autoclaved distilled water plus 5 ml Clorox and 1 drop of soap). Mix well.
  • Seeds are rinsed using 2 1 -liter bottles of sterile distilled water and those less than 4 mm are placed on individual microscope slides. The small end of the seed are cut and the cotyledons pressed out of the seed coat. Cotyledons are transferred to plates containing SB1 medium (25-30 cotyledons per plate). Plates are wrapped with fiber tape and stored for 8 weeks. After this time secondary embryos are cut and placed into SB196 liquid media for 7 days.
  • Plasmid DNA for bombardment are routinely prepared and purified using the method described in the PromegaTM Protocols and Applications Guide, Second Edition (page 106). Fragments of the plasmids carrying a COX Vila subunit polynucleotide (such as the coding sequence of the Zm COX Vila subunit polynucleotide as set forth in nucleotides 81-281 of SEQ ID NO: 1 or in SEQ ID NO: 3) are obtained by gel isolation of double digested plasmids.
  • a COX Vila subunit polynucleotide such as the coding sequence of the Zm COX Vila subunit polynucleotide as set forth in nucleotides 81-281 of SEQ ID NO: 1 or in SEQ ID NO: 3
  • plasmid DNA is digested in 0.5 ml of the specific enzyme mix that is appropriate for the plasmid of interest.
  • the resulting DNA fragments are separated by gel electrophoresis on 1% SeaPlaque GTG agarose (BioWhitaker Molecular Applications) and the DNA fragments containing the COX VIIa subunit polynucleotide are cut from the agarose gel.
  • DNA is purified from the agarose using the GELase digesting enzyme following the manufacturer's protocol.
  • a 50 ⁇ l aliquot of sterile distilled water containing 3 mg of gold particles (3 mg gold) is added to 5 ⁇ l of a 1 ⁇ g/ ⁇ l DNA solution (either intact plasmid or DNA fragment prepared as described above), 50 ⁇ l 2.5M CaCI 2 and 20 ⁇ l of 0.1 M spermidine.
  • the mixture is shaken 3 min on level 3 of a vortex shaker and spun for 10 sec in a bench microfuge. After a wash with 400 ⁇ l 100% ethanol the pellet is suspended by sonication in 40 ⁇ l of 100% ethanol.
  • Five ⁇ l of DNA suspension is dispensed to each flying disk of the Biolistic PDS1000/HE instrument disk. Each 5 ⁇ l aliquot contains approximately 0.375 mg gold per bombardment (i.e., per disk).
  • Tissue is bombarded 1 or 2 shots per plate with membrane rupture pressure set at 1 100 PSI and the chamber evacuated to a vacuum of 27-28 inches of mercury. Tissue is placed approximately 3.5 inches from the retaining/stopping screen.
  • Transformed embryos were selected either using hygromycin (when the hygromycin phosphotransferase, HPT, gene was used as the selectable marker) or chlorsulfuron (when the acetolactate synthase, ALS, gene was used as the selectable marker).
  • the tissue is placed into fresh SB196 media and cultured as described above.
  • the SB196 is exchanged with fresh SB196 containing a selection agent of 30 mg/L hygromycin.
  • the selection media is refreshed weekly.
  • green, transformed tissue may be observed growing from untransformed, necrotic embryogenic clusters. Isolated, green tissue is removed and inoculated into multiwell plates to generate new, clonally propagated, transformed embryogenic suspension cultures.
  • the tissue is divided between 2 flasks with fresh SB196 media and cultured as described above.
  • the SB196 is exchanged with fresh SB196 containing selection agent of 100 ng/ml Chlorsulfuron.
  • the selection media is refreshed weekly.
  • green, transformed tissue may be observed growing from untransformed, necrotic embryogenic clusters. Isolated, green tissue is removed and inoculated into multiwell plates containing SB196 to generate new, clonally propagated, transformed embryogenic suspension cultures.
  • the tissue In order to obtain whole plants from embryogenic suspension cultures, the tissue must be regenerated.
  • Embryos are cultured for 4-6 weeks at 26 0 C in SB196 under cool white fluorescent (Phillips cool white Econowatt F40/CW/RS/EW) and Agro (Phillips F40 Agro) bulbs (40 watt) on a 16:8 hr photoperiod with light intensity of 90-120 uE/m2s. After this time embryo clusters are removed to a solid agar media, SB166, for 1-2 weeks. Clusters are then subcultured to medium SB103 for 3 weeks. During this period, individual embryos can be removed from the clusters and screened for levels of COX VIIA expression and/or activity.
  • Matured individual embryos are desiccated by placing them into an empty, small petri dish (35 x 10 mm) for approximately 4-7 days. The plates are sealed with fiber tape (creating a small humidity chamber). Desiccated embryos are planted into SB71-4 medium where they were left to germinate under the same culture conditions described above. Germinated plantlets are removed from germination medium and rinsed thoroughly with water and then planted in Redi- Earth in 24-cell pack tray, covered with clear plastic dome. After 2 weeks the dome is removed and plants hardened off for a further week. If plantlets looked hardy they are transplanted to 10" pot of Redi-Earth with up to 3 plantlets per pot. After 10 to 16 weeks, mature seeds are harvested, chipped and analyzed for proteins. Media Recipes
  • Na 2 MoO 4 - 2H 2 O 0.025 g 0.0125 g SB1 solid medium comprises: 1 pkg. MS salts (GIBCO/BRL - Cat# 1 11 17-066); 1 ml B5 vitamins 1000X stock; 31.5 g sucrose; 2 ml 2,4-D (20 mg/L final concentration); pH 5.7; and, 8 g TC agar.
  • SB 166 solid medium (per liter) comprises: 1 pkg. MS salts (GIBCO/BRL - Cat# 1 11 17-066); 1 ml B5 vitamins 1000X stock; 60 g maltose; 750 mg MgCI 2 hexahydrate; 5 g activated charcoal; pH 5.7; and, 2 g gelrite.
  • SB 103 solid medium (per liter) comprises: 1 pkg. MS salts (GIBCO/BRL - Cat# 1 11 17-066); 1 ml B5 vitamins 1000X stock; 60 g maltose; 750 mg MgCI2 hexahydrate; pH 5.7; and, 2 g gelrite.
  • SB 71-4 solid medium (per liter) comprises: 1 bottle Gamborg's B5 salts w/ sucrose (GIBCO/BRL - Cat# 21153-036); pH 5.7; and, 5 g TC agar.
  • 2,4-D stock is obtained premade from Phytotech cat# D 295 - concentration is 1 mg/ml.
  • B5 Vitamins Stock (per 100 ml) which is stored in aliquots at -20 0 C comprises: 10 g myoinositol; 100 mg nicotinic acid; 100 mg pyridoxine HCI; and, 1 g thiamine. If the solution does not dissolve quickly enough, apply a low level of heat via the hot stir plate.
  • Chlorsulfuron Stock comprises: 1 mg / ml in 0.01 N Ammonium Hydroxide.
  • Example 8 Vector construction and over expression of ZM-COXVIIa in maize to increase the yield.
  • ZM-COXVIIa The coding sequence of ZM-COXVIIa was amplified by PCR and cloned unidirectionaly in pENTR.D.TOPO vector (Invitrogen) to make an entry vector.
  • the entry vector in combination with other entry vectors were used in a multisite Gateway (Invitrogen) reaction to generate ZM- UBI PRO:ZM-COXVIIa:PINII and ZM-GOS2 PRO:ZM-COXVIIa:PINII.
  • UBhMOPAT and LTP2:RFP are used as herbicide resistance and visible markers, respectively.
  • the expression vectors were quality checked by restriction digestion mapping and transferred into Agrobacterium tumefaciens LB4404JT by electroporation. This Agrobacterium strain was used to transform lntroEF09B maize inbred. Molecular analyses on TO events were performed and single copy transgene expressing events were advanced for further experiments.
  • Example 9 Field Evaluation with Co
  • Progeny seed of multiple transgenic corn events containing ZmG0S2-C0X Vila produced in Example 8 can be planted in the field to evaluate the transgene's ability to enhance yield as compared to the non-transgenic control plants.
  • the plants would be planted at a multiple locations having a variety of environmental stresses.
  • the data to be collected would consist of multiple measurements for yield and plant health/quality.
  • the measured items could include, but are not limited to the following: enhanced vegetative growth, biomass accumulation, accelerated growth rate, stand count, stalk and/or root lodging, grain yield, average kernel weight, total seed number/plant, total seed weight/plant, harvest index, number of seeds filled/plant, primary and secondary ear mass, and grain yield increase.
  • the experimental data demonstrates which of the transgenic corn plants expressing COX Vila gene perform better than the non-transgenic control plants in the specific traits measured.
  • the COXVIIA nucleotide sequences are used to generate variant nucleotide sequences having the nucleotide sequence of the open reading frame with about 70%, 75%, 80%, 85%, 90%, and 95% nucleotide sequence identity when compared to the starting unaltered ORF nucleotide sequence of the corresponding SEQ ID NO. These functional variants are generated using a standard codon table. While the nucleotide sequence of the variants are altered, the amino acid sequence encoded by the open reading frames do not change.
  • Variant amino acid sequences of the COX VIIA polypeptides are generated.
  • one amino acid is altered.
  • the open reading frames are reviewed to determine the appropriate amino acid alteration.
  • the selection of the amino acid to change is made by consulting the protein alignment (with the other orthologs and other gene family members from various species).
  • An amino acid is selected that is deemed not to be under high selection pressure (not highly conserved) and which is rather easily substituted by an amino acid with similar chemical characteristics (i.e., similar functional side-chain).
  • an appropriate amino acid can be changed. Once the targeted amino acid is identified, the procedure outlined in the following section C is followed.
  • Variants having about 70%, 75%, 80%, 85%, 90%, and 95% nucleic acid sequence identity are generated using this method.
  • C. Additional Variant Amino Acid Sequences of COX VIIA Polypeptides In this example, artificial protein sequences are created having 80%, 85%, 90%, and 95% identity relative to the reference protein sequence. This latter effort requires identifying conserved and variable regions from the alignment set forth in Figure 1 and then the judicious application of an amino acid substitutions table. These parts will be discussed in more detail below.
  • H, C, and P are not changed in any circumstance.
  • the changes will occur with isoleucine first, sweeping N-terminal to C-terminal. Then leucine, and so on down the list until the desired target it reached. Interim number substitutions can be made so as not to cause reversal of changes.
  • the list is ordered 1-17, so start with as many isoleucine changes as needed before leucine, and so on down to methionine. Clearly many amino acids will in this manner not need to be changed.
  • L, I and V will involve a 50:50 substitution of the two alternate optimal substitutions.
  • variant amino acid sequences are written as output. Perl script is used to calculate the percent identities. Using this procedure, variants of the COXVIIA polypeptides are generating having about 80%, 85%, 90%, and 95% amino acid identity to the starting unaltered ORF nucleotide sequence of SEQ ID NO: 3.

Abstract

The present invention provides methods and compositions for making and using transgenic plants that exhibit increased yield compared to wild-type plants. Methods of the invention comprise increasing expression of a COX VIIa subunit polypeptide in plants, particularly in monocots. In some embodiments, at least one nucleotide construct comprising a nucleotide sequence encoding the COX VIIa subunit protein or a biologically active fragment or variant thereof is introduced into a plant. The invention further provides methods for producing plants with increased yield, such as increased total seed number, increased total seed weight, increased number of seeds filled, increased harvest index, or any combination thereof, which is desirable in commercial crops, including those used for seed production. A Zm COX VIIa promoter and biologically active fragments or variants thereof are also provided. The Zm COX VIIa promoter and as well as fragments and variants thereof find use in driving expression of polynucleotides of interest in a plant.

Description

OVER-EXPRESSION OF MAIZE COX Vila SUBUNIT FOR ENHANCED
YIELD
Field of the invention The invention relates to the field of molecular biology. More specifically, this invention pertains to increased yield in a plant conferred by expression of a COX Vila subunit polypeptide.
Background of the invention
Grain yield improvements by conventional breeding have nearly reached a plateau in maize. It is natural then to explore some alternative, non-conventional approaches that could be employed to obtain further yield increases. Since the harvest index in maize has remained essentially unchanged during selection for grain yield over the last hundred or so years, the yield improvements have been realized from the increased total biomass production per unit land area (Sinclair, et al., (1998) Crop Science 38:638-643; Duvick, et al., (1999) Crop Science 39:1622-1630; and Tollenaar, et al., (1999) Crop Science 39:1597-1604). This increased total biomass has been achieved by increasing planting density, which has led to adaptive phenotypic alterations, such as a reduction in leaf angle and tassel size, the former to reduce shading of lower leaves and the latter perhaps to increase harvest index (Duvick, et al., (1999) Crop Science 39:1622-1630).
Cereal grain development can be divided into two main stages, grain development and grain filling. The first stage, grain enlargement, involves early, rapid division of the zygote and nucleus. Cell division is followed by the influx of water, which drives cell extension (Briarty, et al., (1979) Annals of Botany 44:641-658). During the second stage (grain filling), cell division slows and then ceases and storage products are accumulated, until maturity when the endosperm serves its function as a starch store (Briarty, et al., (1979) Annals of Botany 44:641-658; and Bewley and Black (1994) Seeds: Physiology of Development and Germination (2d ed. New York and London: Plenum Press Ltd.), pp. 94-108). The pathway of starch synthesis in non-photosynthetic storage tissue involves the conversion of sucrose into ADPglucose (ADPG) (Smith (1999) Curr. Opin. Plant Bio. 2:223-229). Synthesis of ADPG requires a supply of hexosephosphate and ATP.
The mitochondrial respiratory chain, which consists of multiple transmembrane protein complexes, is responsible for ATP synthesis. Electron flow within these transmembrane complexes leads to the transport of protons across the inner mitochondrial membrane and the generation of a membrane potential. ATP is synthesized when protons flow back to the mitochondrial matrix through a channel in an ATP-synthesizing complex. Given the complexity of the ATP synthesis pathway, it is likely that synchronous improvement in different components of the whole pathway may allow increased ATP production, and lead to grain yield improvements.
Methods and compositions are needed in the art which can employ such components of the ATP synthesis pathway to modulate ATP synthesis in plants.
Brief summary of the invention Compositions and methods for increasing yield of a plant are provided. Compositions of the invention include expression cassettes comprising operably linked coding sequences for a cytochrome c oxidase (COX) Vila subunit polypeptide (COX Vila subunit), or a biologically active fragment thereof having COX Vila subunit activity, and an operably linked promoter. In some embodiments, the COX Vila subunit polypeptide is the Zea mays (Zm) COX Vila subunit polypeptide set forth in SEQ ID NO: 2, and the expression cassette comprises the coding sequence for the Zm COX Vila subunit polypeptide as set forth in nucleotides 81-281 of SEQ ID NO: 1 or in SEQ ID NO: 3, or a nucleotide sequence encoding the COX Vila subunit polypeptide set forth in SEQ ID NO: 2. In other embodiments, the expression cassette comprises the coding sequence for a biologically active fragment of a COX Vila subunit polypeptide. The operably linked promoter can be any promoter that drives expression in a plant cell, including, but not limited to, a constitutive promoter, an inducible promoter, or a tissue-preferred promoter. Further provided are plants, plant cells, and plant seeds comprising these expression cassettes. In some embodiments, the expression cassettes are stably incorporated into the genomes of the plants, plant cells, and plant seeds. These expression cassettes find use in the methods of the invention to increase plant yield, particularly seed yield of crop plants as described herein.
Methods of the invention comprise increasing the expression levels of a COX Vila subunit polypeptide within the cells of a plant, particularly expression of a Zm COX Vila subunit polypeptide, or biologically active fragment thereof having COX Vila subunit activity, thereby increasing yield in the plant. In this manner, the methods comprise introducing into a plant of interest at least one expression cassette of the invention, comprising a nucleotide sequence that includes a coding sequence for a COX Vila subunit polypeptide, or a biologically active fragment thereof, where the coding sequence is operably linked to a promoter that drives expression in a plant cell. The methods of the invention find use in producing plants having a phenotype that contributes to increased yield, including, but not limited to, increased total seed number, increased total seed weight, increased number of seeds filled, increased harvest index, and combinations thereof.
Additionally, a promoter sequence is provided. The Zm COX Vila subunit promoter sequence is useful for driving expression of polynucleotides of interest in a plant.
Brief description of the drawing
Figure 1 shows an alignment of the maize COX VIIa subunit protein sequence (SEQ ID NO: 2) with the Triticum aestivum (SEQ ID NO: 5), Oryza sativa (SEQ ID NO: 6), Sorghum sp. (SEQ ID NO: 7), Arabidopsis thaliana (SEQ ID NO: 8), Cyamopsis tetragonolobus (SEQ ID NO: 9), Lilium longiflorum (SEQ ID NO: 10), and Glycine max (SEQ ID NO: 11 ) COX Vila subunit protein sequences. The three underlined domains refer to the alpha-helix, turn and transmembrane domains, respectively. A consensus sequence (SEQ ID NO: 12) is also shown.
