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WO2013067252A1 - Polynucléotides, polypeptides et procédés d'amélioration de la photo-assimilation chez les plantes - Google Patents

Polynucléotides, polypeptides et procédés d'amélioration de la photo-assimilation chez les plantes Download PDF

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Publication number
WO2013067252A1
WO2013067252A1 PCT/US2012/063161 US2012063161W WO2013067252A1 WO 2013067252 A1 WO2013067252 A1 WO 2013067252A1 US 2012063161 W US2012063161 W US 2012063161W WO 2013067252 A1 WO2013067252 A1 WO 2013067252A1
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plant
seq
expression cassette
sequence
plants
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PCT/US2012/063161
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English (en)
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Michael Nuccio
Laura Potter
Jonathan Cohn
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Syngenta Participations Ag
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Priority claimed from PCT/US2011/059123 external-priority patent/WO2012061585A2/fr
Application filed by Syngenta Participations Ag filed Critical Syngenta Participations Ag
Priority to BR112014010642A priority Critical patent/BR112014010642A2/pt
Priority to CA2853490A priority patent/CA2853490A1/fr
Priority to AU2012332343A priority patent/AU2012332343A1/en
Priority to HU1400505A priority patent/HUP1400505A2/hu
Priority to CN201280053974.5A priority patent/CN103998611A/zh
Priority to US14/355,251 priority patent/US20140317783A1/en
Priority to EA201400527A priority patent/EA201400527A1/ru
Priority to MX2014005375A priority patent/MX2014005375A/es
Publication of WO2013067252A1 publication Critical patent/WO2013067252A1/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
    • C12N15/8262—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield involving plant development
    • C12N15/8269—Photosynthesis
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1205—Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1294—Phosphotransferases with paired acceptors (2.7.9)
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/88—Lyases (4.)

Definitions

  • the disclosure relates generally to the field of molecular biology and regards to various polynucleotides, polypeptides and methods of use that may be employed to enhance photoassimilation and yield in transgenic plants.
  • Transgenic plants comprising any one of the polynucleotides or polypeptides described herein may exhibit any one of the traits consisting of increased biomass, increased photoassimilation or increased yield.
  • One embodiment of the invention is an expression cassette comprising at least three polynucleotides selected from the group consisting of a polynucleotide encoding a phosphoenolpyruvate carboxylase, a polynucleotide encoding a fructose- 1, 6-bisphosphate phosphatase, a polynucleotide encoding a NADP-malate dehydrogenase, a polynucleotide encoding a phosphoribulokinase, and a polynucleotide encoding a pyruvate orthophosphate dikinase.
  • the expression cassette may comprises a polynucleotide encoding a fructose- 1, 6- bisphosphate phosphatase, a polynucleotide encoding a phosphoribulokinase and a polynucleotide encoding a phosphoenolpyruvate carboxylase or a polynucleotide encoding a fructose- 1, 6-bisphosphate phosphatase, a polynucleotide encoding a phosphoribulokinase, a polynucleotide encoding a pyruvate orthophosphate dikinase and a polynucleotide encoding a NADP-malate dehydrogenase.
  • the expression cassette may contain polynucleotides encoding polypeptides having at least 70%, 80%, 90% or 95% identity to SEQ ID NO. 1; SEQ ID NO. 2; SEQ ID NO: 3; SEQ ID NO. 4 or SEQ ID NO: 5.
  • the expression cassette may comprise polynucleotides encoding polypeptides comprising SEQ ID NO. 1, SEQ ID NO. 2, and SEQ ID NO. 3 or SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, and SEQ ID NO. 5.
  • the polynucleotides of the expression cassette may be operably linked to one or more light inducible promoters.
  • the polynucleotides of the expression cassette may also comprise the polynucleotides described in SEQ ID NO. 6; SEQ ID NO. 7 and SEQ ID NO. 8 or SEQ ID NO. 9; SEQ ID NO. 10; SEQ ID NO. 11 and SEQ ID NO. 12.
  • Additional embodiments include a method for increasing biomass comprising introducing any one of the expression cassette described into a plant cell; growing the plant cell into a plant; and selecting a transgenic plant having increased biomass.
  • the plant may be a C4 plant and could be selected from the group consisting of sugarcane, maize and sorghum. Alternatively, the plant may be maize.
  • Another embodiment includes a method of making a transgenic plant comprising introducing any of the described expression cassette into a plant; growing the plant cell into a plant; and selecting a plant comprising the expression cassette.
  • the plant may be a C4 plant and could be selected from the group consisting of sugarcane, maize and sorghum. Alternatively, the plant may be maize.
  • FIG. 1 is a plasmid map of 19862 showing SoFBP, SoPRK, and ZmPEPC expression cassettes in a binary vector, "pr-" prefix denotes a promoter; “i-” prefix denotes an intron; “e-” prefix denotes an enhancer; “c-” prefix denotes a coding sequence; “t-” prefix denotes a terminator.
  • FIG. 2 is a plasmid map of 19863 showing SoFBP, SbPPDK, and SbNADP-MD expression cassettes in a binary vector, "pr-" prefix denotes a promoter; “i-” prefix denotes an intron; “e-” prefix denotes an enhancer; “c-” prefix denotes a coding sequence; “t-” prefix denotes a terminator.
  • FIG. 3 describes daily photoassimilation and night time respiration in B027A Fl plants.
  • A Steady state photoassimilation rate and
  • B night time respiration cultivated under closed-chamber conditions. Plants were subject to 16 hour day at 25°C and 8 hour night at 20°C. Relative humidity was 60%. Atmospheric CO 2 was maintained by metered injection at 400 ppm during the day. Photoassimilation is the daily rate of CO 2 injected to maintain the 400 ppm set point. Night time respiration is the CO 2 released during the night as a function of CO 2 assimilated the previous day. Data are for 40 plants.
  • compositions, method or structure may include additional ingredients, steps and/or parts, but only if the additional ingredients, steps and/or parts do not materially alter the basic and novel characteristics of the claimed composition, method or structure.
  • microbe any microorganism (including both eukaryotic and prokaryotic microorganisms), such as fungi, yeast, bacteria, actinomycetes, algae and protozoa, as well as other unicellular structures.
  • conservatively modified variants refer to those nucleic acids that encode identical or conservatively modified variants of the amino acid sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are "silent variations" and represent one species of conservatively modified variation.
  • Every nucleic acid sequence herein that encodes a polypeptide also describes every possible silent variation of the nucleic acid.
  • AUG which is ordinarily the only codon for methionine; one exception is Micrococcus rubens, for which GTG is the methionine codon (Ishizuka, et al , (1993) J. Gen. Microbiol. 139:425-32) can be modified to yield a functionally identical molecule. Accordingly, each silent variation of a nucleic acid, which encodes a polypeptide of the present invention, is implicit in each described polypeptide sequence and incorporated herein by reference.
  • a "control plant” or “control” as used herein may be a non-transgenic plant of the parental line used to generate a transgenic plant herein.
  • a control plant may in some cases be a transgenic plant line that includes an empty vector or marker gene, but does not contain the recombinant polynucleotide of the present invention that is expressed in the transgenic plant being evaluated.
  • a control plant in other cases is a transgenic plant expressing the gene with a constitutive promoter.
  • a control plant is a plant of the same line or variety as the transgenic plant being tested, lacking the specific trait-conferring, recombinant DNA that characterizes the transgenic plant.
  • Such a progenitor plant that lacks that specific trait- conferring recombinant DNA can be a natural, wild-type plant, an elite, non-transgenic plant, or a transgenic plant without the specific trait-conferring, recombinant DNA that characterizes the transgenic plant.
  • the progenitor plant lacking the specific, trait-conferring recombinant DNA can be a sibling of a transgenic plant having the specific, trait-conferring recombinant DNA.
  • Such a progenitor sibling plant may include other recombinant DNA
  • amino acid sequences one of skill will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide or protein sequence which alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a "conservatively modified variant" when the alteration results in the substitution of an amino acid with a chemically similar amino acid.
  • any number of amino acid residues selected from the group of integers consisting of from 1 to 15 can be so altered.
  • 1, 2, 3, 4, 5, 7 or 10 alterations can be made.
