WO2004063379A1 - Genes homologues de zea mays revoluta/if1 et leurs utilisations - Google Patents

Genes homologues de zea mays revoluta/if1 et leurs utilisations Download PDF

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WO2004063379A1
WO2004063379A1 PCT/US2004/000126 US2004000126W WO2004063379A1 WO 2004063379 A1 WO2004063379 A1 WO 2004063379A1 US 2004000126 W US2004000126 W US 2004000126W WO 2004063379 A1 WO2004063379 A1 WO 2004063379A1
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plant
seq
nucleic acid
nucleotide sequence
acid molecule
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PCT/US2004/000126
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Wesley B. Bruce
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Pioneer Hi-Bred International, Inc.
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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)
    • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
    • C12N15/8218—Antisense, co-suppression, viral induced gene silencing [VIGS], post-transcriptional induced gene silencing [PTGS]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • 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 the genetic manipulation of plants, particularly the modulation of gene activity and development in plants.
  • the shoot apical meristem In Arabidopsis, the shoot apical meristem (SAM) is the source of all above ground postembryonic organs (e.g. stems, leaves and flowers). However, the majority of these organs are not initiated directly by the SAM, but are instead initiated by lateral meristems.
  • the lateral meristems include flower meristems (FM), which form on the flanks of the SAM and lateral shoot meristems (LSM), which develop in leaf axils. Each LSM gives rise to a side branch containing leaves and flowers (Smyth et al., Early flower development in Arabidopsis, Plant Cell, 2, 755-767, (1990)).
  • the overall morphology and reproductive capability of a plant is determined in part by the number, position and growth of lateral meristems.
  • the morphology of FM and LSM formation has been described in Arabidopsis (Grbic and Bleeker, Axillary meristem development in Arabidopsis, Plant J. 21 , 215-223, (2000)) and many other species (Steeves and Wales, Patterns in Plant Development, New York; Cambridge University Press (1989)), and several mutations affecting FM and LSM formation have been reported.
  • These mutants include the tomato lateral suppressor (Is) mutant (Schumacher et al., The lateral suppressor (Ls) gene of tomato encodes a new member of the VHIID protein family, Proc Natl Acad.
  • the present invention provides maize REVOLUTA nucleotide and amino acid sequences.
  • the sequences of the invention find use in modulating development and developmental pathways, particularly, modulating meristem development.
  • the present invention provides an isolated polypeptide selected from the group consisting of (a) a polypeptide comprising an amino acid sequence set forth in SEQ ID NO: 2, 4 or 6; (b) a polypeptide encoded by a nucleotide sequence comprising the sequence set forth in SEQ ID NO: 1 , 3, or 5; (c) a polypeptide encoded by a nucleotide sequence that hybridizes under stringent conditions to the complement of a nucleotide sequence comprising the sequences set forth in SEQ ID NOS: 1 , 3, or 5, wherein said stringent conditions comprise hybridization in 50% formamide, 1 M NaCl, 1 % SDS at 37 ° C, and a wash in 0.1 X SSC at 60 ° C to 65 ° C; (d) a polypeptide comprising an amino acid sequence having at least 70% sequence identity to the sequence set forth in SEQ ID NO:2, 4 or 6, wherein said sequence modulates meristem development; and, (e) a polypeptide comprising at least
  • the present invention further provides an isolated nucleic acid molecule selected from the group consisting of (a) a nucleic acid molecule comprising a nucleotide sequence set forth in SEQ ID NO: 1 , 3, or 5; (b) a nucleic acid molecule comprising a nucleotide sequence encoding an amino acid sequence set forth in SEQ ID NO: 2, 4 or 6; (c) a nucleic acid molecule comprising at least 15 consecutive nucleotides of the complement of SEQ ID NO: 1 , 3, or 5; (d) a nucleic acid molecule comprising at least 15 consecutive nucleotides of SEQ ID NO:1 , 3, or 5; (e) a nucleic acid molecule comprising a nucleotide sequence that hybridizes under stringent conditions to the complement of nucleotide sequence set forth in SEQ ID NO:1 , 3, or 4, wherein said stringent conditions comprise hybridization in 50% formamide, 1 M NaCl, 1% SDS at 37 ° C, and
  • compositions of the invention further include host cells, plants, plant cells and seeds having stably incorporated into their genome at least one nucleotide construct comprising a nucleotide sequence of the invention. Methods for making such cells, plants, and seeds are further provided. It is recognized that a variety of promoters will be useful in the invention, the choice of which will depend in part upon the desired level of expression of the disclosed nucleotide sequences. It is recognized that the levels of expression can be controlled to modulate the levels of expression in the plant cell.
  • the methods and compositions can be used to modulate plant development. More specifically, methods and compositions of the invention may be used for modulating meristem development.
  • the method involves stably transforming a plant with a REVOLUTA nucleotide sequence and expressing said sequence in a plant or plant cell.
  • Method are also provided for modulating the level of a polypeptide in a plant or plant cell comprising introducing into the plant or plant cell a nucleic acid molecule of the invention encoding said polypeptide and expressing the nucleic acid molecule for a time sufficient to modulate the level of said polypeptide.
  • the level of a polypeptide in a plant or plant cell is modulated by providing a plant comprising an mRNA having the sequence set forth in SEQ ID NO:1 , 3, or 5, and introducing into the plant or plant cell a nucleotide sequence comprising at least 15 consecutive nucleotides of the complement of SEQ ID NO:1 , 3, or 5, wherein the nucleotide sequence inhibits expression of the mRNA in the plant or plant cell.
  • Figure 1 Alignment of ZmREVI peptide, Rice REV-like protein, and Arabidopsis REV/IFL protein and determination of the consensus sequence for the three molecules. conserveed domains are identified for the aligned proteins.
  • Line 1 is SEQ ID NO: 2
  • ZmREVI cmstls.pk008.b16:fis_pep
  • Line 2 is SEQ ID NO: 9
  • Rice REV-like protein Line 3 is SEQ ID NO: 8
  • Arabidopsis REV/IFL protein Line 4 is the Consensus sequence
  • the domains of the HD-ZIP REV protein are indicated in the consensus seq as follows: homeodomain (aa #57-87) indicated by underlined bold-italics, ZIP (aa).
  • Line 1 is SEQ ID NO: 2
  • ZmREVI cmstls.pk008.b16:fis_pep
  • Line 2 is SEQ ID NO: 9, Rice REV-like protein
  • the protein match between the two plant species is 65.3% identical and 76.3% positive as determined by Vector NTI suite 5.5 program, AlignX.
  • compositions of the present invention comprise nucleic acid and amino acid sequences.
  • nucleotide sequence SEQ ID NO:1 , 3, or 5
  • amino acid sequence SEQ ID NO:2, 4 or 6
  • Methods are therefore provided for the expression of the REVOLUTA sequences in host cells, host plants, or plant cells to modulate plant development and/or developmental pathways.
  • the invention finds use in controlling or modulating cell division, differentiation, and development.
  • sequences of the present invention find use in modulating meristem development.
  • modulating meristem development is intended any alteration in the meristem phenotype including, for example, an alteration in the formation and maintenance of the meristem and/or an alteration of size and appearance of lateral shoot meristems. Such modulations in meristem development may result in an increase or a decrease in meristem size and/or an increase or decrease in the yield of leaves, tassel, and/or ears.
  • compositions of the invention include nucleic acid and amino acid sequences that are involved in modulating plant development and developmental pathways.
  • the present invention provides for isolated nucleic acid molecules comprising nucleotide sequences encoding the amino acid sequences shown in SEQ ID NO: 2, 4 or 6. Further provided are polypeptides having an amino acid sequence encoded by a nucleic acid molecule described herein, and fragments and variants thereof.
  • the invention encompasses isolated or substantially purified nucleic acid or protein compositions.
  • An "isolated” or “purified” nucleic acid molecule or protein, or biologically active portion thereof, is substantially or essentially free from components that normally accompany or interact with the nucleic acid molecule or protein as found in its naturally occurring environment.
  • an isolated or purified nucleic acid molecule 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" nucleic acid is free of sequences (preferably protein encoding sequences) that naturally flank the nucleic acid (i.e., sequences located at the 5' and 3' ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived.
  • the isolated nucleic acid molecule can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kb of nucleotide sequences that naturally flank the nucleic acid molecule in genomic DNA of the cell from which the nucleic acid 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.
  • 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 the disclosed nucleotide sequences and proteins encoded thereby are also encompassed by the present invention.
  • fragment is intended a portion of the nucleotide sequence or a portion of the amino acid sequence and hence protein encoded thereby.
  • Fragments of a nucleotide sequence may encode protein fragments that retain the biological activity of the native protein and hence bind receptors, influence signal transduction, and/or retain the ability to modulate meristem development (i.e., prevent meristem enlargement).
  • fragments of a nucleotide sequence that are useful as hybridization probes generally do not encode fragment proteins retaining biological activity.
  • fragments of a nucleotide sequence may range from at least about 20 nucleotides, about 50 nucleotides, about 100 nucleotides, about 150, about 200, about 250, about 300, about 350, about 400, about 450, about 500 and up to the full-length nucleotide sequence encoding the proteins of the invention.
  • the fragment can comprises nucleotides 1-50, 50-100, 100-150, 150- 200, 200-250, 250-300, 300-350, 350-400, 400-450, and 450-500.
  • Other fragments of the polypeptide of SEQ ID NO:2 include amino acids 1-20, amino acids 21-40, amino acids 41-60, amino acids 61-80, and amino acids 81-94.
  • a fragment of a REVOLUTA sequence that encodes a biologically active portion of a REVOLUTA protein of the invention will encode at least 15, 25, 30, 50, 60, 70, 80, 90, or 100 contiguous amino acids, or up to the total number of amino acids present in a full-length REVOLUTA protein of the invention. Fragments of a REVOLUTA nucleotide sequence that are useful as hybridization probes or PCR primers generally need not encode a biologically active portion of a REVOLUTA protein. Thus, a fragment of a REVOLUTA nucleotide sequence may encode a biologically active portion of a REVOLUTA protein, or it may be a fragment that can be used as a hybridization probe or PCR primer using methods disclosed below.
