CORN WITH INCREASED YIELD AND NITROGEN UTILIZATION EFFICIENCY

Abstract

The subject invention relates in part to the use of insect-protected corn to modify fertility recommendations for given yield targets on any transgenic corn type.

Description

BACKGROUND OF THE INVENTION

[0001] Current fertility recommendations for corn were developed over a long period of time with traditional corn susceptible to insect attack, or with corn protected from insects via application of chemical insecticides. Subject to the price of chemical fertilizers, typical current practice is for farmers to over-saturate their fields with fertilizers to a lesser or greater degree.

FIELD OF THE INVENTION

[0002] This invention is in the field of genetically engineered plants having improved yield.

BRIEF SUMMARY OF THE INVENTION

[0003] The subject invention concerns the surprising discovery that transgenic corn that produces Bacillus thuringiensis (Bt) insecticidal toxins to provide in-plant protection against feeding damage by above-ground and below-ground insect pests exhibits desirable agronomic characteristics apart from the protection against insect feeding damage.

[0004] The subject invention relates in part to the use of insect-protected transgenic corn to modify fertility recommendations for a given yield target. Specifically, it is found that transgenic corn commercially adopted as HERCULEX-XTRA; thus containing genes that encode Cry34Ab1, Cry35Ab1, and Cry1F insecticidal proteins, produces increased grain yield, as measured by kernel weight and number, when grown in field conditions and compared to isogenic control populations. It is additionally found that these enhanced yields are obtained with a further advantage of increased efficiency of nitrogen fertilizer utilization.

[0005] The present invention also includes methods of modulating nitrogen utilization efficiency (NUE) in a plant cell, comprising: (a) introducing into a plant cell a recombinant expression cassette comprising a Cry protein operably linked to a promoter that drives expression in a plant; and (b) culturing the plant cell under plant cell growing conditions; wherein the nitrogen uptake in the plant cell is modulated.

[0006] Other methods include increasing yield in a plant, wherein such methods comprise the steps of: (a) introducing into a plant cell a construct comprising a Cry protein operably linked to a promoter functional in a plant cell, so as to yield transformed plant cells; and, (b) regenerating a transgenic plant from said transformed plant cell, wherein said Cry protein is expressed in the cells of said transgenic plant at levels sufficient to increase yield in said transgenic plant; wherein increased yield comprises enhanced root growth, increased seed size, increased seed weight, seed with increased embryo size, increased leaf size, increased seedling vigor, enhanced silk emergence, increased ear size, nitrogen utilization or chlorophyll content.

[0007] Seeds and plants made from these methods may also be included.

BRIEF DESCRIPTION OF THE SEQUENCES

[0008] SEQ ID NO:1: cry34 plant-optimized gene sequence

[0009] SEQ ID NO:2: cry35 plant-optimized gene sequence

[0010] SEQ ID NO:3: truncated cry1F sequence encoding core toxin

[0011] SEQ ID NO:4: truncated/core-toxin Cry1F protein sequence

[0012] SEQ ID NO:5: native/full-length cry1F sequence

[0013] SEQ ID NO:6: native/full-length Cry1F protein sequence

[0014] SEQ ID NO:7: native cry34 sequence

[0015] SEQ ID NO:8: Cry34 protein sequence

[0016] SEQ ID NO:9: native cry35 sequence

[0017] SEQ ID NO:10: Cry35 protein sequence

DETAILED DISCLOSURE OF THE INVENTION

[0018] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Unless mentioned otherwise, the techniques employed or contemplated herein are standard methodologies well known to one of ordinary skill in the art. The materials, methods and examples are illustrative only and not limiting. The following is presented by way of illustration and is not intended to limit the scope of the invention.

[0019] The present inventions now will be described more fully hereinafter with reference to the accompanying drawings, in which some, but not all embodiments of the invention are shown. Indeed, these inventions may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will satisfy applicable legal requirements. Like numbers refer to like elements throughout.

[0020] Many modifications and other embodiments of the inventions set forth herein will come to mind to one skilled in the art to which these inventions pertain having the benefit of the teachings presented in the foregoing descriptions and the associated drawings. Therefore, it is to be understood that the inventions are not to be limited to the specific embodiments disclosed and that modifications and other embodiments are intended to be included within the scope of the appended claims. Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation.

[0021] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of botany, microbiology, tissue culture, molecular biology, chemistry, biochemistry and recombinant DNA technology, which are within the skill of the art. Such techniques are explained fully in the literature. See, e.g., Langenheim and Thimann, (1982) Botany: Plant Biology and Its Relation to Human Affairs, John Wiley; Cell Culture and Somatic Cell Genetics of Plants, vol. 1, Vasil, ed. (1984); Stanier, et al., (1986) The Microbial World, 5^th ed., Prentice-Hall; Dhringra and Sinclair, (1985) Basic Plant Pathology Methods, CRC Press; Maniatis, et al., (1982) Molecular Cloning: A Laboratory Manual; DNA Cloning, vols. I and II, Glover, ed. (1985); Oligonucleotide Synthesis, Gait, ed. (1984); Nucleic Acid Hybridization, Hames and Higgins, eds. (1984); and the series Methods in Enzymology, Colowick and Kaplan, eds, Academic Press, Inc., San Diego, Calif.

[0022] As used herein "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. Generally, 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. As used herein, the term "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, Solanum, Petunia, Digitalis, Majorana, Ciahorium, Helianthus, Lactuca, Bromus, Asparagus, Antirrhinum, Heterocallis, Nemesis, Pelargonium, Panieum, Pennisetum, Ranunculus, Senecio, Salpiglossis, Cucumis, Browaalia, Glycine, Pisum, Phaseolus, Lolium, Oryza, Avena, Hordeum, Secale, Allium, and Triticum. Plants of the invention include, but are not limited to, rice, wheat, peanut, sugarcane, sorghum, corn, cotton, soybean, vegetable, ornamental, conifer, alfalfa, spinach, tobacco, tomato, potato, sunflower, canola, barley or millet Brassica sp., safflower, sweet potato, cassava, coffee, coconut, pineapple, citrus trees, cocoa, tea, banana, palm, avocado, fig, guava, mango, olive, papaya, cashew, macadamia, almond, sugar beet, sugarcane, buckwheat, triticale, spelt, linseed, sugar cane, oil seed rape, canola, cress, Arabidopsis, cabbages, soya, pea, beans, eggplant, bell pepper, Tagetes, lettuce, Calendula, melon, pumpkin, squash and zucchini or oat plant. A particularly preferred plant is Zea mays.

[0023] As used herein, "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.

[0024] As used herein, "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. Thus, DNAs or RNAs with backbones modified for stability or for other reasons are "polynucleotides" as that term is intended herein. Moreover, 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. The term 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.

[0025] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers.

[0026] As used herein "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. 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, developmentally regulated, and inducible promoters constitute the class of "non-constitutive" promoters. A "constitutive" promoter is a promoter, which is active under most environmental conditions, for example, the ubiquitin gene promoter Ubil.

[0027] Transgenic corn varieties with insect protection traits have been available for several years. Multiple events are available for control of above-ground and below-ground feeding insects. These events are transformed with genes that produce Bt proteins and are marketed under names such as HERCULEX, YIELDGARD and AGRISURE. They are widely recognized to be efficacious against insect damage and are recommended for planting by University field extension services, and by their manufacturers, in locations where insect damage is expected to adversely affect corn yields. The growing recommendations (e.g. fertilizer application rates) for all of these insect protected corn varieties are identical to those recommended for nonprotected corn varieties.

[0028] While insect-protected corn varieties such as HERCULEX-XTRA are currently planted in many corn growing regions globally, farming methods of growing them with reduced fertilizer recommendations were not previously taught or suggested and are a subject of the present invention. Fertilization rates, ranges, and amounts as described elsewhere herein and as specifically exemplified in the Examples can be used to define methods of the subject invention. Various units and rates can be used to express such rates and/or ranges--some of which are used in the Examples (e.g., pounds of nitrogen fertilizer per acre).

[0029] Nitrogen fertilizer inputs are the third most costly input (behind land and seed) in corn production, and the cost may vary greatly depending on the price of natural gas required to produce nitrogen fertilizer. Nitrogen use efficiency (NUE) represents an important target for maize (corn) breeding programs. Previous research demonstrates that genetic variability exists for NUE and its components, N uptake efficiency (NUpE) and N utilization efficiency (NUtE). Thus, widespread adoption of the subject invention, having a result of less natural gas devoted to fertilizer production, can reduce costs for farmers as well as for consumers who use natural gas for heating and cooking.

[0030] Grain yield in corn is largely affected by the ability of the plant to take up nitrogen (N) from the soil and utilize it for growth and reproduction. Plant responses to nitrogen fertilizer are observed across a wide range of application rates. The subject invention demonstrates that grain yield and its components, kernel weight and number, are increased across a range of N application rates by the presence of the transgenic insecticidal proteins Cry34Ab1, Cry35Ab1 and Cry1F. These enhancements are accompanied by prolonged stay-green after flowering, and they correlate with increased N uptake efficiency before and after flowering. Further, it is surprisingly seen that the plants of the subject invention have increased grain yield per unit of plant nitrogen, both within and between N application rates. The effect is not seen with nontransgenic isogenic lines protected from insect attack by application of chemical insecticides, thus demonstrating that these benefits may be in addition to the root protection afforded by the transgenic insect control.

[0031] The subject invention stems in part from our observation that yields of genetically similar corn lines, with and without the insect resistance traits, unexpectedly respond differently to inputs, particularly nitrogen fertilizer. The insect-protected plants have less root damage from below-ground insect feeding. In addition, when damaged by root feeding insects such as corn root worm, the damaged plants regrow quicker and produce a larger root mass and have improved overall plant health. This combination of factors allows the rate of nitrogen and other fertilizers to be reduced to obtain the same amount of yield as is obtained with non insect-protected corn lines grown with higher fertility amounts.