  • Conservatively modified variants typically provide similar biological activity as the unmodified polypeptide sequence from which they are derived.
  • substrate specificity, enzyme activity or ligand/receptor binding is generally at least 30%, 40%, 50%, 60%, 70%, 80% or 90%, preferably 60-90% of the native protein for its native substrate.
  • Conservative substitution tables providing functionally similar amino acids are well known in the art.
  • I Isoleucine
  • L Leucine
  • M Methionine
  • V Valine
  • nucleic acid encoding a protein comprising the information for translation into the specified protein.
  • a nucleic acid encoding a protein may comprise non-translated sequences (e.g., introns) within translated regions of the nucleic acid or may lack such intervening non-translated sequences (e.g., as in cDNA).
  • the information by which a protein is encoded is specified by the use of codons.
  • amino acid sequence is encoded by the nucleic acid using the "universal" genetic code.
  • variants of the universal code such as is present in some plant, animal and fungal mitochondria, the bacterium Mycoplasma capricolumn (Yamao, et al, (1985)Proc. Natl. Acad. Sci. USA 82:2306-9) or the ciliate Macronucleus, may be used when the nucleic acid is expressed using these organisms.
  • nucleic acid sequences of the present invention may be expressed in both monocotyledonous and dicotyledonous plant species, sequences can be modified to account for the specific codon preferences and GC content preferences of monocotyledonous plants or dicotyledonous plants as these preferences have been shown to differ (Murray, et al , (1989) Nucleic Acids Res. 17:477-98 and herein incorporated by reference).
  • the maize preferred codon for a particular amino acid might be derived from known gene sequences from maize. Maize codon usage for 28 genes from maize plants is listed in Table 4 of Murray, et al. , supra.
  • heterologous in reference to a nucleic acid is a nucleic acid that originates from a foreign species, or, if from the same species, is substantially modified from its native form in composition and/or genomic locus by deliberate human intervention.
  • a promoter operably linked to a heterologous structural gene is from a species different from that from which the structural gene was derived or, if from the same species, one or both are substantially modified from their original form.
  • a heterologous protein may originate from a foreign species or, if from the same species, is substantially modified from its original form by deliberate human intervention.
  • host cell is meant a cell, which comprises a heterologous nucleic acid sequence of the invention, which contains a vector and supports the replication and/or expression of the expression vector.
  • Host cells may be prokaryotic cells such as E. coli, or eukaryotic cells such as yeast, insect, plant, amphibian or mammalian cells.
  • host cells are monocotyledonous or dicotyledonous plant cells, including but not limited to maize, sorghum, sunflower, soybean, wheat, alfalfa, rice, cotton, canola, barley, millet and tomato.
  • a particularly preferred monocotyledonous host cell is a maize host cell.
  • hybridization complex includes reference to a duplex nucleic acid structure formed by two single-stranded nucleic acid sequences selectively hybridized with each other.
  • the term "introduced” in the context of inserting a nucleic acid into a cell by any means, such as, “transfection”, “transformation” or “transduction” and includes reference to the incorporation of a nucleic acid into a eukaryotic or prokaryotic cell where the nucleic acid may be incorporated into the genome of the cell (e.g., chromosome, plasmid, plastid or mitochondrial DNA), converted into an autonomous replicon, as part of a mini-chromosome or transiently expressed (e.g., transfected mRNA).
  • transfection e.g., chromosome, plasmid, plastid or mitochondrial DNA
  • transiently expressed e.g., transfected mRNA
  • gene stack refers to the introduction of two or more genes into the genome of an organism. It may be desirable to stack the genes as described herein with genes conferring insect resistance, disease resistance, increased yield or any other beneficial trait (e.g. increased plant height, etc) known in the art. Alternatively, transgenic plants comprising a gene, polypeptide or polynucleotide as described herein may be stacked with native trait alleles that confer additional traits, such as, improved water use, increased disease resistance and the like. Traits may be stacked by introducing expression cassettes with multiple genes or breeding/crossing plants with one or more traits with other plants containing one or more additional traits.
  • isolated refers to material, such as a nucleic acid or a protein, which is substantially or essentially free from components which normally accompany or interact with it as found in its naturally occurring environment.
  • the isolated material optionally comprises material not found with the material in its natural environment.
  • Nucleic acids, which are “isolated”, as defined herein, are also referred to as “heterologous” nucleic acids.
  • NUE nucleic acid means a nucleic acid comprising a polynucleotide (“NUE polynucleotide”) encoding a full length or partial length NUE polypeptide.
  • nucleic acid includes reference to a deoxyribonucleotide or ribonucleotide polymer in either single- or double-stranded form, and unless otherwise limited, encompasses known analogues having the essential nature of natural nucleotides in that they hybridize to single-stranded nucleic acids in a manner similar to naturally occurring nucleotides (e.g., peptide nucleic acids).
  • nucleic acid library is meant a collection of isolated DNA or RNA molecules, which comprise in one case a substantial representation of the entire transcribed fraction of a genome of a specified organism. Construction of exemplary nucleic acid libraries, such as genomic and cDNA libraries, is taught in standard molecular biology references such as Berger and Kimmel, (1987) Guide To Molecular Cloning Techniques, from the series Methods in Enzymology, vol. 152, Academic Press, Inc., San Diego, Calif.; Sambrook, et al, (1989) Molecular Cloning: A Laboratory Manual, 2nd ed., vols.
  • nucleic acid library as defined herein may also be understood to represent libraries comprising a prescribed faction or rather not substantially representing an entire genome of a specified organism. For example, small RNAs, mRNAs and methylated DNA.
  • a nucleic acid library as defined herein might also encompass variants of a particular molecule (e.g. a collection of variants for a particular protein).
  • operably linked includes reference to a functional linkage between a first sequence, such as a promoter and a second sequence, wherein the promoter sequence initiates and mediates transcription of the DNA corresponding to the second sequence.
  • operably linked means that the nucleic acid sequences being linked are contiguous and, where necessary to join two protein coding regions, contiguous and in the same reading frame.
  • plant includes reference to whole plants, plant organs (e.g., leaves, stems, roots, etc.), seeds and plant cells and progeny of same.
  • Plant cell as used herein includes, without limitation, seeds, suspension cultures, embryos, meristematic regions, callus tissue, leaves, roots, shoots, gametophytes, sporophytes, pollen and microspores.
  • the class of plants which can be used in the methods of the invention, is generally as broad as the class of higher plants amenable to transformation techniques, including both monocotyledonous and dicotyledonous plants including species from the genera: Cucurbita, Rosa, Vitis, Juglans, Fragaria, Lotus, Medicago, Onobrychis, Trifolium, Trigonella, Vigna, Citrus, Linum, Geranium, Manihot, Daucus, Arabidopsis, Brassica, Raphanus, Sinapis, Atropa, Capsicum, Datura, Hyoscyamus, Lycopersicon, Nicotiana, Solarium, Petunia, Digitalis, Majorana, Ciahorium, Helianthus, Lactuca, Bromus, Asparagus, Antirrhinum, Heterocallis, Nemesis, Pelargonium, Panieum, Pennisetum, Ranunculus, Senecio, Salpiglossis, Cucumis, Bro
  • a C4 plant as defined herein, is one that utilizes the C 4 carbon fixation pathway such that the CO 2 is first bound to a phosphoenopyruvate in a mesophyll cell resulting in the formation of four-carbon compound that is shuttled to the bundle sheath cell where it decarboxylated to liberate the CO 2 to be utilized in the C3 pathway.
  • C4 plants include, but are not limited to, members of the Poaceae family (also called Gramineae or true grasses), such as, sugarcane, maize, sorghum, amaranth, millet; members of the sedge family Cyperaceae; and numerous families of Eudicots, including the daisies Asteracae; cabbages Brassicaceae; and spurges Euphorbiaceae.
  • members of the Poaceae family also called Gramineae or true grasses
  • sugarcane sugarcane
  • maize maize
  • sorghum amaranth
  • millet members of the sedge family Cyperaceae
  • Eudicots including the daisies Asteracae; cabbages Brassicaceae; and spurges Euphorbiaceae.
