WO2014164668A1 - Compositions et procédés permettant d'accroître le nombre et le poids des semences et/ou le rendement chez les plantes - Google Patents

Compositions et procédés permettant d'accroître le nombre et le poids des semences et/ou le rendement chez les plantes Download PDF

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WO2014164668A1
WO2014164668A1 PCT/US2014/023172 US2014023172W WO2014164668A1 WO 2014164668 A1 WO2014164668 A1 WO 2014164668A1 US 2014023172 W US2014023172 W US 2014023172W WO 2014164668 A1 WO2014164668 A1 WO 2014164668A1
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Prior art keywords
plant
gene
expression cassette
promoter
expression
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PCT/US2014/023172
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English (en)
Inventor
Jean Paul OLIVER
Carolyn Hutcheon
Asha Palta
George Singletary
Stefan DITTMAR
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Targeted Growth, Inc.
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Publication of WO2014164668A1 publication Critical patent/WO2014164668A1/fr

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

Definitions

  • the invention generally relates to methods for increasing crop yield. More specifically, the present invention relates to methods and compositions for increasing plant seed weight, seed size, seed number and/or yield by expressing a gene of interest in plants under the control of specific promoter-intron combinations.
  • 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 for expression of a gene of interest.
  • the expression cassettes of the present invention avoid deleterious effects of strong promoters
  • the expression cassettes of the present invention can be used to express a gene of interest, e.g., a plant REVOLUTA gene, to increase seed number, seed size, and'Or seed yield, without causing any negative pleiotropic effects in the plant
  • the plant is selected from the group consisting of Zea mays, Oryza sativa.
  • Glycine max Canola, Hordeum vidgare, Triticum spp, Sorghum bicolor, Vitis vinifera, Lycopersicon esculentum, Zinnia elegans, Populus trichocarpa, Lotus japonicus, Brachypodium distachyon, and P yscomiirella patens.
  • the expression cassettes comprise a plant promoter sequence, an intron sequence, and a gene of interest.
  • the plant promoter sequence is a constitutive plant promoter.
  • the gene of interest is a gene encoding a functional plant REVOLUTA polypeptide, such as a polypeptide capable of carrying out all functions of a wild type REVOLUTA protein.
  • the plant promoter sequence, the intron sequence, and the gene of interest are operatively linked.
  • the intron attenuates the strength of the plant promoters to avoid deleterious effects of strong promoters. For example, the strength of the plant promoter with the intron is altered, reduced, delayed, and/or limited when compared to the strength of the plant promoter without the intron or when compared to the strength of the plant promoter with its endogenous! ⁇ ' associated intron.
  • the intron is heterologous to the plant promoter.
  • the intron is a modified one derived from the intron that is endogenous ly associated with the plant promoter so it gives a different expression level relative to the unmodified, endogenous intron.
  • the gene of interest when expressed in the plant under the control of the same promoter sequence but without the control of the intron sequence or with its endogenously associated intron would lead to one or more negative pleiotropic effects in the plant.
  • the negative pleiotropic effects include, but are not limited to, reduced axillary branches and wrinkled leaves.
  • the gene of interest when expressed in a maize plant under the control of the promoter sequence and the intron sequence would lead to increased yield.
  • the increased yield is due to increased seed weight, seed size, and/or seed number.
  • the intron sequence is derived from a plant alcohol dehydrogenase gene.
  • the intron is derived from a rice or a maize plant.
  • the intron comprises SEQ ID NO: 1, or functional fragments or variants thereof, including but are not limited to (!) sequences having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%), 99.5% or more nucleic acid identity to SEQ ID NO: 1; (2) sequences derived from a plant alcohol dehydrogenase gene of plant species other than maize, wherein the plant alcohol dehydrogenase gene of another plant species shares at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% amino acid identity to the maize alcohol dehydrogenase gene; (3) sequences that can hybridize to SEQ ID NO: 1 under stringent
  • said 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 dodecyi 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 dodecyi sulfate, at 55°C to 65°C; (4) functional fragments of any one or more of the intron sequences disclosed herein.
  • the fragment comprises about 20bp, 25bp, 30bp, 35bp, 40bp, 45bp, 50bp, 55bp, 60bp, 65bp, 70bp, 75bp, 80bp, 85bp, 90bp, 95bp, lOObp, l lObp, 120bp, 130hp, 140bp, 150bp, 160bp, 170bp, 180bp, 190bp, 200bp, 210bp, 220bp, 230bp, 240bp, 250bp, 260bp, 270bp, 280bp, 290bp, 300bp, 310bp, 320bp, 330bp, 340bp, 350bp, 360bp, 370bp, 380bp, 390bp, 400bp, 410bp, 420bp, 430bp, 440bp, 450bp, 460bp, 470bp,
  • the gene of interest encodes a plant growth and/or development protein, such as a HD-Zip transcription factor, a NAC-containing transcription factor, a BHLH transcription factor, a MYB transcription factor, an APETALA2-like transcription factor, a SBP-like transcription factor, a SCL. transcription factor, an ARF transcription factor, an F-box protein, AINTEGUMENTA (Mizukami and Fischer (2000) Proc. Natl Acad. Sci. USA, 97: 942-947), DWARF4 (Choe, S. et al. (2001) Plant J. 26: 573- 582), AHK ( iefler, M. et al.
  • a plant growth and/or development protein such as a HD-Zip transcription factor, a NAC-containing transcription factor, a BHLH transcription factor, a MYB transcription factor, an APETALA2-like transcription factor, a SBP-like transcription factor, a SCL. transcription factor, an ARF transcription factor
  • the HD-Zip transcription factor can be the REVOLUTA (REV), PHABULOSA (PHB), PHAV OLUTA (PHV), ⁇ 8, or ATHB15;
  • the NAC-containing transcription factor can be NAC1, CUC1 , or CUC2;
  • the BHLH transcription factor can be TCP2, TCP3, TCP4, TCP 10, or TCP24;
  • the MYB transcription factor can be MYB33, MYB65, or GAMYB;
  • the APETALA2-like transcription factor can be AP2, ⁇ 1 , ⁇ 2, ⁇ 3, or GL15;
  • the SBP-like transcription factor can be SPL3, SPL4, or SPL5,
  • the SCL transcription factor can be SCL6-IL or SCL6-III,
  • the ARF transcription factor can be ARF6, ARF 10, ARF16, ARF17, or ARF18;
  • the F-box protein can be TIR1.
  • the gene of interest is an interference RNA or other non-coding
  • the gene of interest encodes a REV LUTA protein.
  • the gene of interest is a corn REVOLUTA protein or functional fragments or functional variants thereof.
