[go: up one dir, main page]

WO2014150879A1 - Compositions et procédés pour améliorer le nombre de semences végétales et/ou le rendement d'une plante - Google Patents

Compositions et procédés pour améliorer le nombre de semences végétales et/ou le rendement d'une plante Download PDF

Info

Publication number
WO2014150879A1
WO2014150879A1 PCT/US2014/024447 US2014024447W WO2014150879A1 WO 2014150879 A1 WO2014150879 A1 WO 2014150879A1 US 2014024447 W US2014024447 W US 2014024447W WO 2014150879 A1 WO2014150879 A1 WO 2014150879A1
Authority
WO
WIPO (PCT)
Prior art keywords
plant
gene
promoter
seq
expression cassette
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/US2014/024447
Other languages
English (en)
Inventor
Jim Roberts
Robin GUILFOIL
Paul Olivier
Carolyn Hutcheon
Stefan DITTMAR
George Singletary
Asha Palta
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Targeted Growth Inc
Original Assignee
Targeted Growth Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Targeted Growth Inc filed Critical Targeted Growth Inc
Publication of WO2014150879A1 publication Critical patent/WO2014150879A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

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

Definitions

  • the invention generally relates to methods for increasing crop yield. More specifically, the present invention relates to methods and compositions for increasing plant seed weight, seed number and/or seed size by expressing one or more genes coding for leucine-rich-repeat receptor serine/threonine/tyrosine dual specificity kinase brassinosteroid insensitive 1 (BRI1) or truncated BRI1 (BRIIACT) polypeptide in the plant.
  • BRI1 leucine-rich-repeat receptor serine/threonine/tyrosine dual specificity kinase brassinosteroid insensitive 1
  • BRI1 truncated BRI1
  • the most important trait as a target for crop improvement is yield.
  • Efforts to improve crop yields by developing new plant varieties can be divided into two approaches. One is to reduce crop yield losses by breeding or engineering crop varieties with increased resistance to abiotic stress conditions such as drought, cold, or salt or to biotic stress conditions resulting from pests or disease-causing pathogens. While this approach has value, it does not provide fundamentally improved crop yield in the absence of stress conditions and in fact, such resistance may direct plant resources that otherwise would be available for increased yield in the plant.
  • the second approach is to breed or engineer new crop varieties in which the basic yield capacity is increased.
  • the spatial, temporal, and level of expression of a transgene may be critical to the outcome. Some genes have to be expressed at a specific level, in a specific tissue, during a specific time in order to achieve the desired activity. Thus, there is a great need to identify elements that can be used to achieve strength specificity, spatial specificity, and/or temporal specificity of gene expression.
  • the present invention provides expression cassettes.
  • the expression cassettes comprise a promoter.
  • the expression cassettes comprise a polynucleotide, wherein the promoter and the polynucleotide are operatively linked.
  • the promoter is a plant promoter. In some embodiments, the plant promoter has a preferred expression in one or more certain plant tissues. In some embodiments, the plant promoter is a plant seed-preferred promoter. In some embodiments, the plant seed preferred promoter has expression in the embryo sac, early embryo, early endosperm, aleurone and/or basal endosperm transfer cell layer (BETL). In some embodiments, the plant promoter is embryo sac-specific or preferred. In some embodiments, the plant promoter is embryo-specific or preferred. In some embodiments, the plant promoter is endosperm-specific or preferred. In some embodiments, the plant promoter is aleurone- specific or preferred. In some embodiments, the plant promoter is basal endosperm transfer cell layer (BETL)-specific or preferred.
  • BETL basal endosperm transfer cell layer
  • the expression is at the early stage of seed development.
  • the expression starts about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days or more after pollination.
  • the plant promoter is selected from the group consisting of the promoters derived from Zea mays early endosperm 5 gene (e.g., SEQ ID NO: 1), Zea mays early endosperm 1 gene (e.g., SEQ ID NO: 48), Zea mays early endosperm 2 gene (e.g., SEQ ID NO: 49), GRMZM2G124663 (e.g., SEQ ID NO: 37), GRMZM2G006585 (e.g., SEQ ID NO: 38), GRMZM2G120008 (EMBl) (e.g., SEQ ID NO: 82), GRMZM2G157806 (EMB3) (e.g., SEQ ID NO: 83), GRMZM2G176390 (EMB4) (e.g., SEQ ID NO: 84), GRMZM2G472234 (EMB5) (e.g., SEQ ID NO: 85), GRMZM2G13
  • the plant promoter has at least 80% identity to any one of SEQ ID NOs. 1, 24-50, 72-73, 75-78, and 82-87 and wherein the promoter can drive gene expression in the embryo sac, early embryo, early endosperm, aleurone and/or basal endosperm transfer cell layer (BETL).
  • BETL basal endosperm transfer cell layer
  • the cassettes further comprise an intron.
  • the intron is between the plant promoter and the gene of interest.
  • the cassettes further comprise a gene termination sequence.
  • the polynucleotide can activate the brassmosteroid signaling pathway in a plant when it is expressed in the plant. In some embodiments, the polynucleotide can activate the brassmosteroid signaling pathway when expressed in the plant, with or without the presence of brassmosteroid.
  • the polynucleotide is selected from the group consisting of (1) polynucleotides encoding brassmosteroid insensitive 1 (BRI1) polypeptides; (2) polynucleotides encoding positive regulators of the brassmosteroid signaling pathway such as Brassmosteroid Signaling Kinase (BSK), BSU1 phosphatase and/or homologs, PP2A phosphatase, BZR1 transcription factor, BZR2 transcription factor, atypical bHLH ATBS1 transcription factor; (3) polynucleotides reducing activity of negative regulators of the brassmosteroid signaling pathway such as Brassmosteroid Kinase Inhibitor 1 (BKIl) and/or homologs, Brassmosteroid insensitive 2 (BIN2)/glycogen synthase 3 (GSK3) kinase and/or homologs ; (4) polynucleotides encoding polypeptides that increase level of brassmosteroid or functionally
  • the gene of interest is a gene encoding a brassmosteroid insensitive 1 (BRIl) polypeptide.
  • the BRIl is derived from a dicotyledonous plant. In some embodiments, the BRIl is derived from a monocotyledonous plant. In some embodiments, the BRIl is derived from a cereal crop plant. In some embodiments, the BRIl is derived from maize, rice, wheat, barley, sorghum, pea, tomato, Arabidopsis thaliana, soybean, Strawberry, Brassica napus, potato, or Brachypodium distachyon. In some embodiments, the BRIl is any one of SEQ ID NOs.
  • the plant promoter and the gene of interest are operatively linked.
  • the BRIl polypeptide comprises an amino acid sequence having at least 80% identity to any one of SEQ ID NOs. 15, 17, 19, 21, 23, and 51-58, functional variants thereof, or functional fragments thereof.
  • the functional fragment of BRIl polypeptide comprises an amino acid sequence having at least 80% identity to any one of SEQ ID NOs. 15, 17, 19, 21, 23, and 51-58, wherein the functional fragment of BRIl polypeptide can activate the brassmosteroid signaling pathway when expressed in a plant, with or without the presence of brassmosteroid.
  • the functional fragment of BRIl polypeptide comprises at least amino acids corresponding to the brassmosteroid binding domain and kinase domain of any one of SEQ ID NOs. 15, 17, 19, 21, 23, and 51-58, wherein the functional fragment of BRIl polypeptide can activate the brassmosteroid signaling pathway when expressed in a plant, with or without the presence of brassmosteroid.
  • the functional fragment of BRIl polypeptide comprises at least amino acids corresponding to the kinase domain of any one of SEQ ID NOs. 15, 17, 19, 21, 23, and 51-58, wherein the functional fragment of BRIl polypeptide can activate the brassmosteroid signaling pathway when expressed in a plant, with or without the presence of brassmosteroid.
  • the functional fragment of BRIl polypeptide comprises amino acids having at least 80% identity to amino acids 1 to 1082 of SEQ ID NO: 51.
  • the BRIl polypeptide is encoded by a mutant polynucleotide sequence having at least one mutation compared to any one of SEQ ID NOs. 15, 17, 19, 21, 23, and 51-58, wherein the polynucleotide sequence encodes a BRI1 polypeptide, and wherein the BRI1 polypeptide can activate the brassinosteroid signaling pathway when expressed in a plant, with or without the presence of brassinosteroid.
  • the mutation is within the region corresponding to amino acids 1083 to 1122 of SEQ ID NO: 51.
  • the polypeptide further comprises a tag.
  • the tag is a FLAG tag.
  • the FLAG tag comprises the sequence of SEQ ID NO: 13.
  • the gene of interest is a gene encoding a brassinosteroid insensitive 1 (BRI1) polypeptide variant, or a BRI1 ACT variant, such as CT variant ZmBIRl (SEQ ID NO: 79), Juxtamembrane variant of full-length ZmBRIl protein (SEQ ID NO: 80), or Juxtamembrane variant of ZmBRIl ACT protein (SEQ ID NO: 81).
  • BRI1 ACT variant such as CT variant ZmBIRl (SEQ ID NO: 79), Juxtamembrane variant of full-length ZmBRIl protein (SEQ ID NO: 80), or Juxtamembrane variant of ZmBRIl ACT protein (SEQ ID NO: 81).
  • the plant promoter is heterologous to the gene encoding the BRI1 polypeptide.
  • the intron sequence is heterologous to the plant promoter and/or the BRIl polypeptide.
  • the present invention provides expression vectors.
  • the expression vectors are used for expressing a gene of interest in a plant.
  • the expression vectors comprise any one of the expression cassettes of the present invention as described herein.
  • the present invention provides non-human transgenic cells.
  • the non-human transgenic cells comprise the expression cassette of the present invention as described herein.
  • non-human transgenic cells are selected from the group consisting of plant cells, animal cells, bacterial cells, and fungal cells.
  • the present invention provides organisms comprising the transgenic cell of the present invention.
  • the present invention provides transformed plants, plant parts or plant cells.
  • the transformed plants, plant parts or plant cells comprise the expression cassette of the present invention as described herein.
  • the expression cassette is stably incorporated into the genome of the plants, plant parts, or plant cells.
  • the plant is a dicotyledonous plant.
  • the plant is a monocotyledonous plant.
  • the plant is a crop species.
  • the plant is a cereal crop species.
  • the plant is selected from the group consisting of Zea mays, Oryza sativa, Glycine max, Canola, Hordeum vulgare, Sorghum bicolor, Triticum spp., Solanum tuberosum, Brachypodium distachyon, Pisum sativum, Lycopersicon esculentum, and Fragaria x ananassa.
  • the present invention provides seeds of the transformed plants as described herein.
  • the seeds comprise one or more expression cassettes of the present invention.
  • the present invention further provides methods of increasing seed number and/or yield in a plant compared to a wild-type control plant.
  • the methods comprise incorporating into a plant cell an expression cassette of the present invention.
  • the methods further comprise regenerating a transformed plant from said plant cell.
  • the plant cell is stably transformed with the expression cassette.
  • the expression cassette is incorporated into the plant cell by transformation or homologous recombination.
  • the expression cassettes comprise a maize early endosperm 5 (EEP5) promoter, and a truncated maize BRIl gene.
  • EEP5 maize early endosperm 5
  • the truncated maize BRIl gene encodes a maize BRIl polypeptide that can activate the brassinosteroid signaling pathway, with or without the presence of brassinosteroid.
  • the maize EEP5 promoter comprises SEQ ID NO: 1
  • the maize truncated maize BRIl gene comprises SEQ ID NO: 8.
  • the present invention further provides progeny plants of the plant of the present invention.
  • the present invention further provides methods of producing hybrid seeds.
  • the methods comprise crossing the plant or a progeny plant of the present invention with a different plant of the same species, and harvesting the resultant seed.
  • the present invention further provides methods of breeding plants to produce a plant having an expression cassette of the present invention.
  • the methods comprise i) making a cross between a plant with an expression cassette of the present invention with a second plant to produce an Fl plant.
  • the methods further comprise ii) backcrossing the Fl plant to the second plant.
  • the methods further comprise iii) repeating the backcrossing step to generate a near isogenic or isogenic line, wherein the expression cassette of the present invention is integrated into the genome of the second plant and the near isogenic or isogenic line derived from the second plant with the expression cassette.
  • the present invention further provides methods of expressing a gene encoding a BRI1 polypeptide in a plant.
  • the methods comprise incorporating into a plant cell the expression cassette of the present invention.
  • FIG. 1 depicts recombinant vector TG Zm 131 for expressing Zm BRIIACT gene in maize.
  • the Zm BRIIACT gene is driven by the Os Actin promoter.
  • the Zm ADH1 intron was introduced right before the translation initiation site.
  • FIG. 2 depicts recombinant vector TG Zm 132 for expressing Zm BRIlACT-flag gene in maize.
  • the Zm BRIlACT-flag gene is driven by the Os Actin promoter.
  • the Zm ADH1 intron was introduced right before the translation initiation site.
  • FIG. 3 depicts recombinant vector TG Zm 133 for expressing Zm BRIlACT-flag in maize.
  • the Zm BRIlACT-flag gene is driven by the Zm Legumin I A promoter.
  • FIG. 4 depicts recombinant vector TG Zm 135 for expressing Zm BRIlACT-flag in maize.
  • the Zm BRIlACT-flag gene is driven by the Hv LTP2 promoter.
  • FIG. 5 depicts recombinant vector TG Zm 149 for expressing Zm BRIlACT-flag in maize.
  • the Zm BRIlACT-flag gene is driven by the Zm EEP5 promoter.
  • FIG. 6 depicts recombinant vector TG Zm 150 for expressing Zm BRIlACT-flag in maize.
  • the Zm BRIlACT-flag gene is driven by the Zm LEC1 promoter.
  • FIG. 7 depicts recombinant vector TG Zm 220 for expressing Zm BRIlACT-flag in maize.
  • the Zm BRIlACT-flag gene is driven by the Zm OLE promoter.
  • FIG. 9 depicts the percent ear grain weight of the Null Plants (i.e., control plants without the expression cassette of the present invention) compared to ear grain weight of the transgenic plants for TG Zm 131, 132, 133, 135, 149 and 150 events in year 1 isolated crossing block (ICB) trials.
  • Each symbol represents the mean for a single event.
  • Each event includes results at > 3 locations or at 2 locations if the event was tested at least twice at one of the locations.
  • FIG. 12 depicts an overall summary of the effect of TG Zm 149, 150 and 133 constructs on ear grain production, as a percentage ear grain weight of the Null Plants, in year 2. Each symbol is the mean +/- standard error of events tested for each construct. The number of events evaluated for each construct is indicated by the value subtending the standard error bar. Each event includes results at > 3 locations.
  • FIG. 13 depicts an alignment of full-length Brassinosteroid Insensitive 1 (BRI1) polypeptides from Pisum sativum (Ps; SEQ ID NO: 21), Glycine max (Gm; SEQ ID NO: 52), Fragaria x ananassa (Fa; SEQ ID NO: 53), Arabidopsis thaliana (At; SEQ ID NO: 17), Brassica napus (Bn; SEQ ID NO: 54), Lycopersicon esculentum (Le; SEQ ID NO: 19), Solarium tuberosum (St; SEQ ID NO: 55), Zea mays (Zm; SEQ ID NO: 51), Sorghum bicolor (Sb; SEQ ID NO: 56), Hordeum vulgare (Hv; SEQ ID NO: 23), Triticum aestivum (Ta; SEQ ID NO: 57), Brachypodium distachyon (Bd; SEQ ID NO: 58) and Oryza sativa (Os; SEQ ID
  • FIG. 14 depicts the expression cassette TG Gm 38 for expressing At BRIIACT gene in soy.
  • the At BRIIACT gene is driven by the At LEC2 promoter.
  • FIG. 15 depicts recombinant vector TG 68 for expressing At BRIIACT gene in Canola.
  • the At BRIIACT gene is driven by the At LEC2 promoter.
  • FIG. 16 depicts recombinant vector TG 70 for expressing At BRIIACT gene in
  • the At BRIIACT gene is driven by the LFAH12 promoter.
  • FIG. 17 depicts the relative AtBRIlACT gene expression determined by qPCR in 19 DAP canola embryo tissue.
  • Sequence listing for SEQ ID NO. 1-87 is part of this application and is provided at least at the end of the specification.
  • the contents of the text file submitted electronically are incorporated herein by reference in their entirety: A computer readable format copy of the Sequence Listing (filename: TARG-031-01WO_ST25.txt, date recorded: March 11, 2014, file size 428 kilobyte).
  • AtLEC2 promoter 75 AtLEC2 promoter 76 LFAH12 promoter
  • the invention provides compositions and methods to produce plants having increased seed number and/or yield.
  • plant refers to any living organism belonging to the kingdom Plantae (i.e., any genus/species in the Plant Kingdom). This includes familiar organisms such as but not limited to trees, herbs, bushes, grasses, vines, ferns, mosses and green algae.
  • the term refers to both monocotyledonous plants, also called monocots, and dicotyledonous plants, also called dicots.
  • Examples of particular plants include but are not limited to corn, potatoes, roses, apple trees, sunflowers, wheat, rice, bananas, tomatoes, soybeans, peas, Canola, barleys, sorghums, wheats, potatoes, Brachypodium distachyon, strawberries, tobaccos, opo, pumpkins, squash, lettuce, cabbage, oak trees, guzmania, geraniums, hibiscus, clematis, poinsettias, sugarcane, taro, duck weed, pine trees, Kentucky blue grass, zoysia, coconut trees, brassica leafy vegetables (e.g.
  • berries e.g., tomatoes, barberries, currants, elderberries, gooseberries, honeysuckles, mayapples, nannyberries, Oregon-grapes, see-buckthorns, hackberries, bearberries, lingonberries, strawberries, sea grapes, lackberries, cloudberries, loganberries, raspberries, salmonberries, thimbleberries, and wineberries
  • cereal crops e.g., corn, rice, wheat, barley, sorghum, millets, oats, ryes, triticales, buckwheats, fonio, quinoa, oil palm), pome fruit (e.g., apples, pears), stone fruits (e.g., coffees, jujubes, mangos, olives, coconuts, oil palms, pistachios, almonds, apricots, cherries, damsons, nectarines, peaches and plums), vine (e.g.,
  • plant part refers to any part of a plant including but not limited to the embryo, shoot, root, stem, seed, stipule, leaf, petal, flower, ovule, bract, branch, petiole, internode, bark, pubescence, tiller, rhizome, frond, blade, ovule, pollen, stamen, and the like.
  • the two main parts of plants grown in some sort of media, such as soil, are often referred to as the "above-ground” part, also often referred to as the "shoots”, and the "below-ground” part, also often referred to as the "roots”.
  • a or “an” refers to one or more of that entity; for example, "a gene” refers to one or more genes or at least one gene. As such, the terms “a” (or “an”), “one or more” and “at least one” are used interchangeably herein.
  • reference to “an element” by the indefinite article “a” or “an” does not exclude the possibility that more than one of the elements are present, unless the context clearly requires that there is one and only one of the elements.
  • the invention provides isolated, chimeric, recombinant or synthetic polynucleotide sequences.
  • polynucleotide As used herein, the terms “polynucleotide”, “polynucleotide sequence”, “nucleic acid sequence”, “nucleic acid fragment”, and “isolated nucleic acid fragment” are used interchangeably herein. These terms encompass nucleotide sequences and the like.
  • a polynucleotide may be a polymer of R A or DNA that is single- or double-stranded, that optionally contains synthetic, non-natural or altered nucleotide bases.
  • a polynucleotide in the form of a polymer of DNA may be comprised of one or more segments of cDNA, genomic DNA, synthetic DNA, or mixtures thereof.
  • Nucleotides are referred to by a 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.
  • 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
  • chimeric or “recombinant” when describing a nucleic acid sequence or a protein sequence refers to a nucleic acid or a protein sequence that links at least two heterologous polynucleotides or two heterologous polypeptides into a single macromolecule, or that re-arranges one or more elements of at least one natural nucleic acid or protein sequence.
  • the term “recombinant” can refer 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.
  • a "synthetic nucleotide sequence” or “synthetic polynucleotide sequence” is a nucleotide sequence that is not known to occur in nature or that is not naturally occurring. Generally, such a synthetic nucleotide sequence will comprise at least one nucleotide difference when compared to any other naturally occurring nucleotide sequence. It is recognized that a genetic regulatory element of the present invention comprises a synthetic nucleotide sequence. In some embodiments, the synthetic nucleotide sequence shares little or no extended homology to natural sequences. Extended homology in this context generally refers to 100% sequence identity extending beyond about 25 nucleotides of contiguous sequence. A synthetic genetic regulatory element of the present invention comprises a synthetic nucleotide sequence.
