Nucleic acid molecules for conferring insecticidal properties to plants

Abstract

The present disclosure relates to a nucleic acid sequence that confers expression of an insecticidal protein when introduced into a cell, as well as related compositions and methods of use thereof. In some aspects, the present disclosure provides a plant comprising the nucleic acid sequence.

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims priority to U.S. Provisional Application No. 63 / 183,672, filed May 4, 2021, the entire content of which is incorporated herein by reference.

[0002] The present invention generally relates to nucleic acid sequences that confer the expression of insecticidal proteins when introduced into cells or plants, as well as compositions and methods.

[0003] Sequence Listing This application is accompanied by a Sequence Listing in ASCII text format named "82347 - PCT_ST25.txt", created on March 14, 2022, which is about 395 kilobytes in size. This Sequence Listing is incorporated herein by reference in its entirety. This Sequence Listing is submitted with this specification via EFS - Web and complies with 37 C.F.R. §§ 1.824(a)(2)-(6) and (b).

Background Art

[0004] Plant pests are a major cause of loss of important crops worldwide, including corn. Plant pests are mainly controlled by the intensive application of chemical insecticides. Good pest control can be achieved in this way, but these chemicals can sometimes affect beneficial organisms. Another problem resulting from the widespread use of chemical insecticides is the emergence of resistant insect varieties. This has been partially mitigated by various resistance management techniques, but the need for alternative pest control strategies is increasing. One such alternative includes the expression of foreign genes encoding insecticidal proteins in transgenic plants. This approach provides an effective means of control against selected pests, and transgenic plants expressing insecticidal toxins are commercially available, enabling farmers to reduce the application of chemical insecticides.

[0005] The Bacillus thuringiensis (Bt)Cry protein (also known as delta-endotoxin) is a protein that forms a crystalline matrix in Bacillus, known to possess insecticidal activity when ingested by certain insects. The gene encoding the Cry protein has been isolated, and its expression in crops has been shown to provide another tool for controlling economically important pests.

[0006] While the use of transgenic plants expressing the Cry protein is another tool in the insect control toolbox, it remains susceptible to disruption due to resistance. Currently, pests resistant to the Cry protein expressed in certain transgenic plants are known. For example, the fall armyworm (Spodoptera frugiperda) has been recorded in certain countries as being resistant in the field to Cry1F, Cry1A.105, and Cry2Ab2. Consequently, there is a need for additional insecticidal proteins to address the resistance problem.

[0007] Developing novel insecticidal protein expression cassettes for use in transgenic plants is a challenging task because the expression cassette must express sufficient protein within the transgenic plant to have the desired activity (e.g., insecticidal activity) without causing negative effects on the plant itself (e.g., reduced yield, sterility, stunted growth).

[0008] Provided herein are nucleic acid sequences and related compositions, as well as methods of use, for addressing the aforementioned needs. [Overview of the project] [Means for solving the problem]

[0009] In some embodiments, the present disclosure provides nucleic acid molecules that express one or more insecticidal proteins. As described herein, an expression cassette (SEQ ID NO: 1) encoding the eCry1Gb.1Ig protein (SEQ ID NO: 4) was constructed. When this expression cassette is transformed into plants, it confers insecticidal activity against Lepidoptera species, such as Spodoptera frugiperda (fall armyworm).

[0010] Therefore, in some embodiments, the present disclosure relates to a nucleic acid molecule or the nucleic acid sequence that is at least 90% identical to SEQ ID NO: 1 (for example, at least 90% identical to SEQ ID NO: 1, at least 91% identical to SEQ ID NO: 1, at least 92% identical to SEQ ID NO: 1, at least 93% identical to SEQ ID NO: 1, at least 94% identical to SEQ ID NO: 1, at least 95% identical to SEQ ID NO: 1, at least 96% identical to SEQ ID NO: 1, at least 97% identical to SEQ ID NO: 1, at least 98% identical to SEQ ID NO: 1, at least 99% identical to SEQ ID NO: 1, or at least 99.5% identical to SEQ ID NO: 1). Complementary bodiesThe present invention provides that, in some embodiments, the nucleic acid molecule encodes the same protein encoded by SEQ ID NO: 1. In some embodiments, the nucleic acid sequence comprises SEQ ID NO: 1 or any one of 8-31, or any one or more of the variants in Table 3. In some embodiments, the nucleic acid molecule encodes a protein that is insecticidal against one or more lepidopteran pests, for example, insecticidal against at least Spodoptera frugiperda (fall armyworm). In some embodiments, the nucleic acid molecule encodes a protein that is insecticidal against at least two (e.g., two, three, or four) of the following species: Spodoptera frugiperda (white fall armyworm), Mythimna separata (sea armyworm), Spodoptera litura (beet armyworm / oriental leaf worm), and Ostrinia furnacalis (Asian corn borer). In some embodiments, the nucleic acid molecule is isolated.

[0011] In some embodiments, the Disclosure relates to a nucleic acid molecule or the nucleic acid sequence that is at least 95% identical to SEQ ID NO: 1 (for example, at least 95% identical to SEQ ID NO: 1, at least 96% identical to SEQ ID NO: 1, at least 97% identical to SEQ ID NO: 1, at least 98% identical to SEQ ID NO: 1, at least 99% identical to SEQ ID NO: 1, or at least 99.5% identical to SEQ ID NO: 1). Complementary bodiesThe nucleic acid sequence provides a polypeptide that includes the sequence of SEQ ID NO: 4, or a polypeptide that includes the sequences of SEQ ID NOs: 4 and 6. In some embodiments, the nucleic acid sequence includes SEQ ID NO: 3 or SEQ ID NOs: 3 and 5, or any of the aforementioned variants thereof, including one or more silent mutations. In some embodiments, the nucleic acid sequence includes SEQ ID NO: 1 or any one of SEQ ID NOs: 8-31, or any one or more of the aforementioned variants thereof, including the variants in Table 3 or one or more silent mutations, or other mutations that do not substantially affect the function of SEQ ID NO: 1.