Detailed description of the invention
The present invention provides methods and compositions for increasing the level of a COX Vila subunit in a plant, thereby increasing yield in a plant or plant part thereof, compared to a wild-type or control plant. The COX Vila subunit is a member of the COX complex in mitochondrial membranes involved in oxidizing the reduced form of cytochrome c, which then contributes to the proton-motive gradient used to drive ATP synthesis in the cell. Methods of the invention comprise genetically altering a plant to express or overexpress a COX Vila subunit or a biologically active fragment or variant thereof. Increasing expression of the COX Vila subunit or fragment or variant thereof within the cells of a plant, particularly the vegetative cells, results in a plant with increased yield. "Increasing expression" means an increase in the amount of COX Vila subunit mRNA or polypeptide. For example, increased expression includes, but is not limited to, at least a 20% increase in the amount of COX Vila subunit mRNA or polypeptide within the cells of a plant as compared to the endogenous COX Vila subunit mRNA or polypeptide, such as, but not limited to, at least a 30%, 50%, 75%, 100% or 200% increase of COX Vila subunit mRNA or polypeptide. By "increased yield" is intended increased total seed number, increased total seed weight, increased number of seeds filled, increased harvest index (i.e., the ratio of grain weight to total plant weight), or any combination thereof.
According to the present invention, a COX Vila subunit or a biologically active fragment or variant thereof is a polypeptide that has COX Vila subunit activity. Without being bound by theory, it is believed that the COX Vila subunit is important in stabilizing the integrity of the COX complex, as well as playing a regulatory role in the oxidation of cytochrome c (Poyton and McEwen (1996) Annu. Rev. Biochem. 65:563-607). The COX Vila subunit may be a target for greater selective pressure for specific isoforms due to high energy needs of certain tissues or organisms, suggesting that this Vila subunit may be one of the rate-limiting subunits for COX activity (Jobson, et al., (2004) Proc. Natl. Acad. Sci. USA 101 :18064-18068), and important for enhanced energetics in cell function. A COX Vila subunit or a biologically active fragment or variant thereof that has COX Vila subunit activity increases yield in a plant, at least for total seed number, total seed weight, number of seeds filled, harvest index, and combinations thereof. Other increases in plant yield are also possible.
A polypeptide that has COX Vila subunit activity is referred to as a "COX Vila subunit," a "COX Vila subunit polypeptide," or a "COX Vila subunit protein," and a polynucleotide that encodes a polypeptide that has COX Vila subunit activity is referred to as a "COX Vila subunit polynucleotide."
In particular, COX Vila subunit polynucleotides for use in the methods of the present invention include, for example, the coding sequence of the Zm COX Vila subunit ortholog as set forth in nucleotides 81-281 of SEQ ID NO: 1 or in SEQ ID NO: 3, sequences encoding the Zm COX Vila subunit protein set forth in SEQ ID NO: 2, and fragments and variants thereof as defined below. COX Vila subunit polypeptides of the present invention include, for example, the Zm COX Vila subunit protein set forth in SEQ ID NO: 2 and biologically active fragments and variants thereof as defined herein below. The Zm COX Vila genomic sequence is set forth in SEQ ID NO: 4. The Zm COX Vila promoter sequence is set forth as nucleotides 1 through 1012 of SEQ ID NO: 4.
The Zm COX Vila subunit protein (SEQ ID NO: 2) shares a high degree of sequence identity with COX Vila subunit proteins isolated from other plant species, including, for example, COX Vila subunit proteins from Triticum aestivum (SEQ ID NO: 5), Oryza sativa (SEQ ID NO: 6), Sorghum sp. (SEQ ID NO: 7), Arabidopsis thaliana (SEQ ID NO: 8), Cyamopsis tetragonolobus (SEQ ID NO: 9), Lilium longiflorum (SEQ ID NO: 10), and Glycine max (SEQ ID NO: 11 ) (see the alignment in Figure 1 ).
According to the present invention, the term "increased yield" also means any improvement in the yield of any measured plant product, such as total seed number, total seed weight, number of seeds filled, harvest index, and the like. The increase in yield can comprise a 0.1%, 0.5%, 1%, 3%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or greater increase in measured plant product. Alternatively, the increased plant yield can comprise about a 0.5 fold, 1 fold, 2 fold, 4 fold, 8 fold, 16 fold or 32 fold increase in measured plant products. For example, an increase in the total seed weight of rice or corn from a transgenic crop comprising the COX Vila subunit polypeptide, as compared to the total seed weight from rice or corn without the COX Vila subunit polypeptide, cultivated under the same conditions, would be considered an increased yield. By increasing yield, particularly within the seeds, the nutritional value of a crop plant can be increased.
The methods of the present invention comprise increasing the expression of a COX Vila subunit in plants, particularly expression of the Zm COX Vila subunit or biologically active fragment or variant thereof having COX Vila subunit activity. Thus, in some embodiments, the methods comprise introducing into a plant of interest at least one nucleotide construct comprising a nucleotide sequence encoding the COX Vila subunit protein or a biologically active fragment or variant thereof operably linked to a promoter that drives expression in a plant cell. In particular embodiments, the COX Vila subunit is the coding sequence of the Zm COX Vila subunit ortholog as set forth in nucleotides 81-281 of SEQ ID NO: 1 or in SEQ ID NO: 3, or biologically active fragment or variant thereof and the plant is a monocot, such as maize or rice.
Increasing expression of a COX Vila subunit, for example, the Zm COX Vila subunit protein or biologically active fragment or variant thereof, within a plant increases yield in the plant.
Though the coding sequences for the Zm COX Vila subunit described herein and biologically active fragments and variants thereof can be used to increase yield in any plant of interest, the
Zm COX Vila subunit coding sequence, and fragments and variants thereof, find particular use in increasing yield in a monocot plant, for example maize or rice, as this COX Vila subunit has evolved to function within the monocot cellular environment.
An "isolated" or "purified" polynucleotide or protein, or biologically active portion thereof, is substantially or essentially free from components that normally accompany or interact with the polynucleotide or protein as found in its naturally occurring environment. Thus, an isolated or purified polynucleotide or protein 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. Optimally, an "isolated" polynucleotide is free of sequences (optimally protein encoding sequences) that naturally flank the polynucleotide (i.e., sequences located at the 5' and 3' ends of the polynucleotide) in the genomic DNA of the organism from which the polynucleotide is derived. For example, in various embodiments, the isolated polynucleotide can contain less than about 5 kb, 4 kb, 3 kb,
2 kb, 1 kb, 0.5 kb or 0.1 kb of nucleotide sequence that naturally flank the polynucleotide in genomic DNA of the cell from which the polynucleotide is derived. A protein that is substantially free of cellular material includes preparations of protein having less than about 30%, 20%, 10%, 5% or 1% (by dry weight) of contaminating protein. When the protein of the invention or biologically active portion thereof is recombinantly produced, optimally culture medium represents less than about 30%, 20%, 10%, 5% or 1% (by dry weight) of chemical precursors or non-protein-of-interest chemicals.
The use of fragments and variants of COX Vila subunit polynucleotides and polypeptides encoded thereby is also encompassed by the present invention. Depending on the context, "fragment" refers to a portion of the polynucleotide or a portion of the amino acid sequence and hence protein encoded thereby. Fragments of a polynucleotide may encode protein fragments that retain the biological activity of the original protein and hence confer COX Vila subunit activity. Thus, fragments of a nucleotide sequence may range from at least about 20 nucleotides, about 50 nucleotides, about 100 nucleotides, and up to the full-length polynucleotide encoding a COX Vila subunit polypeptide.
A fragment of a COX Vila subunit polynucleotide that encodes a biologically active portion of a COX Vila subunit polypeptide will encode at least 15, 25, 30 or 50 contiguous amino acids, or up to the total number of amino acids present in a full-length COX Vila subunit polypeptide (for example, 67 amino acids for the Zm COX Vila subunit polypeptide of SEQ ID NO: 2). A biologically active portion of a COX Vila subunit polypeptide can be prepared by isolating a portion of a COX Vila subunit polynucleotide, expressing the encoded portion of the COX Vila subunit polypeptide (e.g., by recombinant expression in vitro), and assessing the activity of the encoded portion of the COX Vila subunit polypeptide. Polynucleotides that are fragments of a COX Vila subunit polynucleotide comprise at least 16, 20, 50, 75, 100, 150, 200, 250, 300, 350, 400 or 450 contiguous nucleotides, or up to the number of nucleotides present in a full- length COX Vila subunit polynucleotide (for example, 485 contiguous nucleotides for the Zm COX Vila subunit nucleotide sequence of SEQ ID NO: 1 or 201 contiguous nucleotides for the Zm COX Vila subunit coding sequence of SEQ ID NO: 3).
The term "variants" refers to substantially similar sequences. For polynucleotides, a variant comprises a polynucleotide having deletions (i.e., truncations) at the 5' and/or 3' end; deletion and/or addition of one or more nucleotides at one or more internal sites in the native polynucleotide; and/or substitution of one or more nucleotides at one or more sites in the native polynucleotide. As used herein, a "native" polynucleotide or polypeptide comprises a naturally occurring nucleotide sequence or amino acid sequence, respectively. For polynucleotides, conservative variants include those sequences that, because of the degeneracy of the genetic code, encode the amino acid sequence of a COX Vila subunit polypeptide, for example, the Zm COX Vila subunit of SEQ ID NO: 2. 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 polynucleotides also include synthetically derived polynucleotides, such as those generated, for example, by using site-directed mutagenesis or "shuffling." Generally, variants of a particular polynucleotide, for example, the Zm COX Vila subunit sequence set forth in SEQ ID NO: 1 , or the Zm COX Vila subunit coding sequence set forth in nucleotides 81-281 of SEQ ID NO: 1 or in SEQ ID NO: 3, have at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to that particular polynucleotide as determined by sequence alignment programs and parameters as described elsewhere herein.
Variants of a particular polynucleotide (i.e., the reference polynucleotide) can also be evaluated by comparison of the percent sequence identity between the polypeptide encoded by a variant polynucleotide and the polypeptide encoded by the reference polynucleotide.
Thus, for example, in one embodiment, the variant of a COX Vila subunit polynucleotide is an isolated polynucleotide that encodes a COX Vila subunit polypeptide having a given percent identity to the Zm COX Vila subunit polypeptide of SEQ ID NO: 2. Percent sequence identity between any two polypeptides can be calculated using sequence alignment programs and parameters described elsewhere herein. Where any given pair of polynucleotides used to practice the invention is evaluated by comparison of the percent sequence identity shared by the two polypeptides they encode, the percent sequence identity between the two encoded polypeptides is at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity.
"Variant" protein is intended to mean a protein derived from a native and/or original protein by deletion (so-called truncation) of one or more amino acids at the N-terminal and/or C-terminal end of the protein; deletion and/or addition of one or more amino acids at one or more internal sites in the protein; or substitution of one or more amino acids at one or more sites in the protein. Variant proteins encompassed by the present invention are biologically active, that is they continue to possess the desired COX Vila subunit activity as described herein (i.e., increasing yield in a plant, at least for total seed number, total seed weight, number of seeds filled, harvest index, and combinations thereof). Biologically active variants of a COX Vila subunit polypeptide, for example, the Zm COX Vila subunit protein shown in SEQ ID NO: 2, will have at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the amino acid sequence for the native protein as determined by sequence alignment programs and parameters described elsewhere herein. A biologically active variant of a COX Vila subunit polypeptide, for example, the Zm COX Vila subunit protein, may differ from that polypeptide by as few as 1-15 amino acid residues, as few as 1-10, such as 6-10, as few as 5, as few as 4, 3, 2 or even 1 amino acid residue.
The COX Vila subunit polypeptides for use in practicing the invention may be altered in various ways including amino acid substitutions, deletions, truncations, and insertions. Methods for such manipulations are generally known in the art. For example, amino acid sequence variants and fragments of the Zm COX Vila subunit protein of SEQ ID NO: 2 can be prepared by mutations in the encoding polynucleotide, for example, the sequence set forth in SEQ ID NO: 1 , or the coding sequence set forth in nucleotides 81-281 of SEQ ID NO: 1 or in SEQ ID NO: 3. Methods for mutagenesis and polynucleotide alterations are well known in the art. See, for example, Kunkel (1985) Proc. Natl. Acad. Sci. USA 82:488-492; Kunkel, et al., (1987) Methods in Enzymol. 154:367-382; U.S. Patent No. 4,873,192; Walker and Gaastra, eds. (1983) Techniques in Molecular Biology (MacMillan Publishing Company, New York) and the references cited therein. Guidance as to amino acid substitutions that do not affect biological activity of the protein of interest may be found in the model of Dayhoff, et al., (1978) Atlas of Protein Sequence and Structure (Natl. Biomed. Res. Found., Washington, D. C), herein incorporated by reference. Conservative substitutions, such as exchanging one amino acid with another having similar properties, may be made. One skilled in the art will appreciate that the activity of a COX Vila subunit polypeptide can be evaluated by routine screening assays (see, e.g., Aggeler and Capaldi (1990) J. Biol. Chem. 265:16389-93; Calder and McEwen (1991 ) MoI. Microbiol. 5:1769-77), including the assays disclosed herein below.
Variant COX Vila subunit polynucleotides and COX Vila subunits for use in the methods of the invention also encompass sequences and proteins derived from a mutagenic and recombinogenic procedure such as DNA shuffling. With such a procedure, one or more different COX Vila subunit polypeptide coding sequences can be manipulated to create a new COX Vila subunit polypeptide possessing the desired properties. In 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. For example, using this approach, sequence motifs encoding a domain of interest may be shuffled between the Zm COX Vila subunit sequence of SEQ ID NO: 1 or SEQ ID NO: 3 and other known COX Vila subunit genes to obtain a new gene coding for a COX Vila subunit protein with an improved property of interest, such as a particular type of increased yield in a plant, for example, increased total seed number, increased total seed weight, increased number of seeds filled, increased harvest index, or any combination thereof. Strategies for such DNA shuffling are known in the art. See, for example, Stemmer (1994) Proc. Natl. Acad. Sci. USA 91 :10747-10751 ; Stemmer (1994) Nature 370:389-391 ; Crameri, et al., (1997) Nature Biotech. 15:436-438; Moore, et al., (1997) J. MoI. Biol. 272:336-347; Zhang, et al., (1997) Proc. Natl. Acad. Sci. USA 94:4504- 4509; Crameri, et al., (1998) Nature 391 :288-291 ; and U.S. Patent Nos. 5,605,793 and 5,837,458.
Compositions of the invention also include isolated nucleic acid molecules comprising the Zm COX Vila promoter nucleotide sequence set forth as nucleotides 1 through 1012 of SEQ ID
NO: 4. By "promoter" is intended a regulatory region of DNA usually comprising a TATA box capable of directing RNA polymerase Il to initiate RNA synthesis at the appropriate transcription initiation site for a particular coding sequence. A promoter may additionally comprise other recognition sequences generally positioned upstream or 5' to the TATA box, referred to as upstream promoter elements, which influence the transcription initiation rate. It is recognized that having identified the nucleotide sequence for the promoter region disclosed herein, it is within the state of the art to isolate and identify additional regulatory elements in the 5'-untranslated region upstream from the particular promoter region defined herein. Thus for example, the promoter region disclosed herein may further comprise upstream regulatory elements that confer tissue-preferred expression of heterologous nucleotide sequences operably linked to the disclosed promoter sequence. See particularly, Australian Patent No.
AU-A-77751/94 and U.S. Patent Nos. 5,466,785 and 5,635,618.
Fragments and variants of the disclosed Zm COX Vila promoter nucleotide sequence are also encompassed by the present invention. By "fragment" is intended a portion of the nucleotide sequence. Fragments of a promoter nucleotide sequence may retain biological activity and hence retain their transcriptional regulatory activity. Thus, for example, less than the entire promoter sequence disclosed herein may be utilized to drive expression of an operably linked nucleotide sequence of interest, such as a nucleotide sequence encoding a heterologous protein. Alternatively, fragments of a promoter nucleotide sequence that are useful as hybridization probes generally do not retain biological activity. Thus, fragments of a promoter nucleotide sequence may range from at least about 20 nucleotides, about 50 nucleotides, about 100 nucleotides, and up to the full-length promoter nucleotide sequence of the invention.
Thus, a fragment of a Zm COX Vila promoter nucleotide sequence may encode a biologically active portion of the Zm COX Vila promoter, or it may be a fragment that can be used as a hybridization probe or PCR primer using methods disclosed herein. A biologically active portion of a Zm COX Vila promoter can be prepared by isolating a portion of the Zm COX Vila promoter nucleotide sequence of the invention, and assessing the activity of the portion of the Zm COX Vila promoter. Nucleic acid molecules that are fragments of a Zm COX Vila promoter nucleotide sequence comprise at least 16, 20, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1012 nucleotides or up to the number of nucleotides present in a full-length Zm COX Vila promoter nucleotide sequence disclosed herein (i.e., 1012 nucleotides for the Zm COX Vila promoter set forth as nucleotides 1-1012 of SEQ ID NO: 4). Assays to determine the activity of a promoter sequence are well known in the art. For example, a Zm COX Vila promoter fragment or variant may be operably linked to the nucleotide sequence encoding any reporter protein, such as the β-glucuronidase protein (GUS reporter) or the luciferase protein. The DNA construct is inserted into the genome of a plant or plant cell, and the mRNA or protein level of the reporter sequence is determined. See, for example, Eulgem, et al., (1999) EMBO Journal 18:4689-4699.