  • an "isolated” or “purified” nucleic acid molecule or polynucleotide, or biologically active portion thereof is substantially or essentially free from components that normally accompany or interact with the nucleic acid molecule or polynucleotide as found in its naturally occurring environment.
  • an isolated or purified nucleic acid molecule or polynucleotide is substantially free of other cellular material or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized.
  • nucleic acid refers to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides, or analogs thereof. This term refers to the primary structure of the molecule, and thus includes double- and single-stranded DNA, as well as double- and single-stranded RNA. It also includes modified nucleic acids such as methylated and/or capped nucleic acids, nucleic acids containing modified bases, backbone modifications, and the like.
  • nucleic acid and nucleotide sequence are used interchangeably.
  • genes comprising the isolated (e.g., wild type, endogenous to the organism), chimeric, recombinant or synthetic genes.
  • gene refers to any segment of DNA associated with a biological function.
  • genes include, but are not limited to, coding sequences and/or the regulatory sequences required for their expression.
  • Genes can also include nonexpressed DNA segments that, for example, form recognition sequences for other proteins.
  • Genes can be obtained from a variety of sources, including cloning from a source of interest or synthesizing from known or predicted sequence information, and may include sequences designed to have desired parameters.
  • the invention provides homologous and orthologous polynucleotides and polypeptides.
  • the term “homologous” or “homologue” or “ortholog” is known in the art and refers to related sequences that share a common ancestor or family member and are determined based on the degree of sequence identity.
  • the terms “homology”, “homologous”, “substantially similar” and “corresponding substantially” are used interchangeably herein. They refer to nucleic acid fragments wherein changes in one or more nucleotide bases do not affect the ability of the nucleic acid fragment to mediate gene expression or produce a certain phenotype.
  • a functional relationship may be indicated in any one of a number of ways, including, but not limited to: (a) degree of sequence identity and/or (b) the same or similar biological function. Preferably, both (a) and (b) are indicated.
  • the degree of sequence identity may vary, but in some embodiments, is at least 50% (when using standard sequence alignment programs known in the art), at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%>, at least about 97%, at least about 98%>, or at least 98.5%, or at least about 99%, or at least 99.5%, or at least 99.8%, or at least 99.9%.
  • Homology can be determined using software programs readily available in the art, such as those discussed in Current Protocols in Molecular Biology (F.M. Ausubel et al., eds., 1987) Supplement 30, section 7.718, Table 7.71.
  • Some alignment programs are MacVector (Oxford Molecular Ltd, Oxford, U.K.), ALIGN Plus (Scientific and Educational Software, Pennsylvania) and AlignX (Vector NTI, Invitrogen, Carlsbad, CA).
  • Another alignment program is Sequencher (Gene Codes, Ann Arbor, Michigan), using default parameters. Where a particular sequence is said to have a specific percent identity to a reference sequence of a defined length, the percent identity is relative to the reference sequence.
  • a sequence that is 50% identical to a referenc sequence that is 100 amino acids (or 100 nucleotides long) can be a 50 amino acid polypeptide or a 50 nucleotide sequence that is completely identical to a 50 amino acid long portion of the reference polypeptide or a 50 nucleotides long portion of the reference nucleotide sequence. It might also be a 100 amino acid long polypeptide, or a 100 nucleotide sequence, which is 50% identical to the reference polypeptide or the reference nucleotide seqeunce over its entire length. Of course, other sequences unspecified may also meet the same criteria.
  • the invention provides polynucleotides with nucleotide change when compared to a wild-type reference sequence.
  • nucleotide change refers to, e.g., nucleotide substitution, deletion, and/or insertion, as is well understood in the art.
  • mutations contain alterations that produce silent substitutions, additions, or deletions, but do not alter the properties or activities of the encoded protein or how the proteins are made.
  • the invention provides polypeptides with protein modification when compared to a wild-type reference sequence.
  • protein modification refers to, e.g., amino acid substitution, amino acid modification, deletion, and/or insertion, as is well understood in the art.
  • the invention provides polynucleotides and polypeptides derived from wild-type reference sequences.
  • the term "derived from” refers to the origin or source, and may include naturally occurring, recombinant, unpurified, or purified molecules.
  • a nucleic acid or an amino acid derived from an origin or source may have all kinds of nucleotide changes or protein modification as defined elsewhere herein.
  • the term "agent”, as used herein, means a biological or chemical compound such as a simple or complex organic or inorganic molecule, a peptide, a protein or an oligonucleotide that modulates the function of a nucleic acid or polypeptide.
  • a vast array of compounds can be synthesized, for example oligomers, such as oligopeptides and oligonucleotides, and synthetic organic and inorganic compounds based on various core structures, and these are also included in the term "agent".
  • various natural sources can provide compounds for screening, such as plant or animal extracts, and the like. Compounds can be tested singly or in combination with one another.
  • the invention provides portions or fragments of the nucleic acid sequences and polypeptide sequences of the present invention.
  • the term "at least a portion” or “fragment” of a nucleic acid or polypeptide means a portion having the minimal size characteristics of such sequences, or any larger fragment of the full length molecule, up to and including the full length molecule.
  • fragments of a nucleotide sequence may range from at least about 10 nucleotides, at least about 15 nucleotides, at least about 20 nucleotides, at least about 30 nucleotides, at least about 40 nucleotides, at least about 50 nucleotides, at least about 60 nucleotides, at least about 70 nucleotides, at least about 80 nucleotides, at least about 90 nucleotides, at least about 100 nucleotides, at least about 150 nucleotides, at least about 200 nucleotides, at least about 250 nucleotides, at least about 300 nucleotides, at least about 350 nucleotides, at least about 400 nucleotides, at least about 450 nucleotides, at least about 500 nucleotides, at least about 550 nucleotides, at least about 600 nucleotides, and up to the full-length polynucleotide of the invention.
  • a fragment of a polynucleotide of the invention may encode a biologically active portion of a genetic regulatory element.
  • a biologically active portion of a genetic regulatory element can be prepared by isolating a portion of one of the polynucleotides of the invention that comprises the genetic regulatory element and assessing activity as described herein.
  • a portion of a polypeptide may be 4 amino acids, 5 amino acids, 6 amino acids, 7 amino acids, and so on, going up to the full length polypeptide.
  • the length of the portion to be used will depend on the particular application.
  • a portion of a nucleic acid useful as hybridization probe may be as short as 12 nucleotides; in some embodiments, it is 20 nucleotides.
  • a portion of a polypeptide useful as an epitope may be as short as 4 amino acids.
  • a portion of a polypeptide that performs the function of the full-length polypeptide would generally be longer than 4 amino acids.
  • sequence identity in the context of two nucleic acid or polypeptide sequences includes reference to the residues in the two sequences which are the same when aligned for maximum correspondence over a specified comparison window.
  • percentage of sequence identity is used in reference to proteins it is recognized that residue positions which are not identical often differ by conservative amino acid substitutions, where amino acid residues are substituted for other amino acid residues with similar chemical properties (e.g., charge or hydrophobicity) and therefore do not change the functional properties of the molecule.
  • sequences differ in conservative substitutions
  • percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution.
  • Sequences which differ by such conservative substitutions are said to have "sequence similarity" or “similarity.” Means for making this adjustment are well-known to those of skill in the art. Typically this involves scoring a conservative substitution as a partial rather than a full mismatch, thereby increasing the percentage sequence identity. Thus, for example, where an identical amino acid is given a score of 1 and a non-conservative substitution is given a score of zero, a conservative substitution is given a score between zero and 1. The scoring of conservative substitutions is calculated, e.g., according to the algorithm of Meyers and Miller, Computer Applic. Biol. Sci., 4: 11-17 (1988).
  • the invention provides sequences substantially complementary to the nucleic acid sequences of the present invention.
  • substantially complementary means that two nucleic acid sequences have at least about 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more sequence complementarities to each other.
  • primers and probes must exhibit sufficient complementarity to their template and target nucleic acid, respectively, to hybridize under stringent conditions. Therefore, the primer and probe sequences need not reflect the exact complementary sequence of the binding region on the template and degenerate primers can be used.
  • a non-complementary nucleotide fragment may be attached to the 5'- end of the primer, with the remainder of the primer sequence being complementary to the strand.
  • non-complementary bases or longer sequences can be interspersed into the primer, provided that the primer has sufficient complementarity with the sequence of one of the strands to be amplified to hybridize therewith, and to thereby form a duplex structure which can be extended by polymerizing means.
  • the non-complementary nucleotide sequences of the primers may include restriction enzyme sites. Appending a restriction enzyme site to the end(s) of the target sequence would be particularly helpful for cloning of the target sequence.
  • a substantially complementary primer sequence is one that has sufficient sequence complementarity to the amplification template to result in primer binding and second-strand synthesis. The skilled person is familiar with the requirements of primers to have sufficient sequence complementarity to the amplification template.
  • the invention provides biologically active variants or functional variants of the nucleic acid sequences and polypeptide sequences of the present invention.
  • a biologically active variant or “functional variant” with respect to a protein refers to an amino acid sequence that is altered by one or more amino acids with respect to a reference sequence, while still maintains substantial biological activity of the reference sequence.
  • the variant can have "conservative" changes, wherein a substituted amino acid has similar structural or chemical properties, e.g., replacement of leucine with isoleucine.
  • the following table shows exemplary conservative amino acid substitutions.
  • the variant has one or more amino acid substitutaions, wherein one or more or all subsitutations are acidic amino acid, such as Aspartic acid, Asparagine, Glutamc acid, or Glutamine.
  • a variant can have "nonconservative" changes, e.g., replacement of a glycine with a tryptophan.
  • Analogous minor variations can also include amino acid deletion or insertion, or both. Guidance in determining which amino acid residues can be substituted, inserted, or deleted without eliminating biological or immunological activity can be found using computer programs well known in the art, for example, DNASTAR software.
  • a variant comprises a polynucleotide having deletions (i.e., truncations) at the 5' and/or 3' end; deletion and/or addition of one or more nucleotides at one or more internal sites in the reference polynucleotide; and/or substitution of one or more nucleotides at one or more sites in the reference polynucleotide.
  • a "reference" polynucleotide comprises a nucleotide sequence produced by the methods disclosed herein.
  • Variant polynucleotides also include synthetically derived polynucleotides, such as those generated, for example, by using site directed mutagenesis but which still comprise genetic regulatory element activity.
  • variants of a particular polynucleotide or nucleic acid molecule, or polypeptide of the invention will have at least about 60%, 65%, 70%>, 75%, 80%, 85%, 90%, 91%, 91.5%, 92%, 92.5%, 93%, 93.5%, 94%, 94.5%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%), 99.9%) or more sequence identity to that particular polynucleotide/polypeptides as determined by sequence alignment programs and parameters as described elsewhere herein.
  • Variant polynucleotides also encompass sequences derived from a mutagenic and recombinogenic procedure such as DNA shuffling.
  • Strategies for such DNA shuffling are known in the art. See, for example, Stemmer (1994) PNAS 91 : 10747-10751; Stemmer (1994) Nature 370:389-391; Crameri et al. (1997) Nature Biotech. 15:436-438; Moore et al. (1997) J. Mol. Biol. 272:336-347; Zhang et al. (1997) PNAS 94:4504-4509; Crameri et al. (1998) Nature 391 :288-291; and U.S. Patent Nos.
  • oligonucleotide primers can be designed for use in PCR reactions to amplify corresponding DNA sequences from cDNA or genomic DNA extracted from any plant of interest.
  • Methods for designing PCR primers and PCR cloning are generally known in the art and are disclosed in Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2nd ed., Cold Spring Harbor Laboratory Press, Plainview, New York). See also Innis et al, eds. (1990) FCR Protocols: A Guide to Methods and Applications (Academic Press, New York); Innis and Gelfand, eds.
  • PCR PCR Strategies
  • nested primers single specific primers
  • degenerate primers gene-specific primers
  • vector-specific primers partially-mismatched primers
  • the invention provides primers that are derived from the nucleic acid sequences and polypeptide sequences of the present invention.
  • the term "primer” as used herein refers to an oligonucleotide which is capable of annealing to the amplification target allowing a DNA polymerase to attach, thereby serving as a point of initiation of DNA synthesis when placed under conditions in which synthesis of primer extension product is induced, i.e., in the presence of nucleotides and an agent for polymerization such as DNA polymerase and at a suitable temperature and pH.
  • the (amplification) primer is preferably single stranded for maximum efficiency in amplification.
  • the primer is an oligodeoxyribonucleotide.
  • the primer must be sufficiently long to prime the synthesis of extension products in the presence of the agent for polymerization.
  • the exact lengths of the primers will depend on many factors, including temperature and composition (A/T vs. G/C content) of primer.
  • a pair of bi-directional primers consists of one forward and one reverse primer as commonly used in the art of DNA amplification such as in PCR amplification.
  • the invention provides polynucleotide sequences that can hybridize with the nucleic acid sequences of the present invention.
  • stringency or “stringent hybridization conditions” refer to hybridization conditions that affect the stability of hybrids, e.g., temperature, salt concentration, pH, formamide concentration and the like. These conditions are empirically optimized to maximize specific binding and minimize non-specific binding of primer or probe to its target nucleic acid sequence.
  • the terms as used include reference to conditions under which a probe or primer will hybridize to its target sequence, to a detectably greater degree than other sequences (e.g. at least 2-fold over background). Stringent conditions are sequence dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures.
  • stringent conditions are selected to be about 5° C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH.
  • Tm is the temperature (under defined ionic strength and pH) at which 50% of a complementary target sequence hybridizes to a perfectly matched probe or primer.
  • stringent conditions will be those in which the salt concentration is less than about 1.0 M Na + ion, typically about 0.01 to 1.0 M Na + ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30° C for short probes or primers (e.g. 10 to 50 nucleotides) and at least about 60° C for long probes or primers (e.g. greater than 50 nucleotides).
  • Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide.
  • exemplary low stringent conditions or “conditions of reduced stringency” include hybridization with a buffer solution of 30% formamide, 1 M NaCl, 1% SDS at 37° C and a wash in 2xSSC at 40° C.
  • Exemplary high stringency conditions include hybridization in 50% formamide, 1M NaCl, 1% SDS at 37° C, and a wash in 0.1 xSSC at 60° C. Hybridization procedures are well known in the art and are described by e.g. Ausubel et al, 1998 and Sambrook et al, 2001.
  • stringent conditions are hybridization in 0.25 M Na 2 HP0 4 buffer (pH 7.2) containing 1 mM Na 2 EDTA, 0.5-20% sodium dodecyl sulfate at 45°C, such as 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19% or 20%, followed by a wash in 5xSSC, containing 0.1% (w/v) sodium dodecyl sulfate, at 55°C to 65°C.
  • promoter refers to a DNA sequence capable of controlling the expression of a coding sequence or functional R A.
  • the promoter sequence consists of proximal and more distal upstream elements, the latter elements often referred to as enhancers.
  • an “enhancer” is a DNA sequence that can stimulate promoter activity, and may be an innate element of the promoter or a heterologous element inserted to enhance the level or tissue specificity of a promoter. 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.
  • promoters may direct the expression of a gene in different tissues or cell types, or at different stages of development, or in response to different environmental conditions. It is further recognized that since in most cases the exact boundaries of regulatory sequences have not been completely defined, DNA fragments of some variation may have identical promoter activity.
  • promoter emcompasses any given promoter sequence, as well as functional variants or fragments thereof that still fully or substantially maintain the promoter activity of the given promoter sequence, e.g., still having at least 50%, 60%, 70%, 80%, 90%, 95%, or more, or 100% of the promoter activity of the given promoter sequence.
  • the invention provides plant promoters.
  • a plant promoter As used herein, a
  • plant promoter is a promoter capable of initiating transcription in plant cells whether or not its origin is a plant cell, e.g. it is well known that Agrobacterium promoters are functional in plant cells.
  • plant promoters include promoter DNA obtained from plants, plant viruses and bacteria such as Agrobacterium and Bradyrhizobium bacteria.
  • a plant promoter can be a constitutive promoter or a non-constitutive promoter.
  • the invention provides constitutive promoters.
  • a "constitutive promoter” is a promoter which is active under most conditions and/or during most development stages.
  • constitutive promoters include, CaMV 35 S promoter, opine promoters, ubiquitin promoter, alcohol dehydrogenase promoter, etc.
  • the invention provides non-constitutive promoters.
  • a "non-constitutive promoter” is a promoter which is active under certain conditions, in certain types of cells, and/or during certain development stages.
  • tissue specific, tissue preferred, cell type specific, cell type preferred, inducible promoters, and promoters under development control are non-constitutive promoters.
  • promoters under developmental control include promoters that preferentially initiate transcription in certain tissues, such as stems, leaves, roots, or seeds.
  • the invention provides inducible promoters.
  • inducible or “repressible” promoter is a promoter which is under chemical or environmental factors control. Examples of environmental conditions that may effect transcription by inducible promoters include anaerobic conditions, or certain chemicals, or the presence of light.
