WO2015009666A1 - Suppression du silençage génique par des protéines gwar - Google Patents

Suppression du silençage génique par des protéines gwar Download PDF

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WO2015009666A1
WO2015009666A1 PCT/US2014/046605 US2014046605W WO2015009666A1 WO 2015009666 A1 WO2015009666 A1 WO 2015009666A1 US 2014046605 W US2014046605 W US 2014046605W WO 2015009666 A1 WO2015009666 A1 WO 2015009666A1
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plant
dna construct
recombinant dna
sequence
seq
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PCT/US2014/046605
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Stephen M. Allen
Milo Aukerman
Mary PORTILLO-BOCANEGRA
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E. I. Du Pont De Nemours And Company
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Publication of WO2015009666A1 publication Critical patent/WO2015009666A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
    • C12N15/8251—Amino acid content, e.g. synthetic storage proteins, altering amino acid biosynthesis
    • C12N15/8254—Tryptophan or lysine

Definitions

  • the field of invention relates to plant breeding and genetics and, in particular, relates to recombinant DNA constructs useful in plants for suppression of gene silencing.
  • a method to suppress the gene silencing of beneficial transgenes would be useful for the genetic engineering of plants.
  • Activation tagging can be utilized to identify genes with the ability to affect a trait. This approach has been used in the model plant species Arabidopsis thaliana (Weigel, D., et al. (2000) Plant Physiol. 122:1003-1013). Insertions of transcriptional enhancer elements can dominantly activate and/or elevate the expression of nearby endogenous genes. SUMMARY
  • a plant comprising in its genome a recombinant DNA construct comprising a polynucleotide operably linked to at least one heterologous regulatory element, wherein said polynucleotide encodes a polypeptide having an amino acid sequence of at least 80% sequence identity, based on the Clustal W method of alignment, when compared to SEQ ID NO:5, 8, 13, 15, 17, 19, 21 , 23, 25 or 27, and wherein said plant exhibits suppression of gene silencing when compared to a control plant not comprising said recombinant DNA construct.
  • the at least one heterologous regulatory element may be a tissue-specific promoter.
  • the plant may be selected from the group consisting of: Arabidopsis, maize, soybean, sunflower, sorghum, canola, wheat, alfalfa, cotton, rice, barley, millet, sugar cane and switchgrass.
  • a said seed comprising in its genome a recombinant DNA construct comprising a polynucleotide operably linked to at least one heterologous regulatory element, wherein said polynucleotide encodes a polypeptide having an amino acid sequence of at least 80% sequence identity, based on the Clustal W method of alignment, when compared to SEQ ID NO:5, 8, 13, 15, 17, 19, 21 , 23, 25 or 27, and wherein a plant produced from said seed exhibits suppression of gene silencing when compared to a control plant not comprising said recombinant DNA construct.
  • a method of suppressing gene silencing in a plant comprising: (a) introducing into a regenerable plant cell a recombinant DNA construct comprising a polynucleotide operably linked to at least one heterologous regulatory sequence, wherein the polynucleotide encodes a polypeptide having an amino acid sequence of at least 80% sequence identity, based on the Clustal W method of alignment, when compared to SEQ ID NO:5, 8, 13, 15, 17, 19, 21 , 23, 25 or 27; (b)
  • transgenic plant from the regenerable plant cell of (a), wherein the transgenic plant comprises in its genome the recombinant DNA construct and exhibits suppression of gene silencing when compared to a control plant not comprising the recombinant DNA construct; and optionally (c) obtaining a progeny plant derived from the transgenic plant of (b), wherein said progeny plant comprises in its genome the recombinant DNA construct and exhibits suppression of gene silencing when compared to a control plant not comprising the recombinant DNA construct.
  • a method of selecting for suppression of gene silencing in a plant
  • transgenic plant comprising: (a) obtaining a transgenic plant, wherein the transgenic plant comprises in its genome a recombinant DNA construct comprising a polynucleotide operably linked to at least one heterologous regulatory element, wherein said polynucleotide encodes a polypeptide having an amino acid sequence of at least 80% sequence identity, based on the Clustal W method of alignment, when compared to SEQ ID NO:5, 8, 13, 15, 17, 19, 21 , 23, 25 or 27; (b) growing the transgenic plant of part (a) under conditions wherein the polynucleotide is expressed; and (c) selecting the transgenic plant of part (b) with suppression of gene silencing compared to a control plant not comprising the recombinant DNA construct.
  • the at least one heterologous regulatory element may be a tissue-specific promoter.
  • the plant may be selected from the group consisting of: Arabidopsis, maize, soybean, sunflower, sorghum, canola, wheat, alfalfa, cotton, rice, barley, millet, sugar cane and switchgrass.
  • a plant comprising in its genome an endogenous polynucleotide operably linked to at least one heterologous regulatory element, wherein said endogenous polynucleotide encodes a polypeptide having an amino acid sequence of at least 80% sequence identity, based on the Clustal W method of alignment, when compared to SEQ ID NO:5, 8, 13, 15, 17, 19, 21 , 23, 25 or 27, and wherein said plant exhibits suppression of gene silencing when compared to a control plant not comprising the heterologous regulatory element.
  • the at least one heterologous regulatory element may be a tissue-specific promoter.
  • the plant may be selected from the group consisting of: Arabidopsis, maize, soybean, sunflower, sorghum, canola, wheat, alfalfa, cotton, rice, barley, millet, sugar cane and switchgrass.
  • a method of producing a transgenic plant with an increase in gene silencing comprising the steps of: (a) introducing into a regenerable plant cell a recombinant DNA construct comprising an isolated polynucleotide operably linked, in sense or antisense orientation, to a heterologous promoter functional in a plant, wherein the polynucleotide comprises: (i) the nucleotide sequence of SEQ ID NO:3, 4, 6, 7, 12, 14, 16, 18, 20, 22, 24 or 26; (ii) a nucleotide sequence with at least 90% sequence identity, based on the Clustal W method of alignment, when compared to SEQ ID NO:3, 4, 6, 7, 12, 14, 16, 18, 20, 22, 24 or 26; (iii) a nucleotide sequence of at least 100 contiguous nucleotides of SEQ ID NO:3, 4, 6, 7, 12, 14, 16, 18, 20, 22, 24 or 26; (iv) a nucleotide sequence that can hybridize under stringent conditions with the nucleotide sequence of (
  • a plant comprising in its genome a recombinant DNA construct comprising an isolated polynucleotide operably linked, in sense or antisense orientation or both, to a heterologous promoter functional in a plant, wherein the polynucleotide comprises: (a) the nucleotide sequence of SEQ ID NO:3, 4, 6, 7, 12, 14, 16, 18, 20, 22, 24 or 26; (b) a nucleotide sequence with at least 90% sequence identity, based on the Clustal W method of alignment, when compared to SEQ ID NO:3, 4, 6, 7, 12, 14, 16, 18, 20, 22, 24 or 26; (c) a nucleotide sequence of at least 100 contiguous
  • nucleotide sequence that can hybridize under stringent conditions with the
  • the plant may be selected from the group consisting of: Arabidopsis, tomato, maize, soybean, sunflower, sorghum, canola, wheat, alfalfa, cotton, rice, barley, millet, sugar cane and switchgrass.
  • a seed comprising in its genome a recombinant DNA construct comprising an isolated polynucleotide operably linked, in sense or antisense orientation, to a heterologous promoter functional in a plant, wherein the polynucleotide comprises: (a) the nucleotide sequence of SEQ ID NO:3, 4, 6, 7, 12, 14, 16, 18, 20, 22, 24 or 26; (b) a nucleotide sequence with at least 90% sequence identity, based on the Clustal W method of alignment, when compared to SEQ ID NO:3, 4, 6, 7, 12, 14, 16, 18, 20, 22, 24 or 26; (c) a nucleotide sequence of at least 100 contiguous
  • nucleotide sequence that can hybridize under stringent conditions with the
  • a method of producing a seed comprising: (a) crossing a first plant with a second plant, wherein at least one of the first plant and the second plant comprises a recombinant DNA construct comprising a polynucleotide operably linked to at least one heterologous regulatory element, wherein the polynucleotide encodes a polypeptide having an amino acid sequence of at least 80% sequence identity, based on the Clustal W method of alignment, when compared to SEQ ID NO:5, 8, 13, 15, 17, 19, 21 , 23, 25 or 27; and (b) selecting a seed of the crossing of step (a), wherein the seed comprises the recombinant DNA construct.
  • the polypeptide may be over-expressed in at least one tissue of a plant produced from the seed of step (b), or may be over-expressed during at least one condition of abiotic stress, or both.
  • the seed may be selected from the group consisting of: maize, soybean, sunflower, sorghum, canola, wheat, alfalfa, cotton, rice, barley, millet, sugar cane and switchgrass.
  • An isolated polynucleotide comprising: (a) a nucleotide sequence encoding a polypeptide with suppression of gene silencing activity, wherein the polypeptide has an amino acid sequence of at least 80% sequence identity when compared to SEQ ID NO:25 or 27, based on the Clustal W method of alignment; or (b) the full complement of the nucleotide sequence of (a).
  • the polypeptide may have a GW-like motif, a RAWUL domain, or both.
  • the polynucleotide may encode a polypeptide comprising the amino acid sequence of SEQ ID NO:25 or 27.
  • the polynucleotide may comprise the nucleotide sequence of SEQ ID NO:24 or 26.
  • the polynucleotide may be present in a recombinant DNA construct, wherein the polynucleotide is operably linked to at least one heterologous regulatory sequence.
  • FIG. 1 A - 1 D show the multiple alignment of the amino acid sequences of the GWAR polypeptides of SEQ ID NOs:5, 8, 13, 15, 17, 19, 21 , 23, 25 and 27.
