WO2000050585A1 - Nouveaux polynucleotides et polypeptides adc, leurs utilisations, dont les procedes d'amelioration des semences - Google Patents

Nouveaux polynucleotides et polypeptides adc, leurs utilisations, dont les procedes d'amelioration des semences Download PDF

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Publication number
WO2000050585A1
WO2000050585A1 PCT/US2000/004718 US0004718W WO0050585A1 WO 2000050585 A1 WO2000050585 A1 WO 2000050585A1 US 0004718 W US0004718 W US 0004718W WO 0050585 A1 WO0050585 A1 WO 0050585A1
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nucleic acid
adc
plant
seed
sequence
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PCT/US2000/004718
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English (en)
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Diane K. Jofuku
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Ceres, Inc.
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Priority to AU33759/00A priority Critical patent/AU3375900A/en
Priority to US09/959,625 priority patent/US7126043B1/en
Priority to EP00911946A priority patent/EP1155123A4/fr
Priority to CA002363599A priority patent/CA2363599A1/fr
Publication of WO2000050585A1 publication Critical patent/WO2000050585A1/fr
Priority to US11/524,633 priority patent/US20070094750A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
    • Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants

Definitions

  • the present invention is directed to plant genetic engineering. In particular, it relates to new methods for modulating mass and other properties of plant seeds.
  • APETALA2 In Arabidopsis, a floral homeotic gene APETALA2 (AP2) controls three critical aspects of flower ontogeny - the establishment of the floral meristem (Irish and Wales, Plant Cell 2:741-753 (1990); Huala and Wales, Plant Cell 4:901-913 (1992); Bowman et al, Development 119:721-743 (1993); Schultz and Haughn, Development 119:745-765 (1993); Shannon and Meeks- Wagner, Plant Cell 5:639-655 (1993)), the specification of floral organ identity (Komaki et al, Development 104:195-203 (1988)); Bowman et al, Plant Cell 1 :37-52 (1989); Kunststoff et al, Plant Cell 1 :1195-1208 (1989)), and the temporal and spatial regulation of floral homeotic gene expression (Bowman et al, Plant Cell 3:749-758 (1991); Drews et al, Cell 65:91-1002 (1991)).
  • AP2 performs this function in cooperation with at least three other floral meristem genes, APETALA1 (API), LEAFY (LEY), and CAULIFLOWER (CAL) (Irish and Wales (1990); Bowman, Flowering Newsletter 14:7-19 (1992); Huala and Wales (1992); Bowman et al, (1993); Schultz and Haughn, (1993); Shannon and Meeks- Wagner, ( 1993)).
  • a second function of AP2 is to regulate floral organ development. In Arabidopsis, the floral meristem produces four concentric rings or whorls of floral organs - sepals, petals, stamens, and carpels.
  • Ap2 plays a critical role in the regulation of Arabidopsis flower development. Yet, little is known about how it carries out its functions at the cellular and molecular levels.
  • a spatial and combinatorial model has been proposed to explain the role o ⁇ AP2 and other floral homeotic genes in the specification of floral organ identity(,see, e.g., Coen and Carpenter, supra).
  • One central premise of this model is that AP2 and a second floral homeotic gene AGAMOUS (AG) are mutually antagonistic genes. That is, AP2 negatively regulates AG gene expression in sepals and petals, and conversely, AG negatively regulates AP2 gene expression in stamens and carpels.
  • AP2 encodes a putative nuclear factor that bears no significant similarity to any known fungal, or animal regulatory protein.
  • the present invention relates to AP2 domain containing ("ADC) polynucleotides and polypeptides, including variants thereof, such as mutants, fragments, and fusions.
  • ADC AP2 domain containing
  • Such polynucleotides of the invention can be used to construct ribozyme, antisense, and expression constructs and vectors.
  • host cells comprising these constructs and vectors to modulate expression of ADC polypeptides in any number of cell types, including, without limitation, bacterial, yeast, insect, mammalian, and plant.
  • the present invention provides methods of modulating seed mass and other traits in plants, such as oat, wheat, rice, and maize, for example.
  • the methods involve providing a plant comprising a recombinant expression cassette containing an ADC nucleic acid linked to a plant promoter.
  • the plant is either selfed or crossed with a second plant to produce a plurality of seeds. Seeds with the desired trait (e.g. , altered mass) are then selected.
  • transcription of the ADC nucleic acid inhibits expression of an endogenous ADC gene or activity the encoded protein.
  • the step of selecting includes the step of selecting seed with increased mass or another trait.
  • the seed may have, for instance, increased protein content, carbohydrate content, or oil content. In the case of increased oil content, the types of fatty acids may or may not be altered as compared to the parental lines.
  • the ADC nucleic acid may be linked to the plant promoter in the sense or the antisense orientation.
  • expression of the ADC nucleic acid may enhance expression of an endogenous ADC gene or ADC activity and the step of selecting includes the step of selecting seed with decreased mass. This embodiment is particularly useful for producing seedless varieties of crop plants.
  • the two plants may be the same or different species.
  • the plants may be any higher plants, for example, members of the families Brassicaceae or Solanaceae.
  • either the female or the male parent plant can comprise the expression cassette containing the ADC nucleic acid.
  • both parents contain the expression cassette.
  • the plant promoter may be a constitutive promoter, for example, the CaMV 35S promoter.
  • the promoter may be a tissue-specific promoter. Examples of tissue specific expression useful in the invention include fruit- specific, seed-specific (e.g., ovule-specific, embryo-specific, endosperm-specific,
  • the invention also provides seed produced by the methods described above.
  • the seed of the invention comprise a recombinant expression cassette containing an ADC nucleic acid. If the expression cassette is used to inhibit expression of endogenous ADC expression, the seed will have a mass at least about 20% greater than the average mass of
  • the seed will have a mass at least about 20% less than the average mass of seeds of the same plant variety which lack the recombinant expression cassette.
  • Other traits such as protein content, carbohydrate content, and oil content can be altered in the same manner.
  • nucleic acid sequence refers to a single or double-stranded polymer of deoxyribonucleotide or ribonucleotide bases read from the 5' to the 3' end. It includes chromosomal DNA, self-replicating plasmids, infectious polymers of DNA or RNA and DNA or RNA that performs a primarily structural role.