  • yield may include reference to bushels per acre of a grain crop at harvest, as adjusted for grain moisture (15% typically for maize, for example), and the volume of biomass generated (for forage crops such as alfalfa and plant root size for multiple crops). Grain moisture is measured in the grain at harvest. The adjusted test weight of grain is determined to be the weight in pounds per bushel, adjusted for grain moisture level at harvest. Biomass is measured as the weight of harvestable plant material generated.
  • Yield can be affected by many properties including without limitation, plant height, pod number, pod position on the plant, number of internodes, incidence of pod shatter, grain size, efficiency of nodulation and nitrogen fixation, efficiency of nutrient assimilation, carbon assimilation, plant architecture, percent seed germination, seedling vigor, and juvenile traits. Yield can also be affected by efficiency of germination (including germination in stressed conditions), growth rate (including growth rate in stressed conditions), ear number, seed number per ear, seed size, composition of seed (starch, oil, protein) and characteristics of seed fill. Yield of a plant of the can be measured in a number of ways, including test weight, seed number per plant, seed weight, seed number per unit area (i.e.
  • corn yield may be measured as production of shelled corn kernels per unit of production area, for example in bushels per acre or metric tons per hectare, often reported on a moisture adjusted basis, for example at 15.5 percent moisture.
  • a bushel of corn is defined by law in the State of Iowa as 56 pounds by weight, a useful conversion factor for corn yield is: 100 bushels per acre is equivalent to 6.272 metric tons per hectare.
  • Other measurements for yield are common practice in the art. In certain embodiments of the invention yield may be increased in stressed and/or non-stressed conditions.
  • polynucleotide includes reference to a deoxyribopolynucleotide, ribopolynucleotide or analogs thereof that have the essential nature of a natural ribonucleotide in that they hybridize, under stringent hybridization conditions, to substantially the same nucleotide sequence as naturally occurring nucleotides and/or allow translation into the same amino acid(s) as the naturally occurring nucleotide(s).
  • a polynucleotide can be full-length or a subsequence of a native or heterologous structural or regulatory gene. Unless otherwise indicated, the term includes reference to the specified sequence as well as the complementary sequence thereof.
  • DNAs or RNAs with backbones modified for stability or for other reasons are "polynucleotides" as that term is intended herein.
  • DNAs or RNAs comprising unusual bases, such as inosine or modified bases, such as tritylated bases, to name just two examples are polynucleotides as the term is used herein. It will be appreciated that a great variety of modifications have been made to DNA and RNA that serve many useful purposes known to those of skill in the art.
  • polynucleotide as it is employed herein embraces such chemically, enzymatically or metabolically modified forms of polynucleotides, as well as the chemical forms of DNA and RNA characteristic of viruses and cells, including inter alia, simple and complex cells.
  • polypeptide peptide
  • protein protein
  • amino acid polymers in which one or more amino acid residue is an artificial chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers.
  • promoter includes reference to a region of DNA upstream from the start of transcription and involved in recognition and binding of RNA polymerase and other proteins to initiate transcription.
  • a "plant promoter” is a promoter capable of initiating transcription in plant cells. Exemplary plant promoters include, but are not limited to, those that are obtained from plants, plant viruses and bacteria which comprise genes expressed in plant cells such Agrobacterium or Rhizobium. Examples are promoters that preferentially initiate transcription in certain tissues, such as leaves, roots, seeds, fibres, xylem vessels, tracheids or sclerenchyma.
  • tissue preferred Such promoters are referred to as "tissue preferred.”
  • a "cell type” specific promoter primarily drives expression in certain cell types in one or more organs, for example, vascular cells in roots or leaves.
  • An “inducible” or “regulatable” promoter is a promoter, which is under environmental control. Examples of environmental conditions that may affect transcription by inducible promoters include anaerobic conditions or the presence of light.
  • Another type of promoter is a developmentally regulated promoter, for example, a promoter that drives expression during pollen development.
  • Tissue preferred, cell type specific, developmental ⁇ regulated and inducible promoters constitute the class of "non- constitutive" promoters.
  • a “constitutive” promoter is a promoter, which is active under most environmental conditions in most cells.
  • any suitable promoter sequence can be used by the nucleic acid construct of the present invention.
  • the promoter is a constitutive promoter, a tissue-specific, or a light inducible promoter.
  • Suitable constitutive promoters include, for example, CaMV 35S promoter (Odell et al, Nature 313:810-812, 1985); Arabidopsis At6669 promoter (see PCT Publication No. WO04081173A2); maize Ubi 1 (Christensen et al, Plant Mol. Biol. 18:675-689, 1992); rice actin (McElroy et al, Plant Cell 2: 163-171, 1990); pEMU (Last et al, Theor. Appl. Genet. 81 :581-588, 1991); CaMV 19S (Nilsson et al , Physiol.
  • tissue-specific promoters include, but not limited to, leaf-specific promoters [such as described, for example, by Yamamoto et al , Plant J. 12:255-265, 1997; Kwon et al, Plant Physiol. 105:357-67, 1994; Yamamoto et al, Plant Cell Physiol. 35:773-778, 1994; Gotor et al , Plant J. 3:509-18, 1993; Orozco et al , Plant Mol. Biol. 23: 1129-1138, 1993; and Matsuoka et al, Proc. Natl. Acad. Sci.
  • seed-preferred promoters e.g., from seed specific genes (Simon, et al , Plant Mol. Biol. 5. 191, 1985; Scofield, et al, J. Biol. Chem. 262: 12202, 1987; Baszczynski, et al , Plant Mol. Biol. 14: 633, 1990), Brazil Nut albumin (Pearson' et al, Plant Mol. Biol. 18: 235-245, 1992), legumin (Ellis, et al Plant Mol. Biol. 10: 203-214, 1988), Glutelin (rice) (Takaiwa, et al, Mol. Gen. Genet.
  • endosperm specific promoters e.g., wheat LMW and HMW, glutenin-1 (Mol Gen Genet 216:81-90, 1989; NAR 17:461-2), wheat a, b and g gliadins (EMB03: 1409-15, 1984), Barley ltrl promoter, barley B l, C, D hordein (Theor Appl Gen 98:1253-62, 1999; Plant J 4:343-55, 1993; Mol Gen Genet 250:750-60, 1996), Barley DOF (Mena et al , The Plant Journal, 116(1): 53-62, 1998), Biz2 (EP99106056.7), Synthetic promoter (Vicente-Carbajosa et al, Plant J.
  • Suitable abiotic stress-inducible promoters include, but not limited to, salt-inducible promoters such as RD29A (Yamaguchi-Shinozalei et al , Mol. Gen. Genet. 236:331-340, 1993); drought-inducible promoters such as maize rabl7 gene promoter (Pla et. al., Plant Mol. Biol. 21:259-266, 1993), maize rab28 gene promoter (Busk et. al, Plant J. 11 : 1285- 1295, 1997) and maize Ivr2 gene promoter (Pelleschi et. al, Plant Mol. Biol. 39:373-380, 1999); heat-inducible promoters such as heat tomato hsp80-promoter from tomato (U.S. Pat. No. 5,187,267).
  • salt-inducible promoters such as RD29A (Yamaguchi-Shinozalei et al , Mol. Gen. Gene
  • Light inducible promoters have enhanced expression during irradiation with light, while substantially reduced expression or no expression in the absence of light.
  • Examples of light inducible promoter include, but are not limited to, the SSU small subunit gene promoter Berry-Lowe, (1982) J. Mol. Appl. Gen.
  • nucleic acid refers to a deoxyribonucleotide or ribonucleotide polymer in either single- or double-stranded form, or sense or anti-sense, and unless otherwise limited, encompasses known analogues of natural nucleotides that hybridize to nucleic acids in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence includes the complementary sequence thereof.
  • a "structural gene” is that portion of a gene comprising a DNA segment encoding a protein, polypeptide or a portion thereof, and excluding the 5' sequence which drives the initiation of transcription.
  • the structural gene may alternatively encode a nontranslatable product.
  • the structural gene may be one which is normally found in the cell or one which is not normally found in the cell or cellular location wherein it is introduced, in which case it is termed a "heterologous gene".