  • the invention provides tissue specific promoters.
  • tissue specific is a promoter that initiates transcription only in certain tissues. Unlike constitutive expression of genes, tissue-specific expression is the result of several interacting levels of gene regulation. As such, in the art sometimes it is preferable to use promoters from homologous or closely related plant species to achieve efficient and reliable expression of transgenes in particular tissues. This is one of the main reasons for the large amount of tissue-specific promoters isolated from particular plants and tissues found in both scientific and patent literature.
  • tissue-preferred promoters As used herein, a "tissue preferred" promoter is a promoter that initiates transcription mostly, but not necessarily entirely or solely in certain tissues.
  • the invention provides cell type specific promoters.
  • a "cell type specific" promoter is a promoter that primarily drives expression in certain cell types in one or more organs, for example, vascular cells in roots, leaves, stalk cells, and stem cells.
  • the invention provides cell type preferred promoters.
  • a "cell type preferred" promoter is a promoter that primarily drives expression mostly, but not necessarily entirely or solely in certain cell types in one or more organs, for example, vascular cells in roots, leaves, stalk cells, and stem cells.
  • the invention provides seed-preferred promoters.
  • the seed-preferred promoters have expression in embryo sac, early embryo, early endosperm, aleurone and/or basal endosperm transfer cell layer (BETL).
  • the invention provides basal endosperm transfer cell layer (BETL)-specific/preferred promoters.
  • BETL basal endosperm transfer cell layer
  • Transfer cells cells specialized for a special purpose. Annu Rev Plant Biol 54: 431 ⁇ 154.
  • Transfer cells typically have a dense cytoplasm that is rich in small, spherical mitochondria.
  • Sabelli and Larkins The Development endosperm in Grasses, Plant Physiology, 2009, 149:14-26
  • Thompson et al. 2001, Development and functions of seed transfer cells. Plant Sci. Apr; 160(5):775-783
  • intron is any nucleotide sequence within a gene that is removed by RNA splicing while the final mature RNA product of a gene is being generated.
  • the term refers to both the DNA sequence within a gene, and the corresponding sequence in RNA transcripts.
  • the invention provides recombinant genes comprising 3' non-coding sequences or 3' untranslated regions.
  • the "3' non-coding sequences" or “3' untranslated regions” refer to DNA sequences located downstream of a coding sequence and include polyadenylation recognition sequences and other sequences encoding regulatory signals capable of affecting mRNA processing or gene expression.
  • the polyadenylation signal is usually characterized by affecting the addition of polyadenylic acid tracts to the 3' end of the mRNA precursor.
  • the use of different 3' non-coding sequences is exemplified by Ingelbrecht, I. L., et al. (1989) Plant Cell 1 :671-680.
  • the invention provides recombinant genes in which a gene of interest is operably linked to a promoter sequence.
  • operably linked refers to the association of nucleic acid sequences on a single nucleic acid fragment so that the function of one is regulated by the other.
  • a promoter is operably linked with a coding sequence when it is capable of regulating the expression of that coding sequence (i.e., that the coding sequence is under the transcriptional control of the promoter). Coding sequences can be operably linked to regulatory sequences in a sense or antisense orientation.
  • the complementary R A regions of the invention can be operably linked, either directly or indirectly, 5' to the target m NA, or 3' to the target m NA, or within the target mR A, or a first complementary region is 5' and its complement is 3' to the target mRNA.
  • the invention provides recombinant expression cassettes and recombinant constructs.
  • a recombinant construct comprises an artificial combination of nucleic acid fragments, e.g., regulatory and coding sequences that are not found together in nature.
  • a chimeric 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 found in nature.
  • Such construct may be used by itself or may be used in conjunction with a vector. If a vector is used then the choice of vector is dependent upon the method that will be used to transform host cells as is well known to those skilled in the art.
  • a plasmid vector can be used.
  • the skilled artisan is well aware of the genetic elements that must be present on the vector in order to successfully transform, select and propagate host cells comprising any of the isolated nucleic acid fragments of the invention.
  • the skilled artisan will also recognize that different independent transformation events will result in different levels and patterns of expression (Jones et al, (1985) EMBO J. 4:2411-2418; De Almeida et al, (1989) Mol. Gen. Genetics 218:78-86), and thus that multiple events must be screened in order to obtain lines displaying the desired expression level and pattern.
  • Vectors can be plasmids, viruses, bacteriophages, pro-viruses, phagemids, transposons, artificial chromosomes, and the like, that replicate autonomously or can integrate into a chromosome of a host cell.
  • a vector can also be a naked RNA polynucleotide, a naked DNA polynucleotide, a polynucleotide composed of both DNA and RNA within the same strand, a poly-lysine-conjugated DNA or RNA, a peptide-conjugated DNA or RNA, a liposome-conjugated DNA, or the like, that is not autonomously replicating.
  • expression refers to the production of a functional end-product e.g., an mRNA or a protein (precursor or mature).
  • the expression cassettes or recombinant constructs comprise at least one selectable or screenable marker.
  • the selectable or screenable marker is a plant selectable or screenable marker.
  • plant selectable or screenable marker refers to a genetic marker functional in a plant cell. A selectable marker allows cells containing and expressing that marker to grow under conditions unfavorable to growth of cells not expressing that marker. A screenable marker facilitates identification of cells which express that marker.
  • the invention provides inbred plants comprising recombinant sequences.
  • inbred inbred plant
  • inbred plant is used in the context of the present invention. This also includes any single gene conversions of that inbred.
  • single allele converted plant refers to those plants which are developed by a plant breeding technique called backcrossing wherein essentially all of the desired morphological and physiological characteristics of an inbred are recovered in addition to the single allele transferred into the inbred via the backcrossing technique.
  • sample includes a sample from a plant, a plant part, a plant cell, or from a transmission vector, or a soil, water or air sample.
  • the invention provides offsprings comprising recombinant sequences.
  • the term "offspring” refers to any plant resulting as progeny from a vegetative or sexual reproduction from one or more parent plants or descendants thereof.
  • an offspring plant may be obtained by cloning or selfing of a parent plant or by crossing two parent plants and include selfmgs as well as the Fl or F2 or still further generations.
  • An Fl is a first-generation offspring produced from parents at least one of which is used for the first time as donor of a trait, while offspring of second generation (F2) or subsequent generations (F3, F4, etc.) are specimens produced from selfmgs of Fl's, F2's etc.
  • An Fl may thus be (and usually is) a hybrid resulting from a cross between two true breeding parents (true-breeding is homozygous for a trait), while an F2 may be (and usually is) an offspring resulting from self- pollination of said Fl hybrids.
  • the invention provides methods for crossing a first plant comprising recombinant sequences with a second plant.
  • crossing refers to the process by which the pollen of one flower on one plant is applied (artificially or naturally) to the ovule (stigma) of a flower on another plant.
  • the invention provides plant cultivars comprising recombinant sequences.
  • cultivar refers to a variety, strain or race of plant that has been produced by horticultural or agronomic techniques and is not normally found in wild populations.
  • the present invention provides methods for obtaining plant genotypes comprising recombinant genes.
  • genotype refers to the genetic makeup of an individual cell, cell culture, tissue, organism (e.g., a plant), or group of organisms.
  • the present invention provides homozygotes comprising recombinant genes.
  • homozygote refers to an individual cell or plant having the same alleles at one or more loci.
  • the present invention provides homozygous plants comprising recombinant genes.
  • homozygous refers to the presence of identical alleles at one or more loci in homologous chromosomal segments.
  • the transgenic cell or organism is hemizygous for the gene of interest which is under control of promoters of the present invention.
  • hemizygous refers to a cell, tissue or organism in which a gene is present only once in a genotype, as a gene in a haploid cell or organism, a sex-linked gene in the heterogametic sex, or a gene in a segment of chromosome in a diploid cell or organism where its partner segment has been deleted.
  • the present invention provides heterozygotes comprising recombinant genes.
  • heterozygote and “heterozygous” refer to a diploid or polyploid individual cell or plant having different alleles (forms of a given gene) present at least at one locus.
  • the cell or organism is heterozygous for the gene of interest which is under control of the synthetic regulatory element.
  • heterologous polynucleotide or a “heterologous nucleic acid” or an “exogenous DNA segment” refer to a polynucleotide, nucleic acid or DNA segment that originates from a source foreign to the particular host cell, or, if from the same source, is modified from its original form.
  • a heterologous gene in a host cell includes a gene that is endogenous to the particular host cell, but has been modified.
  • the terms refer to a DNA segment which is foreign or heterologous to the cell, or homologous to the cell but in a position within the host cell nucleic acid in which the element is not ordinarily found. Exogenous DNA segments are expressed to yield exogenous polypeptides.
  • the cell or organism has at least one heterologous trait.
  • heterologous trait refers to a phenotype imparted to a transformed host cell or transgenic organism by an exogenous DNA segment, heterologous polynucleotide or heterologous nucleic acid.
  • Various changes in phenotype are of interest to the present invention, including but not limited to modifying the fatty acid composition in a plant, altering the amino acid content of a plant, altering a plant's pathogen defense mechanism, increasing a plant's yield of an economically important trait (e.g., grain yield, forage yield, etc.) and the like.
  • the invention provides methods for obtaining plant lines comprising recombinant genes.
  • line is used broadly to include, but is not limited to, a group of plants vegetatively propagated from a single parent plant, via tissue culture techniques or a group of inbred plants which are genetically very similar due to descent from a common parent(s).
  • a plant is said to "belong” to a particular line if it (a) is a primary transformant (TO) plant regenerated from material of that line; (b) has a pedigree comprised of a TO plant of that line; or (c) is genetically very similar due to common ancestry (e.g., via inbreeding or selfing).
  • TO primary transformant
  • the term "pedigree” denotes the lineage of a plant, e.g. in terms of the sexual crosses affected such that a gene or a combination of genes, in heterozygous (hemizygous) or homozygous condition, imparts a desired trait to the plant.
  • the invention provides open-pollinated populations comprising recombinant genes.
  • the terms "open-pollinated population” or “open-pollinated variety” refer to plants normally capable of at least some cross-fertilization, selected to a standard, that may show variation but that also have one or more genotypic or phenotypic characteristics by which the population or the variety can be differentiated from others.
  • a hybrid which has no barriers to cross-pollination, is an open-pollinated population or an open-pollinated variety.
  • the invention provides self-pollination populations comprising recombinant genes.
  • self-crossing means the pollen of one flower on one plant is applied (artificially or naturally) to the ovule (stigma) of the same or a different flower on the same plant.
  • the invention provides ovules and pollens comprising recombinant genes.
  • ovule refers to the female gametophyte
  • polyen means the male gametophyte
  • the transgenic plants comprising recombinant genes have one or more preferred phenotypes.
  • phenotype refers to the observable characters of an individual cell, cell culture, organism (e.g., a plant), or group of organisms which results from the interaction between that individual's genetic makeup (i.e., genotype) and the environment.
  • plant tissue refers to any part of a plant.
  • plant organs include, but are not limited to the leaf, stem, root, tuber, seed, branch, pubescence, nodule, leaf axil, flower, pollen, stamen, pistil, petal, peduncle, stalk, stigma, style, bract, fruit, trunk, carpel, sepal, anther, ovule, pedicel, needle, cone, rhizome, stolon, shoot, pericarp, endosperm, placenta, berry, stamen, and leaf sheath.
  • the invention provides methods for obtaining plants comprising recombinant genes through transformation.
  • transformation refers to the transfer of nucleic acid (i.e., a nucleotide polymer) into a cell.
  • genetic transformation refers to the transfer and incorporation of DNA, especially recombinant DNA, into a cell.
  • transformants comprising recombinant genes.
  • transformant refers to a cell, tissue or organism that has undergone transformation.
  • the original transformant is designated as “TO” or “To.”
  • Selfing the TO produces a first transformed generation designated as “Tl” or “Ti.”
  • transgene comprising recombinant promoters.
  • transgene refers to a nucleic acid that is inserted into an organism, host cell or vector in a manner that ensures its function.
  • transgenic plants comprising recombinant promoters.
  • transgenic refers to cells, cell cultures, organisms (e.g., plants), and progeny which have received a foreign or modified gene by one of the various methods of transformation, wherein the foreign or modified gene is from the same or different species than the species of the organism receiving the foreign or modified gene.
  • transgenic events comprising recombinant promoters.
  • transformation event refers to the movement of a transposon from a donor site to a target site.
  • the present invention provides plant varieties comprising recombinant genes.
  • the term "variety" refers to a subdivision of a species, consisting of a group of individuals within the species that are distinct in form or function from other similar arrays of individuals.
  • the invention provides novel nucleotide sequences of genetic regulatory elements. It is recognized that from such nucleotide sequences, a nucleic acid molecule can be synthesized or produced using a number of methods known in the art.
  • producing a nucleic acid molecule is intended to comprise the making of a nucleic acid molecule by any known method including, but not limited to, chemical synthesis of the entire nucleic acid molecule or part or parts thereof, modification of a pre-existing nucleic acid molecule, such as, for example, a DNA molecule comprising a genetic regulatory element of the present invention, by molecular biology methods such as, for example, restriction endonuclease digestion, DNA amplification by polymerase and ligation, and the combination of chemical synthesis and modification.
  • the present invention provides organisms recombinant genes.
  • an "organism” refers any life form that has genetic material comprising nucleic acids including, but not limited to, prokaryotes, eukaryotes, and viruses.
  • Organisms of the present invention include, for example, plants, animals, fungi, bacteria, and viruses, and cells and parts thereof.
  • the invention provides coding sequences of a gene of interest that are operably linked with the promoters of the present invention.
  • coding sequence refers to a DNA sequence that codes for a specific amino acid sequence.
  • gene of interest is intended any nucleotide sequence that can be expressed when operably linked to a promoter.
  • a gene of interest of the present invention may, but need not, encode a protein. Unless stated otherwise or readily apparent from the context, when a gene of interest of the present invention is said to be operably linked to a promoter of the invention, the gene of interest does not by itself comprise a functional promoter.
  • 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.
  • operably linked refers to the association of nucleic acid sequences on a single nucleic acid fragment so that the function of one is regulated by the other.
  • a promoter is operably linked with a coding sequence when it is capable of regulating the expression of that coding sequence (i.e., that the coding sequence is under the transcriptional control of the promoter).
  • Coding sequences can be operably linked to regulatory sequences in a sense or antisense orientation.
  • the complementary RNA regions of the invention can be operably linked, either directly or indirectly, 5' to the target mRNA, or 3' to the target mRNA, or within the target mRNA, or a first complementary region is 5' and its complement is 3' to the target mRNA.
  • the transgenes of the present invention comprise at least one reporter gene.
  • a reporter or a “reporter gene” refers to a nucleic acid molecule encoding a detectable marker.
  • the reporter gene can be, for example, luciferase (e.g., firefly luciferase or Renilla luciferase), GUS ( ⁇ -glucuronidase), ⁇ -galactosidase, chloramphenicol acetyl transferase (CAT), or a fluorescent protein (e.g., green fluorescent protein (GFP), red fluorescent protein (DsRed), yellow fluorescent protein, blue fluorescent protein, cyan fluorescent protein, or variants thereof.
  • GFP green fluorescent protein
  • DsRed red fluorescent protein
  • yellow fluorescent protein blue fluorescent protein
  • cyan fluorescent protein or variants thereof.
  • Reporter genes are detectable by a reporter assay. Reporter assays can measure the level of reporter gene expression or activity by any number of means, including, for example, measuring the level of reporter mRNA, the level of reporter protein, or the amount of reporter protein activity. Reporter assays are known in the art or otherwise disclosed herein.
  • Brassinosteroids and the BR signaling pathway Brassinosteroids and the BR signaling pathway
  • Brassinosteroids are plant steroid hormones that are important for many developmental processes in the plant, including seed germination and dormancy, cell elongation and division, photomorphogenesis, plant architecture, senescence, male fertility, and xylem differentiation (Divi and Krishna (2009) New Biotechnology 26: 131-136; Vriet et al. (2012) The Plant Cell 24: 842-857). BRs have also been implicated in tolerance to biotic and abiotic stresses, such as drought, extreme temperatures, salinity, and pathogen attack. Historically, BRs have been applied to plants exogenously to improve plant growth (Kripach et al. (2000) Ann.Bot. 86: 441-447). However, the expense of synthesizing the hormones together with inconsistent results, have encouraged the exploration of more efficacious routes to influence BR levels in a plant.
  • the level of BRs in a plant is determined by both BR genes involved in the synthesis and catabolism of the hormones and by genes involved in signaling to upregulate or downregulate the synthesis of BRs.
  • the first essential step in the BR pathway is the biosynthesis of steroidal compounds, beginning with campesterol, which through multiple intermediates eventually results in the brassinosteroid hormone brassinolide.
  • Critical biosynthetic enzymes identified in this pathway, listed in the order of action, are DET2 (DWF6, steroid 5 -reductase), DIM1 (DWF1), CYP90B1 (DWF4, cytochrome P450 monooxygenase), CDP (DWF3, 23 -hydroxylase), and ROT3.
  • BR signaling pathway has been elucidated through numerous studies (reviewed in Clouse (2011) The Plant Cell 23: 1219-1230). BRs are perceived at the cell surface by BRASSINOSTEROID INSENSITIVE 1 (BRIl), a member of the large family of leucine-rich repeat receptor-like kinases (LRR-RLKs) found in plants (Belkhadir and Chory (2006) Science 314: 11410-1411). Binding of BR to BRIl leads to phosphorylation and activation of the BRIl kinase domain. Activation of the receptor then leads to a cascade of events that involve other LRR-RLKs, downstream kinases, phosphatases and ultimately, to BR responsive transcription factors that regulate genes controlling a wide spectrum of plant development.