  • promoter refers to a region or sequence determinants located upstream or downstream from the start of transcription and which are involved in recognition and binding of RNA polymerase and other proteins to initiate transcription.
  • a "plant promoter” is a promoter capable of initiating transcription in plant cells.
  • plant includes whole plants, plant organs (e.g., leaves, stems, flowers,
  • the class of plants which can be used in the method of the invention is generally as broad as the class of higher plants amenable to transformation techniques, including angiosperms (monocotyledonous and dicotyledonous plants), as well as gymnosperms. It includes plants of a variety of ploidy levels, including polyploid, diploid, haploid and hemizygous.
  • a polynucleotide sequence is "heterologous to" an organism or a second polynucleotide sequence if it originates from a foreign species, or, if from the same species, is modified from its original form.
  • a promoter operably linked to a heterologous coding sequence refers to a coding sequence from a species different from that from which the promoter was derived, or, if from the same species, a coding sequence
  • a modified ADC coding sequence which is heterologous to an operably linked ADC promoter does not include the T-DNA insertional mutants (e.g., ap2-10) as described in Jofuku et al. The Plant Cell 6:1211-1225 (1994).
  • a polynucleotide "exogenous to" an individual plant is a polynucleotide which is
  • ADC (AP2 domain containing) nucleic acid or "ADC polynucleotide sequence” of the invention is a subsequence or full length polynucleotide sequence of a gene which, encodes an polypeptide containing an AP2 domain.
  • a class of these nucleic acids encode polypeptides which, when present in a transgenic plant, can be used to
  • Native ADC polynucleotides are defined by their ability to hybridize under defined conditions to the exemplified nucleic acids or PCR products derived from them.
  • An ADC polynucleotide (e.g., those shown in the Sequence Listing) is typically at least about 30-40 nucleotides to about 3000, usually less than about 5000 nucleotides in length. Usually the nucleic acids are from about 100 to
  • 135 about 2000 nucleotides, often from about 500 to about 1700 nucleotides in length.
  • ADC nucleic acids are a class of plant regulatory genes that encode ADC polypeptides, which are distinguished by the presence of one or more of a repeated amino acid repeated motif, referred to here as the "AP2 domain".
  • a repeated amino acid repeated motif referred to here as the "AP2 domain”.
  • a motif is at least 50 amino acids; more typically, at least 54
  • the scope of the invention includes native ADC nucleic acids, allelic variants, and other variants, such as mutants, fragments, and fusions.
  • ADC polypeptides includes those native oat, wheat, rice, and corn sequences disclosed in the Sequence Listing.
  • An "allelic variant” is a sequence that is a variant of native polynucleotides shown
  • allelic variants can be produced by genetic engineering methods.
  • a preferred allelic variant is one that is found in a naturally occurring plant, including a laboratory strain. Allelic variants are either silent or
  • a silent allele is one that does not affect the phenotype of the organism.
  • An expressed allele results in a detectable change in the phenotype of the trait represented by the locus. Alleles can occur in any portion of the genome, including regulatory regions as well as structural genes.
  • the inserted polynucleotide sequence need not be identical, but may be only "substantially identical" to a sequence of the gene from which it was derived. As explained below, these substantially identical variants are specifically covered by the term ADC nucleic acid.
  • ADC nucleic acid specifically includes those full length sequences substantially identical (determined as described below) with an ADC polynucleotide sequence and that encode proteins that
  • variants can be those that encode dominant negative mutants as described below.
  • nucleic acid sequences or polypeptides are said to be “identical” if the sequence of nucleotides or amino acid residues, respectively, in the two sequences is the
  • complementary to is used herein to mean that the complementary sequence is identical to all or a portion of a reference polynucleotide sequence.
  • Sequence comparisons between two (or more) polynucleotides or polypeptides are typically performed by comparing sequences of the two sequences over a "comparison
  • a “comparison window” refers to a segment of at least about 20 contiguous positions, usually about 50 to about 200, more usually about 100 to about 150 in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned.
  • Optimal alignment of sequences for comparison may be conducted by the local homology algorithm of Smith and Waterman Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman and WunschJ Mol Biol 48:443 (1970), by the search for similarity method of Pearson and Lipman Proc. Natl Acad. Sci. (U.S.A.) 85 : 2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT,
  • Percentage of sequence identity is determined by comparing two optimally
  • 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
  • substantially identical of polynucleotide sequences means that a polynucleotide comprises a sequence that has at least about 60% sequence identity
  • 205 preferably at least about 80%, more preferably at least about 85%. and most preferably, at least about 90, 92%, 95%, 98%, of 99% compared to a reference sequence using the programs described above (preferably BLAST) using standard parameters.
  • a reference sequence preferably BLAST
  • One of skill will recognize that these values can be appropriately adjusted to determine corresponding identity of proteins encoded by two nucleotide sequences by taking into account codon
  • amino acid sequences for these purposes normally means sequence identity of at least about 35%, preferably at least about 60%, more preferably at least about 70% or about 80%, and most preferably at least about 90, 92%, 95%, 98%, of 99%.
  • Polypeptides which are "substantially similar" share sequences as noted above except that residue
  • amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic- hydroxyl side chains is serine and threonine; a group of amino acids having amide-
  • side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino acids having sulfur-containing side chains is cysteine and methionine.
  • Preferred conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine,
  • 225 lysine-arginine, alanine-valine, and asparagine-glutamine.
  • nucleotide sequences are substantially identical is if two molecules hybridize to each other, or a third nucleic acid, under stringent conditions.
  • Stringent conditions are sequence dependent and will be different in different circumstances. Usually, stringent conditions are selected to be about 15° C lower than the
  • Tm thermal melting point
  • stringent conditions will be those in which the salt concentration is about 0.02 molar at pH 7 and the temperature is at least
  • genomic DNA or cDNA comprising ADC nucleic acids of the invention can be identified in standard Southern blots under stringent conditions using the nucleic acid sequences disclosed here.
  • stringent conditions for such hybridizations are those which include at least one wash in 0.2X SSC
  • nucleic acids of the invention at a temperature of at least about 50°C, usually about 55°C to about 60°C, for 20 minutes, or equivalent conditions.