[0032] The present invention can comprise transgenic plants that accumulate the insecticidal proteins Cry34Ab1 and Cry35Ab1 as well as CryF1. These proteins protect the root system of maize from damage from corn rootworm feeding and facilitate improved N uptake and utilization, thereby providing increased grain yield. Fertilizer application rates can be determined from the subject disclosure. Exemplary application rates, included within the subject invention, are demonstrated. Some preferred embodiments are also further specified in the Claims. Additionally, Herculex can be used in other crop species such as canola, wheat, rice, barley and other non-legume crops.

[0033] The present invention also includes methods of increasing nitrogen uptake efficiency in a plant by administering to a plant an expression cassette containing at least one Bacillus thuringiensis insect-resistance gene which functions to improve expression of at least one insecticidal portion of a protein or amino acid sequence variant thereof from a nucleic acid coding sequence in a plant cell. Such method can further increase in the yield of the plant. The increase in yield can include an increase in the kernel number per plant and/or an increase in the kernel mass per plant. Nitrogen utilization can also be modulated. Additionally, the increase in nitrogen can occur during the flowering and grain filling periods of development.

[0034] The Bacillus thuringiensis insect-resistance gene used can include a Cry protein. The insect-resistance gene can be selected from the group consisting of, e.g., a cry34 gene, a cry35 gene, a cry1F gene, and a cry3A gene. The Bacillus thuringiensis insect-resistance gene can comprise, e.g., Cry34Ab, Cry35Ab, and/or Cry1F. Sequences of the relevant proteins and genes of HERCULEX products are readily determinable. Unless otherwise indicated herein, the Cry1F protein and gene are as described in U.S. Pat. No. 6,218,188 (preferably the truncated, plant-optimized version described therein), and the Cry34/35 genes and proteins are as described in U.S. Pat. No. 6,340,593 (preferably the 149B1 genes and proteins). Other genes and proteins that can be used according to the subject invention are also known in the art. See e.g. U.S. Pat. Nos. 7,179,965; 7,524,810; 7,939,651; 6,893,872; and 6,900,371. The Crickmore et al. website of the official Bacillus thuringiensis nomenclature committee is also well-known in the art and provides links to many, publically available Cry protein and gene sequences. Truncated and/or core-toxin fragments of Cry1F, for example, can be used, as is known in the art. Variants thereof are included. The official nomenclature committee uses boundaries of at least 45% identity (e.g. Cry1F), 78% identity (e.g. Cry1Fa), and 95% protein sequence identity (Cry1Fa1) as primary, secondary, and tertiary ranks, respectively. Such boundaries, as well as 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, and/or 99% sequence identity (with an exemplified or suggested protein or gene sequence) can be used according to the subject invention in some embodiments.

[0035] Either monocotyledonous plants or dicotyledonous plants may be used in the present invention. The present invention also includes method of growing transgenic corn plants having an increased yield by using a reduced amount of fertilizer, wherein the transgenic corn is insect resistant due to expression of an insect-resistance gene, and wherein the reduced amount of fertilizer is relative to fertilizer recommended for use on non-transgenic corn, wherein said non-transgenic corn is optionally protected by granular chemical insecticide to control rootworms. The increase in year can include an increase in the kernel number per plant or can include an increase in the kernel mass per plant. Such transgenic corn plants can yield corn comparable to corn yield from said non-transgenic corn grown using said recommended amounts of fertilizer. The fertilizer used can be a nitrogenous fertilizer. The nitrogenous fertilizer can be applied at any rate of less than 150 pounds per acre. Alternatively the nitrogenous fertilizer can be applied at a rate of less than 50, 60, 70, 75, 80, 90, 100, 110, 120, 125, 130, 140 pounds per acre. In general, it may be preferable to have some nitrogenous fertilizer added to a crop. The nitrogenous fertilizer can be applied to a field after planting said corn plants in the field but prior to emergence.

[0036] The present invention also includes methods of increasing yield of monocotyledonous plants or dicotyledonous plants due to nitrogen utilization.

[0037] Likewise, by means of the present invention, other agronomic genes can be expressed in plants of the present invention. More particularly, plants can be genetically engineered to express various phenotypes of agronomic interest. Exemplary genes implicated in this regard include, but are not limited to, those categorized below:

[0038] 1. Genes that Confer Resistance to Pests or Disease and that Encode:

[0039] A. Plant disease resistance genes. Plant defenses are often activated by specific interaction between the product of a disease resistance gene (R) in the plant and the product of a corresponding avirulence (Avr) gene in the pathogen. A plant variety can be transformed with cloned resistance genes to engineer plants that are resistant to specific pathogen strains. See, for example, Jones et al., Science 266:789 (1994) (cloning of the tomato Cf-9 gene for resistance to Cladosporium fulvum); Martin et al., Science 262:1432 (1993) (tomato Pto gene for resistance to Pseudomonas syringae pv. tomato encodes a protein kinase); Mindrinos et al., Cell 78:1089 (1994) (Arabidopsis RSP2 gene for resistance to Pseudomonas syringae).

[0040] B. A gene conferring resistance to a pest, such as soybean cyst nematode. See e.g., PCT Application WO 96/30517; PCT Application WO 93/19181.

[0041] C. A Bacillus thuringiensis protein, a derivative thereof or a synthetic polypeptide modeled thereon. See, for example, Geiser et al., Gene 48:109 (1986), who disclose the cloning and nucleotide sequence of a Bt delta-endotoxin gene. Moreover, DNA molecules encoding delta-endotoxin genes can be purchased from American Type Culture Collection, Manassas, Va., for example, under ATCC Accession Nos. 40098, 67136, 31995 and 31998.

[0042] D. A lectin. See, for example, the disclosure by Van Damme et al., Plant Molec. Biol. 24:25 (1994), who disclose the nucleotide sequences of several Clivia miniata mannose-binding lectin genes.

[0043] E. A vitamin-binding protein such as avidin. See PCT application US93/06487. The application teaches the use of avidin and avidin homologues as larvicides against insect pests.

[0044] F. An enzyme inhibitor, for example, a protease or proteinase inhibitor or an amylase inhibitor. See, for example, Abe et al., J. Biol. Chem. 262:16793 (1987) (nucleotide sequence of rice cysteine proteinase inhibitor); Huub et al., Plant Molec. Biol. 21:985 (1993) (nucleotide sequence of cDNA encoding tobacco proteinase inhibitor I); Sumitani et al., Biosci. Biotech. Biochem. 57:1243 (1993) (nucleotide sequence of Streptomyces nitrosporeus alpha-amylase, inhibitor); and U.S. Pat. No. 5,494,813 (Hepher and Atkinson, issued Feb. 27, 1996).

[0045] G. An insect-specific hormone or pheromone such as an ecdysteroid or juvenile hormone, a variant thereof, a mimetic based thereon, or an antagonist or agonist thereof. See, for example, the disclosure by Hammock et al., Nature 344:458 (1990), of baculovirus expression of cloned juvenile hormone esterase, an inactivator of juvenile hormone.

[0046] H. An insect-specific peptide or neuropeptide which, upon expression, disrupts the physiology of the affected pest. For example, see the disclosures of Regan, J. Biol. Chem. 269:9 (1994) (expression cloning yields DNA coding for insect diuretic hormone receptor); and Pratt et al., Biochem. Biophys. Res. Comm. 163:1243 (1989) (an allostatin is identified in Diploptera puntata). See also U.S. Pat. No. 5,266,317 to Tomalski et al., who disclose genes encoding insect-specific, paralytic neurotoxins.

[0047] I. An insect-specific venom produced in nature by a snake, a wasp, etc. For example, see Pang et al., Gene 116:165 (1992), for disclosure of heterologous expression in plants of a gene coding for a scorpion insectotoxic peptide.

[0048] J. An enzyme responsible for a hyperaccumulation of a monoterpene, a sesquiterpene, a steroid, hydroxamic acid, a phenylpropanoid derivative or another non-protein molecule with insecticidal activity.

[0049] K. An enzyme involved in the modification, including the post-translational modification, of a biologically active molecule; for example, a glycolytic enzyme, a proteolytic enzyme, a lipolytic enzyme, a nuclease, a cyclase, a transaminase, an esterase, a hydrolase, a phosphatase, a kinase, a phosphorylase, a polymerase, an elastase, a chitinase and a glucanase, whether natural or synthetic. See PCT application WO 93/02197 in the name of Scott et al., which discloses the nucleotide sequence of a callase gene. DNA molecules which contain chitinase-encoding sequences can be obtained, for example, from the ATCC under Accession Nos. 39637 and 67152. See also Kramer et al., Insect Biochem. Molec. Biol. 23:691 (1993), who teach the nucleotide sequence of a cDNA encoding tobacco hornworm chitinase; and Kawalleck et al., Plant Molec. Biol. 21:673 (1993), who provide the nucleotide sequence of the parsley ubi4-2 polyubiquitin gene.

[0050] L. A molecule that stimulates signal transduction. For example, see the disclosure by Botella et al., Plant Molec. Biol. 24:757 (1994), of nucleotide sequences for mung bean calmodulin cDNA clones; and Griess et al., Plant Physiol. 104:1467 (1994), who provide the nucleotide sequence of a maize calmodulin cDNA clone.

[0051] M. A hydrophobic moment peptide. See PCT application WO 95/16776 (disclosure of peptide derivatives of Tachyplesin which inhibit fungal plant pathogens) and PCT application WO 95/18855 (teaches synthetic antimicrobial peptides that confer disease resistance).

[0052] N. A membrane permease, a channel former or a channel blocker. For example, see the disclosure of Jaynes et al., Plant Sci. 89:43 (1993), of heterologous expression of a cecropin-β, lytic peptide analog to render transgenic tobacco plants resistant to Pseudomonas solanacearum.