[0012] In some embodiments, the Disclosure provides recombinant nucleic acid vectors comprising nucleic acid molecules from any of the embodiments described above or any other embodiments described herein (e.g., one of SEQ ID NOs: 1 or 8-31, or one or more of the variants in Table 3). In some embodiments, the vector is a binary vector. In some embodiments, the vector is a plasmid. In some embodiments, the vector is present in a host cell.

[0013] In some embodiments, the Disclosure provides transgenic host cells comprising nucleic acid molecules from any of the embodiments described above or any other embodiments described herein (e.g., one of SEQ ID NOs: 1 or 8-31, or one or more of the variants in Table 3). In some embodiments, the cells are plant cells, yeast cells, bacterial cells, or insect cells. In some embodiments, the cells are bacterial cells or plant cells. In some embodiments, the cells are bacterial cells, and the bacterial cells are Escherichia coli, Bacillus thuringiensis, Bacillus subtilis, Bacillus megaterium, Bacillus cereus, Agrobacterium species, or Pseudomonas species cells. In some embodiments, the cells are plant cells, and the plant cells are cells of maize, sorghum, wheat, sunflower, tomato, cruciferous plants, oats, grass, pasture grass, pepper, potato, cotton, rice, soybean, sugarcane, sugar beet, tobacco, barley, or rapeseed. In some embodiments, the plant cells are maize cells. In some embodiments, the plant cells are present in a plant. In some embodiments, the plant cells are isolated. In some embodiments, the plant cells can regenerate a plant. In some embodiments, the plant cells cannot regenerate an entire plant.

[0014] In some embodiments, the Disclosure provides transgenic plants comprising nucleic acid molecules from any of the embodiments described above or any other embodiments described herein (e.g., one of SEQ ID NOs: 1 or 8-31, or one or more of the mutants in Table 3). In some embodiments, the plant is a monocotyledonous plant. In some embodiments, the plant is a dicotyledonous plant. In some embodiments, the plant is selected from the group consisting of maize, sorghum, wheat, sunflower, tomato, cruciferous plants, oat, turfgrass, pasture grass, pepper, potato, cotton, rice, soybean, sugarcane, sugar beet, tobacco, barley, or rapeseed. In some embodiments, the plant is a maize plant. In some embodiments, the plant is an entire plant. In some embodiments, the plant is a transgenic maize plant comprising nucleic acid molecules comprising one of SEQ ID NOs: 1 or 8-31, or one or more of the mutants in Table 3. In some embodiments, the plant is insecticidal against at least Spodoptera frugiperda (fall armyworm). In some embodiments, the plant is insecticidal against at least two (e.g., two, three, or four) of the following: Spodoptera frugiperda (fall armyworm), Mythimna separata (sea armyworm), Spodoptera litura (beet armyworm / oriental leaf worm), and Ostrinia furnacalis (Asian corn borer). In some embodiments, the plant has enhanced insecticidal properties, for example, against at least Spodoptera frugiperda (fall armyworm), compared to a control plant, for example, a plant that does not contain the nucleic acid molecule. In some embodiments, the disclosure provides offspring of any generation of a plant, the offspring containing the nucleic acid molecule. In some embodiments, the disclosure provides vegetative propagates of a plant, the vegetative propagates containing the nucleic acid molecule. In some embodiments, the disclosure provides plant parts of a plant, the plant parts containing the nucleic acid molecule.In some embodiments, the plant part is an embryo, pollen, ovule, seed, leaf, flower, branch, fruit, grain, spike, rachis, bark, stem, root, root tip, anther, tuber, or rhizome. In some embodiments, the plant part is a seed.

[0015] In some embodiments, the Disclosure provides a method for producing transgenic plants having enhanced insecticidal properties, comprising introducing nucleic acid molecules from any of the embodiments described above or any other embodiments described herein (for example, nucleic acid molecules comprising any one of SEQ ID NOs: 1 or 8-31, or any one or more of the mutants in Table 3) into a plant to produce transgenic plants, wherein the nucleic acid molecules express an effective insect control amount of protein. In some embodiments, an effective insect control amount of protein is effective in controlling at least Spodoptera frugiperda (fall armyworm). In some embodiments, an effective insect control amount of protein is effective in controlling at least two (e.g., two, three, or four) of the following: Spodoptera frugiperda (white fall armyworm), Mythimna separata (sea armyworm), Spodoptera litura (beet armyworm / oriental leaf worm), and Ostrinia furnacalis (Asian corn borer).

[0016] In some embodiments, the Disclosure provides a method for producing transgenic plants having enhanced insecticidal properties, comprising: (a) providing a nucleic acid molecule of any of the embodiments described above or any other embodiment described herein (for example, a nucleic acid molecule comprising any one of SEQ ID NOs: 1 or 8-31 or any one or more of the mutants in Table 3); (b) introducing the nucleic acid molecule of step (a) into a plant, tissue culture, or plant cell to obtain a transgenic plant, transgenic tissue culture, or transgenic cell having enhanced insecticidal properties; and (c) growing the transgenic plant or regenerating a transgenic plant from the transgenic tissue culture or transgenic plant cell to thus produce a transgenic plant having enhanced insecticidal properties. In some embodiments, the enhanced insecticidal properties are enhanced insecticidal properties against at least Spodoptera frugiperda (fall armyworm). In some embodiments, the enhanced insecticidal properties are enhanced insecticidal properties against at least two (e.g., two, three, or four) of the following: Spodoptera frugiperda (white fall armyworm), Mythimna separata (sea armyworm), Spodoptera litura (beet armyworm / oriental leaf worm), and Ostrinia furnacalis (Asian corn borer). In some embodiments, the transgenic plant is a transgenic maize plant.