  • LRR-RLKs leucine-rich repeat receptor-like kinases
  • the BRIl receptor kinase is organized into several domains: the extracellular portion of BRIl is responsible for ligand binding and receptor oligomerization; a transmembrane domain makes a single pass through the membrane; an intracellular catalytic kinase domain is flanked by a juxtamembrane domain and a C-terminal domain (Wang et al (2005) Developmental Cell 8: 855-865). The C-terminal domain was determined to negatively regulate BRIl function. A truncation of the C-terminal domain (BRIl ACT) leads to a hypersensitive receptor with enhanced BR signaling (Wang et al 2005).
  • Non-limiting examples of BRIl genes/proteins are listed in the Sequence Listings and Figures of the present application, and also described in US 6245969, US6765085, US 7135625, US2013/0007910, Yamamuro (2000) The Plant Cell 12: 1591-1605 , Chono (2003) Plant Physiology 133: 1209-1219, Li and Chory (1997) Cell 90: 929-938, Montoya (2002) The Plant Cell 14: 3163-3176, Nomura (2003) The Plant Journal 36: 291-300, Chai (2013) Plant Growth Regul. 69(1): 63-69, Holton (2007) Plant Cell 19 (5), 1709-1717, each of which is herein incorporated by reference in its entirety.
  • the brassinosteroid (BR) signaling pathway is defined by positive and negative regulators (Clouse (2011) The Plant Cell 23: 1219-1230 and references therein) and the details of how these players participate in the cascade is being revealed from work in Arabidopsis.
  • the BRIl receptor exists as a homodimer with a basal level of activity.
  • BRIl activity is inhibited in trans by the BRIl kinase inhibitor 1 (BKI1) protein and in cis by its own C-terminal tail (CT).
  • BKI1 BRIl kinase inhibitor 1
  • CT C-terminal tail
  • BKI1 dissociates from the membrane and receptor, BRIl autophosphorylates key serine and threonine residues in the activation loop of its kinase domain, the auto-inhibition of BRIl by its CT domain is alleviated by phosphorylation of serine/threonine residues in the CT region and further auto- phosphorylation of BRIl may occur. Jallais et al.
  • BKI1 Somatic Embryogenesis Receptor Kinase subfamily of leucine-rich repeat receptor-like kinases (LRR-RLKs) like BAK1, BKK1 and SERK1.
  • SERK Somatic Embryogenesis Receptor Kinase
  • LRR-RLKs leucine-rich repeat receptor-like kinases
  • MAKRs Membrane Associated Kinase Regulators
  • BR-signaling kinases (BSK1, BSK2 and BSK3) have been identified as substrates for BRIl and are positive regulators of the BR signaling pathway (Tang W, et al. (2008) Mol Cell Proteomics 7:728-738, Tang W. et al. (2008) Science 321 : 557-560, WO2010/011285).
  • BRIl phosphorylates BSK1 on Ser 230, allowing its release from the BRIl receptor complex and association with the downstream phosphatase, BSU1 (BRIl Suppressor 1).
  • BSK1 BSU1
  • BSU1 is a nuclear-localized serine/threonine phosphatase and has 3 other homologs in Arabidopsis (Mora-Garcia, S. et al. (2004) Genes Dev. 18: 448-460).
  • BSU1 is a positive regulator of the BR signaling pathway because it inactivates a critical negative regulator of the BR signaling cascade, the glycogen synthase 3 (GSK3)-like kinase, BIN2 (Brassinosteroid insensitive 2).
  • GSK3 glycogen synthase 3
  • BIN2 Brassinosteroid insensitive 2
  • BSU1 dephosphorylates BIN2 on Tyr 200, rendering the kinase unable to inactivate the downstream transcription factors responsible for expression of BR-responsive genes (Kim, T.W. et al. (2009) Nat.
  • the GSK3/BIN2 kinase phosphorylates the BZR1 (Brassinazole-resistant 1) and BZR2 (BES1, BRIl EMS suppressor 1) transcription factors (Wang et al. 2002 Developmental Cell, Vol. 2, 505-513, US 6921848).
  • the phosphorylation inactivates the transcription factors through possible mechanisms such as degradation, interaction with 14-3-3 proteins for nucleocytoplasmic shuttling and/or inhibition of DNA binding and dimerization.
  • the inactive phosphorylated BZR1 and BZR2 transcription factors must be dephosphorylated to become active.
  • cytoplasmic protein phosphatase 2 A performs this function (Tang, W. et al. (2011) Nat. Cell Biol. 13: 124-131).
  • PP2A is a positive regulator of BR signaling.
  • the regulatory subunit of PP2A binds a putative PEST (pro-glu- ser-thr) domain in BZR1.
  • PEST pro-glu- ser-thr domain in BZR1.
  • BZR1 and BZR2 Three different types of mutations of BZR1 and BZR2 have been shown to make these transcription factors hypersensitive to the BR-hormone and/or constitutively active.
  • BZR1 binding motifs are found in the promoters of these BR biosynthesis genes, and when bound, BZR1 blocks transcription. This negative feedback pathway is critical for the overall growth and development of the plant.
  • the BZR1 and BZR2 transcription factors are also known to associate with other transcription factors for function.
  • an atypical basic helix-loop-helix (bHLH) transcription factor, ATBS1 is hypothesized to be a positive regulator of BR signaling by heterodimerizing and sequestering other bHLH transcription factors that are negative regulators of BR signaling.
  • BR gene expression has conferred tolerance to various stresses.
  • a knock-out of OsGSKl a protein kinase involved in the negative regulation of BR signaling, gave rice plants with increased tolerance to heat, cold, salt and drought stress (Koh (2007) Plant Mol Biol 65:453-466).
  • Li and coworkers Li (2007) Plant Physiology 145: 87-97) found that expressing a hydroxysteroid dehydrogenase, a gene induced by BR, in Arabidopsis and canola gave tolerance to salt stress and other BR-like effects such as increased plant growth and seed yield.
  • a BR biosynthesis mutant and a BR signaling mutant exhibited hypersensitivity to salt stress (Zeng (2010) J Plant Growth Regulation 29:44-52).
  • BR biosynthesis genes Modification in either BR biosynthesis genes or BR signaling genes also affects seed size and seed yield.
  • OsDWARFl l Mutation in a rice P450 cytochrome involved in BR biosynthesis, CYP724B (OsDWARFl l), reduces seed length (Tanabe (2005) The Plant Cell 17: 776-790), while mutation in another P450 cytochrome, CYP90B2 (OsDWARF4), gives increases in above-ground biomass and grain yield under dense planting conditions (Sakamoto (2006) Nature Biotechnology 24: 105-109).
  • the Os dwf4 mutant has erect leaves, which were hypothesized to increase access to light for lower leaves with a concomitant increase in photosynthesis for greater carbohydrate synthesis and partitioning to seed.
  • BR biosynthesis or BR signaling genes have been mostly constitutive. There are only a few instances of targeting BR biosynthesis or signaling genes with tissue-specific promoters. BR biosynthesis or signaling genes have been expressed in epidermal and vascular tissues to determine how shoot growth occurs (Savaldi-Goldstein (2007) Nature 446: 199-202). Divi and Krishna expressed the BR biosynthesis gene MDWF4 in Arabidopsis seeds with the seed-specific oleosin promoter and saw that the seeds could overcome inhibition of germination caused by exogenous abscisic acid and were more cold- tolerant than the non-transgenic control seeds (Divi and Krishna (2010) Journal of Plant Growth Regulation 29: 385-393).
  • grain yield can be affected by expression of a BRIl polypeptide, such as a hypersensitive BRIl receptor specifically in a sub-region of the seed.
  • Any suitable way to activate the brassinosteroid signaling pathway in a plant can be used.
  • the manipulation of positive and negative regulators provide different avenues to obtain a more active BR signaling pathway, similar to the truncation of the BRIl C-terminal domain that leads to a hypersensitive receptor.
  • a BRIl ACT with the gain-of-function mutation corresponding to Y831F may be overexpressed constitutively or in a tissue-specific manner.
  • BKIl and MAKRs that are specific to BRIl inhibition can be modified, such as mutation to their plasma-membrane localization motifs and/or mutations to mimic phosphorylation of the conserved Tyr residue corresponding to Tyr 211, so that they no longer regulate BR signaling.
  • BKIland/or its functional homo logs can be reduced by RNAi or the BKIl -binding domain in the BRIl receptor can be removed or altered in such a way to disrupt BKIl binding.
  • the SERK subfamily of co-receptors either the full-length versions or truncated versions, may be overexpressed or co-expressed with BRIl or BRIIACT.
  • the overexpression of the positive regulator BSK with phospho-mimicking mutations of the conserved Ser residue corresponding to Ser 230 may create a constitutively active BSK.
  • the BSK without its plasma membrane anchor could be overexpressed and this modification can be combined with the phospho-mimicking mutations.
  • Targeted expression of the positive regulators BSU1 and/or its homologs (BSL1, BSL2, BSL3) and PP2A phosphatase in seed may activate the BR pathway.
  • the knockdown or knock-out of the critical negative regulator BIN2 kinase and/or its functional homologs may lead to more activation of the BR pathway.
  • the atypical basic helix-loop-helix (bHLH) transcription factor, ATBS1, which is hypothesized to be a positive regulator, may be overexpressed constitutively or in a tissue-specific manner.
  • Transgenic overexpression of BR biosynthesis enzymes is not effective sometimes because of the negative feedback inhibition by the BR transcription factor BZR1.
  • Uncoupling the expression of a BR biosynthetic enzyme from negative feedback inhibition by BZR1 through the use of a promoter that is temporally and spatially specific to seed development and growth could result in enhanced biosynthesis of BR.
  • BR enzymes that modify BR hormones for inactivation could be repressed.
  • one or more mutant BRIl polypeptides can be expressed in the plant to activate the brassinosteroids signaling pathway.
  • the mutant BRIl polypeptides can be made by deleting one or more amino acid in the C-terminal of wild type BRIl polypeptides. In some embodiments, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more amino acids in the C- terminal of
  • wild type BRIl polypeptides can be deleted.
  • the deletion is within the region corresponding to amino acid 1083 to 1122 of SEQ ID NO: 51.
  • the truncated BRIl polypeptides can still activate the brassinosteroid signaling pathway, with or without presence of brassinosteroid.
  • the mutant BRIl polypeptides can be made by replacing one or more amino acid in the C-terminal of wild type BRIl polypeptides. In some embodiments, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more amino acids in the C-terminal of wild type BRIl polypeptides can be replaced.
  • the C-terminal domain (CT) of BRIl contains multiple serine and threonine residues that are potentially phosphorylated. Work on the Arabidopsis BRIl CT domain identified Ser 1162 and Thr 1180 as residues that are phosphorylated (Wang et al (2005) Developmental Cell 8: 855-865).
  • Combinations of these CT Ser and Thr residues were changed to the acidic amino acid Asp, which can mimic phosphorylation in certain cases.
  • These BRIl CT mutants were able to increase BRIl kinase activity and enhance hypocotyl elongation compared to the wild type BRIl, suggesting that introducing mutations into the CT region that mimic phosphorylation can lead to a more activated receptor.
  • the replacement of one or more amino acids is within the region corresponding to amino acid 1083 to 1122 of SEQ ID NO: 51.
  • the mutant BRIl polypeptides with amino acid replacement can still activate the brassinosteroid signaling pathway, with or without presence of brassinosteroid.
  • Promoters that can drive gene expression in a plant seed-preferred manner with expression in the embryo sac, early embryo, early endosperm, aleurone and/or basal endosperm transfer cell layer (BETL) can be used in the present invention.
  • Such promoters include, but are not limited to promoters that are naturally linked to Zea mays early endosperm 5 gene (e.g., SEQ ID NO: 1), Zea mays early endosperm 1 gene (e.g., SEQ ID NO: 48), Zea mays early endosperm 2 gene (e.g., SEQ ID NO: 49), GRMZM2G124663 (e.g., SEQ ID NO: 37), GRMZM2G006585 (e.g., SEQ ID NO: 38), GRMZM2G120008 (e.g., SEQ ID NO: 82), GRMZM2G157806 (e.g., SEQ ID NO: 83), GRMZM2G176390 (e.
  • Functional variants or functional fragments of the promoters described herein can also be used. Fragments and variants of the disclosed promoter nucleotide sequences are also encompassed by the present invention. In particular, fragments and variants of the promoters may be used in the DNA constructs of the invention.
  • fragment refers to a portion of the nucleic acid sequence. Fragments of the promoters may retain the biological activity of initiating transcription, more particularly driving transcription in a plant seed-preferred manner with expression in the embryo sac, early embryo, early endosperm, aleurone and/or basal endosperm transfer cell layer (BETL). Alternatively, certain useful fragments of a nucleotide sequence, such as those that are useful as hybridization probes or in hairpin constructs targeting the promoter of interest, may not necessarily retain biological activity.
  • Fragments of a nucleotide sequence for the promoter regions may range from at least about 17 nucleotides, about 50 nucleotides, about 100 nucleotides, about 200 nucleotides, about 300 nucleotides, about 400 nucleotides, about 500 nucleotides, about 600 nucleotides, about 700 nucleotides, about 800 nucleotides, about 900 nucleotides, about lk nucleotides, about 1.5k nucleotides, about 2k nucleotides, about 2.5k nucleotides, about 3k nucleotides, about 5k nucleotides, about 10k nucleotides, or more, e.g., up to the full length of the promoters disclosed herein, or to the full length of the same promoters that one can isolate from plants.
  • variants here is intended to mean sequences having substantial similarity with a promoter sequence disclosed herein.
  • a variant comprises a deletion and/or addition of one or more nucleotides at one or more internal sites within the native polynucleotide and/or a substitution of one or more nucleotides at one or more sites in the native polynucleotide.
  • a "native" nucleotide sequence comprises a naturally occurring nucleotide sequence.
  • naturally occurring variants can be identified with the use of well-known molecular biology techniques, such as, for example, with polymerase chain reaction (PCR) and hybridization techniques as outlined herein.
  • the plant promoter has at least 80% identity to any one of SEQ
  • BETL basal endosperm transfer cell layer
  • the plant promoter can hybridize to any one of SEQ ID NOs. 1 and 24-50, 72-73, 75-78, 82-87, under stringent hybridization conditions, and wherein the promoter can drive gene expression in the embryo sac, early embryo, early endosperm, aleurone and/or basal endosperm transfer cell layer (BETL).
  • Variant nucleotide sequences also include synthetically derived nucleotide sequences, such as those generated, for example, by using site-directed mutagenesis.
  • variants of a particular nucleotide sequence of the embodiments will have at least 40%, 50%>, 60%>, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, to 95%, 96%, 97%, 98%, 99% or more sequence identity to that particular nucleotide sequence as determined by sequence alignment programs described elsewhere herein using default parameters.
  • Biologically active variants are also encompassed by the embodiments.
  • Biologically active variants include, for example, the native promoter sequences of the embodiments having one or more nucleotide substitutions, deletions or insertions. Promoter activity may be measured by using techniques such as Northern blot analysis, reporter activity measurements taken from transcriptional fusions, and the like.
  • nucleotide sequences also encompass sequences derived from a mutagenic and recombinogenic procedure such as DNA shuffling.
  • the present invention provides expression cassettes and expression vectors comprising one or more plant promoters and one ore more genes of interest of the present application.
  • the backbone of the expression vectors can be any expression vectors suitable for producing transgenic plant, which are well known in the art.
  • the expression vector is suitable for expressing transgene in a cereal crop plant.
  • the expression vector is an Agrobacterium binary vector (see, Karimi et al, Plant Physiol 145: 1183-1191; Komari et al, Methods Mol Biol 343: 15-42; Bevan MW (1984) Nucleic Acids Res 12: 1811-1821; Becker (1992), Plant Mol Biol 20: 1195-1197; Datla et al, (1992), Gene 122: 383-384; Hajdukiewicz (1994) Plant Mol Biol 25:989-994; Xiang (1999), Plant Mol Biol 40: 711-717; Chen et al, (2003) Mol Breed 11 : 287-293; Weigel et al, (2000) Plant Physiol 122: 1003-1013).
  • Agrobacterium binary vector see, Karimi et al, Plant Physiol 145: 1183-1191; Komari et al, Methods Mol Biol 343: 15-42; Bevan MW (1984) Nucleic Acids Res 12: 1811-1821
  • the expression vector is a co-integrated vector (also called hybrid Ti plasmids). More expression vectors and methods of using them can be found in U.S. Patent Nos. 4940838, 5464763, 5149645, 5501967, 6265638, 4693976, 5635381, 5731179, 5693512, 6162965, 5693512, 5981840, 6420630, 6919494, 6329571, 6215051, 6369298, 5169770, 5376543, 5416011, 5569834, 5824877, 5959179, 5563055, and 5968830. Each of the references mentioned herein is incorporated by reference in its entirety.
  • the expression cassettes or the expression vectors comprise at least one nucleic acid sequence encoding a gene of interest.
  • the gene of interest is operably linked to the promoters described herein.
  • the gene of interest is a gene that can activate the brassinosteroid signaling pathway when it is expressed in the plant.
  • the expression cassettes comprise a second gene of interest, such as a gene associated with one or more agronomically important traits.
  • agronomically important traits include resistance to biotic and/or abiotic stresses.
  • the phrase "biotic stress” or “biotic pressure” refers to a situation where damage is done to plants by other living organisms, such as bacteria, viruses, fungi, parasites, insects, weeds, animals and human.
  • the phrase "abiotic stress” or “abiotic pressure” refers to the negative impact of non-living factors on plants in a specific environment. The non-living variable must influence the environment beyond its normal range of variation to adversely affect the population performance or individual physiology of plants in a significant way.
  • Non-limiting examples of stressors are high winds, extreme temperatures, drought, flood, and other natural disasters, such as tornados and wildfires.
  • the trait is associated with increased biomass production, production of specific biofuels, increased food production, improved food quality, increased seed oil content, altered fatty acid composition, etc.
  • the gene of interest is a REVOLUTA gene or a dominant negative KRP gene, as described in WO 2007/016319 and WO 2007/079353, each of which is incorporated herein by reference in its entirety.
  • the gene of interest is an acyl-acyl carrier protein (acyl-ACP) thioesterase (Hawkins and Kridl 1998 The Plant Journal 13: 743-752; Facciotti 1999 Nature Biotechnology 17: 593 - 597; WO 1996/06936), an acyl-acyl carrier protein (acyl-ACP) desaturase (Knutzon 1992 PNAS 89: 2624-2628; Liu 2002 Plant Physiology 129: 1732-1743; Rousselin 2002 Plant Breeding 121 : 108-116), a fatty acid elongase (Millar and Kunststoff 1997 The Plant Journal 12: 121-131), a fatty acid desaturase (Okuley 1994 The Plant Cell 6: 147- 158; Hitz 1994 Plant Phys.
  • acyl-ACP acyl-acyl carrier protein
  • acyl-ACP acyl-ACP desaturase
  • Knutzon 1992 PNAS 89: 2624-2628 Liu 2002 Plant Physio
  • the gene of interest encodes a polypeptide. In some other embodiments, the gene of interest encodes a microRNA. In some other embodiments, the gene of interest encodes an interference RNA. The agronomically important traits are achieved by expression of the polypeptide, and/or the expression of the interference RNA in the plant.
  • RNA interference is the process of sequence-specific, post-transcriptional gene silencing or transcriptional gene silencing in animals and plants, initiated by double- stranded RNA (dsRNA) that is homologous in sequence to the silenced gene.
  • dsRNA or "dsRNA molecule” or “double-strand RNA effector molecule” refers to an at least partially double-strand ribonucleic acid molecule containing a region of at least about 19 or more nucleotides that are in a double-strand conformation.
  • the double-stranded RNA effector molecule may be a duplex double-stranded RNA formed from two separate RNA strands or it may be a single RNA strand with regions of self-complementarity capable of assuming an at least partially double-stranded hairpin conformation (i.e., a hairpin dsRNA or stem-loop dsRNA).
  • the dsRNA consists entirely of ribonucleotides or consists of a mixture of ribonucleotides and deoxynucleotides, such as RNA/DNA hybrids.
  • the dsRNA may be a single molecule with regions of self-complementarity such that nucleotides in one segment of the molecule base pair with nucleotides in another segment of the molecule.