  • the gene of interest is a REVOLUTA protein or functional fragments or functional variants thereof derived from other plant species, such as those described in U.S. Patent No. 7816139 or those known to a person skilled in the art.
  • the gene encodes a REVOLUTA protein comprising SEQ ID NO: 4 or functional fragments or functional variants thereof.
  • the plant promoter sequence is a constitutive promoter sequence.
  • the constitutive promoter sequence is selected from the group consisting of viral promoters, ubiquitin promoters, and actin promoters such as ones described in Hermann et al. (The banana actin 1 promoter drives near-constitutive transgene expression in vegetative tissues of banana (Musa spp.), Plant Cell Reports 20: 525-530 (2001), SEQ ID NO: 50), He et al.
  • the actin promoter is a rice actin promoter or a corn actin promoter.
  • the rice actin promoter comprises SEQ ID NO: 5, or functional fragments or functional variants thereof.
  • the corn actin promoter comprises SEQ ID NO: 2, or functional fragments or functional variants thereof.
  • the viral promoter is a a promoter derived from Cauliflower mosaic vims (CaMV), Mirabilis mosaic virus (MMV), or Figwort mosaic virus (FMV), such as the Cauliflower mosaic virus (CaMV) 35S promoter (e.g., SEQ ID NO: 66), Mirabilis mosaic vims sub-genomic transcript 13 promote (e.g., SEQ ID NO: 67), Figwort mosaic virus promoter (e.g., SEQ ID NO: 68), Figwort mosaic virus sub-genomic transcript 3 promoter (e.g., SEQ ID NO: 69), or Figwort mosaic virus sub-genomic transcript 10 promoter (e.g., SEQ ID NO: 70).
  • Cauliflower mosaic vims Cauliflower mosaic vims
  • MMV Mirabilis mosaic virus
  • FMV Figwort mosaic virus
  • the plant promoter sequence is heterologous to the intron sequence and/or the gene of interest. In some embodiments, the intron sequence is heterologous to the gene of interest.
  • the present invention also provides expression vectors for expressing a gene of interest in a plant comprising any one of the expression cassettes of the present invention.
  • the vectors further comprise a gene termination sequence.
  • the present invention also provides non-human transgenic cells.
  • the non-human transgenic ceil comprises the expression cassettes described herein.
  • the transgenic cell is a maize plant cell, an animal cell, a bacterial cell, and a fungal ceil.
  • the present invention also provides organisms comprising the expression cassettes described herein.
  • the organism is a maize plant.
  • the expression cassette is incorporated into the plant cell by transformation or homologous recombination.
  • the present invention also provides parts of the organisms comprising the expression cassettes described herein.
  • the part is a seed, wherein the seed comprises the expression cassette.
  • the present invention also provides progeny plants of the transgenic plants described herein.
  • the progeny plants have the expression cassette described herein.
  • the present invention also provides methods for producing transgenic plants, plant parts, or plant cells.
  • the methods are used to produce seeds.
  • the methods comprise crossing a transgenic plant or the progeny of the transgenic plant described herein as a donor with a recipient plant.
  • the donor plant and the recipient plant belong to the same or different varieties.
  • the present invention also provides methods of increasing seed weight, seed size, seed number and/or yield in a plant compared to a wild-type control plant.
  • the methods comprise incorporating into a plant cell the expression cassette of the present invention.
  • the expression cassette comprises a rice actin promoter, a maize ADH1 intron, and a maize REV gene.
  • the rice actin promoter comprises SEQ ID NO: 5.
  • the maize ADH1 intron comprises SEQ ID NO: 1.
  • the maize REV gene comprises a nucleic acid sequence encoding a protein comprising SEQ ID NO: 4.
  • the present invention also provides methods of producing hybrid seed comprising crossing the plant of the present invention or a progeny plant of the present invention with a different plant of the same species, and harvesting the resultant seed.
  • the present invention also provides methods of breeding plants to produce a plant having an expression cassette of the present invention.
  • the methods comprise making a cross between a plant with an expression cassette of the present invention with a second plant to produce an F l plant.
  • the methods further comprise backcrossing the Fl plant to the second plant.
  • the methods further comprise 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 invention also provides products derived from the transgenic plants described herein. Any and all products made using the seeds, plants and parts thereof obtained from the transgenic plants or from any line produced using the transgenic plants described herein as a direct or indirect parent are also part of the invention. Examples of such products include but are not limited to corn meal, com flour, corn starch, corn syrup, corn sweetener and com oil.
  • the origin of the corn used in such corn products can be determined by tracking the source of the com used to make the products and/or by using protein (isozyme, ELISA, etc.) and/or DNA (RFLP, PGR, SSR, SNP, EST, etc.) testing.
  • the present invention also provides expression cassettes to express a REVOLUTA gene in a plant to achieve increased seed size, seed number, and/or seed yield, wherein the expression cassettes comprise a plant actin promoter.
  • a plant actin promoter is a constitutive promoter, it has the abilit ⁇ ' to express a REVOLUTA gene in a plant at an appropriate level therefore to achieve desired phenotypes, such as increased seed size, seed number, and/or seed yield, without causing any negative pleiotropic effect or a particular negative pleiotropic effect in the plant.
  • the REVOLUTA. gene encodes a functional REVOLUTA. polypeptide, such as a polypeptide capable of carrying out all functions of a wild type REVOLUTA protein.
  • the plant actin promoter and the gene encoding a REVOLUTA protein are operatively linked, in some embodiments, the expression cassette further comprises an intron.
  • the intron is homologous or heterologous to the actin promoter.
  • the intron is an actin intron.
  • the actin promoter is a rice actin promoter or a com actin promoter.
  • the rice actin promoter comprises SEQ I D NO: 5, functional variants thereof, functional fragments thereof, or orthoiogs thereof.
  • the com actin promoter comprises SEQ ID NO: 2, functional variants thereof, functional fragments thereof, or orthoiogs thereof.
  • the intron comprises SEQ ID NO: I or functional fragments or variants thereof.
  • the present invention also provides methods of increasing seed number and/or yield in a plant compared to a wild-type control plant, said methods comprise incorporating into a plant cell the expression cassette in which a REVOLUTA gene is under the control of a plant aetin promoter, with or without an mtrori.
  • FIG. 1 A depicts a TG_Zm 179 corn callus event with strong GUS expression.
  • FIG. IB shows a representative TG Zm 180 corn callus event with weak GUS expression
  • FIG. 1 C shows a representative TG_Zm 180 corn callus event with undetectable GUS expression.
  • FIG. 2 depicts strong GUS expression for TG Zm 179 in Tl embryo, endosperm and aleurone at 21 days after pollination (DAP).