  • Other means by which nucleic acids of the invention can be identified are described in more detail below.
  • This invention relates to plant ADC genes, such as those from oat, wheat, rice, and 245 corn.
  • the invention provides molecular strategies for controlling seed size and total seed protein using ADC overexpression and antisense gene constructs.
  • transgenic plants containing antisense constructs have dramatically increased seed mass, seed protein, or seed oil.
  • overexpression of ADC using a constructs of the invention leads to reduced seed size and total seed protein.
  • data presented here demonstrate that 250 a number of agronomically important traits including seed mass, total seed protein, and oil content, can be controlled in species of agricultural importance. Isolation of ADC nucleic acids
  • Standard techniques are used for cloning, DNA and RNA isolation, amplification and purification. Generally enzymatic reactions involving DNA ligase, DNA polymerase, restriction endonucleases and the like are performed according to the manufacturer's specifications. These techniques and various other techniques are generally performed according to Sambrook et al. , Molecular Cloning - A Laboratory Manual, Cold Spring
  • ADC nucleic acids may be accomplished by a number of techniques. For instance, oligonucleotide probes based on the sequences disclosed here can be used to identify the desired gene in a cDNA or genomic DNA library. To construct genomic libraries, large segments of genomic DNA are generated by random
  • RNA is isolated from the desired organ, such as flowers, and a cDNA library which contains the ADC gene transcript is prepared from the mRNA.
  • cDNA may be prepared from mRNA extracted from other tissues in which ADC genes or
  • the cDNA or genomic library can then be screened using a probe based upon the sequence of a cloned ADC gene disclosed here. Probes may be used to hybridize with genomic DNA or cDNA sequences to isolate homologous genes in the same or different plant species. Alternatively, antibodies raised against an ADC polypeptide can be used to
  • the nucleic acids of interest can be amplified from nucleic acid samples using amplification techniques.
  • amplification technique for instance, polymerase chain reaction (PCR) technology can be used to amplify the sequences of the ADC genes directly from genomic DNA, from cDNA, from genomic libraries or cDNA libraries.
  • PCR polymerase chain reaction
  • 280 amplification methods may also be useful, for example, to clone nucleic acid sequences that code for proteins to be expressed, to make nucleic acids to use as probes for detecting the presence of the desired mRNA in samples, for nucleic acid sequencing, or for other purposes.
  • nucleic acids of the invention are characterized by the presence of sequence encoding an AP2 domain or fragments thereof. Thus, these nucleic acids can be identified by their ability to specifically hybridize to sequences encoding AP2 domain disclosed here. Primers which specifically amplify AP2 domains of the exemplified genes are particularly useful for identification of particular ADC polynucleotides. Primers
  • PCR primers are used under standard PCR conditions (described for instance in Innis et al.) using the nucleic acids as described above as a template.
  • the PCR products generated by any of the reactions can then be used to identify nucleic acids of the invention (e.g., from a cDNA library) by their ability to hybridize to these products.
  • Hybridization Buffer consisting of: 0.25M Phosphate Buffer (pH 7.2), 1 mM EDTA, 1 % Bovine Serum Albumin, 7% SDS. Hybridization is then followed by a first wash with 2.0XSSC + 0.1% SDS or 0.39M Na+ (Wash Buffer A) and subsequent washes with 0.2XSSC + 0.1% SDS or 0.042M Na+ (Wash Buffer B). Hybridization temperature will be from about 45°C to about 78°C, usually from about 50°C to about
  • primers that amplify regions more specific to particular ADC genes can 315 be used.
  • the PCR products produced by these primers can be used in the hybridization conditions described above to isolate nucleic acids of the invention.
  • Polynucleotides may also be synthesized by well-known techniques as described in the technical literature. See, e.g., Carruthers et al. Cold Spring Harbor Symp. Quant. Biol 47:411-418 (1982), andAdams et ⁇ /.,JAm. Chem. Soc. 105:661 (1983). Double stranded DNA fragments may then be obtained either by synthesizing the complementary strand and annealing the strands together under appropriate conditions, or by adding the complementary strand using DNA polymerase with an appropriate primer sequence.
  • Standard nucleic acid hybridization techniques using the conditions disclosed above can then be used to identify full length cDNA or genomic clones.
  • the DNA primers based on the sequences shown in the Sequence Listing can be used in an inverse PCR reaction to specifically amplify flanking AP2 gene sequences.
  • Such a technique is single primer PCR (SPPCR).
  • SPPCR single primer PCR
  • a typical SPPCR reaction is as follows: 1-5 ⁇ g of template plant DNA, 10 pmol of a selected primer, and 1.25 U of Taq DNA polymerase in standard IX PCR reaction buffer as specified by the manufacturer (Promega, Madison, WI).
  • PCR reaction conditions of twenty (20) cycles of denaturation at 94°C for 30 sec, primer-template annealing at 55°C for 30 sec, synthesis at 72°C for 1 min., 30 sec, two cycles (2) of denaturation at 94°C for 30 sec, primer-template annealing at 30°C for 15 sec, 35°C for 15 sec, 40°C for 15 sec, 45°C for 15 se , 50°C for 15 sec. 55°C for 15 sec, 60°C for 15 se , 65°C for 15 sec, and synthesis at 72°C for 1 min., 30 sec.
  • ADC activity can be modulated in the plant cell at the gene, transcriptional. posttranscriptional, translational, or posttranslational. Techniques for modulating ADC activity at each of these levels are generally well known to one of skill and are discussed briefly below. Methods for introducing genetic mutations into plant genes are well known. For instance, seeds or other plant material can be treated with a mutagenic chemical substance,
  • Such chemical substances include, but are not limited to, the following: diethyl sulfate, ethylene imine, ethyl methanesulfonate and N-nitroso-N- ethylurea.
  • ionizing radiation from sources such as, for example, X-rays or gamma rays can be used. Desired mutants are selected by assaying for increased seed mass, oil content and other properties.