The Zm COX Vila subunit polynucleotide and Zm COX Vila promoter sequence for use in the methods of the invention can be used to isolate corresponding COX Vila subunit sequences and promoter sequences from other plants, including other monocots. In this manner, methods such as PCR, hybridization, and the like can be used to identify such sequences based on their sequence homology to the Zm COX Vila subunit sequence set forth in SEQ ID NO: 1 , the Zm COX Vila subunit coding sequence set forth in nucleotides 81-281 of SEQ ID NO: 1 or in SEQ ID NO: 3, or the Zm COX Vila promoter sequence set forth as nucleotides 1 through 1012 of SEQ ID NO: 4. Sequences isolated based on their sequence identity to the entire Zm COX Vila subunit nucleotide sequence or Zm COX Vila promoter sequence set forth herein, or to variants and fragments thereof, are encompassed by the present invention. Such sequences include sequences that are orthologs of the disclosed sequences. "Orthologs" is intended to mean genes derived from a common ancestral gene and which are found in different species as a result of speciation. Genes found in different species are considered orthologs when their nucleotide sequences and/or their encoded protein sequences share at least 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence identity. Functions of orthologs are often highly conserved among species. Thus, isolated polynucleotides that encode for a COX Vila subunit polypeptide, or isolated polynucleotides that confer promoter activity, and which hybridize under stringent conditions to the respective Zm COX Vila subunit sequence of SEQ ID NO: 1 , the Zm COX Vila subunit coding sequence set forth in nucleotides 81-281 of SEQ ID NO: 1 or in SEQ ID NO: 3, the Zm COX Vila promoter sequence set forth as nucleotides 1 through 1012 of SEQ ID NO: 4, or to variants or fragments thereof, can be used to practice the present invention. In a PCR approach, 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 and are disclosed in Sambrook, et al., (1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, New York). See also, Innis, et ai, eds. (1990) PCR Protocols: A Guide to Methods and Applications (Academic Press, New York); Innis and Gelfand, eds. (1995) PCR Strategies (Academic Press, New York); and Innis and Gelfand, eds. (1999) PCR Methods Manual (Academic Press, New York). Known methods of PCR include, but are not limited to, methods using paired primers, nested primers, single specific primers, degenerate primers, gene-specific primers, vector-specific primers, partially mismatched primers, and the like.
In hybridization techniques, all or part of a known polynucleotide is used as a probe that selectively hybridizes to other corresponding polynucleotides 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 32P, or any other detectable marker. Thus, for example, probes for hybridization can be made by labeling synthetic oligonucleotides based on the Zm COX Vila subunit nucleotide sequence of SEQ ID NO: 1 , or the Zm COX Vila subunit coding sequence set forth in nucleotides 81-281 of SEQ ID NO: 1 or in SEQ ID NO: 3. Methods for preparation of probes for hybridization and for construction of cDNA and genomic libraries are generally known in the art and are disclosed in Sambrook, et al., (1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, New York).
For example, the entire Zm COX Vila subunit polynucleotide disclosed in SEQ ID NO: 1 , nucleotides 81-281 of SEQ ID NO: 1 , or SEQ ID NO: 3, or one or more portions thereof, may be used as a probe capable of specifically hybridizing to corresponding COX Vila subunit polynucleotides and messenger RNAs. To achieve specific hybridization under a variety of conditions, such probes include sequences that are unique among COX Vila subunit polynucleotide sequences and are optimally at least about 10 nucleotides in length, and most optimally at least about 20 nucleotides in length. Such probes may be used to amplify corresponding COX Vila subunit polynucleotides from a chosen plant by PCR. This technique may be used to isolate additional COX Vila subunit coding sequences from a desired plant or as a diagnostic assay to determine the presence of COX Vila subunit coding sequences in a plant. Hybridization techniques include hybridization screening of plated DNA libraries (either plaques or colonies; see, for example, Sambrook, et al., (1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, New York).
Hybridization of such sequences may be carried out under stringent conditions. By "stringent conditions" or "stringent hybridization conditions" is intended conditions under which a probe will hybridize to its target sequence to a detectably greater degree than to other sequences (e.g., at least 2-fold over background). Stringent conditions are sequence-dependent and will be different in different circumstances. By controlling the stringency of the hybridization and/or washing conditions, target sequences that are 100% complementary to the probe can be identified (homologous probing). Alternatively, stringency conditions can be adjusted to allow some mismatching in sequences so that lower degrees of similarity are detected (heterologous probing). Generally, a probe is less than about 1000 nucleotides in length, optimally less than 500 nucleotides in length.
Typically, stringent conditions will be those in which the salt concentration is less than about 1.5 M Na ion, typically about 0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 300C for short probes (e.g., 10 to 50 nucleotides) and at least about 600C for long probes (e.g., greater than 50 nucleotides). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. Exemplary low stringency conditions include hybridization with a buffer solution of 30 to 35% formamide, 1 M NaCI, 1% SDS (sodium dodecyl sulphate) at 37°C, and a wash in 1X to 2X SSC (2OX SSC = 3.0 M NaCI/0.3 M trisodium citrate) at 50 to 55°C. Exemplary moderate stringency conditions include hybridization in 40 to 45% formamide, 1.0 M NaCI, 1 % SDS at 37°C, and a wash in 0.5X to 1X SSC at 55 to 600C. Exemplary high stringency conditions include hybridization in 50% formamide, 1 M NaCI, 1% SDS at 37°C, and a wash in 0.1 X SSC at 60 to 65°C. Optionally, wash buffers may comprise about 0.1% to about 1% SDS. Duration of hybridization is generally less than about 24 hours, usually about 4 to about 12 hours. The duration of the wash time will be at least a length of time sufficient to reach equilibrium.
Specificity is typically the function of post-hybridization washes, the critical factors being the ionic strength and temperature of the final wash solution. For DNA-DNA hybrids, the Tm can be approximated from the equation of Meinkoth and Wahl (1984) Anal. Biochem. 138:267-284: Tm = 81.50C + 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 of the hybrid in base pairs. The Tm is the temperature (under defined ionic strength and pH) at which 50% of a complementary target sequence hybridizes to a perfectly matched probe. Tm is reduced by about 1 °C for each 1 % of mismatching; thus, Tm, hybridization, and/or wash conditions can be adjusted to hybridize to sequences of the desired identity. For example, if sequences with >90% identity are sought, the Tm can be decreased 100C. Generally, stringent conditions are selected to be about 5°C lower than the thermal melting point (Tm) for the specific sequence and its complement at a defined ionic strength and pH. However, severely stringent conditions can utilize a hybridization and/or wash at 1 , 2, 3 or 4°C lower than the thermal melting point (Tm); moderately stringent conditions can utilize a hybridization and/or wash at 6, 7, 8, 9 or 100C lower than the thermal melting point (Tm); low stringency conditions can utilize a hybridization and/or wash at 11 , 12, 13, 14, 15 or 200C lower than the thermal melting point (Tm). Using the equation, hybridization and wash compositions, and desired Tm, those of ordinary skill will understand that variations in the stringency of hybridization and/or wash solutions are inherently described. If the desired degree of mismatching results in a Tm of less than 45°C (aqueous solution) or 32°C (formamide solution), it is optimal to increase the SSC concentration so that a higher temperature can be used. An extensive guide to the hybridization of nucleic acids is found in Tijssen (1993) Laboratory Techniques in Biochemistry and Molecular Biology— Hybridization with Nucleic Acid Probes, Part I, Chapter 2 (Elsevier, New York); and Ausubel, et al., eds. (1995) Current Protocols in Molecular Biology, Chapter 2 (Greene Publishing and Wiley-lnterscience, New York). See Sambrook, et al., (1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, New York).
The following terms are used to describe the sequence relationships between two or more polynucleotides or polypeptides: (a) "reference sequence", (b) "comparison window", (c) "sequence identity", and, (d) "percentage of sequence identity."
(a) As used herein, "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 of a full-length cDNA or gene sequence, or the complete cDNA or gene sequence.
(b) As used herein, "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 of the two polynucleotides. Generally, the comparison window is at least 20 contiguous nucleotides in length, and optionally can be 30, 40, 50, 100 or longer. Those of skill in the art understand that to avoid a high similarity to a reference sequence due to inclusion of gaps in the polynucleotide sequence a gap penalty is typically introduced and is subtracted from the number of matches.
Methods of alignment of sequences for comparison are well known in the art. Thus, the determination of percent sequence identity between any two sequences can be accomplished using a mathematical algorithm. Non-limiting examples of such mathematical algorithms are the algorithm of Myers and Miller (1988) CABIOS 4:11-17; the local alignment algorithm of
Smith, et al., (^W ) AdV. Appl. Math. 2:482; the global alignment algorithm of Needleman and
Wunsch (1970) J. MoI. Biol. 48:443-453; the search-for-local alignment method of Pearson and Lipman (1988) Proc. Natl. Acad. Sci. 85:2444-2448; the algorithm of Karlin and Altschul (1990)
Proc. Natl. Acad. Sci. USA 87:2264-2268, modified as in Karlin and Altschul (1993) Proc. Natl.
Acad. Sci. USA 90:5873-5877.
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, Mountain View, California); the ALIGN program (Version 2.0) and GAP, BESTFIT, BLAST, FASTA, and TFASTA in the GCG Wisconsin Genetics Software Package, Version 10 (available from Accelrys Inc., 9685 Scranton Road, San Diego, California, USA). Alignments using these programs can be performed using the default parameters. The CLUSTAL program is well described by Higgins, et al., (1988) Gene 73:237-244 (1988); Higgins, et al., (1989) CABIOS 5:151-153; Corpet, et al., (1988) Nucleic Acids Res. 16:10881-90; Huang, et al., (1992) CABIOS 8:155-65; and Pearson, et al., (1994) Meth. MoI. Biol. 24:307-331. The ALIGN program is based on the algorithm of Myers and Miller (1988) supra. A PAM 120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used with the ALIGN program when comparing amino acid sequences. The BLAST programs of Altschul, et al., (1990) J. MoI. Biol. 215:403 are based on the algorithm of Karlin and Altschul (1990) supra. BLAST nucleotide searches can be performed with the BLASTN program, score = 100, wordlength = 12, to obtain nucleotide sequences homologous to a nucleotide sequence encoding a COX Vila subunit for use in the methods of the present invention. BLAST protein searches can be performed with the BLASTX program, score = 50, wordlength = 3, to obtain amino acid sequences homologous to a COX Vila subunit for use in the methods of the present invention. To obtain gapped alignments for comparison purposes, Gapped BLAST (in BLAST 2.0) can be utilized as described in Altschul, et al., (1997) Nucleic Acids Res. 25:3389. Alternatively, PSI-BLAST (in BLAST 2.0) can be used to perform an iterated search that detects distant relationships between molecules. See, Altschul, et al., (1997) supra. When utilizing BLAST, Gapped BLAST, PSI-BLAST, the default parameters of the respective programs (e.g., BLASTN for nucleotide sequences, BLASTX for proteins) can be used. BLAST software is publicly available on the NCBI website. Alignment may also be performed manually by inspection.
Unless otherwise stated, sequence identity/similarity values provided herein refer to the value obtained using GAP Version 10 using the following parameters: % identity and % similarity for a nucleotide sequence using GAP Weight of 50 and Length Weight of 3, and the nwsgapdna.cmp scoring matrix; % identity and % similarity for an amino acid sequence using GAP Weight of 8 and Length Weight of 2, and the BLOSUM62 scoring matrix; or any equivalent program thereof. By "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 GAP Version 10.
GAP uses the algorithm of Needleman and Wunsch (1970) J. MoI. Biol. 48:443-453, to find the alignment of two complete sequences that maximizes the number of matches and minimizes the number of gaps. GAP considers all possible alignments and gap positions and creates the alignment with the largest number of matched bases and the fewest gaps. It allows for the provision of a gap creation penalty and a gap extension penalty in units of matched bases. GAP must make a profit of gap creation penalty number of matches for each gap it inserts. If a gap extension penalty greater than zero is chosen, GAP must, in addition, make a profit for each gap inserted of the length of the gap times the gap extension penalty. Default gap creation penalty values and gap extension penalty values in Version 10 of the GCG Wisconsin Genetics Software Package for protein sequences are 8 and 2, respectively. For nucleotide sequences the default gap creation penalty is 50 while the default gap extension penalty is 3. The gap creation and gap extension penalties can be expressed as an integer selected from the group of integers consisting of from 0 to 200. Thus, for example, the gap creation and gap extension penalties can be 0, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65 or greater.
GAP presents one member of the family of best alignments. There may be many members of this family, but no other member has a better quality. GAP displays four figures of merit for alignments: Quality, Ratio, Identity, and Similarity. The Quality is the metric maximized in order to align the sequences. Ratio is the quality divided by the number of bases in the shorter segment. Percent Identity is the percent of the symbols that actually match. Percent Similarity is the percent of the symbols that are similar. Symbols that are across from gaps are ignored. A similarity is scored when the scoring matrix value for a pair of symbols is greater than or equal to 0.50, the similarity threshold. The scoring matrix used in Version 10 of the GCG Wisconsin Genetics Software Package is BLOSUM62 (see, Henikoff and Henikoff (1989) Proc. Natl. Acad. Sci. USA 89:10915).
(c) As used herein, "sequence identity" or "identity" in the context of two polynucleotides or polypeptide sequences makes reference to the residues in the two sequences that are the same when aligned for maximum correspondence over a specified comparison window. When 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 of the molecule. When sequences differ in conservative substitutions, the percent sequence identity may be adjusted upwards to correct for the conservative nature of the 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, for example, as implemented in the program PC/GENE (Intelligenetics, Mountain View, California).
(d) As used herein, "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 of the 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.
The use of the term "polynucleotide" is not intended to be limited to polynucleotides comprising DNA. Those of ordinary skill in the art will recognize that polynucleotides can comprise ribonucleotides and combinations of ribonucleotides and deoxyribonucleotides. Such deoxyribonucleotides and ribonucleotides include both naturally occurring molecules and synthetic analogues. Thus, polynucleotides also encompass all forms of sequences including, but not limited to, single-stranded forms, double-stranded forms, hairpins, stem-and-loop structures, and the like.
The COX Vila subunit polynucleotide, for example, the Zm COX Vila subunit polynucleotide or fragment or variant thereof, can be provided in expression cassettes for expression in the plant of interest. The cassette will include 5' and 3' regulatory sequences operably linked to the COX Vila subunit polynucleotide. Operably linked" is intended to mean a functional linkage between two or more elements. For example, an operable linkage between a polynucleotide of interest and a regulatory sequence (i.e., a promoter) is functional link that allows for expression of the polynucleotide of interest. Operably linked elements may be contiguous or non-contiguous. When used to refer to the joining of two protein coding regions, by "operably linked" is intended that the coding regions are in the same reading frame. The cassette may additionally contain at least one additional gene to be cotransformed into the plant. Alternatively, the additional gene(s) can be provided on multiple expression cassettes. Such an expression cassette is provided with a plurality of restriction sites and/or recombination sites for insertion of the COX Vila subunit polynucleotide to be under the transcriptional regulation of the regulatory regions. The expression cassette may additionally contain other genes, including other selectable marker genes.
The expression cassette will include in the 5'-3' direction of transcription a transcriptional and translational initiation region (i.e., a promoter), a COX Vila subunit polynucleotide of the invention, for example, SEQ ID NO: 1 , nucleotides 81-281 of SEQ ID NO: 1 , SEQ ID NO: 3, or fragment or variant thereof, and a transcriptional and translational termination region (i.e., termination region) functional in plants. The regulatory regions (i.e., promoters, transcriptional regulatory regions, and translational termination regions) and/or the COX Vila subunit polynucleotide may be native/analogous to the host cell or to each other. Alternatively, the regulatory regions and/or the COX Vila subunit polynucleotide may be heterologous to the host cell or to each other. As used herein, "heterologous" in reference to a sequence is a sequence that originates from a foreign species, or, if from the same species, is substantially modified from its native form in composition and/or genomic locus by deliberate human intervention. For example, a promoter operably linked to a heterologous polynucleotide is from a species different from the species from which the polynucleotide was derived, or, if from the same/analogous species, one or both are substantially modified from their original form and/or genomic locus, or the promoter is not the native promoter for the operably linked polynucleotide. While it may be optimal to express the COX Vila subunit polynucleotides using heterologous promoters, the native promoter sequences may be used. Such constructs can change expression levels of the encoded polypeptide in the plant or plant cell. Thus, the phenotype of the plant or cell can be altered.
The termination region may be native with the transcriptional initiation region, may be native with the operably linked COX Vila subunit polynucleotide of interest, may be native with the plant host, or may be derived from another source (i.e., foreign or heterologous) to the promoter, the COX Vila subunit polynucleotide of interest, the plant host, or any combination thereof. Convenient termination regions for use in the present invention include those available from the Ti-plasmid of A. tumefaciens, such as the octopine synthase and nopaline synthase termination regions. See also, Guerineau, et al., (1991 ) MoI. Gen. Genet. 262:141- 144; Proudfoot (1991 ) Cell 64:671-674; Sanfacon, et al., (1991 ) Genes Dev. 5:141-149; Mogen, et al., (1990) Plant Cell 2:1261-1272; Munroe, et al., (1990) Gene 91 :151-158; Ballas, et al., (1989) Nucleic Acids Res. 17:7891-7903; and Joshi, et al., (1987) Nucleic Acids Res. 15:9627-9639. Also included is the terminator region of the protease inhibitor Il gene from Solanum tuberosum (nucleotides 2 through 310; see, An, et al., (1989) Plant Ce// 1 :1 15-122).