  • a heterologous gene may be derived in whole or in part from any source known to the art, including a bacterial genome or episome, eukaryotic, nuclear or plasmid DNA, cDNA, viral DNA or chemically synthesized DNA.
  • a structural gene may contain one or more modifications that could affect biological activity or its characteristics, the biological activity or the chemical structure of the expression product, the rate of expression or the manner of expression control. Such modifications include, but are not limited to, mutations, insertions, deletions and substitutions of one or more nucleotides.
  • the structural gene may constitute an uninterrupted coding sequence or it may include one or more introns, bounded by the appropriate splice junctions.
  • the structural gene may be translatable or non-translatable, including in an anti-sense orientation.
  • the structural gene may be a composite of segments derived from a plurality of sources and from a plurality of gene sequences (naturally occurring or synthetic, where synthetic refers to DNA that is chemically synthesized).
  • Derivative from is used to mean taken, obtained, received, traced, replicated or descended from a source (chemical and/or biological).
  • a derivative may be produced by chemical or biological manipulation (including, but not limited to, substitution, addition, insertion, deletion, extraction, isolation, mutation and replication) of the original source.
  • “Chemically synthesized”, as related to a sequence of DNA, means that portions of the component nucleotides were assembled in vitro.
  • Manual chemical synthesis of DNA may be accomplished using well established procedures (Caruthers, Methodology of DNA and RNA Sequencing, (1983), Weissman (ed.), Praeger Publishers, New York, Chapter 1); automated chemical synthesis can be performed using one of a number of commercially available machines.
  • recombinant includes reference to a cell or vector, that has been modified by the introduction of a heterologous nucleic acid or that the cell is derived from a cell so modified.
  • recombinant cells express genes that are not found in identical form within the native (non-recombinant) form of the cell or express native genes that are otherwise abnormally expressed, under expressed or not expressed at all as a result of deliberate human intervention or may have reduced or eliminated expression of a native gene.
  • the term “recombinant” as used herein does not encompass the alteration of the cell or vector by naturally occurring events (e.g., spontaneous mutation, natural transformation/transduction/transposition) such as those occurring without deliberate human intervention.
  • an "expression cassette” is a nucleic acid construct, generated recombinantly or synthetically, with a series of specified nucleic acid elements, which permit transcription of a particular nucleic acid in a target cell.
  • the expression cassette can be incorporated into a plasmid, chromosome, mitochondrial DNA, plastid DNA, virus or nucleic acid fragment.
  • the expression cassette portion of an expression vector includes, among other sequences, a nucleic acid to be transcribed and a promoter.
  • amino acid residue or “amino acid residue” or “amino acid” are used interchangeably herein to refer to an amino acid that is incorporated into a protein, polypeptide or peptide (collectively “protein”).
  • the amino acid may be a naturally occurring amino acid and, unless otherwise limited, may encompass known analogs of natural amino acids that can function in a similar manner as naturally occurring amino acids.
  • sequences include reference to hybridization, under stringent hybridization conditions, of a nucleic acid sequence to a specified nucleic acid target sequence to a detectably greater degree (e.g., at least 2-fold over background) than its hybridization to non-target nucleic acid sequences and to the substantial exclusion of non- target nucleic acids.
  • Selectively hybridizing sequences typically have about at least 40% sequence identity, preferably 60-90% sequence identity and most preferably 100% sequence identity (i.e., complementary) with each other.
  • stringent conditions or “stringent hybridization conditions” include reference to conditions under which a probe 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. By controlling the stringency of the hybridization and/or washing conditions, target sequences can be identified which can be up to 100% complementary to the probe (homologous probing). Alternatively, stringency conditions can be adjusted to allow some mismatching in sequences so that lower degrees of similarity are detected (heterologous probing). Optimally, the probe is approximately 500 nucleotides in length, but can vary greatly in length from less than 500 nucleotides to equal to the entire length of the target sequence.
  • stringent conditions will be those in which the salt concentration is less than about 1.5 M Na ion, typically about 0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30° C. for short probes (e.g., 10 to 50 nucleotides) and at least about 60° C. for long probes (e.g., greater than 50 nucleotides).
  • Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide or Denhardt's.
  • Exemplary low stringency conditions include hybridization with a buffer solution of 30 to 35% formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulphate) at 37° C.
  • Exemplary moderate stringency conditions include hybridization in 40 to 45% formamide, 1 M NaCl, 1% SDS at 37° C. and a wash in 0.5x to lxSSC at 55 to 60° C.
  • Exemplary high stringency conditions include hybridization in 50% formamide, 1 M NaCl, 1% SDS at 37° C. and a wash in O. lxSSC at 60 to 65° C. Specificity is typically the function of post-hybridization washes, the critical factors being the ionic strength and temperature of the final wash solution.
  • the T m is the temperature (under defined ionic strength and pH) at which 50% of a complementary target sequence hybridizes to a perfectly matched probe.
  • T m is reduced by about 1° C. for each 1% of mismatching; thus, T m , hybridization and/or wash conditions can be adjusted to hybridize to sequences of the desired identity. For example, if sequences with >90% identity are sought, the T m can be decreased 10° C.
  • stringent conditions are selected to be about 5° C. lower than the thermal melting point (T m ) for the specific sequence and its complement at a defined ionic strength and pH.
  • severely stringent conditions can utilize a hybridization and/or wash at 1, 2, 3 or 4° C. lower than the thermal melting point (T m ); moderately stringent conditions can utilize a hybridization and/or wash at 6, 7, 8, 9 or 10° C.
  • T m thermal melting point
  • low stringency conditions can utilize a hybridization and/or wash at 11, 12, 13, 14, 15 or 20° C. lower than the thermal melting point (T m ).
  • T m thermal melting point
  • high stringency is defined as hybridization in 4xSSC, 5xDenhardt's (5 g Ficoll, 5 g polyvinypyrrolidone, 5 g bovine serum albumin in 500 ml of water), 0.1 mg/ml boiled salmon sperm DNA, and 25 mM Na phosphate at 65° C. and a wash in O. lxSSC, 0.1% SDS at 65° C.
  • transgenic plant includes reference to a plant, which comprises within its genome a heterologous polynucleotide.
  • the heterologous polynucleotide is stably integrated within the genome such that the polynucleotide is passed on to successive generations.
  • the heterologous polynucleotide may be integrated into the genome alone or as part of a recombinant expression cassette.
  • Transgenic is used herein to include any cell, cell line, callus, tissue, plant part or plant, the genotype of which has been altered by the presence of heterologous nucleic acid including those transgenics initially so altered as well as those created by sexual crosses or asexual propagation from the initial transgenic.
  • transgenic does not encompass the alteration of the genome (chromosomal or extra-chromosomal) by conventional plant breeding methods or by naturally occurring events such as random cross-fertilization, non-recombinant viral infection, non-recombinant bacterial transformation, non-recombinant transposition or spontaneous mutation.
  • vector includes reference to a nucleic acid used in transfection of a host cell and into which can be inserted a polynucleotide. Vectors are often replicons. Expression vectors permit transcription of a nucleic acid inserted therein.
  • Plant tissue includes differentiated and undifferentiated tissues or plants, including but not limited to roots, stems, shoots, leaves, pollen, seeds, tumor tissue and various forms of cells and culture such as single cells, protoplast, embryos, and callus tissue.
  • the plant tissue may be in plants or in organ, tissue or cell culture.
  • Preferential transcription or “preferred transcription” interchangeably refers to the expression of gene products that are preferably expressed at a higher level in one or a few plant tissues (spatial limitation) and/or to one or a few plant developmental stages (temporal limitation) while in other tissues/developmental stages there is a relatively low level of expression.
  • transformation refers to the transfer of a nucleic acid fragment into the genome of a host cell, resulting in genetically stable inheritance.
  • Transiently transformed refers to cells in which transgenes and foreign DNA have been introduced (for example, by such methods as Agrobacterium-mediated transformation or biolistic bombardment), but not selected for stable maintenance.
  • Stably transformed refers to cells that have been selected and regenerated on a selection media following transformation.
  • Transformed / transgenic / recombinant refer to a host organism such as a bacterium or a plant into which a heterologous nucleic acid molecule has been introduced.