  • the regions of self-complementarity are linked by a region of at least about 3-4 nucleotides, or about 5, 6, 7, 9 to 15 nucleotides or more, which lacks complementarity to another part of the molecule and thus remains single-stranded (i.e., the "loop region").
  • Such a molecule will assume a partially double-stranded stem-loop structure, optionally, with short single stranded 5' and/or 3' ends.
  • the regions of self- complementarity of the hairpin dsRNA or the double-stranded region of a duplex dsRNA will comprise an Effector Sequence and an Effector Complement (e.g., linked by a single- stranded loop region in a hairpin dsRNA).
  • the Effector Sequence or Effector Strand is that strand of the double-stranded region or duplex which is incorporated in or associates with RISC.
  • the double-stranded RNA effector molecule will comprise an at least 19 contiguous nucleotide effector sequence, preferably 19 to 29, 19 to 27, or 19 to 21 nucleotides, which is a reverse complement to the targeted gene, or an opposite strand replication intermediate, or the anti-genomic plus strand or non-mRNA plus strand sequences of the targeted gene.
  • said double-stranded RNA effector molecules are provided by providing to a plant, plant tissue, or plant cell an expression construct comprising one or more double-stranded RNA effector molecules.
  • suitable double-strand RNA effector molecule based on the nucleotide sequences of the targeted gene.
  • the dsRNA effector molecule of the invention is a "hairpin dsRNA", a “dsRNA hairpin”, “short-hairpin RNA” or “shRNA”, i.e., an RNA molecule of less than approximately 400 to 500 nucleotides (nt), or less than 100 to 200 nt, in which at least one stretch of at least 15 to 100 nucleotides (e.g., 17 to 50 nt, 19 to 29 nt) is based paired with a complementary sequence located on the same RNA molecule (single RNA strand), and where said sequence and complementary sequence are separated by an unpaired region of at least about 4 to 7 nucleotides (or about 9 to about 15 nt, about 15 to about 100 nt, about 100 to about 1000 nt) which forms a single-stranded loop above the stem structure created by the two regions of base complementarity.
  • the shRNA molecules comprise at least one stem-loop structure comprising a double-stranded stem region of about 17 to about 100 bp; about 17 to about 50 bp; about 40 to about 100 bp; about 18 to about 40 bp; or from about 19 to about 29 bp; homologous and complementary to a target sequence to be inhibited; and an unpaired loop region of at least about 4 to 7 nucleotides, or about 9 to about 15 nucleotides, about 15 to about 100 nt, about 100 to about 1000 nt, which forms a single-stranded loop above the stem structure created by the two regions of base complementarity.
  • the nucleic acid sequence encoding the gene of interest is also operably linked to a plant 3' non-translated region (3' UTR).
  • a plant 3' non-translated sequence is not necessarily derived from a plant gene.
  • it can be a terminator sequence derived from viral or bacterium gene, or T-DNA.
  • the 3' non-translated regulatory DNA sequence can include from about 20 to 50, about 50 to 100, about 100 to 500, or about 500 to 1,000 nucleotide base pairs and may contain plant transcriptional and translational termination sequences in addition to a polyadenylation signal and any other regulatory signals capable of effecting mRNA processing or gene expression.
  • Non-limiting examples of suitable 3' non- translated sequences are the 3' transcribed non-translated regions containing a polyadenylation signal from the nopaline synthase (NOS) gene of Agrobacterium tumefaciens (Bevan et al, 1983, Nucl. Acid Res., 11 :369), or terminator for the T7 transcript from the octopine synthase gene of Agrobacterium tumefaciens.
  • More suitable 3' non-translated sequences include, 3 'UTR of the potato cathepsin D inhibitor gene (GenBank Acc. No.: X74985), 3 'UTR of the field bean storage protein gene VfLEIB3 (GenBank Acc.
  • the expression cassettes or the expression vectors of the present invention can further comprise nucleic acids encoding one or more selection markers.
  • the selection marker can be a positive selectable marker, a negative selectable marker, or combination thereof.
  • a "positive selectable marker gene” encodes a protein that allows growth on selective medium of cells that carry the marker gene, but not of cells that do not carry the marker gene. Selection is for cells that grow on the selective medium (showing acquisition of the marker) and is used to identify transformants.
  • a common example is a drug-resistance marker such as NPT (neomycin phosphotransferase), whose gene product detoxifies kanamycin by phosphorylation and thus allows growth on media containing the drug.
  • Neo gene Pratrykus et al, 1985
  • kanamycin resistance and can be selected for using kanamycin, G418, etc.
  • bar gene which codes for bialaphos (basta) resistance
  • mutant aroA gene which encodes an altered EPSP synthase protein (Hinchee et al, 1988), thus conferring glyphosate resistance
  • a nitrilase gene such as bxn from Klebsiella ozaenae, which confers resistance to bromoxynil (Stalker et al., 1988)
  • a mutant acetolactate synthase gene ALS
  • a methotrexate resistant DHFR gene Thillet et al., 1988
  • methotrexate resistant DHFR gene Thillet et al., 1988
  • Additional positive selectable marker genes include those genes that provide resistance to environmental factors such as excess moisture, chilling, freezing, high temperature, salt, and oxidative stress. Of course, when it is desired to introduce such a trait into a plant as a "gene of interest", the selectable marker cannot be one that provides for resistance to an environmental factor.
  • Markers useful in the practice of the claimed invention include: an "antifreeze” protein such as that of the winter flounder (Cutler et al, 1989) or synthetic gene derivatives thereof; genes which provide improved chilling tolerance, such as that conferred through increased expression of glycerol-3 -phosphate acetyltransferase in chloroplasts (Murata et al., 1992; Wo Iter et al, 1992); resistance to oxidative stress conferred by expression of superoxide dismutase (Gupta et al, 1993), and may be improved by glutathione reductase (Bowler et al, 1992); genes providing "drought resistance” and “drought tolerance”, such as genes encoding for mannitol dehydrogenase (Lee and Saier, 1982) and trehalose-6-phosphate synthase (Kaasen et al., 1992).
  • an "antifreeze” protein such as that of the winter flounder (Cutler
  • a "negative selectable marker gene” encodes a protein that prevents the growth of a plant or plant cell on selective medium of plants that carry the marker gene, but not of plants that do not carry the marker gene. Selection of plants that grow on the selective medium provides for the identification of plants that have eliminated or evicted the selectable marker genes.
  • An example is CodA ⁇ Escherichia coli cytosine deaminase), whose gene product deaminates 5- fluorocytosine (which is normally non-toxic as plants do not metabolize cytosine) to the toxic 5- fluorouracil.
  • haloalkane dehalogenase dhlA gene of Xanthobacter autotrophicus GJ10 which encodes a dehalogenase, which hydro lyzes dihaloalkanes, such as 1, 2-dichloroethane (DCE), to a halogenated alcohol and an inorganic halide (Naested et al., 1999, Plant J. 18 (5): 571-6).
  • DCE 1, 2-dichloroethane
  • nucleic acid sequence can be included into the expression vectors of the present invention to facilitate the transcription, translation, and post- translational modification, so that expression and accumulation of a gene of interest in a plant cell are increased.
  • additional nucleic acid sequence can enhance either the expression, or the stability of the protein.
  • nucleic acid is an intron that has positive effect on gene expression, which has been also known as intron-mediated enhancement (IME, see Mascarenhas et al, (1990). Plant Mol. Biol. 15: 913-920). IME has been observed in a wide range of eukaryotes, including vertebrates, invertebrates, fungi, and plants (see references 17-26), suggesting that it reflects a fundamental feature of gene expression.
  • introns have a larger influence than do promoters in determining the level and pattern of expression.
  • Non-limiting IME in plants have been described in Rose et al. (The Plant Cell 20:543-551 (2008)); Lee et al. (Plant Physiology 145: 1294-1300 (2007)); Casas-Mollano et al. (Journal of Experimental Botany Volume 57, Number 12 Pp. 3301-3311); Jeong et al. (Plant Physiology 140: 196-209 (2006)); Clancy et al. (Plant Physiol, October 2002, Vol. 130, pp. 918-929); Jeon et al. (Plant Physiol, July 2000, Vol. 123, pp.
  • any one of the IME described herein can be included in the expression vectors of the present invention.
  • an intron (SEQ ID NO: 5) of ADHI (Alcohol Dehydrogenase 1) gene can be included upstream of the initiator methionine to increase expression (see Callis et al, Genes Dev. 1987 1 : 1183-1200; Mascarenhas et al, Plant Mol Biol 1990 15: 913-920).
  • the expression cassettes or the expression vectors comprise at least one enhancer sequence.
  • the enhancer sequence is a transcriptional enhancer sequence and/or a translation enhancer sequence.
  • the enhancer is a non-translated leader sequence.
  • the enhancer sequence is the intron sequence associated with an alcohol dehydrogenase (ADH) gene, the Heat Shock Protein 70 leader sequence (U.S. Pat. No. 5,659,122), or the petE enhancer sequence (WO 97/20056).
  • ADH alcohol dehydrogenase
  • the heat Shock Protein 70 leader sequence U.S. Pat. No. 5,659,122
  • the petE enhancer sequence WO 97/20056.
  • the alcohol dehydrogenase gene is a plant gene.
  • the plant alcohol dehydrogenase gene is the ADH gene in rice or maize.
  • the intron of the plant ADH gene comprises the sequence of SEQ ID NO: 5.
  • the enhancer is associated with a viral gene.
  • the enhancer sequence is the 5' proximal region of the genome of a potyvirus (e.g., WO/1998/044097), or viral non-translated leader sequence associated with virus selected from the group consisting of Tobacco Mosaic Virus (TMV), Maize Chlorotic Mottle Virus (MCMV), Alfalfa Mosaic Virus (AMV), Picornavirus, Potyvirus, and AMV RNA4.
  • the viral enhancer sequence can suppress gene silencing, such as the sequence associated with the gene encoding Pl ⁇ HC-Pro, the 2b protein of cucumber mosaic virus (CMV), the enhancer sequences derived from the CaMV (U.S. Pat. Nos. 5,164,316; 5,196,525; 5,322,938; 5,530,196; 5,352,605; 5,359,142; and 5,858,742 for example), and HC-Pro of potato virus Y (PVY) (e.g., U.S. Pat. No. 6395962).
  • enhancer sequences include shrunken- 1 (Shi) intron/exon enhancer sequence (U.S. Pat. No.
  • the expression cassettes or the expression vectors of the present invention can be transformed into a plant to increase the seed number and/or yield, using the transformation methods described separately below.
  • the present invention provides transgenic plants transformed with the expression vectors as described herein.
  • the plant can be any plant in which an increased seed number and/or yield is preferred by breeders for any reasons, e.g., for economical/agricultural interests.
  • the present invention provides methods of making and using the expression cassettes described herein.
  • the expression cassettes are used to express a gene of interest in a plant or plant cell, said method comprising incorporating into a plant cell a polynucleotide construct comprising a nucleic acid molecule operably linked to a gene of interest.
  • the gene of interest is a gene which when expressed will lead to activation of the brassinosteroid signaling pathway in a plant when expressed in the plant, with or without the presence of brassinosteroid.
  • the present invention provides methods for increasing seed number and/or yield in a plant.
  • the methods comprise introducing the expression cassettes of the present invention into a plant, a plant part, or a plant cell.
  • the methods can increase the average seed number per plant, per 100 or 1000 plants, or per acre, or increase yield by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%,
  • the present invention provides methods for expressing a gene encoding a BRI1 polypeptide in a plant, plant part, or plant cell.
  • the methods comprise introducing the expression cassettes of the present invention into a plant, a plant part, or a plant cell.
  • Any transgenic plant incorporated with the expression cassette generated from the present invention can be used as a donor to produce more transgenic plants through plant breeding methods well known to those skilled in the art.
  • the goal in general is to develop new, unique and superior varieties and hybrids.
  • selection methods e.g., molecular marker assisted selection
  • breeding methods can be combined with breeding methods to accelerate the process. Additional breeding methods have been known to one of ordinary skill in the art, e.g., methods discussed in Chahal and Gosal (Principles and procedures of plant breeding: biotechnological and conventional approaches, CRC Press, 2002, ISBN 084931321X, 9780849313219), Taji et al.
  • said method comprises (i) crossing any one of the plants of the present invention comprising the expression cassette as a donor to a recipient plant line to create a Fl population; (ii) selecting offsprings that have expression cassette.
  • the offsprings can be further selected by testing the expression of the gene of interest.
  • the transgenic plant with the expression cassette can serve as a male or female parent in a cross pollination to produce offspring plants, wherein by receiving the transgene from the donor plant, the offspring plants have the expression cassette.
  • protoplast fusion can also be used for the transfer of the transgene from a donor plant to a recipient plant.
  • Protoplast fusion is an induced or spontaneous union, such as a somatic hybridization, between two or more protoplasts (cells in which the cell walls are removed by enzymatic treatment) to produce a single bi- or multi-nucleate cell.
  • the fused cell that may even be obtained with plant species that cannot be interbred in nature, is tissue cultured into a hybrid plant exhibiting the desirable combination of traits. More specifically, a first protoplast can be obtained from a plant having the expression cassette.
  • a second protoplast can be obtained from a second plant line, optionally from another plant species or variety, preferably from the same plant species or variety, that comprises commercially desirable characteristics, such as, but not limited to disease resistance, insect resistance, valuable grain characteristics (e.g., increased seed number, see weight and/or seed size) etc.
  • the protoplasts are then fused using traditional protoplast fusion procedures, which are known in the art to produce the cross.
  • embryo rescue may be employed in the transfer of the expression cassette from a donor plant to a recipient plant.
  • Embryo rescue can be used as a procedure to isolate embryos from crosses wherein plants fail to produce viable seed.
  • the fertilized ovary or immature seed of a plant is tissue cultured to create new plants (see Pierik, 1999, In vitro culture of higher plants, Springer, ISBN 079235267x, 9780792352679, which is incorporated herein by reference in its entirety).
  • the recipient plant is an elite line having one or more certain agronomically important traits.
  • agronomically important traits include but are not limited to those that result in increased biomass production, production of specific biofuels, increased food production, improved food quality, increased seed oil content, etc.
  • Additional examples of agronomically important traits includes pest resistance, vigor, development time (time to harvest), enhanced nutrient content, novel growth patterns, flavors or colors, salt, heat, drought and cold tolerance, and the like.
  • Agronomically important traits do not include selectable marker genes (e.g., genes encoding herbicide or antibiotic resistance used only to facilitate detection or selection of transformed cells), hormone biosynthesis genes leading to the production of a plant hormone (e.g., auxins, gibberellins, cytokinins, abscisic acid and ethylene that are used only for selection), or reporter genes (e.g. luciferase, ⁇ -glucuronidase, chloramphenicol acetyl transferase (CAT, etc.).
  • the recipient plant can be a plant with increased seed weight and/or seed size which is due to a trait not related to the expression cassette in the donor plant.
  • the recipient plant can also be a plant with preferred carbohydrate composition, e.g., composition preferred for nutritional or industrial applications, especially those plants in which the preferred composition is present in seeds.
  • molecular markers are designed and made, based on the promoters or the genes of interest of the present application.
  • the molecular markers are selected from Isozyme Electrophoresis, Restriction Fragment Length Polymorphisms (RFLPs), Randomly Amplified Polymorphic DNAs (RAPDs), Arbitrarily Primed Polymerase Chain Reaction (AP-PCR), DNA Amplification Fingerprinting (DAF), Sequence Characterized Amplified Regions (SCARs). Amplified Fragment Length Polymorphisms (AFLPs), and Simple Sequence Repeats (SSRs) which are also referred to as Microsatellites, etc.
  • RFLPs Restriction Fragment Length Polymorphisms
  • RAPDs Randomly Amplified Polymorphic DNAs
  • AP-PCR Arbitrarily Primed Polymerase Chain Reaction
  • DAF DNA Amplification Fingerprinting
  • SCARs Sequence Characterized Amplified Regions
  • AFLPs
  • the molecular markers can be used in molecular marker assisted breeding.
  • the molecular markers can be utilized to monitor the transfer of the genetic material.
  • the transferred genetic material is a gene of interest, such as genes that contribute to one or more favorable agronomic phenotypes when expressed in a plant cell, a plant part, or a plant.
  • the present invention provides transgenic plants having an expression cassette of the present invention.
  • the present invention also provides seeds, fruits, plant populations, plant parts, plant cells and/or plant tissue cultures derived from the transgenic plants as described herein.
  • the composition of the medium particularly the plant hormones and the nitrogen source (nitrate versus ammonium salts or amino acids) have profound effects on the morphology of the tissues that grow from the initial explant.
  • an excess of auxin will often result in a proliferation of roots, while an excess of cytokinin may yield shoots.
  • a balance of both auxin and cytokinin will often produce an unorganized growth of cells, or callus, but the morphology of the outgrowth will depend on the plant species as well as the medium composition.
  • cultures grow pieces are typically sliced off and transferred to new media (subcultured) to allow for growth or to alter the morphology of the culture.
  • the skill and experience of the tissue culturist are important in judging which pieces to culture and which to discard.
  • shoots emerge from a culture they may be sliced off and rooted with auxin to produce plantlets which, when mature, can be transferred to potting soil for further growth in the greenhouse as normal plants.
  • the transgenic plants of the present invention can be used for many purposes.
  • the transgenic plant is used as a donor plant of genetic material which can be transferred to a recipient plant to produce a plant which has the transferred genetic material.
  • Any suitable method known in the art can be applied to transfer genetic material from a donor plant to a recipient plant. In most cases, such genetic material is genomic material.
  • the whole genome of the transgenic plants of the present invention is transferred into a recipient plant. This can be done by crossing the transgenic plants to a recipient plant to create a Fl plant.
  • the Fl plant can be further selfed and selected for one, two, three, four, or more generations to give plants with the expression cassette of the present invention.
  • At least the parts containing the transgene of the donor plant's genome are transferred. This can be done by crossing the transgenic plants to a recipient plant to create a Fl plant, followed with one or more backcrosses to one of the parent plants to plants with the desired genetic background. The progeny resulting from the backcrosses can be further selfed and selected to give plants with increased seed number and/or yield.
  • the recipient plant is an elite line having one or more certain agronomically important traits.
  • the transgenic plants have increased seed number, and/or yield compared to a wild type plant that does not have the expression cassette of the present invention.
  • the transgenic plants of the present invention have an increased average seed number per plant, per 100 plants, or per acre, or increased yield by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%
  • the transgenic plants of the present invention may have altered metabolic profiles compared to a wild type plant.
  • a plant with altered metabolic profiles can be selected by methods well known to one skilled in the art.