[0017] In some embodiments, the Disclosure provides a method for producing transgenic seeds, comprising: (a) obtaining a fertile transgenic plant of any of the embodiments described above or any other embodiments described herein (e.g., including any one of SEQ ID NOs: 1 or 8-31 or any one or more of the mutants in Table 3); and (b) growing the plant under appropriate conditions to produce transgenic seeds. In some embodiments, the transgenic seeds are transgenic maize seeds.

[0018] In some embodiments, the Disclosure provides a method for producing offspring of any generation of a fertile transgenic plant having enhanced insecticidal properties, comprising: (a) obtaining a fertile transgenic plant having enhanced insecticidal properties comprising nucleic acids of any of the embodiments described above or any other embodiments described herein (e.g., nucleic acids comprising any one of SEQ ID NOs: 1 or 8-31 or any one or more of the mutants in Table 3); (b) collecting transgenic seeds from the transgenic plant; (c) planting the collected transgenic seeds; and (d) growing offspring transgenic plants from the seeds, wherein the offspring have enhanced insecticidal properties compared to non-transgenic plants. In some embodiments, the offspring plants are maize plants.

[0019] In some embodiments, the Disclosure provides a method for producing transgenic plants having enhanced insecticidal properties, comprising the step of sexually crossing a first parent plant with a second parent plant to produce a first generation of offspring plants containing nucleic acid molecules, wherein the first or second parent plant is a plant of any of the embodiments described above or any other embodiment described herein (e.g., including any one of SEQ ID NOs: 1 or 8-31 or any one or more of the mutants in Table 3). In some embodiments, the enhanced insecticidal property is enhanced insecticidal property at least against Spodoptera frugiperda (fall armyworm). In some embodiments, the enhanced insecticidal properties are enhanced insecticidal properties against at least two (e.g., two, three, or four) of the following species: Spodoptera frugiperda (white fall armyworm), Mythimna separata (sea armyworm), Spodoptera litura (beet armyworm / oriental leaf worm), and Ostrinia furnacalis (Asian corn borer).

[0020] In some embodiments, the Disclosure provides a method for producing transgenic plants having enhanced insecticidal properties, comprising: (a) a mating step of sexually crossing a first parent plant with a second parent plant, wherein the first or second parent plant is a plant of any of the embodiments described above or any other embodiment described herein (e.g., including any one of SEQ ID NOs. 1 or 8-31 or any one or more of the mutants in Table 3); and (b) a selection step of selecting first-generation offspring plants having enhanced insecticidal properties, wherein the selected offspring plants contain nucleic acid molecules. In some embodiments, the enhanced insecticidal properties are enhanced insecticidal properties against at least Spodoptera frugiperda (fall armyworm). In some embodiments, the enhanced insecticidal properties are enhanced against at least two (e.g., two, three, or four) of the following: Spodoptera frugiperda (white fall armyworm), Mythimna separata (sea armyworm), Spodoptera litura (beet armyworm / oriental leaf worm), and Ostrinia furnacalis (Asian corn borer). In some embodiments, the first generation offspring plants are maize plants. In some embodiments, the method further includes (a) self-pollinating first-generation offspring plants to produce a plurality of second-generation offspring plants, and (b) selecting plants from the second-generation offspring plants that have enhanced insecticidal properties, wherein the selected second-generation offspring plants contain nucleic acid molecules.

[0021] In some embodiments, the Disclosure provides a method for controlling lepidopteran pests, comprising having the pests ingest a plant or plant part containing a nucleic acid molecule of any of the embodiments described above or any other embodiments described herein (e.g., one of SEQ ID NOs: 1 or 8-31, or one or more of the variants in Table 3). In some embodiments, the lepidopteran pest is Spodoptera frugiperda (fall armyworm). In some embodiments, the lepidopteran pests are at least two (e.g., two, three, or four) of the following: Spodoptera frugiperda (white fall armyworm), Mythimna separata (sea armyworm), Spodoptera litura (beet armyworm / oriental leaf worm), and Ostrinia furnacalis (Asian corn borer). In some embodiments, the plant or plant part is a maize plant or a maize plant part.

[0022] In some embodiments, the Disclosure provides a method for producing a commercial plant product, the method comprising using a plant of any of the embodiments described above or any other embodiment described herein (e.g., including any one of SEQ ID NOs: 1 or 8-31 or any one or more of the variants in Table 3) and producing the commercial plant product therefrom. In some embodiments, the plant is a maize plant. In some embodiments, the commercial plant product is a grain, starch, seed oil, syrup, grain flour, edible flour, starch, cereal, or protein.

[0023] In some embodiments, the Disclosure provides a method for detecting the presence of nucleic acid molecules in a sample, the method comprising: (a) contacting the sample with a pair of primers that produce an amplicon useful for diagnosing nucleic acid molecules when used in a nucleic acid amplification reaction with DNA containing a nucleic acid molecule of any of the embodiments described above or any other embodiment described herein (e.g., SEQ ID NO: SEQ ID NO: 1 or 8-31, or one or more variants in Table 3); (b) carrying out the nucleic acid amplification reaction to thereby produce an amplicon; and (c) detecting the amplicon. In some embodiments, the primer pair is a first primer and a second primer, the first primer comprising at least 10 consecutive nucleotides complementary to any one of SEQ ID NO: 1 or 8-31, or one or more variants in Table 3; and the second primer comprising at least 10 consecutive nucleotides complementary to any one of SEQ ID NO: 1 or 8-31, or one or more reverse complementary sequences in Table 3. In some embodiments, the first and second primers are 10-30 nucleotides long. In some embodiments, the sample is a sample obtained from a maize plant part or cell.