[0053] O. A viral-invasive protein or a complex toxin derived therefrom. For example, the accumulation of viral coat proteins in transformed plant cells imparts resistance to viral infection and/or disease development effected by the virus from which the coat protein gene is derived, as well as by related viruses. See Beachy et al., Ann. rev. Phytopathol. 28:451 (1990). Coat protein-mediated resistance has been conferred upon transformed plants against alfalfa mosaic virus, cucumber mosaic virus, tobacco streak virus, potato virus X, potato virus Y, tobacco etch virus, tobacco rattle virus and tobacco mosaic virus. Id.

[0054] P. An insect-specific antibody or an immunotoxin derived therefrom. Thus, an antibody targeted to a critical metabolic function in the insect gut would inactivate an affected enzyme, killing the insect. Cf. Taylor et al., Abstract #497, Seventh Int'l Symposium on Molecular Plant-Microbe Interactions (Edinburgh, Scotland) (1994) (enzymatic inactivation in transgenic tobacco via production of single-chain antibody fragments).

[0055] Q. A virus-specific antibody. See, for example, Tavladoraki et al., Nature 366:469 (1993), who show that transgenic plants expressing recombinant antibody genes are protected from virus attack.

[0056] R. A developmental-arrestive protein produced in nature by a pathogen or a parasite. Thus, fungal endo alpha-1,4-D-polygalacturonases facilitate fungal colonization and plant nutrient release by solubilizing plant cell wall homo-alpha-1,4-D-galacturonase. See Lamb et al., Bio/Technology 10:1436 (1992). The cloning and characterization of a gene which encodes a bean endopolygalacturonase-inhibiting protein is described by Toubart et al., Plant J. 2:367 (1992).

[0057] S. A developmental-arrestive protein produced in nature by a plant. For example, Logemann et al., Bio/Technology 10:305 (1992), have shown that transgenic plants expressing the barley ribosome-inactivating gene have an increased resistance to fungal disease.

[0058] 2. Genes that Confer Resistance to an Herbicide:

[0059] A. An herbicide that inhibits the growing point or meristem, such as an imidazolinone or a sulfonylurea. Exemplary genes in this category code for mutant ALS and AHAS enzyme as described, for example, by Lee et al., EMBO J. 7:1241 (1988); and Miki et al., Theon. Appl. Genet. 80:449 (1990), respectively.

[0060] B. Glyphosate (resistance conferred by, e.g., mutant 5-enolpyruvylshikimate-3-phosphate synthase (EPSPs) genes (via the introduction of recombinant nucleic acids and/or various forms of in vivo mutagenesis of native EPSPs genes), aroA genes and glyphosate acetyl transferase (GAT) genes, respectively), other phosphono compounds such as glufosinate (phosphinothricin acetyl transferase (PAT) genes from Streptomyces species, including Streptomyces hygroscopicus and Streptomyces viridichromogenes), and pyridinoxy or phenoxy proprionic acids and cyclohexones (ACCase inhibitor-encoding genes), See, for example, U.S. Pat. No. 4,940,835 to Shah, et al. and U.S. Pat. No. 6,248,876 to Barry et. al., which disclose nucleotide sequences of forms of EPSPs which can confer glyphosate resistance to a plant. A DNA molecule encoding a mutant aroA gene can be obtained under ATCC accession number 39256, and the nucleotide sequence of the mutant gene is disclosed in U.S. Pat. No. 4,769,061 to Comai. European patent application No. 0 333 033 to Kumada et al., and U.S. Pat. No. 4,975,374 to Goodman et al., disclose nucleotide sequences of glutamine synthetase genes which confer resistance to herbicides such as L-phosphinothricin. The nucleotide sequence of a PAT gene is provided in European application No. 0 242 246 to Leemans et al., DeGreef et al., Bio/Technology 7:61 (1989), describe the production of transgenic plants that express chimeric bar genes coding for PAT activity. Exemplary of genes conferring resistance to phenoxy proprionic acids and cyclohexones, such as sethoxydim and haloxyfop are the Acc1-S1, Acc1-S2 and Acc1-S3 genes described by Marshall et al., Theon. Appl. Genet. 83:435 (1992). GAT genes capable of conferring glyphosate resistance are described in WO 2005012515 to Castle et. al. Genes conferring resistance to 2,4-D, fop and pyridyloxy auxin herbicides are described in WO 2005107437 and U.S. patent application Ser. No. 11/587,893, both assigned to Dow AgroSciences LLC.

[0061] C. An herbicide that inhibits photosynthesis, such as a triazine (psbA and Is+ genes) or a benzonitrile (nitrilase gene). Przibila et al., Plant Cell 3:169 (1991), describe the transformation of Chlamydomonas with plasmids encoding mutant psbA genes. Nucleotide sequences for nitrilase genes are disclosed in U.S. Pat. No. 4,810,648 to Stalker, and DNA molecules containing these genes are available under ATCC Accession Nos. 53435, 67441, and 67442. Cloning and expression of DNA coding for a glutathione S-transferase is described by Hayes et al., Biochem. J. 285:173 (1992).

[0062] 3. Genes that Confer or Contribute to a Value-Added Trait, Such as:

[0063] A. Modified fatty acid metabolism, for example, by transforming a plant with an antisense gene of stearyl-ACP desaturase to increase stearic acid content of the plant. See Knultzon et al., Proc. Natl. Acad. Sci. U.S.A. 89:2624 (1992).

[0064] B. Decreased phytate content-I) Introduction of a phytase-encoding gene would enhance breakdown of phytate, adding more free phosphate to the transformed plant. For example, see Van Hartingsveldt et al., Gene 127:87 (1993), for a disclosure of the nucleotide sequence of an Aspergillus niger phytase gene. 2) A gene could be introduced that reduced phytate content. In maize for example, this could be accomplished by cloning and then reintroducing DNA associated with the single allele which is responsible for maize mutants characterized by low levels of phytic acid. See Raboy et al., Maydica 35:383 (1990).

[0065] C. Modified carbohydrate composition effected, for example, by transforming plants with a gene coding for an enzyme that alters the branching pattern of starch. See Shiroza et al., J. Bacteol. 170:810 (1988) (nucleotide sequence of Streptococcus mutants fructosyltransferase gene); Steinmetz et al., Mol. Gen. Genet. 20:220 (1985) (nucleotide sequence of Bacillus subtilis levansucrase gene); Pen et al., Bio/Technology 10:292 (1992) (production of transgenic plants that express Bacillus lichenifonnis alpha-amylase); Elliot et al., Plant Molec. Biol. 21:515 (1993) (nucleotide sequences of tomato invertase genes); Sogaard et al., J. Biol. Chem. 268:22480 (1993) (site-directed mutagenesis of barley alpha-amylase gene); and Fisher et al., Plant Physiol. 102:1045 (1993) (maize endosperm starch branching enzyme II).

[0066] D. Abiotic Stress Tolerance which includes resistance to non-biological sources of stress conferred by traits such as nitrogen utilization efficiency, altered nitrogen responsiveness, drought resistance cold, and salt resistance. Genes that affect abiotic stress resistance (including but not limited to flowering, ear and seed development, enhancement of nitrogen utilization efficiency, altered nitrogen responsiveness, drought resistance or tolerance, cold resistance or tolerance, and salt resistance or tolerance) and increased yield under stress.

[0067] All patents, patent applications, provisional applications, and publications referred to or cited herein are incorporated by reference in their entirety, including all figures and tables, to the extent they are not inconsistent with the explicit teachings of this specification.

[0068] Unless specifically indicated or implied, the terms "a", "an", and "the" signify "at least one" as used herein.

[0069] The present invention is explained in greater detail in the Examples that follow. These examples are intended as illustrative of the invention and are not to be taken are limiting thereof. All percentages are by weight and all solvent mixture proportions are by volume unless otherwise noted.

EXAMPLE 1

[0070] Corn hybrids derived from the IBMRIL population (Lee, M.; Sharopova, N.; Beavis, W. D.; Grant, D.; Katt, M.; Blair, D.; and Amel Hallauer, A. (2002) Expanding the genetic map of maize with the intermated B73×Mo17 (IBM) population. Plant Molec. Biol. 48:453-461) were crossed to HERCULEX-XTRA and non-HERCULEX-XTRA isogenic testers. One hundred female inbreds were used in this experiment. These inbreds consisted of 99 recombinant inbred lines from the intermated B73×Mo17 population (IBMRILs) and one Dow AgroSciences proprietary inbred (DASM7). The genotypes were consistent between 2008 and 2009 with the exception of MO329 which was grown only in 2008 and MO379 which was grown only in 2009. Furthermore, the hybrids formed from the parental inbred lines, B73 and Mo17, were only grown in 2009. The IBMRILs were chosen based on previous data obtained in 2006 and 2007, and were selected to minimize any confounding effects due to differences in maturity. Each of the female lines was crossed to both DASV8 and its near isogenic line, DASV8XT, which contains the HERCULEX-XTRA traits, to create a total of 200 hybrids.

[0071] A split˜block design with three replications was used in which N rate and female parent were the whole plot treatments. Male parent was included as a split˜plot within each N rate/female parent subplot. Plots were planted on May 7, 2008 and May 21, 2009 on the University of Illinois Cruse Farm in Champaign, Ill. All plots received an in˜furrow application of chlorpyrifos (Lorsban 15G) at a rate of 1.3 lbs a.i. per acre in 2008 and tefluthrin (Force 3G) at a rate of 0.099 lbs a.i. per acre in 2009. N was applied as ammonium sulfate ((NH₄)₂SO₄) in a diffuse band after emergence and incorporated between V2 and V3 plant growth stage. The N rates used in the study were 0 and 225 lbs per acre. Each experimental unit consisted of two rows spaced 2.5 feet apart. The rows were 15 feet long in 2008 and 17.5 feet long in 2009. Plots were thinned to an approximate density of 32,000 plants acre. At flowering, four (2008) or five (2009) representative plants were sampled and weighed. A shredded aliquot was dried to constant weight and ground in a Wiley mill to pass through a 20 mesh screen. Dried, ground stover samples were analyzed for total N concentration using a combustion technique (NA2000 N˜Protein, Fisons Instruments, San Carlos, Calif.). A similar sampling approach was used at the R6 plant growth stage except that the ears were removed, dried, and shelled to allow for calculation of per-plant grain weight at physiological maturity. At harvest, all plants within a single row of the two row plot were harvested. A subsample of the grain was analyzed for protein concentration using near-infrared transmittance spectroscopy (Foss 1241 NIT grain analyzer; FOSS, Eden Prairie, Minn.). Three-hundred kernels from each plot were counted using an electronic seed counter and weighed to obtain an estimate of individual kernel weight.