  • the nucleic acid molecule can be stably integrated into the genome of the host or the nucleic acid molecule can also be present as an extrachromosomal molecule. Such an extrachromosomal molecule can be auto-replicating.
  • Transformed cells, tissues, or plants are understood to encompass not only the end product of a transformation process, but also transgenic progeny thereof.
  • non-transformed refers to a wild- type organism, e.g., a bacterium or plant, which does not contain the heterologous nucleic acid molecule.
  • translational enhancer sequence refers to that DNA sequence portion of a gene between the promoter and coding sequence that is transcribed into RNA and is present in the fully processed mRNA upstream (5') of the translation start codon.
  • the translational enhancer sequence may affect processing of the primary transcript to mRNA, mRNA stability or translation efficiency.
  • “Visible marker” refers to a gene whose expression does not confer an advantage to a transformed cell but can be made detectable or visible. Examples of visible markers include but are not limited to ⁇ -glucuronidase (GUS), lucif erase (LUC) and green fluorescent protein (GFP).
  • Wild-type refers to the normal gene, virus, or organism found in nature without any mutation or modification.
  • plant material means plant cells, plant protoplasts, plant cell tissue cultures from which plants can be regenerated, plant calli, plant clumps, and plant cells that are intact in plants or parts of plants such as embryos, pollen, ovules, seeds, leaves, flowers, branches, fruit, kernels, ears, cobs, husks, stalks, roots, root tips, anthers, tubers, rhizomes and the like.
  • Protein extract refers to partial or total protein extracted from a plant part. Plant protein extraction methods are well known in the art.
  • Plant sample refers to either intact or non-intact (e.g. milled seed or plant tissue, chopped plant tissue, lyophilized tissue) plant tissue. It may also be an extract comprising intact or non-intact seed or plant tissue.
  • sequence relationships between two or more nucleic acids or polynucleotides or polypeptides are used to describe the sequence relationships between two or more nucleic acids or polynucleotides or polypeptides: (a) “reference sequence,” (b) “comparison window,” (c) “sequence identity,” (d) “percentage of sequence identity” and (e) “substantial identity.”
  • reference sequence is a defined sequence used as a basis for sequence comparison.
  • a reference sequence may be a subset or the entirety of a specified sequence; for example, as a segment of a full-length cDNA or gene sequence or the complete cDNA or gene sequence.
  • comparison window means includes reference to a contiguous and specified segment of a polynucleotide sequence, wherein the polynucleotide sequence may be compared to a reference sequence and wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences.
  • the comparison window is at least 20 contiguous nucleotides in length, and optionally can be 30, 40, 50, and 100 or longer.
  • the BLAST family of programs which can be used for database similarity searches includes: BLASTN for nucleotide query sequences against nucleotide database sequences; BLASTX for nucleotide query sequences against protein database sequences; BLASTP for protein query sequences against protein database sequences; TBLASTN for protein query sequences against nucleotide database sequences; and TBLASTX for nucleotide query sequences against nucleotide database sequences.
  • GAP uses the algorithm of Needleman and Wunsch, supra, to find the alignment of two complete sequences that maximizes the number of matches and minimizes the number of gaps. GAP considers all possible alignments and gap positions and creates the alignment with the largest number of matched bases and the fewest gaps. It allows for the provision of a gap creation penalty and a gap extension penalty in units of matched bases. GAP must make a profit of gap creation penalty number of matches for each gap it inserts. If a gap extension penalty greater than zero is chosen, GAP must, in addition, make a profit for each gap inserted of the length of the gap times the gap extension penalty. Default gap creation penalty values and gap extension penalty values in Version 10 of the Wisconsin Genetics Software Package are 8 and 2, respectively. The gap creation and gap extension penalties can be expressed as an integer selected from the group of integers consisting of from 0 to 100. Thus, for example, the gap creation and gap extension penalties can be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40 and 50 or greater.
  • GAP presents one member of the family of best alignments. There may be many members of this family, but no other member has a better quality. GAP displays four figures of merit for alignments: Quality, Ratio, Identity and Similarity.
  • the Quality is the metric maximized in order to align the sequences. Ratio is the quality divided by the number of bases in the shorter segment.
  • Percent Identity is the percent of the symbols that actually match.
  • Percent Similarity is the percent of the symbols that are similar. Symbols that are across from gaps are ignored.
  • a similarity is scored when the scoring matrix value for a pair of symbols is greater than or equal to 0.50, the similarity threshold.
  • the scoring matrix used in Version 10 of the Wisconsin Genetics Software Package is BLOSUM62 (see, Henikoff and Henikoff, (1989) Proc. Natl Acad. Sci. USA 89: 10915).
  • sequence identity/similarity values refer to the value obtained using the BLAST 2.0 suite of programs using default parameters (Altschul, et al , (1997) Nucleic Acids Res. 25:3389-402).
  • BLAST searches assume that proteins can be modeled as random sequences. However, many real proteins comprise regions of nonrandom sequences, which may be homopolymeric tracts, short-period repeats, or regions enriched in one or more amino acids. Such low-complexity regions may be aligned between unrelated proteins even though other regions of the protein are entirely dissimilar.
  • a number of low-complexity filter programs can be employed to reduce such low-complexity alignments. For example, the SEG (Wooten and Federhen, (1993) Comput. Chem. 17: 149-63) and XNU (Claverie and States, (1993) Comput. Chem. 17: 191-201) low-complexity filters can be employed alone or in combination.
  • 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 When percentage of sequence identity is used in reference to proteins it is recognized that residue positions which are not identical often differ by conservative amino acid substitutions, where amino acid residues are substituted for other amino acid residues with similar chemical properties (e.g., charge or hydrophobicity) and therefore do not change the functional properties of the molecule. Where sequences differ in conservative substitutions, the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution.
  • Sequences which differ by such conservative substitutions, are said to have "sequence similarity" or "similarity.” Means for making this adjustment are well known to those of skill in the art. Typically this involves scoring a conservative substitution as a partial rather than a full mismatch, thereby increasing the percentage sequence identity. Thus, for example, where an identical amino acid is given a score of 1 and a non-conservative substitution is given a score of zero, a conservative substitution is given a score between zero and 1. The scoring of conservative substitutions is calculated, e.g., according to the algorithm of Meyers and Miller, (1988) Computer Applic. Biol. Sci. 4: 11-17, e.g., as implemented in the program PC/GENE (Intelligenetics, Mountain View, Calif., USA).
  • percentage of sequence identity means the value determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity.
  • polynucleotide sequences means that a polynucleotide comprises a sequence that has between 50-100% sequence identity, such as, at least 50% sequence identity, at least 60% sequence identity, at least 70%, at least 80%, more preferably at least 90% and at least 95%, compared to a reference sequence using one of the alignment programs described using standard parameters.
  • sequence identity such as, at least 50% sequence identity, at least 60% sequence identity, at least 70%, at least 80%, more preferably at least 90% and at least 95%
  • sequence identity such as, at least 50% sequence identity, at least 60% sequence identity, at least 70%, at least 80%, more preferably at least 90% and at least 95%.
  • nucleotide sequences are substantially identical is if two molecules hybridize to each other under stringent conditions.
  • the degeneracy of the genetic code allows for many amino acids substitutions that lead to variety in the nucleotide sequence that code for the same amino acid, hence it is possible that the DNA sequence could code for the same polypeptide but not hybridize to each other under stringent conditions. This may occur, e.g., when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code.
  • One indication that two nucleic acid sequences are substantially identical is that the polypeptide, which the first nucleic acid encodes, is immunologically cross reactive with the polypeptide encoded by the second nucleic acid.
  • plant biomass refers to the amount (measured in grams of air-dry or dry tissue) of a tissue produced from the plant in a growing season, which could also determine or affect the plant yield or the yield per growing area.
  • Yield is normally defined as the measurable produce of economic value from a crop. This may be defined in terms of quantity and/or quality. Yield is directly dependent on several factors, for example, the number and size of the organs, plant architecture (for example, the number of branches), seed production, leaf senescence and more. Root development, nutrient uptake, stress tolerance and early vigor may also be important factors in determining yield. In addition it is greatly desirable in agriculture to develop crops that may show increased yield in optimal growth conditions as well as in non-optimal growth conditions (e.g. drought, under abiotic stress conditions). Optimizing the abovementioned factors may therefore contribute to increasing crop yield.