  • a biologically active portion of a REVOLUTA protein can be prepared by isolating a portion of one of the REVOLUTA nucleotide sequences of the invention, expressing the encoded portion of the REVOLUTA protein (e.g., by recombinant expression in vitro), and assessing the activity of the encoded portion of the REVOLUTA protein.
  • Nucleic acid molecules that are fragments of a REVOLUTA nucleotide sequence comprise at least 16, 20, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500 nucleotides, or up to the number of nucleotides present in a full-length REVOLUTA nucleotide sequence disclosed herein.
  • variants are intended substantially similar sequences.
  • conservative variants include those sequences that, because of the degeneracy of the genetic code, encode the amino acid sequence of one of the REVOLUTA polypeptides of the invention.
  • 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 as outlined below.
  • Variant nucleotide sequences also include synthetically derived nucleotide sequences, such as those generated, for example, by using site-directed mutagenesis but which still encode a REVOLUTA protein of the invention.
  • variants of a particular nucleotide sequence of the invention will have at least about 40%, 50%, 60%, 65%, 70%, generally at least about 75%, 80%, 85%, preferably at least about 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, and more preferably at least about 98%, 99% or more sequence identity to that particular nucleotide sequence as determined by sequence alignment programs described elsewhere herein using default parameters.
  • variant protein is intended a protein derived from the native protein by deletion (so-called truncation) or addition of one or more amino acids to the N- terminal and/or C-terminal end of the native protein; deletion or addition of one or more amino acids at one or more sites in the native protein; or substitution of one or more amino acids at one or more sites in the native protein.
  • Variant proteins encompassed by the present invention are biologically active, that is they continue to possess the desired biological activity of the native protein, that is, bind receptors, influence signal transduction, and/or retain the ability to modulate meristem development (i.e., prevent meristem enlargement), as described herein. Such variants may result from, for example, genetic polymorphism or from human manipulation.
  • Biologically active variants of a native REVOLUTA protein of the invention will have at least about 40%, 50%, 60%, 65%, 70%, generally at least about 75%, 80%, 85%, preferably at least about 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, and more preferably at least about 98%, 99% or more sequence identity to the amino acid sequence for the native protein as determined by sequence alignment programs described elsewhere herein using default parameters.
  • a biologically active variant of a protein of the invention may differ from that protein 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 proteins of 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 of the REVOLUTA proteins can be prepared by mutations in the DNA. Methods for mutagenesis and nucleotide sequence alterations are well known in the art. See, for example, Kunkel, Proc. Natl. Acad. Sci. USA 82:488-492, (1985); Kunkel et al., Methods in Enzymol. 154:367-382, (1987); U.S. Patent No. 4,873,192; Walker and Gaastra, eds.
  • the genes and nucleotide sequences of the invention include both the naturally occurring sequences as well as mutant forms.
  • the proteins of the invention encompass both naturally occurring proteins as well as variations and modified forms thereof.
  • Such variants will continue to possess the desired activity (i.e., bind receptors, influence signal transduction, and/or retain the ability to modulate meristem development (i.e., prevent meristem enlargement)).
  • the mutations that will be made in the DNA encoding the variant must not place the sequence out of reading frame and preferably will not create complementary regions that could produce secondary mRNA structure. See, EP Patent Application Publication No. 75,444.
  • deletions, insertions, and substitutions of the protein sequences encompassed herein are not expected to produce radical changes in the characteristics of the protein. However, when it is difficult to predict the exact effect of the substitution, deletion, or insertion in advance of doing so, one skilled in the art will appreciate that the effect will be evaluated by routine screening assays. Plants exhibiting modulated meristem development can be selected using visual observation. See, for example, U.S. Patent No. 5,637,785, 6,002,069, 5,859,338, 6,025,483, and Fletcher et al., Science 283:1911-1914, (1999) all of which are herein incorporated by reference. Alternatively, assays to measure receptor binding are also routine. See, for example, Trotochaud et al. , Science 289:613-617 (2000), which is herein incorporated by reference.
  • Variant nucleotide sequences and proteins also encompass sequences and proteins derived from a mutagenic and recombinogenic procedure such as DNA shuffling. With such a procedure, one or more different REVOLUTA coding sequences can be manipulated to create a new REVOLUTA sequence 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 REVOLUTA gene of the invention and other known sequences involved in meristem maintenance or development (such as other REVOLUTA homologs) to obtain a new gene coding for a protein with an improved property of interest, such as an increased K m in the case of an enzyme.
  • Strategies for such DNA shuffling are known in the art. See, for example, Stemmer, Proc. Natl. Acad. Sci. USA 91 :10747-10751 , (1994); Stemmer, Nature 370:389-391 , (1994); Crameri et al., Nature Biotech. 15:436-438, (1997); Moore et al, J. Mol.
  • 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 sequences.
  • the comparison window is at least 20 contiguous nucleotides in length, and optionally can be 30, 40, 50, 100, or longer.
  • Computer implementations of these mathematical algorithms can be utilized for comparison of sequences to determine sequence identity. Such implementations include, but are not limited to: CLUSTAL in the PC/Gene program (available from Intelligenetics, Mountain View, California); the ALIGN program (Version 2.0) and GAP, BESTFIT, BLAST, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Version 8 (available from Genetics Computer Group GCG® programs (Accelrys, Inc., San Diego, CA), USA). Alignments using these programs can be performed using the default parameters.
  • the CLUSTAL program is well described by Higgins et al., Gene 73:237-244 (1988); Higgins et al., CABIOS 5:151-153, (1989); Corpet et al, Nucleic Acids Res. 16:10881-90, (1988); Huang et al., CABIOS 8:155-65, (1992); and Pearson et al., Meth. Mol. Biol. 24:307-331 , (1994).
  • the ALIGN program is based on the algorithm of Myers and Miller (1988) supra. A PAM120 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
  • 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 using GAP Weight of 50 and Length Weight of 3; % similarity using Gap Weight of 12 and Length Weight of 4, or any equivalent program.
  • equivalent program is intended any sequence comparison program that, for any two sequences in question, generates an alignment having identical nucleotide or amino acid residue matches and an identical percent sequence identity when compared to the corresponding alignment generated by GAP Version 10.
  • GAP uses the algorithm of Needleman and Wunsch, J. Mol. Biol. 48:443- 453, (1970), to find the alignment of two complete sequences that maximizes the number of matches and minimizes the number of gaps.
  • 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 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.
  • 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 Wisconsin Genetics Software Package is BLOSUM62 (see Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915, (1989)).
  • sequence identity or “identity” in the context of two nucleic acid 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, e.g., 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 sequences means that a polynucleotide comprises a sequence that has at least 70% sequence identity, preferably at least 80%, more preferably at least 90%, and most preferably at least 95%, compared to a reference sequence using one of the alignment programs described using standard parameters.
  • sequence identity preferably at least 80%, more preferably at least 90%, and most preferably at least 95%.
  • nucleotide sequences are substantially identical if two molecules hybridize to each other under stringent conditions.
  • stringent conditions are selected to be about 5°C lower than the thermal melting point (T m ) for the specific sequence at a defined ionic strength and pH.
  • T m thermal melting point
  • stringent conditions encompass temperatures in the range of about 1 °C to about 20°C lower than the T m , depending upon the desired degree of stringency as otherwise qualified herein.
  • Nucleic acids that do not hybridize to each other under stringent conditions are still substantially identical if the polypeptides they encode are substantially identical. This may occur, e.g., when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code.
  • One indication that two nucleic acid sequences are substantially identical is when the polypeptide encoded by the first nucleic acid is immunologically cross reactive with the polypeptide encoded by the second nucleic acid.
  • substantially identical in the context of a peptide indicates that a peptide comprises a sequence with at least 70% sequence identity to a reference sequence, preferably 80%, more preferably 85%, most preferably at least 90% or 95% sequence identity to the reference sequence over a specified comparison window.
  • optimal alignment is conducted using the homology alignment algorithm of Needleman and Wunsch, J. Mol. Biol. 48:443- 453, (1970).
  • An indication that two peptide sequences are substantially identical is that one peptide is immunologically reactive with antibodies raised against the second peptide.
  • a peptide is substantially identical to a second peptide, for example, where the two peptides differ only by a conservative substitution.
  • nucleotide sequences of the invention can be used to isolate corresponding sequences from other organisms, particularly other plants, more particularly other monocots. In this manner, methods such as PCR, hybridization, and the like can be used to identify such sequences based on their sequence homology to the sequences set forth herein. Sequences isolated based on their sequence identity to the entire REVOLUTA sequences set forth herein or to fragments thereof are encompassed by the present invention. Such sequences include sequences that are orthologs of the disclosed sequences.
  • orthologs 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 substantial identity as defined elsewhere herein. Functions of orthologs are often highly conserved among species. Thus, isolated sequences that encode for a REVOLUTA protein and which hybridize under stringent conditions to the REVOLUTA sequences disclosed herein, or to fragments thereof, are encompassed by the present invention.
  • 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., Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, New York (1989)).
  • PCR Protocols A Guide to Methods and Applications (Academic Press, New York, (1990)); Innis and Gelfand, eds., PCR Strategies (Academic Press, New York (1995)); and Innis and Gelfand, eds., PCR Methods Manual (Academic Press, New York (1999)).
  • 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.
  • hybridization techniques all or part of a known nucleotide sequence is used as a probe that selectively hybridizes to other corresponding nucleotide sequences present in a population of cloned genomic DNA fragments or cDNA fragments (i.e., genomic or cDNA libraries) from a chosen organism.
  • the hybridization probes may be genomic DNA fragments, cDNA fragments, RNA fragments, or other oligonucleotides, and may be labeled with a detectable group such as 32 P, or any other detectable marker.
  • probes for hybridization can be made by labeling synthetic oligonucleotides based on the REVOLUTA sequences of the invention.
  • the entire REVOLUTA sequence disclosed herein, or one or more portions thereof, may be used as a probe capable of specifically hybridizing to corresponding REVOLUTA sequences and messenger RNAs.
  • probes include sequences that are unique among REVOLUTA sequences and are preferably at least about 10 nucleotides in length, and most preferably at least about 15, about 20 or about 50 nucleotides in length.