Methods are known in the art for increasing expression of a polypeptide of interest in a plant or plant cell, for example, by inserting into the polypeptide coding sequence one or two G/C-rich codons (such as GCG or GCT) immediately adjacent to and downstream of the initiating methionine ATG codon. Where appropriate, the COX Vila subunit polynucleotides may be optimized for increased expression in the transformed plant. See, for example, Campbell and Gowri (1990) Plant Physiol. 92:1-1 1 for a discussion of host-preferred codon usage. Methods are available in the art for synthesizing plant-preferred genes. See, for example, U.S. Patent Nos. 5,380,831 , and 5,436,391 , and Murray, et al., (1989) Nucleic Acids Res. 17:477-498, herein incorporated by reference. Embodiments comprising such modifications are also a feature of the invention.
Additional sequence modifications are known to enhance gene expression in a particular plant host. These include elimination of sequences encoding spurious polyadenylation signals, exon-intron splice site signals, transposon-like repeats, and other such well-characterized sequences that may be deleterious to gene expression. The G-C content of the sequence may be adjusted to levels average for a given plant host, as calculated by reference to known genes expressed in the host cell. When possible, the sequence is modified to avoid predicted hairpin secondary mRNA structures. The expression cassettes may additionally contain 5' leader sequences. Such leader sequences can act to enhance translation. Translation leaders are known in the art and include: picornavirus leaders, for example, EMCV leader (Encephalomyocarditis 5' noncoding region) (Elroy-Stein, et al., (1989) Proc. Natl. Acad. Sci. USA 86:6126-6130); potyvirus leaders, for example, TEV leader (Tobacco Etch Virus) (GaIMe, et ah, (1995) Gene 165(2):233- 238), MDMV leader (Maize Dwarf Mosaic Virus) {Virology 154:9-20), and human immunoglobulin heavy-chain binding protein (BiP) (Macejak, et ai, (1991 ) Nature 353:90-94); untranslated leader from the coat protein mRNA of alfalfa mosaic virus (AMV RNA 4) (Jobling, et al., (1987) Nature 325:622-625); tobacco mosaic virus leader (TMV) (GaIMe, et al., (1989) in Molecular Biology of RNA, ed. Cech (Liss, New York), pp. 237-256); and maize chlorotic mottle virus leader (MCMV) (Lommel, et al., (1991 ) Virology 81 :382-385). See also, Della-Cioppa, et al., (1987) Plant Physiol. 84:965-968.
In preparing the expression cassette, the various polynucleotide fragments may be manipulated, so as to provide for sequences to be in the proper orientation and, as appropriate, in the proper reading frame. Toward this end, adapters or linkers may be employed to join the fragments or other manipulations may be involved to provide for convenient restriction sites, removal of superfluous material such as the removal of restriction sites, or the like. For this purpose, in vitro mutagenesis, primer repair, restriction, annealing, resubstitutions, for example, transitions and transversions, may be involved. Standard recombinant DNA and molecular cloning techniques used herein are well known in the art and are described more fully, for example, in Sambrook, et al., Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press; Plainview, New York).
A number of promoters can be used in the practice of the invention, including the native promoter of the COX Vila subunit polynucleotide sequence of interest. The promoters can be selected for increased expression. The COX Vila subunit polynucleotides of interest can be combined with constitutive, tissue-preferred, or other promoters for expression in plants.
Such constitutive promoters include, for example, the core promoter of the Rsyn7 promoter and other constitutive promoters disclosed in WO 99/43838 and U.S. Patent No. 6,072,050; the core CaMV 35S promoter (Odell, et al., (1985) Nature 313:810-812); rice actin (McElroy, et al., (1990) Plant Cell 2:163-171 ); ubiquitin (UBI) (Christensen, et al., (1989) Plant MoI. Biol. 12:619-632 and Christensen, et al., (1992) Plant MoI. Biol. 18:675-689); pEMU (Last, et al., (1991 ) Theor. Appl. Genet. 81 :581-588); MAS (Velten, et al., (1984) EMBO J. 3:2723-2730); ALS promoter (U.S. Patent No. 5,659,026), and the like. Other constitutive promoters include, for example, those described in U.S. Patent Nos. 5,608,149; 5,608,144; 5,604,121 ; 5,569,597; 5,466,785; 5,399,680; 5,268,463; 5,608,142; and 6,177,611.
Tissue-preferred promoters can be utilized to target enhanced COX Vila subunit polypeptide expression within a particular plant tissue. Tissue-preferred promoters include those disclosed in Yamamoto, et al., (1997) Plant J. 12(2):255-265; Kawamata, et al., (1997) Plant Cell Physiol. 38(7):792-803; Hansen, et al., (1997) MoI. Gen Genet. 254(3):337-343; Russell, et al., (1997) Transgenic Res. 6(2):157-168; Rinehart, et al., (1996) Plant Physiol. 1 12(3):1331-1341 ; Van Camp, et al., (1996) Plant Physiol. 112(2):525-535; Canevascini, et al., (1996) Plant Physiol. 112(2):513-524; Yamamoto, et al., (1994) Plant Cell Physiol. 35(5):773-778; Lam (1994) Results Probl. Cell Differ. 20:181-196; Orozco, et al., (1993) Plant MoI Biol. 23(6):1129- 1138; Matsuoka, et al., (1993) Proc Natl. Acad. Sci. USA 90(20):9586-9590; and Guevara- Garcia, et al., (1993) Plant J. 4(3):495-505.
The expression cassette can also comprise a selectable marker gene for the selection of transformed cells. Selectable marker genes are utilized for the selection of transformed cells or tissues. Marker genes include genes encoding antibiotic resistance, such as those encoding neomycin phosphotransferase Il (NEO) and hygromycin phosphotransferase (HPT), as well as genes conferring resistance to herbicidal compounds, such as glufosinate ammonium, bromoxynil, imidazolinones, and 2,4-dichlorophenoxyacetate (2,4-D). Additional selectable markers include phenotypic markers such as β-galactosidase and fluorescent proteins such as green fluorescent protein (GFP) (Su, et al., (2004) Biotechnol. Bioeng. 85:610-9 and Fetter, et al., (2004) Plant Cell 16:215-28), cyanofluorescent protein (CYP) (Bolte, et al., (2004) J. Cell Science 117:943-54 and Kato, et al., (2002) Plant Physiol 129:913-42), and yellow fluorescent protein (PhiYFP™ from Evrogen, see, Bolte, et al., (2004) J. Cell Science 117:943-54). For additional selectable markers, see generally, Yarranton (1992) Curr. Opin. Biotech. 3:506-511 ; Christopherson, et al., (1992) Proc. Natl. Acad. Sci. USA 89:6314-6318; Yao, et al., (1992) Cell 71 :63-72; Reznikoff (1992) MoI. Microbiol. 6:2419-2422; Barkley, et al., (1980) in The Operon, pp. 177-220; Hu, et al., (1987) Ce// 48:555-566; Brown, et al., (1987) Ce// 49:603-612; Figge, et al., (1988) Ce// 52:713-722; Deuschle, et al., (1989) Proc. Natl. Acad. Sci. USA 86:5400-5404; Fuerst, et al., (1989) Proc. Natl. Acad. Sci. USA 86:2549-2553; Deuschle, et al., (1990) Science 248:480- 483; Gossen (1993) Ph.D. Thesis, University of Heidelberg; Reines, et al., (1993) Proc. Natl. Acad. Sci. USA 90:1917-1921 ; Labow, et al., (1990) MoI. Cell. Biol. 10:3343-3356; Zambretti, et al., (1992) Proc. Natl. Acad. Sci. USA 89:3952-3956; Bairn, et al., (1991) Proc. Natl. Acad. Sci. USA 88:5072-5076; Wyborski, et al., (1991 ) Nucleic Acids Res. 19:4647-4653; Hillenand- Wissman (1989) Topics MoI. Struc. Biol. 10:143-162; Degenkolb, et al., (1991) Antimicrob. Agents Chemother. 35:1591-1595; Kleinschnidt, et al., (1988) Biochemistry 27:1094-1104; Bonin (1993) Ph.D. Thesis, University of Heidelberg; Gossen, et al., (1992) Proc. Natl. Acad. ScL USA 89:5547-5551 ; Oliva, et al., (1992) Antimicrob. Agents Chemother. 36:913-919; Hlavka, et al., (1985) Handbook of Experimental Pharmacology, Vol. 78 (Springer-Verlag, Berlin); Gill, et al., (1988) Nature 334:721-724. Such disclosures are herein incorporated by reference. The above list of selectable marker genes is not meant to be limiting. Any selectable marker gene can be used in the present invention.
The present invention also provides a method for increasing the concentration and/or activity of a COX Vila subunit polypeptide, for example, the Zm COX Vila subunit protein of SEQ ID NO: 2 or biologically active fragment or variant thereof, in a plant. In general, concentration and/or activity is increased by at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% relative to a wild-type or control plant, plant part, or cell that did not have a COX Vila subunit sequence of the invention introduced. Increasing the concentration and/or activity of a COX Vila subunit polypeptide in the present invention may occur during and/or subsequent to growth of the plant to the desired stage of development. In specific embodiments, COX Vila subunit polypeptides such as the Zm COX Vila subunit protein or fragment or variant thereof are increased in monocots, including, but not limited to, maize and rice.
The expression level of the COX Vila subunit polypeptide can be measured directly, for example, by assaying for the level of the COX Vila subunit polypeptide in the plant, or indirectly, for example, by measuring the COX Vila subunit activity of the polypeptide in the plant. Methods for determining COX Vila subunit activity of a polypeptide of interest are described elsewhere herein. Additionally, the level of the COX Vila subunit mRNA can be measured by methods well known to one of skill in the art, including, for example, Northern analysis.
In specific embodiments, the COX Vila subunit polypeptide or polynucleotide is introduced into the plant cell. As discussed elsewhere herein, many methods are known the art for providing a polypeptide to a plant including, but not limited to, direct introduction of the polypeptide into the plant and introducing into the plant (transiently or stably) a polynucleotide construct encoding a polypeptide having COX Vila subunit activity. Subsequently, a plant cell having the introduced sequence of the invention is selected using methods known to those of skill in the art such as, but not limited to, Southern blot analysis, DNA sequencing, PCR analysis, or phenotypic analysis (for increased yield, such as increased total seed number, increased total seed weight, increased number of seeds filled, increased harvest index, or any combination thereof). A plant or plant part modified by the foregoing embodiments is grown under plant forming conditions for a time sufficient to increase the concentration and/or activity of the COX Vila subunit polypeptide, for example, the Zm COX Vila subunit protein or fragment or variant thereof, in the plant. Plant forming conditions are well known in the art and discussed briefly elsewhere herein.
It is also recognized that the level and/or activity of the COX Vila subunit polypeptide may be increased by employing a polynucleotide that is not capable of directing, in a transformed plant, the expression of a protein or an RNA. For example, COX Vila subunit polynucleotides such as the Zm COX Vila subunit gene may be used to design polynucleotide constructs that can be employed in methods for altering or mutating a genomic nucleotide sequence in an organism. Such polynucleotide constructs include, but are not limited to, RNA:DNA vectors, RNA:DNA mutational vectors, RNA:DNA repair vectors, mixed-duplex oligonucleotides, self- complementary RNA:DNA oligonucleotides, and recombinogenic oligonucleobases. Such nucleotide constructs and methods of use are known in the art. See, U.S. Patent Nos. 5,565,350; 5,731 ,181 ; 5,756,325; 5,760,012; 5,795,972; and 5,871 ,984; all of which are herein incorporated by reference. See also, WO 98/49350, WO 99/07865, WO 99/25821 , and Beetham, et al., (1999) Proc. Natl. Acad. Sci. USA 96:8774-8778; herein incorporated by reference. Thus, the level and/or activity of a COX Vila subunit polypeptide, for example, the Zm COX Vila subunit protein of SEQ ID NO: 2 or fragment or variant thereof, may be increased by altering the gene encoding the COX Vila subunit polypeptide or its promoter. See, for example, Kmiec, U.S. Patent 5,565,350; Zarling, et al., PCT/US93/03868. Thus mutagenized plants that carry mutations in COX Vila subunit genes, where the mutations increase expression of the COX Vila subunit gene, for example, the Zm COX Vila subunit gene, or increase the COX Vila subunit activity of the encoded COX Vila subunit polypeptide, for example, the Zm COX Vila subunit protein, are provided.
It is therefore recognized that methods of the present invention do not depend on the incorporation of an entire polynucleotide into the genome, only that the plant or cell thereof is altered as a result of the introduction of the polynucleotide into a cell. In one embodiment of the invention, the genome may be altered following the introduction of a COX Vila subunit polynucleotide, such as the Zm COX Vila subunit sequence of SEQ ID NO: 1 , or the Zm COX Vila subunit coding sequence set forth in nucleotides 81-281 of SEQ ID NO: 1 or in SEQ ID NO: 3, into a cell. For example, the polynucleotide, or any part thereof, may incorporate into the genome of the plant. Alterations to the genome of the present invention include, but are not limited to, additions, deletions, and substitutions of nucleotides into the genome. While the methods of the present invention do not depend on additions, deletions, and substitutions of any particular number of nucleotides, it is recognized that such additions, deletions, or substitutions comprises at least one nucleotide. Accordingly, in some embodiments, the methods of the invention involve introducing a COX
Vila subunit polypeptide or polynucleotide into a plant. "Introducing" is intended to mean presenting to the plant the COX Vila subunit polynucleotide or polypeptide in such a manner that the sequence gains access to the interior of a cell of the plant. The methods of the invention do not depend on a particular method for introducing a sequence into a plant, only that the polynucleotide or polypeptides gains access to the interior of at least one cell of the plant. Methods for introducing COX Vila subunit polynucleotide or polypeptides into plants are known in the art including, but not limited to, stable transformation methods, transient transformation methods, and virus-mediated methods.
"Stable transformation" is intended to mean that the nucleotide construct introduced into a plant integrates into the genome of the plant and is capable of being inherited by the progeny thereof. "Transient transformation" is intended to mean that a polynucleotide is introduced into the plant and does not integrate into the genome of the plant or a polypeptide is introduced into a plant.
Transformation protocols as well as protocols for introducing COX Vila subunit polypeptides or polynucleotide sequences into plants may vary depending on the type of plant or plant cell targeted for transformation. In some embodiments, the methods of the present invention involve transformation protocols suitable for introducing COX Vila subunit polypeptides or polynucleotide sequences into monocots.
Suitable methods of introducing COX Vila subunit polypeptides and polynucleotides into plant cells include microinjection (Crossway, et al., (1986) Biotechniques 4320-334), electroporation
(Riggs, et al., (1986) Proc. Natl. Acad. Sci. USA 83:5602-5606, Agrobacterium-meώated transformation (U.S. Patent No. 5,563,055 and U.S. Patent No. 5,981 ,840), direct gene transfer (Paszkowski, et al., (1984) EMBO J. 3:2717-2722), and ballistic particle acceleration
(see, e.g., U.S. Patent Nos. 4,945,050; U.S. Patent No. 5,879,918; U.S. Patent No. 5,886,244; and, 5,932,782; Tomes, et al., (1995) in Plant Cell, Tissue, and Organ Culture: Fundamental
Methods, ed. Gamborg and Phillips (Springer-Verlag, Berlin); McCabe, et al., (1988)
Biotechnology 6:923-926); and Led transformation (WO 00/28058). Also see, Weissinger, et al., (1988) Ann. Rev. Genet. 22:421-477; Sanford, et al., (1987) Particulate Science and
Technology 5:27-37 (onion); Datta, et al., (1990) Biotechnology 8:736-740 (rice); Klein, et al., (1988) Proc. Natl. Acad. Sci. USA 85:4305-4309 (maize); Klein, et al., (1988) Biotechnology
6:559-563 (maize); U.S. Patent Nos. 5,240,855; 5,322,783; and, 5,324,646; Klein, et al.,
(1988) Plant Physiol. 91 :440-444 (maize); Fromm, et al., (1990) Biotechnology 8:833-839 (maize); Hooykaas-Van Slogteren, et al., (1984) Nature (London) 31 1 :763-764; U.S. Patent No. 5,736,369 (cereals); Bytebier, et al., (1987) Proc. Natl. Acad. Sci. USA 84:5345-5349 (Liliaceae); De Wet, et al., (1985) in The Experimental Manipulation of Ovule Tissues, ed. Chapman, et al., (Longman, New York), pp. 197-209 (pollen); Kaeppler, et al., (1990) Plant Cell Reports 9:415-418 and Kaeppler, et al., (1992) Theor. Appl. Genet. 84:560-566 (whisker- mediated transformation); D'Halluin, et al., (1992) Plant Ce// 4:1495-1505 (electroporation); Li, et al., (1993) Plant Cell Reports 12:250-255 and Christou and Ford (1995) Annals of Botany 75:407-413 (rice); Osjoda, et al., (1996) Nature Biotechnology 14:745-750 (maize via Agrobacterium tumefaciens); all of which are herein incorporated by reference.