  • Seed yield is a particularly important trait, since the seeds of many plants are important for human and animal nutrition. Crops such as, corn, rice, wheat, canola and soybean account for over half the total human caloric intake whether through direct consumption of the seeds themselves or through consumption of livestock raised on processed seeds. Plant seeds are also a source of sugars, oils and many kinds of metabolites used in various industrial processes. Seeds consist of an embryo (the source of new shoots and roots) and an endosperm (the source of nutrients for embryo growth during germination and during early growth of seedlings). The development of a seed involves many genes, and requires the transfer of metabolites from the roots, leaves and stems into the developing seed. The endosperm assimilates the metabolic precursors of carbohydrates, oils and proteins and synthesizes them into storage macromolecules to fill out the grain.
  • plant yield is relative to the amount of plant biomass a particular plant may produce.
  • a larger plant with a greater leaf area can typically absorb more light, nutrients and carbon dioxide than a smaller plant and therefore will likely gain a greater weight during the same period (Fasoula & Tollenaar 2005 Maydica 50:39).
  • Increased plant biomass may also be highly desirable in processes such as the conversion of biomass (e.g. corn, grasses, sorghum, cane) to fuels such as for example ethanol or butanol.
  • Plant breeders are often interested in improving specific aspects of yield depending on the crop or plant in question, and the part of that plant or crop which is of relative economic value.
  • a plant breeder may look specifically for improvements in plant biomass (weight) of one or more parts of a plant, which may include aboveground (harvestable) parts and/or harvestable parts below ground. This is particularly relevant where the aboveground parts or below ground parts of a plant are for consumption.
  • an improvement in seed yield is highly desirable.
  • Increased seed yield may manifest itself in many ways with each individual aspect of seed yield being of varying importance to a plant breeder depending on the crop or plant in question and its end use.
  • Various systems, computer program products and methods for using a model of biological process can predict candidate components such as genes and/or combinations of genes that enhance the biological process. For example, please see the methods as disclosed in WO2012/061585, published on 10 May 2012 and hereby incorporated by reference.
  • One may select a candidate component based on the phenotypic outcome and the determined sensitivity for the purpose of producing a biological product that exhibits or will exhibit the phenotypic outcome.
  • a candidate gene may be selected based on a phenotypic outcome in which the gene is predicted to cause and based on the determined sensitivity.
  • a single candidate gene that is relatively insensitive to variations to the optimal expression level may cause the predicted phenotypic outcome or a phenotypic outcome that is acceptably close (based on a predefined difference) to the predicted phenotypic outcome even when the optimal expression levels are not achieved in the biological product during, for example, laboratory experimentation and/or manufacturing.
  • the polynucleotide sequence of the selected candidate gene(s) identified by the invention can be synthesized or isolated and introduced into expression cassettes, which contain genetic regulatory elements to target the expression level and cell type(s).
  • at least one expression cassette may be introduced into a binary vector and transformed into plants. The sensitivity and actual phenotypic outcome can then be determined.
  • one embodiment uses the invention to identify three or four candidate genes which are introduced into expression cassettes and transformed into plants using methods known to one skilled in the art. The examples also describe known methods for measuring the phenotypic outcome of the transgenic plants.
  • One embodiment of the invention includes an expression cassette, cell, or plant comprising alone or in any combination a phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31), a fructose- 1, 6-bisphosphate phosphatase (FBP, EC 3.1.3.11), a NADP-malate dehydrogenase (NADPMD, EC 1.1.1.82), a phosphoribulokinase (PRK, EC 2.7.1.19), and a pyruvate, orthophosphate dikinase (PPDK, EC 2.7.9.1).
  • PEPC phosphoenolpyruvate carboxylase
  • FBP fructose- 1, 6-bisphosphate phosphatase
  • NADPMD NADP-malate dehydrogenase
  • PRK phosphoribulokinase
  • PPDK pyruvate, orthophosphate dikinase
  • Another embodiment of the invention includes an expression cassette, cell or plant comprising any two genes in combination comprising a phosphoenolpyruvate carboxylase (PEPC), a fructose-1, 6-bisphosphate phosphatase (FBP), a NADP-malate dehydrogenase (NADPMD), a phosphoribulokinase (PRK), and a pyruvate, orthophosphate dikinase (PPDK).
  • PEPC phosphoenolpyruvate carboxylase
  • FBP fructose-1, 6-bisphosphate phosphatase
  • NADPMD NADP-malate dehydrogenase
  • PRK phosphoribulokinase
  • PPDK pyruvate, orthophosphate dikinase
  • Yet another embodiment of the invention includes an expression cassette, cell or plant comprising any three genes in combination comprising a phosphoenolpyruvate carboxylase (PEPC), a fructose-1, 6-bisphosphate phosphatase (FBP), a NADP-malate dehydrogenase (NADPMD), a phosphoribulokinase (PRK), and a pyruvate, orthophosphate dikinase (PPDK).
  • expression cassettes, cells or plant comprising a fructose-1, 6-bisphosphate phosphatase (FBP), a phosphoribulokinase (PRK) and a phosphoenolpyruvate carboxylase (PEPC).
  • Yet another embodiment of the invention includes an expression cassette, cell or plant comprising any four genes in combination comprising a phosphoenolpyruvate carboxylase (PEPC), a fructose-1, 6-bisphosphate phosphatase (FBP), a NADP-malate dehydrogenase (NADP-MD), phosphoribulokinase (P K), and a pyruvate, orthophosphate dikinase (PPDK).
  • PEPC phosphoenolpyruvate carboxylase
  • FBP fructose-1, 6-bisphosphate phosphatase
  • NADP-MD NADP-malate dehydrogenase
  • P K phosphoribulokinase
  • PPDK orthophosphate dikinase
  • expression cassettes, cells or plant comprising a fructose-1, 6- bisphosphate phosphatase (FBP), a phosphoribulokinase (PRK), a NADP-malate dehydrogenase (NADP-MD) and a phosphoenolpyruvate carboxylase (PEPC).
  • FBP fructose-1, 6- bisphosphate phosphatase
  • PRK phosphoribulokinase
  • NADP-MD NADP-malate dehydrogenase
  • PEPC phosphoenolpyruvate carboxylase
  • Yet another embodiment of the invention includes an expression cassette, cell or plant comprising a phosphoenolpyruvate carboxylase (PEPC), a fructose-1, 6-bisphosphate phosphatase (FBP), a NADP-malate dehydrogenase (NADP-MD), phosphoribulokinase (PRK), and a pyruvate, orthophosphate dikinase (PPDK).
  • PEPC phosphoenolpyruvate carboxylase
  • FBP fructose-1, 6-bisphosphate phosphatase
  • NADP-MD NADP-malate dehydrogenase
  • PRK phosphoribulokinase
  • PPDK orthophosphate dikinase
  • One embodiment of the invention can also include an expression cassette, cell or plant comprising SEQ ID NO. 6, SEQ ID NO. 7, and SEQ ID NO. 8 or polynucleotides have 50, 60, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity or polynucleotides having capable of hybridizing under low, medium or high stringent hybridization conditions to SEQ ID NO. 6, SEQ ID NO. 7, and SEQ ID NO. 8.
  • Another embodiment of the invention includes an expression cassette, cell or plant comprising any two of the sequences SEQ ID NO. 6, SEQ ID NO. 7, and SEQ ID NO. 8 or polynucleotides have 50, 60, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity or polynucleotides having capable of hybridizing under low, medium or high stringent hybridization conditions to SEQ ID NO. 6, SEQ ID NO. 7, and SEQ ID NO. 8.
  • Yet another embodiment of the invention includes an expression cassette, cell or plant comprising one of the sequences SEQ ID NO. 6, SEQ ID NO. 7, and SEQ ID NO. 8 or polynucleotides have 50, 60, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity or polynucleotides having capable of hybridizing under low, medium or high stringent hybridization conditions to SEQ ID NO. 6, SEQ ID NO. 7, and SEQ ID NO. 8.