  • homologous recombination can be used to induce targeted gene disruptions by specifically deleting or altering the ADC gene in vivo (see, generally, Grewal and Klar, Genetics 146: 1221-1238 (1997) and Xu et al, Genes Dev. 10: 2411- 2422 ( 1996)). Homologous recombination has been demonstrated in plants (Puchta et al. , Experientia 50: 277-284 (1994), Swoboda et al, EMBO J. 13: 484-489 (1994); and
  • mutations in selected portions of an ADC gene sequences are made in vitro and then introduced into the desired plant using standard techniques. Since the efficiency of
  • 365 homologous recombination is known to be dependent on the vectors used, use of dicistronic gene targeting vectors as described by Mountford et al. Proc. Natl. Acad. Sci. USA 91 : 4303-4307 (1994); and Vaulont et al. Transgenic Res. 4: 247-255 (1995) are conveniently used to increase the efficiency of selecting for altered ADC gene expression in transgenic plants.
  • the mutated gene will interact with the target wild-type gene in such
  • RNA/DNA sequence is designed to align with the sequence of the target ADC gene and to
  • 375 contain the desired nucleotide change.
  • Introduction of the chimeric oligonucleotide on an extrachromosomal T-DNA plasmid results in efficient and specific ADC gene conversion directed by chimeric molecules in a small number of transformed plant cells. This method is described in Cole-Strauss et al. Science 273:1386-1389 (1996) and Yoon et al. Proc. Natl. Acad. Sci. USA 93: 2071-2076 (1996).
  • Gene expression can be inactivated using recombinant DNA techniques by transforming plant cells with constructs comprising transposons or T-DNA sequences.
  • ADC mutants prepared by these methods are identified according to standard techniques. For instance, mutants can be detected by PCR or by detecting the presence or absence of ADC mRNA, e.g., by Northern blots. Mutants can also be selected by assaying for
  • isolated nucleic acid sequences prepared as described herein can also be used in a number of techniques to control endogenous ADC gene expression at various levels. Subsequences from the sequences disclosed here can be used to control, transcription, RNA accumulation, translation, and the like.
  • a number of methods can be used to inhibit gene expression in plants.
  • antisense technology can be conveniently used.
  • a nucleic acid segment from the desired gene is cloned and operably linked to a promoter such that the antisense strand of RNA will be transcribed.
  • the construct is then transformed into plants and the antisense strand of RNA is produced.
  • plant cells it has been suggested
  • the nucleic acid segment to be introduced generally will be substantially identical
  • the sequence need not be perfectly identical to inhibit expression.
  • the vectors of the present invention can be designed such that the inhibitory effect applies to other genes within a family of genes exhibiting homology or substantial homology to the target gene.
  • the introduced sequence also need not be full length
  • 410 relative to either the primary transcription product or fully processed mRNA.
  • higher homology can be used to compensate for the use of a shorter sequence.
  • the introduced sequence need not have the same intron or exon pattern, and homology of non-coding segments may be equally effective. Normally, a sequence of between about 30 or 40 nucleotides and about full length nucleotides should be used,
  • a sequence of at least about 100 nucleotides is preferred, a sequence of at least about 200 nucleotides is more preferred, and a sequence of about 500 to about 1700 nucleotides is especially preferred.
  • a number of gene regions can be targeted to suppress ADC gene expression.
  • the targets can include, for instance, the coding regions (e.g., regions flanking the PA2
  • the constructs can be designed to eliminate the ability of regulatory proteins to bind to ADC gene sequences that are required for its cell- and/or tissue-specific expression.
  • transcriptional regulatory sequences can be located either 5'-, 3'-, or within the coding region of the gene and can be either promote (positive
  • sequences can be identified using standard deletion analysis, well known to those of skill in the art. Once the sequences are identified, an antisense construct targeting these sequences is introduced into plants to control AP2 gene transcription in particular tissue, for instance, in developing ovules and/or seed.
  • Oligonucleotide-based triple-helix formation can be used to disrupt ADC gene expression.
  • Triplex DNA can inhibit DNA transcription and replication, generate site- specific mutations, cleave DNA, and induce homologous recombination (see, e.g., Havre and GlazerJ Virology 67:7324-7331 (1993); Sca lon et al. FASEB J. 9:1288-1296 (1995); Giovannangeli et al. Biochemistry 35:10539-10548 (1996); Chan and Glazer J Mol
  • Triple helix DNAs can be used to target the same sequences identified for antisense regulation.
  • Catalytic RNA molecules or ribozymes can also be used to inhibit expression of ADC genes. It is possible to design ribozymes that specifically pair with virtually any target RNA and cleave the phosphodiester backbone at a specific location, thereby
  • ribozyme functionally inactivating the target RNA.
  • the ribozyme In carrying out this cleavage, the ribozyme is not itself altered, and is thus capable of recycling and cleaving other molecules, making it a true enzyme.
  • the inclusion of ribozyme sequences within antisense RNAs confers RNA-cleaving activity upon them, thereby increasing the activity of the constructs.
  • ribozymes can be used to target the same sequences identified for antisense regulation.
  • RNAs from avocado sunblotch viroid and the satellite RNAs from tobacco ringspot virus, lucerne transient streak virus, velvet tobacco mottle 450 virus, solanum nodiflorum mottle virus and subterranean clover mottle virus.
  • RNA-specific ribozymes The design and use of target RNA-specific ribozymes is described in Zhao and Pick Nature 365:448- 451 (1993); Eastham and Ahlering J Urology 156:1186-1 188 (1996); Sokol and Murray Transgenic Res. 5:363-371 (1996); Sun et al. Mol. Biotechnology 7:241-251 (1997); and Haseloffet ⁇ /. N ⁇ twre, 334:585-591 (1988).
  • the suppressive effect may occur where the introduced sequence contains no coding sequence per se, but only intron or untranslated sequences homologous to sequences present in the primary transcript of the endogenous sequence.
  • 465 sequence generally will be substantially identical to the endogenous sequence intended to be repressed. This minimal identity will typically be greater than about 65%, but a higher identity might exert a more effective repression of expression of the endogenous sequences. Substantially greater identity of more than about 80% is preferred, though about 95% to absolute identity would be most preferred. As with antisense regulation, the
  • the introduced sequence needing less than absolute identity, also need not be full length, relative to either the primary transcription product or fully processed mR ⁇ A. This may be preferred to avoid concurrent production of some plants
  • ADC activity may be modulated by eliminating the proteins that are required for ADC cell-specific gene expression.
  • expression of regulatory proteins and/or the sequences that control ADC gene expression can be modulated using the methods described here.