Efficient Brassica transformation methods that do not involve the use of Agrobacterium are well know in the art. For example, U.S. Patents 6,051 ,756 and 6,495,741 , and published U.S. Patent Applications 2003/0093840 and 2004/0045056 (all of which are herein incorporated by reference) describe transformation of seedling hypocotyls by particle bombardment. United States Patent 6,297,056 (herein incorporated by reference) describes the transformation of cotyledonary petioles. United States Patent 6,515,206 and published U.S. Patent Application 2003/0200568 (both of which are herein incorporated by reference) describe the use of transformation of plastids in true leaves. Chen and Beversdorf (Theor. Appl. Genet. (1994) 88:187-192) describe a biolistic transformation procedure of microspore-derived hypocotyls involving DNA imbibition. Fukuoka, et al., Plant Cell Reports (1998) 17:323-328 describe biolistic transformation of fresh microspores. Nehlin et al. (Plant Physiol. (2000) 156:175-183) describe transient biolistic transformation of pre-incubated microspores.
In specific embodiments, increased total seed number, increased total seed weight, increased number of seeds filled, increased harvest index, or any combination thereof, compared to a wild-type or control plant can be provided to a plant using a variety of transient transformation methods. Such transient transformation methods include, but are not limited to, the introduction of the COX Vila subunit polypeptide, for example, the Zm COX Vila subunit protein of SEQ ID NO: 2 or biologically active fragment or variant thereof, directly into the plant or the introduction of a transcript into the plant. Such methods include, for example, microinjection or particle bombardment. See, for example, Crossway, et al., (1986) MoI Gen.
Genet. 202:179-185; Nomura, et al., (1986) Plant Sci. 44:53-58; Hepler, et al., (1994) Proc.
Natl. Acad. Sci. 91 :2176-2180 and Hush, et al., (1994) The Journal of Cell Science 107:775-
784, all of which are herein incorporated by reference. Alternatively, a COX Vila subunit polynucleotide, for example, the Zm COX Vila subunit sequence of SEQ ID NO: 1 , the Zm
COX Vila subunit coding sequence set forth in nucleotides 81-281 of SEQ ID NO: 1 or in SEQ
ID NO: 3, or fragment or variant thereof encoding a COX Vila subunit polypeptide, can be transiently transformed into the plant using techniques known in the art. Such techniques include viral vector systems and the precipitation of the polynucleotide in a manner that precludes subsequent release of the DNA. Thus, the transcription from the particle-bound DNA can occur, but the frequency with which it is released to become integrated into the genome is greatly reduced. Such methods include the use of particles coated with polyethylimine (PEI; Sigma #P3143).
In other embodiments, COX Vila subunit polynucleotides may be introduced into plants by contacting plants with a virus or viral nucleic acids. Generally, such methods involve incorporating a nucleotide construct within a viral DNA or RNA molecule. It is recognized that a COX Vila subunit polypeptide of interest may be initially synthesized as part of a viral polyprotein, which later may be processed by proteolysis in vivo or in vitro to produce the desired recombinant protein. Further, it is recognized that useful promoters may include promoters utilized for transcription by viral RNA polymerases. Methods for introducing polynucleotides into plants and expressing a polypeptide encoded thereby, involving viral DNA or RNA molecules, are known in the art. See, for example, U.S. Patent Nos. 5,889,191 , 5,889,190, 5,866,785, 5,589,367, 5,316,931 , and Porta, et al., (1996) Molecular Biotechnology 5:209-221 ; herein incorporated by reference.
Methods are known in the art for the targeted insertion of a polynucleotide at a specific location in the plant genome. In one embodiment, the insertion of the polynucleotide at a desired genomic location is achieved using a site-specific recombination system. See, for example, WO 99/25821 , WO 99/25854, WO 99/25840, WO 99/25855, and WO 99/25853, all of which are herein incorporated by reference. Briefly, a polynucleotide can be contained in a transfer cassette flanked by two non-recombinogenic recombination sites. The transfer cassette is introduced into a plant having stably incorporated into its genome a target site that is flanked by two non-recombinogenic recombination sites that correspond to the sites of the transfer cassette. An appropriate recombinase is provided and the transfer cassette is integrated at the target site. The polynucleotide of interest is thereby integrated at a specific chromosomal position in the plant genome.
The cells that have been transformed may be grown into plants in accordance with conventional ways. See, for example, McCormick, et al., (1986) Plant Cell Reports 5:81-84. These plants may then be grown, and either pollinated with the same transformed strain or different strains, and the resulting progeny having expression of the desired phenotypic characteristic, for example, increased total seed number, increased total seed weight, increased number of seeds filled, increased harvest index, or any combination thereof, identified. Two or more generations may be grown to ensure that expression of the desired phenotypic characteristic is stably maintained and inherited and then seeds harvested to ensure expression of the desired phenotypic characteristic has been achieved. In this manner, the present invention provides transformed seed (also referred to as "transgenic seed") having a polynucleotide described herein, for example, an expression cassette comprising the Zm COX Vila subunit sequence of SEQ ID NO: 1 , the Zm COX Vila subunit coding sequence set forth in nucleotides 81-281 of SEQ ID NO: 1 or in SEQ ID NO: 3, or fragment or variant thereof encoding a COX VIIa subunit polypeptide, stably incorporated into their genome.
Plants of the invention may be produced by any suitable method, including breeding. Plant breeding can be used to introduce desired characteristics (e.g., a stably incorporated transgene) into a particular plant line of interest, and can be performed in any of several different ways. Pedigree breeding starts with the crossing of two genotypes, such as an elite line of interest and one other elite inbred line having one or more desirable characteristics (i.e., having stably incorporated a polynucleotide of interest, having a modulated activity and/or level of the polypeptide of interest, etc.) which complements the elite plant line of interest. If the two original parents do not provide all the desired characteristics, other sources can be included in the breeding population. In the pedigree method, superior plants are selfed and selected in successive filial generations. In the succeeding filial generations the heterozygous condition gives way to homogeneous lines as a result of self-pollination and selection. Typically in the pedigree method of breeding, five or more successive filial generations of selfing and selection is practiced: F1 → F2; F2→ F3; F3 → F4; F4 → F5, etc. After a sufficient amount of inbreeding, successive filial generations will serve to increase seed of the developed inbred. In specific embodiments, the inbred line comprises homozygous alleles at about 95% or more of its loci.
In addition to being used to create a backcross conversion, backcrossing can also be used in combination with pedigree breeding to modify an elite line of interest and a hybrid that is made using the modified elite line. As discussed previously, backcrossing can be used to transfer one or more specifically desirable traits from one line, the donor parent, to an inbred called the recurrent parent, which has overall good agronomic characteristics yet lacks that desirable trait or traits. However, the same procedure can be used to move the progeny toward the genotype of the recurrent parent but at the same time retain many components of the nonrecurrent parent by stopping the backcrossing at an early stage and proceeding with selfing and selection. For example, an F1 , such as a commercial hybrid, is created. This commercial hybrid may be backcrossed to one of its parent lines to create a BC1 or BC2. Progeny are selfed and selected so that the newly developed inbred has many of the attributes of the recurrent parent and yet several of the desired attributes of the non-recurrent parent. This approach leverages the value and strengths of the recurrent parent for use in new hybrids and breeding.
In certain embodiments, the COX Vila subunit polynucleotides of the present invention can be stacked with any combination of polynucleotide sequences of interest in order to create plants with a desired trait. A "trait," as used herein, refers to the phenotype derived from a particular sequence or groups of sequences. For example, the Zm COX Vila subunit polynucleotides of the present invention may be stacked with any other polynucleotides encoding polypeptides having COX Vila subunit activity, such as COX Vila subunit polynucleotides from Pinguicula (GenBank Accession No. AY601869), Utricularia (GenBank Accession No. AY601870), Arabidopsis thaliana GenBank Accession No. AK1 18629), Pinus taeda (GenBank Accession No. BF778888), Ceratopteris richardii (GenBank Accession No. BE643431 ), Anopheles gambiae (GenBank Accession No. XM_311038), Drosophila melanogaster (GenBank Accession No. AE003678), Bombyx mori (GenBank Accession No. CK536562), Mus musculusH (GenBank Accession No. NM_009944), Bos taurusH (GenBank Accession No. NIVM 76674), Tetraodon nigroviridis (GenBank Accession No. CAAE01014645), Xenopus laevis (GenBank Accession No. BC07861 1 ), Gallus gallus (GenBank Accession No. XM_419878), Danio rerio (GenBank Accession No. CO355138), Mus musculusL (GenBank Accession No. NM_009945), Bos taurusl (GenBank Accession No. NM_175807), Oryza sativa (GenBank Accession No. XM_479340), Medicago truncatula (GenBank Accession No. AC137510), Populus balsamifera (GenBank Accession No. AC149425), Zea mays (GenBank Accession No. AY1 1 1015), Malus domestica (GenBank Accession No. CN994265), Picea glauca (GenBank Accession No. CO237069), Rosa chinensis (GenBank Accession No. BI978872), Callinectes sapidus (GenBank Accession No. CV086499), Glossina morsitans morsitans (GenBank Accession No. BX567633), Ambystoma mexicanum (GenBank Accession No. CN046134), Oryzias latipes (GenBank Accession No. BJ514640), and Oncorhynchus mykiss (GenBank Accession No. BX308258). The combinations generated can also include multiple copies of any one of the polynucleotides of interest.
The polynucleotides of the present invention can also be stacked with any other gene or combination of genes to produce plants with a variety of desired trait combinations including, but not limited to, traits desirable for animal feed such as high oil genes (e.g., U.S. Patent No. 6,232,529); balanced amino acids (e.g., hordothionins (U.S. Patent Nos. 5,990,389; 5,885,801 ; 5,885,802; and 5,703,409); barley high lysine (Williamson, et ah, (1987) Eur. J. Biochem. 165:99-106; and WO 98/20122) and high methionine proteins (Pedersen, et al., (1986) J. Biol. Chem. 261 :6279; Kirihara, et al., (1988) Gene 71 :359; and Musumura, et al., (1989) Plant MoI. Biol. 12:123)); increased digestibility (e.g., modified storage proteins (U.S. Application Serial No. 10/053,410, filed November 7, 2001 ); and thioredoxins (U.S. Application Serial No. 10/005,429, filed December 3, 2001 )); the disclosures of which are herein incorporated by reference.
The polynucleotides of the present invention can also be stacked with traits desirable for disease or herbicide resistance (e.g., fumonisin detoxification genes (U.S. Patent No. 5,792,931 ); avirulence and disease resistance genes (Jones, et al., (1994) Science 266:789; Martin, et al., (1993) Science 262:1432; Mindrinos, et al., (1994) Cell 78:1089); acetolactate synthase (ALS) mutants that lead to herbicide resistance such as the S4 and/or Hra mutations; inhibitors of glutamine synthase such as phosphinothricin or basta (e.g., bar gene); and glyphosate resistance (e.g., the EPSPS gene and the GAT gene; see, for example, U.S. Publication No. 20040082770 and WO 03/092360)); and traits desirable for processing or process products such as high oil (e.g., U.S. Patent No. 6,232,529 ); modified oils (e.g., fatty acid desaturase genes (U.S. Patent No. 5,952,544; WO 94/1 1516)); modified starches (e.g., ADPG pyrophosph tases (AGPase), starch synthases (SS), starch branching enzymes (SBE), and starch debranching enzymes (SDBE)); and polymers or bioplastics (e.g., U.S. Patent No. 5.602,321 ; beta-ketothiolase, polyhydroxybutyrate synthase, and acetoacetyl-CoA reductase (Schubert, et al., (1988) J. Bacteriol. 170:5837-5847) facilitate expression of polyhydroxyalkanoat.es (PHAs)); the disclosures of which are herein incorporated by reference. One could also combine the polynucleotides of the present invention with polynucleotides providing agronomic traits such as male sterility (e.g., see U.S. Patent No. 5,583,210), stalk strength, flowering time, increased nitrogen storage capacity, or transformation technology traits such as cell cycle regulation or gene targeting (e.g., WO 99/61619, WO 00/17364, and WO 99/25821 ); the disclosures of which are herein incorporated by reference.
These stacked combinations can be created by any method including, but not limited to, crossbreeding plants by any conventional or TopCross methodology, or genetic transformation. If the sequences are stacked by genetically transforming the plants, the polynucleotide sequences of interest can be combined at any time and in any order. For example, a transgenic plant comprising one or more desired traits can be used as the target to introduce further traits by subsequent transformation. The traits can be introduced simultaneously in a co-transformation protocol with the polynucleotides of interest provided by any combination of transformation cassettes. For example, if two sequences will be introduced, the two sequences can be contained in separate transformation cassettes {trans) or contained on the same transformation cassette (cis). Expression of the sequences can be driven by the same promoter or by different promoters. In one embodiment, it is desirable to introduce a transformation cassette that will result in the overexpression of the polynucleotide of interest. This may be combined with any combination of other overexpression cassettes to generate the desired combination of traits in the plant. It is further recognized that polynucleotide sequences can be stacked at a desired genomic location using a site-specific recombination system. See, for example, WO 99/25821 , WO 99/25854, WO 99/25840, WO 99/25855, and WO 99/25853, all of which are herein incorporated by reference.
As used herein, the term "plant" includes plant cells, plant protoplasts, plant cell tissue cultures from which plants can be regenerated, plant calli, plant clumps, and plant cells that are intact in plants or parts of plants such as embryos, pollen, ovules, seeds, leaves, flowers, branches, fruit, kernels, ears, cobs, husks, stalks, roots, root tips, anthers, and the like. Grain is intended to mean the mature seed produced by commercial growers for purposes other than growing or reproducing the species. Progeny, variants, and mutants of the regenerated plants are also included within the scope of the invention, provided that these parts comprise the introduced polynucleotides. Thus, the invention provides transgenic seeds produced by the plants of the invention.
A "subject plant or plant cell" is one in which a genetic alteration, such as transformation, has been effected as to a COX Vila subunit gene of interest, or is a plant or plant cell that is descended from a plant or cell so altered and which comprises the alteration. A "control" or "control plant" or "control plant cell" provides a reference point for measuring changes in phenotype of the subject plant or plant cell.
A control plant or plant cell may comprise, for example: (a) a wild-type plant or cell, that is, of the same genotype as the starting material for the genetic alteration which resulted in the subject plant or cell; (b) a plant or plant cell of the same genotype as the starting material but which has been transformed with a null construct (that is, with a construct that has no known effect on the trait of interest, such as a construct comprising a marker gene); (c) a plant or plant cell that is a non-transformed segregant among progeny of a subject plant or plant cell; (d) a plant or plant cell genetically identical to the subject plant or plant cell but which is not exposed to conditions or stimuli that would induce expression of the gene of interest; or (e) the subject plant or plant cell itself, under conditions in which the COX Vila subunit gene of interest is not expressed.
The present invention may be used for transformation of any plant species, including, but not limited to, monocots and dicots. Examples of plant species of interest 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 {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 (Solarium tuberosum), peanuts (Arachis hypogaea), cotton (Gossypium barbadense, Gossypium hirsutum), sweet potato (Ipomoea batatus), cassava (Manihot esculenta), coffee (Coffea spp.), coconut (Cocos nucifera), pineapple (Ananas comosus), citrus trees (Citrus spp.), cocoa (Theobroma cacao), tea (Camellia sinensis), banana (Musa spp.), avocado (Persea americana), fig (Ficus casica), guava (Psidium guajava), mango (Mangifera indica), olive (Olea europaea), papaya (Carica papaya), cashew (Anacardium occidentale), macadamia (Macadamia integrifolia), almond (Prunus amygdalus), sugar beets (Beta vulgaris), sugarcane (Saccharum spp.), oats, barley, vegetables, ornamentals, and conifers.
Vegetables include tomatoes (Lycopersicon esculentum), lettuce (e.g., Lactuca sativa), green beans (Phaseolus vulgaris), lima beans (Phaseolus limensis), peas (Lathyrus spp.), and members of the genus Cucumis such as cucumber (C. sativus), cantaloupe (C. cantalupensis), and musk melon (C. melo). Ornamentals include azalea (Rhododendron spp.), hydrangea (Macrophylla 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 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). In specific embodiments, plants of the present invention are crop plants (for example, corn, alfalfa, sunflower, Brassica, soybean, cotton, safflower, peanut, sorghum, wheat, millet, tobacco, etc.).
In other embodiments, plants of interest are monocots, for example, corn (Zea mays), 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)), wheat (Triticum aestivum), sugarcane (Saccharum spp.), oats, and barley. Other plants of interest include grain plants that provide seeds of interest, oil-seed plants, and leguminous plants. Seeds of interest include grain seeds, such as corn, wheat, barley, rice, sorghum, rye, etc. Oil-seed plants include cotton, soybean, safflower, sunflower, Brassica, maize, alfalfa, palm, coconut, etc. Leguminous plants include beans and peas. Beans include guar, locust bean, fenugreek, soybean, garden beans, cowpea, mungbean, lima bean, fava bean, lentils, chickpea, etc.
The nucleotide sequence for the Zm COX Vila promoter disclosed in the present invention as well as variants and fragments thereof are useful in the genetic manipulation of any plant when assembled within a DNA construct such that the promoter sequence is operably linked to a nucleotide sequence encoding a heterologous protein of interest. In this manner, the nucleotide sequence of the Zm COX Vila promoter of the invention, or fragment or variant thereof, is provided in expression cassettes along with heterologous nucleotide sequences for expression in the plant of interest.