  • the Clustal W method of alignment was used with the default parameters. Residues that are identical to the residue of SEQ ID NO:5 at a given position are enclosed in a box.
  • FIG. 2 shows the percent sequence identity and the divergence values for each pair of amino acids sequences of GWAR polypeptide displayed in FIG. 1 A - 1 D.
  • SEQ ID NO:1 is the nucleotide sequence of the 4x35S enhancer element from the pHSbarENDs2 activation tagging vector.
  • SEQ ID NO:2 is the nucleotide sequence poly-linker in the pHSbarENDs2 vector.
  • SEQ ID NO:3 is the genomic nucleotide sequence of the GWAR1 locus (At5g 13250).
  • SEQ ID NO:4 is the nucleotide sequence of the protein-coding region of GWAR1 .
  • SEQ ID NO:5 is the amino acid sequence of GWAR1 .
  • SEQ ID NO:6 is the genomic nucleotide sequence of the GWAR2 locus (At1 g28080).
  • SEQ ID NO:7 is the nucleotide sequence of the protein-coding region of GWAR2.
  • SEQ ID NO:8 is the amino acid sequence of GWAR2.
  • SEQ ID NO:9 is the nucleotide sequence of the attB1 site.
  • SEQ ID NO:10 is the nucleotide sequence of the attB2 site.
  • SEQ ID NO:1 1 is the nucleotide sequence of the ubiquitin promoter with 5' UTR and first intron from Zea mays.
  • the Sequence Listing contains the one letter code for nucleotide sequence characters and the three letter codes for amino acids as defined in conformity with the lUPAC-IUBMB standards described in Nucleic Acids Res. 73:3021 -3030 (1985) and in the Biochemical J. 219 (No. 2 ⁇ :345-373 (1984) which are herein incorporated by reference.
  • the symbols and format used for nucleotide and amino acid sequence data comply with the rules set forth in 37 C.F.R. ⁇ 1 .822.
  • GWAR polypeptide refers to a polypeptide having both a GW-like motif and a RAWUL domain (GW And RAWUL).
  • the At5g13250 protein, containing these domains, has been designated “GWAR1 ".
  • the At1g28080 gene, with similarity to GWAR1 has been designated “GWAR2”.
  • a monocot of the current disclosure includes the
  • a dicot of the current disclosure includes the following families:
  • full complement and “full-length complement” are used interchangeably herein, and refer to a complement of a given nucleotide sequence, wherein the complement and the nucleotide sequence consist of the same number of nucleotides and are 100% complementary.
  • EST is a DNA sequence derived from a cDNA library and therefore is a sequence which has been transcribed.
  • An EST is typically obtained by a single sequencing pass of a cDNA insert.
  • the sequence of an entire cDNA insert is termed the "Full-Insert Sequence” (“FIS").
  • FIS Frull-Insert Sequence
  • a "Contig” sequence is a sequence assembled from two or more sequences that can be selected from, but not limited to, the group consisting of an EST, FIS and PCR sequence.
  • a sequence encoding an entire or functional protein is termed a
  • CCS Complete Gene Sequence
  • a “trait” refers to a physiological, morphological, biochemical, or physical characteristic of a plant or a particular plant material or cell. In some instances, this characteristic is visible to the human eye, such as seed or plant size, or can be measured by biochemical techniques, such as detecting the protein, starch, or oil content of seed or leaves, or by observation of a metabolic or physiological process, e.g. by measuring tolerance to water deprivation or particular salt or sugar concentrations, or by the observation of the expression level of a gene or genes, or by agricultural observations such as osmotic stress tolerance or yield.
  • Agronomic characteristic is a measurable parameter including but not limited to, abiotic stress tolerance, greenness, yield, growth rate, biomass, fresh weight at maturation, dry weight at maturation, fruit yield, seed yield, total plant nitrogen content, fruit nitrogen content, seed nitrogen content, nitrogen content in a vegetative tissue, total plant free amino acid content, fruit free amino acid content, seed free amino acid content, free amino acid content in a vegetative tissue, total plant protein content, fruit protein content, seed protein content, protein content in a vegetative tissue, drought tolerance, nitrogen uptake, root lodging, harvest index, stalk lodging, plant height, ear height, ear length, salt tolerance, early seedling vigor and seedling emergence under low temperature stress.
  • Abiotic stress may be at least one condition selected from the group consisting of: drought, water deprivation, flood, high light intensity, high temperature, low temperature, salinity, etiolation, defoliation, heavy metal toxicity, anaerobiosis, nutrient deficiency, nutrient excess, UV irradiation, atmospheric pollution (e.g., ozone) and exposure to chemicals (e.g., paraquat) that induce production of reactive oxygen species (ROS).
  • ROS reactive oxygen species
  • “Increased stress tolerance" of a plant is measured relative to a reference or control plant, and is a trait of the plant to survive under stress conditions over prolonged periods of time, without exhibiting the same degree of physiological or physical deterioration relative to the reference or control plant grown under similar stress conditions.
  • a plant with "increased stress tolerance” can exhibit increased tolerance to one or more different stress conditions.
  • Stress tolerance activity of a polypeptide indicates that over-expression of the polypeptide in a transgenic plant confers increased stress tolerance to the transgenic plant relative to a reference or control plant.
  • Transgenic refers to any cell, cell line, callus, tissue, plant part or plant, the genome of which has been altered by the presence of a heterologous nucleic acid, such as a recombinant DNA construct, including those initial transgenic events as well as those created by sexual crosses or asexual propagation from the initial transgenic event.
  • a heterologous nucleic acid such as a recombinant DNA construct
  • the term “transgenic” as used herein does not encompass the alteration of the genome (chromosomal or extra-chromosomal) by conventional plant breeding methods or by naturally occurring events such as random cross- fertilization, non-recombinant viral infection, non-recombinant bacterial
  • Gene as it applies to plant cells encompasses not only chromosomal DNA found within the nucleus, but organelle DNA found within subcellular components (e.g., mitochondrial, plastid) of the cell.
  • Plant includes reference to whole plants, plant organs, plant tissues, plant propagules, seeds and plant cells and progeny of same.
  • Plant cells include, without limitation, cells from seeds, suspension cultures, embryos, meristematic regions, callus tissue, leaves, roots, shoots, gametophytes, sporophytes, pollen, and microspores.
  • Propagule includes all products of meiosis and mitosis able to propagate a new plant, including but not limited to, seeds, spores and parts of a plant that serve as a means of vegetative reproduction, such as corms, tubers, offsets, or runners. Propagule also includes grafts where one portion of a plant is grafted to another portion of a different plant (even one of a different species) to create a living organism. Propagule also includes all plants and seeds produced by cloning or by bringing together meiotic products, or allowing meiotic products to come together to form an embryo or fertilized egg (naturally or with human intervention).
  • Progeny comprises any subsequent generation of a plant.
  • Transgenic plant includes reference to a plant which comprises within its genome a heterologous polynucleotide.
  • heterologous polynucleotide For example, the heterologous
  • polynucleotide is stably integrated within the genome such that the polynucleotide is passed on to successive generations.
  • the heterologous polynucleotide may be integrated into the genome alone or as part of a recombinant DNA construct.
  • Gene stacking can be accomplished by many means including but not limited to co-transformation, retransformation, and crossing lines with different transgenes.
  • Transgenic plant also includes reference to plants which comprise more than one heterologous polynucleotide within their genome. Each heterologous polynucleotide may confer a different trait to the transgenic plant.
  • Heterologous with respect to sequence means a sequence that originates from a foreign species, or, if from the same species, is substantially modified from its native form in composition and/or genomic locus by deliberate human
  • nucleic acid sequence is a polymer of RNA or DNA that is single- or double-stranded, optionally containing synthetic, non-natural or altered nucleotide bases.
  • Nucleotides are referred to by their single letter designation as follows: “A” for adenylate or deoxyadenylate (for RNA or DNA, respectively), “C” for cytidylate or deoxycytidylate, “G” for guanylate or deoxyguanylate, “U” for uridylate, “T” for deoxythymidylate, “R” for purines (A or G), “Y” for pyrimidines (C or T), "K” for G or T, “H” for A or C or T, “I” for inosine, and “N” for any nucleotide.
  • Polypeptide”, “peptide”, “amino acid sequence” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers.
  • the terms “polypeptide”, “peptide”, “amino acid sequence”, and “protein” are also inclusive of modifications including, but not limited to, glycosylation, lipid attachment, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation and ADP-ribosylation.
  • mRNA essential RNA
  • mRNA RNA that is without introns and that can be translated into protein by the cell.
  • cDNA refers to a DNA that is complementary to and synthesized from a mRNA template using the enzyme reverse transcriptase.
  • the cDNA can be single- stranded or converted into the double-stranded form using the Klenow fragment of DNA polymerase I.
  • Coding region refers to the portion of a messenger RNA (or the
  • Non-coding region refers to all portions of a messenger RNA or other nucleic acid molecule that are not a coding region, including but not limited to, for example, the promoter region, 5' untranslated region (“UTR”), 3' UTR, intron and terminator.
  • the terms “coding region” and “coding sequence” are used interchangeably herein.
  • the terms “non-coding region” and “non-coding sequence” are used interchangeably herein.
  • “Mature” protein refers to a post-translationally processed polypeptide; i.e., one from which any pre- or pro-peptides present in the primary translation product have been removed.
  • Precursor protein refers to the primary product of translation of mRNA; i.e., with pre- and pro-peptides still present. Pre- and pro-peptides may be and are not limited to intracellular localization signals.
  • isolated refers to materials, such as nucleic acid molecules and/or proteins, which are substantially free or otherwise removed from components that normally accompany or interact with the materials in a naturally occurring environment.
  • Isolated polynucleotides may be purified from a host cell in which they naturally occur. Conventional nucleic acid purification methods known to skilled artisans may be used to obtain isolated polynucleotides. The term also embraces recombinant polynucleotides and chemically synthesized polynucleotides.