  • the present invention includes an expression cassette, cell or plant comprising at least one of the sequences SEQ ID NO. 6, SEQ ID NO. 7, or SEQ ID NO. 8 or polynucleotides have 50, 60, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity or polynucleotides having capable of hybridizing under low, medium or high stringent hybridization conditions to SEQ ID NO. 6, SEQ ID NO. 7, and SEQ ID NO. 8.
  • Yet another embodiment of the invention includes an expression cassette, cell or plant comprising the sequences SEQ ID NO. 9, SEQ ID NO. 10, and SEQ ID NO. 11, and SEQ ID NO. 12 or polynucleotides have 50, 60, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity or polynucleotides having capable of hybridizing under low, medium or high stringent hybridization conditions to SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. Hand SEQ ID NO. 12.
  • Another embodiment of the invention includes an expression cassette, cell, plant, or mammal comprising two of the sequences SEQ ID NO. 9, SEQ ID NO. 10, and SEQ ID NO. 11, and SEQ ID NO. 12 or polynucleotides have 50, 60, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity or polynucleotides having capable of hybridizing under low, medium or high stringent hybridization conditions to SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. Hand SEQ ID NO. 12.
  • One embodiment of the invention also includes an expression cassette, cell, plant, or mammal comprising one of the sequences SEQ ID NO. 9, SEQ ID NO. 10, and SEQ ID NO. 11, and SEQ ID NO. 12 or polynucleotides have 50, 60, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity or polynucleotides having capable of hybridizing under low, medium or high stringent hybridization conditions to SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. Hand SEQ ID NO. 12.
  • An embodiment of the invention includes an expression cassette, cell, plant or mammal plant comprising at least one of the sequences SEQ ID NO. 9, SEQ ID NO. 10, and SEQ ID NO. 11, and SEQ ID NO. 12 or polynucleotides have 50, 60, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity or polynucleotides having capable of hybridizing under low, medium or high stringent hybridization conditions to SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. Hand SEQ ID NO. 12.
  • Implementations of the invention may be made in hardware, firmware, software, or any suitable combination thereof. Implementations of the invention may also be implemented as instructions stored on a machine readable medium, which may be read and executed by one or more processors.
  • a tangible machine-readable medium may include any tangible, non-transitory, mechanism for storing or transmitting information in a form readable by a machine (e.g., a computing device).
  • a tangible machine- readable storage medium may include read only memory, random access memory, magnetic disk storage media, optical storage media, flash memory devices, and other tangible storage media.
  • Intangible machine-readable transmission media may include intangible forms of propagated signals, such as carrier waves, infrared signals, digital signals, and other intangible transmission media.
  • firmware, software, routines, or instructions may be described in the above disclosure in terms of specific exemplary implementations of the invention, and performing certain actions. However, it will be apparent that such descriptions are merely for convenience and that such actions in fact result from computing devices, processors, controllers, or other devices executing the firmware, software, routines, or instructions.
  • Implementations of the invention may be described as including a particular feature, structure, or characteristic, but every aspect or implementation may not necessarily include the particular feature, structure, or characteristic. Further, when a particular feature, structure, or characteristic is described in connection with an aspect or implementation, it will be understood that such feature, structure, or characteristic may be included in connection with other implementations, whether or not explicitly described. Thus, various changes and modifications may be made to the provided description without departing from the scope or spirit of the invention. As such, the specification and drawings should be regarded as exemplary only, and the scope of the invention to be determined solely by the appended claims.
  • SEQ ID NO: 1 depicts a polypeptide sequence, Zea mays phosphoenolpyruvate carboxylase
  • SEQ ID NO: 2 depicts a polypeptide sequence, Spinacia oleracea fructose- 1, 6-bisphosphate phosphatase
  • SEQ ID NO: 3 depicts a polypeptide sequence
  • SEQ ID NO: 4 depicts a polypeptide sequence
  • SEQ ID NO: 5 depicts a polypeptide sequence, Sorghum bicolor engineered pyruvate, orthophosphate dikinase
  • SEQ ID NO: 6 depicts a polynucleotide sequence
  • SoFBP in expression cassette ZmPRK-1 depicts a polynucleotide sequence
  • SoPRK in expression cassette ZmSBP depicts a polynucleotide sequence
  • ZmPEPC in expression cassette ZmPGK depicts a polynucleotide sequence
  • SoFBP in expression cassette ZmPRK-2 depicts a polynucleotide sequence
  • SEQ ID NO: 11 depicts a polynucleotide sequence
  • SbPPDK in expression cassette ZmPEPC
  • SEQ ID NO: 12 depicts a polynucleotide sequence, SbNADP-MD in expression cassette ZmPGK
  • This example describes a genetic engineering strategy to enhance photoassimilation in maize and other NADP malic-type C4 species.
  • a computer model output was organized into 3 and 4 gene combination solutions.
  • a 3-gene and a 4-gene combination were each selected for trait development.
  • the BRENDA database (brenda.enzymes.org) was queried for sequence information on phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31), fructose-1, 6-bisphosphate phosphatase (FBPase, EC 3.1.3.11), phosphoribulokinase (PRK, EC 2.7.1.19), NADP-malate dehydrogenase (NADPME, EC 1.1.1.82) and pyruvate, orthophosphate dikinase (PPDK, EC 2.7.9.1).
  • PPDK orthophosphate dikinase
  • the sorghum gDNA and cDNA sequence were pulled from the sorghum genome database using the maize PPDK cDNA and protein sequence as the queries.
  • the sorghum cDNA was expanded through alignment with corresponding ESTs. The sequences were compiled into a contig that was broken into exons and aligned with the gDNA. There are 19 exons, and all but one defines introns bordered by GT...AG sequence. There were several places where sorghum PPDK gDNA and cDNA sequence diverged; in most instances the cDNA sequence was substituted for the gDNA sequence.
  • the maize and sorghum protein sequences were also aligned and used to further refine the gDNA sequence.
  • Flaveria brownie PPDK residue substitutions were introduced.
  • the result is the SbPPDK-engineered sequence, SEQ ID NO 5.
  • the gDNA sequence was also modified to silence Xhol, SanDI, Ncol, Sacl, RsrII, and Xmal restriction endonuclease sites by base substitution. An Ncol site was added at the translation start codon and a Sacl site was added after the translation stop codon.
  • the promoter consists of 5'- non-transcribed sequence, the first intron, and a 5 '-untranslated sequence that is made up of the first and part of the second exon.
  • the promoter terminates with a translational enhancer derived from the tobacco mosaic virus omega sequence (Gallie, D. R., Walbot, V. (1992) Nucleic Acids Res 20(17): 4631-4638) and a maize-optimized sequence (Kozak, M. (2002) Gene 299: 1-34).
  • the terminator consists of 3 '-untranslated sequence starting just after the translation stop codon and 3 '-non-transcribed sequence.
  • a three-gene and a four-gene expression cassette binary vector containing the candidate genes selected by the method of the present invention will each be used to reduce the C4 photosynthesis model output to practice.
  • the three gene C4 photosynthesis enhancement construct is shown in Table 2; the four gene C4 photosynthesis enhancement construct is shown in Table 3.
  • the gene number indicates order, starting at the right border of the T-DNA and extending to the left border.
  • the three gene binary vector is 19862 and is shown in Figure 1.
  • the four gene binary vector is 19863 and is shown in Figure 2.
  • Constructs 19862 and 19863 were used for Agrobacterium-mediated maize transformation. Transformation of immature maize embryos was performed essentially as described in Negrotto et al., 2000, Plant Cell Reports 19: 798-803. For this example, all media constituents were essentially as described in Negrotto et al., supra. However, various media constituents known in the art may be substituted.
  • Vectors used in this example contain the phosphomannose isomerase (PMI) gene for selection of transgenic lines (Negrotto et al., supra), as well as the selectable marker phosphinothricin acetyl transferase (PAT) (U.S. Patent No. 5,637,489).