  • probes may be used to amplify corresponding REVOLUTA sequences from a chosen plant by PCR. This technique may be used to isolate additional coding sequences from a desired plant or as a diagnostic assay to determine the presence of coding sequences in a plant.
  • Hybridization techniques include hybridization screening of plated DNA libraries (either plaques or colonies; see, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, New York (1989)). 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.
  • 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, preferably 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°C for short probes (e.g., 10 to 50 nucleotides) and at least about 60°C for long probes (e.g., greater than 50 nucleotides).
  • Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide.
  • Exemplary moderate stringency conditions include hybridization in 40 to 45% formamide, 1.0 M NaCl, 1 % SDS at 37°C, and a wash in 0.5X to 1X SSC at 55 to 60°C.
  • Exemplary high stringency conditions include hybridization in 50% formamide, 1 M NaCl, 1 % SDS at 37°C, and a wash in 0.1X 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.
  • T m 81.5°C + 16.6 (log M) + 0.41 (%GC) - 0.61 (% form) - 500/L; where M is the molarity of monovalent cations, %GC is the percentage of guanosine and cytosine nucleotides in the DNA, % form is the percentage of formamide in the hybridization solution, and L is the length 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°C. Generally, 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.
  • the REVOLUTA sequences of the present invention are predicted to limit the growth of meristem structures by regulating cell division in the meristem.
  • fragments and variants of the REVOLUTA sequences that encode REVOLUTA polypeptides of the invention are not limited to retaining the native biological activity of the protein. It is recognized that the sequences of the invention encoding the REVOLUTA polypeptides may be modified such that expression of the polypeptide results in meristem development. Consequently, sequences encoding dominant-negative REVOLUTA polypeptide are encompassed by the present invention.
  • the variants and fragment of the REVOLUTA polypeptide interfere with the function of the normal, endogenous REVOLUTA protein. Thus, the action of the endogenous REVOLUTA polypeptide can be block without inactivating the structural REVOLUTA gene sequence or its RNA.
  • a fragment of a REVOLUTA nucleotide sequence that encodes a REVOLUTA polypeptide having dominate negative activity will encode at least 15, 25, 30, 50, 60, 70, 80, 90, or 100 contiguous amino acids, or up to the total number of amino acids present in a full-length REVOLUTA protein of the invention.
  • a variant of a REVOLUTA protein of the invention that has dominant negative activity will have at least about 40%, 50%, 60%, 65%, 70%, generally at least about 75%, 80%, 85%, preferably at least about 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, and more preferably at least about 98%, 99% or more sequence identity to the amino acid sequence for the native protein as determined by sequence alignment programs described elsewhere herein using default parameters.
  • a variant of a REVOLUTA polypeptide having dominate negative activity may differ from that protein 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.
  • Functional assays to identify REVOLUTA sequences having dominant negative activity include, expression of the REVOLUTA fragment or variant in a plant or plant cell and visually assaying for a modulation in meristem development, particularly an increase in lateral shoots.
  • nucleic acid sequences of the present invention can be expressed in a host cell such as bacteria, yeast, insect, mammalian, or preferably plant cells. It is expected that those of skill in the art are knowledgeable in the numerous expression systems available for expression of a nucleic acid encoding a protein of the present invention. No attempt to describe in detail the various methods known for the expression of proteins in prokaryotes or eukaryotes will be made.
  • a host cells may be prokaryotic cells such as E. coli, or eukaryotic cells such as yeast, insect, amphibian, or mammalian cells.
  • host cells are monocotyledonous or dicotyledonous plant cells.
  • a particularly preferred monocotyledonous host cell is a maize host cell.
  • the REVOLUTA sequences of the invention are provided in expression cassettes or nucleotide constructs for expression in the plant of interest.
  • the cassette will include 5' and 3' regulatory sequences operably linked to a REVOLUTA sequence of the invention.
  • operably linked is intended a functional linkage between a promoter and a second sequence, wherein the promoter sequence initiates and mediates transcription of the DNA sequence corresponding to the second sequence.
  • operably linked means that the nucleic acid sequences being linked are contiguous and, where necessary to join two protein coding regions, contiguous and in the same reading frame.
  • the cassette may additionally contain at least one additional gene to be cotransformed into the organism. Alternatively, the additional gene(s) can be provided on multiple expression cassettes.
  • Such an expression cassette is provided with a plurality of restriction sites for insertion of the REVOLUTA sequence to be under the transcriptional regulation of the regulatory regions.
  • the expression cassette may additionally contain selectable marker genes.
  • the expression cassette will include in the 5'-3' direction of transcription, a transcriptional and translational initiation region, a REVOLUTA sequence of the invention, and a transcriptional and translational termination region functional in plants.
  • the transcriptional initiation region, the promoter may be native or analogous or foreign or heterologous to the plant host. Additionally, the promoter may be the natural sequence or alternatively a synthetic sequence. By “foreign” is intended that the transcriptional initiation region is not found in the native plant into which the transcriptional initiation region is introduced.
  • a chimeric gene comprises a coding sequence operably linked to a transcription initiation region that is heterologous to the coding sequence.
  • the native promoter sequences may be used. Such constructs would change expression levels of the REVOLUTA RNA/protein in the plant or plant cell. Thus, the phenotype of the plant or plant cell is altered.
  • the termination region may be native with the transcriptional initiation region, may be native with the operably linked DNA sequence of interest, or may be derived from another source. Convenient termination regions are available from the Ti-plasmid of A. tumefaciens, such as the octopine synthase and nopaline synthase termination regions. See also Guerineau et al., Mol. Gen. Genet.
  • the gene(s) may be optimized for increased expression in the transformed plant. That is, the genes can be synthesized using plant- preferred codons for improved expression.
  • Additional sequence modifications are known to enhance gene expression in a cellular 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 cellular 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 in the expression cassette construct. 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., PNAS USA 86:6126-6130, (1989)); potyvirus leaders, for example, TEV leader (Tobacco Etch Virus) (Allison et al, MDMV leader (Maize Dwarf Mosaic Virus); Virology 754:9-20, (1986)), and human immunoglobulin heavy-chain binding protein (BiP), (Macejak et al.
  • picornavirus leaders for example, EMCV leader (Encephalomyocarditis 5' noncoding region) (Elroy-Stein et al., PNAS USA 86:6126-6130, (1989)
  • potyvirus leaders for example, TEV leader (Tobacco Etch Virus) (Allison et al, MDMV leader (Maize Dwarf Mosaic Virus); Virology 7
  • AMV RNA 4 alfalfa mosaic virus
  • TMV tobacco mosaic virus leader
  • MCMV maize chlorotic mottle virus leader
  • the various DNA fragments may be manipulated, so as to provide for the DNA sequences in the proper orientation and, as appropriate, in the proper reading frame.
  • adapters or linkers may be employed to join the DNA fragments or other manipulations may be involved to provide for convenient restriction sites, removal of superfluous DNA, removal of restriction sites, or the like.
  • in vitro mutagenesis, primer repair, restriction, annealing, resubstitutions, e.g., transitions and transversions may be involved.
  • the expression cassette will comprise a selectable marker gene for the selection of transformed cells.
  • Marker genes include genes encoding antibiotic resistance, such as those encoding neomycin phosphotransferase II (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).
  • NEO neomycin phosphotransferase II
  • HPT hygromycin phosphotransferase
  • a number of promoters can be used in the practice of the invention.
  • the promoters can be selected based on the desired outcome. That is, the nucleic acids can be combined with constitutive, tissue-preferred, or other promoters for expression in plants.
  • constitutive promoters include, for example, the core promoter of the Rsyn7 (PCT Application Serial No. US99/03863); Scp1 promoter (U.S. Patent No. 6,072,050), rice actin (McElroy et al., Plant Cell 2:163-171 , (1990)); ubiquitin (Christensen et al., Plant Mol. Biol. 72:619-632, (1989); and Christensen et al., Plant Mol Biol.
  • the gene from an inducible promoter.
  • chemical-regulated promoters can be used to modulate the expression of a gene in a plant through the application of an exogenous chemical regulator.
  • the promoter may be a chemical-inducible promoter, where application of the chemical induces gene expression, or a chemical-repressible promoter, where application of the chemical represses gene expression.
  • Chemical-inducible promoters are known in the art and include, but are not limited to, the maize ln2-2 promoter, which is activated by benzenesulfonamide herbicide safeners, the maize GST promoter, which is activated by hydrophobic electrophilic compounds that are used as pre-emergent herbicides, and the tobacco PR-1a promoter, which is activated by salicylic acid.
  • Other chemical-regulated promoters of interest include steroid-responsive promoters (see, for example, the glucocorticoid-inducible promoter in Schena et al., Proc. Natl. Acad. Sci. USA 88:10421-10425, (1991 ) and McNellis et al., Plant
  • the promoter is a drought-inducible promoter.
  • drought-inducible promoter is intended a promoter that is inducible under conditions of osmotic stress.
  • the RAB-17 promoter is induced by drought as well as other stresses, presumably as a result of its regulation by the plant hormone ABA .
  • the promoter of the Arabidopsis Atmybl gene can be used as a general ABA-responsive, drought and stress-induced promoter (Urao et al., Plant Cell 5:1529-1539, (1993)).
  • drought-inducible promoters include 26-g from Pisum sativum (Guerrero et al., Plant Physiol 88:401-408, (1988); Guerrero et al, Plant Mol Biol 15: 11-26, (1990)), trg-31 from tobacco (Guerrero et al., Plant Mol Biol 27:929-935, (1993)) btg-26 from Brassica napus (U.S. Patent No.
  • tissue-preferred promoters can be utilized to target enhanced REVOLUTA sequence expression within a particular plant tissue.
  • Tissue- preferred promoters include, for example, shoot meristem-preferred promoters as described in Atanassova et al., Plant J. 2:291 , (1992), U.S. Patent No. 6,239, 329; which is herein incorporated by reference.
  • the regulatory elements of the KNOTTED1 gene can be used to direct shoot meristem-preferred expression (Dorien et al, Plant Molecule Biology 48:423-441 , (2002) and Tomoaki et al, Genes and Development 75:581-590, (2001 ), both of which are herein incorporated by reference).