  • FIG. 2B shows strong GUS expression for TG Zm 179 in T2 7 DAP immature/developing ear.
  • FIG. 3 depicts strong GUS expression for TG_Zm 179 in TO leaf, stalk, root, pollen, anther and silk.
  • FIG. 5 depicts a grand summary of the effect of TG_Zm 137 construct on ear grain production, as a percentage of null ear grain weight, across two years compared with other efficacious and non-efficacious constructs.
  • 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.
  • the total number of corn ears (each measured individually) involved in calculating the mean is shown atop the standard error bar. In general, half of the ears tested were Null and hal f Transgenic.
  • FIG. 6 depicts the percent of null ear grain weight (y-axis) plotted against the percent of null kernel number/ear (x-axis) for TG Zm 137 events across four locations in year 1 . There is good correlation between the transgene effect on kernel number per ear and effect on ear grain weight, as reflected in the R 2 value.
  • FIG. 7 depicts REVOLUTA transgene levels measured by qPCR relative to event 138-01 using the AACT method. Transgene copy number is mdicated below each event name and was determined using the CopyCaller assay (Applied Biosystems).
  • FIG. 8 depicts GUS transgene levels measured by qPCR relative to event TG Zm
  • FIG. 9 depicts transformation efficiencies for various constitutive promoter-intron- Zm REV constructs. (1) and (2) are 2 rounds of transformation initiated for TG Zm 21 1 .
  • Sequence listings for SEQ ID NOs: 1- 71 are part of this application and are incorporated by reference herein. Sequence listings are provided at least at the end of this document,
  • the invention provides compositions and methods to produce plants having increased seed size, seed weight, 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 moiiocotyledonous plants, also called monocots, and dicotyledonous plants, also called dicots.
  • Examples of particular plants include but are not limited to com, potatoes, roses, apple trees, sunflowers, wheat, rice, barley, soybean, canola, sorghum, grapes, Populus irichocarpa, Lotus japonicas, Brachypodium distachyon, and Physcomitrella patens bananas, tomatoes, 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 RNA 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 cytidyiate or deoxycytidylate, “G” for guanylate or deoxyguanylate, “U” for uri.dyl.ate, “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 cytidyiate or deoxycytidylate
  • G for guanylate or deoxyguanylate
  • U for uri.dyl.ate
  • T for deoxythymid
  • 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 deoxyribonueleotides, 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, or any isolated mutants), 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 regulator ⁇ ' 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 orthoiogous 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 (P.M. Ausubel et ai, eds., 1987) Supplement 30, section 7.718, Table 7.71 , Some alignment programs are Mac Vector (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.
  • nucleotide change refers to, e.g., nucleotide substitution, deletion, and/or insertion, as is well understood in the art. For example, 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 invention provides agents to make and use the biological materials of the present invention.
  • 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.
  • the invention provides sequences having high similarity or identity to the nucleic acid sequences and polypeptide sequences of the present invention.
  • 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.
  • sequence identity or “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.
  • 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 f - 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.
  • 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 regulator ⁇ ' element activity.
  • variants of a particular polynucleotide or nucleic acid molecule 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 as determined by sequence alignment programs and parameters as described elsewhere herein.
  • Variant polynucleotides also encompass sequences derived from a mutagenic and recombinogemc 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, Plamview, New York). See also Innis et al., eds.
  • PCR Protocols A Guide to Methods and Applications (Academic Press, New York); Innis and Gelfand, eds. (1995) PCR Strategies (Academic Press, New York); and Innis and Gelfand, eds. (1999) PCR Methods Manual (Academic Press, New York).
  • Known methods of PCR include, but are not limited to, methods using paired primers, nested primers, single specific primers, degenerate primers, gene-specific primers, vector-specific primers, partially-mismatched primers, and the like.
  • 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 2*SSC 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 *SSC at 60° C.
  • Hybridization procedures are well known in the art and are described by e.g. Ausubel et al., 1998 and Sambrook et ai, 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 5*SSC, containing 0.1% (w7v) 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 RNA.
  • 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 DN A segments.
  • promoters may direct the expression of a gene in different tissues or cel l 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 regulator ⁇ ' sequences have not been completely defined, DNA fragments of some variation may have identical promoter activity.
  • the invention provides plant promoters.
  • a "plant promoter” is a promoter capable of initiating transcription in plant cel ls 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 35S 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, ceil 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 ceils.
  • 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 ceils in roots, leaves, stalk ceils, and stem cells.
  • 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 introns containing enhancer like characteristics. Such intron effects are commonly refered to as intron mediated enhancement (IME). Unlike traditional enhancers, introns associated with IME are not comprised of discrete sequences. Rather, an enhancing intron involves a region that is transcribed and preferentially located near the 5' end of the start of transcription.
  • intron mediated enhancement IME
  • an enhancing intron involves a region that is transcribed and preferentially located near the 5' end of the start of transcription.
  • 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 poiyadenylic acid tracts to the 3' end of the mRNA precursor.
  • the use of different 3' non-coding sequences is exemplified by mgelbreeht, I. L, et al. (1989) Plant Cell 1 :671-680.
  • RNA transcript refers to the product resulting from RNA polymerase-eatalyzed transcription of a D ' NA sequence. When the RNA transcript is a perfect complementary copy of the DNA sequence, it is referred to as the primary transcript. An RNA transcript is referred to as the mature RNA when it is an RNA sequence derived from post-transcriptional processing of the primary transcript.
  • Messenger RNA (mRNA) refers to the RNA that is without introns and that can be translated into protein by the cell.
  • cDNA refers to a DNA that is complementary to and synthesized from an mRNA template using the enzyme reverse transcriptase.
  • the cDNA can be single-stranded or converted into the double-stranded form using the Klenow fragment of DNA polymerase I.
  • Sense RN A refers to RNA transcript that includes the mRNA. and can be translated into protein within a cell or in vitro.
  • Antisense RNA refers to an RNA transcript that is complementary to ail or part of a target primary transcript or mRNA, and that blocks the expression of a target gene (U.S. Pat. No. 5,107,065). The complementarity of an antisense RNA may be with any part of the specific gene transcript, i.e., at the 5' non- coding sequence, 3' non-coding sequence, introns, or the coding sequence.
  • RNA refers to antisense RNA, ribozyme RNA, or other RNA that may not be translated but yet has an effect on cellular processes.
  • complement and “reverse complement” are used interchangeably herein with respect to mRNA transcripts, and are meant to define the antisense RNA of the message.
  • the invention provides recombinant genes in which a gene of interest is operabiy linked to a promoter sequence.