  • Another method is use of engineered tRNA suppression of ADC mRNA translation.
  • This method involves the use of suppressor tRNAs to transactivate target genes containing premature stop codons (see, Betzner et al. Plant J.11:587-595 (1997); and Choisne et al.
  • Plant J 11 : 597-604 (1997).
  • a plant line containing a constitutively expressed ADC gene that contains an amber stop codon is first created. Multiple lines of plants, each containing
  • tRNA suppressor gene constructs under the direction of cell-type specific promoters are also generated.
  • the tRNA gene construct is then crossed into the ADC line to activate ADC activity in a targeted manner.
  • These tRNA suppressor lines could also be used to target the expression of any type of gene to the same cell or tissue types.
  • ADC proteins e.g., AP2
  • ADC proteins are believed to form multimers in vivo.
  • an alternative method for inhibiting ADC function is through use of dominant negative mutants.
  • This approach involves transformation of plants with constructs encoding mutant ADC polypeptides that form defective multimers with endogenous wild- type ADC proteins and thereby inactivate the protein.
  • the mutant polypeptide may vary from the naturally occurring sequence at the primary structure level by amino acid
  • the native ADC proteins may exist in both a phosphorylated and a nonphosphorylated form. Thus, activity may also be regulated by protein kinase signal transduction cascades. In addition, such genes may be regulated by and/or play a role in
  • mutant forms of the ADC proteins used in dominant negative strategies can include substitutions at amino acid residues targeted for phosphorylation so as to decrease phosphorylation of the
  • mutant ADC forms can be designed so that they are hyperphosphorylated. Glycosylation events are known to affect protein activity in a cell- and/or tissue-specific manner (see. Meshi and Iwabuchi Plant Cell Physiol 36: 1405-1420 (1995); Meynial-Salles and Combes J Biotech. 46: 1-14 (1996)). Thus, mutant forms of the
  • 520 ADC proteins can also include those in which amino acid residues that are targeted for glycosylation are altered in the same manner as that described for phosphorylation mutants.
  • ADC polypeptide may carry out some of its functions through its interactions with other transcription factors/proteins (e.g., AINTEGUMENTA, Elliott et al Plant Cell 8:
  • one simple method for suppressing ADC activity is to suppress the activities of proteins that are required for ADC activity.
  • ADC activity can thus be controlled by "titrating " out transcription factors/proteins required for ADC
  • 530 activity This can be done by overexpressing domains ADC proteins that are involved in proteimprotein interactions in plant cells (e.g. , AP2 domains or the putative transcriptional activation domain as described in Jofuku et al, Plant Cell 6: 121 1-1225 (1994)). This strategy has been used to modulate gene activity (Lee et al, Exptl Cell Res. 234: 270-276 (1997); Thiesen Gene Expression 5: 229-243 (1996); and Waterman et al, Cancer Res.
  • ADC proteins that are involved in proteimprotein interactions in plant cells
  • Isolated sequences prepared as described herein can also be used to introduce expression of a particular ADC nucleic acid to enhance or increase endogenous gene expression. Enhanced expression will generally lead to smaller seeds or seedless fruit.
  • the desired gene from a different species may be overexpression of a gene.
  • Modified protein chains can also be readily designed utilizing various recombinant DNA techniques well known to those skilled in the art and described in detail, below.
  • the chains can vary from the naturally occurring sequence at the primary structure level by amino acid substitutions, additions, deletions, and the like.
  • 555 modifications can be used in a number of combinations to produce the final modified protein chain.
  • Polypeptide variants of the native ADC sequences shown in the Sequence Listing and the polynucleotides that encode such variants are within the scope of the invention.
  • Variants including mutants, fragments, and fusions will exhibit at least about 35% sequence identity to those native polypeptides shown in the Sequence Listing or fragments thereof; more typically, at least about 60%; even more typically, at least about 70%. Sequence identity is used for polypeptides as defined above for polynucleotides. More preferably, the variants will exhibit at least about 85% sequence identity; even more
  • 565 preferably, at least about 90% sequence identity; more preferably at least about 95%, 96%, 97%, 98%, or 99% sequence identity.
  • the variants will exhibit at least one of the structural properties of a native ADC protein.
  • Such structural properties include, without limitation, 3 -dimensional structure, serine-rich acidically charged regions and alpha-helical structure.
  • variants are functional, in that variants exhibit at least one of the activities of the native protein.
  • activities include, without limitation, protein-protein interaction, DNA interaction, biological activity, immunological activity, signal transduction activity, transcription activity, etc. More specifically, the activities include DNA binding, activation of transcription or transcription factors, multimer formation, nuclear localization,
  • the variants are capable of exhibiting at least about 60% of the activity of the native protein; more typically, about 70%; even more typically, at least about 80%, 85%, 90% or 95% of at least one activity of the native protein.
  • Mutants of the native polypeptides comprise amino acid additions, deletions, or
  • substitutions are preferred to maintain the function or activity of the polypeptide. Such substitutions include conservation of charge, polarity, hydrophobicity, size, etc For example, one or more amino acid residues within the sequence can be substituted with another amino acid of similar polarity that acts as a functional equivalent, for example providing a hydrogen bond in an enzymatic catalysis. Substitutes for
  • an amino acid within an exemplified sequence are preferably made among the members of the class to which the amino acid belongs.
  • the nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and methionine.
  • the polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine.
  • the positively charged (basic) amino acids include
  • the negatively charged (acidic) amino acids include aspartic acid and glutamic acid. Other examples of conservative substitutions are described above.
  • Fragments of the native and mutant polypeptides of the invention comprise deletion of the amino acids at either termini. Fragments of particular interest are those that include only one of domains included in the native ADC polypeptides. Further, fusions of native
  • polypeptides, mutants, and fragment can comprise additional individual amino acids or amino acid sequences inserted into the polypeptide in the middle thereof and/or at the N- terminal and/or C-terminal ends thereof.
  • Chimeras can be constructed of fragments of the instant invention and other ADC sequences, such as Arabidopsis AP2 and RAP2 genes.
  • chimeras comprising fragment of the instant ADC polypeptides with domains from
  • a leucine zipper can be fused to an ADC sequence.