  • Recombinant refers to an artificial combination of two otherwise separated segments of sequence, e.g., by chemical synthesis or by the manipulation of isolated segments of nucleic acids by genetic engineering techniques.
  • Recombinant also includes reference to a cell or vector, that has been modified by the introduction of a heterologous nucleic acid or a cell derived from a cell so modified, but does not encompass the alteration of the cell or vector by naturally occurring events (e.g., spontaneous mutation, natural
  • Recombinant DNA construct refers to a combination of nucleic acid fragments that are not normally found together in nature. Accordingly, a
  • recombinant DNA construct may comprise regulatory sequences and coding sequences that are derived from different sources, or regulatory sequences and coding sequences derived from the same source, but arranged in a manner different than that normally found in nature.
  • the terms "recombinant DNA construct” and “recombinant construct” are used interchangeably herein.
  • regulatory sequences refer to nucleotide sequences located upstream (5' non-coding sequences), within, or downstream (3' non-coding sequences) of a coding sequence, and which influence the transcription, RNA processing or stability, or translation of the associated coding sequence. Regulatory sequences may include, but are not limited to, promoters, translation leader sequences, introns, and polyadenylation recognition sequences. The terms “regulatory sequence” and “regulatory element” are used interchangeably herein.
  • Promoter refers to a nucleic acid fragment capable of controlling
  • Promoter functional in a plant is a promoter capable of controlling
  • tissue-specific promoter and “tissue-preferred promoter” are used interchangeably, and refer to a promoter that is expressed predominantly but not necessarily exclusively in one tissue or organ, but that may also be expressed in one specific cell.
  • “Developmentally regulated promoter” refers to a promoter whose activity is determined by developmental events.
  • “Operably linked” refers to the association of nucleic acid fragments in a single fragment so that the function of one is regulated by the other.
  • a promoter is operably linked with a nucleic acid fragment when it is capable of regulating the transcription of that nucleic acid fragment.
  • “Expression” refers to the production of a functional product.
  • expression of a nucleic acid fragment may refer to transcription of the nucleic acid fragment (e.g., transcription resulting in mRNA or functional RNA) and/or translation of mRNA into a precursor or mature protein.
  • Phenotype means the detectable characteristics of a cell or organism.
  • “Introduced” in the context of inserting a nucleic acid fragment (e.g., a recombinant DNA construct) into a cell means “transfection” or “transformation” or “transduction” and includes reference to the incorporation of a nucleic acid fragment into a eukaryotic or prokaryotic cell where the nucleic acid fragment may be incorporated into the genome of the cell (e.g., chromosome, plasmid, plastid or mitochondrial DNA), converted into an autonomous replicon, or transiently expressed (e.g., transfected mRNA).
  • a nucleic acid fragment e.g., a recombinant DNA construct
  • a “transformed cell” is any cell into which a nucleic acid fragment (e.g., a recombinant DNA construct) has been introduced.
  • Transformation refers to both stable transformation and transient transformation.
  • “Stable transformation” refers to the introduction of a nucleic acid fragment into a genome of a host organism resulting in genetically stable inheritance. Once stably transformed, the nucleic acid fragment is stably integrated in the genome of the host organism and any subsequent generation.
  • Transient transformation refers to the introduction of a nucleic acid fragment into the nucleus, or DNA-containing organelle, of a host organism resulting in gene expression without genetically stable inheritance.
  • Allele is one of several alternative forms of a gene occupying a given locus on a chromosome. When the alleles present at a given locus on a pair of
  • homologous chromosomes in a diploid plant are the same that plant is homozygous at that locus. If the alleles present at a given locus on a pair of homologous chromosomes in a diploid plant differ that plant is heterozygous at that locus. If a transgene is present on one of a pair of homologous chromosomes in a diploid plant that plant is hemizygous at that locus.
  • chloroplast transit peptide is an amino acid sequence which is translated in conjunction with a protein and directs the protein to the chloroplast or other plastid types present in the cell in which the protein is made (Lee et al. (2008) Plant Cell 20:1603-1622).
  • chloroplast transit peptide and “plastid transit peptide” are used interchangeably herein.
  • Chloroplast transit sequence refers to a nucleotide sequence that encodes a chloroplast transit peptide.
  • a “signal peptide” is an amino acid sequence which is translated in conjunction with a protein and directs the protein to the secretory system (Chrispeels (1991 ) Ann. Rev. Plant Phys. Plant Mol. Biol.
  • a vacuolar targeting signal can further be added, or if to the endoplasmic reticulum, an endoplasmic reticulum retention signal (supra) may be added.
  • any signal peptide present should be removed and instead a nuclear localization signal included (Raikhel (1992) Plant Phys. 700:1627-1632).
  • a "mitochondrial signal peptide” is an amino acid sequence which directs a precursor protein into the mitochondria (Zhang and Glaser (2002) Trends Plant Sci 7:14-21 ).
  • Sequence alignments and percent identity calculations may be determined using a variety of comparison methods designed to detect homologous sequences including, but not limited to, the Megalign® program of the LASERGENE®
  • the Clustal W method of alignment may be used.
  • Standard recombinant DNA and molecular cloning techniques used herein are well known in the art and are described more fully in Sambrook, J., Fritsch, E.F. and Maniatis, T. Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory Press: Cold Spring Harbor, 1989 (hereinafter "Sambrook”).
  • a codon for the amino acid alanine, a hydrophobic amino acid may be substituted by a codon encoding another less hydrophobic residue, such as glycine, or a more hydrophobic residue, such as valine, leucine, or isoleucine.
  • the protein of the current disclosure may also be a protein which comprises an amino acid sequence comprising deletion, substitution, insertion and/or addition of one or more amino acids in an amino acid sequence presented in SEQ ID NO:5, 8, 13, 15, 17, 19, 21 , 23, 25 or 27.
  • the substitution may be conservative, which means the replacement of a certain amino acid residue by another residue having similar physical and chemical characteristics.
  • conservative substitution include replacement between aliphatic group-containing amino acid residues such as lie, Val, Leu or Ala, and replacement between polar residues such as Lys-Arg, Glu-Asp or Gln-Asn replacement.
  • Proteins derived by amino acid deletion, substitution, insertion and/or addition can be prepared when DNAs encoding their wild-type proteins are subjected to, for example, well-known site-directed mutagenesis (see, e.g., Nucleic Acid Research, Vol. 10, No. 20, p.6487-6500, 1982, which is hereby incorporated by reference in its entirety).
  • site-directed mutagenesis see, e.g., Nucleic Acid Research, Vol. 10, No. 20, p.6487-6500, 1982, which is hereby incorporated by reference in its entirety.
  • the term "one or more amino acids” is intended to mean a possible number of amino acids which may be deleted, substituted, inserted and/or added by site-directed mutagenesis.
  • Site-directed mutagenesis may be accomplished, for example, as follows using a synthetic oligonucleotide primer that is complementary to single-stranded phage DNA to be mutated, except for having a specific mismatch (i.e., a desired mutation).
  • the above synthetic oligonucleotide is used as a primer to cause synthesis of a complementary strand by phages, and the resulting duplex DNA is then used to transform host cells.
  • the transformed bacterial culture is plated on agar, whereby plaques are allowed to form from phage-containing single cells.
  • 50% of new colonies contain phages with the mutation as a single strand, while the remaining 50% have the original sequence.
  • the resulting plaques are allowed to hybridize with a synthetic probe labeled by kinase treatment.
  • plaques hybridized with the probe are picked up and cultured for collection of their DNA.
  • Techniques for allowing deletion, substitution, insertion and/or addition of one or more amino acids in the amino acid sequences of biologically active peptides such as enzymes while retaining their activity include site-directed mutagenesis mentioned above, as well as other techniques such as those for treating a gene with a mutagen, and those in which a gene is selectively cleaved to remove, substitute, insert or add a selected nucleotide or nucleotides, and then ligated.
  • the protein of the present disclosure may also be a protein which is encoded by a nucleic acid comprising a nucleotide sequence comprising deletion,
  • nucleotide sequence of SEQ ID NO:3, 4, 6, 7, 12, 14, 16, 18, 20, 22, 24 or 26 may be accomplished by site- directed mutagenesis or other techniques as mentioned above.
  • the protein of the present disclosure may also be a protein which is encoded by a nucleic acid comprising a nucleotide sequence hybridizable under stringent conditions with the complementary strand of the nucleotide sequence of SEQ ID NO:3, 4, 6, 7, 12, 14, 16, 18, 20, 22, 24 or 26.
  • under stringent conditions means that two sequences hybridize under moderately or highly stringent conditions. More specifically, moderately stringent conditions can be readily determined by those having ordinary skill in the art, e.g., depending on the length of DNA. The basic conditions are set forth by Sambrook et al., Molecular Cloning: A Laboratory Manual, third edition, chapters 6 and 7, Cold Spring Harbor Laboratory Press, 2001 and include the use of a prewashing solution for nitrocellulose filters 5xSSC, 0.5% SDS, 1 .0 mM EDTA (pH 8.0), hybridization conditions of about 50% formamide, 2xSSC to 6xSSC at about 40-50 °C (or other similar hybridization solutions, such as Stark's solution, in about 50% formamide at about 42 °C) and washing conditions of, for example, about 40- 60 °C, 0.5-6xSSC, 0.1 % SDS.
  • moderately stringent conditions include hybridization (and washing) at about 50 °C and 6xSSC. Highly stringent conditions can
  • such conditions include hybridization and/or washing at higher temperature and/or lower salt concentration (such as hybridization at about 65 °C, 6xSSC to 0.2xSSC, preferably 6xSSC, more preferably 2xSSC, most preferably 0.2xSSC), compared to the moderately stringent conditions.