  • operbiy 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 operabiy 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 operabiy linked to regulator ⁇ ' sequences in a sense or antisense orientation.
  • the complementary RNA regions of the invention can be operabiy 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 mRN A.
  • the invention provides recombinant expression cassettes and recombinant constructs.
  • the phrases "recombinant construct”, “expression construct”, “chimeric construct”, “construct”, and “recombinant DNA construct” are used interchangeably herein.
  • a recombinant construct comprises an artificial combination of nucleic acid fragments, e.g., regulator ⁇ ' 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 regulator 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 plasm Id 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.
  • 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 poiy-iysme-eonju gated 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 sellings 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 sellings 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.
  • the terms “beterozygote” 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 celi 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 ceil or organism has at least one heterologous trait.
  • heterologous trait refers to a phenotype imparted to a transformed host ceil 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.
  • 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 pbenotypic 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.
  • the invention provides plant tissue comprising recombinant genes.
  • 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.
  • the term ''transformation refers to the transfer of nucleic acid (i.e., a nucleotide polymer) into a cell.
  • the term “genetic transformation” refers to the transfer and incorporation of DNA, especially recombinant DNA, into a cell.
  • the invention provides transformants comprising recombinant genes.
  • trans formant refers to a cell, tissue or organism that has undergone transformation.
  • the original transformatit is designated as “TO” or “To.”
  • Selfing the TO produces a first transformed generation designated as “Tl” or “Tj .”
  • 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 the 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. As used herein "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 regulator ⁇ ' 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 f 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), ⁇ -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, including enhanced variants such as enhanced GFP (eGFP)).
  • GFP green fluorescent protein
  • DsRed red fluorescent protein
  • eGFP red fluorescent protein
  • cyan fluorescent protein or variants thereof, including enhanced variants such as enhanced GFP (eGFP)
  • 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. REVOLUTA
  • REVOLUTA is a homeodomain leucine zipper transcription factor belonging to subfamily III (HD-ZIP III) that has multiple functions in plant development. It controls meristem and organ growth, establishes cell fate and polarity, and controls vascular development (Talbert et al 1995, Development, 121(9): 2723-2735; Otsuga et al 2001, Plant Journal, 25(2): 223-236; Zhong and Ye 1999, Plant Cell, 1 1 (11): 2139-2152).
  • miRNAs originate from distinct loci within a plant's genome and are short non-coding RNAs (20-24 nucleotides (nt) in length) whose function is to repress the expression of defined target genes (Rhoades et al , Cell 1 10:513-520, 2002; Bonnet et al, Proc. Natl. Acad. Sci. USA, 101 :1151 1 - 1 1516, 2004; Reinhart et al, Genes Dev. 16:1616-1626, 2002).
  • miRNAs are generated from longer precursor molecules by a Dicer-like (DCL) ribonuciease and get incorporated into ribonucleoprotein silencing complexes that effect repression of target mRNAs via base pairing of the small RNA and its target mRNA (Chen, Science 303:2022-2025, 2004; Bao et al, Dev. Cell. 7:653-662, 2004).
  • DCL Dicer-like
  • REV and the other four members of the HD-Zip III family have miRNA binding sites in their START (sterol lipid binding) domains that are complementary to the miRNAs designated 165 and 166.
  • REVOLUTA nucleic acid sequences and polypeptide sequences that can be used in the present invention include, but are not limited to:
  • wild type protein SEQ ID NO: 4; wild type cds, SEQ ID NO: 3
  • wild type protein SEQ ID NO: 8; wild type cds, SEQ ID NO: 9
  • REVOLUTA sequences isolated ixom other plant species which are homologous to the REVOLUTA sequences described in (1), such as sequences sharing at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more identity to the REVOLUTA sequences in (1), wherein the sequences have REVOLUTA functions;
  • REVOLUTA sequences isolated from other plant species which when expressed in an Arabidopsis thaliana or maize REVOLUTA loss-of-function mutant can complement the endogenous wild type REVOLUTA function.
  • Expression cassettes and vectors The present invention provides expression cassettes and expression vectors for expression of a gene of interest.
  • the expression cassettes and expression vectors comprise a pl ant promoter sequence, an intron sequence, and a gene of interest.
  • the gene of interest when expressed in a maize plant under the control of the same promoter sequence but without the control of the intron sequence would lead to one or more negative pleiotropic effects in the plant.
  • introduction of one or more intron sequences between the promoter sequence and the gene of interest can lead to expression of the gene of interest in the plant without causing any negative pleiotropic effects.
  • expression of the gene of interest brings in one or more desired phenotvpes when the negative pleiotropic effects are eliminated. Therefore, the present invention provides a method to express a gene of interest in a plant to achieve desired effects while avoiding negative pleiotropic effects.
  • the plant promoter is heterologous to the intron sequence and/or the gene of interest.
  • endogenous genes are usually under the control of their own promoters, and the position, timing, and amount of their expressions are highly controlled and balanced during development to achieve their functions as programmed, which is a result of evolution.
  • a gene of interest under a non-native promoter e.g., a promoter heterologous to the gene, such as a plant promoter derived from the same plant species, but not endogenously linked with the gene of interest, or a promoter derived from other species, may cause expression in the wrong tissues, cells or cell compartments, wrong timing of expression, and/or wrong expression level, which in turn leads to one or more negative pleiotropic effects.
  • a promoter heterologous to the gene such as a plant promoter derived from the same plant species, but not endogenously linked with the gene of interest, or a promoter derived from other species
  • a promoter heterologous to the gene such as a plant promoter derived from the same plant species, but not endogenously linked with the gene of interest, or a promoter derived from other species, may cause expression in the wrong tissues, cells or cell compartments, wrong timing of expression, and/or wrong expression level, which in turn leads to one or more negative pleiotropic effects.
  • the site of insertion of the expression cassette can influence how the gene of interest is expressed from its endogenous promoter.
  • some synthetic plant promoters may also cause negative pleiotropic effects because they have not been selected by evolution and may not be compatible in plants.
  • such a promoter is a constitutive promoter. Expression of certain genes under a constitutive promoter is more likely to cause negative pleiotropic effects because the gene will be expressed constantly in all plant cells under most conditions and/or during most development stages. Examples of such constitutive promoters include, but are not limited to, CaMV 35S promoter, opine promoters, ubiquitin promoter, alcohol dehydrogenase promoter, and actin promoters such as ones described in Hermann et al, (The banana actinl promoter drives near-constitutive transgene expression in vegetative tissues of banana (Musa spp.), Plant Cell Reports 20: 525-530 (2001), SEQ ID NO: 50), He et al.