[0024] In some embodiments, the Disclosure provides a method for detecting the presence of nucleic acid molecules in a sample, the method comprising: (a) hybridizing the sample with DNA containing a nucleic acid molecule of any of the embodiments described above or any other embodiment described herein (e.g., including one of SEQ ID NOs: 1 or 8-31, or one or more of the variants in Table 3) under high stringency conditions, and contacting it with a probe that does not hybridize under high stringency conditions with DNA of a control maize plant that does not contain this nucleic acid molecule; (b) subjecting the sample and probe to high stringency hybridization conditions; and (c) detecting the hybridization of the probe to the nucleic acid molecule. In some embodiments, the probe contains one of SEQ ID NOs: 1 or 8-31, or one or more of the variants in Table 3, or at least 10 consecutive nucleotides complementary to their reverse complementary sequence. In some embodiments, the probe is 10-50 nucleotides long. In some embodiments, the sample is a sample obtained from a maize plant part or cell.

[0025] In some embodiments, the Disclosure provides a pair of polynucleotide primers in a sample for producing an amplicon useful for diagnosing the presence of a nucleic acid molecule in the sample, comprising a first polynucleotide primer and a second polynucleotide primer in the sample, which function together in the presence of a nucleic acid molecule (e.g., including one of SEQ ID NOs: 1 or 8-31, or one or more of the variants in Table 3) in any of the embodiments described above or any other embodiments described herein. In some embodiments, the sample is a sample obtained from a maize plant part or cells. In some embodiments, the first polynucleotide primer comprises at least 10 consecutive nucleotides complementary to one of SEQ ID NOs: 1 or 8-31, or one or more of the variants in Table 3, and the second polynucleotide primer comprises at least 10 consecutive nucleotides complementary to one of SEQ ID NOs: 1 or 8-31, or one or more of the reverse complementary sequences in Table 3. In some embodiments, the first and second primers are 10-30 nucleotides long.

[0026] In some embodiments, a kit for detecting a nucleic acid molecule of any of the above-described embodiments, or any other embodiment described herein (e.g., any one of SEQ ID NO: 1 or 8 - 31, or one or more of the variants in Table 3) is provided. The kit contains at least one nucleic acid molecule of a continuous nucleotide of a length sufficient to function as a primer or a probe in a nucleic acid detection method, and is useful for diagnosing the presence of the nucleic acid molecule during amplification of a target nucleic acid sequence in a sample or hybridization to the target nucleic acid sequence, followed by detection of the amplicon or detection of hybridization to the target sequence. In some embodiments, the at least one nucleic acid molecule contains at least 10 consecutive nucleotides complementary to any one of SEQ ID NO: 1 or 8 - 31, or one or more of the variants in Table 3. In some embodiments, the at least one nucleic acid molecule contains a pair of primers. The first polynucleotide primer contains at least 10 consecutive nucleotides complementary to any one of SEQ ID NO: 1 or 8 - 31, or one or more of the variants in Table 3, and the second polynucleotide primer contains at least 10 consecutive nucleotides complementary to the reverse complementary sequence of any one of SEQ ID NO: 1 or 8 - 31, or one or more of the variants in Table 3. In some embodiments, the first and second primers are 10 - 30 nucleotides in length. In some embodiments, the at least one nucleic acid molecule contains a probe containing at least 10 consecutive nucleotides complementary to any one of SEQ ID NO: 1 or 8 - 31, or one or more of the variants in Table 3, or its reverse complementary sequence. In some embodiments, the probe is 10 - 50 nucleotides in length.

[0027] In some embodiments, the disclosure provides a method that includes introducing a modification into a nucleic acid molecule, transgenic host cell, or transgenic plant of any one of the above-described embodiments, thereby producing a modified nucleic acid molecule, transgenic host cell, or modified transgenic plant. In some embodiments, the modification is a deletion, insertion, substitution, duplication, or inversion, or a combination thereof. In some embodiments, the modification includes a partial or complete deletion of a selectable marker coding sequence (e.g., PMI) present in the nucleic acid molecule. In some embodiments, the modification is introduced using a nuclease or homologous recombination, or a combination thereof. In some embodiments, the nuclease is a CRISPR-Cas nuclease. In some embodiments, the method further includes producing a plant from the modified transgenic host cell and self-pollinating or crossing this plant with another plant, thereby producing a modified transgenic progeny plant. In some embodiments, the method further includes self-pollinating or crossing the modified transgenic plant with another plant, thereby producing a modified transgenic progeny plant. In some embodiments, the method further includes self-pollinating or outcrossing the modified transgenic progeny plant over at least one additional generation.

BRIEF DESCRIPTION OF THE DRAWINGS

[0028] [Figure 1] A diagram of binary vector 24795, the nucleic acid sequence of which is SEQ ID NO: 2.

DETAILED DESCRIPTION OF THE INVENTION

[0029] Brief Description of the Sequences in the Sequence Listing SEQ ID NO: 1 is the nucleic acid sequence of an expression cassette encoding the eCry1Gb.1Ig protein (SEQ ID NO: 4) and PMI (SEQ ID NO: 6) as a selectable marker.

[0030] Sequence ID 2 is the nucleic acid sequence of binary vector 24795, which contains the expression cassette for Sequence ID 1.

[0031] Sequence ID 3 is the nucleic acid sequence of the coding sequence that encodes eCry1Gb.1Ig.

[0032] Sequence ID 4 is the amino acid sequence of eCry1Gb.1Ig.

[0033] Sequence ID 5 is the nucleic acid sequence of the coding sequence that encodes PMI.

[0034] Sequence ID 6 is the amino acid sequence of PMI.

[0035] Sequence ID 7 is a nucleic acid sequence of a coding sequence that encodes a PMI having a silent mutation at one nucleotide position compared to Sequence ID 5.

[0036] Sequence ID 8 is the nucleic acid sequence of an expression cassette that encodes the eCry1Gb.1Ig protein (Sequence ID 4) and PMI (Sequence ID 6) as a selectable marker, and contains a silent mutation in Sequence ID 7.

[0037] Sequence ID 9 is a nucleic acid sequence of an expression cassette that encodes the eCry1Gb.1Ig protein (Sequence ID 4) and PMI (Sequence ID 6) as a selectable marker, and contains additional mutations compared to Sequence ID 1.