Synthetic hybrid promoter regions are known in the art. Such regions comprise upstream promoter elements of one nucleotide sequence operably linked to the promoter element of another nucleotide sequence. In an embodiment of the invention, heterologous gene expression is controlled by a synthetic hybrid promoter comprising the Zm COX Vila promoter sequence of the invention, or a variant or fragment thereof, operably linked to upstream promoter element(s) from a heterologous promoter. Upstream promoter elements have been identified and may be used to generate a synthetic promoter. See, for example, Rushton, et al., (1998) Curr. Opin. Plant Biol. 1 :31 1-315. Alternatively, a synthetic Zm COX VIIa promoter sequence may comprise duplications of upstream promoter elements found within the Zm COX Vila promoter sequence. It is recognized that the promoter sequence of the invention may be used with its native Zm COX Vila subunit coding sequence. A DNA construct comprising the Zm COX Vila promoter operably linked with its native Zm COX Vila subunit coding sequence may be used to transform any plant of interest to bring about a desired phenotypic change, such as increased yield, for example, reflected in increased total seed number, increased total seed weight, increased number of seeds filled, increased harvest index, or any combination thereof. Where the promoter and its native gene are naturally occurring within the plant, i.e., in maize, transformation of the plant with these operably linked sequences also results in either a change in phenotype, such as increased yield, or the insertion of operably linked sequences within a different region of the chromosome thereby altering the plant's genome. In another embodiment of the invention, expression cassettes will comprise a transcriptional initiation region comprising the Zm COX Vila promoter nucleotide sequence disclosed herein, or variant or fragment thereof, operably linked to the heterologous nucleotide sequence whose expression is to be controlled by the Zm COX Vila promoter of the invention.
The promoter nucleotide sequence and methods disclosed herein are useful in regulating expression of any heterologous nucleotide sequence in a host plant in order to vary the phenotype of a plant. Various changes in phenotype are of interest, including, but not limited to, modifying the fatty acid composition in a plant, altering the amino acid content of a plant, altering a plant's pathogen defense mechanism, and the like. These results can be achieved by providing expression of heterologous products or increased expression of endogenous products in plants. Alternatively, the results can be achieved by providing for a reduction of expression of one or more endogenous products, particularly enzymes or cofactors in the plant. These changes result in a change in phenotype of the transformed plant.
Item
1. An expression cassette comprising a nucleotide sequence selected from the group consisting of:
(a) the nucleotide sequence set forth in SEQ ID NO: 3; (b) a nucleotide sequence encoding the amino acid sequence set forth in SEQ ID
NO: 2;
(c) a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 3, wherein said nucleotide sequence encodes a polypeptide having COX Vila subunit activity; (d) a nucleotide sequence comprising at least 147 consecutive nucleotides of SEQ
ID NO: 3, wherein said nucleotide sequence encodes a polypeptide having COX Vila subunit activity; and
(e) a nucleotide sequence encoding an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 2, wherein said nucleotide sequence encodes a polypeptide having COX Vila subunit activity; wherein said nucleotide sequence is operably linked to a promoter that drives expression in a plant.
2. The expression cassette of item 1 , wherein said promoter is a constitutive promoter or a tissue-preferred promoter. 3. The expression cassette of item 2, wherein said promoter is a constitutive promoter.
4. A plant comprising the expression cassette of any one of items 1 to 3.
5. The plant of item 4, wherein said plant is a monocot. 6. The plant of item 5, wherein said monocot is maize, wheat, rice, barley, sorghum, or rye.
7. The plant of item 4, wherein said plant has a phenotype selected from the group consisting of: (a) increased total seed number;
(b) increased total seed weight;
(c) increased number of seeds filled;
(d) increased harvest index; and
(e) any combination of (a)-(d). 8. The plant of any one of items 4 to 7, wherein said expression cassette is stably incorporated into the genome of said plant.
9. A seed having stably incorporated into its genome the expression cassette of any one of items 1 to 3.
10. A method for increasing the level of a COX Vila subunit polypeptide in a plant comprising introducing into said plant the expression cassette of any one of items 1 to
3.
1 1. The method of item 10, wherein said plant is a monocot.
12. The method of item 11 , wherein said monocot is maize, wheat, rice, barley, sorghum, or rye. 13. The method of item 10, wherein the yield of said plant is increased.
14. The method of item 13, wherein said plant has a phenotype selected from the group consisting of:
(a) increased total seed number;
(b) increased total seed weight; (c) increased number of seeds filled;
(d) increased harvest index; and
(e) any combination of (a)-(d).
15. The method of any one of items 10 to 14, wherein said expression cassette is stably integrated into the genome of said plant. 16. A method for increasing yield in a plant comprising increasing expression of a COX Vila subunit polypeptide in said plant, wherein said COX Vila subunit polypeptide has COX Vila subunit activity and is selected from the group consisting of: (a) a polypeptide comprising an amino acid sequence having at least 80% sequence identity to the sequence set forth in SEQ ID NO: 2; and (b) a polypeptide comprising a functional fragment of the amino acid sequence set forth in SEQ ID NO: 2. 17. The method of item 16, wherein said polypeptide comprises an amino acid sequence having at least 90% sequence identity to the sequence set forth in SEQ ID NO: 2.
18. The method of item 17, wherein said polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 2. 19. The method of any one of item 16 to 18, wherein said plant is a monocot.
20. The method of item 19, wherein said monocot is maize, wheat, rice, barley, sorghum, or rye.
21. The method of any one of items 16 to 18, wherein said plant has a phenotype selected from the group consisting of: (a) increased total seed number;
(b) increased total seed weight;
(c) increased number of seeds filled;
(d) increased harvest index; and
(e) any combination of (a)-(d). 22. The method of any one of items 16 to 21 , comprising introducing into said plant an expression cassette comprising a nucleotide sequence selected from the group consisting of:
(a) the nucleotide sequence set forth in SEQ ID NO: 3;
(b) a nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO: 2;
(c) a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 3, wherein said nucleotide sequence encodes a polypeptide having COX Vila subunit activity;
(d) a nucleotide sequence comprising at least 147 consecutive nucleotides of SEQ ID NO: 3, wherein said nucleotide sequence encodes a polypeptide having COX
Vila subunit activity; and
(e) a nucleotide sequence encoding an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 2, wherein said nucleotide sequence encodes a polypeptide having COX Vila subunit activity; wherein said nucleotide sequence is operably linked to a promoter that drives expression in a plant.
23. The method of item 22, wherein said promoter is a constitutive promoter or a tissue- preferred promoter.
24. The method of item 23, wherein said promoter is a constitutive promoter. 25. The method of any one of item 22 to 24, comprising:
(a) transforming a plant cell with said expression cassette; and
(b) regenerating a transformed plant from the transformed plant cell of (a). 26. The method of item 25, wherein said expression cassette is stably incorporated into the genome of said plant.
27. An isolated polynucleotide comprising a nucleotide sequence selected from the group consisting of: (a) the nucleotide sequence set forth as nucleotides 1 through 1012 of SEQ ID NO:
4;
(b) a nucleotide sequence with at least 90% sequence identity to the nucleotide sequence set forth as nucleotides 1 through 1012 of SEQ ID NO: 4, wherein said sequence drives expression in a plant cell; (c) a nucleotide sequence with at least 95% sequence identity to the nucleotide sequence set forth as nucleotides 1 through 1012 of SEQ ID NO: 4, wherein said sequence drives expression in a plant cell; and
(d) a nucleotide sequence with at least 98% sequence identity to the nucleotide sequence set forth as nucleotides 1 through 1012 of SEQ ID NO: 4, wherein said sequence drives expression in a plant cell.
28. An expression cassette comprising the polynucleotide of item 27 operably linked to a heterologous polynucleotide of interest.
29. A vector comprising the expression cassette of item 28.
30. A plant cell having stably incorporated into its genome the expression cassette of item 28.
31. The plant cell of item 30, wherein said plant cell is from a monocot.
32. The plant cell of item 31 , wherein said monocot is maize, wheat, rice, barley, sorghum, or rye.
33. The plant cell of item 30, wherein said plant cell is from a dicot. 34. The plant cell of item 33, wherein said dicot is soybean, Brassica, sunflower, cotton, or alfalfa.
35. A plant having stably incorporated into its genome the expression cassette of item 28.
36. The plant of item 35, wherein said plant is a monocot.
37. The plant of item 36, wherein said monocot is maize, wheat, rice, barley, sorghum, or rye.
38. The plant of item 35, wherein said plant is a dicot.
39. The plant of item 38, wherein said dicot is soybean, Brassica, sunflower, cotton, or alfalfa.
40. A transformed seed of the plant of any one of item 35 to 39, wherein said seed comprises said expression cassette stably integrated into its genome.
The following examples are offered by way of illustration and not by way of limitation. Experimental
Example 1: Cloning of Maize COX Vila Gene
The cDNA that encoded the COX Vila polypeptide from maize was identified by sequence homology from a collection of ESTs generated from a maize cDNA library using BLAST 2.0 (Altschul, et al., (1990) J. MoI. Biol. 215:403) against the NCBI DNA sequence database. From the EST plasmid, the maize COX Vila cDNA fragment was amplified by PCR using Hifi Taq DNA polymerase in standard conditions with maize COX W/a-specific primers that included the AttB site for Gateway recombination cloning. A PCR fragment of the expected length was amplified and purified using standard methods as described by Sambrook, et al., (1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, New York). The first step of the Gateway procedure, the BP reaction, was then performed, during which the PCR fragment recombined in vivo with the pDONR201 plasmid to produce the "entry clone." Plasmid pDONR201 was purchased from Invitrogen, as part of the Gateway® technology (Invitrogen, Carlsbad, CA).
Example 2: Vector Construction (pG0S2::ZmC0XVIIa)
The entry clone was subsequently used in an LR reaction with a destination vector used for Oryza sativa transformation. This vector contains as functional elements within the T-DNA borders, a plant selectable marker, a screenable marker, and a Gateway cassette intended for LR in vivo recombination with the sequence of interest already cloned in the entry clone. Upstream of this Gateway cassette is the rice GOS2 promoter (Hensgens, et al., (1993) Plant MoI. Biol. 23:643-669) that confers moderate constitutive expression on the gene of interest. After the LR recombination step, the resulting expression vector pGOS2::ZmCOXVIIa was transformed into Agrobacterium tumefaciens strain LBA4044 and subsequently into Oryza sativa var. Nipponbare plants as described herein in Example 3. Transformed rice plants were grown and examined for various growth characteristics as described herein in Example 4.
Example 3: Rice Transformation Method Genetic Confirmation of the COX VIIA Gene
For the genetic confirmation of the COX Vila cloning, it was necessary to transform the wild- type genomic DNA obtained from the COX Vila region into Cox Vila mutants, confirming not only the COX Vila gene but also the genomic region that contains the complete set of regulatory elements for COX Vila expression. Two fragments of genomic DNA were prepared for the transformation, one covering 6 kb upstream and 3 kb downstream regions, and another covering 3 kb upstream and 2.5 kb downstream regions. In order to confirm possible tissue- specific expression of the COX Vila gene, the presence of the COX Vila transcript in various tissues was analyzed by RNA blot analysis and in situ hybridization.
High-velocity ballistic bombardment using metal particles coated with the nucleic acid constructs was used to transform wild-type rice and Cox Vila mutants (Klein, et al., (1987) Nature 327:70-73; U.S. Patent No. 4,945,050, incorporated by reference herein). A Biolistic PDS-1000/He (BioRAD Laboratories, Hercules, CA) was used for these complementation experiments. The particle bombardment technique was used to transform wild-type rice and Cox Vila mutants with two genomic DNA fragments: a 10.0 kb Muni fragment from wild-type rice that includes the 4.5 kb upstream and 3.8 kb downstream region of the COX VIIA gene, and a 5.1 kb EcoRI fragment from wild-type rice that includes the 1.7 kb upstream and 1.7 kb downstream region of the COX VIIA gene. The bacterial hygromycin B phosphotransferase (Hpt II) gene from Streptomyces hygroscopicus (which confers resistance to the antibiotic) was used as the selectable marker for rice transformation. In the vector, pML18, the Hpt Il gene was engineered with the 35S promoter from Cauliflower Mosaic Virus and the termination and polyadenylation signals from the octopine synthase gene of Agrobacterium tumefaciens. pML18 is described in WO 97/47731 , the disclosure of which is hereby incorporated by reference.
Embryogenic callus cultures derived from the scutellum of germinating rice seeds served as source material for transformation experiments. This material is generated by germinating sterile rice seeds on a callus initiation media (MS salts, Nitsch and Nitsch vitamins, 1.0 mg/l 2,4-D and 10 μM AgNO3) in the dark at 27-28°C. Embryogenic callus proliferating from the scutellum of the embryos is then transferred to CM media (N6 salts, Nitsch and Nitsch vitamins, 1 mg/l 2,4-D; Chu, et al., (1985) Sci. Sinica 18:659-668). Callus cultures are maintained on CM by routine sub-culture at two week intervals and used for transformation within 10 weeks of initiation. Callus is prepared for transformation by subculturing 0.5-1.0 mm pieces approximately 1 mm apart, arranged in a circular area of about 4 cm in diameter, in the center of a circle of Whatman #541 paper placed on CM media. The plates with callus are incubated in the dark at 27-28°C for 3-5 days. Prior to bombardment, the filters with callus are transferred to CM supplemented with 0.25 M mannitol and 0.25 M sorbitol for 3 hr in the dark. The petri dish lids are then left ajar for 20-45 minutes in a sterile hood to allow moisture on tissue to dissipate.
Each of the genomic DNA fragment was co-precipitated with pML18 containing the selectable marker for rice transformation onto the surface of gold particles. To accomplish this, a total of 10 μg of DNA at a 2:1 ratio of trait:selectable marker DNAs were added to a 50 μl aliquot of gold particles that had been resuspended at a concentration of 60 mg ml"1. Calcium chloride (50 μl of a 2.5 M solution) and spermidine (20 μl of a 0.1 M solution) were then added to the gold-DNA suspension as the tube was vortexing for 3 min. The gold particles were centrifuged in a microfuge for 1 second and the supernatant removed. The gold particles were then washed twice with 1 ml of absolute ethanol and resuspended in 50 μl of absolute ethanol and sonicated (bath sonicator) for one second to disperse the gold particles. The gold suspension was incubated at -700C for five minutes and sonicated (bath sonicator) to disperse the particles. Six μl of the DNA-coated gold particles was then loaded onto mylar macrocarrier disks and the ethanol was allowed to evaporate.
At the end of the drying period, a petri dish containing the tissue was placed in the chamber of the PDS-1000/He. The air in the chamber was then evacuated to a vacuum of 28-29 inches Hg. The macrocarrier was accelerated with a helium shock wave using a rupture membrane that bursts when the He pressure in the shock tube reaches 1080-1100 psi. The tissue was placed approximately 8 cm from the stopping screen and the callus was bombarded two times. Two to four plates of tissue were bombarded in this way with the DNA-coated gold particles. Following bombardment, the callus tissue was transferred to CM media without supplemental sorbitol or mannitol.
Three to five days after bombardment, the callus tissue was transferred to SM media (CM medium containing 50 mg/l hygromycin). To accomplish this, callus tissue was transferred from plates to sterile 50 ml conical tubes and weighed. Molten top-agar at 400C was added using 2.5 ml of top agar/100 mg of callus. Callus clumps were broken into fragments of less than 2 mm diameter by repeated dispensing through a 10 ml pipette. Three ml aliquots of the callus suspension were plated onto fresh SM media and the plates were incubated in the dark for 4 weeks at 27-28°C. After 4 weeks, transgenic callus events were identified, transferred to fresh SM plates and grown for an additional 2 weeks in the dark at 27-28°C.
Growing callus was transferred to RM 1 media (MS salts, Nitsch and Nitsch vitamins, 2% sucrose, 3% sorbitol, 0.4% gelrite +50 ppm hyg B) for 2 weeks in the dark at 25°C. After 2 weeks the callus was transferred to RM2 media (MS salts, Nitsch and Nitsch vitamins, 3% sucrose, 0.4% gelrite + 50 ppm hyg B) and placed under cool white light (-40 μEm"2s"1) with a
12 hr photoperiod at 25°C and 30-40% humidity. After 2-4 weeks in the light, callus began to organize and form shoots. Shoots were removed from surrounding callus/media and gently transferred to RM3 media (1/2 x MS salts, Nitsch and Nitsch vitamins, 1% sucrose + 50 ppm hygromycin B) in phytatrays (Sigma Chemical Co., St. Louis, MO) and incubation was continued using the same conditions as described in the previous step. Plants were transferred from RM3 to 4" pots containing Metro mix 350 after 2-3 weeks, when sufficient root and shoot growth had occurred. The seed obtained from the transgenic plants was examined for genetic complementation of the Cox Vila mutation with the wild-type genomic DNA containing the COX VIIA gene.