  • the native ADC polypeptides of the instant invention comprising sequences shown in the Sequence Listing, comprise a number of domains, elements, regions, and motifs. To construct a variant that retains or exhibits enhanced ADC polypeptide
  • Native ADC polypeptides of the invention can include any one of the following domains, elements, regions, or motifs:
  • Some of the native polypeptides of the invention can comprise an amino terminal
  • serine-rich acidic domain 625 serine-rich acidic domain.
  • Such a domain can be identified by sequence similarity to the serine-rich acidic domain in Arabidopsis AP2, amino acids 14-50, as numbered in copending application U.S. Ser. No. 09/026,039, filed February 19, 1998.
  • This domain is analogous to regions that function as activation domains in a number of RNA polymerase transcription factors. Consequently, changes to this region can modulate activation
  • Such a domain within a variant can be either longer or shorter than those included in a native protein.
  • a domain, modified or unmodified as compared to the native is at least about 20 amino acids; more typically, at least about 25 amino acids; even more typically, at least about 30 amino acids; even more typically, at least about 37 amino acids.
  • KKSR motif capable of nuclear localization of the polypeptide can be included in the native polypeptide sequences of the invention. If nuclear localization is undesired in a variant, such a domain can be deleted or modified to diminish activity. This domain or modification of those found in the native protein can be utilized to enhance or retain
  • such a domain is at least about 4 amino acids; more typically, at least about 7 amino acids; even more typically, at least about 10 amino acids.
  • All native ADC polypeptides of the invention include at least one AP2 domain, some can include two domains. Both copies of this domain or core region are capable of forming amphipathic ⁇ -helical structures. This domain can be responsible for conferring 645 DNA binding or multimer formation or protein-protein interaction activities.
  • the first block referred to as the YRG element
  • YRG tyrosine-arginine-glycine
  • a modified element can be included in a variant that is longer or shorter than the element in a native ADC polypeptide. Typically, the length is between at least about 15
  • An AP2 domain also includes a second block of amino acids, referred to herein as the RAYD element (arginine-alanine-tyrosine-aspartic acid).
  • RAYD element arginine-alanine-tyrosine-aspartic acid. This element is capable of forming an amphipathic alpha helix with alternating charges. This element can be responsible for DNA binding, multimer formation, or protein-protein
  • the domain is altered so an alpha-helix cannot be formed, such as an inclusion of a proline residue
  • substitutions are made to the native sequence; specifically, in the RAYD motif.
  • such a element whether unchanged or modified from the native sequence is at least about 35 amino acids; more typically, at least about 40 amino acid; even more typically, about 42, 43, or 44 amino acids in length.
  • the core region within the RAYD element is predicted to form an amphipathic alpha helix. Typically, this core region is about 12 amino acids;
  • 670 more typically, about 15 amino acids; even more typically, about 18 amino acids in length.
  • invariant amino acid residues within the YRG and RAYD elements that may also play a role in the structure or function of these ADC proteins.
  • glycine residue at position 40 within the RAYD elements is invariant in all AP2 domain containing proteins, and has been shown to be important for AP2 function (Jofuku).
  • This glycine is at position 1 ofall the polypeptides sequences in the Sequence Listing. Mutation of this glycine can result in a variant that is able to act as a double negative mutant.
  • polypeptides comprising two AP2 domains can contain a conserved WEAR/WESH amino acid sequence motif located in the YRG element
  • the linker region is at least about 20, 22, 24, 25 or 26 amino acids in length. Examples of the conserved amino acid sequence are shown in Klucher et al., Plant
  • the full-length native sequences can comprise a carboxyl terminal tail.
  • this tail can include motifs such as a string of negatively or positively charged residues.
  • motifs such as a string of negatively or positively charged residues.
  • a poly-glutamine motif which is usually, at least about 3 amino acids; more usually, at least about 4 amino acids.
  • mutants of interest are those that have additions, substitutions, and deletions in the sequences flanking the domains described above. Further, fragments comprising the domains described above are of interest also. Fusions of such fragments with other AP2 and RAP2 genes, of Arabidopsis, for example, are included within the invention.
  • sequence coding for the desired polypeptide for example a cDNA sequence encoding a full length protein, will preferably be combined with transcriptional and translational initiation regulatory sequences which will direct the transcription of the sequence from the gene in the intended tissues of the transformed plant.
  • transcriptional and translational initiation regulatory sequences which will direct the transcription of the sequence from the gene in the intended tissues of the transformed plant.
  • a plant promoter fragment may be employed
  • constitutive promoters are referred to herein as “constitutive” promoters and are active under most environmental conditions and states of development or cell differentiation.
  • constitutive promoters include the cauliflower mosaic virus (CaMV) 35S transcription initiation region, the 1'- or 2'- promoter derived from T-DNA of Agrobacterium
  • genes include for example, the AP2 gene, ACT11 from Arabidopsis (Huang etal Plant Mol. Biol 33:125-139 (1996)), Cat3 from Arabidopsis (GenBankNo. U43147, Zhong et al. , Mol Gen. Genet. 251 : 196-203 (1996)), the gene encoding stearoyl- acyl carrier protein desaturase from Brassica napus (Genbank No. X74782, Solocombe et
  • the plant promoter may direct expression of the ADC nucleic acid in a specific tissue or may be otherwise under more precise environmental or developmental
  • tissue-specific promoter may drive expression of operably linked sequences in tissues other than the target tissue.
  • tissue-specific promoter may drive expression of operably linked sequences in tissues other than the target tissue.
  • promoter is one that drives expression preferentially in the target tissue, but may also lead to some expression in other tissues as well.