[0038] Sequence ID 10 is a nucleic acid sequence of an expression cassette that encodes the eCry1Gb.1Ig protein (Sequence ID 4) and PMI (Sequence ID 6) as a selectable marker, and contains additional mutations compared to Sequence ID 1.

[0039] Sequence ID 11 is a nucleic acid sequence of an expression cassette that encodes the eCry1Gb.1Ig protein (Sequence ID 4) and PMI (Sequence ID 6) as a selectable marker, and contains additional mutations compared to Sequence ID 1.

[0040] Sequence ID 12 is a nucleic acid sequence of an expression cassette that encodes the eCry1Gb.1Ig protein (Sequence ID 4) and PMI (Sequence ID 6) as a selectable marker, and contains additional mutations compared to Sequence ID 1.

[0041] Sequence ID 13 is a nucleic acid sequence of an expression cassette that encodes the eCry1Gb.1Ig protein (Sequence ID 4) and PMI (Sequence ID 6) as a selectable marker, and contains additional mutations compared to Sequence ID 1.

[0042] Sequence ID 14 is a nucleic acid sequence of an expression cassette that encodes the eCry1Gb.1Ig protein (Sequence ID 4) and PMI (Sequence ID 6) as a selectable marker, and contains additional mutations compared to Sequence ID 1.

[0043] Sequence ID 15 is a nucleic acid sequence of an expression cassette that encodes the eCry1Gb.1Ig protein (Sequence ID 4) and PMI (Sequence ID 6) as a selectable marker, and contains additional mutations compared to Sequence ID 1.

[0044] Sequence ID 16 is a nucleic acid sequence of an expression cassette that encodes the eCry1Gb.1Ig protein (Sequence ID 4) and PMI (Sequence ID 6) as a selectable marker, and contains additional mutations compared to Sequence ID 1.

[0045] Sequence ID 17 is a nucleic acid sequence of an expression cassette that encodes the eCry1Gb.1Ig protein (Sequence ID 4) and PMI (Sequence ID 6) as a selectable marker, and contains additional mutations compared to Sequence ID 1.

[0046] Sequence ID 18 is a nucleic acid sequence of an expression cassette that encodes the eCry1Gb.1Ig protein (Sequence ID 4) and PMI (Sequence ID 6) as a selectable marker, and contains additional mutations compared to Sequence ID 1.

[0047] Sequence ID 19 is a nucleic acid sequence of an expression cassette that encodes the eCry1Gb.1Ig protein (Sequence ID 4) and PMI (Sequence ID 6) as a selectable marker, and contains additional mutations compared to Sequence ID 1.

[0048] Sequence ID 20 is a nucleic acid sequence of an expression cassette that encodes the eCry1Gb.1Ig protein (Sequence ID 4) and PMI (Sequence ID 6) as a selectable marker, and contains additional mutations compared to Sequence ID 1.

[0049] Sequence ID 21 is a nucleic acid sequence of an expression cassette that encodes the eCry1Gb.1Ig protein (Sequence ID 4) and PMI (Sequence ID 6) as a selectable marker, and contains additional mutations compared to Sequence ID 1.

[0050] Sequence ID 22 is a nucleic acid sequence of an expression cassette that encodes the eCry1Gb.1Ig protein (Sequence ID 4) and PMI (Sequence ID 6) as a selectable marker, and contains additional mutations compared to Sequence ID 1.

[0051] Sequence ID 23 is a nucleic acid sequence of an expression cassette that encodes the eCry1Gb.1Ig protein (Sequence ID 4) and PMI (Sequence ID 6) as a selectable marker, and contains additional mutations compared to Sequence ID 1.

[0052] Sequence ID 24 is a nucleic acid sequence of an expression cassette that encodes the eCry1Gb.1Ig protein (Sequence ID 4) and PMI (Sequence ID 6) as a selectable marker, and contains additional mutations compared to Sequence ID 1.

[0053] Sequence ID 25 is a nucleic acid sequence of an expression cassette that encodes the eCry1Gb.1Ig protein (Sequence ID 4) and PMI (Sequence ID 6) as a selectable marker, and contains additional mutations compared to Sequence ID 1.

[0054] Sequence ID 26 is a nucleic acid sequence of an expression cassette that encodes the eCry1Gb.1Ig protein (Sequence ID 4) and PMI (Sequence ID 6) as a selectable marker, and contains additional mutations compared to Sequence ID 1.

[0055] Sequence ID 27 is a nucleic acid sequence of an expression cassette that encodes the eCry1Gb.1Ig protein (Sequence ID 4) and PMI (Sequence ID 6) as a selectable marker, and contains additional mutations compared to Sequence ID 1.

[0056] Sequence ID 28 is a nucleic acid sequence of an expression cassette that encodes the eCry1Gb.1Ig protein (Sequence ID 4) and PMI (Sequence ID 6) as a selectable marker, and contains additional mutations compared to Sequence ID 1.

[0057] Sequence ID 29 is a nucleic acid sequence of an expression cassette that encodes the eCry1Gb.1Ig protein (Sequence ID 4) and PMI (Sequence ID 6) as a selectable marker, and contains additional mutations compared to Sequence ID 1.

[0058] Sequence ID 30 is a nucleic acid sequence of an expression cassette that encodes the eCry1Gb.1Ig protein (Sequence ID 4) and PMI (Sequence ID 6) as a selectable marker, and contains additional mutations compared to Sequence ID 1.

[0059] Sequence ID 31 is a nucleic acid sequence of an expression cassette that encodes the eCry1Gb.1Ig protein (Sequence ID 4) and PMI (Sequence ID 6) as a selectable marker, and contains additional mutations compared to Sequence ID 1.

[0060] Sequence numbers 32-75 are shown in Table 3.

[0061] Detailed explanation This description is not intended to be a detailed catalog of all different ways in which the present invention may be carried out, or of all features that may be added to the present invention. For example, features illustrated in one embodiment may be incorporated into other embodiments, and features illustrated in a particular embodiment may be omitted from that embodiment. Thus, in some embodiments, any features or combinations of features described herein may be excluded or omitted. In addition, numerous variations and additions to the various embodiments suggested herein will be apparent to those skilled in the art in light of this disclosure without departing from this disclosure. Accordingly, the following description is intended to illustrate some specific embodiments of this disclosure and not exhaustively specify all permutations, combinations, and variations thereof.