[0072] The phenotypic data were analyzed using the MIXED procedure of SAS. Nitrogen rate, female parent, and male parent were treated as fixed effects while replication was considered random.

[0073] Genetic utilization (GU) is defined as grain weight (kg) per unit of plant N (kg) under nonfertilized conditions, and has units of kg/kg plantN. Nitrogen Use Efficiency (NUE) was calculated as the ability to produce grain (kg) per unit of added fertilizer N (kg) and has units of kg/kgN. Nitrogen Uptake Efficiency (NUpE) was calculated as the difference in plant nitrogen content at high and low N application levels, divided by the difference in the applied levels of N. This is a measure of the efficiency of fertilizer N uptake into the plant per unit of fertilizer N (kg) and has units of kg plantN/kgN. Nitrogen Utilization Efficiency (NUtE) is defined as the grain weight (kg) per unit of N taken up by plant and has units of kg/kg plantN

[0074] The fields in which these hybrids were grown were treated with chemical pesticides at rates recommended by the manufacturer to control corn rootworms. Thus control of root damage was not dependent upon efficacy of the Cry34Ab1 and Cry35Ab1 HERCULEX-XTRA genes. The data summarized in Table 1 show that when averaged across the 99 IBMRIL female parents, in both 2008 and 2009, the hybrids with the HERCULEX-XTRA trait (DASV8XT) had significantly better Nitrogen Uptake Efficiency (NUpE) than did the hybrids which did not contain the HERCULEX-XTRA genes.

[0075] The values of Nitrogen Use Efficiency (NUE) and Nitrogen Utilization Efficiency (NUtE) were found not to correlate with the presence/absence of the HERCULEX-XTRA traits in 2008 and 2009 (Table 1)

TABLE-US-00001 TABLE 1 N use components of corn lines having the HERCULEX-XTRA traits (DASV8XT) compared to isogenic lines without the traits (DASV8) at Champaign, IL in 2008 and 2009, at two N application rates. N rate Male 2008 2009 Lbs/acre parent GU^a NUE^b NUpE^c NUtE^d GU^a NUE^b NUpE^c NUtE^d 0 DASV8 49.5a 67.4a 0 DASV8XT 46.b 66.0a 225 DASV8 12.9a 0.32a 40.7a 30.5a 0.56a 55.4a 225 DASV8XT 14.0b 0.35b 42.4a 29.2b 0.60b 49.3b Means within a column followed by the same letter are not significantly different at P ≦ 0.05. ^aGenetic Utilization (GU). Defined as grain weight (kg) per unit of plant N (kg) under nonfertilized conditions. Units are kg/kgplantN. ^bN Use Efficiency (NUE) Defined as ability to produce grain (kg) per unit of added fertilizer N (kg). Units are kg/kgN. ^cN Uptake Efficiency (NUpE). Defined as efficiency of fertilizer N uptake into the plant (kg) per unit of fertilizer N (kg). Units are kgplantN/kgN. ^dN Utilization Efficiency (NUtE). Defined as grain weight (kg) per unit of N taken up by plant (kg). Units are kg/kgplantN.

[0076] The increased Nitrogen Uptake Efficiency seen with plants having the HERCULEX-XTRA traits was reflected in increased plant N content at the R6 physiological state (Table 2) in both 2008 and 2009.

TABLE-US-00002 TABLE 2 Effect of N level and presence of the HERCULEX-XTRA trait on N content at physiological maturity (R6) at Champaign, IL in 2008 and 2009. 2008 2009 N rate N content N content lbs/acre Male parent gm/plant gm/plant 0 DASV8 0.7a 0.7a 0 DASV8XT 0.8b 0.8b 225 DASV8 1.8c 2.9c 225 DASV8XT 1.9d 3.1d Means within a column followed by the same letter are not significantly different at P ≦ 0.05.

[0077] Further, the HERCULEX-XTRA containing hybrids had significantly better yield traits than did the hybrids which did not contain the HERCULEX-XTRA genes (Table 3). Yield was increased based on both an increase in kernel number per plant and an increase in kernel mass Improved yield correlated with higher N content at the R6 stage in both years of the study. The data Table 2 and Table 3 taken together demonstrate that improved yield correlated with higher N content at the R6 stage. Thus, one skilled in the field of crop physiology will realize that the HERCULEX-XTRA traits are effective in causing an increase in N uptake during the flowering and grain filling periods on development.

TABLE-US-00003 TABLE 3 Grain yield and yield components of corn lines having the HERCULEX-XTRA traits (DASV8XT) compared to isogenic lines without the traits (DASV8) at Champaign, IL in 2008 and 2009, at two N application rates. 2008 2009 Grain Kernel Kernel Grain Kernel Kernel N rate Male Repro. yield weight Number Repro. yield weight Number Lbs/acre parent Success % Bu/acre mg/kernel Per plant Success % Bu/acre mg/kernel Per plant o DASV8 93a 69a 229a 218a 74a 73a 218a 323a o DASV8XT 95b 88b 258b 240b 83b 85b 235b 315b 225 DASV8 99c 130c 216c 408c 98c 218c 264c 605c 225 DASV8XT 99c 154d 251b 418d 99c 224d 275d 593d Means within a column followed by the same letter are not significantly different at P ≦ 0.05.

[0078] Ma et al. (Ma, B. L.; Meloche, F.; and Wei, L: (2009) Agronomic assessment of Bt trait and seed or soil-applied insecticides on the control of corn rootworm and yield. Field Crops Research. 111^th edition: 189-196) showed little to no yield effect of Cry3Bb1 events (active against corn rootworms) in a commercial hybrid compared with an isogenic non-transgenic hybrid treated with Force 3G insecticide to control corn rootworms. Likewise, Vigna (Vigna, M. M. (2008) Comparison of the effect of corn rootworm technology and seed-applied insecticide (clothianidin) to nitrogen status in corn. MA Thesis, Iowa State University, Ames) found no difference in yield or N content of transgenic hybrids containing either Cry3Bb1 or Cry34Ab1+Cry35Ab1 (event DAS59122-7) with isogenic non-transgenic controls treated with Poncho 1250. Thus, the HERCULEX-XTRA transgenes may have broader effects than simply limiting rootworm damage.

[0079] Increasing nitrogen uptake efficiency and yield by production of HERCULEX-XTRA proteins in plants could have a dramatic effect on agriculture if also effective in crop species such as canola, wheat, rice, barley and other non-legume crops.

TABLE-US-00004 TABLE 4 Analysis of variance results for grain yield and N use traits measured in 2008. Source of Variation Trait N rate Female N rate × female Male parent N rate × male Female × male N rate × female × male R1 whole shoot biomass 0.0051 0.0041 0.0163 <0.0001 0.0150 n.s.* n.s. R1 whole shoot N content 0.0018 n.s. n.s. n.s. n.s. n.s. n.s. R6 grain weight 0.0004 <0.0001 n.s. <0.0001 0.0043 n.s. n.s. R6 whole shoot biomass 0.0054 <0.0001 n.s. 0.0424 n.s. n.s. n.s. R6 total N content 0.0016 0.0083 n.s. <0.0001 n.s. n.s. n.s. Harvest index <0.0001 <0.0001 0.0318 n.s. n.s. 0.0386 n.s. Nitrogen harvest index <0.0001 0.0003 n.s. n.s. n.s. n.s. n.s. Grain protein concentration <0.0001 <0.0001 <0.0001 0.0188 0.0011 0.0948 n.s. Yield <0.0001 <0.0001 0.0524 <0.0001 0.0516 0.0325 n.s. Kernel weight 0.0004 <0.0001 <0.0001 <0.0001 0.0857 0.0005 n.s. Kernel number 0.0010 <0.0001 n.s. <0.0001 0.0193 n.s. n.s. Reproductive success 0.0189 n.s. 0.0259 0.0164 0.0027 n.s. n.s. NUE -- 0.0279 -- 0.0018 -- n.s. -- NUpE -- n.s. -- 0.0011 -- n.s. -- NUtE -- 0.0385 -- n.s. -- n.s. -- GU -- 0.0004 -- <0.0001 -- n.s. -- *Non-significant source variation (n.s.).

TABLE-US-00005 TABLE 5 Analysis of variance results for grain yield and N use traits measured in 2009. Source of Variation Trait N rate Female N rate × female Male parent N rate × male Female × male N rate × female × male R1 whole shoot biomass 0.0200 <0.0001 n.s.* <0.0001 n.s. 0.0153 n.s. R1 whole shoot N content 0.0014 0.0117 0.0831 <0.0001 <0.0001 0.0905 n.s. R6 grain weight 0.0023 <0.0001 0.0001 0.0015 0.0017 0.0870 0.0489 R6 whole shoot biomass 0.0012 <0.0001 0.0007 <0.0001 0.0598 0.0013 0.0178 R6 total N content 0.0003 <0.0001 0.0001 <0.0001 0.0004 <0.0001 <0.0001 Harvest index 0.0057 <0.0001 <0.0001 0.1091 <0.0001 n.s. n.s. Nitrogen harvest index 0.0513 0.0002 <0.0001 n.s. <0.0001 n.s. n.s. Grain protein concentration 0.0005 <0.0001 <0.0001 <0.0001 n.s. 0.0329 n.s. Yield 0.0005 <0.0001 <0.0001 <0.0001 0.0007 0.0003 0.0440 Kernel weight 0.0009 <0.0001 0.0015 <0.0001 <0.0001 <0.0001 n.s. Kernel number 0.0008 <0.0001 0.0016 <0.0001 n.s. 0.0024 n.s. Reproductive success 0.0016 <0.0001 <0.0001 <0.0001 <0.0001 0.0060 0.0005 NUE -- <0.0001 -- 0.0003 -- 0.0140 -- NUpE -- <0.0001 -- <0.0001 -- 0.0002 -- NUtE -- <0.0001 -- <0.0001 -- <0.0001 -- GU -- <0.0001 -- 0.0735 -- n.s. -- *Non-significant source variation (n.s.).