  • the promoter and or other regulatory elements of the REVOLUTA gene could be used for meristem-preferred expression. See, for example, Genbank Accession No. AC024594 (Rice) and AB005246 (Arabidopsis), both of which are herein incorporated by reference.
  • the promoter can encompass up to .5Kb, 1 Kb, 2Kb, or more of the sequences flanking the coding region of these genes. Such promoters can be modified, if necessary, for weak expression.
  • the methods of the invention involve introducing a nucleotide construct into a plant.
  • introducing is intended presenting to the plant the nucleotide construct in such a manner that the construct 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 nucleotide construct to a plant, only that the nucleotide construct gains access to the interior of at least one cell of the plant.
  • Methods for introducing nucleotide constructs 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 that the nucleotide construct introduced into a plant integrates into the genome of the plant and is capable of being inherited by progeny thereof.
  • transient transformation is intended that a nucleotide construct introduced into a plant does not integrate into the genome of the plant.
  • the nucleotide constructs of the invention may be introduced into plants by contacting plants with a virus or viral nucleic acids. Generally, such methods involve incorporating a nucleotide construct of the invention within a viral DNA or RNA molecule. It is recognized that the a REVOLUTA sequence of the invention 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 promoters of the invention also encompass promoters utilized for transcription by viral RNA polymerases. Methods for introducing nucleotide constructs into plants and expressing a protein encoded therein, involving viral DNA or RNA molecules, are known in the art.
  • Transformation protocols as well as protocols for introducing nucleotide sequences into plants may vary depending on the type of plant or plant cell, i.e., monocot or dicot, targeted for transformation.
  • Suitable methods of introducing nucleotide sequences into plant cells and subsequent insertion into the plant genome include microinjection (Crossway et al., Biotechniques 4:320-334, (1986)), elecfroporation (Riggs et al., Proc. Natl. Acad. Sci. USA 83:5602-5606, (1986), Agrobacterium-med a ⁇ ed transformation (Townsend et al., U.S. Patent No. 5,563,055; Zhao et al., U.S. Patent No. 5,981 ,840), direct gene transfer (Paszkowski et al., EMBO J.
  • the nucleic acid sequences of the present invention can be stacked with any combination of polynucleotide sequences of interest in order to create plants with a desired phenotype.
  • the polynucleotides of the present invention may be stacked with any other polynucleotides of the present invention, such as any combination of ZmREVI and ZmREV2 or with other genes.
  • 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.
  • polynucleotides of the present invention can also be stacked with traits desirable for insect, disease or herbicide resistance (e.g. Bacillus thuringiensis toxic proteins (U.S. Patent Nos.
  • modified oils e.g., fatty acid desaturase genes (U.S. Patent No. 5,952,544; WO 94/11516)
  • modified starches e.g., ADPG pyrophosphorylases (AGPase), starch synthases (SS), starch branching enzymes (SBE) and starch debranching enzymes (SDBE)
  • polymers or bioplastics e.g., U.S. patent No. 5.602,321; beta-ketothiolase, polyhydroxybutyrate synthase, and acetoacetyl-CoA reductase (Schubert et al., J. Bacteriol.
  • PHAs polyhydroxyalkanoates
  • stacked combinations can be created by any method including but not limited to cross breeding plants by any conventional or TopCross methodology, or genetic transformation.
  • the polynucleotide sequences of interest can be combined at any time and in any order.
  • 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.
  • 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.
  • the cells that have been transformed may be grown into plants in accordance with conventional ways. See, for example, McCormick et al, Plant
  • the present invention may be used for transformation of any plant species, including, but not limited to, monocots and dicots. Examples of plants of interest include, but are not limited to, corn (Zea mays), Brassica sp. (e.g., ⁇ . 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 (Seta ⁇ a italica), finger millet (Eleusine coracana)), sunflower (Helianthus annuus), safflower (Carthamus tinctorius), wheat (Triticum aestivum), soybean (Glycine max), tobacco (Nicotiana tabacum), potato (Solanum tuberosum), peanuts (Arachis hypogaea), cotton (Gossypium barbadense, Gossypium hirsutum), sweet potato (Ipomoea batatus), cassava (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 hyb da), 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 (Chamaecypahs nootkatensis).
  • pines such as loblolly pine (Pinus taeda), slash pine
  • plants of the present invention are crop plants (for example, corn, alfalfa, sunflower, Brassica, soybean, cotton, safflower, peanut, sorghum, wheat, millet, tobacco, etc.), more preferably corn and soybean plants, yet more preferably corn plants.
  • crop plants for example, corn, alfalfa, sunflower, Brassica, soybean, cotton, safflower, peanut, sorghum, wheat, millet, tobacco, etc.
  • 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 (Chamaecypahs nootkatensis).
  • pines such as loblolly pine (Pinus taeda), slash pine
  • plants of the present invention are crop plants (for example, corn, alfalfa, sunflower, Brassica, soybean, cotton, safflower, peanut, sorghum, wheat, millet, tobacco, etc.), more preferably corn and soybean plants, yet more preferably corn plants.
  • crop plants for example, corn, alfalfa, sunflower, Brassica, soybean, cotton, safflower, peanut, sorghum, wheat, millet, tobacco, etc.
  • Prokaryotic cells may be used as hosts for expression. Prokaryotes most frequently are represented by various strains of E. coli; however, other microbial strains may also be used. Commonly used prokaryotic control sequences which are defined herein to include promoters for transcription initiation, optionally with an operator, along with ribosome binding sequences, include such commonly used promoters as the beta lactamase (penicillinase) and lactose (lac) promoter systems (Chang et al., Nature 798:1056, (1977)), the tryptophan (trp) promoter system (Goeddel et al., Nucleic Acids Res.
  • promoters for transcription initiation optionally with an operator, along with ribosome binding sequences
  • promoters include such commonly used promoters as the beta lactamase (penicillinase) and lactose (lac) promoter systems (Chang et al., Nature 798:1056, (1977)), the tryptophan
  • selection markers include genes specifying resistance to ampicillin, tetracycline, or chloramphenicol.
  • Bacterial vectors are typically of plasmid or phage origin. Appropriate bacterial cells are infected with phage vector particles or transfected with naked phage vector DNA. If a plasmid vector is used, the bacterial cells are transfected with the plasmid vector DNA. Expression systems for expressing a protein of the present invention are available using Bacillus sp. and Salmonella (Palva et al, Gene 22:229-235, (1983)); Mosbach et al., Nature 302:543-545, (1983)).
  • eukaryotic expression systems such as yeast, insect cell lines, plant and mammalian cells, are known to those of skill in the art.
  • a polynucleotide of the present invention can be expressed in these eukaryotic systems.
  • transformed/transfected plant cells as discussed infra, are employed as expression systems for production of the proteins of the instant invention.
  • yeasts for production of eukaryotic proteins are Saccharomyces cerevisiae and Pichia pastoris.
  • Vectors, strains, and protocols for expression in Saccharomyces and Pichia are known in the art and available from commercial suppliers (e.g., Invitrogen).
  • Suitable vectors usually have expression control sequences, such as promoters, including 3-phosphoglycerate kinase or alcohol oxidase, and an origin of replication, termination sequences and the like as desired.
  • a protein of the present invention once expressed, can be isolated from yeast by lysing the cells and applying standard protein isolation techniques to the lists.
  • the monitoring of the purification process can be accomplished by using Western blot techniques or radioimmunoassay of other standard immunoassay techniques.
  • sequences of the present invention can also be ligated to various expression vectors for use in transfecting cell cultures of, for instance, mammalian, insect, or plant origin.
  • Illustrative cell cultures useful for the production of the peptides are mammalian cells.
  • a number of suitable host cell lines capable of expressing intact proteins have been developed in the art, and include the HEK293, BHK21 , and CHO cell lines.
  • Expression vectors for these cells can include expression control sequences, such as an origin of replication, a promoter (e.g. the CMV promoter, a HSV tk promoter or pgk (phosphoglycerate kinase) promoter), an enhancer (Queen et al, Immunol. Rev.
  • ribosome binding sites such as ribosome binding sites, RNA splice sites, polyadenylation sites (e.g., an SV40 large T Ag poly A addition site), and transcriptional terminator sequences.
  • polyadenylation sites e.g., an SV40 large T Ag poly A addition site
  • transcriptional terminator sequences e.g., an SV40 large T Ag poly A addition site
  • Appropriate vectors for expressing proteins of the present invention in insect cells are usually derived from the SF9 baculovirus.
  • suitable insect cell lines include mosquito larvae, silkworm, armyworm, moth and Drosophila cell lines such as a Schneider cell line (See, Schneider, J. Embryol. Exp. Morphol. 27:353-365, (1987)).
  • polyadenylation or transcription terminator sequences are typically incorporated into the vector.
  • An example of a terminator sequence is the polyadenylation sequence from the bovine growth hormone gene. Sequences for accurate splicing of the transcript may also be included.
  • An example of a splicing sequence is the VP1 intron from SV40 (Sprague et al., J. Virol. 45:773-781 , (1983)).
  • gene sequences to control replication in the host cell may be incorporated into the vector such as those found in bovine papilloma virus type-vectors (Saveria- Campo, DNA Cloning Vol. II a Practical Approach, D.M. Glover, Ed., IRL Press, Arlington, Virginia, pp. 213-238, (1985)).
  • Animal and lower eukaryotic (e.g., yeast) host cells are competent or rendered competent for transfection by various means.
  • eukaryotic (e.g., yeast) host cells are competent or rendered competent for transfection by various means.
  • methods of introducing DNA into animal cells include: calcium phosphate precipitation, fusion of the recipient cells with bacterial protoplasts containing the DNA, treatment of the recipient cells with liposomes containing the DNA, DEAE dextrin, electroporation, biolistics, and micro-injection of the DNA directly into the cells.
  • the transfected cells are cultured by means well known in the art (Kuchler, Biochemical Methods in Cell Culture and Virology, Dowden, Hutchinson and Ross, Inc., (1997)).
  • antisense constructions complementary to at least a portion of the messenger RNA (mRNA) for the REVOLUTA sequences can be constructed.