  • Genes regulating plant cell growth and/or division are more likely to cause negative pleiotropic effects when expressed under the control of a heterologous promoter.
  • One such gene of interest is REVQLUTA.
  • the present invention provides a simple solution to the issue by including an intron sequence in the expression cassettes or expression vectors.
  • genes of interest which when expressed in a plant may cause negative pleiotropic effects include, but are not limited to heterologous genes to a given plant species, genes involved in chromosome modification, genes involved in homeostasis regulation, genes involved in cell wall synthesis and modification, genes involved in stress responses, etc., such as those described in Branch et al. (The pleiotropic effects of the bar gene and glufosinate on the Arabidopsis transeriptome, Plant Biotechnol J. 2009 Apr;7(3):266-82), arlowski et al. (The over-expression of an alfalfa RING-H2 gene induces pleiotropic effects on plant growth and development, Plant Mol Biol.
  • genes are involved in processes such as ovule development (AINTEGUMENTA), brassinosteroid synthesis (DWARF4), cytokinin sensing (AHK), ubiquitination (GW2), auxin transport (AVP1), cell proliferation in leaf primordia (GRF5), inflorescence architecture (KNAT1 ) and cytokinin metabolism (C X). More examples of these types of genes are in Van Daele et al. 2012. Overexpression of KRP cell cycle inhibitors can also be detrimental to plant development (Barroco et al.
  • the gene of interest is toxic to the plant cell, plant part or plant when expressed at high level under other commonly used promoters.
  • a gene is toxic to the plant cell, plant part or plant if its expression induces any negative effect or alteration to cell growth, cell division, physiology, and/or morphology of the plant cell, plant part or plant.
  • a desired expression level can be achieved by the materials and methods described in the present invention, so that the gene of interest is expressed at a desired level which leads to a desired phenotype without causing toxicity or only causing bearable toxicity to the plant ceil, plant part, or plant,
  • Introns are known to regulate gene expression through many mechanisms (Morelo and Breviario (2008) Plant Spliceosomal Introns: Not Only Cut and Paste, Current Genomics 9:227-238).
  • the degree to which gene expression is modulated by the intron may depend upon the promoter-intron combination.
  • the rice aetin constitutive promoter-Zm ADH 1 intron combination may be attenuating the strength of REV expression sufficiently for seed yield increase and avoidance of negative pleiotropic effects.
  • the intron sequence in the expression cassettes and expression vectors of the present invention is derived from a plant alcohol dehydrogenase gene (ADH).
  • ADH plant alcohol dehydrogenase gene
  • the plant alcohol dehydrogenase gene can be the genes described in Strommer (The plant ADH gene family, Plant J, 2011 Apr. 66(1): 128-42) and Gaut (Patterns of genetic diversification within the Adh gene family in the grasses (Poaceae), Mol. Biol. Evol. 16: 1086-1097 (1999)), such as the maize ADH1 gene.
  • the intron can be derived from the maize ADH1 gene (e.g., SEQ ID NO: 1 ), or functional fragments or variants thereof.
  • Other maize ADH1 introns used in gene expression enhancement have been described (Callis, J. et al.
  • Introns increase gene expression in cultured maize cells, Genes & Dev 1 : 1 183-1200; Mascarenhas, D. et al. (1990) Intron-mediated enhancement of heterologous gene expression in maize, Plant Molecular Biology 15: 913-920; Luehrsen, K.R. and Walbot, V. (1991 ) Intron enhancement of gene expression and the splicing efficiency of introns in maize cells, Molecular and General Genetics MGG 225: 81-93).
  • ADH1 introns include intron 1 (SEQ ID NO: 57), intron 2 (SEQ ID NO: 58), intron 3 (SEQ ID NO: 59), intron 8 (SEQ ID NO: 60) and intron 9 (SEQ ID NO: 61).
  • the gene of interest is associated with one or more agronomicaily important traits.
  • agronomicaily important traits include any phenotype in a plant or plant part that is useful or advantageous for human use. Examples of agronomicaily important traits include but are not limited to those that result in increased biornass production, increased food production, improved food quality, decrease in cracking, quicker color change when the fruit matures 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 include resistance to hiotic 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 bio fuels, 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 2007/079353 and WO 2007/016319, each of which is incorporated herein by reference in its entirety.
  • the gene of interest is a sucrose isomerase gene, as described in WO/2012/1 19152, which is incorporated herein by reference in its entirety.
  • the gene of interest is an acyl-acyl carrier protein (acyl-ACP) thioesterase (Hawkins and Kridi 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- 1 16), 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
  • 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 microRNA or 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 double-stranded RNA
  • 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 RN A 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 backbone of the expression vectors can be any expression vectors suitable for producing transgenic plant, which are well known in the art. in some embodiments, the expression vector is suitable for expressing transgene in maize.
  • the expression vector is an Agrobacterium binary vector (see, Karimi et al, Plant Physiol 145: 1183-1191; omari et al., Methods Mol Biol 343: 15-42; lies an MW (1984) Nucleic Acids Res 12: 1811-1821; Becker (1992), rhaii Mol Biol 20: 1195-1197; Datia 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 1 1 : 287-293; Weigel et al, (2000) Plant Physiol 122: 1003-1013).
  • 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, 541601 1, 5569834, 5824877, 5959179, 5563055, and 5968830. Each of the references mentioned herein is incorporated by reference in its entirety.
  • 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 teraiinator sequence derived from viral or bacterium gene, or T-DNA.
  • the 3' non-translated regulatory D A 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 regulator ⁇ ' 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 cathepsm D inhibitor gene (GenBank Acc, No,: X74985), 3'UTR of the field bean storage protein gene V1LEIB3 (GenBank Acc.
  • the expression cassettes or the expression vectors of the present invention 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 cany 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 drag-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 Panet al., 1985
  • bar gene which codes for biaiaphos (basta) resistance
  • a mutant aroA gene which encodes an altered EPSP synthase protein (Hinchee et al., 1988), thus conferring glvphosate 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.
  • 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 acetylfransferase in ehloropiasts (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 marmitol dehydrogenase (Lee and Saier, 1982) and trehalose-6-phosphate synthase (Kaasen et al, 1992).
  • an "antifreeze” protein such as that of
  • 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 cany the marker gene. Selection of plants that grow on the selective medium provides for the dentification of plants that have eliminated or evicted the selectable marker genes.
  • CodA Erichia coli cytosine deaminase
  • 5- fluorocytosine which is normally non-toxic as plants do not metabolize cytosine
  • dhlA haioalkane dehaiogenase gene of Xanthobacter autotrophicus GJ10 which encodes a dehaiogenase, which hydroiyzes dihaloalkanes, such as 1, 2-dichloroethane (DCE), to a halogenated alcohol and an inorganic halide
  • DCE 1, 2-dichloroethane
  • the expression cassettes or the expression vectors of the present invention can be transformed into a maize plant to express a gene of interest without causing any negative pieiotropic effects.