  • highly stringent conditions may include hybridization as defined above, and washing at approximately 65-68 °C, 0.2xSSC, 0.1 % SDS.
  • SSPE (I xSSPE is 0.15 M NaCI, 10 mM NaH2PO4, and 1 .25 mM EDTA, pH 7.4) can be substituted for SSC (1 xSSC is 0.15 M NaCI and 15 mM sodium citrate) in the hybridization and washing buffers; washing is performed for 15 minutes after hybridization is completed.
  • hybridization kit which uses no radioactive substance as a probe.
  • Specific examples include hybridization with an ECL direct labeling & detection system (Amersham).
  • Stringent conditions include, for example, hybridization at 42 °C for 4 hours using the hybridization buffer included in the kit, which is supplemented with 5% (w/v) Blocking reagent and 0.5 M NaCI, and washing twice in 0.4% SDS, 0.5xSSC at 55 °C for 20 minutes and once in 2xSSC at room temperature for 5 minutes.
  • the present disclosure includes recombinant DNA constructs (including suppression DNA constructs).
  • a recombinant DNA construct comprises a
  • polynucleotide operably linked to at least one regulatory sequence (e.g., a promoter functional in a plant; the regulatory sequence may be heterologous), wherein the polynucleotide comprises (i) a nucleic acid sequence encoding an amino acid sequence of at least 50%, 51 %, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61 %, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, based on the Clustal V method of alignment, when compared to SEQ ID NO:5,
  • a recombinant DNA construct comprises a
  • polynucleotide operably linked to at least one regulatory sequence (e.g., a promoter functional in a plant; the regulatory sequence may be heterologous), wherein said polynucleotide comprises (i) a nucleic acid sequence of at least 50%, 51 %, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61 %, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, based on the Clustal V method of alignment, when compared to SEQ ID NO:3, 4, 6, 7, 12, 14, 16, 18,
  • a suppression DNA construct may comprise at least one regulatory sequence (e.g., a promoter functional in a plant; the regulatory sequence may be heterologous) operably linked to (a) all or part of: (i) a nucleic acid sequence encoding a polypeptide having an amino acid sequence of at least 50%, 51 %, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61 %, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, based on the Clustal V
  • the suppression DNA construct may comprise a cosuppression construct, antisense construct, viral-suppression construct, hairpin suppression construct, stem-loop suppression construct, double-stranded RNA-producing construct, RNAi construct, or small RNA construct (e.g., an siRNA construct or an miRNA construct).
  • a codon for the amino acid alanine, a hydrophobic amino acid may be substituted by a codon encoding another less hydrophobic residue, such as glycine, or a more hydrophobic residue, such as valine, leucine, or isoleucine.
  • “Suppression DNA construct” is a recombinant DNA construct which when transformed or stably integrated into the genome of the plant, results in “silencing” of a target gene in the plant.
  • the target gene may be endogenous or transgenic to the plant.
  • “Silencing,” as used herein with respect to the target gene, refers generally to the suppression of levels of mRNA or protein/enzyme expressed by the target gene, and/or the level of the enzyme activity or protein functionality.
  • suppression include lowering, reducing, declining, decreasing, inhibiting, eliminating or preventing.
  • RNAi-based approaches RNAi-based approaches
  • small RNA-based approaches RNAi-based approaches
  • a suppression DNA construct may comprise a region derived from a target gene of interest and may comprise all or part of the nucleic acid sequence of the sense strand (or antisense strand) of the target gene of interest.
  • the region may be 100% identical or less than 100% identical (e.g., at least 50%, 51 %, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61 %, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical) to all or part of the sense strand (or antisense strand) of the
  • a suppression DNA construct may comprise 100, 200, 300, 400, 500, 600, 700, 800, 900 or 1000 contiguous nucleotides of the sense strand (or antisense strand) of the gene of interest, and combinations thereof.
  • RNAi RNA interference
  • small RNA constructs such as siRNA (short interfering RNA) constructs and miRNA (microRNA) constructs.
  • Suppression of gene expression may also be achieved by use of artificial miRNA precursors, ribozyme constructs and gene disruption.
  • a modified plant miRNA precursor may be used, wherein the precursor has been modified to replace the miRNA encoding region with a sequence designed to produce a miRNA directed to the nucleotide sequence of interest.
  • Gene disruption may be achieved by use of transposable elements or by use of chemical agents that cause site-specific mutations.
  • Antisense inhibition refers to the production of antisense RNA transcripts capable of suppressing the expression of the target gene or gene product.
  • Antisense RNA refers to an RNA transcript that is complementary to all or part of a target primary transcript or mRNA and that blocks the expression of a target isolated nucleic acid fragment (U.S. Patent No. 5,107,065).
  • the complementarity of an antisense RNA may be with any part of the specific gene transcript, i.e., at the 5' non-coding sequence, 3' non-coding sequence, introns, or the coding sequence.
  • Codon refers to the production of sense RNA transcripts capable of suppressing the expression of the target gene or gene product.
  • Sense RNA refers to RNA transcript that includes the mRNA and can be translated into protein within a cell or in vitro. Cosuppression constructs in plants have been previously designed by focusing on overexpression of a nucleic acid sequence having homology to a native mRNA, in the sense orientation, which results in the reduction of all RNA having homology to the overexpressed sequence (see Vaucheret et al., Plant J. 16:651 -659 (1998); and Gura, Nature 404:804-808 (2000)).
  • RNA interference refers to the process of sequence-specific post- transcriptional gene silencing in animals mediated by short interfering RNAs
  • RNA silencing (Fire et al., Nature 391 :806 (1998)).
  • PTGS post-transcriptional gene silencing
  • quelling in fungi.
  • the process of post- transcriptional gene silencing is thought to be an evolutionarily-conserved cellular defense mechanism used to prevent the expression of foreign genes and is commonly shared by diverse flora and phyla (Fire et al., Trends Genet. 15:358 (1999)).
  • Small RNAs play an important role in controlling gene expression. Regulation of many developmental processes, including flowering, is controlled by small RNAs. It is now possible to engineer changes in gene expression of plant genes by using transgenic constructs which produce small RNAs in the plant.
  • Small RNAs appear to function by base-pairing to complementary RNA or DNA target sequences. When bound to RNA, small RNAs trigger either RNA cleavage or translational inhibition of the target sequence. When bound to DNA target sequences, it is thought that small RNAs can mediate DNA methylation of the target sequence. The consequence of these events, regardless of the specific mechanism, is that gene expression is inhibited.
  • MicroRNAs are noncoding RNAs of about 19 to about 24 nucleotides (nt) in length that have been identified in both animals and plants (Lagos-Quintana et al., Science 294:853-858 (2001 ), Lagos-Quintana et al., Curr. Biol. 12:735-739 (2002); Lau et al., Science 294:858-862 (2001 ); Lee and Ambros, Science 294:862-864 (2001 ); Llave et al., Plant Cell 14:1605-1619 (2002);
  • MicroRNAs appear to regulate target genes by binding to complementary sequences located in the transcripts produced by these genes. It seems likely that miRNAs can enter at least two pathways of target gene regulation: (1 ) translational inhibition; and (2) RNA cleavage. MicroRNAs entering the RNA cleavage pathway are analogous to the 21 -25 nt short interfering RNAs (siRNAs) generated during RNA interference (RNAi) in animals and posttranscriptional gene silencing (PTGS) in plants, and likely are incorporated into an RNA-induced silencing complex (RISC) that is similar or identical to that seen for RNAi.
  • siRNAs short interfering RNAs
  • PTGS posttranscriptional gene silencing
  • miRNA-star sequence and “miRNA * sequence” are used interchangeably herein and they refer to a sequence in the miRNA precursor that is highly complementary to the miRNA sequence.
  • miRNA and miRNA * are used interchangeably herein and they refer to a sequence in the miRNA precursor that is highly complementary to the miRNA sequence.
  • sequences form part of the stem region of the miRNA precursor hairpin structure.
  • a method for the suppression of a target sequence comprising introducing into a cell a nucleic acid construct encoding a miRNA substantially complementary to the target.
  • the miRNA comprises about 19, 20, 21 , 22, 23, 24 or 25 nucleotides.
  • the miRNA comprises 21 nucleotides.
  • the nucleic acid construct encodes the miRNA.
  • the nucleic acid construct encodes a polynucleotide precursor which may form a double-stranded RNA, or hairpin structure comprising the miRNA.
  • the nucleic acid construct comprises a modified endogenous plant miRNA precursor, wherein the precursor has been modified to replace the endogenous miRNA encoding region with a sequence designed to produce a miRNA directed to the target sequence.
  • the plant miRNA precursor may be full-length of may comprise a fragment of the full-length precursor.
  • the endogenous plant miRNA precursor is from a dicot or a monocot.
  • the endogenous miRNA precursor is from Arabidopsis, tomato, maize, soybean, sunflower, sorghum, canola, wheat, alfalfa, cotton, rice, barley, millet, sugar cane or switchgrass.
  • the miRNA template i.e.
  • the polynucleotide encoding the miRNA), and thereby the miRNA may comprise some mismatches relative to the target sequence.
  • the miRNA template has > 1 nucleotide mismatch as compared to the target sequence, for example, the miRNA template can have 1 , 2, 3, 4, 5, or more mismatches as compared to the target sequence. This degree of mismatch may also be described by determining the percent identity of the miRNA template to the complement of the target sequence.
  • the miRNA template may have a percent identity including about at least 70%, 75%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% as compared to the complement of the target sequence.
  • the miRNA template (i.e. the polynucleotide encoding the miRNA) and thereby the miRNA, may comprise some mismatches relative to the miRNA-star sequence.