[0062] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art in which the present invention pertains. The terms used in the description of the present invention herein are for the purpose of describing specific embodiments only and are not intended to limit the invention.

[0063] All publications, patent applications, patents, and other references cited herein are incorporated by reference in their entirety with respect to the teachings relating to the documents and / or paragraphs in which such references are presented.

[0064] The nucleotide sequences provided herein are presented from left to right, in the 5' to 3' direction, and are presented using standard codes for representing nucleotide bases as described in 37 CFR §§1.821-1.825 and World Intellectual Property Organization (WIPO) Standard ST.25, e.g., adenine (A), cytosine (C), thymine (T), and guanine (G).

[0065] Amino acids are similarly represented using WIPO standard ST.25, for example: alanine (Ala;A), arginine (Arg;R), asparagine (Asn;N), aspartic acid (Asp;D), cysteine ​​(Cys;C), glutamine (Gln;Q), glutamic acid (Glu;E), glycine (Gly;G), histidine (His;H), isoleucine (Ile;1), leucine (Leu;L), lysine (Lys;K), methionine (Met;M), phenylalanine (Phe;F), proline (Pro;P), serine (Ser;S), threonine (Thr;T), tryptophan (Trp;W), tyrosine (Tyr;Y), and valine (Val;V).

[0066] Unless otherwise indicated by context, the various features of the Disclosure described herein are specifically intended to be used in any combination. Furthermore, the Disclosure is also intended to exclude or omit any feature or combination of features described herein in some embodiments. For example, if the specification describes a composition comprising components A, B, and C, it is specifically intended that A, B, C, or any combination thereof may be omitted, discarded individually, or abandoned in any combination.

[0067] definition For clarity, certain terms used herein are defined and presented below.

[0068] As used herein and in the appended claims, the singular forms "a," "an," and "the" refer to multiple objects unless the context explicitly indicates otherwise. For example, a reference to "a plant" refers to one or more plants, including their equivalents known to those skilled in the art.

[0069] As used herein, the word "or" also includes "and / or" unless the context explicitly indicates otherwise.

[0070] The term “approximately” is used herein to mean roughly, nearly, approximately, or within that range. When the term “approximately” is used in conjunction with a numerical range, it modifies that range by extending the boundary above and below the set numerical value. Generally, the term “approximately” is used herein to mean a change of 20 percent above or below a given value, preferably 10 percent above or below (higher or lower). With respect to temperature, the term “approximately” means ±1°C, preferably ±0.5°C. When the term “approximately” is used in the context of this disclosure (for example, in combination with a value of temperature or molecular weight), an exact value (i.e., without “approximately”) is preferred.

[0071] As used herein, the terms “about X and Y,” “about X and about Y,” “X to Y,” and “about X to about Y” (and similar terms) should be interpreted as including X and Y unless the context indicates otherwise.

[0072] The words "comprises," "comprising," "includes," "including," "having," and their conjugations all mean "includes, but not limited to." The term "consisting of" means "includes, and limited to." The term "consisting essentially of" means that a composition, method, or structure may include additional components, processes, and / or parts, but only if the additional components, processes, and / or parts do not substantially alter the basic and novel properties of the claimed composition, method, or structure.

[0073] Units, prefixes, and symbols may be represented in SI-approved form. Unless otherwise specified, nucleic acids are drawn with a 5' to 3' orientation from left to right, and amino acid sequences are drawn with an N-terminus to C-terminus orientation from left to right. Amino acids may be represented herein by either their commonly known three-letter symbols or the single-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Similarly, nucleotides may be represented by their commonly accepted single-letter codes.

[0074] The “activity” of the insecticidal proteins in this disclosure means that the insecticidal proteins function as orally active pest (e.g., insect) control agents, have toxic effects (e.g., inhibit the ability of pests to survive, grow, and / or reproduce), and / or can interfere with or prevent pest feeding (which may or may not cause death of insects). When the insecticidal proteins of this disclosure are delivered to pests, the result is typically death of the pests or the pests not feeding on a source that makes the insecticidal proteins available to them.

[0075] As used herein, the terms “chimeric polynucleotide” or “chimeric protein” (or similar terms) refer to a molecule comprising two or more polynucleotides or proteins of different origins assembled into a single molecule, or fragments thereof. The terms “chimeric construct,” “chimeric gene,” “chimeric polynucleotide,” or “chimeric nucleic acid” refer to any construct or molecule containing, but not limited to, (1) a polynucleotide (e.g., DNA) comprising a regulatory polynucleotide and a coding polynucleotide that are not found together in nature (i.e., at least one of the polynucleotides in the construct is heterogeneous to at least one of the other polynucleotides), or (2) a polynucleotide encoding a portion of a protein that is not naturally adjacent, or (3) a portion of a promoter that is not naturally adjacent. Furthermore, a chimeric construct, chimeric gene, chimeric polynucleotide, or chimeric nucleic acid may comprise a regulatory polynucleotide and a coding polynucleotide from different sources, or from the same source, but may be arranged in a manner different from that found in nature. In some embodiments of this disclosure, a chimeric construct, chimeric gene, chimeric polynucleotide, or chimeric nucleic acid includes an expression cassette containing the polynucleotide of this disclosure under the control of a regulatory polynucleotide, particularly functionally controlled in plants or bacteria. The terms “chimeric” and “hybrid” with respect to polynucleotides or proteins are used interchangeably herein.