  • Antisense nucleotides are constructed to hybridize with the corresponding mRNA. Modifications of the antisense sequences may be made as long as the sequences hybridize to and interfere with expression of the corresponding mRNA. In this manner, antisense constructions having 70%, preferably 80%, more preferably 85% sequence identity to the corresponding antisense sequences may be used. Furthermore, portions of the antisense nucleotides may be used to disrupt the expression of the target gene.
  • sequences of at least 50 nucleotides, 100 nucleotides, 200 nucleotides, or greater may be used.
  • the sequences may range from nucleotides 1-370 or 370-740 of SEQ ID NO:1 ; from nucleotides 1-141 to 141-282 of SEQ ID NO:3; or comprise the nucleotide sequence encoding the conserved C-terminal domain of REVOLUTA as outlined in Figure 2.
  • the nucleotide sequences of the present invention may also be used in the sense orientation to suppress the expression of endogenous genes in plants.
  • Methods for suppressing gene expression in plants using nucleotide sequences in the sense orientation are known in the art.
  • the methods generally involve transforming plants with a DNA construct comprising a promoter that drives expression in a plant operably linked to at least a portion of a nucleotide sequence that corresponds to the transcript of the endogenous gene.
  • a nucleotide sequence has substantial sequence identity to the sequence of the transcript of the endogenous gene, preferably greater than about 65% sequence identity, more preferably greater than about 85% sequence identity, most preferably greater than about 95% sequence identity. See, U.S. Patent Nos. 5,283,184 and 5,034,323; herein incorporated by reference.
  • nucleotide constructs are not intended to limit the present invention to nucleotide constructs comprising DNA.
  • nucleotide constructs particularly polynucleotides and oligonucleotides, comprised of ribonucleotides and combinations of ribonucleotides and deoxyribonucleotides may also be employed in the methods disclosed herein.
  • the nucleotide constructs of the present invention encompass all nucleotide constructs that can be employed in the methods of the present invention for transforming plants including, but not limited to, those comprised of deoxyribonucleotides, ribonucleotides, and combinations thereof.
  • nucleotide constructs of the invention also encompass all forms of nucleotide constructs including, but not limited to, single-stranded forms, double-stranded forms, hairpins, stem-and-loop structures, and the like.
  • the methods of the invention may employ a nucleotide construct that is capable of directing, in a transformed plant, the expression of at least one protein, or at least one RNA, such as, for example, an antisense RNA that is complementary to at least a portion of an mRNA.
  • nucleotide construct is comprised of a coding sequence for a protein or an RNA operably linked to 5' and 3' transcriptional regulatory regions.
  • methods of the invention may employ a nucleotide construct that is not capable of directing, in a transformed plant, the expression of a protein or an RNA.
  • methods of the present invention do not depend on the incorporation of the entire nucleotide construct into the genome, only that the plant or cell thereof is altered as a result of the introduction of the nucleotide construct into a cell.
  • the genome may be altered following the introduction of the nucleotide construct into a cell.
  • the nucleotide construct, 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 in the genome. While the methods of the present invention do not depend on additions, deletions, or substitutions of any particular number of nucleotides, it is recognized that such additions, deletions, or substitutions comprise at least one nucleotide.
  • nucleotide constructs of the invention also encompass nucleotide constructs that may be employed in methods for altering or mutating a genomic nucleotide sequence in an organism, including, but not limited to, chimeric vectors, chimeric mutational vectors, chimeric repair vectors, mixed-duplex oligonucleotides, self-complementary chimeric oligonucleotides, and recombinogenic oligonucleobases.
  • nucleotide constructs and methods of use such as, for example, chimeraplasty, are known in the art.
  • Chimeraplasty involves the use of such nucleotide constructs to introduce site-specific changes into the sequence of genomic DNA within an organism. See, U.S.
  • the present invention provides methods and compositions for modulating the level and/or activity of the REVOLUTA polypeptides in a host cell, particularly, a plant or plant cell.
  • the level and/or activity of the REVOLUTA polypeptide of the present invention is modulated by altering, in vivo or in vitro, the promoter of a gene to up- or down- regulate gene expression.
  • the coding regions of native genes of the present invention can be altered via substitution, addition, insertion, or deletion to decrease activity of the encoded enzyme. See, e.g., U.S. Patent 5,565,350 and PCT/US93/03868.
  • an isolated nucleic acid e.g., a vector
  • a plant cell comprising the promoter operably linked to a polynucleotide of the present invention is selected for by means known to those of skill in the art such as, but not limited to, Southern blot, DNA sequencing, or PCR analysis using primers specific to the promoter and to the gene and detecting amplicons produced therefrom.
  • a plant or plant part altered or modified by the foregoing embodiments is grown under plant forming conditions for a time sufficient to modulate the level and/or activity of polypeptides of the present invention in the plant. Plant forming conditions are well known in the art and discussed briefly, supra.
  • the level and/or activity of the polypeptide of the invention may be modulated in a plant or plant cell by contacting the plant or plant cell with the polypeptide of the invention in the form of a chemical spray.
  • the activity and/or level of the REVOLUTA polypeptide is modulated (increased or decreased) by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% relative to a native control plant, plant part, or cell lacking the aforementioned recombinant expression cassette. Modulation in the present invention may occur during and/or subsequent to growth of the plant to the desired stage of development. Modulating nucleic acid expression temporally and/or in particular tissues can be controlled by employing the appropriate promoter operably linked to a polynucleotide of the present invention in, for example, sense or antisense orientation as discussed in greater detail, supra.
  • Induction of expression of a polynucleotide of the present invention can also be controlled by exogenous administration of an effective amount of inducing compound.
  • Inducible promoters and inducing compounds, which activate expression from these promoters are well known in the art.
  • the polypeptides of the present invention are modulated in monocots, particularly maize.
  • the present invention further provides compositions and methods for modulating plant development and/or developmental pathways. Accordingly, the present invention
  • REVOLUTA sequences of the invention find use in controlling or modulating cell division, differentiation, and development.
  • the sequences of the present invention find use in modulating meristem development.
  • modulating meristem development is intended any alteration in the meristem phenotype including, for example, an alteration in the formation and maintenance of the meristem and/or an alteration of size and appearance of shoot and floral meristems. Such modulations in meristem development may result in an increased or a decreased yield of leaves, tassel, and/or ears.
  • expression of the REVOLUTA sequences of the invention in a manner that results in a decreased activity or level of the REVOLUTA polypeptide can result in: 1 ) the generation of additional leaves before flowering begins, thereby providing plants having greater energy production and thus increasing yield; 2) an increase in the number of seed-bearing carpels, thereby allowing for the production of additional seeds; 3) the generation of a thicker stem thereby preventing crop lodging, increasing wind resistance ect.; 4) an alteration of the fruit of the plants (i.e., an increase in size, shape, and/or the number of fruit per plant); and, 5) an increase in the number of flowers. Plants exhibiting modulated meristem development as described above can be selected using visual observation.
  • an antisense construct of a REVOLUTA sequences can be expressed in a plant or plant cell to reduce the level or activity of the endogenous REVOLUTA sequence.
  • the level of a polypeptide in a host cell is modulated and comprises providing a plant cell comprising an mRNA having the sequence set forth in SEQ ID NO:1 , 3, 5 or a variant or fragment thereof; and, introducing a nucleotide sequence comprising at least 15 consecutive nucleotides of SEQ ID NO:1 , 3, or 5 into the plant or plant cell, wherein the expression of the nucleotide sequence inhibits expression of the mRNA in the plant or plant cell.
  • transformation with a REVOLUTA sequence can result in cosuppression of the endogenous REVOLUTA sequence.
  • modifications can be made to the REVOLUTA sequences of the invention that result in the REVOLUTA sequence having a dominant negative activity.
  • transformation of a plant or plant cell with the appropriate REVOLUTA sequences will result in a decrease level or activity of the endogenous REVOLUTA sequences, resulting in meristem enlargement.
  • the polypeptide having dominant negative activity can be contacted with the plant or plant cell in the form of a chemical spray.
  • methods are provided to decrease the level and/or activity of the endogenous REVOLUTA sequence in a plant or plant cell.
  • the decrease in REVOLUTA activity or level is predicted to cause a decrease in meristem growth.
  • Such an alteration in growth may present as a shorter ear and as a decrease in spikelet formation.
  • Such a phenotype finds use in producing plants having a higher kernel carrying potential and an enhanced yield stability under a variety of stress-prone conditions (i.e., drought, over population, etc.).
  • down regulation of the expression of the REVOLUTA sequences of the invention may find use in increasing grain yields under drought conditions.
  • Many factors have been identified that contribute to higher plant performance under water deficiencies. Improved maize yield performance under drought stress was accompanied by shorter ears (possibly due to better silking synchronizations resulting in efficient pollinations or quicker escape from the stress by shortened ear development duration).
  • phenotypic changes in drought-tolerant maize plants include increased kernel carrying capacity due to reduced barrenness under drought conditions.
  • drought tolerant maize have a reduction in the number of spikelets formed on the ear and thus overall seed set is facilitated by reducing water and carbon requirements per spikelet.
  • each spikelet is ultimately more successful in forming grain, especially under drought conditions.
  • each spikelet is ultimately more successful in forming grain, especially under drought conditions.
  • decreasing the level and/or reducing the functional activity of the REVOLUTA sequences of the invention in a plant or plant cell may find use in producing plants having a higher kernel carrying capacity on a per square foot of land and further enhance yield stability in a variety of stress- prone conditions (i.e., drought or overpopulated).
  • the polypeptide is contacted with the plant in the form of a chemical spray at time of drought.
  • the dominant negative form of the REVOLUTA sequence is expressed in the plant.
  • the dominant negative form of the REVOLUTA sequence may be operably linked to a drought-inducible promoter, such as the drought-inducible promoter RAB17 promoter (Vilardell et al., Plant Mol Biol 14:423-432, (1990)).
  • a drought-inducible promoter such as the drought-inducible promoter RAB17 promoter (Vilardell et al., Plant Mol Biol 14:423-432, (1990)).
  • Such promoters are known in the art and have been described elsewhere herein.
  • the REVOLUTA polypeptides and variants and fragments thereof of the present invention can be presented to the plant or plant cell as a chemical spray.