  • the miRNA template has > 1 nucleotide mismatch as compared to the miRNA-star sequence, for example, the miRNA template can have 1 , 2, 3, 4, 5, or more mismatches as compared to the miRNA-star sequence. This degree of mismatch may also be described by determining the percent identity of the miRNA template to the complement of the miRNA-star sequence.
  • the miRNA template may have a percent identity including about at least 70%, 75%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% as compared to the complement of the miRNA-star sequence.
  • a recombinant DNA construct (including a suppression DNA construct) of the present disclosure may comprise at least one regulatory sequence.
  • the at least one regulatory sequence may be heterologous.
  • a regulatory sequence may be a promoter.
  • promoters can be used in recombinant DNA constructs of the present disclosure.
  • the promoters can be selected based on the desired outcome, and may include constitutive, tissue-specific, inducible, or other promoters for expression in the host organism. Promoters that cause a gene to be expressed in most cell types at most times are commonly referred to as "constitutive promoters".
  • constitutive expression of the candidate gene under control of the 35S or UBI promoter may have pleiotropic effects, although candidate gene efficacy may be estimated when driven by a constitutive promoter.
  • Use of tissue-specific and/or stress-specific promoters may eliminate undesirable effects.
  • Suitable constitutive promoters for use in a plant host cell include, for example, the core promoter of the Rsyn7 promoter and other constitutive promoters disclosed in WO 99/43838 and U.S. Patent No. 6,072,050; the core CaMV 35S promoter (Odell et al., Nature 313:810-812 (1985)); rice actin (McElroy et al., Plant Cell 2:163-171 (1990)); ubiquitin (Christensen et al., Plant Mol. Biol. 12:619-632 (1989) and Christensen et al., Plant Mol. Biol. 18:675-689 (1992)); pEMU (Last et al., Theor. Appl.
  • tissue-specific or developmentally regulated promoter it may be desirable to use a tissue-specific or developmentally regulated promoter.
  • a tissue-specific or developmentally regulated promoter is a DNA sequence which regulates the expression of a DNA sequence selectively in the cells/tissues of a plant critical to tassel development, seed set, or both, and limits the expression of such a DNA sequence to the period of tassel development or seed maturation in the plant. Any identifiable promoter may be used in the methods of the present disclosure which causes the desired temporal and spatial expression.
  • Promoters which are seed or embryo-specific and may be useful in the disclosure include soybean Kunitz trypsin inhibitor (Kti3, Jofuku and Goldberg, Plant Cell 1 :1079-1093 (1989)), patatin (potato tubers) (Rocha-Sosa, M., et al. (1989) EMBO J. 8:23-29), convicilin, vicilin, and legumin (pea cotyledons) (Rerie, W.G., et al. (1991 ) Mol. Gen. Genet. 259:149-157; Newbigin, E.J., et al. (1990) Planta 180:461 -470; Higgins, T.J.V., et al.
  • Promoters of seed-specific genes operably linked to heterologous coding regions in chimeric gene constructions maintain their temporal and spatial expression pattern in transgenic plants.
  • Such examples include Arabidopsis thaliana 2S seed storage protein gene promoter to express enkephalin peptides in
  • Arabidopsis and Brassica napus seeds (Vanderkerckhove et al ., Bio/Technology 7:L929-932 (1989)), bean lectin and bean beta-phaseolin promoters to express luciferase (Riggs et al., Plant Sci. 63:47-57 (1989)), and wheat glutenin promoters to express chloramphenicol acetyl transferase (Colot et al., EMBO J 6:3559- 3564 (1987)).
  • Inducible promoters selectively express an operably linked DNA sequence in response to the presence of an endogenous or exogenous stimulus, for example by chemical compounds (chemical inducers) or in response to environmental, hormonal, chemical, and/or developmental signals.
  • Inducible or regulated promoters include, for example, promoters regulated by light, heat, stress, flooding or drought, phytohormones, wounding, or chemicals such as ethanol, jasmonate, salicylic acid, or safeners.
  • Promoters for use in the current disclosure include the following: 1 ) the stress-inducible RD29A promoter (Kasuga et al . (1999) Nature Biotechnol. 17:287- 91 ); 2) the barley promoter, B22E; expression of B22E is specific to the pedicel in developing maize kernels ("Primary Structure of a Novel Barley Gene Differentially Expressed in Immature Aleurone Layers". Klemsdal, S.S. et al., Mol. Gen. Genet.
  • Zag2 transcripts can be detected 5 days prior to pollination to 7 to 8 days after pollination ("DAP"), and directs expression in the carpel of developing female inflorescences and Ciml which is specific to the nucleus of developing maize kernels. Ciml transcript is detected 4 to 5 days before pollination to 6 to 8 DAP.
  • Other useful promoters include any promoter which can be derived from a gene whose expression is maternally associated with developing female florets.
  • stalk-specific promoters include the alfalfa S2A promoter (GenBank Accession No. EF030816; Abrahams et al., Plant Mol. Biol. 27:513-528 (1995)) and S2B promoter (GenBank Accession No. EF030817) and the like, herein incorporated by reference.
  • Promoters may be derived in their entirety from a native gene, or be composed of different elements derived from different promoters found in nature, or even comprise synthetic DNA segments.
  • endogenous promoter operably linked to at least one enhancer element; e.g., a 35S, nos or ocs enhancer element.
  • Promoters for use in the current disclosure may include: RIP2, ml_IP15, ZmCORI , Rab17, CaMV 35S, RD29A, B22E, Zag2, SAM synthetase, ubiquitin, CaMV 19S, nos, Adh, sucrose synthase, R-allele, the vascular tissue preferred promoters S2A (Genbank accession number EF030816) and S2B (Genbank accession number EF030817), and the constitutive promoter GOS2 from Zea mays.
  • promoters include root preferred promoters, such as the maize NAS2 promoter, the maize Cyclo promoter (US 2006/0156439, published July 13, 2006), the maize ROOTMET2 promoter (WO05063998, published July 14, 2005), the CR1 BIO promoter (WO06055487, published May 26, 2006), the CRWAQ81
  • Recombinant DNA constructs of the present disclosure may also include other regulatory sequences, including but not limited to, translation leader sequences, introns, and polyadenylation recognition sequences.
  • a recombinant DNA construct of the present disclosure further comprises an enhancer or silencer.
  • An intron sequence can be added to the 5' untranslated region, the protein- coding region or the 3' untranslated region to increase the amount of the mature message that accumulates in the cytosol. Inclusion of a spliceable intron in the transcription unit in both plant and animal expression constructs has been shown to increase gene expression at both the mRNA and protein levels up to 1000-fold. Buchman and Berg, Mol. Cell Biol. 8:4395-4405 (1988); Callis et al., Genes Dev. 1 :1 183-1200 (1987).
  • Any plant can be selected for the identification of regulatory sequences and GWAR polypeptide genes to be used in recombinant DNA constructs and other compositions (e.g. transgenic plants, seeds and cells) and methods of the present disclosure.
  • suitable plants for the isolation of genes and regulatory sequences and for compositions and methods of the present disclosure would include but are not limited to alfalfa, apple, apricot, Arabidopsis, artichoke, arugula, asparagus, avocado, banana, barley, beans, beet, blackberry, blueberry, broccoli, brussels sprouts, cabbage, canola, cantaloupe, carrot, cassava, castorbean, cauliflower, celery, cherry, chicory, cilantro, citrus, Clementines, clover, coconut, coffee, corn, cotton, cranberry, cucumber, Douglas fir, eggplant, endive, escarole, eucalyptus, fennel, figs, garlic, gourd, grape, grapefruit, honey dew, j
  • compositions persimmon, pine, pineapple, plantain, plum, pomegranate, poplar, potato, pumpkin, quince, radiata pine, radicchio, radish, rapeseed, raspberry, rice, rye, sorghum, Southern pine, soybean, spinach, squash, strawberry, sugarbeet, sugarcane, sunflower, sweet potato, sweetgum, switchgrass, tangerine, tea, tobacco, tomato, triticale, turf, turnip, a vine, watermelon, wheat, yams, and zucchini.
  • a composition of the present disclosure includes a transgenic microorganism, cell, plant, and seed comprising the recombinant DNA construct.
  • the cell may be eukaryotic, e.g., a yeast, insect or plant cell, or prokaryotic, e.g., a bacterial cell.
  • composition of the present disclosure is a plant comprising in its genome any of the recombinant DNA constructs (including any of the suppression DNA constructs) of the present disclosure (such as any of the constructs discussed above).
  • Compositions also include any progeny of the plant, and any seed obtained from the plant or its progeny, wherein the progeny or seed comprises within its genome the recombinant DNA construct (or suppression DNA construct).
  • Progeny includes subsequent generations obtained by self-pollination or out-crossing of a plant.
  • Progeny also includes hybrids and inbreds.
  • mature transgenic plants can be self- pollinated to produce a homozygous inbred plant.
  • the inbred plant produces seed containing the newly introduced recombinant DNA construct (or suppression DNA construct). These seeds can be grown to produce plants with the desired
  • the seeds may be maize seeds.
  • the plant may be a monocotyledonous or dicotyledonous plant, for example, a maize or soybean plant.
  • the plant may also be sunflower, sorghum, canola, wheat, alfalfa, cotton, rice, barley, millet, sugar cane or switchgrass.
  • the plant may be a hybrid plant or an inbred plant.
  • the recombinant DNA construct may be stably integrated into the genome of the plant.
  • a plant for example, a maize, rice or soybean plant
  • a recombinant DNA construct comprising a polynucleotide operably linked to at least one regulatory sequence (the regulatory sequence may be heterologous)
  • said polynucleotide encodes a polypeptide having an amino acid sequence of at least 50%, 51 %, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61 %, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, based on the Clustal
  • a plant for example, a maize, rice or soybean plant
  • a recombinant DNA construct comprising a polynucleotide operably linked to at least one regulatory sequence (the regulatory sequence may be heterologous), wherein said polynucleotide encodes a GWAR polypeptide, and wherein said plant exhibits suppression of gene silencing when compared to a control plant not comprising said recombinant DNA construct.