  • metabolic profiles can be screened using quantitative chemical analysis methods, e.g., using gas chromatography (GC) analysis, or high performance liquid chromatography analysis (or high pressure liquid chromatography, HPLC).
  • GC gas chromatography
  • HPLC high pressure liquid chromatography
  • the expression cassettes of the present invention can be transformed into a plant.
  • the most common method for the introduction of new genetic material into a plant genome involves the use of living cells of the bacterial pathogen Agrobacterium tumefaciens to literally inject a piece of DNA, called transfer or T-DNA, into individual plant cells (usually following wounding of the tissue) where it is targeted to the plant nucleus for chromosomal integration.
  • Agrobacterium-mediated plant transformation involves as a first step the placement of DNA fragments cloned on plasmids into living Agrobacterium cells, which are then subsequently used for transformation into individual plant cells.
  • Agrobacterium- mediated plant transformation is thus an indirect plant transformation method.
  • Methods of Agrobacterium-mediated plant transformation that involve using vectors with no T-DNA are also well known to those skilled in the art and can have applicability in the present invention. See, for example, U.S. Patent No. 7,250,554, which utilizes P-DNA instead of T-DNA in the transformation vector.
  • a third direct method uses fibrous forms of metal or ceramic consisting of sharp, porous or hollow needle-like projections that literally impale the cells, and also the nuclear envelope of cells.
  • silicon carbide and aluminum borate whiskers have been used for plant transformation (Mizuno et al., 2004; Petolino et al., 2000; US5302523 US Application 20040197909) and also for bacterial and animal transformation (Kaepler et al, 1992; Raloff, 1990; Wang, 1995).
  • plant transformation Mizuno et al., 2004; Petolino et al., 2000; US5302523 US Application 20040197909
  • bacterial and animal transformation Korean epler et al, 1992; Raloff, 1990; Wang, 1995
  • a selection method For efficient plant transformation, a selection method must be employed such that whole plants are regenerated from a single transformed cell and every cell of the transformed plant carries the DNA of interest.
  • These methods can employ positive selection, whereby a foreign gene is supplied to a plant cell that allows it to utilize a substrate present in the medium that it otherwise could not use, such as mannose or xylose (for example, refer US 5767378; US 5994629). More typically, however, negative selection is used because it is more efficient, utilizing selective agents such as herbicides or antibiotics that either kill or inhibit the growth of nontransformed plant cells and reducing the possibility of chimeras. Resistance genes that are effective against negative selective agents are provided on the introduced foreign DNA used for the plant transformation.
  • nptll neomycin phosphotransferase
  • herbicides and herbicide resistance genes have been used for transformation purposes, including the bar gene, which confers resistance to the herbicide phosphinothricin (White et al, Nucl Acids Res 18: 1062 (1990), Spencer et al, Theor Appl Genet 79: 625-631(1990), US 4795855, US 5378824 and US 6107549).
  • the dhfr gene which confers resistance to the anticancer agent methotrexate, has been used for selection (Bourouis et al, EMBO J. 2(7): 1099-1104 (1983).
  • Genes can be introduced in a site directed fashion using homologous recombination.
  • Homologous recombination permits site specific modifications in endogenous genes and thus inherited or acquired mutations may be corrected, and/or novel alterations may be engineered into the genome.
  • Homologous recombination and site-directed integration in plants are discussed in, for example, U.S. Patent Nos. 5,451,513; 5,501,967 and 5,527,695.
  • Transgenic plants can now be produced by a variety of different transformation methods including, but not limited to, electroporation; microinjection; microprojectile bombardment, also known as particle acceleration or biolistic bombardment; viral-mediated transformation; and Agrobacterium-mediated transformation. See, for example, U.S. Patent Nos. 5,405,765; 5,472,869; 5,538,877; 5,538,880; 5,550,318; 5,641,664; 5,736,369 and 5,736,369; International Patent Application Publication Nos.
  • Microprojectile bombardment is also known as particle acceleration, biolistic bombardment, and the gene gun (Biolistic® Gene Gun).
  • the gene gun is used to shoot pellets that are coated with genes (e.g., for desired traits) into plant seeds or plant tissues in order to get the plant cells to then express the new genes.
  • the gene gun uses an actual explosive (.22 caliber blank) to propel the material. Compressed air or steam may also be used as the propellant.
  • the Biolistic® Gene Gun was invented in 1983-1984 at Cornell University by John Sanford, Edward Wolf, and Nelson Allen. It and its registered trademark are now owned by E. I. du Pont de Nemours and Company. Most species of plants have been transformed using this method.
  • Agrobacterium tumefaciens is a naturally occurring bacterium that is capable of inserting its DNA (genetic information) into plants, resulting in a type of injury to the plant known as crown gall. Most species of plants can now be transformed using this method, including cucurbitaceous species.
  • a transgenic plant formed using Agrobacterium transformation methods typically contains a single gene on one chromosome, although multiple copies are possible. Such transgenic plants can be referred to as being hemizygous for the added gene.
  • a more accurate name for such a plant is an independent segregant, because each transformed plant represents a unique T-DNA integration event (U.S. Patent No. 6,156,953).
  • a transgene locus is generally characterized by the presence and/or absence of the transgene.
  • a heterozygous genotype in which one allele corresponds to the absence of the transgene is also designated hemizygous (U.S. Patent No. 6,008,437).
  • the expression cassettes can be introduced into an expression vector suitable for corn transformation, such as the vectors described by Sidorov and Duncan, 2008 (Agrobacterium-Mediated Maize Transformation: Immature Embryos Versus Callus, Methods in Molecular Biology, 526:47-58), Frame et al., 2002 (Agrobacterium tumefaciens- Mediated Transformation of Maize Embryos Using a Standard Binary Vector System, Plant Physiology, May 2002, Vol. 129, pp. 13-22), Ahmadabadi et al, 2007 (A leaf-based regeneration and transformation system for maize (Zea mays L.), TransgenicRes. 16, 437- 448), U.S. Patent Nos. 6,420,630, 6,919,494 and 7,682,829, or similar experimental procedures well known to those skilled in the art.
  • an expression vector suitable for corn transformation such as the vectors described by Sidorov and Duncan, 2008 (Agrobacterium-Mediated Maize Transformation: Immature
  • Classic breeding methods can be included in the present invention to introduce one or more recombinant expression cassettes of the present invention into other plant varieties, or other close-related species that are compatible to be crossed with the transgenic plant of the present invention.
  • Open-Pollinated Populations The improvement of open-pollinated populations of such crops as rye, many maizes and sugar beets, herbage grasses, legumes such as alfalfa and clover, and tropical tree crops such as cacao, coconuts, oil palm and some rubber, depends essentially upon changing gene-frequencies towards fixation of favorable alleles while maintaining a high (but far from maximal) degree of heterozygosity. Uniformity in such populations is impossible and trueness-to-type in an open-pollinated variety is a statistical feature of the population as a whole, not a characteristic of individual plants. Thus, the heterogeneity of open-pollinated populations contrasts with the homogeneity (or virtually so) of inbred lines, clones and hybrids.
  • Interpopulation improvement utilizes the concept of open breeding populations; allowing genes to flow from one population to another. Plants in one population (cultivar, strain, ecotype, or any germplasm source) are crossed either naturally (e.g., by wind) or by hand or by bees (commonly Apis mellifera L. or Megachile rotundata F.) with plants from other populations. Selection is applied to improve one (or sometimes both) population(s) by isolating plants with desirable traits from both sources.
  • Mass Selection In mass selection, desirable individual plants are chosen, harvested, and the seed composited without progeny testing to produce the following generation. Since selection is based on the maternal parent only, and there is no control over pollination, mass selection amounts to a form of random mating with selection. As stated herein, the purpose of mass selection is to increase the proportion of superior genotypes in the population.
  • Synthetics A synthetic variety is produced by crossing inter se a number of genotypes selected for good combining ability in all possible hybrid combinations, with subsequent maintenance of the variety by open pollination. Whether parents are (more or less inbred) seed-propagated lines, as in some sugar beet and beans (Vicia) or clones, as in herbage grasses, clovers and alfalfa, makes no difference in principle. Parents are selected on general combining ability, sometimes by test crosses or topcrosses, more generally by polycrosses. Parental seed lines may be deliberately inbred (e.g. by selfmg or sib crossing). However, even if the parents are not deliberately inbred, selection within lines during line maintenance will ensure that some inbreeding occurs. Clonal parents will, of course, remain unchanged and highly heterozygous.
  • the number of parental lines or clones that enter a synthetic vary widely. In practice, numbers of parental lines range from 10 to several hundred, with 100-200 being the average. Broad based synthetics formed from 100 or more clones would be expected to be more stable during seed multiplication than narrow based synthetics.
  • Pedigreed varieties A pedigreed variety is a superior genotype developed from selection of individual plants out of a segregating population followed by propagation and seed increase of self pollinated offspring and careful testing of the genotype over several generations. This is an open pollinated method that works well with naturally self pollinating species. This method can be used in combination with mass selection in variety development. Variations in pedigree and mass selection in combination are the most common methods for generating varieties in self pollinated crops.
  • Hybrids A hybrid is an individual plant resulting from a cross between parents of differing genotypes. Commercial hybrids are now used extensively in many crops, including corn (maize), sorghum, sugarbeet, sunflower and broccoli. Hybrids can be formed in a number of different ways, including by crossing two parents directly (single cross hybrids), by crossing a single cross hybrid with another parent (three-way or triple cross hybrids), or by crossing two different hybrids (four- way or double cross hybrids).
  • hybrids most individuals in an out breeding (i.e., open-pollinated) population are hybrids, but the term is usually reserved for cases in which the parents are individuals whose genomes are sufficiently distinct for them to be recognized as different species or subspecies.
  • Hybrids may be fertile or sterile depending on qualitative and/or quantitative differences in the genomes of the two parents.
  • Heterosis, or hybrid vigor is usually associated with increased heterozygosity that results in increased vigor of growth, survival, and fertility of hybrids as compared with the parental lines that were used to form the hybrid. Maximum heterosis is usually achieved by crossing two genetically different, highly inbred lines.
  • hybrids The production of hybrids is a well-developed industry, involving the isolated production of both the parental lines and the hybrids which result from crossing those lines.
  • hybrid production process see, e.g., Wright, Commercial Hybrid Seed Production 8: 161-176, In Hybridization of Crop Plants.
  • Corn is used as human food, livestock feed, and as raw material in industry.
  • the food uses of corn in addition to human consumption of corn kernels, include both products of dry- and wet-milling industries.
  • the principal products of corn dry-milling are grits, meal and flour.
  • Corn meal is flour ground to fine, medium, and coarse consistencies from dried corn. In the United States, the finely ground corn meal is also referred to as corn flour.
  • corn flour denotes corn starch in the United Kingdom.
  • Corn meal has a long shelf life and is used to produce an assortment of products, including but not limited to tortillas, taco shells, bread, cereal and muffins.
  • the corn wet-milling industry can provide corn starch, corn syrups, corn sweeteners and dextrose for food use.
  • Corn syrup is used in foods to soften texture, add volume, prevent crystallization of sugar and enhance flavor. Corn syrup is distinct from high-fructose corn syrup (HFCS), which is created when corn syrup undergoes enzymatic processing, producing a sweeter compound that contains higher levels of fructose.
  • HFCS high-fructose corn syrup
  • Corn oil is recovered from corn germ, which is a by-product of both dry- and wet- milling industries. Corn oil which is high in mono and poly unsaturated fats, is used for reducing fat and trans fat in numerous food products.
  • Corn including both grain and non-grain portions of the plant, is also used extensively as livestock feed, primarily for beef cattle, dairy cattle, hogs and poultry.
  • Corn ethanol is ethanol produced from corn as a biomass through industrial fermentation, chemical processing and distillation. Corn is the main feedstock used for producing ethanol fuel in the United States.
  • the industrial applications of corn starch and flour are based on functional properties, such as viscosity, film formation, adhesive properties, and ability to suspend particles. Corn starch and flour also have application in the paper and textile industries. Other industrial uses include applications in adhesives, building materials, foundry binders, laundry starches, explosives, oil-well muds and other mining applications.
  • TILLING Targeting Induced Local Lesions in Genomes
  • TILLING® was introduced in 2000, using the model plant Arabidopsis thaliana.
  • TILLING® has since been used as a reverse genetics method in other organisms such as zebrafish, corn, wheat, rice, soybean, tomato and lettuce.
  • TILLING is used to isolate genes encoding BRI polypeptides that can activate the brassinosteroid signaling pathway when expressed in a plant.
  • the method combines a standard and efficient technique of mutagenesis with a chemical mutagen (e.g., Ethyl methanesulfonate (EMS)) with a sensitive DNA screening- technique that identifies single base mutations (also called point mutations) in a target gene.
  • EcoTILLING is a method that uses TILLING® techniques to look for natural mutations in individuals, usually for population genetics analysis. See Comai, et al, 2003. Efficient discovery of DNA polymorphisms in natural populations by Ecotilling. The Plant Journal 37, 778-786. Gilchrist et al. 2006. Use of Ecotilling as an efficient SNP discovery tool to survey genetic variation in wild populations of Populus trichocarpa. Mol. Ecol.
  • the TILLING® method relies on the formation of heteroduplexes that are formed when multiple alleles (which could be from a heterozygote or a pool of multiple homozygotes and heterozygotes) are amplified in a PCR, heated, and then slowly cooled. A "bubble” forms at the mismatch of the two DNA strands (the induced mutation in TILLING® or the natural mutation or SNP in EcoTILLING), which is then cleaved by single stranded nucleases. The products are then separated by size on several different platforms.
  • TILLING® centers exists over the world that focus on agriculturally important species: UC Davis (USA), focusing on Rice; Purdue University (USA), focusing on Maize; University of British Columbia (CA), focusing on Brassica napus; John Innes Centre (UK), focusing on Brassica rapa; Fred Hutchinson Cancer Research, focusing on Arabidopsis; Southern Illinois University (USA), focusing on Soybean; John Innes Centre (UK), focusing on Lotus and Medicago ; and INRA (France), focusing on Pea and Tomato.
  • Each recombinant expression vector comprises:
  • the promoters used for expression in maize plants for these examples are as follows the Oryza sativa constitutive promoter, Actin.
  • Zm ADH1 Alcohol Dehydrogenase 1 intron (SEQ ID NO: 5) was optionally included upstream of the initiator methionine.
  • the constructs in superbinary Agrobacterium were maintained on minimal medium containing the antibiotics spectinomycin, rifampicin and tetracycline. Agrobacterium was streaked on LB medium with antibiotics and grown for 1-2 days.
  • Greenhouse-grown plants of a hybrid (such as Hi-II) crossed with an inbred were used as the donor material and ears were harvested 9-12 days after pollination. These were surface-sterilized with bleach solution and rinsed with sterile Milli-Q water. Immature zygotic embryos were aseptically excised from the F2 kernels.
  • the Agrobacterium from LB bacterial medium was collected and suspended in liquid infection medium and acetosyringone added to a final concentration of 100 ⁇ .
  • Zygotic embryos were immersed in the Agrobacterium suspension to start the bacterial infection process. Subsequently, the embryos were cultured with the scutellum side up onto the surface of co-cultivation medium and incubated in the dark for 4 days.
  • Embryos were transferred to resting medium for 3 days followed by culturing these on selection medium containing an enzyme inhibitor used as a selection marker. Explants were sub-cultured to fresh medium every 2 weeks and maintained in the dark at 28°C. Callus resistant to the enzyme inhibitor was selected and cultured on regeneration media to initiate shoot regeneration. In most cases, multiple shoots from subcultured callus of a single source-embryo were carried through the regeneration process to produce replicate plants, or "clones", of a single “event". Although it is recognized that multiple clones derived from a single Agrobacterium- fGcted embryo do not always represent identical transgenic events of equal patterns for T-DNA integration into the maize genome, commonly this is the case.
  • the regenerated plants were transferred to 25 x 150 mm test tubes containing growth and rooting medium. Callus and leaves of regenerated plants were confirmed to be transformed. Plantlets with healthy roots were transferred into 4 inch pots containing Metro- mix 360 and maintained in the greenhouse. At 4-5 leaf stage, plants were transferred to 3 gallon pots and grown to maturity. The plants were self-pollinated or crossed with the inbred parent, and Tl seed was collected -35 days post-pollination.
  • Recombinant vectors TGZm 131, 132, 133, 135, 149 and 150 were transformed into maize. Regenerated transgenic TO plants were crossed to the recurrent inbred parent to generate Tl seeds segregating 1 : 1 for the transgene. Plants grown from Tl seed were crossed with tester inbreds in a nursery to generate Fl hybrid seed that were planted in isolated crossing block (ICB) trials to measure seed yield.
  • IOB isolated crossing block
  • Isolated Crossing Block (ICB)
  • seed being planted are Fl hybrid seed. They are produced by crossing one type of plant (A), which carries the gene of interest and typically contains 75% elite corn breeding germplasm background (i.e., recurring parent), with a second type (B) that is a commercial elite inbred of a counter heterotic group. In other cases, seed being planted are BC1 or BC2, meaning that they contain, on average, 75 or 87.5% elite corn breeding germplasm background (i.e., recurring parent).
  • the events in the ICB trials employ a visual color marker gene which helps segregate Null seed (yellow, lacks transgene) from Transgenic seed (reddish, contains transgene).
  • rows are planted with a mix of Null and Transgenic seed, so plants of these two types occur at random positions within each row. These are considered female rows.
  • Female plots consist of four female rows 30 inches apart. Planted adjacent to each female plot are two male rows, which are planted with the recurring parent or a mix of commercial hybrids. At all ICB trial locations, plants in female rows are pollinated by pollen released from the plants in the male rows. Therefore, each ICB field is planted in a pattern of two male rows for every four female rows.
  • ears are harvested from plants within each female row, dried with heated forced-air to reduce moisture levels to a point safe for grain storage and equilibration with ambient conditions, and segregated as originating from a Null or Transgenic plant. This is done on the basis of whether ears contain color marked seed (Transgenic), or not (Null). Individual ears are shelled and the grain weighed immediately for determination of fresh weight (as is basis, following equilibration with ambient conditions) or after oven drying at 103°C for 24 hours to determine grain dry weight. A grain subsample is collected from each ear for determination of 100-kernel weight, which in turn allows estimation of the number of kernels on each ear. Alternatively, all kernels from each individual ear are counted and 100-kernel weight is calculated from the ear grain weight and kernel number. Thus, grain productivity per ear, expressed as the core kernel weight and kernel number yield components, is determined.