[0076] In the context of this disclosure, a “chimeric” protein is a protein created by fusing all or part of at least two different proteins. Chimeric proteins may also be further modified to include the addition, substitution, and / or deletion of one or more amino acids. In some embodiments of this disclosure, a chimeric protein is a chimeric Cry protein, which comprises all or part of two different Cry proteins fused together in a single polypeptide. In some embodiments, a chimeric Cry protein further includes additional modifications such as the addition, substitution, and / or deletion of one or more amino acids. A “chimeric insecticidal protein” is a chimeric protein having insecticidal activity.

[0077] As used herein, “codon-optimized” sequence means a nucleotide sequence in which codons are selected to reflect a particular codon bias that a host cell or organism may have. This is typically done in a manner that preserves the amino acid sequence of the polypeptide encoded by the optimized nucleotide sequence. In certain embodiments, the DNA sequence of a recombinant DNA construct includes a sequence that is codon-optimized for the cell in which the construct is expressed (e.g., an animal, plant, or fungal cell). For example, a construct expressed in a plant cell may have all or part of its sequence (e.g., a first repressor or gene expression element) that is codon-optimized for expression in the plant. See, for example, U.S. Patent No. 6,121,014, incorporated herein by reference. In some embodiments, the polynucleotides of this disclosure are codon-optimized for expression in plant cells (e.g., dicotyledonous or monocotyledonous plant cells) or bacterial cells.

[0078] "Controlling" insects means, through toxic effects, inhibiting the ability of pests to survive, grow, feed, and / or reproduce, and / or limiting insect-related damage or loss to crops, and / or protecting the productivity of crops when grown in the presence of pests. "Controlling" insects may or may not mean killing insects, but in some embodiments of this disclosure, "controlling" insects means killing insects.

[0079] As used herein, “control plant” or “control” may be a non-transgenic plant of the parental line used to generate the transgenic plant herein. The control plant may, in some cases, be a transgenic plant line that contains an empty vector or marker gene but does not contain the recombinant polynucleotide of this disclosure expressed in the transgenic plant being evaluated. Generally, the control plant is a plant of the same line or variety as the transgenic plant being tested, lacking the recombinant DNA that confers the specific traits characterizing the transgenic plant. Such a precursor plant lacking the recombinant DNA that confers the specific traits may be a natural wild-type plant, a selected non-transgenic plant, or a transgenic plant that does not contain the recombinant DNA that confers the specific traits characterizing the transgenic plant. A precursor plant lacking the recombinant DNA that confers the specific traits may be a sibling of the transgenic plant that has the recombinant DNA that confers the specific traits. Such a precursor sibling plant may contain other recombinant DNA.

[0080] With respect to this disclosure, "corresponding to" means that if the amino acid sequence of a reference sequence matches a second amino acid sequence different from the reference sequence (e.g., a variant or homologous sequence), then amino acids corresponding to specific listed positions in the second amino acid sequence will match those positions in the reference amino acid sequence, but will not necessarily be at the exact numerical positions with respect to a particular reference amino acid sequence of this disclosure.

[0081] As used herein, the term “Cry protein” means the crystalline delta-endotoxin-type insecticidal protein of Bacillus thuringiensis. The term “Cry protein” may also refer to any insecticidal fragment or toxin (e.g., without the N-terminal peptidyl fragment and / or C-terminal protoxin tail), including the protoxin form, partially processed forms, and mature toxin form.

[0082] "Delivering" or "being delivered" a composition or toxin means bringing the composition or toxin into contact with an insect to produce a toxic effect and control the insect. Compositions or toxins can be delivered orally by insect ingestion through many recognized methods, such as transgenic plant expression.

[0083] The term "domain" refers to a set of amino acids conserved at specific positions along the alignment of evolutionarily related protein sequences. While amino acids at other positions may vary between homologs, those highly conserved at specific positions likely indicate amino acids essential to the protein's structure, stability, or function. Identified by their high degree of conservation in the aligned sequences of a family of protein homologs, they can be used as identifiers to determine whether any given polypeptide belongs to a pre-identified group of polypeptides.

[0084] The “engineered” protein in this disclosure refers to a protein having a sequence different at at least one amino acid position compared to at least one corresponding parent protein. An engineered protein may be a mutant protein containing one or more modifications, such as deletions, additions, and / or substitutions at one or more amino acid positions compared to the parent protein. An engineered protein may be a chimeric protein, which may contain, for example, one or more exchanged or shuffled domains or fragments from at least two parent proteins.

[0085] "Effective insect control dose" means a concentration of one or more toxins that inhibits the ability of insects to survive, grow, feed, and / or reproduce through toxic action, or that limits insect-related damage or loss to a crop. "Effective insect control dose" may or may not mean killing insects, but it is preferable that it means killing insects. "Insecticidal activity" is defined as the toxic biological activity that can control insects, preferably by killing them. A transgenic plant having "enhanced insecticidal properties" is a plant that expresses one or more proteins at an effective insect control dose, thereby, in some embodiments, the plant is insecticidal against an increased range of insect species compared to an untransformed plant of the same species. This increased range of insect species includes lepidopteran pests, such as insect-plant pests such as Spodoptera frugiperda (fall armyworm).

[0086] The term “event” refers to the original transformant and / or the offspring of the transformant, which contain heterologous DNA. The term “event” also refers to the offspring produced by sexual crossing between the transformant and another maize lineage. Even after repeated backcrossing with a recurrent parent, the inserted DNA and flanking DNA from the transformed parent are present in the offspring of the cross at the same chromosomal location. The term “event” also refers to the DNA derived from the original transformant, which contains the inserted DNA and the flanking genome sequence immediately adjacent to the inserted DNA that is expected to be passed on to the offspring as a result of sexual crossing between one parent lineage containing the inserted DNA (e.g., offspring obtained from self-pollination of the original transformant) and a parent lineage that does not contain the inserted DNA. Typically, transformation of plant tissue produces multiple events, each corresponding to the insertion of a DNA construct at a different location in the genome of the plant cell.