  • a plant for example, a maize, rice or soybean plant comprising in its genome a recombinant DNA construct comprising a polynucleotide operably linked to at least one regulatory element (the regulatory element may be heterologous), wherein said polynucleotide comprises a nucleotide sequence, wherein the nucleotide sequence is: (a) hybridizable under stringent conditions with a DNA molecule comprising the full complement of SEQ ID NO:3, 4, 6, 7, 12, 14, 16, 18, 20, 22, 24 or 26; or (b) derived from SEQ ID NO:3, 4, 6, 7, 12, 14, 16, 18, 20, 22, 24 or 26 by alteration of one or more nucleotides by at least one method selected from the group consisting of: deletion, substitution, addition and insertion; and wherein said plant exhibits suppression of gene silencing, when compared to a control plant not comprising said recombinant DNA construct.
  • a plant for example, a maize, rice or soybean plant
  • a polynucleotide (optionally an endogenous polynucleotide) operably linked to at least one heterologous regulatory element
  • said polynucleotide encodes a polypeptide having an amino acid sequence of at least 50%, 51 %, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61 %, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, based on the Clustal V method of alignment
  • the GWAR polypeptide may be from Arabidopsis thaliana, Zea mays, Glycine max, Glycine tabacina, Glycine soja, Glycine tomentella, Oryza sativa, Brassica napus, Sorghum bicolor, Saccharum officinarum,or Triticum aestivum.
  • suppression DNA construct may comprise at least a promoter functional in a plant as a regulatory sequence.
  • the promoter may be heterologous.
  • control plant e.g., compositions or methods as described herein.
  • the second hybrid line would typically be measured relative to the first hybrid line (i.e., the first hybrid line is the control or reference plant).
  • a plant comprising a recombinant DNA construct (or suppression DNA construct) the plant may be assessed or measured relative to a control plant not comprising the recombinant DNA construct (or suppression DNA construct) but otherwise having a comparable genetic background to the plant (e.g., sharing at least 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity of nuclear genetic material compared to the plant comprising the
  • SCARs Characterized Amplified Regions
  • Amplified Fragment Length Amplified Fragment Length
  • AFLP®s Polymorphisms
  • SSRs Simple Sequence Repeats
  • a suitable control or reference plant to be utilized when assessing or measuring an agronomic characteristic or phenotype of a transgenic plant would not include a plant that had been previously selected, via mutagenesis or transformation, for the desired agronomic characteristic or phenotype.
  • the plant used in the methods may be a monocotyledonous or
  • dicotyledonous plant for example, a maize or soybean plant.
  • the plant may also be sunflower, sorghum, canola, wheat, alfalfa, cotton, rice, barley, millet, sugar cane or sorghum.
  • the seed may be a maize or soybean seed, for example, a maize hybrid seed or maize inbred seed.
  • Methods include but are not limited to the following:
  • a method for transforming a cell (or microorganism) comprising transforming a cell (or microorganism) with any of the isolated polynucleotides or recombinant DNA constructs of the present disclosure.
  • the cell (or microorganism) transformed by this method is also included.
  • the cell is eukaryotic cell, e.g., a yeast, insect or plant cell, or prokaryotic, e.g., a bacterial cell.
  • the microorganism may be Agrobacterium, e.g. Agrobacterium tumefaciens or
  • a method for producing a transgenic plant comprising transforming a plant cell with any of the isolated polynucleotides or recombinant DNA constructs
  • the disclosure is also directed to the transgenic plant produced by this method, and transgenic seed obtained from this transgenic plant.
  • the transgenic plant obtained by this method may be used in other methods of the present disclosure.
  • a method for isolating a polypeptide of the disclosure from a cell or culture medium of the cell wherein the cell comprises a recombinant DNA construct comprising a polynucleotide of the disclosure operably linked to at least one regulatory sequence (the regulatory sequence may be heterologous), and wherein the transformed host cell is grown under conditions that are suitable for expression of the recombinant DNA construct.
  • a method of altering the level of expression of a polypeptide of the disclosure in a host cell comprising: (a) transforming a host cell with a recombinant DNA construct of the present disclosure; and (b) growing the transformed host cell under conditions that are suitable for expression of the recombinant DNA construct wherein expression of the recombinant DNA construct results in production of altered levels of the polypeptide of the disclosure in the transformed host cell.
  • a method of suppressing gene silencing in a plant comprising: (a) introducing into a regenerable plant cell a recombinant DNA construct comprising a polynucleotide operably linked to at least one regulatory sequence (for example, a promoter functional in a plant; the regulatory sequence may be heterologous), wherein the polynucleotide encodes a polypeptide having an amino acid sequence of at least 50%, 51 %, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61 %, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%,
  • the method may further comprise (c) obtaining a progeny plant derived from the transgenic plant, wherein said progeny plant comprises in its genome the recombinant DNA construct and exhibits suppression of gene silencing when compared to a control plant not comprising the recombinant DNA construct.
  • a method of suppressing gene silencing comprising: (a) introducing into a regenerable plant cell a recombinant DNA construct comprising a polynucleotide operably linked to at least one regulatory element, wherein said polynucleotide comprises a nucleotide sequence, wherein the nucleotide sequence is: (a) hybridizable under stringent conditions with a DNA molecule comprising the full complement of SEQ ID NO:3, 4, 6, 7, 12, 14, 16, 18, 20, 22, 24 or 26; or (b) derived from SEQ ID NO:3, 4, 6, 7, 12, 14, 16, 18, 20, 22, 24 or 26 by alteration of one or more nucleotides by at least one method selected from the group consisting of: deletion, substitution, addition and insertion; and (b) regenerating a transgenic plant from the regenerable plant cell after step (a), wherein the transgenic plant comprises in its genome the recombinant DNA construct and exhibits suppression of gene silencing when compared to a control plant not comprising the recombin
  • the method may further comprise (c) obtaining a progeny plant derived from the transgenic plant, wherein said progeny plant comprises in its genome the recombinant DNA construct and exhibits suppression of gene silencing, when compared to a control plant not comprising the recombinant DNA construct.
  • a method of increasing gene silencing in a plant comprising: (a) introducing into a regenerable plant cell a suppression DNA constructcomprising at least one regulatory sequence (for example, a promoter functional in a plant; the regulatory sequence may be heterologous) operably linked to all or part of (i) a nucleic acid sequence encoding a polypeptide having an amino acid sequence of at least 50%, 51 %, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61 %, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or
  • step (b) regenerating a transgenic plant from the regenerable plant cell after step (a), wherein the transgenic plant comprises in its genome the suppression DNA construct and exhibits increased gene silencing when compared to a control plant not comprising the suppression DNA construct.
  • the method may further comprise
  • a method of increasing gene silencing in a plant comprising: (a) introducing into a regenerable plant cell a suppression DNA construct comprising at least one regulatory sequence (for example, a promoter functional in a plant; the regulatory sequence may be heterologous) operably linked to a region derived from all or part of a sense strand or antisense strand of a target gene of interest, said region having a nucleic acid sequence of at least 50%, 51 %, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61 %, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%,
  • a method of selecting for (or identifying) suppression of gene silencing in a plant comprising (a) obtaining a transgenic plant, wherein the transgenic plant comprises in its genome a recombinant DNA construct comprising a polynucleotide operably linked to at least one regulatory sequence (for example, a promoter functional in a plant; the regulatory sequence may be heterologous), wherein said polynucleotide encodes a polypeptide having an amino acid sequence of at least 50%, 51 %, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61 %, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%,
  • a method of selecting for (or identifying) suppression of gene silencing in a plant comprising: (a) obtaining a transgenic plant, wherein the transgenic plant comprises in its genome a recombinant DNA construct comprising a polynucleotide operably linked to at least one regulatory element, wherein said polynucleotide encodes a polypeptide having an amino acid sequence of at least 50%, 51 %, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61 %, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 9
  • a method of selecting for (or identifying) suppression of gene silencing in a plant comprising: (a) obtaining a transgenic plant, wherein the transgenic plant comprises in its genome a recombinant DNA construct comprising a polynucleotide operably linked to at least one regulatory element, wherein said polynucleotide comprises a nucleotide sequence, wherein the nucleotide sequence is: (i) hybridizable under stringent conditions with a DNA molecule comprising the full complement of SEQ ID NO:3, 4, 6, 7, 12, 14, 16, 18, 20, 22, 24 or 26; or (ii) derived from SEQ ID NO:3, 4, 6, 7, 12, 14, 16, 18, 20, 22, 24 or 26 by alteration of one or more nucleotides by at least one method selected from the group consisting of: deletion, substitution, addition and insertion; and (b) selecting (or identifying) a progeny plant of the transgenic plant of step (a), wherein the progeny plant comprises in its genome the recombinant DNA construct
  • a method of selecting for (or identifying) increased gene silencing in a plant comprising (a) obtaining a transgenic plant, wherein the transgenic plant comprises in its genome a suppression DNA construct comprising at least one regulatory sequence (for example, a promoter functional in a plant; the regulatory sequence may be heterologous) operably linked to all or part of (i) a nucleic acid sequence encoding a polypeptide having an amino acid sequence of at least 50%, 51 %, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61 %, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%,
  • a method of selecting for (or identifying) increased gene silencing in a plant comprising (a) obtaining a transgenic plant, wherein the transgenic plant comprises in its genome a suppression DNA construct comprising at least one regulatory sequence (for example, a promoter functional in a plant; the regulatory sequence may be heterologous) operably linked to a region derived from all or part of a sense strand or antisense strand of a target gene of interest, said region having a nucleic acid sequence of at least 50%, 51 %, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61 %, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %,
  • step (b) selecting (or identifying) a progeny plant derived from the transgenic plant of step (a), wherein the progeny plant comprises in its genome the suppression DNA construct and wherein the progeny plant exhibits increased gene silencing compared to a control plant not comprising the suppression DNA construct.