  • expression vectors of the present invention can be transformed into a maize plant to increase the seed weight, seed number, and/or seed size.
  • the present invention provides transgenic maize plants transformed with the expression vectors as described herein.
  • the plant can be any maize plant in which an increased seed weight and/or seed size 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 and vectors of described herein.
  • the expression cassettes are used to express a gene of interest in a plant or plant cell. In some embodiments, the expression cassettes are used to express a gene of interest in a plant or plant cell without causing any negative pleiotropic effects. In some embodiments, said methods comprises incorporating into a plant cell a polynucleotide construct comprising a nucleic acid molecule of the present invention.
  • 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 08493 ! 32 I X. 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 FI population; (ii) selecting offsprings that have expression cassette.
  • the offsprings can be further selected by testing the expression of the gene of interest,
  • said methods comprise molecular stacking of transgenes.
  • 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 (ceils 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 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 embryo's 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, ⁇ -gluc ronidase, 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 any element of the expression cassettes 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 Microsateilites, 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 Amp
  • 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 the expression cassettes of the present invention, which comprise a recombinant nucleic acid sequence comprising a promoter, an intron, and a gene of interest.
  • the transgenic plants having the expression cassettes do not have any negative pleiotropic effect.
  • the transgenic plants have at least one desired phenotype due to the expression of the gene of interest.
  • the transgenic plants have one or more agronomically important traits due to the expression of the gene of interest.
  • the transgenic plants would have one or more negative pleiotropic effects if the gene of interest is under the control of the same promoter sequence, but without the control of the intron sequence.
  • the transgenic plants have one or more desired phenotypes compared to a wild type control plant of the same speci es.
  • the transgenic plants have increased seed size, seed number and/or yield compared to the wild type control plant.
  • Thepresent invention also provides a seed, a fruit, a plant population, a plant part, a plant cell and/or a plant tissue culture derived from the transgenic plants as described herein.
  • Modem plant tissue culture is performed under aseptic conditions under filtered air.
  • Living plant materials from the environment are naturally contaminated on their surfaces (and sometimes interiors) with microorganisms, so surface sterilization of starting materials (explants) in chemical solutions (usually alcohol or bleach) is required.
  • Explants are then usually placed on the surface of a solid culture medium, but are sometimes placed directly into a liquid medium, particularly when cell suspension cultures are desired.
  • Solid and liquid media are generally composed of inorganic salts plus a few organic nutrients, vitamms and plant hormones.
  • Solid media are prepared from liquid media with the addition of a gelling agent, usually purified agar.
  • 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. For example, 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. As 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 cuiturist are important in judging which pieces to culture and which to discard. As shoots emerge from a culture, they may be sliced off and rooted with auxin to produce plantiets 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 materia] 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 an 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 an expression cassette of the present invention.
  • the recipient plant is an elite line having one or more certain agronomically important traits.
  • 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.
  • Agrobacteri urn-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.
  • biolistic bombardment uses ultrafine particles, usually tungsten or gold, that are coated with DNA and then sprayed onto the surface of a plant tissue with sufficient force to cause the particles to penetrate plant cells, including the thick cell wall, membrane and nuclear envelope, but without killing at least some of them (US 5,204,253, US 5,015,580).
  • 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.
  • a selection method For efficient plant transformation, a selection method must be employed such that whole pl ants 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 a!., Theor Appl Genet 79: 625-631(1990), US 4795855, US 5378824 and US 6107549).
  • the diifr gene which confers resistance to the anticancer agent methotrexate, has been used for selection (Bourouis et al. , EM BO J. 2(7) : 1099- 1 104 ( 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, eleetroporation; 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.
  • Agrobacierium twnefaciens 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 Agrobacierium 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.), TramgeriicR.es. 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. Each of the references above is incorporated herein by reference in its entirety. Breeding Methods
  • 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-Po llinated 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.
  • Jnterpopuiation 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) populations) 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 poly crosses. Parental seed lines may be deliberately inbred (e.g. by selfing 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 pol linating 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 com meal is also referred to as corn flour.
  • corn flour denotes corn starch in the United Kingdom, Com 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 com 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. Com oil which is high in mono and poly unsaturated fats, is used for reducing fat and trans fat in numerous food products.
  • Com including both grain and non-grain portions of the plant, is also used extensively as livestock feed, primarily for beef cattle, dair cattle, hogs and poultry .
  • Com ethanol is ethanol produced from corn as a biomass through industrial fermentation, chemical processing and distillation. Com is the main feedstock used for producing ethanol fuel in the United States.
  • the industrial applications of com starch and flour are based on functional properties of pastes, such as viscosity, film formation, adhesive properties, and ability to suspend particles. Com 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.
  • a rice actiii promoter-Zm ADHl intron-ZmREV construct (TG Zm 137) was built and transformed into corn.
  • Previous work in Arabidopsis and canola demonstrated that constitutive overexpression of REV leads to negative pleiotropic effects, such as reduced axillary branches and leaf polarity defects (in Arabidopsis) and wrinkled leaves (in canola), see WO/2007/079353, which is incorporated by reference in its entirety.
  • REV controls meristem and organ growth, establishes cell fate and polarity, and plays a role in vascular development (Taihert et al 1995, Development, 121(9): 2723-2735; Otsuga et ai 2001, Plant Journal, 25(2): 223-236; Zhong and Ye 1999, Plant Cell 11(1 1): 2139-2152; Juarez et al. 2004, Nature, 428: 84-88).
  • this maize construct was expected to impact corn growth and development such as organ polarity and would 1) show that the ZmREV transgene was functional in corn and 2) be a benchmark against which other promoter-ZmREV constructs could be compared.
  • ZmRLDl Japanese et al 2004, microRNA- mediated repression of roiled leaf! specifies maize leaf polarity. Nature 428:84-88
  • ZmREV2 SEQ ID NO: 2 of WO2004063379
  • ZmRLDl was used in the present invention, as the microRNA mutant version of this corn REV gave polarity phenotypes in corn (Juarez et al 2004) and thus suggested that this REV gene was operational in com.
  • the rice actin promoter was chosen as the constitutive monocot promoter to drive
  • the rice actin promoter is used in conjunction with the actin intron for enhancement of gene expression (McElroy et al., Moi Gen Genetics (1991) 231 : 150-160).
  • the Zm ADH l intron was used with the Os actin promoter under the assumption that the ADHl intron would also enhance expression of REV through intron-mediated enhancement (I E).