  • promoters under developmental control include promoters that initiate transcription only (or primarily only) in certain tissues, such as fruit, seeds, or flowers. Promoters that direct expression of nucleic acids in ovules, flowers or seeds are
  • a seed-specific promoter is one which directs expression in seed tissues, such promoters may be, for example, ovule- specific, embryo-specific, endosperm-specific, integument-specific, seed coat-specific, or some combination thereof. Examples include a promoter from the ovule-specific BEL1 gene described in Reiser et al Cell 83:735-742 (1995) (GenBank No. U39944). Other promoters from the ovule-specific BEL1 gene described in Reiser et al Cell 83:735-742 (1995) (GenBank No. U39944). Other promoter from the ovule-specific BEL1 gene described in Reiser et al Cell 83:735-742 (1995) (GenBank No. U39944). Other promoter from the ovule-specific BEL1 gene described in Reiser et al Cell 83:735-742 (1995) (GenBank No. U39944). Other promoter from the ovule-specific BEL1 gene described
  • suitable seed specific promoters are derived from the following genes: MAC1 from maize (Sheridan etal. Genetics 142:1009-1020 (1996), Ca ⁇ from maize (GenBankNo. L05934, Abler et al Plant Mol. Biol. 22:10131-1038 (1993), the gene encoding oleosin 18kD from maize (GenBankNo. J05212, Lee etal. Plant Mol. Biol. 26:1981-1987 (1994)), vivparous- 1 from Arabidopsis (Genbank No. U93215), the gene encoding oleosin from Arabidopsis (GenbankNo. Z 17657), Atmycl from Arabidopsis (Urao etal.
  • Plant Mol. Biol. 32:571-576 (1996), the 2s seed storage protein gene family from Arabidopsis (Conceicao e al. Plant 5:493-505 (1994)) the gene encoding oleosin 20kD from Brassica napus (GenBank No. M63985), napA from Brassica napus (GenBank No. J02798, Josefsson et al. JBL 26:12196-1301 (1987), the napin gene family from Brassica napus (Sjodahl et al. Planta 197:264-271 (1995), the gene encoding the 2S storage protein from Brassica napus (Dasgupta et al.
  • oleosin A Genebank No. U09118
  • oleosin B Genebank No. U09119
  • soybean genes encoding low molecular weight sulphur rich protein from soybean
  • a polyadenylation region at the 3 '-end of the coding region should be included.
  • the polyadenylation region can be derived from the natural gene, from a variety of other plant genes, or from T-DNA.
  • the vector comprising the sequences (e.g., promoters or coding regions) from genes of the invention will typically comprise a marker gene which confers a selectable phenotype on plant cells.
  • the marker may encode biocide resistance, particularly antibiotic resistance, such as resistance to kanamycin, G418, bleomycin, hygromycin, or herbicide resistance, such as resistance to chlorosulfuron or Basta.
  • DNA constructs of the invention may be introduced into the genome of the desired plant host by a variety of conventional techniques.
  • the DNA construct may be introduced directly into the genomic DNA of the plant cell using techniques such as electroporation and microinjection of plant cell protoplasts, or the DNA constructs can be introduced directly to plant tissue using ballistic methods, such as DNA particle bombardment.
  • Microinjection techniques are known in the art and well described in the scientific and patent literature.
  • the introduction of DNA constructs using polyethylene glycol precipitation is described in Paszkowski et al. Embo J. 3:2717-2722 (1984).
  • Electroporation techniques are described in Fromm et al. Proc. Natl. Acad. Sci. USA 82:5824 (1985).
  • Ballistic transformation techniques are described in Klein et al.
  • the DNA constructs may be combined with suitable T-DNA flanking regions and introduced into a conventional Agrobacterium tumefaciens host vector.
  • the virulence functions of the Agrobacterium tumefaciens host will direct the insertion of the construct and adjacent marker into the plant cell DNA when the cell is infected by the
  • Agrobacterium tumefaciens-mediated transformation techniques including disarming and use of binary vectors, are well described in the scientific literature. See, for example Horsch et al. Science 233 :496-498 (1984), and Fraley et al. Proc. N ⁇ tl Ac ⁇ d. Sci. USA 80:4803 (1983).
  • 785 techniques can be cultured to regenerate a whole plant which possesses the transformed genotype and thus the desired phenotype such as increased seed mass.
  • Such regeneration techniques rely on manipulation of certain phytohormones in a tissue culture growth medium, typically relying on a biocide and/or herbicide marker which has been introduced together with the desired nucleotide sequences. Plant regeneration from cultured
  • nucleic acids of the invention can be used to confer desired traits on essentially any plant.
  • the invention has use over a broad range of plants, including species from the genera Anacardium, Arachis, Asparagus, Atropa, Avena, Brassica, Citrus, Citrullus, Capsicum, Carthamus, Cocos, Coffea, Cucumis, Cucurbita, Daucus, Elaeis, Fragaria,
  • Increasing seed size, protein, amino acid, and oils content is particularly desirable in crop plants in which seed are used directly for animal or human consumption or for industrial purposes.
  • Examples include soybean, canola, and grains such as rice, wheat, corn, rye, and the like. Decreasing seed size, or producing seedless varieties, is particularly important in plants grown for their fruit and in which large seeds may be
  • Examples include cucumbers, tomatoes, melons, and cherries.
  • the expression cassette is stably incorporated in transgenic plants and confirmed to be operable, it can be introduced into other plants by sexual crossing. Any of a number of standard breeding techniques can be used, depending upon the species to be crossed.
  • transgenic expression of the nucleic acids of the invention leads to phenotypic changes in seeds and fruit
  • plants comprising the expression cassettes discussed above must be sexually crossed with a second plant to obtain the final product.
  • the seed of the invention can be derived from a cross between two transgenic plants of the invention, or a cross between a plant of the invention and another plant. The desired
  • Seed obtained from plants of the present invention can be analyzed according to well known procedures to identify seed with the desired trait. Increased or decreased size can be determined by weighing seeds or by visual inspection. Protein content is
  • Oil content is determined using standard procedures such as gas chromatography. These procedures can also be used to determine whether the types of fatty acids and other lipids are altered in the plants of the invention.
  • the seed mass will be at least about 10%, often about 20% greater than the average seed mass of plants of the same variety that lack the expression cassette.
  • the mass can be about 50% greater and preferably at least about 75% to about 100% greater. Increases in other properties e.g. , protein and oil will usually be proportional to the increases in mass.
  • protein or oil content can increase by about 10%, 20%, 50%, 75% or 100%, or in approximate proportion to the increase in mass.
  • seed of the invention in which AP2 expression is enhanced will have the expression cassettes of the invention and decreased seed mass.
  • Seed mass will be at least about 20% less than the average seed mass of plants of the same variety that lack the
  • the mass will be about 50% less and preferably at least about 75% less or the seed will be absent.