[0080] The invention has been described with reference to various specific and preferred embodiments and techniques. However, it should be understood that many variations and modifications may be made while remaining within the spirit and scope of the invention.

Sequence CWU 1

1

101369DNAArtificial Sequencecry34 plant-optimized gene sequence 1atgtccgccc gcgaggtgca catcgacgtg aacaacaaga ccggccacac cctccagctg 60gaggacaaga ccaagctcga cggcggcagg tggcgcacct ccccgaccaa cgtggccaac 120gaccagatca agaccttcgt ggccgaatcc aacggcttca tgaccggcac cgagggcacc 180atctactact ccatcaacgg cgaggccgag atcagcctct acttcgacaa cccgttcgcc 240ggctccaaca aatacgacgg ccactccaac aagtcccagt acgagatcat cacccagggc 300ggctccggca accagtccca cgtgacctac accatccaga ccacctcctc ccgctacggc 360cacaagtcc 36921149DNAArtificial Sequencecry35 plant-optimized gene sequence 2atgctcgaca ccaacaaggt gtacgagatc agcaaccacg ccaacggcct ctacgccgcc 60acctacctct ccctcgacga ctccggcgtg tccctcatga acaagaacga cgacgacatc 120gacgactaca acctcaagtg gttcctcttc ccgatcgacg acgaccagta catcatcacc 180tcctacgccg ccaacaactg caaggtgtgg aacgtgaaca acgacaagat caacgtgtcc 240acctactcct ccaccaactc catccagaag tggcagatca aggccaacgg ctcctcctac 300gtgatccagt ccgacaacgg caaggtgctc accgccggca ccggccaggc cctcggcctc 360atccgcctca ccgacgagtc ctccaacaac ccgaaccagc aatggaacct gacgtccgtg 420cagaccatcc agctcccgca gaagccgatc atcgacacca agctcaagga ctacccgaag 480tactccccga ccggcaacat cgacaacggc acctccccgc agctcatggg ctggaccctc 540gtgccgtgca tcatggtgaa cgacccgaac atcgacaaga acacccagat caagaccacc 600ccgtactaca tcctcaagaa gtaccagtac tggcagaggg ccgtgggctc caacgtcgcg 660ctccgcccgc acgagaagaa gtcctacacc tacgagtggg gcaccgagat cgaccagaag 720accaccatca tcaacaccct cggcttccag atcaacatcg acagcggcat gaagttcgac 780atcccggagg tgggcggcgg taccgacgag atcaagaccc agctcaacga ggagctcaag 840atcgagtact cccacgagac gaagatcatg gagaagtacc aggagcagtc cgagatcgac 900aacccgaccg accagtccat gaactccatc ggcttcctca ccatcacctc cctggagctc 960taccgctaca acggctccga gatccgcatc atgcagatcc agacctccga caacgacacc 1020tacaacgtga cctcctaccc gaaccaccag caggccctgc tgctgctgac caaccactcc 1080tacgaggagg tggaggagat caccaacatc ccgaagtcca ccctcaagaa gctcaagaag 1140tactacttc 114931815DNAArtificial Sequencetruncated cry1F sequence encoding core toxin 3atggagaaca acatacagaa tcagtgcgtc ccctacaact gcctcaacaa tcctgaagta 60gagattctca acgaagagag gtcgactggc agattgccgt tagacatctc cctgtccctt 120acacgtttcc tgttgtctga gtttgttcca ggtgtgggag ttgcgtttgg cctcttcgac 180ctcatctggg gcttcatcac tccatctgat tggagcctct ttcttctcca gattgaacag 240ttgattgaac aaaggattga gaccttggaa aggaatcggg ccatcactac ccttcgtggc 300ttagcagaca gctatgagat ctacattgaa gcactaagag agtgggaagc caatcctaac 360aatgcccaac tgagagaaga tgtgcgtata cgctttgcta acacagatga tgctttgatc 420acagccatca acaacttcac ccttaccagc ttcgagatcc ctcttctctc ggtctatgtt 480caagctgcta acctgcactt gtcactactg cgcgacgctg tgtcgtttgg gcaaggttgg 540ggactggaca tagctactgt caacaatcac tacaacagac tcatcaatct gattcatcga 600tacacgaaac attgtttgga tacctacaat cagggattgg agaacctgag aggtactaac 660actcgccaat gggccaggtt caatcagttc aggagagacc ttacacttac tgtgttagac 720atagttgctc tctttccgaa ctacgatgtt cgtacctatc cgattcaaac gtcatcccaa 780cttacaaggg agatctacac cagttcagtc attgaagact ctccagtttc tgcgaacata 840cccaatggtt tcaacagggc tgagtttgga gtcagaccac cccatctcat ggacttcatg 900aactctttgt ttgtgactgc agagactgtt agatcccaaa ctgtgtgggg aggacactta 960gttagctcac gcaacacggc tggcaatcgt atcaactttc ctagttacgg ggtcttcaat 1020cccgggggcg ccatctggat tgcagatgaa gatccacgtc ctttctatcg gaccttgtca 1080gatcctgtct tcgtccgagg aggctttggc aatcctcact atgtactcgg tcttagggga 1140gtggcctttc aacaaactgg tacgaatcac acccgcacat tcaggaactc cgggaccatt 1200gactctctag atgagatacc acctcaagac aacagcggcg caccttggaa tgactactcc 1260catgtgctga atcatgttac ctttgtgcgc tggccaggtg agatctcagg ttccgactca 1320tggagagcac caatgttctc ttggacgcat cgtagcgcta cccccacaaa caccattgat 1380ccagagagaa tcactcagat tcccttggtg aaggcacaca cacttcagtc aggaactaca 1440gttgtaagag ggccggggtt cacgggagga gacattcttc gacgcactag tggaggacca 1500ttcgcgtaca ccattgtcaa catcaatggg caacttcccc aaaggtatcg tgccaggata 1560cgctatgcct ctactaccaa tctaagaatc tacgttacgg ttgcaggtga acggatcttt 1620gctggtcagt tcaacaagac aatggatacc ggtgatccac ttacattcca atctttctcc 1680tacgccacta tcaacaccgc gttcaccttt ccaatgagcc agagcagttt cacagtaggt 1740gctgatacct tcagttcagg caacgaagtg tacattgaca ggtttgagtt gattccagtt 1800actgccacac tcgag 18154605PRTArtificial Sequencetruncated / core-toxin Cry1F protein sequence 4Met Glu Asn Asn Ile Gln Asn Gln Cys Val Pro Tyr Asn Cys Leu Asn 1 5 10 15 Asn Pro Glu Val Glu Ile Leu Asn Glu Glu Arg Ser Thr Gly Arg Leu 20 25 30 Pro Leu Asp Ile Ser Leu Ser Leu Thr Arg Phe Leu Leu Ser Glu Phe 35 40 45 Val Pro Gly Val Gly Val Ala Phe Gly Leu Phe Asp Leu Ile Trp Gly 50 55 60 Phe Ile Thr Pro Ser Asp Trp Ser Leu Phe Leu Leu Gln Ile Glu Gln 65 70 75 80 Leu Ile Glu Gln Arg Ile Glu Thr Leu Glu Arg Asn Arg Ala Ile Thr 85 90 95 Thr Leu Arg Gly Leu Ala Asp Ser Tyr Glu Ile Tyr Ile Glu Ala Leu 100 105 110 Arg Glu Trp Glu Ala Asn Pro Asn Asn Ala Gln Leu Arg Glu Asp Val 115 120 125 Arg Ile Arg Phe Ala Asn Thr Asp Asp Ala Leu Ile Thr Ala Ile Asn 130 135 140 Asn Phe Thr Leu Thr Ser Phe Glu Ile Pro Leu Leu Ser Val Tyr Val 145 150 155 160 Gln Ala Ala Asn Leu His Leu Ser Leu Leu Arg Asp Ala Val Ser Phe 165 170 175 Gly Gln Gly Trp Gly Leu Asp Ile Ala Thr Val Asn Asn His Tyr Asn 180 185 190 Arg Leu Ile Asn Leu Ile His Arg Tyr Thr Lys His Cys Leu Asp Thr 195 200 205 Tyr Asn Gln Gly Leu Glu Asn Leu Arg Gly Thr Asn Thr Arg Gln Trp 210 215 220 Ala Arg Phe Asn Gln Phe Arg Arg Asp Leu Thr Leu Thr Val Leu Asp 225 230 235 240 Ile Val Ala Leu Phe Pro Asn Tyr Asp Val Arg Thr Tyr Pro Ile Gln 245 250 255 Thr Ser Ser Gln Leu Thr Arg Glu Ile Tyr Thr Ser Ser Val Ile Glu 260 265 270 Asp Ser Pro Val Ser Ala Asn Ile Pro Asn Gly Phe Asn Arg Ala Glu 275 280 285 Phe Gly Val Arg Pro Pro His Leu Met Asp Phe Met Asn Ser Leu Phe 290 295 300 Val Thr Ala Glu Thr Val Arg Ser Gln Thr Val Trp Gly Gly His Leu 305 310 315 320 Val Ser Ser Arg Asn Thr Ala Gly Asn Arg Ile Asn Phe Pro Ser Tyr 325 330 335 Gly Val Phe Asn Pro Gly Gly Ala Ile Trp Ile Ala Asp Glu Asp Pro 340 345 350 Arg Pro Phe Tyr Arg Thr Leu Ser Asp Pro Val Phe Val Arg Gly Gly 355 360 365 Phe Gly Asn Pro His Tyr Val Leu Gly Leu Arg Gly Val Ala Phe Gln 370 375 380 Gln Thr Gly Thr Asn His Thr Arg Thr Phe Arg Asn Ser Gly Thr Ile 385 390 395 400 Asp Ser Leu Asp Glu Ile Pro Pro Gln Asp Asn Ser Gly Ala Pro Trp 405 410 415 Asn Asp Tyr Ser His Val Leu Asn His Val Thr Phe Val Arg Trp Pro 420 425 430 Gly Glu Ile Ser Gly Ser Asp Ser Trp Arg Ala Pro Met Phe Ser Trp 435 440 445 Thr His Arg Ser Ala Thr Pro Thr Asn Thr Ile Asp Pro Glu Arg Ile 450 455 460 Thr Gln Ile Pro Leu Val Lys Ala His Thr Leu Gln Ser Gly Thr Thr 465 470 475 480 Val Val Arg Gly Pro Gly Phe Thr Gly Gly Asp Ile Leu Arg Arg Thr 485 490 495 Ser Gly Gly Pro Phe Ala Tyr Thr Ile Val Asn Ile Asn Gly Gln Leu 500 505 510 Pro Gln Arg Tyr Arg Ala Arg Ile Arg Tyr Ala Ser Thr Thr Asn Leu 515 520 525 Arg Ile Tyr Val Thr Val Ala Gly Glu Arg Ile Phe Ala Gly Gln Phe 530 535 540 Asn Lys Thr Met Asp Thr Gly Asp Pro Leu Thr Phe Gln Ser Phe Ser 545 550 555 560 Tyr Ala Thr Ile Asn Thr Ala Phe