  • Beta-giucuronidase (GUS) reporter assays were performed to compare the effectiveness of the Os actin promoter ⁇ Os actin intron-GUS (TG Zm 179) and Os actin promoter-Zm ADHl intron-GUS (TG_Zm 180) constructs.
  • Corn was stably transformed with both GUS constructs.
  • Ten TO events for each construct were regenerated and advanced to the greenhouse.
  • Different transformed corn tissues were analyzed for GUS staining, a measure of the potency and spatial specificity of the promoter in a given construct.
  • TG Zm 179 showed strong GUS expression in Tl zygotic embryos 9, 14, and 21 days after pollination (DAP), Tl zygotic endosperm 9, 14 and 21 DAP (FIG. 2 A) and in 3, 5 and 7 DAP T2 immature/developing ears (FIG. 2B). Additionally, strong GUS expression was also seen for TG_Zm 179 in T l silk, anther, pollen, leaf, stem and root (FIG. 3). A summary of TG Zm 179 expression is shown in FIG. 4.
  • TG_Zm 180 did not show any visible GUS staining in any corn tissue analyzed with the exception of callus for one event (FIG. IB).
  • the GUS in TG_Zm 1 80 might still be expressed in the plant, but at a low level which was not detectable by the GUS staining protocol.
  • Example 7 shows that expression of a transgene from the Os actin promoter-Zm ADH1 intron can be detected by qPCR.
  • TG_Zm 137 construct without any apparent negative alterations to the corn plant (see data below).
  • Os actin promoter-Zm ADH1 intron combination may drive a low level of ZmREV expression sufficient for seed yield increase but below the threshold of REV expression whereby negative pleiotropic effects are triggered.
  • Transgenic Zea mays corn plants (each called a "TG_Zm 137 event” herein) were generated following the procedure described in WO2012065166, which is herein incorporated by reference in its entirety.
  • the REVOLUTA gene from Zea mays was operably linked to the constitutive rice actin promoter, with the corn ADH1 intron between the promoter and gene (Os actin promoter-Zm ADH1 intron-ZmREV).
  • Isolated crossing block (ICB) trials were conducted in Year 1 and Year 2 to assess the influence of ZmREV on productivity of grain per plant and two underlying yield components, kernel number per ear and average kernel weight.
  • ICB trials took place at five United States locations.
  • Year 2 the trials took place at five United States locations.
  • 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 ro ws with rows 30 inches apart. Planted adjacent to each female plot are two male rows, which are planted with an inbred that has previously been backcrossed with this genetic background (i.e., recurring parent) or a mix of commercial hybrids. Plants in female rows are pollinated by pollen released from the plants in the male rows.
  • ears were harvested from plants within each female row and segregated as originating from a Null or Transgenic plant. Individual ears were shelled and the grain dried and weighed. The average grain weight of Null ears and Transgenic ears, as well as the ratio of Transgenic to Null, was calculated for each event. After determining the degree that grain yield was impacted by the transgene within each event (i.e., "% of Null Ear Grain Weight"), an overall value of "% of Null Ear Grain Weight" was calculated for each construct by averaging corresponding values of each event within the construct. A grain subsample was collected from each ear for determination of average kernel dry weight, which in turn allows estimation of the number of kernels on each ear.
  • FIG. 5 shows a grand summary of the effect of TG Zm 137 construct on ear grain production, as a percentage of null ear grain weight, across Year 1 and Year 2 compared with other efficacious and non-efficacious constructs.
  • Data from events tested at three to five unique locations for two years within the corn belt were used for calculating the average effect of various transgenes on grain production in com. The calculations were done as described above.
  • the TG_Zm 137 construct gave a mean 5% increase in grain weight in Year 1 and a mean 2% increase in grain weight in Year 2. There was moderate drought in Year 1 and severe drought in Year 2, which could explain the lower mean % increase in grain weight for TG Zm 137 in Year 2.
  • FIG. 6 shows that the increase in grain weight was correlated with an increase in kernel number in Year 1.
  • the same analysis in Year 2 also confirmed that the increase in grain weight was correlated with an increase in kernel number.
  • transgenic Zea mays corn plants are generated, following the procedure described in WO2012065166.
  • the REVOLUTA gene from Zea mays (Juarez et al 2004) is operably linked to a constitutive promoter with or without an intron between the promoter and gene.
  • Some constitutive promoter-intron-Zm REV combinations are listed in Table 3. ICB trials are conducted to assess the effect of each constitutive promoter-intron-ZmREV construct on grain yield.
  • Phenotypic observations such as flowering time, plant height, stand establishment, and resistance to abiotic and biotic stresses are recorded.
  • Various tissues are collected from transgenic events of each construct and expression of ZmREV is measured by qPCR and/or Western blot assays.
  • GUS staining a measure of the potency and spatial specificity of the promoter in a given construct.
  • the expression level of GUS in corn tissues containing these constructs is also compared by qPCR and/or Western blot assays.
  • Os actin promoter-Zm ADHI intron-ZmREV is stacked with other expression cassettes that give increases in seed yield, such as those described in WO 2007/079353, WO 2007/016319, WO 2012/065166 and WO 2012/1 19152.
  • Molecular stacks of efficacious constructs are made and transformed into corn, transgenic events are generated and !CB trials conducted to assess the influence of stack constructs on productivity of grain per plant and two underlying yield components, kernel number per ear and average kernel weight.
  • transgene expression is determined using the AACT method and the relative expression levels are compared to: 1) the background signal of the transgene of interest primer/probe in an unrelated event (e.g.
  • transgenic but does not carry the transgene of interest whose expression is measured), 2) the transgene level in an event containing the transgene of interest when using a transgene-specific primer/probe, or 3) the endogenous gene level + transgene level in an event containing the transgene of interest when using an endogenous gene-specific primer/probe.
  • TG_Zm 179 and 180 events were used and expression was determined for TG_Zm 179 and 180 events relative to expression in event TG Zm 180-A002.
  • Levels of transgene expression in TG Zm 179 events are, on average, 50-200 fold those seen in TG_Zm 180 events (FIG. 8) indicating that the Os actin promoter is considerably stronger when used with the Os actin intron than with the Zm ADH1 intron.
  • Cora zygotic embryos were transformed with various constitutive promoter-intron- Ziii REV constructs (Table 6) as described in WO2012065166.
  • the transformation efficiency for each construct was calculated by dividing the number of selectable marker-positive callus events by the total number of zygotic embryos that were inoculated with Agrohacterium. The transformation efficiencies are shown in FIG. 9.