  • ZmEEP5-ZmBRIl ACT-FLAG gives increased ear grain weight
  • TG ZM 149 ZmEEP5 promoter-Zm BRIl ACT-FLAG significantly increased ear grain weight, with three of seven events having a significant increase in ear grain weight at least at one location compared to the null sibling control (FIG. 8).
  • event 149-S002 significantly increased ear grain weight (24% and 29%) at 2 out of 3 locations tested.
  • the promoter used is crucial, as constructs containing Zm BRIl ACT driven by other promoters were either (on average) neutral for ear grain weight (TG Zm 131, 132, 135, 150) or was, in fact, negative for ear grain weight (TG Zm 133) (FIG. 9).
  • the promoters that were neutral for ear grain weight included Os actin (constitutive, TG Zm 131, 132), Hv LTP2 (aleurone, TG Zm 135) and Zm LEC1 (early embryo, TG Zm 150).
  • the expression of Zm BRIl ACT-FLAG in endosperm using the Zm LeguminlA promoter is detrimental to ear grain weight.
  • Zm EEP5 promoter drives expression in the embryo sac, early embryo, early endosperm, aleurone and/or the basal endosperm transfer cell layer and is efficacious in giving ear grain weight increase when operably linked to Zm BRIl ACT-FLAG.
  • TG ZM 149 ZmEEP5 promoter-Zm BRI1 ACT-FLAG again significantly increased ear grain weight, with five of nine events having a significant increase in ear grain weight at least at one location compared to the null sibling control (FIG. 10).
  • TG Zm 149 gives a mean increase in ear grain weight across all events (including those, on average, affected by the transgene, as well as those unaffected) of about +2.5% compared to TG Zm 150, which gave a mean ear grain weight decrease across all events of about -1% and TG Zm 133, which was again a clearly detrimental construct with a mean ear grain weight decrease across all events of about -11.7% (FIG. 12).
  • the increase in ear grain weight for TG Zm 149 was initially determined to come from an increase in seed number. However, other reasons may be possible which can be analyzed later.
  • ZmEEP5 promoter-Zm BRI1 ACT-FLAG is stacked with other expression cassettes that give increases in seed yield, such as those described in WO 2007/079353, WO
  • the ZmEEP5 type promoter can be, but is not limited to, ZmEEP5 (SEQ ID NO: 1), OsPR602 or OsPR9a (Li et al (2008) Plant Biotechnology Journal 6: 465-476, SEQ ID NOs: 24 and 25).
  • the monocot BRIIACT gene can be ZmBRIlACT (SEQ ID NOs: 8, 9), OsBRIlACT (SEQ ID NO: 59), HvBRIlACT (SEQ ID NO: 63), SbBRIlACT (SEQ ID NO: 68), TaBRIlACT (SEQ ID NO: 69) or BdBRIlACT (SEQ ID NO: 70).
  • Agrobacterium host EHA105 carrying the resulting binary vector containing a monocot BRIIACT cassette is selected by kanamycin antibiotic.
  • the binary clones are restriction digested and sequenced to verify that the cassettes have been cloned at the appropriate sites and that the junctions are intact.
  • Each binary vector containing a monocot BRIIACT cassette or an empty binary vector (to serve as control) is transformed into rice calli by Agrobacterium-mediated transformation and transgenic calli is selected on hygromycin.
  • Shoot and root formation of the transformed calli is induced by hormones and the resulting TO plantlets are grown up in the greenhouse.
  • TO tissue is collected and Southerns done to confirm presence of the BRIIACT transgene and to determine transgene insert number.
  • single insert events expressing BRIIACT are taken forward. About 15-30 TO events are obtained for each BRIIACT construct.
  • Tl mean seed number and weight is measured. These preliminary analyses are compared to the corresponding measurements from transgenic events carrying the empty vector. It is expected that Tl mean seed yield from transgenic BRIIACT events is increased by about 1-100% or more compared to the empty vector events.
  • Reproducibility of the increased seed yield is determined in subsequent generations and in newly created rice hybrids in the greenhouse and field.
  • a cassette containing a dicot BRIIACT gene operably linked to a ZmEEP5 type or seed-preferred promoter and a suitable 3 ' terminator is constructed and co-transformed with a cassette carrying a suitable selectable marker.
  • the ZmEEP5 type or seed-preferred promoter can be, but is not limited to, the promoters of AT3G10590 (SEQ ID NO: 26), AT4G18870 (SEQ ID NO: 27), AT4G21080 (SEQ ID NO: 28), AT5G23650 (SEQ ID NO: 29), AT3G05860 (SEQ ID NO: 30), AT5G42910 (SEQ ID NO: 31), AT2G26320 (SEQ ID NO: 32), AT3G03260 (SEQ ID NO: 33), AT5G26630 (SEQ ID NO: 34) (Le (2010) PNAS 107: 8063-8070), AtIPT4 (SEQ ID NO: 35) and AtIPT8 (SEQ ID NO: 36) (Miyawaki (2004) The
  • the seed-preferred promoter can also be, but is not limited to, the promoter for AtLEC2 (SEQ ID NO: 75).
  • the dicot BRIIACT gene can be Glycine max BRIIACT (SEQ ID NO: 64), Arabidopsis thaliana BRIIACT (Wang et al (2005) Developmental Cell 8: 855-865, SEQ ID NO: 60), Brassica napus BRIIACT (SEQ ID NO: 66), Lycopersicon esculentum BRIIACT (SEQ ID NO: 61) or Pisum sativum BRIIACT (SEQ ID NO: 62).
  • a recombinant expression cassette was constructed with At BRIIACT gene operably linked to the AtLEC2 seed-preferred promoter and a suitable 3' terminator (TGGm_38; FIG. 14). Soybean tissue was transformed and plants recovered as described in Li et al. (In Vitro CeU.Dev.Biol.— Plant (2011) 47:274-281).
  • TO tissue is collected and Southerns done to confirm presence of the BRIIACT transgene and to determine transgene insert number. Typically single insert events expressing BRIIACT are taken forward. About 15-30 TO events are obtained for each BRIIACT construct.
  • Tl mean seed number and weight is measured for each transgenic event. These preliminary analyses are compared to the corresponding measurements from transgenic events carrying the empty vector. It is expected that Tl mean seed yield from transgenic BRIIACT events is increased by 1-100% or more compared to the empty vector events.
  • Reproducibility of the increased seed yield is determined in subsequent generations in the greenhouse and field.
  • BRIIACT BRIIACT with a ZmEEP5 type or seed-preferred promoter in the dicotyledonous crop, canola (Brassica napus), will lead to increased average seed yield
  • constructs are made for Agrobacterium-rnQdiated transformation into canola as described in WO 2007/079353.
  • An expression cassette containing a dicot BRIIACT gene operably linked to a ZmEEP5 type or seed-preferred promoter and a suitable 3 ' terminator is constructed and inserted into a binary vector containing a suitable selectable marker expression cassette.
  • Each recombinant expression vector comprises:
  • the ZmEEP5 type or seed-preferred promoter can be, but is not limited to, the promoters of AT3G10590 (SEQ ID NO: 26), AT4G18870 (SEQ ID NO: 27), AT4G21080 (SEQ ID NO: 28), AT5G23650 (SEQ ID NO: 29), AT3G05860 (SEQ ID NO: 30), AT5G42910 (SEQ ID NO: 31), AT2G26320 (SEQ ID NO: 32), AT3G03260 (SEQ ID NO: 33), AT5G26630 (SEQ ID NO: 34) (Le (2010) PNAS 107: 8063-8070), AtIPT4 (SEQ ID NO: 35) and AtIPT8 (SEQ ID NO: 36) (Miyawaki (2004) The Plant Journal 37: 128-138).
  • the seed-preferred promoter can be, but is not limited to, the promoters of AtLEC2 (SEQ ID NO:
  • the dicot BRIIACT gene can be Glycine max BRIIACT (SEQ ID NO: 64), Arabidopsis thaliana BRIIACT (Wang et al (2005) Developmental Cell 8: 855-865, SEQ ID NO: 60), Brassica napus BRIIACT (SEQ ID NO: 66), Lycopersicon esculentum BRIIACT (SEQ ID NO: 61) or Pisum sativum BRIIACT (SEQ ID NO: 62).
  • FIG. 17 summarizes qPCR and Southern data for a subset of TG68 and TG70 individual events. Typically single insert events expressing BRIl ACT are taken forward. About 15-30 TO events are obtained for each BRIl ACT construct.
  • Tl mean seed number and weight is measured for each transgenic event. These preliminary analyses are compared to the corresponding measurements from transgenic events carrying the empty vector. It is expected that Tl mean seed yield from transgenic BRIl ACT events is increased by 1-100% or more compared to the empty vector events.
  • Reproducibility of the increased seed yield is determined in subsequent generations in the greenhouse and field.
  • a hypersensitive ZmBRIlACT with the embryo sac, early embryo, early endosperm, aleurone and/or the basal endosperm transfer cell layer-preferred promoter ZmEEP5 leads to an increase in ear grain weight.
  • the ZmBRIlACT is a more active receptor and shows enhanced BR downstream signaling (Wang et al (2005) Developmental Cell 8: 855-865). It is well known that the binding of brassinosteroid hormone to the full-length BRIl receptor kinase leads to its activation. BR hormone is expressed in the developing seed (Xiong, Y. et al. (2011) Plant Mol Biol Rep 29:835-847), so expressing the full-length BRIl receptor in seed will result in activation of the BR signaling pathway, leading to increased seed yield.
  • An expression construct containing a ZmEEP5 -full-length ZmBRIl-3' UTR cassette is built and transformed into corn as described above. Regenerated transgenic TO plants are crossed to the recurrent inbred parent to generate Tl seeds segregating 1 : 1 for the transgene. Plants grown from Tl seed are crossed with tester inbreds in a nursery to generate Fl hybrid seed that are planted in isolated crossing block (ICB) trials to measure ear grain weight compared to the null sibling controls. It is expected that plants containing the ZmEEP5-full- length ZmBRIl-3' UTR transgene has increased ear grain weight compared to the control null plants not having the transgene.
  • ICB isolated crossing block
  • BRIIACT hexaploid wheat
  • tetraploid wheat T. turgidum L. var.
  • constructs are made for Agrobacterium-mQdiatQd transformation into wheat by floral dip methods (Zale et al, Plant Cell Rep 28(6):903-913 (2009)), by Agrobacterium-mQdiatQd transformation of dissected explants (Tamas-Nyittrai et al, Methods in Molecular Biology 877:357-384 (2012)) or by biolistic approach (Zhang et al, Plant Cell Rep 19:241-250 (2000)).
  • a cassette containing a monocot BRIIACT gene operably linked to a ZmEEP5 type promoter and a suitable 3 ' terminator is cloned into the binary vector pAL156 (Tamas-Nyittrai et al., Methods in Molecular Biology 877:357-384 (2012)) or the JT based vector system.
  • the ZmEEP5 type promoter can be, but is not limited to, ZmEEP5 (SEQ ID NO: 1), OsPR602 or OsPR9a (Li et al (2008) Plant Biotechnology Journal 6: 465-476, SEQ ID NOs: 24 and 25), TdPR60 (SEQ ID NO: 47), TdPR91 (SEQ ID NO: 72), or TdGL7 (SEQ ID NO: 73).
  • the monocot BRIIACT gene can be ZmBRIlACT (SEQ ID NOs: 8, 9), OsBRIlACT (SEQ ID NO: 59), HvBRIlACT (SEQ ID NO: 63), SbBRIlACT (SEQ ID NO: 68), TaBRIlACT (SEQ ID NO: 69) or BdBRIlACT (SEQ ID NO: 70).
  • An Agrobacterium host carrying the resulting binary vector containing a monocot BRIIACT cassette is selected by the bar gene, which confers resistance to the herbicide phosphinothricin (White et al, Nucl Acids Res 18: 1062 (1990), Spencer et al, Theor Appl Genet 79: 625-631(1990), US4795855, US5378824 and US 6107549).
  • the binary clones are restriction digested and sequenced to verify that the cassettes have been cloned at the appropriate sites and that the junctions are intact.
  • Each binary vector containing a monocot BRIIACT cassette or an empty binary vector (to serve as control) is transformed into wheat immature embryo-derived scutella by Agrobacterium-mQdiatQd transformation and transgenic calli is selected on phosphinothricin.
  • Shoot and root formation of the transformed calli is induced by hormones and the resulting TO plantlets are grown up in the greenhouse.
  • TO tissue is collected and Southerns done to confirm presence of the BRIIACT transgene and to determine transgene insert number.
  • single insert events expressing BRIIACT are taken forward. About 15-30 TO events are obtained for each BRIIACT construct.
  • Tl mean seed number and weight is measured. These preliminary analyses are compared to the corresponding measurements from transgenic events carrying the empty vector. It is expected that Tl mean seed yield from transgenic BRIl ACT events is increased by about 1 %, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25% or more compared to the empty vector events.
  • tissues were ground under liquid nitrogen and ground tissue powder lysed in appropriate cold lysis buffer (50 mM Tris-HCl [pH 7.5], 10 mM EDTA, 150 mM NaCl, 50 mM NaF, 500 nM microcystin, 10% glycerol, and 0.5% Triton X-100) containing plant protease and phosphatase inhibitors (Sigma). Equal amounts of extract were mixed with 2X SDS buffer and separated by Tris-Glycine based SDS PAGE, and transferred to nitrocellulose membrane. Monoclonal anti-FLAG M2 antibodies (Sigma) were used to detect FLAG fusion proteins on immunoblots.
  • Goat anti-mouse HRP-conjugated secondary antibodies were used to detect bound mouse anti-FLAG M2 on the immunoblot. Signals were detected by incubating immunoblots with Super Signal Fempto Chemiluminescent solution and exposed to X-ray film. Immunoblots were reprobed with anti-PSTAIR (Millipore) to help control for protein loading between samples. Coomassie stained gels were also used to monitor loading of each sample.
  • Extracts were also subjected to immunoprecipitation experiments to monitor transgenic BRI kinase activity.
  • mouse anti-FLAG M2 affinity gel (Sigma) was added and incubated at 4°C. Beads were washed several times in lysis buffer and then washed in in vitro kinase buffer (50 mM HEPES [pH 7.4], 10 mM MgCl 2 , 10 mM MnCl 2 , 1 mM DTT) and then subjected to in vitro kinase assay using kinase buffer supplemented with 10 ⁇ ATP, 1 ⁇ (10 ⁇ ) [ ⁇ -32 ⁇ ] ATP in final volume of 24 ⁇ .
  • Reactions were incubated at 30°C and terminated by adding 8 ⁇ of 4X SDS loading buffer and boiling samples for 5 minutes. Samples were separated by Tris-Glycine SDS PAGE, stained, destained and dried and exposed to phosphoimager or X- ray film.
  • a hypersensitive ZmBRI 1 ACT with the embryo sac, early embryo, early endosperm, aleurone and/or basal endosperm transfer cell layer (BETL)-preferred promoter ZmEEP5 leads to an increase in ear grain weight.
  • the ZmBRI 1 ACT is a more active receptor and shows enhanced BR downstream signaling (Wang et al 2005 Developmental Cell 8: 855-865).
  • ZmBRI 1 The portion of ZmBRI 1 highlighted in FIG. 13 spans a region of 41 aa and contains 8 potential Serine/Threonine phosphorylation sites.
  • S1087, S1088, S1092, T1094, S1095, T1098, Ti l 12 and Tl 113 are all potential sites of phosphorylation, either individually or in various or all possible combinations.
  • CT variants to test examples include S1087D, S1088D, S1092D, T1094D, S1095D, T1098D, T1112D and T1113D.
  • CT variants can also be grouped in groups of 4 such as S1087D+S1088D+S1092D+T1094D, and S1095D+T1098D+T1112D +T1113D.
  • the CT variant could encompass all serine and threonine residues converted to the phospho- mimicking residues simultaneously,
  • CT variants can also include pairs of phosphomimic substitutions, groups of three, four, five, six and seven phosphomimic combinations. Additional CT variants also include shorter C- terminal deletions of SEQ ID NO: 51 (the full length ZmBRI polypeptide). CT variants having amino acid sequences such as (a.a.1-1111), (a.a.1-1097), (a.a.1-1086) that show similar or increased activity compared to wild type ZmBRI are further tested in plants.
  • An expression construct containing a ZmEEP5-CT variant ZmBRIl-3' UTR cassette is built and transformed into corn as described above.
  • the CT variant ZmBRIl is represented by SEQ ID NO: 79.
  • Regenerated transgenic TO plants are crossed to the recurrent inbred parent to generate Tl seeds segregating 1 : 1 for the transgene.
  • Plants grown from Tl seed are crossed with tester inbreds in a nursery to generate Fl hybrid seed that are planted in isolated crossing block (ICB) trials to measure ear grain weight compared to the null sibling controls. It is expected that plants containing the ZmEEP5- CT variant ZmBRIl -3' UTR transgene has increased ear grain weight compared to the control null plants not having the transgene.
  • ZmBRIl ACT is a more active receptor and shows enhanced BR downstream signaling (Wang et al. 2005).
  • LRR-RLKs have recently been shown to be dual specificity kinases (Oh, M.H. et al. 2009 Proc. Natl. Acad. Sci. USA 106: 658-663) and many of these LRR-RLKs, including AtBRIl and AtBAKl, undergo autophosphorylation on serine, threonine and tyrosine residues. Autophosphorylation of Y956 within the kinase domain of AtBRIl is required for kinase activity while phosphorylation of Y831 within the juxta-membrane region of AtBRIl is not essential for kinase activation but may play a role in further stimulation of kinase activity (Oh et al. 2009). Y831 of AtBRIl is conserved throughout most LRR RLKs. In particular, residue Y760 in ZmBRIl is the equivalent of AtBRIl Y831.
  • An expression construct containing a ZmEEP5 -full-length ZmBRIl (Y760F)-3' UTR cassette along with a ZmEEP5 -ZmBRIl ACT plus (Y760F)-3' UTR cassette are built and transformed into corn as described above.
  • the full-length ZmBRIl (Y760F) is represented by SEQ ID NO: 80.
  • the ZmBRIl ACT plus (Y760F) is represented by SEQ ID NO: 81.
  • Regenerated transgenic TO plants are crossed to the recurrent inbred parent to generate Tl seeds segregating 1 : 1 for the transgene.
  • Plants grown from Tl seed are crossed with tester inbreds in a nursery to generate Fl hybrid seed that are planted in isolated crossing block (ICB) trials to measure ear grain weight compared to the null sibling controls. It is expected that plants containing the ZmEEP5 -full-length ZmBRIl (Y760F)-3' UTR transgene or the ZmEEP5 -ZmBRI 1 ACT plus (Y760F)-3' UTR transgene has increased ear grain weight compared to the control null plants not having the transgene.
  • TILLING® Targeting Induced Local Lesions IN Genomes
  • TILLING® is a method in molecular biology that allows directed identification of mutations in a specific gene. TILLING® may be used to identify mutations of interest in key domains of BRI1 such as the juxtamembrane, kinase and CT domains. It is possible that nucleotide mutations induced by EMS can result in stop codons close to and within the C-terminal region of BRI1.
  • Primers designed to amplify a fragment of the TaBRI 1 genomic locus encompassing the C-terminal region of TaBRI 1 are used to screen a mutant wheat population such as that described in Slade, A. J. et al. (2005) (A reverse genetic, nontransgenic approach to wheat crop improvement by TILLING, Nat Biotechnol 23: 75-81) or in Uauy, C.F. et al. (2009) (A modified TILLING approach to detect induced mutations in tetraploid and hexaploid wheat, BMC Plant Biol 9:115). Mutations resulting in premature stop codons within the C-terminal domain of TaBRI 1 are identified.
  • Desired TaBRI 1 CT mutants are backcrossed to clean up unrelated genomic lesions caused by the EMS mutagen. Backcrossed TaBRI 1 CT mutants are tested in the field for increased biomass, increased growth, increased seed yield and/or other agronomic traits. The TaBRI 1 CT mutations are also introgressed into other desired wheat backgrounds to create other TaBRI 1 CT mutant varieties.
  • TILLING® Targeting Induced Local Lesions IN Genomes
  • TILLING® is a method in molecular biology that allows directed identification of mutations in a specific gene. TILLING® may be used to identify mutations of interest in key domains of BRIl like the juxtamembrane, kinase and CT domains. It is possible that nucleotide mutations induced by EMS can result in stop codons close to and within the C-terminal region of BRIl .
  • Primers designed to amplify a fragment of the ZmBRI 1 genomic locus encompassing the C-terminal region of ZmBRI 1 are used to screen a mutant corn population such as that described in Weil, C.F. and Monde, R.-A. (Getting the point— mutations in maize, The Plant Genome [A Supplement to Crop Science] January 2007 No. 1 : S60-S67) or in Weil, C.F. (TILLING in grass species, Plant Physiology, January 2009, Vol. 149, pp. 158-164).
  • primers designed to amplify a fragment of the OsBRIl genomic locus encompassing the C-terminal region of OsBRIl are used to screen a mutant rice population such as that described in Till, B.J. (Discovery of chemically induced mutations in rice by TILLING, BMC Plant Biology 2007, 7:19). Mutations resulting in premature stop codons within the C-terminal domain of ZmBRIl or OsBRIl are identified. Desired ZmBRIl CT and OsBRIl CT mutants are backcrossed to clean up unrelated genomic lesions caused by the EMS mutagen.
  • ZmBRIl CT and OsBRIl CT mutants are tested in the field for increased biomass, increased growth, increased seed yield and/or other agronomic traits.
  • the ZmBRIl CT and OsBRIl CT mutations are also introgressed into other desired backgrounds to create other ZmBRIl CT and OsBRIl CT mutant varieties.