  • a method of producing seed comprising any of the preceding methods, and further comprising obtaining seeds from said progeny plant, wherein said seeds comprise in their genome said recombinant DNA construct (or suppression DNA construct).
  • said regenerable plant cell may comprise a callus cell, an embryogenic callus cell, a gametic cell, a meristematic cell, or a cell of an immature embryo.
  • the regenerable plant cells may derive from an inbred maize plant.
  • said regenerating step may comprise the following: (i) culturing said transformed plant cells in a media comprising an embryogenic promoting hormone until callus organization is observed; (ii) transferring said transformed plant cells of step (i) to a first media which includes a tissue organization promoting hormone; and (iii) subculturing said transformed plant cells after step (ii) onto a second media, to allow for shoot elongation, root development or both.
  • a plant comprising in its genome a recombinant DNA construct comprising a polynucleotide operably linked to at least one heterologous regulatory element, wherein said polynucleotide encodes a polypeptide having an amino acid sequence of at least 50%, 51 %, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61 %, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, based on the Clustal W method of alignment, when compared to SEQ ID NO:5, 8, 13, 15, 17, 19, 21
  • the at least one heterologous regulatory element may be a tissue-specific promoter.
  • the plant may be selected from the group consisting of: Arabidopsis, maize, soybean, sunflower, sorghum, canola, wheat, alfalfa, cotton, rice, barley, millet, sugar cane and switchgrass.
  • a seed comprising in its genome a recombinant DNA construct comprising a polynucleotide operably linked to at least one heterologous regulatory element, wherein said polynucleotide encodes a polypeptide having an amino acid sequence of at least 50%, 51 %, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61 %, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, based on the Clustal W method of alignment, when compared to SEQ ID NO:5, 8, 13, 15, 17, 19, 21
  • a method of suppressing gene silencing in a plant comprising: (a)
  • a recombinant DNA construct comprising a polynucleotide operably linked to at least one heterologous regulatory sequence, wherein the polynucleotide encodes a polypeptide having an amino acid sequence of at least 50%, 51 %, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61 %, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, based on the Clustal W method of alignment, when compared to SEQ ID NO:5, 8, 13, 15, 17, 19, 21
  • transgenic plant comprising: (a) obtaining a transgenic plant, wherein the transgenic plant comprises in its genome a recombinant DNA construct comprising a polynucleotide operably linked to at least one heterologous regulatory element, wherein said polynucleotide encodes a polypeptide having an amino acid sequence of at least 50%, 51 %, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61 %, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, based on the Clustal W method
  • the at least one heterologous regulatory element may be a tissue-specific promoter.
  • the plant may be selected from the group consisting of: Arabidopsis, maize, soybean, sunflower, sorghum, canola, wheat, alfalfa, cotton, rice, barley, millet, sugar cane and switchgrass.
  • a plant comprising in its genome an endogenous polynucleotide operably linked to at least one heterologous regulatory element, wherein said endogenous polynucleotide encodes a polypeptide having an amino acid sequence of at least 50%, 51 %, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61 %, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, based on the Clustal W method of alignment, when compared to SEQ ID NO:5, 8, 13, 15, 17, 19, 21 , 23, 25 or 27, and
  • the at least one heterologous regulatory element may be a tissue-specific promoter.
  • the plant may be selected from the group consisting of: Arabidopsis, maize, soybean, sunflower, sorghum, canola, wheat, alfalfa, cotton, rice, barley, millet, sugar cane and switchgrass.
  • a method of producing a transgenic plant with an increase in gene silencing comprising the steps of: (a) introducing into a regenerable plant cell a recombinant DNA construct comprising an isolated polynucleotide operably linked, in sense or antisense orientation, to a promoter functional in a plant, wherein the polynucleotide comprises: (i) the nucleotide sequence of SEQ ID NO:3, 4, 6, 7, 12, 14, 16, 18, 20, 22, 24 or 26; (ii) a nucleotide sequence with at least 90% sequence identity, based on the Clustal W method of alignment, when compared to SEQ ID NO:3, 4, 6, 7, 12, 14, 16, 18, 20, 22, 24 or 26; (iii) a nucleotide sequence of at least 100 contiguous nucleotides of SEQ ID NO:3, 4, 6, 7, 12, 14, 16, 18, 20, 22, 24 or 26; (iv) a nucleotide sequence that can hybridize under stringent conditions with the nucleotide sequence of (i); or
  • Arabidopsis tomato, maize, soybean, sunflower, sorghum, canola, wheat, alfalfa, cotton, rice, barley, millet, sugar cane and switchgrass.
  • a plant comprising in its genome a recombinant DNA construct comprising an isolated polynucleotide operably linked, in sense or antisense orientation or both, to a promoter functional in a plant, wherein the polynucleotide comprises: (a) the nucleotide sequence of SEQ ID NO:3, 4, 6, 7, 12, 14, 16, 18, 20, 22, 24 or 26; (b) a nucleotide sequence with at least 90% sequence identity, based on the Clustal W method of alignment, when compared to SEQ ID NO:3, 4, 6, 7, 12, 14, 16, 18, 20, 22, 24 or 26; (c) a nucleotide sequence of at least 100 contiguous nucleotides of SEQ ID NO:3, 4, 6, 7, 12, 14, 16, 18, 20, 22, 24 or 26; (d) a nucleotide sequence that can hybridize under stringent conditions with the nucleotide sequence of (a); or (e) a modified plant miRNA precursor, wherein the precursor has been modified to replace the miRNA encoding region with a sequence designed
  • a seed comprising in its genome a recombinant DNA construct comprising an isolated polynucleotide operably linked, in sense or antisense orientation, to a promoter functional in a plant, wherein the polynucleotide comprises: (a) the nucleotide sequence of SEQ ID NO:3, 4, 6, 7, 12, 14, 16, 18, 20, 22, 24 or 26; (b) a nucleotide sequence with at least 90% sequence identity, based on the Clustal W method of alignment, when compared to SEQ ID NO:3, 4, 6, 7, 12, 14, 16, 18, 20, 22, 24 or 26; (c) a nucleotide sequence of at least 100 contiguous nucleotides of SEQ ID NO:3, 4, 6, 7, 12, 14, 16, 18, 20, 22, 24 or 26; (d) a nucleotide sequence that can hybridize under stringent conditions with the nucleotide sequence of (a); or (e) a modified plant miRNA precursor, wherein the precursor has been modified to replace the miRNA encoding region with a sequence designed to produce
  • a recombinant DNA construct for producing a plant that exhibits suppression of gene silencing when compared to a control plant not comprising said recombinant DNA construct
  • the recombinant DNA construct comprises a polynucleotide operably linked to at least one heterologous regulatory element, wherein the polynucleotide encodes a polypeptide having an amino acid sequence of at least 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, based on the Clustal V or the Clustal W method of alignment, using the respective default parameters, when compared to SEQ ID NO:5, 8, 13, 15, 17, 19, 21 , 23, 25 or 27.
  • the polypeptide may be over- expressed in at least one tissue of the plant, or during at least one condition of abiotic stress, or both.
  • the plant may be selected from the group consisting of: maize, soybean, sunflower, sorghum, canola, wheat, alfalfa, cotton, rice, barley, millet, sugar cane and switchgrass.
  • a method of producing a plant that exhibits suppression of gene silencing comprises: (a) obtaining a seed comprising a recombinant DNA construct comprising a polynucleotide operably linked to at least one heterologous regulatory element, wherein the polynucleotide encodes a polypeptide having an amino acid sequence of at least 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, based on the Clustal V or the Clustal W method of alignment, using the respective default parameters, when compared to SEQ ID NO:5, 8, 13, 15, 17, 19, 21 , 23, 25 or 27; and (b) growing a plant from the seed of step (a) wherein the plant exhibits suppression of gene silencing, when compared to a control plant not comprising the recombinant DNA construct.
  • the polypeptide may be over-expressed in at least one tissue of the plant, or during at least one condition of abiotic stress, or both.
  • the plant may be selected from the group consisting of: maize, soybean, sunflower, sorghum, canola, wheat, alfalfa, cotton, rice, barley, millet, sugar cane and switchgrass.
  • a method of producing seed comprising: (a) crossing a first plant with a second plant, wherein at least one of the first plant and the second plant comprises a recombinant DNA construct comprising a polynucleotide operably linked to at least one heterologous regulatory element, wherein the polynucleotide encodes a polypeptide having an amino acid sequence of at least 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, based on the Clustal V or the Clustal W method of alignment, using the respective default parameters, when compared to SEQ ID NO:5, 8, 13, 15, 17, 19, 21 , 23, 25 or 27; and (b) selecting a seed of the cross of step (a), wherein the seed comprises the recombinant DNA construct.
  • a plant grown from the seed may exhibit suppression of gene silencing, when compared to a control plant not comprising the recombinant DNA construct.
  • the polypeptide may be over- expressed in at least one tissue of the plant, or during at least one condition of abiotic stress, or both.
  • the plant may be selected from the group consisting of: maize, soybean, sunflower, sorghum, canola, wheat, alfalfa, cotton, rice, barley, millet, sugar cane and switchgrass.
  • a method of producing oil or a seed by-product, or both, from a seed comprising extracting oil or a seed by-product, or both, from a seed that comprises a recombinant DNA construct comprising a polynucleotide operably linked to at least one heterologous regulatory element, wherein the polynucleotide encodes a polypeptide having an amino acid sequence of at least 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, based on the Clustal V or the Clustal W method of alignment, using the respective default parameters, when compared to SEQ ID NO:5, 8, 13, 15, 17, 19, 21 , 23, 25 or 27.