Example 4: Overexpression of a COX Vila Sequence to Increase Yield in Rice Evaluation of TO, T1 , and T2 Rice Plants Transformed with pG0S2::ZmC0XVIIa Approximately 15 to 20 independent TO transformants were generated. The primary transformants were transferred from tissue culture chambers to a greenhouse for growing and harvest of T1 seed. Six events of which the T1 progeny segregated 3/1 for presence/absence of the transgene were retained. "Null plants" or "Null segregants" or "Nullizygotes" are the plants treated in the same way as a transgenic plant, but from which the transgene has segregated. Null plants can also be described as the homozygous negative transformants. For each of these events, approximately 10 T1 seedlings containing the transgene (hetero- and homozygotes), and approximately 10 T1 seedlings lacking the transgene (nullizygotes), were selected by PCR.
Based on the results of the T1 evaluation (described herein), four events that showed improved growth and yield characteristics at the T1 level were chosen for further characterization in the T2 generation. To this extent, seed batches from the positive T1 plants (both hetero- and homozygotes), were screened by monitoring marker expression. For each chosen event, the heterozygote seed batches were then selected for T2 evaluation. An equal number of positive and negative plants within each seed batch were transplanted for evaluation in the greenhouse (i.e., for each event 40 plants, of which 20 were positives for the transgene and 20 were negative for the transgene). For the four events, a total of 160 plants were evaluated in the T2 generation. Both T1 and T2 plants were transferred to a greenhouse and evaluated for vegetative growth parameters, as described herein.
Statistical Analyses on Transgenic T1 & T2 lines A two-factor ANOVA (analyses of variance) corrected for the unbalanced design was used as a statistical evaluation model for the numeric values of the observed plant phenotypic characteristics. The numerical values were submitted to a t-test and an F-test. The p-value was obtained by comparing the t-value to the t-distribution or, alternatively, by comparing the F-value to the F-distribution. The p-value stands for the probability that the null hypothesis (i.e., no effect of the transgene) is correct. A t-test was performed on all the values of all plants per event. Such a t-test was repeated for each event and for each growth characteristic. The t-test was carried out to check for an effect of the gene within one transformation event, also described herein as "line-specific effect." In the t-test, the threshold for a significant line-specific effect is set at 10% probability level. Therefore, data with a p-value of the t-test under 10% means that the phenotype observed in the transgenic plants of that line was caused by the presence of the transgene. Within one population of transformation events, some events may be under or below this threshold. This difference may be due to the difference in the position of the transgene within the rice genome (i.e., a gene might only have an effect in certain positions of the genome). Therefore, the "line- specific effect" is sometimes referred to as the "position-dependent effect."
An F-test was carried out on all the values measured for all plants of all events. An F-test was repeated for each growth characteristic. The F-test was conducted to check for an effect of the gene over all the transformation events and to verify an overall effect of the gene, also described herein as the "gene effect." In the F-test, the threshold for a significant global gene effect is set at 5% probability level. Therefore, data with a p-value of the F-test under 5% means that the observed phenotype was caused by more than just the presence of the gene, and/or the position of the transgene within the genome. A "gene effect" is an indication for the wide applicability of the gene in transgenic plants.
Vegetative Growth Measurements
The selected plants were grown in a greenhouse. Each plant received a unique barcode label to link the phenotyping data unambiguously to the corresponding plant. The selected plants were grown on soil in 10 cm diameter, clear-bottom pots under the following environmental settings: photoperiod=11.5 hours; daylight intensity=30,000 lux or more; daytime temperature=28° C or higher; night-time temperature=22°C; and relative humidity=60-70%. Transgenic plants and the corresponding nullizygotes were grown side-by-side at random positions. From the stage of sowing until the stage of maturity (i.e., the stage were there is no more increase in biomass), the plants were passed weekly through a digital imaging cabinet. At each time point digital images (2048x1536 pixels, 16 million colors) were taken of each plant from at least 6 different angles. The parameters described herein were derived in an automated way from the digital images using image analysis software.
Plants were also passed through a root-imaging system that digitally photographs the root morphology and mass from the base of the clear-bottom pots. Plant above-ground area and root mass were determined by counting the total number of pixels from plant parts discriminated from the background. The above-ground value was averaged for the pictures taken on the same time point from the different angles and was converted to a physical surface value expressed in square mm by calibration. Experiments have shown that the above-ground plant area, which corresponds to the total maximum area, measured this way correlates with the biomass of plant parts above-ground.
In addition to digital images during the growth of the plants, when the plants reached maturity and senescence the number of panicles per plant and the total number of florets per plant were counted by hand. Dried florets were collected and those with filled seeds were mechanically separated from empty florets using an enclosed air-driven blower system. Dehusked seeds were then collected and counted using a seed counter and weighed using a standard balance. Harvest index was calculated using a ratio of the total weight of seeds produced per plant with the biomass calculated from digital images as described herein. Thousand kernel weight was calculated from the ratio of total seed weight per plant and the number of filled seeds per plant times 1000. The time to flower interval was recorded as the number of days between sowing and the emergence of the first panicle, extrapolated by the size of the panicles in the earliest imaging that a panicle was detected and the date of that imaging.
Overall Effects of Zm COX VIIa in Rice On the average of five events examined, pG0S2::ZmC0XVIIa transgenic plants in the T1 generation showed a statistically significant increase of 10% in total seed number per plant, a 34% increase in the number of seeds filled per plant, a 36% increase in total seed weight per plant, and a 31% increase in harvest index with p-values less than 0.002, as compared to the nullizygotes. These data show that the constitutively expressed Zm COX Vila gene confers a strong positive effect on several important yield traits in a plant.
Example 5: Overexpression of a COX Vila Sequence to Increase Yield In Maize
Immature maize embryos from greenhouse donor plants are bombarded with a plasmid containing a COX Vila subunit polynucleotide (such as the coding sequence of the Zm COX Vila subunit polynucleotide as set forth in nucleotides 81-281 of SEQ ID NO: 1 or in SEQ ID NO: 3) operably linked to a UBI promoter and the selectable marker gene PAT (Wohlleben, et al., (1988) Gene 70:25-37), which confers resistance to the herbicide Bialaphos. Alternatively, the selectable marker gene is provided on a separate plasmid. Transformation is performed as follows. Media recipes follow below. Preparation of Target Tissue
The ears are husked and surface sterilized in 30% Clorox bleach plus 0.5% Micro detergent for 20 minutes, and rinsed two times with sterile water. The immature embryos are excised and placed embryo axis side down (scutellum side up), 25 embryos per plate, on 560Y medium for 4 hours and then aligned within the 2.5 cm target zone in preparation for bombardment.
A plasmid vector comprising the COX Vila subunit sequence operably linked to a UBI promoter is made. This plasmid DNA plus plasmid DNA containing a PAT selectable marker is precipitated onto 1.1 μm (average diameter) tungsten pellets using a CaCI2 precipitation procedure as follows: 100 μl prepared tungsten particles in water; 10 μl (1 μg) DNA in Tris EDTA buffer (1 μg total DNA); 100 μl 2.5 M CaCI2; and 10 μl 0.1 M spermidine.
Each reagent is added sequentially to the tungsten particle suspension, while maintained on the multitube vortexer. The final mixture is sonicated briefly and allowed to incubate under constant vortexing for 10 minutes. After the precipitation period, the tubes are centrifuged briefly, liquid removed, washed with 500 ml 100% ethanol, and centrifuged for 30 seconds. Again the liquid is removed, and 105 μl 100% ethanol is added to the final tungsten particle pellet. For particle gun bombardment, the tungsten/DNA particles are briefly sonicated and 10 μl spotted onto the center of each macrocarrier and allowed to dry about 2 minutes before bombardment.
The sample plates are bombarded at level #4 in a particle gun. All samples receive a single shot at 650 PSI, with a total of ten aliquots taken from each tube of prepared particles/DNA.
Following bombardment, the embryos are kept on 560Y medium for 2 days, then transferred to 560R selection medium containing 3 mg/liter Bialaphos, and subcultured every 2 weeks. After approximately 10 weeks of selection, selection-resistant callus clones are transferred to 288J medium to initiate plant regeneration. Following somatic embryo maturation (2-4 weeks), well- developed somatic embryos are transferred to medium for germination and transferred to the lighted culture room. Approximately 7-10 days later, developing plantlets are transferred to 272V hormone-free medium in tubes for 7-10 days until plantlets are well established. Plants are then transferred to inserts in flats (equivalent to 2.5" pot) containing potting soil and grown for 1 week in a growth chamber, subsequently grown an additional 1-2 weeks in the greenhouse, then transferred to classic 600 pots (1.6 gallon) and grown to maturity. Plants are monitored and scored for increased yield, such as increased total seed number, increased total seed weight, increased number of seeds filled, increased harvest index, or any combination thereof. Bombardment medium (560Y) comprises 4.0 g/l N6 basal salts (SIGMA C-1416), 1.0 ml/l Eriksson's Vitamin Mix (1000X SIGMA-151 1 ), 0.5 mg/l thiamine HCI, 120.0 g/l sucrose, 1.0 mg/l 2,4-D, and 2.88 g/l L-proline (brought to volume with D-I H2O following adjustment to pH 5.8 with KOH); 2.0 g/l Gelrite (added after bringing to volume with D-I H2O); and 8.5 mg/l silver nitrate (added after sterilizing the medium and cooling to room temperature). Selection medium (560R) comprises 4.0 g/l N6 basal salts (SIGMA C-1416), 1.0 ml/l Eriksson's Vitamin Mix (1000X SIGMA-151 1 ), 0.5 mg/l thiamine HCI, 30.0 g/l sucrose, and 2.0 mg/l 2,4-D (brought to volume with D-I H2O following adjustment to pH 5.8 with KOH); 3.0 g/l Gelrite (added after bringing to volume with D-I H2O); and 0.85 mg/l silver nitrate and 3.0 mg/l Bialaphos(both added after sterilizing the medium and cooling to room temperature).
Plant regeneration medium (288J) comprises 4.3 g/l MS salts (GIBCO 1 1117-074), 5.0 ml/l MS vitamins stock solution (0.100 g nicotinic acid, 0.02 g/l thiamine HCL, 0.10 g/l pyridoxine HCL, and 0.40 g/l glycine brought to volume with polished D-I H2O) (Murashige and Skoog (1962) Physiol. Plant. 15:473), 100 mg/l myo-inositol, 0.5 mg/l zeatin, 60 g/l sucrose, and 1.0 ml/l of 0.1 mM abscisic acid (brought to volume with polished D-I H2O after adjusting to pH 5.6); 3.0 g/l Gelrite (added after bringing to volume with D-I H2O); and 1.0 mg/l indoleacetic acid and 3.0 mg/l Bialaphos (added after sterilizing the medium and cooling to 600C). Hormone-free medium (272V) comprises 4.3 g/l MS salts (GIBCO 1 11 17-074), 5.0 ml/l MS vitamins stock solution (0.100 g/l nicotinic acid, 0.02 g/l thiamine HCL, 0.10 g/l pyridoxine HCL, and 0.40 g/l glycine brought to volume with polished D-I H2O), 0.1 g/l myo-inositol, and 40.0 g/l sucrose (brought to volume with polished D-I H2O after adjusting pH to 5.6); and 6 g/l bacto-agar (added after bringing to volume with polished D-I H2O), sterilized and cooled to 600C.
Example 6: Agrobacterium-mediated Transformation
For Λgrobacfem/m-mediated transformation of maize with a COX Vila subunit polynucleotide (such as the coding sequence of the Zm COX Vila subunit polynucleotide as set forth in nucleotides 81-281 of SEQ ID NO: 1 or in SEQ ID NO: 3), the method of Zhao is employed (U.S. Patent No. 5,981 ,840, and PCT patent publication WO98/32326; the contents of which are hereby incorporated by reference). Briefly, immature embryos are isolated from maize and the embryos contacted with a suspension of Agrobacterium, where the bacteria are capable of transferring the COX Vila subunit polynucleotide to at least one cell of at least one of the immature embryos (step 1 : the infection step). In this step the immature embryos are immersed in an Agrobacterium suspension for the initiation of inoculation. The embryos are co-cultured for a time with the Agrobacterium (step 2: the co-cultivation step). The immature embryos are cultured on solid medium following the infection step. Following this co-cultivation period an optional "resting" step is contemplated. In this resting step, the embryos are incubated in the presence of at least one antibiotic known to inhibit the growth of Agrobacterium without the addition of a selective agent for plant transformants (step 3: resting step). The immature embryos are cultured on solid medium with antibiotic, but without a selecting agent, for elimination of Agrobacterium and for a resting phase for the infected cells. Next, inoculated embryos are cultured on medium containing a selective agent and growing transformed callus is recovered (step 4: the selection step). The immature embryos are cultured on solid medium with a selective agent resulting in the selective growth of transformed cells. The callus is then regenerated into plants (step 5: the regeneration step), and calli grown on selective medium are cultured on solid medium to regenerate the plants.
Example 7: Soybean Embryo Transformation
Culture Conditions
Soybean embryogenic suspension cultures (cv. Jack) are maintained in 35 ml liquid medium SB196 (see recipes below) on rotary shaker, 150 rpm, 26°C with cool white fluorescent lights on 16:8 hr day/night photoperiod at light intensity of 60-85 μE/m2/s. Cultures are subcultured every 7 days to two weeks by inoculating approximately 35 mg of tissue into 35 ml of fresh liquid SB196 (the preferred subculture interval is every 7 days).
Soybean embryogenic suspension cultures are transformed with the plasmids and DNA fragments described in the following examples by the method of particle gun bombardment (Klein, et al., (1987) Nature 327:70).
Soybean Embrvogenic Suspension Culture Initiation Soybean cultures are initiated twice each month with 5-7 days between each initiation.
Pods with immature seeds from available soybean plants 45-55 days after planting are picked, removed from their shells and placed into a sterilized magenta box. The soybean seeds are sterilized by shaking them for 15 minutes in a 5% Clorox solution with 1 drop of ivory soap (95 ml of autoclaved distilled water plus 5 ml Clorox and 1 drop of soap). Mix well. Seeds are rinsed using 2 1 -liter bottles of sterile distilled water and those less than 4 mm are placed on individual microscope slides. The small end of the seed are cut and the cotyledons pressed out of the seed coat. Cotyledons are transferred to plates containing SB1 medium (25-30 cotyledons per plate). Plates are wrapped with fiber tape and stored for 8 weeks. After this time secondary embryos are cut and placed into SB196 liquid media for 7 days. Preparation of DNA for Bombardment
Either an intact plasmid or a DNA plasmid fragment containing the genes of interest and the selectable marker gene are used for bombardment. Plasmid DNA for bombardment are routinely prepared and purified using the method described in the Promega™ Protocols and Applications Guide, Second Edition (page 106). Fragments of the plasmids carrying a COX Vila subunit polynucleotide (such as the coding sequence of the Zm COX Vila subunit polynucleotide as set forth in nucleotides 81-281 of SEQ ID NO: 1 or in SEQ ID NO: 3) are obtained by gel isolation of double digested plasmids. In each case, 100 μg of plasmid DNA is digested in 0.5 ml of the specific enzyme mix that is appropriate for the plasmid of interest. The resulting DNA fragments are separated by gel electrophoresis on 1% SeaPlaque GTG agarose (BioWhitaker Molecular Applications) and the DNA fragments containing the COX VIIa subunit polynucleotide are cut from the agarose gel. DNA is purified from the agarose using the GELase digesting enzyme following the manufacturer's protocol.
A 50 μl aliquot of sterile distilled water containing 3 mg of gold particles (3 mg gold) is added to 5 μl of a 1 μg/μl DNA solution (either intact plasmid or DNA fragment prepared as described above), 50 μl 2.5M CaCI2 and 20 μl of 0.1 M spermidine. The mixture is shaken 3 min on level 3 of a vortex shaker and spun for 10 sec in a bench microfuge. After a wash with 400 μl 100% ethanol the pellet is suspended by sonication in 40 μl of 100% ethanol. Five μl of DNA suspension is dispensed to each flying disk of the Biolistic PDS1000/HE instrument disk. Each 5 μl aliquot contains approximately 0.375 mg gold per bombardment (i.e., per disk).
Tissue Preparation and Bombardment with DNA
Approximately 150-200 mg of 7 day old embryonic suspension cultures are placed in an empty, sterile 60 x 15 mm petri dish and the dish covered with plastic mesh. Tissue is bombarded 1 or 2 shots per plate with membrane rupture pressure set at 1 100 PSI and the chamber evacuated to a vacuum of 27-28 inches of mercury. Tissue is placed approximately 3.5 inches from the retaining/stopping screen.
Selection of Transformed Embryos
Transformed embryos were selected either using hygromycin (when the hygromycin phosphotransferase, HPT, gene was used as the selectable marker) or chlorsulfuron (when the acetolactate synthase, ALS, gene was used as the selectable marker).
Hvgromvcin (HPT) Selection
Following bombardment, the tissue is placed into fresh SB196 media and cultured as described above. Six days post-bombardment, the SB196 is exchanged with fresh SB196 containing a selection agent of 30 mg/L hygromycin. The selection media is refreshed weekly. Four to six weeks post selection, green, transformed tissue may be observed growing from untransformed, necrotic embryogenic clusters. Isolated, green tissue is removed and inoculated into multiwell plates to generate new, clonally propagated, transformed embryogenic suspension cultures.