  • actin promoter-ADH intron-monocot REV gene-3 ' terminator is ligated into the binary vector pMH20 at the Smal site.
  • the actin promoter can be Os actin (SEQ ID NO: 5), corn actin (SEQ ID NO: 2) or banana actin (SEQ ID NO: 50).
  • the ADH intron can be from maize (SEQ ID NOs: 1 , 57-61 ).
  • the monocot REV gene can be OsREVl (SEQ ID NOs: 14, 15) or OsREV2 (SEQ ID NOs: 16, 17).
  • a weak constitutive promoter such as the rice E1F5 promoter described in Park et al. (Analysis of five novel putative constitutive gene promoters in transgenic rice plants, Journal of Experimental Botany 61 : 2459-2467 (2010); SEQ ID NO: 64) may be used to express rice RE V.
  • Agrohacterium host EHA105 carrying the resulting binary vector containing actin promoter-ADH intron-monocot REV cassette or weak constitutive promoter-monocot REV cassette is selected by kanamvcin 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.
  • a binary vector containing actin promoter-ADH intron-monocot REV cassette (or weak constitutive promoter-monocot REV cassette) or a control empty binary vector is transformed into rice calli by Agrobacterium- ediated transformation and transgenic calli is selected on hygromycin.
  • 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 REV events is increased by 1-100% 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.
  • soy (Glycine max)
  • constructs are made for biolistic transformation into soy as described in WO 2007/079353.
  • a cassette containing a weak constitutive promoter-dicot REV gene-3 ' terminator is constructed and co-transformed with a cassette carrying a suitable selectable marker.
  • the weak constitutive promoter can be Agrobacterium tumefaciens nopaline synthase (Depicker et al. (1982) J.
  • a strong constitutive promoter can be rendered weak by the use of a heterologous intron between the promoter and REV gene in dicots as described by Leon et al. (1991 ) (Transient Gene Expression in Protoplasts of Phaseolus vulgaris Isolated from a Ceil Suspension Culture, Plant Physiol. 95: 968-972) and Maas et al.
  • the constitutive promoter can be cauliflower mosaic vims (CaMV) 35S (Kay et al (1987) Science 230: 1299-1302; SEQ ID NO: 66) or figwort mosaic virus (FMV) (Maiti, LB. et al. (1997) Transgenic Research 6: 143-156; Bhattacharyya, S. et al.
  • CaMV cauliflower mosaic vims
  • FMV figwort mosaic virus
  • the dicot REV gene can be GmREV A (SEQ ID NOs: 10, 1 1) or GmREV B (SEQ ID NOs: 12, 13).
  • TO tissue is collected and Southerns done to confirm presence of the REV transgene and to determine transgene insert number. Typically single insert events expressing REV are taken forward. About 15-30 TO events are obtained for each REV construct.
  • TI 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 TI mean seed yield from transgenic REV events is increased by 1-100% compared to the empty vector events.
  • Reproducibility of the increased seed yield is determined in subsequent generations in the greenhouse and field.
  • constructs are made for Agrobacterium-medi&tQd transformation into canoia as described in WO 2007/079353.
  • a cassette containing a weak constitutive promoter-dicot REV gene-3' tenninator is constructed and inserted into a binary vector containing a suitable selectable marker expression cassette.
  • the weak constitutive promoter can be AgrohacJerium tumefaciens nopaline synthase (Depicker et al. (1982) J.
  • a strong constitutive promoter can be rendered weak by the use of a heterologous intron between the promoter and REV gene in dicots as described by Leon et al. (1991) (Transient Gene Expression in Protoplasts of Phaseolus vulgaris Isolated from a Cell Suspension Culture, Plant Physiol. 95 : 968-972) and Maas et al.
  • the constitutive promoter can be cauliflower mosaic virus (CaMV) 35S (Kay et al . (1987) Science 230: 1299-1302; SEQ ID NO: 66) or figwort mosaic virus (FMV) (Maiti, LB. et al. (1997) Transgenic Research 6: 143-156; Bhattacharvya, S.
  • CaMV cauliflower mosaic virus
  • FMV figwort mosaic virus
  • the dicot REV gene can be BnREV (SEQ ID NOs: 8,
  • TO tissue is collected and Southerns done to confirm presence of the REV transgene and to determine transgene insert number. Typically single insert events expressing REV are taken forward. About 15-30 TO events are obtained for each REV construct.
  • T! 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 REV events is increased by 1-100% compared to the empty vector events.
  • Reproducibility of the increased seed yield is determined in subsequent generations in the greenhouse and field.

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Abstract

L'invention concerne des matériaux et des procédés utilisables dans le cadre de l'expression génique et permettant de diriger et/ou de réguler l'expression d'un acide nucléique fonctionnellement lié chez une plante sans aucun effet pléiotrope négatif. L'invention concerne également des compositions et des procédés permettant un accroissement du nombre de semences et/ou du rendement chez une plante par rapport à ce que l'on observe chez une plante témoin de type sauvage.
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WO2020010333A1 (fr) * 2018-07-06 2020-01-09 Fte Genetics, Inc. Cultivars de soja à haut rendement uniques
CN113234731A (zh) * 2021-05-10 2021-08-10 山东农业大学 编码大豆ARF转录因子的GmARF16基因及应用
CN114703226A (zh) * 2022-04-11 2022-07-05 中国水稻研究所 水稻OsUBC27基因或其编码的蛋白在提高水稻产量中的应用

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WO2007138070A2 (fr) * 2006-05-30 2007-12-06 Cropdesign N.V. Plantes possédant des caractéristiques de rendement amélioré et procédé de fabrication
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WO2012119152A1 (fr) * 2011-03-03 2012-09-07 Targeted Growth, Inc. L'expression d'isomères de sucrose augmente le poids des graines, le nombre de graines et/ou la dimension des graines

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Cited By (5)

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Publication number Priority date Publication date Assignee Title
WO2020010333A1 (fr) * 2018-07-06 2020-01-09 Fte Genetics, Inc. Cultivars de soja à haut rendement uniques
CN113234731A (zh) * 2021-05-10 2021-08-10 山东农业大学 编码大豆ARF转录因子的GmARF16基因及应用
CN113234731B (zh) * 2021-05-10 2022-03-25 山东农业大学 编码大豆ARF转录因子的GmARF16基因及应用
CN114703226A (zh) * 2022-04-11 2022-07-05 中国水稻研究所 水稻OsUBC27基因或其编码的蛋白在提高水稻产量中的应用
CN114703226B (zh) * 2022-04-11 2022-09-13 中国水稻研究所 水稻OsUBC27基因或其编码的蛋白在提高水稻产量中的应用

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