Thr Phe Pro Met Ser Gln Ser Ser 565 570 575 Phe Thr Val Gly Ala Asp Thr Phe Ser Ser Gly Asn Glu Val Tyr Ile 580 585 590 Asp Arg Phe Glu Leu Ile Pro Val Thr Ala Thr Leu Glu 595 600 605 53522DNABacillus thuringiensis 5atggagaata atattcaaaa tcaatgcgta ccttacaatt gtttaaataa tcctgaagta 60gaaatattaa atgaagaaag aagtactggc agattaccgt tagatatatc cttatcgctt 120acacgtttcc ttttgagtga atttgttcca ggtgtgggag ttgcgtttgg attatttgat 180ttaatatggg gttttataac tccttctgat tggagcttat ttcttttaca gattgaacaa 240ttgattgagc aaagaataga aacattggaa aggaaccggg caattactac attacgaggg 300ttagcagata gctatgaaat ttatattgaa gcactaagag agtgggaagc aaatcctaat 360aatgcacaat taagggaaga tgtgcgtatt cgatttgcta atacagacga cgctttaata 420acagcaataa ataattttac acttacaagt tttgaaatcc ctcttttatc ggtctatgtt 480caagcggcga atttacattt atcactatta agagacgctg tatcgtttgg gcagggttgg 540ggactggata tagctactgt taataatcat tataatagat taataaatct tattcataga 600tatacgaaac attgtttgga cacatacaat caaggattag aaaacttaag aggtactaat 660actcgacaat gggcaagatt caatcagttt aggagagatt taacacttac tgtattagat 720atcgttgctc tttttccgaa ctacgatgtt agaacatatc caattcaaac gtcatcccaa 780ttaacaaggg aaatttatac aagttcagta attgaggatt ctccagtttc tgctaatata 840cctaatggtt ttaatagggc ggaatttgga gttagaccgc cccatcttat ggactttatg 900aattctttgt ttgtaactgc agagactgtt agaagtcaaa ctgtgtgggg aggacactta 960gttagttcac gaaatacggc tggtaaccgt ataaatttcc ctagttacgg ggtcttcaat 1020cctggtggcg ccatttggat tgcagatgag gatccacgtc ctttttatcg gacattatca 1080gatcctgttt ttgtccgagg aggatttggg aatcctcatt atgtactggg gcttagggga 1140gtagcatttc aacaaactgg tacgaaccac acccgaacat ttagaaatag tgggaccata 1200gattctctag atgaaatccc acctcaggat aatagtgggg caccttggaa tgattatagt 1260catgtattaa atcatgttac atttgtacga tggccaggtg agatttcagg aagtgattca 1320tggagagctc caatgttttc ttggacgcac cgtagtgcaa cccctacaaa tacaattgat 1380ccggagagga ttactcaaat accattggta aaagcacata cacttcagtc aggtactact 1440gttgtaagag ggcccgggtt tacgggagga gatattcttc gacgaacaag tggaggacca 1500tttgcttata ctattgttaa tataaatggg caattacccc aaaggtatcg tgcaagaata 1560cgctatgcct ctactacaaa tctaagaatt tacgtaacgg ttgcaggtga acggattttt 1620gctggtcaat ttaacaaaac aatggatacc ggtgacccat taacattcca atcttttagt 1680tacgcaacta ttaatacagc ttttacattc ccaatgagcc agagtagttt cacagtaggt 1740gctgatactt ttagttcagg gaatgaagtt tatatagaca gatttgaatt gattccagtt 1800actgcaacat ttgaagcaga atatgattta gaaagagcac aaaaggcggt gaatgcgctg 1860tttacttcta taaaccaaat agggataaaa acagatgtga cggattatca tattgatcaa 1920gtatccaatt tagtggattg tttatcagat gaattttgtc tggatgaaaa gcgagaattg 1980tccgagaaag tcaaacatgc gaagcgactc agtgatgagc ggaatttact tcaagatcca 2040aacttcaaag gcatcaatag gcaactagac cgtggttgga gaggaagtac ggatattacc 2100atccaaagag gagatgacgt attcaaagaa aattatgtca cactaccagg tacctttgat 2160gagtgctatc caacgtattt atatcaaaaa atagatgagt cgaaattaaa accctatact 2220cgttatcaat taagagggta tatcgaggat agtcaagact tagaaatcta tttgatccgc 2280tataatgcaa aacacgaaac agtaaatgtg ctaggtacgg gttctttatg gccgctttca 2340gtccaaagtc caatcagaaa gtgtggagaa ccgaatcgat gcgcgccaca ccttgaatgg 2400aatcctgatc tagattgttc ctgcagagac ggggaaaaat gtgcacatca ttcgcatcat 2460ttctccttgg acattgatgt tggatgtaca gacttaaatg aggacttaga tgtatgggtg 2520atattcaaga ttaagacgca agatggccat gcaagactag gaaatctaga gtttctcgaa 2580gagaaaccat tagtcgggga agcactagct cgtgtgaaaa gagcagagaa aaaatggaga 2640gataaacgtg aaaaattgga attggaaaca aatattgttt ataaagaggc aaaagaatct 2700gtagatgctt tatttgtaaa ctctcaatat gatcaattac aagcggatac gaatattgcc 2760atgattcatg cggcagataa acgtgttcat agaattcggg aagcgtatct tccagagtta 2820tctgtgattc cgggtgtaaa tgtagacatt ttcgaagaat taaaagggcg tattttcact 2880gcattcttcc tatatgatgc gagaaatgtc attaaaaacg gtgatttcaa taatggctta 2940tcatgctgga acgtgaaagg gcatgtagat gtagaagaac aaaacaacca ccgttcggtc 3000cttgttgttc cggaatggga agcagaagtg tcacaagaag ttcgtgtctg tccgggtcgt 3060ggctatatcc ttcgtgtcac agcgtacaag gagggatatg gagaaggttg cgtaaccatt 3120catgagatcg agaacaatac agacgaactg aagtttagca actgcgtaga agaggaagtc 3180tatccaaaca acacggtaac gtgtaatgat tatactgcaa atcaagaaga atacgggggt 3240gcgtacactt cccgtaatcg tggatatgac gaaacttatg gaagcaattc ttctgtacca 3300gctgattatg cgtcagtcta tgaagaaaaa tcgtatacag atggacgaag agacaatcct 3360tgtgaatcta acagaggata tggggattac acaccactac cagctggcta tgtgacaaaa 3420gaattagagt acttcccaga aaccgataag gtatggattg agatcggaga aacggaagga 3480acattcatcg tggacagcgt ggaattactc cttatggagg aa 352261174PRTBacillus thuringiensis 6Met Glu Asn Asn Ile Gln Asn Gln Cys Val Pro Tyr Asn Cys Leu Asn 1 5 10 15 Asn Pro Glu Val Glu Ile Leu Asn Glu Glu Arg Ser Thr Gly Arg Leu 20 25 30 Pro Leu Asp Ile Ser Leu Ser Leu Thr Arg Phe Leu Leu Ser Glu Phe 35 40 45 Val Pro Gly Val Gly Val Ala Phe Gly Leu Phe Asp Leu Ile Trp Gly 50 55 60 Phe Ile Thr Pro Ser Asp Trp Ser Leu Phe Leu Leu Gln Ile Glu Gln 65 70 75 80 Leu Ile Glu Gln Arg Ile Glu Thr Leu Glu Arg Asn Arg Ala Ile Thr 85 90 95 Thr Leu Arg Gly Leu Ala Asp Ser Tyr Glu Ile Tyr Ile Glu Ala Leu 100 105 110 Arg Glu Trp Glu Ala Asn Pro Asn Asn Ala Gln Leu Arg Glu Asp Val 115 120 125 Arg Ile Arg Phe Ala Asn Thr Asp Asp Ala Leu Ile Thr Ala Ile Asn 130 135 140 Asn Phe Thr Leu Thr Ser Phe Glu Ile Pro Leu Leu Ser Val Tyr Val 145 150 155 160 Gln Ala Ala Asn Leu His Leu Ser Leu Leu Arg Asp Ala Val Ser Phe 165 170 175 Gly Gln Gly Trp Gly Leu Asp Ile Ala Thr Val Asn Asn His Tyr Asn 180 185 190 Arg Leu Ile Asn Leu Ile His Arg Tyr Thr Lys His Cys Leu Asp Thr 195 200 205 Tyr Asn Gln Gly Leu Glu Asn Leu Arg Gly Thr Asn Thr Arg Gln Trp 210 215 220 Ala Arg Phe Asn Gln Phe Arg Arg Asp Leu Thr Leu Thr Val Leu Asp 225 230 235 240 Ile Val Ala Leu Phe Pro Asn Tyr Asp Val Arg Thr Tyr Pro Ile Gln 245 250 255 Thr Ser Ser Gln Leu Thr Arg Glu Ile Tyr Thr Ser Ser Val Ile Glu 260 265 270 Asp Ser Pro Val Ser Ala Asn Ile Pro Asn Gly Phe Asn Arg Ala Glu 275 280 285 Phe Gly Val Arg Pro Pro His Leu Met Asp Phe Met Asn Ser Leu Phe 290 295 300 Val Thr Ala Glu Thr Val Arg Ser Gln Thr Val Trp Gly Gly His Leu 305 310 315 320 Val Ser Ser Arg Asn Thr Ala Gly Asn Arg Ile Asn Phe Pro Ser Tyr 325 330 335 Gly Val Phe Asn Pro Gly Gly Ala Ile Trp Ile Ala Asp Glu Asp Pro 340 345 350 Arg Pro Phe Tyr Arg Thr Leu Ser Asp Pro Val Phe Val Arg Gly Gly 355 360 365 Phe Gly Asn Pro His Tyr Val Leu Gly Leu Arg Gly Val Ala Phe Gln 370 375 380 Gln Thr Gly Thr Asn His Thr Arg Thr Phe Arg Asn Ser Gly Thr Ile 385 390 395 400 Asp Ser Leu Asp Glu Ile Pro Pro Gln Asp Asn Ser Gly Ala Pro Trp 405 410 415 Asn Asp Tyr Ser His Val Leu Asn His Val Thr Phe Val Arg Trp Pro 420 425 430 Gly Glu Ile Ser Gly Ser Asp Ser Trp Arg Ala Pro Met Phe Ser Trp 435 440 445 Thr His Arg Ser Ala Thr Pro Thr Asn Thr Ile Asp Pro Glu Arg Ile 450 455 460 Thr Gln Ile Pro Leu Val Lys Ala His Thr Leu Gln Ser Gly Thr Thr 465 470 475 480 Val Val Arg Gly Pro Gly Phe Thr Gly Gly Asp Ile Leu Arg Arg Thr 485 490 495 Ser Gly Gly Pro Phe Ala Tyr Thr Ile Val Asn Ile Asn Gly Gln Leu 500 505 510 Pro Gln Arg Tyr Arg Ala Arg Ile Arg Tyr Ala Ser Thr Thr Asn Leu 515 520 525 Arg Ile Tyr Val Thr Val Ala Gly Glu Arg Ile Phe Ala Gly Gln Phe 530 535 540 Asn Lys Thr Met Asp Thr Gly Asp Pro Leu Thr Phe Gln Ser Phe Ser 545 550 555 560 Tyr Ala Thr Ile Asn Thr Ala Phe Thr Phe Pro Met Ser Gln Ser Ser 565 570 575 Phe Thr Val Gly Ala Asp Thr Phe