  • the REVOLUTA polypeptide or variants or fragments thereof are presented to the plant or plant cell during an appropriate stage of development or under the appropriate environmental conditions in amounts sufficient for the polypeptide to modulate meristem or plant organ or plant development.
  • the compositions can be applied to the plant or plant cell by, for example, spraying, atomizing, dusting, scattering, coating or pouring. It is recognized that any means to bring the REVOLUTA polypeptides in contact with the plant can be used in the practice of the invention.
  • the polypeptides of the invention can be formulated with an acceptable carrier into a composition(s) that is, for example, a suspension, a solution, an emulsion, a dusting powder, a dispersible granule, a wettable powder, an emulsifiable concentrate, an aerosol, an impregnated granule, an adjuvant, a coatable paste, and also encapsulations in, for example, polymer substances.
  • a composition(s) that is, for example, a suspension, a solution, an emulsion, a dusting powder, a dispersible granule, a wettable powder, an emulsifiable concentrate, an aerosol, an impregnated granule, an adjuvant, a coatable paste, and also encapsulations in, for example, polymer substances.
  • compositions disclosed above may be obtained by the addition of a surface- active agent, an inert carrier, a preservative, a humectant, a feeding stimulant, an attractant, an encapsulating agent, a binder, an emulsifier, a dye, a UV protectant, a buffer, a flow agent or fertilizers, micronutrient donors or other preparations that influence plant growth.
  • One or more agrochemicals including, but not limited to, herbicides, insecticides, fungicides, bacteriocides, nematocides, molluscicides, acaracides, plant growth regulators, harvest aids, and fertilizers, can be combined with carriers, surfactants, or adjuvants customarily employed in the art of formulation or other components to facilitate product handling and application for particular target mycotoxins.
  • Suitable carriers and adjuvants can be solid or liquid and correspond to the substances ordinarily employed in formulation technology, e.g., natural or regenerated mineral substances, solvents, dispersants, wetting agents, tackifiers, binders, or fertilizers.
  • the active ingredients of the present invention are normally applied in the form of compositions and can be applied to the crop area or plant to be treated, simultaneously or in succession, with other compounds.
  • Suitable surface-active agents include, but are not limited to, anionic compounds such as a carboxylate of, for example, a metal; a carboxylate of a long chain fatty acid; an N-acylsarcosinate; mono or di-esters of phosphoric acid with fatty alcohol ethoxylates or salts of such esters; fatty alcohol sulfates such as sodium dodecyl sulfate, sodium octadecyl sulfate, or sodium cetyl sulfate; ethoxylated fatty alcohol sulfates; ethoxylated alkylphenol sulfates; lignin sulfonates; petroleum sulfonates; alkyl aryl sulfonates such as alkyl-benzene sulfonates or lower alkylnaphtalene sulfonates, e.g., butyl-naphthalene sulfonate;
  • Non-ionic agents include condensation products of fatty acid esters, fatty alcohols, fatty acid amides or fatty-alkyl- or alkenyl-substituted phenols with ethylene oxide, fatty esters of polyhydric alcohol ethers, e.g., sorbitan fatty acid esters, condensation products of such esters with ethylene oxide, e.g. polyoxyethylene sorbitar fatty acid esters, block copolymers of ethylene oxide and propylene oxide, acetylenic glycols such as 2, 4, 7, 9-tetraethyl-5-decyn-4, 7-diol, or ethoxylated acetylenic glycols.
  • a cationic surface-active agent examples include, for instance, an aliphatic mono-, di-, or polyamine such as an acetate, naphthenate, or oleate; or oxygen-containing amine such as an amine oxide of polyoxyethylene alkylamine; an amide-linked amine prepared by the condensation of a carboxylic acid with a di- or polyamine; or a quaternary ammonium salt.
  • inert materials include, but are not limited to, inorganic minerals such as kaolin, phyllosilicates, carbonates, sulfates, phosphates, or botanical materials such as cork, powdered corncobs, peanut hulls, rice hulls, and walnut shells.
  • inorganic minerals such as kaolin, phyllosilicates, carbonates, sulfates, phosphates, or botanical materials such as cork, powdered corncobs, peanut hulls, rice hulls, and walnut shells.
  • compositions of the present invention can be in a suitable form for direct application or as concentrate of primary composition, which requires dilution with a suitable quantity of water or other diluent before application.
  • concentration will vary depending upon the nature of the particular formulation, specifically, whether it is a concentrate or to be used directly.
  • compositions, as well as the polypeptides of the present invention can be treated prior to formulation to prolong the activity when applied to the plant or plant cell as long as the pretreatment is not deleterious to the activity.
  • Such treatment can be by chemical and/or physical means as long as the treatment does not deleteriously affect the properties of the composition(s).
  • chemical reagents include, but are not limited to, halogenating agents; aldehydes such as formaldehyde and glutaraldehyde; anti- infectives, such as zephiran chloride; alcohols, such as isopropanol and ethanol; and histological fixatives, such as Bouin's fixative and Helly's fixative (see, for example, Humason, Animal Tissue Techniques (W.H. Freeman and Co.), (1967).
  • halogenating agents aldehydes such as formaldehyde and glutaraldehyde
  • anti- infectives such as zephiran chloride
  • alcohols such as isopropanol and ethanol
  • histological fixatives such as Bouin's fixative and Helly's fixative (see, for example, Humason, Animal Tissue Techniques (W.H. Freeman and Co.), (1967).
  • the present invention further provides a method of genotyping a plant using the polynucleotide of the present invention.
  • the REVOLUTA sequences of the invention regulate meristem or plant organ or whole plant development. The sequences therefore find use as molecular markers for the selection of plants that will have the desired developmental phenotype. For instance, specific alleles of the REVOLUTA sequences of the invention could be used to trace maize hybrids that form more or fewer ears.
  • the plant is a monocot, such as maize or sorghum. Genotyping provides a means of distinguishing homologs of a chromosome pair and can be used to differentiate segregants in a plant population.
  • RFLPs restriction fragment length polymorphism's
  • RFLPs are the product of allelic differences between DNA restriction fragments resulting from nucleotide sequence variability.
  • RFLPs are typically detected by extraction of genomic DNA and digestion with a restriction enzyme. Generally, the resulting fragments are separated according to size and hybridized with a probe; single copy probes are preferred. Restriction fragments from homologous chromosomes are revealed. Differences in fragment size among alleles represent an RFLP.
  • the present invention further provides a means to follow segregation of a gene or nucleic acid of the present invention as well as chromosomal sequences genetically linked to these genes or nucleic acids using such techniques as RFLP analysis.
  • Linked chromosomal sequences are within 50 centiMorgans (cM), often within 40 or 30 cM, preferably within 20 or 10 cM, more preferably within 5, 3, 2, or 1 cM of a gene of the present invention.
  • the nucleic acid probes employed for molecular marker mapping of plant nuclear genomes selectively hybridize, under selective hybridization conditions, to a gene encoding a polynucleotide of the present invention.
  • the probes are selected from polynucleotides of the present invention.
  • these probes are cDNA probes or restriction enzyme treated (e.g., PST I) genomic clones.
  • the length of the probes is discussed in greater detail, supra, but is typically at least 15 bases in length, more preferably at least 20, 25, 30, 35, 40, or 50 bases in length. Generally, however, the probes are less than about 1 kilobase in length.
  • the probes are single copy probes that hybridize to a unique locus in haploid chromosome compliment.
  • Some exemplary restriction enzymes employed in RFLP mapping are EcoRI, EcoRv, and Ss l.
  • restriction enzyme includes reference to a composition that recognizes and, alone or in conjunction with another composition, cleaves at a specific nucleotide sequence.
  • the method of detecting an RFLP comprises the steps of (a) digesting genomic DNA of a plant with a restriction enzyme; (b) hybridizing a nucleic acid probe, under selective hybridization conditions, to a sequence of a polynucleotide of the present of said genomic DNA; (c) detecting therefrom a RFLP.
  • polymorphic (allelic) variants of polynucleotides of the present invention can be had by utilizing molecular marker techniques well known to those of skill in the art including such techniques as: 1 ) single stranded conformation analysis (SSCA); 2) denaturing gradient gel electrophoresis (DGGE); 3) RNase protection assays; 4) allele-specific oligonucleotides (ASOs); 5) the use of proteins which recognize nucleotide mismatches, such as the E. coli mutS protein; and 6)allele-specific PCR.
  • molecular marker techniques well known to those of skill in the art including such techniques as: 1 ) single stranded conformation analysis (SSCA); 2) denaturing gradient gel electrophoresis (DGGE); 3) RNase protection assays; 4) allele-specific oligonucleotides (ASOs); 5) the use of proteins which recognize nucleotide mismatches, such as the E. coli mutS protein; and
  • the present invention further provides a method of genotyping comprising the steps of contacting, under stringent hybridization conditions, a sample suspected of comprising a polynucleotide of the present invention with a nucleic acid probe.
  • a sample suspected of comprising a polynucleotide of the present invention with a nucleic acid probe.
  • the sample is a plant sample, preferably, a sample suspected of comprising a maize polynucleotide of the present invention (e.g., gene, mRNA).
  • the nucleic acid probe selectively hybridizes, under stringent conditions, to a subsequence of a polynucleotide of the present invention comprising a polymorphic marker. Selective hybridization of the nucleic acid probe to the polymorphic marker nucleic acid sequence yields a hybridization complex. Detection of the hybridization complex indicates the presence of that polymorphic marker in the sample.
  • the nucleic acid probe comprises a polynucleotide of the present invention.
  • Example 1 Isolation of REVOLUTA sequences and Expression Patterns in Maize ZmREV is a homologous sequence clone of the Arabidopsis REV/IFL1 gene product from maize.
  • the Arabidopsis gene encodes a homeodomain- leucine zipper-type transcription factor, class Ill-type (HD-ZIP).
  • HD-Zips are classified as transcriptional factors containing DNA binding homeodomain and protein-protein (dimerization) leucine zipper region.
  • HD-Zips such as Glabrous2 and REV also contain a START domain.