  • PMI phosphomannose isomerase
  • PAT selectable marker phosphinothricin acetyl transferase
  • Agrobacterium strain LBA4404 containing a plant transformation plasmid was grown on YEP (yeast extract (5 g/L), peptone (lOg/L), NaCl (5g/L), 15g/l agar, pH 6.8) solid medium for 2 - 4 days at 28°C. Approximately 0.8X 10 9 Agrobacterium were suspended in LS-inf media supplemented with 100 M As (Negrotto et al, supra). Bacteria were pre- induced in this medium for 30-60 minutes.
  • LSD1M0.5S medium The cultures were selected on this medium for about 6 weeks with a subculture step at about 3 weeks. Surviving calli were transferred to Regl medium supplemented with mannose. Following culturing in the light (16 hour light/ 8 hour dark regiment), green tissues were then transferred to Reg2 medium without growth regulators and incubated for about 1-2 weeks. Plantlets were transferred to Magenta GA-7 boxes (Magenta Corp, Chicago 111.) containing Reg3 medium and grown in the light.
  • Magenta GA-7 boxes Magnenta Corp, Chicago 111.
  • Plants were assayed for PMI, PAT, one candidate gene coding sequence and vector backbone by TaqMan. Plants that were positive for PMI, PAT and the candidate gene coding sequence and negative for vector backbone were transferred to the greenhouse. Expression for all trait expression cassettes was assayed by qRT-PCR. Fertile, single copy events were identified and transferred to the greenhouse.
  • EXAMPLE 5 EVALUATION OF TRANSGENIC PLANTS EXPRESSING CANDIDATE GENES
  • Plant photoassimilation can be assessed in several ways. The following prophetic example described how the transgenic plants described above will be measured for changes in plant photoassimilation.
  • First plant growth between hemizygous trait positive and null seedlings can be compared in V3 seedlings. In this assay, approximately 60 B l plants are germinated in 4.5 inch pots and genotyped. About 17 days after germination the pot soil is saturated with water and the soil surface is sealed to prevent evaporation. Some seedlings are sacrificed to determine shoot mass (in both fresh and dry weight) at time zero. Pot mass is recorded daily to assess plant water demand. After 7 days shoots are harvested and weighed (both fresh and dry weight). Plant water utilization is corrected using a pot with no plant to report natural water loss. This protocol enables plant growth and water utilization to be compared between trait positive and null groups. Improved photoassimilation may enable the trait positive plants to accumulate more aerial biomass relative to null plants.
  • a second method is to measure photoassimilation using an infrared gas analysis (IRGA) instrument.
  • IRGA infrared gas analysis
  • a CIRAS-2 IRGA device can be fixed to a tripod to gently clamp the gas exchange cuvette to leaves and minimize data noise generated by plant handling. Stomatal aperture is very sensitive to touch and plant movement.
  • the environment applied to the leaf patch can be programmed to mimic a growth chamber environment (400 ⁇ mol "1 CO 2 ; 26°C; ambient humidity) to assess steady-state photosynthesis under standard growth conditions. In this way photoassimilation between trait positive and null plants can be directly compared.
  • IRGA is a powerful and common tool to assess photosynthetic activity (e.g. A/Ci curves), it has some caveats.
  • it is an invasive technique requiring direct contact with the leaf. A component of the data generated is leaf response to the instrument. Taken together this creates high (10-15%) coefficients of variation. Hence, it may not be possible to detect small, but significant changes in photoassimilation using this device.
  • hypobaric chambers such as the chambers at the Controlled Environment Systems Research Facility at the University of Guelph, Ontario (Wheeler, R.M., et. al. (2011) Adv Space Res 47: 1600-1607) can be used to monitor with high precision plant CO 2 demand, night time respiration and transpiration of a 30-40 plant population for periods lasting up to several weeks.
  • Transgenic maize events were produced according to Example 4, using binary vectors 19862 and 19863. A total of 32 single-copy, backbone free 19862 events were identified. A total of 22 single-copy, backbone free 19863 events were identified.
  • Messenger RNA produced from each transgene was measured in seedling leaf tissue by qRT-PCR. The qRT-PCR data are reported as the ratio of the gene-specific (coding sequence) signal to that of the endogenous control signal times 1000.
  • Data in the Table below show that all the trait expression cassettes function to produce trait transcript in leaf as expected. Data for the constitutive expression cassettes are included as a benchmark for signal strength. It should be noted that the constitutive cassettes are active in far more leaf cells than the trait cassettes which are restricted to either mesophyll or bundle sheath cells. Table 4.
  • TO seedling leaf tissue was sampled for qRT-PCR analysis roughly two weeks after transfer to soil (V3). Gene-specific TaqMan probes were used to determine transcript abundance. Data are reported relative to EF1A transcript, the internal control. Each event was assayed in quadruplicate. Data are the mean ⁇ standard deviation for each construct.
  • EXAMPLE 7 SEEDLING BIOMASS ACCUMULATION IN A GROWTH CHAMBER
  • Seedling growth can be used to determine if a trait has the potential to cause yield drag. We used this assay to determine if either the 19862 or 19863 traits reduced plant growth. Back-crossed seed were germinated and seedlings were evaluated in a growth chamber according to Example 5. Seedlings for each event were genotyped to establish trait segregation and organize transgenic and null groups. Trait segregation was confirmed as 1 null: 1 hemizygote, as expected, for each event. Data in the Table below summarize the results of several assays. For each event, growth of the transgenic seedlings could not be distinguished from the null seedlings. This indicates the trait is not impeding growth. The wild type plants are included as a benchmark. It should be noted that plants one generation removed from a parent regenerated through tissue culture tend to grow slower than non- transformed or wild type plants. The mean data suggest that the 19862 plants may be growing slower than the wild type plants but the difference is not statistically significant.
  • Transgenic B l seed were germinated in 4.5 inch pots and genotyped. Plants for each event were organized into transgenic and null groups which were grown in a growth chamber. Plants were harvested 24 days after planting. Plants were dried in an oven at 89°C for 5 days then weighed. Data report the mean ⁇ standard deviation for each construct.
  • EXAMPLE 8 EVALUATION OF 19862 EVENTS IN CLOSED CHAMBERS
  • Fl hybrid seed were germinated and genotyped. Plants were organized into transgenic and null groups. Each group was cultivated in a large hypobaric chamber at the Controlled Environment Systems Research Facility at the University of Guelph. Plants were harvested, dried and weighed. Initial biomass was determined for seedlings shortly after genotyping and represent shoot mass at the time beginning of the study. Data are the mean ⁇ standard deviation for each group.

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Abstract

De manière générale, cette invention concerne le domaine de la biologie moléculaire et notamment, divers polynucléotides, polypeptides et procédés qui peuvent être utilisés pour améliorer le rendement chez les plantes transgéniques. Plus spécifiquement, les plantes transgéniques peuvent manifester un rendement accru, une biomasse accrue ou une photo-assimilation accrue.
PCT/US2012/063161 2010-11-04 2012-11-02 Polynucléotides, polypeptides et procédés d'amélioration de la photo-assimilation chez les plantes WO2013067252A1 (fr)

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BR112014010642A BR112014010642A2 (pt) 2010-11-04 2012-11-02 polinucleotídeos, polipeptídeos e métodos para melhorar a fotoassimilação em plantas
CA2853490A CA2853490A1 (fr) 2011-11-03 2012-11-02 Polynucleotides, polypeptides et procedes d'amelioration de la photo-assimilation chez les plantes
AU2012332343A AU2012332343A1 (en) 2011-11-03 2012-11-02 Polynucleotides, polypeptides and methods for enhancing photossimilation in plants
HU1400505A HUP1400505A2 (en) 2011-11-03 2012-11-02 Polynucleotides, polypeptides and methods for enhancing photossimilation in plants
CN201280053974.5A CN103998611A (zh) 2011-11-03 2012-11-02 用于增强植物中光同化作用的多核苷酸、多肽和方法
US14/355,251 US20140317783A1 (en) 2011-11-03 2012-11-02 Polynucleotides, polypeptides and methods for enhancing photossimilation in plants
EA201400527A EA201400527A1 (ru) 2011-11-03 2012-11-02 Полинуклеотиды, полипептиды и способы усиления фотоассимиляции у растений
MX2014005375A MX2014005375A (es) 2011-11-03 2012-11-02 Polinucleotidos, polipeptidos y metodos para mejorar la fotoasimilacion en plantas.

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