Ser Ser Gly Asn Glu Val Tyr Ile 580 585 590 Asp Arg Phe Glu Leu Ile Pro Val Thr Ala Thr Phe Glu Ala Glu Tyr 595 600 605 Asp Leu Glu Arg Ala Gln Lys Ala Val Asn Ala Leu Phe Thr Ser Ile 610 615 620 Asn Gln Ile Gly Ile Lys Thr Asp Val Thr Asp Tyr His Ile Asp Gln 625 630 635 640 Val Ser Asn Leu Val Asp Cys Leu Ser Asp Glu Phe Cys Leu Asp Glu 645 650 655 Lys Arg Glu Leu Ser Glu Lys Val Lys His Ala Lys Arg Leu Ser Asp 660 665 670 Glu Arg Asn Leu Leu Gln Asp Pro Asn Phe Lys Gly Ile Asn Arg Gln 675 680 685 Leu Asp Arg Gly Trp Arg Gly Ser Thr Asp Ile Thr Ile Gln Arg Gly 690 695 700 Asp Asp Val Phe Lys Glu Asn Tyr Val Thr Leu Pro Gly Thr Phe Asp 705 710 715 720 Glu Cys Tyr Pro Thr Tyr Leu Tyr Gln Lys Ile Asp Glu Ser Lys Leu 725 730 735 Lys Pro Tyr Thr Arg Tyr Gln Leu Arg Gly Tyr Ile Glu Asp Ser Gln 740 745 750 Asp Leu Glu Ile Tyr Leu Ile Arg Tyr Asn Ala Lys His Glu Thr Val 755 760 765 Asn Val Leu Gly Thr Gly Ser Leu Trp Pro Leu Ser Val Gln Ser Pro 770 775 780 Ile Arg Lys Cys Gly Glu Pro Asn Arg Cys Ala Pro His Leu Glu Trp 785 790 795 800 Asn Pro Asp Leu Asp Cys Ser Cys Arg Asp Gly Glu Lys Cys Ala His 805 810 815 His Ser His His Phe Ser Leu Asp Ile Asp Val Gly Cys Thr Asp Leu 820 825 830 Asn Glu Asp Leu Asp Val Trp Val Ile Phe Lys Ile Lys Thr Gln Asp 835 840 845 Gly His Ala Arg Leu Gly Asn Leu Glu Phe Leu Glu Glu Lys Pro Leu 850 855 860 Val Gly Glu Ala Leu Ala Arg Val Lys Arg Ala Glu Lys Lys Trp Arg 865 870 875 880 Asp Lys Arg Glu Lys Leu Glu Leu Glu Thr Asn Ile Val Tyr Lys Glu 885 890 895 Ala Lys Glu Ser Val Asp Ala Leu Phe Val Asn Ser Gln Tyr Asp Gln 900 905 910 Leu Gln Ala Asp Thr Asn Ile Ala Met Ile His Ala Ala Asp Lys Arg 915 920 925 Val His Arg Ile Arg Glu Ala Tyr Leu Pro Glu Leu Ser Val Ile Pro 930 935 940 Gly Val Asn Val Asp Ile Phe Glu Glu Leu Lys Gly Arg Ile Phe Thr 945 950 955 960 Ala Phe Phe Leu Tyr Asp Ala Arg Asn Val Ile Lys Asn Gly Asp Phe 965 970 975 Asn Asn Gly Leu Ser Cys Trp Asn Val Lys Gly His Val Asp Val Glu 980 985 990 Glu Gln Asn Asn His Arg Ser Val Leu Val Val Pro Glu Trp Glu Ala 995 1000 1005 Glu Val Ser Gln Glu Val Arg Val Cys Pro Gly Arg Gly Tyr Ile 1010 1015 1020 Leu Arg Val Thr Ala Tyr Lys Glu Gly Tyr Gly Glu Gly Cys Val 1025 1030 1035 Thr Ile His Glu Ile Glu Asn Asn Thr Asp Glu Leu Lys Phe Ser 1040 1045 1050 Asn Cys Val Glu Glu Glu Val Tyr Pro Asn Asn Thr Val Thr Cys 1055 1060 1065 Asn Asp Tyr Thr Ala Asn Gln Glu Glu Tyr Gly Gly Ala Tyr Thr 1070 1075 1080 Ser Arg Asn Arg Gly Tyr Asp Glu Thr Tyr Gly Ser Asn Ser Ser 1085 1090 1095 Val Pro Ala Asp Tyr Ala Ser Val Tyr Glu Glu Lys Ser Tyr Thr 1100 1105 1110 Asp Gly Arg Arg Asp Asn Pro Cys Glu Ser Asn Arg Gly Tyr Gly 1115 1120 1125 Asp Tyr Thr Pro Leu Pro Ala Gly Tyr Val Thr Lys Glu Leu Glu 1130 1135 1140 Tyr Phe Pro Glu Thr Asp Lys Val Trp Ile Glu Ile Gly Glu Thr 1145 1150 1155 Glu Gly Thr Phe Ile Val Asp Ser Val Glu Leu Leu Leu Met Glu 1160 1165 1170 Glu 7372DNABacillus thuringiensis 7atgtcagcac gtgaagtaca cattgatgta aataataaga caggtcatac attacaatta 60gaagataaaa caaaacttga tggtggtaga tggcgaacat cacctacaaa tgttgctaat 120gatcaaatta aaacatttgt agcagaatca aatggtttta tgacaggtac agaaggtact 180atatattata gtataaatgg agaagcagaa attagtttat attttgacaa tccttttgca 240ggttctaata aatatgatgg acattccaat aaatctcaat atgaaattat tacccaagga 300ggatcaggaa atcaatctca tgttacgtat actattcaaa ccacatcctc acgatatggg 360cataaatcat aa 3728123PRTBacillus thuringiensis 8Met Ser Ala Arg Glu Val His Ile Asp Val Asn Asn Lys Thr Gly His 1 5 10 15 Thr Leu Gln Leu Glu Asp Lys Thr Lys Leu Asp Gly Gly Arg Trp Arg 20 25 30 Thr Ser Pro Thr Asn Val Ala Asn Asp Gln Ile Lys Thr Phe Val Ala 35 40 45 Glu Ser Asn Gly Phe Met Thr Gly Thr Glu Gly Thr Ile Tyr Tyr Ser 50 55 60 Ile Asn Gly Glu Ala Glu Ile Ser Leu Tyr Phe Asp Asn Pro Phe Ala 65 70 75 80 Gly Ser Asn Lys Tyr Asp Gly His Ser Asn Lys Ser Gln Tyr Glu Ile 85 90 95 Ile Thr Gln Gly Gly Ser Gly Asn Gln Ser His Val Thr Tyr Thr Ile 100 105 110 Gln Thr Thr Ser Ser Arg Tyr Gly His Lys Ser 115 120 91241DNABacillus thuringiensismisc_feature(18)..(18)any nucleotide 9wcdmtkdvrm wahkcmdndb ygtrawbmkg cwtkctgyhd cywagmawtd cvnwmhasrt 60nchhtmsnwr manrgarcrr nwrgarhatg ttagatacta ataaagttta tgaaataagc 120aatcatgcta atggactata tgcagcaact tatttaagtt tagatgattc aggtgttagt 180ttaatgaata aaaatgatga tgatattgat gattataact taaaatggtt tttatttcct 240attgatgatg atcaatatat tattacaagc tatgcagcaa ataattgtaa agtttggaat 300gttaataatg ataaaataaa tgtttcgact tattcttcaa caaattcaat acaaaaatgg 360caaataaaag ctaatggttc ttcatatgta atacaaagtg ataatggaaa agtcttaaca 420gcaggaaccg gtcaagctct tggattgata cgtttaactg atgaatcctc aaataatccc 480aatcaacaat ggaatttaac ttctgtacaa acaattcaac ttccacaaaa acctataata 540gatacaaaat taaaagatta tcccaaatat tcaccaactg gaaatataga taatggaaca 600tctcctcaat taatgggatg gacattagta ccttgtatta tggtaaatga tccaaatata 660gataaaaata ctcaaattaa aactactcca tattatattt taaaaaaata tcaatattgg 720caacgagcag taggaagtaa tgtagcttta cgtccacatg aaaaaaaatc atatacttat 780gaatggggca cagaaataga tcaaaaaaca acaattataa atacattagg atttcaaatc 840aatatagatt caggaatgaa atttgatata ccagaagtag gtggaggtac agatgaaata 900aaaacacaac taaatgaaga attaaaaata gaatatagtc atgaaactaa aataatggaa 960aaatatcaag aacaatctga aatagataat ccaactgatc aatcaatgaa ttctatagga 1020tttcttacta ttacttcctt agaattatat agatataatg gctcagaaat tcgtataatg 1080caaattcaaa cctcagataa tgatacttat aatgttactt cttatccaaa tcatcaacaa 1140gctttattac ttcttacaaa tcattcatat gaagaagtag aagaaataac aaatattcct 1200aaaagtacac taaaaaaatt aaaaaaatat tatttttaav v 124110383PRTBacillus thuringiensis 10Met Leu Asp Thr Asn Lys Val Tyr Glu Ile Ser Asn His Ala Asn Gly 1 5 10 15 Leu Tyr Ala Ala Thr Tyr Leu Ser Leu Asp Asp Ser Gly Val Ser Leu 20 25 30 Met Asn Lys Asn Asp Asp Asp Ile Asp Asp Tyr Asn Leu Lys Trp Phe 35 40 45 Leu Phe Pro Ile Asp Asp Asp Gln Tyr Ile Ile Thr Ser Tyr Ala Ala 50 55 60 Asn Asn Cys Lys Val Trp Asn Val Asn Asn Asp Lys Ile Asn Val Ser 65 70 75 80 Thr Tyr Ser Ser Thr Asn Ser Ile Gln Lys Trp Gln Ile Lys Ala Asn 85 90 95 Gly Ser Ser Tyr Val Ile Gln Ser Asp Asn Gly Lys Val Leu Thr Ala 100 105 110 Gly Thr Gly Gln Ala Leu Gly Leu Ile Arg Leu Thr Asp Glu Ser Ser 115 120 125 Asn Asn Pro Asn Gln Gln Trp Asn Leu Thr Ser Val Gln Thr Ile Gln 130 135 140 Leu Pro Gln Lys Pro Ile Ile Asp Thr Lys Leu Lys Asp Tyr Pro Lys 145 150 155 160 Tyr Ser Pro Thr Gly Asn Ile Asp Asn Gly Thr Ser Pro Gln Leu Met 165 170 175 Gly Trp Thr Leu Val Pro Cys Ile Met Val Asn Asp Pro Asn Ile Asp 180 185 190 Lys Asn Thr Gln Ile Lys Thr Thr Pro Tyr Tyr Ile Leu Lys Lys Tyr 195 200 205 Gln Tyr Trp Gln Arg Ala Val Gly Ser Asn Val Ala Leu Arg Pro His 210 215 220 Glu Lys Lys Ser Tyr Thr Tyr Glu Trp Gly Thr Glu Ile Asp Gln Lys 225 230 235 240 Thr Thr Ile Ile Asn Thr Leu Gly Phe Gln Ile Asn Ile Asp Ser Gly 245 250 255 Met Lys Phe Asp Ile Pro Glu Val Gly Gly Gly Thr Asp Glu Ile Lys 260 265 270 Thr Gln Leu Asn Glu Glu Leu Lys Ile Glu Tyr Ser His Glu Thr Lys 275 280 285 Ile Met Glu Lys Tyr Gln Glu Gln Ser Glu Ile Asp Asn Pro Thr Asp 290 295 300 Gln Ser Met Asn Ser Ile Gly Phe Leu Thr Ile Thr Ser Leu Glu Leu 305 310 315 320 Tyr Arg Tyr Asn Gly Ser Glu Ile Arg Ile Met Gln Ile Gln Thr Ser 325 330 335 Asp Asn Asp Thr Tyr Asn Val Thr Ser Tyr Pro Asn His Gln Gln Ala 340 345 350 Leu Leu Leu Leu Thr Asn His Ser Tyr Glu Glu Val Glu Glu Ile Thr 355 360 365 Asn Ile Pro Lys Ser Thr Leu Lys Lys Leu Lys Lys Tyr Tyr Phe 370 375 380