  • SEQ ID NO.: 7 Using the nucleic acid sequence (SEQ ID NO.: 7) encoding amino acids 56 - 184 and 156-842 of SEQ ID NO.: 8 from Arabidopsis REV, the sequence set forth as SEQ ID NO: 1 was identified from a maize library that may have included meristematic tissues. The sequence match between the ZmREV (SEQ ID NO: 1) and the Arabidopsis REV gene over the length of the cDNA is 69.2% identical.
  • Lynx Microarray Data shows ZmREV expression at 1.535% (+0.725).
  • the pattern of expression indicates average relative REV expression at 100% in ear, other tissues produced expression levels as follows: 75% in stalk, 63% in kernel, 32% in tassel, 25% in other tissues.
  • M13 forward primers and M13 reverse primers were sequenced using M13 reverse primers.
  • Colony hybridization was conducted as described by Sambrook, J., Fritsch, E.F. and Maniatis, T., (in Molecular Cloning: A Laboratory Manual, 2 nd Edition). The following probes were used in colony hybridization:
  • a Sal-A20 oligonucleotide (SEQ ID NO: 9): TCG ACC CAC GCG TCC GAA AAA AAA AAA AAA AAA, removes clones containing a poly A tail but no cDNA.
  • the image of the autoradiography was scanned into computer and the signal intensity and cold colony addresses of each colony was analyzed. Re- arraying of cold-colonies from 384 well plates to 96 well plates was conducted using Q-bot.
  • the pair of primers 5-2/3-2 and 5-3/3-3 showed that the consensus contig sequence resides on chromosome 9 while l4/e21 clone resides on chromosome 1.
  • These are oat lines carrying individual maize chromosomes, for all ten chromosomes.
  • other primer pairs e.g. ZmREV5-1/ZmREV3-1
  • ZmREV5-1/ZmREV3-1 showed two PCR-generated bands when using genomic DNA from B73 and Mo17. In fact, it was noted that the upper of the two bands showed length polymorphism between these two genotypes.
  • Immature maize embryos from greenhouse donor plants are bombarded with a plasmid containing the REVOLUTA sequence operably linked to the drought-inducible promoter RAB17 promoter (Vilardell et al, Plant Mol Biol
  • the ears are husked and surface sterilized in 30% Clorox bleach plus 0.5%
  • 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 REVOLUTA sequence operably linked to an ubiquitin 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:
  • 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 particle gun #HE34-1 or #HE34-2. All samples receive a single shot at 650 PSI, with a total of ten aliquots taken from each tube of prepared particles/DNA. Subsequent Treatment:
  • 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 drought tolerance. Assays to measure improved drought tolerance are routine in the art and include, for example, increased kernel-earring capacity yields under drought conditions when compared to control maize plants under identical environmental conditions. Alternatively, the transformed plants can be monitored for a modulation in meristem development (i.e., a decrease in spikelet formation on the ear). See, for example, Bruce et al, Journal of Experimental Botany 53: 1 -13, (2002).
  • Bombardment medium comprises 4.0 g/l N6 basal salts (SIGMA C- 1416), 1.0 ml/l Eriksson's Vitamin Mix (1000X SIGMA-1511), 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-l H 2 0 following adjustment to pH 5.8 with KOH); 2.0 g/l Gelrite (added after bringing to volume with D-l H 2 0); 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-1511 ), 0.5 mg/l thiamine HCI, 30.0 g/l sucrose, and 2.0 mg/l 2,4-D (brought to volume with D-l H 2 0 following adjustment to pH 5.8 with KOH); 3.0 g/l Gelrite (added after bringing to volume with D-l H 2 0); 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 11117-074), 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-l H 2 0) (Murashige and Skoog (1962) Physiol. Plant.
  • Hormone-free medium comprises 4.3 g/l MS salts (GIBCO 11117-074), 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-l H 2 0), 0.1 g/l myo-inositol, and 40.0 g/l sucrose (brought to volume with polished D-l H 2 0 after adjusting pH to 5.6); and 6 g/l bacto-agar (added after bringing to volume with polished D-l H 2 0), sterilized and cooled to 60°C.
  • immature embryos are isolated from maize and the embryos contacted with a suspension of Agrobactehum, where the bacteria are capable of transferring the antisense REVOLUTA sequences to at least one cell of at least one of the immature embryos (step 1 : the infection step).
  • the immature embryos are preferably immersed in an Agrobactehum suspension for the initiation of inoculation.
  • the embryos are co-cultured for a time with the Agrobactehum (step 2: the co-cultivation step).
  • the immature embryos are cultured on solid medium following the infection step.
  • an optional "resting" step is contemplated.
  • the embryos are incubated in the presence of at least one antibiotic known to inhibit the growth of Agrobactehum 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 Agrobactehum and for a resting phase for the infected cells.
  • 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 preferably calli grown on selective medium are cultured on solid medium to regenerate the plants. Plants are monitored and scored for a modulation in meristem development. For instance, alterations of size and appearance of the shoot and floral meristems and/or increased yields of leaves, flowers, and/or fruits.
  • REVOLUTA sequences operably linked to an ubiquitin promoter as follows. To induce somatic embryos, cotyledons, 3-5 mm in length dissected from surface- sterilized, immature seeds of the soybean cultivar A2872, are cultured in the light or dark at 26°C on an appropriate agar medium for six to ten weeks. Somatic embryos producing secondary embryos are then excised and placed into a suitable liquid medium. After repeated selection for clusters of somatic embryos that multiplied as early, globular-staged embryos, the suspensions are maintained as described below.
  • Soybean embryogenic suspension cultures can maintained in 35 ml liquid media on a rotary shaker, 150 rpm, at 26°C with fluorescent lights on a 16:8 hour day/night schedule. Cultures are subcultured every two weeks by inoculating approximately 35 mg of tissue into 35 ml of liquid medium.
  • Soybean embryogenic suspension cultures may then be transformed by the method of particle gun bombardment (Klein et al., Nature (London) 327:70-73, (1987), U.S. Patent No. 4,945,050).
  • a Du Pont Biolistic PDS1000/HE instrument helium retrofit
  • a selectable marker gene that can be used to facilitate soybean transformation is a transgene composed of the 35S promoter from Cauliflower Mosaic Virus (Odell et al., Nature 313:810-812, (1985)), the hygromycin phosphotransferase gene from plasmid pJR225 (from E. coli; Gritz et al, Gene 25:179-188, (1983)), and the 3' region of the nopaline synthase gene from the T-DNA of the Ti plasmid of Agrobactehum tumefaciens.
  • the expression cassette comprising an antisense REVOLUTA sequence operably linked to the ubiquitin promoter can be isolated as a restriction fragment. This fragment can then be inserted into a unique restriction site of the vector carrying the marker gene.
  • Approximately 300-400 mg of a two-week-old suspension culture is placed in an empty 60x15 mm petri dish and the residual liquid removed from the tissue with a pipette.
  • approximately 5-10 plates of tissue are normally bombarded.
  • Membrane rupture pressure is set at 1100 psi, and the chamber is evacuated to a vacuum of 28 inches mercury.
  • the tissue is placed approximately 3.5 inches away from the retaining screen and bombarded three times. Following bombardment, the tissue can be divided in half and placed back into liquid and cultured as described above.
  • the liquid media may be exchanged with fresh media, and eleven to twelve days post-bombardment with fresh media containing 50 mg/ml hygromycin. This selective media can be refreshed weekly.
  • Green, transformed tissue may be observed growing from untransformed, necrotic embryogenic clusters. Isolated green tissue is removed and inoculated into individual flasks to generate new, clonally propagated, transformed embryogenic suspension cultures. Each new line may be treated as an independent transformation event. These suspensions can then be subcultured and maintained as clusters of immature embryos or regenerated into whole plants by maturation and germination of individual somatic embryos.
  • Example 7 Sunflower Meristem Tissue Transformation
  • Sunflower meristem tissues are transformed with an expression cassette containing an antisense REVOLUTA sequences operably linked to a ubiquitin promoter as follows (see also European Patent Number EP 0 486233, herein incorporated by reference, and Malone-Schoneberg et al, Plant Science 103:199- 207, (1994)).
  • Mature sunflower seed (Helianthus annuus L.) are dehulled using a single wheat-head thresher. Seeds are surface sterilized for 30 minutes in a 20% Clorox bleach solution with the addition of two drops of Tween 20 per 50 ml of solution. The seeds are rinsed twice with sterile distilled water.
  • Split embryonic axis explants are prepared by a modification of procedures described by Schrammeijer et al. (Schrammeijer et al, Plant Cell Rep. 9:55-60, (1990)). Seeds are imbibed in distilled water for 60 minutes following the surface sterilization procedure. The cotyledons of each seed are then broken off, producing a clean fracture at the plane of the embryonic axis. Following excision of the root tip, the explants are bisected longitudinally between the primordial leaves. The two halves are placed, cut surface up, on GBA medium consisting of Murashige and Skoog mineral elements (Murashige et al, Physiol.
  • the explants are subjected to microprojectile bombardment prior to Agrobactehum treatment (Bidney et al, Plant Mol. Biol. 18:301-313, (1992)). Thirty to forty explants are placed in a circle at the center of a 60 X 20 mm plate for this treatment. Approximately 4.7 mg of 1.8 mm tungsten microprojectiles are resuspended in 25 ml of sterile TE buffer (10 mM Tris HCI, 1 mM EDTA, pH 8.0) and 1.5 ml aliquots are used per bombardment. Each plate is bombarded twice through a 150 mm nytex screen placed 2 cm above the samples in a PDS 1000® particle acceleration device.
  • a binary plasmid vector comprising the expression cassette that contains the REVOLUTA gene operably linked to the ubiquitin promoter is introduced into Agrobactehum strain EHA105 via freeze-thawing as described by Holsters et al.,Mol. Gen. Genet. 163:181-187, (1978).
  • This plasmid further comprises a kanamycin selectable marker gene (i.e, nptll).
  • Bacteria for plant transformation experiments are grown overnight (28°C and 100 RPM continuous agitation) in liquid YEP medium (10 gm/l yeast extract, 10 gm/l Bactopeptone, and 5 gm/l NaCl, pH 7.0) with the appropriate antibiotics required for bacterial strain and binary plasmid maintenance.