Landscapes

  • Genetics & Genomics (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Organic Chemistry (AREA)
  • Biomedical Technology (AREA)
  • Chemical & Material Sciences (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Microbiology (AREA)
  • Physics & Mathematics (AREA)
  • Plant Pathology (AREA)
  • Biophysics (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Cell Biology (AREA)
  • Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

La présente invention concerne des vecteurs d'expression comprenant des polynucléotides codant pour un récepteur à répétitions riches en leucine polypeptide brassinostéroïde insensible 1 (BRI1) à double spécificité sérine/thréonine/tyrosine kinase ou BRI1 tronqué (BRI1ΔCT), et leurs procédés d'utilisation. De plus, l'invention concerne des plantes transgéniques exprimant lesdits polynucléotides. En outre, l'invention concerne des procédés d'augmentation de la dimension moyenne des semences, du poids des semences et/ou du rendement d'une plante.
PCT/US2014/024447 2013-03-15 2014-03-12 Compositions et procédés pour améliorer le nombre de semences végétales et/ou le rendement d'une plante Ceased WO2014150879A1 (fr)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US201361792136P 2013-03-15 2013-03-15
US61/792,136 2013-03-15
US201361806168P 2013-03-28 2013-03-28
US61/806,168 2013-03-28

Publications (1)

Publication Number Publication Date
WO2014150879A1 true WO2014150879A1 (fr) 2014-09-25

Family

ID=51580847

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2014/024447 Ceased WO2014150879A1 (fr) 2013-03-15 2014-03-12 Compositions et procédés pour améliorer le nombre de semences végétales et/ou le rendement d'une plante

Country Status (1)

Country Link
WO (1) WO2014150879A1 (fr)

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109097364A (zh) * 2018-09-03 2018-12-28 深圳广三系农业科技有限公司 一种植物胚乳特异性表达启动子pOsEnS100的鉴定和应用
WO2023019188A1 (fr) * 2021-08-12 2023-02-16 Pairwise Plants Services, Inc. Modification de gènes du récepteur des brassinostéroïdes pour améliorer des caractéristiques de rendement
CN116445523A (zh) * 2023-04-10 2023-07-18 西北农林科技大学 一种小麦抗逆性基因TaBSL3与应用
CN117866984A (zh) * 2024-03-13 2024-04-12 中国科学院遗传与发育生物学研究所 抑制小麦淀粉合成的转录因子TaABI3及其应用
WO2024186950A1 (fr) * 2023-03-09 2024-09-12 Pairwise Plants Services, Inc. Modification de gènes de la voie de signalisation de brassinostéroïdes pour améliorer des caractères de rendement chez des plantes
CN120888563A (zh) * 2025-09-08 2025-11-04 河南省农业科学院中药材研究所 一种忍冬花色素合成基因及其在调控植物花色素含量和抗性中的应用
CN120944848A (zh) * 2025-10-20 2025-11-14 山东大学 小麦TaPPKL3突变体及应用

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20020073446A1 (en) * 1999-03-16 2002-06-13 Neff Michael M. Genetically modified plants having modulated brassinosteroid signaling
US6573438B1 (en) * 1997-03-14 2003-06-03 Syngenta Participations Ag Inbred Maize line 2044BT
US20040088761A1 (en) * 2000-03-31 2004-05-06 Hiroshi Tanaka Gene concerning brassinosteroid-sensitivity of plants and utilization thereof
US20080295196A1 (en) * 2006-12-06 2008-11-27 Abad Mark S Genes and uses for plant improvement
US20090293143A9 (en) * 2004-09-01 2009-11-26 Kunsheng Wu Zea mays ribulose bisphosphate carboxylase activase promoter
US20100313301A1 (en) * 2009-06-09 2010-12-09 Pioneer Hi-Bred International, Inc. Early Endosperm Promoter and Methods of Use
US20110107464A1 (en) * 2008-06-26 2011-05-05 Basf Plant Science Gmbh Plants Having Enhanced Yield-Related Traits and a Method for Making the Same

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6573438B1 (en) * 1997-03-14 2003-06-03 Syngenta Participations Ag Inbred Maize line 2044BT
US20020073446A1 (en) * 1999-03-16 2002-06-13 Neff Michael M. Genetically modified plants having modulated brassinosteroid signaling
US20040088761A1 (en) * 2000-03-31 2004-05-06 Hiroshi Tanaka Gene concerning brassinosteroid-sensitivity of plants and utilization thereof
US20090293143A9 (en) * 2004-09-01 2009-11-26 Kunsheng Wu Zea mays ribulose bisphosphate carboxylase activase promoter
US20080295196A1 (en) * 2006-12-06 2008-11-27 Abad Mark S Genes and uses for plant improvement
US20110107464A1 (en) * 2008-06-26 2011-05-05 Basf Plant Science Gmbh Plants Having Enhanced Yield-Related Traits and a Method for Making the Same
US20100313301A1 (en) * 2009-06-09 2010-12-09 Pioneer Hi-Bred International, Inc. Early Endosperm Promoter and Methods of Use

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
LI ET AL.: "A Putative Leucine-Rich Repeat Receptor Kinase Involved in Brassinosteroid Signal Transduction", CELL, vol. 90, 5 September 1997 (1997-09-05), pages 929 - 938 *
SCHNABLE ET AL.: "The B73 maize genome: complexity, diversity, and dynamics", SCIENCE, vol. 326, 20 November 2009 (2009-11-20), pages 1112 - 1115 *

Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109097364A (zh) * 2018-09-03 2018-12-28 深圳广三系农业科技有限公司 一种植物胚乳特异性表达启动子pOsEnS100的鉴定和应用
CN109097364B (zh) * 2018-09-03 2022-02-11 深圳广三系农业科技有限公司 一种植物胚乳特异性表达启动子pOsEnS100的鉴定和应用
WO2023019188A1 (fr) * 2021-08-12 2023-02-16 Pairwise Plants Services, Inc. Modification de gènes du récepteur des brassinostéroïdes pour améliorer des caractéristiques de rendement
US12365911B2 (en) * 2021-08-12 2025-07-22 Pairwise Plants Services, Inc. Modification of brassinosteroid receptor genes to improve yield traits
WO2024186950A1 (fr) * 2023-03-09 2024-09-12 Pairwise Plants Services, Inc. Modification de gènes de la voie de signalisation de brassinostéroïdes pour améliorer des caractères de rendement chez des plantes
CN116445523A (zh) * 2023-04-10 2023-07-18 西北农林科技大学 一种小麦抗逆性基因TaBSL3与应用
CN116445523B (zh) * 2023-04-10 2024-05-07 西北农林科技大学 一种小麦抗逆性基因TaBSL3与应用
CN117866984A (zh) * 2024-03-13 2024-04-12 中国科学院遗传与发育生物学研究所 抑制小麦淀粉合成的转录因子TaABI3及其应用
CN120888563A (zh) * 2025-09-08 2025-11-04 河南省农业科学院中药材研究所 一种忍冬花色素合成基因及其在调控植物花色素含量和抗性中的应用
CN120944848A (zh) * 2025-10-20 2025-11-14 山东大学 小麦TaPPKL3突变体及应用
CN120944848B (zh) * 2025-10-20 2025-12-23 山东大学 小麦TaPPKL3突变体及应用

Similar Documents

Publication Publication Date Title
DK2281895T3 (en) Methods for increasing stress tolerance in plants and compositions thereof
US9745596B2 (en) Identification and use of KRP mutants in wheat
US10913954B2 (en) Abiotic stress tolerant plants and methods
US10443069B2 (en) Plants and methods to improve agronomic characteristics under abioticstress conditions
WO2014160304A1 (fr) Adaptation améliorée du maïs
US20170142919A1 (en) Identification and the use of krp mutants in plants
US10662435B2 (en) Plants having altered agronomic characteristics under abiotic stress conditions and related constructs and methods involving genes encoding NAC3/ONAC067 polypeptides
WO2014150879A1 (fr) Compositions et procédés pour améliorer le nombre de semences végétales et/ou le rendement d'une plante
US10465203B2 (en) Plants having altered agronomic characteristics under abiotic stress conditions and related constructs and methods involving abiotic stress tolerance genes
CN104004767A (zh) Wrky转录因子多肽基因应用的载体和方法
US20150299723A1 (en) Plants with altered root architecture, related constructs and methods involving genes encoding leucine rich repeat kinase (llrk) polypeptides and homologs thereof
US8394634B2 (en) Plants having altered agronomic characteristics under nitrogen limiting conditions and related constructs and methods involving genes encoding LNT2 polypeptides and homologs thereof
JP2009540822A (ja) 植物の構造及び成長を調節するための植物クロマチンリモデリング遺伝子の使用
WO2013192081A1 (fr) Gène de terminaison de fleur (tmf) et procédés d'utilisation
US10626410B2 (en) Plants having altered agronomic characteristics under nitrogen limiting conditions and related constructs and methods involving low nitrogen tolerance genes
US20170306346A1 (en) Improved agronomic characteristics under water limiting conditions for plants expressing pub10 polypeptides
US20140215653A1 (en) Identification and use of early embryo and/or early endosperm specific promoters for gene expression in maize
TW201718860A (zh) 用於作物產量之增強的基因改質植物
WO2014164668A1 (fr) Compositions et procédés permettant d'accroître le nombre et le poids des semences et/ou le rendement chez les plantes
US20170183680A1 (en) Dominant negative mutant krp-related proteins (krp) in zea mays and methods of their use
US20180066026A1 (en) Modulation of yep6 gene expression to increase yield and other related traits in plants
WO2015102999A9 (fr) Plantes résistantes à la sécheresse, constructions associées et procédés impliquant des gènes codant pour des polypeptides dtp4
US20160032304A1 (en) Slm1, a suppressor of lesion mimic phenotypes
US20110035837A1 (en) Plants having altered agronomic characteristics under nitrogen limiting conditions and related constructs and methods involving genes encoding lnt3 polypeptides
WO2015193653A1 (fr) Gènes et protéines chimériques de résistance à l'oxydation et plantes transgéniques les comprenant

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 14767835

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

32PN Ep: public notification in the ep bulletin as address of the adressee cannot be established

Free format text: NOTING OF LOSS OF RIGHTS PURSUANT TO RULE 112(1) EPC, FORM 1205A DATED 04-01-2016

122 Ep: pct application non-entry in european phase

Ref document number: 14767835

Country of ref document: EP

Kind code of ref document: A1