Claims

1. A method of increasing nitrogen uptake efficiency in a plant comprising administering to a plant an expression cassette containing at least one Bacillus thuringiensis insect-resistance gene which functions to improve expression of at least one insecticidal portion of a protein or amino acid sequence variant thereof from a nucleic acid coding sequence in a plant cell.

2. The method according to claim 1 wherein said method further comprises an increase in the yield of the plant.

3. The method according to claim 2 wherein the increase in yield comprises an increase in the kernel number per plant and/or an increase in the kernel mass per plant.

4. The method according to claim 1 further comprising culturing the plant under plant cell growing conditions wherein the nitrogen utilization is modulated.

5. The method according to claim 4 wherein said Bacillus thuringiensis insect-resistance gene encodes a Cry protein.

6. The method according to claim 5 wherein said insect-resistance gene is selected from the group consisting of a cry34 gene, a cry35 gene, a cry1F gene, and a cry3A gene.

7. The method according to claim 1 wherein the Bacillus thuringiensis insect-resistance gene comprises Cry34Ab1, Cry35Ab1, and Cry1F.

8. The method according to claim 1 wherein the increase in nitrogen occurs during the flowering and grain filling periods of development.

9. The method according to claim 1 wherein the plant is selected from the group consisting of a monocotyledonous plant and a dicotyledonous plant.

10. The method according to claim 1 wherein the plant is a monocotyledonous plant.

11. A method of growing transgenic corn plants having an increased yield by using a reduced amount of fertilizer, wherein the transgenic corn is insect resistant due to expression of an insect-resistance gene, and wherein the reduced amount of fertilizer is relative to fertilizer recommended for use on non-transgenic corn, wherein said non-transgenic corn is optionally protected by granular chemical insecticide to control rootworms.

12. The method according to claim 11 wherein said transgenic corn plants yield corn comparable to corn yield from said non-transgenic corn grown using said recommended amounts of fertilizer.

13. The method according to claim 11 wherein said fertilizer is a nitrogenous fertilizer.

14. The method according to claim 11 wherein said transgenic plant comprises a Bacillus thuringiensis insect-resistance gene.

15. The method according to claim 14 wherein said Bacillus thuringiensis insect-resistance gene encodes a Cry protein.

16. The method according to claim 14 wherein said insect-resistance gene is selected from the group consisting of a cry34 gene, a cry35 gene, a cry1F gene, and a cry3A gene.

17. The method according to claim 14 wherein the Bacillus thuringiensis insect-resistance gene comprises Cry34Ab1, Cry35Ab1, and Cry1F.

18. The method according to claim 13 wherein said nitrogenous fertilizer is applied at a rate of less than 150 pounds per acre.

19. The method according to claim 11 wherein said transgenic corn plants are grown in a field, and said fertilizer is nitrogenous fertilizer applied to said field after planting said corn plants in said field but prior to emergence.

20. A method of modulating nitrogen utilization efficiency (NUE) in a plant cell, comprising: (a) introducing into a plant cell a recombinant expression cassette comprising a Cry protein operably linked to a promoter that drives expression in a plant; and (b) culturing the plant cell under plant cell growing conditions; wherein the nitrogen uptake in the plant cell is modulated.

21. A method for increasing yield in a plant, said method comprising the steps of: (a) introducing into a plant cell a construct comprising a Cry protein operably linked to a promoter functional in a plant cell, so as to yield transformed plant cells; and, (b) regenerating a transgenic plant from said transformed plant cell, wherein said Cry protein is expressed in the cells of said transgenic plant at levels sufficient to increase yield in said transgenic plant; wherein increased yield comprises enhanced root growth, increased seed size, increased seed weight, seed with increased embryo size, increased leaf size, increased seedling vigor, enhanced silk emergence, increased ear size, nitrogen utilization or chlorophyll content.

22. Seed from the transgenic plant of claim 21.

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