  • decreases in other properties e.g., protein and oil will be proportional to the decreases in mass.
  • ADC genes of the instant invention from oat, wheat, rice, and maize are described also in detail in Bouckaert et al. , Arty, Dkt. No.2750- 117P, Client Dkt. No. 00010.001, filed 25 February 1999.
  • Plant DNA 850 For this example, plant DNAs were isolated according to Jofuku and Goldberg (1988),
  • the plant DNAs were isolated from Avena sativa, Triticum aestivum. Oryza sativa, and Zea mays.
  • Oligonucleotide primer pairs were selected from template Arabidopsis gene sequences using default parameters and the PrimerSelect 3.11 software program (Lasergene sequence analysis suite, DNASTAR, Inc., Madison, WI). Selected primer pairs were then used to generate PCR products utilizing genomic DNA from Brassica napus as a template. PCR products were either sequenced
  • Brassica napus gene regions of greater than or equal to 17 nucleotides in length and 70% sequence identity relative to the Arabidopsis gene were selected and the nucleotide sequences translated into the corresponding amino acid sequences using standard genetic codes. Using the deduced amino acid sequences, the corresponding sequences of triplet codons of the Arabidopsis gene region, and genera- and/or
  • oligonucleotide primer pairs were designed for use in identifying similar gene regions that would encode identical peptides in various unrelated plant genera.
  • the DNA sequence of a primer or its reverse complement would be identical to the sequence of triplet codons of the Arabidopsis gene sequence at nucleotide positions 1 and 2.
  • the nucleotide at position 3 of a triplet codon would be identical to the Arabidopsis 875 codon if that codon is preferentially used in a given plant genera and/or species as determined by published codon usage tables.
  • position 3 would be selected (e.g., A, G, C, T) using genera- and/or species-specific codon usage tables such that the designated nucleotide together with nucleotides in positions 1 and 2 will form a triplet codon that will encode an amino acid that is identical to that encoded by the Arabidopsis triplet codon.
  • the selection of an A, G, C, or T residue will not generate a string of homopolynucleotides greater than four (4) nucleotides.
  • a typical PCR reaction consisted of 1 ⁇ g of template plant DNA, 10 pmol of 885 each primer of a selected primer pair, and 1.25 U of Taq DNA polymerase in standard IX PCR reaction buffer as specified by the manufacturer (Promega, Madison, WI).
  • PCR reaction conditions consisted of one (1) initial cycle of denaturation at 94°C for 7 min, thirty-five (35) cycles of denaturation at 94°C for 1 min., primer-template annealing at 58°C for 30 sec, synthesis at 68°C for 4 min., and one (1) cycle of 890 prolonged synthesis at 68°C for 7 min.
  • SEQ ID NO: 8 MAIZE ADC PROTEIN 65 aa GGFDTAHAAARAYDRAAIKFRGVDADINFN SDYDDDMKQVKSLSKEEFVHA RRQSTGFSRGSS

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Abstract

L'invention a trait à des procédés de modulation du poids des semences et d'autres caractéristiques chez les plantes, notamment l'avoine, le blé, le riz et le maïs. Ces procédés consistent à produire des plantes transgéniques comprenant une cassette d'expression de recombinaison contenant un acide nucléique qui comporte un domaine AP2 (ADC), ledit acide étant lié au promoteur de la plante.
PCT/US2000/004718 1999-02-25 2000-02-25 Nouveaux polynucleotides et polypeptides adc, leurs utilisations, dont les procedes d'amelioration des semences WO2000050585A1 (fr)

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AU33759/00A AU3375900A (en) 1999-02-25 2000-02-25 Novel adc polynucleotides and polypeptides, uses thereof including methods for improving seeds
US09/959,625 US7126043B1 (en) 1999-02-25 2000-02-25 ADC polynucleotides and polypeptides, uses thereof including methods for improving seeds
EP00911946A EP1155123A4 (fr) 1999-02-25 2000-02-25 Nouveaux polynucleotides et polypeptides adc, leurs utilisations, dont les procedes d'amelioration des semences
CA002363599A CA2363599A1 (fr) 1999-02-25 2000-02-25 Nouveaux polynucleotides et polypeptides adc, leurs utilisations, dont les procedes d'amelioration des semences
US11/524,633 US20070094750A1 (en) 2000-02-25 2006-09-21 Novel ADC polynucleotides and polypeptides, uses thereof including methods for improving seeds

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WO1998007842A1 (fr) * 1996-08-20 1998-02-26 The Regents Of The University Of California Procedes d'amelioration de semences

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US6329567B1 (en) * 1996-08-20 2001-12-11 The Regents Of The University Of California Methods for improving seeds
US5994622A (en) * 1996-08-20 1999-11-30 The Regents Of The University Of California Methods for improving seeds
US6559357B1 (en) * 1999-01-08 2003-05-06 The Regents Of The University Of California Methods for altering mass and fertility in plants

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Publication number Priority date Publication date Assignee Title
WO1998007842A1 (fr) * 1996-08-20 1998-02-26 The Regents Of The University Of California Procedes d'amelioration de semences

Non-Patent Citations (5)

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Title
JOFUKU ET. AL.: "Control Of Arabidopsis Flower And Seed Development By The Homeotic Gene APETELA2", THE PLANT CELL, vol. 6, September 1994 (1994-09-01), pages 1211 - 1225, XP002929387 *
KLUCHER ET. AL.: "The AINTEGUMENTA Gene Of Arabidopsis Required For Ovule And Female Gametophyte Development Is Related To The Floral Homeotic Gene APETELA2", THE PLANT CELL, vol. 8, February 1996 (1996-02-01), pages 137 - 153, XP002929388 *
OKAMURO ET. AL.: "The AP2 Domain Of APETELA2 Defines A Large New Family Of DNA Binding Proteins In Arabidopsis", PROC. NATL. ACAD. SCI. USA, vol. 94, June 1997 (1997-06-01), pages 7076 - 7081, XP002929386 *
ROTINO ET. AL.: "Genetic Engineering Of Parthenocarpic Plants", NATURE BIOTECHNOLOGY, vol. 15, December 1997 (1997-12-01), pages 1398 - 1401, XP002929389 *
See also references of EP1155123A4 *

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