Chlorsulfuron (ALS) Selection
Following bombardment, the tissue is divided between 2 flasks with fresh SB196 media and cultured as described above. Six to seven days post-bombardment, the SB196 is exchanged with fresh SB196 containing selection agent of 100 ng/ml Chlorsulfuron. The selection media is refreshed weekly. Four to six weeks post selection, green, transformed tissue may be observed growing from untransformed, necrotic embryogenic clusters. Isolated, green tissue is removed and inoculated into multiwell plates containing SB196 to generate new, clonally propagated, transformed embryogenic suspension cultures.
Regeneration of Soybean Somatic Embryos into Plants
In order to obtain whole plants from embryogenic suspension cultures, the tissue must be regenerated.
Embryo Maturation
Embryos are cultured for 4-6 weeks at 260C in SB196 under cool white fluorescent (Phillips cool white Econowatt F40/CW/RS/EW) and Agro (Phillips F40 Agro) bulbs (40 watt) on a 16:8 hr photoperiod with light intensity of 90-120 uE/m2s. After this time embryo clusters are removed to a solid agar media, SB166, for 1-2 weeks. Clusters are then subcultured to medium SB103 for 3 weeks. During this period, individual embryos can be removed from the clusters and screened for levels of COX VIIA expression and/or activity.
Embryo Desiccation and Germination
Matured individual embryos are desiccated by placing them into an empty, small petri dish (35 x 10 mm) for approximately 4-7 days. The plates are sealed with fiber tape (creating a small humidity chamber). Desiccated embryos are planted into SB71-4 medium where they were left to germinate under the same culture conditions described above. Germinated plantlets are removed from germination medium and rinsed thoroughly with water and then planted in Redi- Earth in 24-cell pack tray, covered with clear plastic dome. After 2 weeks the dome is removed and plants hardened off for a further week. If plantlets looked hardy they are transplanted to 10" pot of Redi-Earth with up to 3 plantlets per pot. After 10 to 16 weeks, mature seeds are harvested, chipped and analyzed for proteins. Media Recipes
SB 196 - FN Lite liquid proliferation medium (per liter) -
MS FeEDTA - 10Ox Stock 1 10 ml MS Sulfate - 100x Stock 2 10 ml
FN Lite Halides - 100x Stock 3 10 ml
FN Lite P, B, Mo - 100x Stock 4 10 ml
B5 vitamins (1 ml/L) 1.0 ml
2,4-D (10 mg/L final concentration) 1.0 ml KNO3 2.83 gm
(NhU)2SO4 0.463 gm
Asparagine 1.0 gm
Sucrose (1%) 10 gm pH 5.8
FN Lite Stock Solutions
Stock # 1000 ml 500 ml
1 MS Fe EDTA IOOx Stock
Na2 EDTA* 3.724 g 1.862 g
FeSO4 - 7H2O 2.784 g 1.392 g
* Add first, dissolve in dark bottle while stirring
2 MS Sulfate 100x stock
MgSO4 - 7H2O 37.O g 18.5 g
MnSO4 - H2O 1.69 g 0.845 g
ZnSO4 - 7H2O 0.86 g 0.43 g
CuSO4 - 5H2O 0.0025 g 0.00125 g
3 FN Lite Halides 10Ox Stock
CaCI2 - 2H2O 30.O g 15.O g
Kl 0.083 g 0.0715 g
CoCI2 - 6H2O 0.0025 g 0.00125 g
4 FN Lite P, B, Mo 10Ox Stock
KH2PO4 18.5 g 9.25 g
H3BO3 0.62 g 0.31 g
Na2MoO4 - 2H2O 0.025 g 0.0125 g SB1 solid medium (per liter) comprises: 1 pkg. MS salts (GIBCO/BRL - Cat# 1 11 17-066); 1 ml B5 vitamins 1000X stock; 31.5 g sucrose; 2 ml 2,4-D (20 mg/L final concentration); pH 5.7; and, 8 g TC agar.
SB 166 solid medium (per liter) comprises: 1 pkg. MS salts (GIBCO/BRL - Cat# 1 11 17-066); 1 ml B5 vitamins 1000X stock; 60 g maltose; 750 mg MgCI2 hexahydrate; 5 g activated charcoal; pH 5.7; and, 2 g gelrite.
SB 103 solid medium (per liter) comprises: 1 pkg. MS salts (GIBCO/BRL - Cat# 1 11 17-066); 1 ml B5 vitamins 1000X stock; 60 g maltose; 750 mg MgCI2 hexahydrate; pH 5.7; and, 2 g gelrite.
SB 71-4 solid medium (per liter) comprises: 1 bottle Gamborg's B5 salts w/ sucrose (GIBCO/BRL - Cat# 21153-036); pH 5.7; and, 5 g TC agar.
2,4-D stock is obtained premade from Phytotech cat# D 295 - concentration is 1 mg/ml.
B5 Vitamins Stock (per 100 ml) which is stored in aliquots at -200C comprises: 10 g myoinositol; 100 mg nicotinic acid; 100 mg pyridoxine HCI; and, 1 g thiamine. If the solution does not dissolve quickly enough, apply a low level of heat via the hot stir plate.
Chlorsulfuron Stock comprises: 1 mg / ml in 0.01 N Ammonium Hydroxide.
Example 8: Vector construction and over expression of ZM-COXVIIa in maize to increase the yield.
The coding sequence of ZM-COXVIIa was amplified by PCR and cloned unidirectionaly in pENTR.D.TOPO vector (Invitrogen) to make an entry vector. The entry vector in combination with other entry vectors were used in a multisite Gateway (Invitrogen) reaction to generate ZM- UBI PRO:ZM-COXVIIa:PINII and ZM-GOS2 PRO:ZM-COXVIIa:PINII. In all these final vectors, UBhMOPAT and LTP2:RFP are used as herbicide resistance and visible markers, respectively. The expression vectors were quality checked by restriction digestion mapping and transferred into Agrobacterium tumefaciens LB4404JT by electroporation. This Agrobacterium strain was used to transform lntroEF09B maize inbred. Molecular analyses on TO events were performed and single copy transgene expressing events were advanced for further experiments. Example 9: Field Evaluation with Cox Vila in maize
To further illustrate the influence of this transgene relating to transgenic corn field tests are used. Progeny seed of multiple transgenic corn events containing ZmG0S2-C0X Vila produced in Example 8 can be planted in the field to evaluate the transgene's ability to enhance yield as compared to the non-transgenic control plants. The plants would be planted at a multiple locations having a variety of environmental stresses. The data to be collected would consist of multiple measurements for yield and plant health/quality. The measured items could include, but are not limited to the following: enhanced vegetative growth, biomass accumulation, accelerated growth rate, stand count, stalk and/or root lodging, grain yield, average kernel weight, total seed number/plant, total seed weight/plant, harvest index, number of seeds filled/plant, primary and secondary ear mass, and grain yield increase. The experimental data demonstrates which of the transgenic corn plants expressing COX Vila gene perform better than the non-transgenic control plants in the specific traits measured.
Example 10: Variants of COX VIIA Sequences
A. Variant Nucleotide Sequences of COX VIIA That Do Not Alter the Encoded Amino Acid Sequence
The COXVIIA nucleotide sequences are used to generate variant nucleotide sequences having the nucleotide sequence of the open reading frame with about 70%, 75%, 80%, 85%, 90%, and 95% nucleotide sequence identity when compared to the starting unaltered ORF nucleotide sequence of the corresponding SEQ ID NO. These functional variants are generated using a standard codon table. While the nucleotide sequence of the variants are altered, the amino acid sequence encoded by the open reading frames do not change.
B. Variant Amino Acid Sequences of COX VIIA Polypeptides
Variant amino acid sequences of the COX VIIA polypeptides are generated. In this example, one amino acid is altered. Specifically, the open reading frames are reviewed to determine the appropriate amino acid alteration. The selection of the amino acid to change is made by consulting the protein alignment (with the other orthologs and other gene family members from various species). An amino acid is selected that is deemed not to be under high selection pressure (not highly conserved) and which is rather easily substituted by an amino acid with similar chemical characteristics (i.e., similar functional side-chain). Using the protein alignment set forth in Figure 1 , an appropriate amino acid can be changed. Once the targeted amino acid is identified, the procedure outlined in the following section C is followed. Variants having about 70%, 75%, 80%, 85%, 90%, and 95% nucleic acid sequence identity are generated using this method. C. Additional Variant Amino Acid Sequences of COX VIIA Polypeptides In this example, artificial protein sequences are created having 80%, 85%, 90%, and 95% identity relative to the reference protein sequence. This latter effort requires identifying conserved and variable regions from the alignment set forth in Figure 1 and then the judicious application of an amino acid substitutions table. These parts will be discussed in more detail below.
Largely, the determination of which amino acid sequences are altered is made based on the conserved regions among COX VIIA protein or among the other COX VIIA polypeptides. Based on the sequence alignment, the various regions of the COX VIIA polypeptide that can likely be altered are represented in lower case letters, while the conserved regions are represented by capital letters. It is recognized that conservative substitutions can be made in the conserved regions below without altering function. In addition, one of skill will understand that functional variants of the COX VIIA sequence of the invention can have minor non- conserved amino acid alterations in the conserved domain.
Artificial protein sequences are then created that are different from the original in the intervals of 80-85%, 85-90%, 90-95%, and 95-100% identity. Midpoints of these intervals are targeted, with liberal latitude of plus or minus 1 %, for example. The amino acids substitutions will be effected by a custom Perl script. The substitution table is provided below in Table 1.
Table 1. Substitution Table
Figure imgf000051_0001
Figure imgf000052_0001
First, any conserved amino acids in the protein that should not be changed is identified and "marked off" for insulation from the substitution. The start methionine will of course be added to this list automatically. Next, the changes are made.
H, C, and P are not changed in any circumstance. The changes will occur with isoleucine first, sweeping N-terminal to C-terminal. Then leucine, and so on down the list until the desired target it reached. Interim number substitutions can be made so as not to cause reversal of changes. The list is ordered 1-17, so start with as many isoleucine changes as needed before leucine, and so on down to methionine. Clearly many amino acids will in this manner not need to be changed. L, I and V will involve a 50:50 substitution of the two alternate optimal substitutions.
The variant amino acid sequences are written as output. Perl script is used to calculate the percent identities. Using this procedure, variants of the COXVIIA polypeptides are generating having about 80%, 85%, 90%, and 95% amino acid identity to the starting unaltered ORF nucleotide sequence of SEQ ID NO: 3.
The article "a" and "an" are used herein to refer to one or more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one or more element.
All publications and patent applications mentioned in the specification are indicative of the level of those skilled in the art to which this invention pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.
Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be obvious that certain changes and modifications may be practiced within the scope of the appended claims.

Claims

That which is claimed
1. An expression cassette comprising a nucleotide sequence selected from the group consisting of:
(a) the nucleotide sequence set forth in SEQ ID NO: 3; (b) a nucleotide sequence encoding the amino acid sequence set forth in SEQ ID
NO: 2;
(c) a nucleotide sequence having at least 70% sequence identity to SEQ ID NO: 3, wherein said nucleotide sequence encodes a polypeptide having COX Vila subunit activity; (d) a nucleotide sequence comprising at least 147 consecutive nucleotides of SEQ
ID NO: 3, wherein said nucleotide sequence encodes a polypeptide having COX Vila subunit activity; and
(e) a nucleotide sequence encoding an amino acid sequence having at least 50% sequence identity to SEQ ID NO: 2, wherein said nucleotide sequence encodes a polypeptide having COX Vila subunit activity; wherein said nucleotide sequence is operably linked to a promoter that drives expression in a plant.
2. The expression cassette of claim 1 , wherein said promoter is a constitutive promoter or a tissue-preferred promoter.
3. A plant comprising the expression cassette of claim 1 or 2.
4. The plant of claim 3, wherein said plant is a monocot, preferably wherein said monocot is maize, wheat, rice, barley, sorghum, or rye.
5. The plant of claim 3 or 4, wherein said plant has a phenotype selected from the group consisting of:
(a) increased total seed number; (b) increased total seed weight;
(c) increased number of seeds filled;
(d) increased harvest index; and
(e) any combination of (a)-(d).
6. The plant of any one of claims 3 to 5, wherein said expression cassette is stably incorporated into the genome of said plant.
7. A seed having stably incorporated into its genome the expression cassette of claim 2.
8. A method for increasing the level of a COX Vila subunit polypeptide in a plant comprising introducing into said plant the expression cassette of claim 1 or 2.
9. The method of claim 8, wherein the yield of said plant is increased.
10. A method for increasing yield in a plant comprising increasing expression of a COX Vila subunit polypeptide in said plant, wherein said COX Vila subunit polypeptide has COX Vila subunit activity and is selected from the group consisting of:
(a) a nucleotide sequence encoding a polypeptide having COX Vila subunit activity;
(b) a polypeptide comprising an amino acid sequence having at least 50%, preferably at least 60%, further preferably at least 70%, even further preferably at least 80%, most preferably at least 90% or more sequence identity to the sequence set forth in SEQ ID NO: 2; and
(c) a polypeptide comprising a functional fragment of the amino acid sequence set forth in SEQ ID NO: 2;
(d) a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 2.
1 1. The method of any one of claims 8 to 10, wherein said plant is a monocot, preferably wherein said monocot is maize, wheat, rice, barley, sorghum, or rye.
12. The method of any one of claims 8 to 11 , wherein said plant has a phenotype selected from the group consisting of: (a) increased total seed number;
(b) increased total seed weight;
(c) increased number of seeds filled;
(d) increased harvest index; and
(e) any combination of (a)-(d).
13. The method of any one of claims 8 to 12, comprising introducing into said plant an expression cassette comprising a nucleotide sequence selected from the group consisting of:
(a) the nucleotide sequence set forth in SEQ ID NO: 3; (b) a nucleotide sequence encoding the amino acid sequence set forth in SEQ ID
NO: 2; (c) a nucleotide sequence having at least 70% sequence identity to SEQ ID NO: 3, wherein said nucleotide sequence encodes a polypeptide having COX Vila subunit activity;
(d) a nucleotide sequence comprising at least 147 consecutive nucleotides of SEQ ID NO: 3, wherein said nucleotide sequence encodes a polypeptide having COX
Vila subunit activity; and
(e) a nucleotide sequence encoding an amino acid sequence having at least 50% sequence identity to SEQ ID NO: 2, wherein said nucleotide sequence encodes a polypeptide having COX Vila subunit activity; wherein said nucleotide sequence is operably linked to a promoter that drives expression in a plant.
14. The method of claim 13, wherein said promoter is a constitutive promoter or a tissue- preferred promoter.
15. The method of any one of claims 8 to 14, comprising:
(a) transforming a plant cell with said expression cassette; and
(b) regenerating a transformed plant from the transformed plant cell of (a).
16. The method of any one of claims 8 to 15, wherein said expression cassette is stably incorporated into the genome of said plant.
17. An isolated polynucleotide comprising a nucleotide sequence selected from the group consisting of: (a) the nucleotide sequence set forth as nucleotides 1 through 1012 of SEQ ID NO:
4; (b) a nucleotide sequence with at least 70%, preferably 80%, most preferably 90% sequence identity to the nucleotide sequence set forth as nucleotides 1 through
1012 of SEQ ID NO: 4, wherein said sequence drives expression in a plant cell; (c) a nucleotide sequence with at least 95% sequence identity to the nucleotide sequence set forth as nucleotides 1 through 1012 of SEQ ID NO: 4, wherein said sequence drives expression in a plant cell; and (d) a nucleotide sequence with at least 98% sequence identity to the nucleotide sequence set forth as nucleotides 1 through 1012 of SEQ ID NO: 4, wherein said sequence drives expression in a plant cell.
18. An expression cassette comprising the polynucleotide of claim 17 operably linked to a heterologous polynucleotide of interest, and/or wherein a vector comprising said expression cassette.
19. A plant, plant cell or transformed seed of the plant having stably incorporated into its genome the expression cassette or vector of claim 18.
20. The plant or plant cell of claim 19, wherein said plant or cell is from a monocot, preferably wherein said monocot is maize, wheat, rice, barley, sorghum, or rye, or wherein said plant cell is from a dicot, preferably wherein said dicot is soybean, Brassica, sunflower, cotton, or alfalfa.
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Title
DATABASE UniProt [Online] 1 December 2001 (2001-12-01), "SubName: Full=Putative uncharacterized protein (Putative uncharacterized protein At4g21105/F7J7.1) (At4g21108/At4g21108);" XP002495553 retrieved from EBI accession no. UNIPROT:Q944S8 Database accession no. Q944S8 *
DATABASE UniProt [Online] 17 October 2006 (2006-10-17), "SubName: Full=Os02g0162000 protein;" XP002495554 retrieved from EBI accession no. UNIPROT:Q0E3P3 Database accession no. Q0E3P3 *
JOBSON RICHARD W ET AL: "Adaptive evolution of cytochrome c oxidase: Infrastructure for a carnivorous plant radiation" PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, vol. 101, no. 52, 13 December 2004 (2004-12-13), pages 18064-18068, XP002495551 ISSN: 0027-8424 -& DATABASE UniProt [Online] 5 July 2004 (2004-07-05), "SubName: Full=Putative COX VIIa-like protein; Flags: Fragment;" XP002495552 retrieved from EBI accession no. UNIPROT:Q6J4I2 Database accession no. Q6J4I2 *

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US8722072B2 (en) 2010-01-22 2014-05-13 Bayer Intellectual Property Gmbh Acaricidal and/or insecticidal active ingredient combinations
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