  • the suspension is used when it reaches an OD600 of about 0.4 to 0.8.
  • the Agrobactehum cells are pelleted and resuspended at a final OD600 of 0.5 in an inoculation medium comprised of 12.5 mM MES pH 5.7, 1 gm/l NH4CI, and 0.3 gm/l MgS ⁇ 4.
  • Freshly bombarded explants are placed in an Agrobactehum suspension, mixed, and left undisturbed for 30 minutes. The explants are then transferred to GBA medium and co-cultivated, cut surface down, at 26°C and 18-hour days. After three days of co-cultivation, the explants are transferred to 374B (GBA medium lacking growth regulators and a reduced sucrose level of 1 %) supplemented with 250 mg/l cefotaxime and 50 mg/l kanamycin sulfate. The explants are cultured for two to five weeks on selection and then transferred to fresh 374B medium lacking kanamycin for one to two weeks of continued development.
  • Explants with differentiating, antibiotic-resistant areas of growth that have not produced shoots suitable for excision are transferred to GBA medium containing 250 mg/l cefotaxime for a second 3-day phytohormone treatment.
  • Leaf samples from green, kanamycin-resistant shoots are assayed for the presence of NPTII by ELISA and for the presence of transgene expression by assaying for a modulation in meristem development (i.e., an alteration of size and appearance of shoot and floral meristems).
  • NPTII-positive shoots are grafted to Pioneer® hybrid 6440 in w ' fro-grown sunflower seedling rootstock. Surface sterilized seeds are germinated in 48-0 medium (half-strength Murashige and Skoog salts, 0.5% sucrose, 0.3% gelrite, pH
  • An alternative sunflower transformation protocol allows the recovery of transgenic progeny without the use of chemical selection pressure. Seeds are dehulled and surface-sterilized for 20 minutes in a 20% Clorox bleach solution with the addition of two to three drops of Tween 20 per 100 ml of solution, then rinsed three times with distilled water. Sterilized seeds are imbibed in the dark at 26°C for 20 hours on filter paper moistened with water.
  • the cotyledons and root radical are removed, and the meristem explants are cultured on 374E (GBA medium consisting of MS salts, Shepard vitamins, 40 mg/l adenine sulfate, 3% sucrose, 0.5 mg/l 6-BAP, 0.25 mg/l IAA, 0.1 mg/l GA, and 0.8% Phytagar at pH 5.6) for 24 hours under the dark.
  • the primary leaves are removed to expose the apical meristem, around 40 explants are placed with the apical dome facing upward in a 2 cm circle in the center of 374M (GBA medium with 1.2% Phytagar), and then cultured on the medium for 24 hours in the dark.
  • tungsten particles are resuspended in 150 ⁇ l absolute ethanol. After sonication, 8 ⁇ l of it is dropped on the center of the surface of macrocarrier. Each plate is bombarded twice with 650 psi rupture discs in the first shelf at 26 mm of Hg helium gun vacuum.
  • the plasmid of interest is introduced into Agrobactehum tumefaciens strain EHA105 via freeze thawing as described previously.
  • the pellet of overnight- grown bacteria at 28°C in a liquid YEP medium (10 g/l yeast extract, 10 g/l Bactopeptone, and 5 g/l NaCl, pH 7.0) in the presence of 50 ⁇ g/l kanamycin is resuspended in an inoculation medium (12.5 mM 2-mM 2-(N-morpholino) ethanesulfonic acid, MES, 1 g/l NH CI and 0.3 g/l MgS0 4 at pH 5.7) to reach a final concentration of 4.0 at OD 600.
  • inoculation medium (12.5 mM 2-mM 2-(N-morpholino) ethanesulfonic acid, MES, 1 g/l NH CI and 0.3 g/l MgS0 4 at pH 5.7
  • Particle-bombarded explants are transferred to GBA medium (374E), and a droplet of bacteria suspension is placed directly onto the top of the meristem.
  • the explants are co-cultivated on the medium for 4 days, after which the explants are transferred to 374C medium (GBA with 1% sucrose and no BAP, IAA, GA3 and supplemented with 250 ⁇ g/ml cefotaxime).
  • the plantlets are cultured on the medium for about two weeks under 16-hour day and 26°C incubation conditions.
  • Explants (around 2 cm long) from two weeks of culture in 374C medium are screened for a modulation in meristem or plant organ or whole plant development (i.e., an alteration of size and appearance of shoot and floral meristems). After positive (i.e., an increase in REVOLUTA expression) explants are identified, those shoots that fail to exhibit an increase in REVOLUTA activity are discarded, and every positive explant is subdivided into nodal explants.
  • One nodal explant contains at least one potential node.
  • the nodal segments are cultured on GBA medium for three to four days to promote the formation of auxiliary buds from each node. Then they are transferred to 374C medium and allowed to develop for an additional four weeks.
  • Recovered shoots positive for an increased REVOLUTA expression are grafted to Pioneer hybrid 6440 in ⁇ ro-grown sunflower seedling rootstock.
  • the rootstocks are prepared in the following manner. Seeds are dehulled and surface- sterilized for 20 minutes in a 20% Clorox bleach solution with the addition of two to three drops of Tween 20 per 100 ml of solution, and are rinsed three times with distilled water. The sterilized seeds are germinated on the filter moistened with water for three days, then they are transferred into 48 medium (half-strength MS salt, 0.5% sucrose, 0.3% gelrite pH 5.0) and grown at 26°C under the dark for three days, then incubated at 16-hour-day culture conditions.
  • the upper portion of selected seedling is removed, a vertical slice is made in each hypocotyl, and a transformed shoot is inserted into a V-cut.
  • the cut area is wrapped with parafilm. After one week of culture on the medium, grafted plants are transferred to soil. In the first two weeks, they are maintained under high humidity conditions to acclimatize to a greenhouse environment.

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Abstract

L'invention concerne des méthodes et des compositions pour moduler un développement et/ou des voies de développement. Des compositions de l'invention comprennent de l'acide nucléique et des séquences d'acides aminés. En particulier, l'invention concerne la séquence nucléotidique (SEQ ID NO:1, 3 ou 5) et la séquence d'acides aminés (SEQ ID NO:2, 4 ou 6) pour un polypeptide REVOLUTA de maïs. L'invention concerne des méthodes pour l'expression des séquences REVOLUTA dans un végétal hôte ou dans une cellule végétale pour moduler leur développement végétal et/ou leurs voies de développement. L'invention concerne encore des méthodes utiles dans le contrôle ou la modulation de la division cellulaire, de la différenciation et du développement cellulaire. En particulier, l'invention concerne des séquences utiles pour la modulation du développement d'un méristème. L'invention concerne également des végétaux, des cellules végétales, des tissus végétaux et des graines transformés.
PCT/US2004/000126 2003-01-06 2004-01-05 Genes homologues de zea mays revoluta/if1 et leurs utilisations WO2004063379A1 (fr)

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US60/438,210 2003-01-06

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1959725A2 (fr) * 2005-12-15 2008-08-27 Targeted Growth, Inc. Taille de graines et nombre de graines accrus par la surexpression transgenique d'un gene lie a la croissance et/ou au developpement lors du developpement embryonnaire precoce
EP2439277A1 (fr) 2006-05-30 2012-04-11 CropDesign N.V. Plantes surexprimant polypeptide-SYR ayant des traits liés au rendement améliorés et procédé pour les obtenir

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001033944A1 (fr) * 1999-11-10 2001-05-17 University Of Washington Compositions et procedes de modulation de la division cellulaire d'une plante

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001033944A1 (fr) * 1999-11-10 2001-05-17 University Of Washington Compositions et procedes de modulation de la division cellulaire d'une plante

Non-Patent Citations (3)

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Title
MEYEROWITZ E M: "GENETIC CONTROL OF CELL DIVISION PATTERNS IN DEVELOPING PLANTS", CELL, CELL PRESS, CAMBRIDGE, NA, US, vol. 88, no. 3, 7 February 1997 (1997-02-07), pages 299 - 308, XP000982657 *
OTSUGA D ET AL: "REVOLUTA REGULATES MERISTEM INITIATION AT LATERAL POSITIONS", PLANT JOURNAL, BLACKWELL SCIENTIFIC PUBLICATIONS, OXFORD, GB, vol. 25, no. 2, 2001, pages 223 - 236, XP002938885 *
TALBERT P B ET AL: "THE REVOLUTA GENE IS NECESSARY FOR APICAL MERISTEM DEVELOPMENT AND FOR LIMITING CELL DIVISIONS IN THE LEAVES AND STERMS OF ARABIDOPSISTHALIANA", DEVELOPMENT, COMPANY OF BIOLOGISTS, CAMBRIDGE,, GB, vol. 121, 1995, pages 2723 - 2735, XP002938883 *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1959725A2 (fr) * 2005-12-15 2008-08-27 Targeted Growth, Inc. Taille de graines et nombre de graines accrus par la surexpression transgenique d'un gene lie a la croissance et/ou au developpement lors du developpement embryonnaire precoce
EP1959725A4 (fr) * 2005-12-15 2009-09-09 Targeted Growth Inc Taille de graines et nombre de graines accrus par la surexpression transgenique d'un gene lie a la croissance et/ou au developpement lors du developpement embryonnaire precoce
EP2441841A2 (fr) 2005-12-15 2012-04-18 Targeted Growth, Inc. Taille de graines et nombre de graines accrus par la surexpression transgénique d'un gène lie à la croissance et/ou au développement lors du développement embryonnaire précoce
EP2441841A3 (fr) * 2005-12-15 2012-05-30 Targeted Growth, Inc. Taille de graines et nombre de graines accrus par la surexpression transgénique d'un gène lie à la croissance et/ou au développement lors du développement embryonnaire précoce
EP2439277A1 (fr) 2006-05-30 2012-04-11 CropDesign N.V. Plantes surexprimant polypeptide-SYR ayant des traits liés au rendement améliorés et procédé pour les obtenir
EP2441839A1 (fr) 2006-05-30 2012-04-18 CropDesign N.V. Plantes ayant une expression réduite d'un gen REV caractéristiques de rendement améliorées et procédé de fabrication de celles-ci

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