  • the seed may be obtained from a plant that comprises the recombinant DNA construct, wherein the plant exhibits suppression of gene silencing, when compared to a control plant not comprising the recombinant DNA construct.
  • the polypeptide may be over-expressed in at least one tissue of the plant, or during at least one condition of abiotic stress, or both.
  • the plant may be selected from the group consisting of: maize, soybean, sunflower, sorghum, canola, wheat, alfalfa, cotton, rice, barley, millet, sugar cane and switchgrass.
  • the oil or the by-product, or both, may comprise the recombinant DNA construct.
  • Seed by-products include but are not limited to the following: meal, lecithin, gums, free fatty acids, pigments, soap, stearine, tocopherols, sterols and volatiles.
  • An isolated polynucleotide comprising: (a) a nucleotide sequence encoding a polypeptide with suppression of gene silencing activity, wherein the polypeptide has an amino acid sequence of at least 80% sequence identity when compared to SEQ ID NO:25 or 27, based on the Clustal W method of alignment; or (b) the full complement of the nucleotide sequence of (a).
  • the polypeptide may have a GW-like motif, a RAWUL domain, or both.
  • the polynucleotide may encode a polypeptide comprising the amino acid sequence of SEQ ID NO:25 or 27.
  • the polynucleotide may comprise the nucleotide sequence of SEQ ID NO:24 or 26.
  • the polynucleotide may be present in a recombinant DNA construct, wherein the polynucleotide is operably linked to at least one heterologous regulatory sequence.
  • a recombinant DNA construct for producing a plant that exhibits in increase of gene silencing, when compared to a control plant not comprising said recombinant DNA construct wherein the recombinant DNA construct comprises a polynucleotide operably linked to at least one heterologous regulatory element, wherein the polynucleotide comprises: (a) the nucleotide sequence of SEQ ID NO:3, 4, 6, 7, 12, 14, 16, 18, 20, 22, 24 or 26; (b) a nucleotide sequence with at least 90% sequence identity, based on the Clustal W method of alignment, when compared to SEQ ID NO:3, 4, 6, 7, 12, 14, 16, 18, 20, 22, 24 or 26; (c) a nucleotide sequence of at least 100 contiguous nucleotides of SEQ ID NO:3, 4, 6, 7, 12, 14, 16, 18, 20, 22, 24 or 26; (d) a nucleotide sequence that can hybridize under stringent conditions with the nucleotide sequence of (a); or (e)
  • a regulatory sequence such as one or more enhancers, optionally as part of a transposable element
  • recombinant DNA constructs of the present disclosure into plants may be carried out by any suitable technique, including but not limited to direct DNA uptake, chemical treatment, electroporation, microinjection, cell fusion, infection, vector-mediated DNA transfer, bombardment, or Agrobacterium-mediated transformation.
  • suitable technique including but not limited to direct DNA uptake, chemical treatment, electroporation, microinjection, cell fusion, infection, vector-mediated DNA transfer, bombardment, or Agrobacterium-mediated transformation.
  • the development or regeneration of plants containing the foreign, exogenous isolated nucleic acid fragment that encodes a protein of interest is well known in the art.
  • the regenerated plants may be self-pollinated to provide homozygous transgenic plants. Otherwise, pollen obtained from the regenerated plants is crossed to seed-grown plants of agronomically important lines. Conversely, pollen from plants of these important lines is used to pollinate regenerated plants.
  • a transgenic plant of the present disclosure containing a desired polypeptide is cultivated using methods well known to one skilled in the art.
  • pHSbarENDs An 18.4-kb T-DNA based binary construct was created, pHSbarENDs, that contains four multimerized enhancer elements (SEQ ID NO:1 ) derived from the
  • the construct also contains vector sequences (pUC9) to allow plasmid rescue, transposon sequences (Ds) to remobilize the T-DNA, and the bar gene to allow for glufosinate selection of transgenic plants.
  • pUC9 vector sequences
  • Ds transposon sequences
  • the bar gene to allow for glufosinate selection of transgenic plants.
  • RB right border
  • LB left border
  • pHSbarENDs were substituted with the Pad restriction site at position 5775 with the following poly-linker (SEQ ID NO:2):
  • Activation Tagging construct were screened for rosette leaf phenotypes.
  • the gwar1 -D mutant was identified in this screen, due to its curled and serrated leaf phenotype.
  • DNA from the gwar1 -D mutant was extracted, and genes flanking the T-DNA insert in the mutant line were identified using ligation-mediated PCR (Siebert et al., Nucleic Acids Res. 23:1087-1088 (1995)).
  • a single amplified fragment was identified that contained a T-DNA border sequence and Arabidopsis genomic sequence.
  • a candidate gene was identified by alignment to the sequence of the Arabidopsis genome. Specifically, the annotated gene nearest the 35S enhancer elements/T- DNA RB was the candidate for the gene that is activated in the line.
  • the gene nearest the 35S enhancers was At5g13250, coding for an unknown protein. Because of the presence of a GW-like motif and a RAWUL domain in the At5g13250 protein, we designated this gene as "GWAR1 ".
  • CDS GWAR1 coding sequence
  • the GWAR2 CDS (SEQ ID NO:7) was amplified by RT-PCR with the following: (1 ) a forward primer that introduces the attB1 sequence
  • the vector also contains the RD29a promoter driving expression of the gene for ZS-Yellow (INVITROGENTM), which confers yellow fluorescence to transformed seed.
  • INVITROGENTM ZS-Yellow
  • a MultiSite GATEWAY® LR Recombination Reaction was performed on the entry clone containing the directionally cloned PCR product and pBC-yellow. This amplification allowed for rapid and directional cloning of the candidate gene behind the 35S promoter in pBC-yellow to create the 35S-GWAR1 and 35S-GWAR2 expression constructs.
  • the 35S-GWAR1 and 35S-GWAR2 expression constructs were introduced into wild-type Arabidopsis ecotype Col-0, using the same Agrobacterium-mediated transformation procedure described in Example 1 .
  • Transgenic T1 seeds were selected by yellow fluorescence, and representative T1 lines were selfed to generate a T2 population.
  • T2 individuals were selected and selfed to allow testing for homozygosity of the transgene in the T3 generation. Lines in which either the 35S-GWAR1 or the 35S-GWAR2 transgene was homozygous were identified, and seed of each homozygous line was bulked.
  • the shoots of individual F1 plants were incubated in a solution of 50 mM NaPO 4 pH 7.0, containing 5 mM EDTA, 0.05% Triton X-100, 0.5 mM K4[Fe(CN) 6 ], 0.5 mM K 3 [Fe(CN) 6 ] and 20 mg/ml X-Gluc dissolved in N,N- Dimethylformamide for 12 to 48 hours at 37 °C.
  • F1 and L1 GUS-treated plants were washed once with 50 mM NaPO 4 pH 7.0, followed by three changes of 70% ethanol at 65 °C by 20 min, and several at room temperature.
  • Sequences homologous to the Arabidopsis GWAR polypeptides can be identified using sequence comparison algorithms such as BLAST (Basic Local Alignment Search Tool; Altschul et al., J. Mol. Biol. 215:403-410 (1993); see also the explanation of the BLAST algorithm on the world wide web site for the National Center for Biotechnology Information at the National Library of Medicine of the National Institutes of Health). Sequences encoding homologous GWAR polypeptides can be chemically synthesized or can be PCR-amplified using methods known in the art.
  • BLAST Basic Local Alignment Search Tool
  • Maize plants can be transformed to overexpress an Arabidopsis GWAR polypeptide or the corresponding homologs from various species in order to examine the resulting phenotype.
  • a GATEWAY® entry clone can be used to directionally clone each gene into a maize transformation vector.
  • Expression of the gene in the maize transformation vector can be under control of a constitutive promoter such as the maize ubiquitin promoter (Christensen et al ., (1989) Plant Mol. Biol. 12:619-632 and Christensen et al., (1992) Plant Mol. Biol. 18:675-689)
  • the recombinant DNA construct described above can then be introduced into corn cells by particle bombardment.
  • Techniques for corn transformation by particle bombardment have been described in International Patent Publication WO
  • Agrobacterium-mediated transformation of maize can be performed essentially as described by Zhao et al. in Meth. Mol. Biol. 318:315-323 (2006) (see also Zhao et al., Mol. Breed. 8:323-333 (2001 ) and U.S. Patent No.
  • Overexpression of GWAR Polypeptides in Soybean Soybean plants can be transformed to overexpress an Arabidopsis GWAR polypeptide or the corresponding homologs from various species in order to examine the resulting phenotype.
  • a GATEWAY® entry clone can be used to directionally clone each gene into the PHP27840 vector (PCT Publication No. WO/2012/058528) such that expression of the gene is under control of the SCP1 promoter (International Publication No. 03/033651 ). Soybean embryos may then be transformed with the expression vector comprising sequences encoding the instant polypeptides. Techniques for soybean transformation and regeneration have been described in International Patent Publication WO 2009/006276, the contents of which are herein incorporated by reference.

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Abstract

L'invention concerne des polynucléotides et des polypeptides isolés et des constructions d'ADN recombiné utiles pour obtenir une suppression du silençage génique. L'invention concerne également des compositions (telles que des plantes ou des graines) comprenant ces constructions d'ADN recombiné, ainsi que des procédés qui utilisent ces constructions d'ADN recombiné. Lesdites constructions d'ADN recombiné contiennent un promoteur, fonctionnel dans une plante, lié fonctionnellement à un polynucléotide qui code pour un polypeptide GWAR.
PCT/US2014/046605 2013-07-15 2014-07-15 Suppression du silençage génique par des protéines gwar WO2015009666A1 (fr)

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Publication number Priority date Publication date Assignee Title
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