[0087] As used herein, “expression cassette” means a nucleic acid sequence capable of directing the expression of a specific nucleotide sequence within a suitable host cell, comprising one or more transgenes, each transgene containing a promoter operably linked to the target nucleotide sequence, which is operably linked to a termination signal. Each transgene typically also includes sequences necessary for the proper translation of the nucleotide sequence. An expression cassette containing the target nucleotide sequence may have at least one component that is heterologous to at least one of the other components. An expression cassette may also be naturally occurring but obtained in a recombinant form useful for heterologous expression. However, typically, an expression cassette is heterologous to the host; that is, the specific nucleic acid sequence in the expression cassette does not naturally exist in the host cell and must have been introduced into the host cell or the host cell's ancestor by a transformation event. The expression of the nucleotide sequence in the expression cassette may be under the control of a constitutive or inductive promoter that initiates transcription only when the host cell is exposed to a specific external stimulus. In multicellular organisms such as plants, the promoter may also be specific to a particular tissue or organ or developmental stage.

[0088] An expression cassette containing the target nucleotide sequence can be a chimeric one, meaning that at least one of its components is heterologous to at least one of the other components. An expression cassette may also contain the native promoter that drives its native gene, but it was obtained in a recombinant form useful for heterologous expression. Such use of an expression cassette ensures that it does not exist naturally within the cell into which it is introduced.

[0089] An expression cassette may optionally include one or more transcription regions and / or translation termination regions that are functional in the plant. Various transcription terminators are available for use in expression cassettes and are involved in terminating transcription across the target heterologous nucleotide sequence and modifying mRNA polyadenylation. The termination region may be native to the transcription start region, native to the target nucleotide sequence to which it is operably linked, native to the plant host, or of another source (i.e., foreign or heterologous to the promoter, target nucleotide sequence, plant host, or any combination thereof).

[0090] A “gene” includes a coding nucleic acid sequence and typically also includes other primarily regulatory nucleic acids involved in the control of expression, i.e., the transcription and translation of the coding portion. A gene may also include other 5' and 3' untranslated sequences and a stop sequence. Further elements that may be present are, for example, introns. The regulatory nucleic acid sequence of a gene is usually not operably ligated to the relevant nucleic acid sequence as found in nature, and is therefore a chimeric gene.

[0091] The term “gene source” refers to an individual (e.g., a plant), a group of individuals (e.g., a plant lineage, variety, or family), or a clone derived from a lineage, variety, species, or culture, or the genetic material derived therefrom. Generally, a gene source may be part of an organism or cell, or can be isolated from an organism or cell. Generally, a gene source provides a specific molecular composition for genetic material, which provides a physical basis for some or all of the genetic properties of an organism or cell culture. As used herein, a gene source may include cells, seeds, or tissues from which a new plant can grow, or plant parts, such as leaves, stems, pollen, or cells that can be cultured into a whole plant.

[0092] The term "heterogeneous," when used in relation to genes, polynucleotides, or polypeptides, refers to genes, polynucleotides, or polypeptides that do not exist in their natural environment (i.e., have been modified by humans) or that contain such a portion. For example, heterogeneous genes may include polynucleotides from one species that have been introduced into another species. Heterogeneous genes may also include polynucleotides that are natural to an organism but have been modified in some way (e.g., by mutation, addition in multiple copies, or ligation to a non-natural promoter or enhancer polynucleotide). Heterogeneous genes may further include plant gene polynucleotides, which include plant genes in cDNA form, and the cDNA may be expressed in either sense orientation (to produce mRNA) or antisense orientation (to produce an antisense RNA transcript complementary to the mRNA transcript). In one aspect of this disclosure, heterogenes are distinguished from endogenous plant genes in that heterogeneous polynucleotides are typically bound to polynucleotides containing regulatory elements, such as promoters that are not naturally bound to the plant gene polynucleotides in the gene or chromosome of the protein encoded by the heterogene, or to a portion of the chromosome not found in nature (e.g., a gene expressed at a locus where the gene is not normally expressed). Furthermore, “heterogeneous” polynucleotides refer to polynucleotides that do not naturally bind to the host cell into which they are introduced, and include multiple copies of naturally occurring polynucleotides that do not exist in nature.

[0093] The terms “increase,” “increasing,” “increased,” “enhance,” “enhanced,” “enhancing,” and “enhancement,” as used herein, describe an increase in the control of plant pests, for example, by bringing the pests into contact with the plants of this disclosure (e.g., by transgenic expression or by topical application). An increase in control may refer to the level of control of plant pests in the absence of the nucleic acid molecules of this disclosure (e.g., plants that do not contain the nucleic acid molecules). Accordingly, in the embodiments, the terms “increasing,” “increasing,” “increased,” “enhancing,” “enhanced,” and “enhanced,” as well as similar terms, can indicate an increase of at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 125%, 150%, 200%, 300%, 400%, 500% or more when compared to a suitable control (e.g., a plant, plant part, or plant cell that does not contain the nucleic acid molecule).

[0094] With respect to two nucleic acid or amino acid sequences, the terms “identity” or “identical” refer to the percentage of identical nucleotides or amino acids in the linear polynucleotide or amino acid sequence of the reference ("query") sequence (or its complementary strand) compared to the test ("control") sequence when the two sequences are globally aligned. Unless otherwise specified, the sequence identity used herein refers to values ​​obtained using the Needleman-Wunsch algorithm ((1970) J.Mol.Biol.48:443-453), which is performed using the EMBOSS Needle alignment tool or any equivalent program with the default matrix file EBLOSUM62, using the default parameters (Gap Open=10, Gap Extend=0.5, End Gap Penalty=False, End Gap Open=10, End Gap Extend=0.5) for proteins, or the default parameters (Gap Open=10, Gap Extend=0.5, End Gap Penalty=False, End Gap Open=10, End Gap Extend=0.5) for DNAful nucleic acids. EMBOSS Needle is available from EMBL-EBI, for example, at the following website: ebi.ac.uk / Tools / psa / emboss_needle / and is described in the following publication: “The EMBL-EBI search and sequence analysis tools APIs in 2019.” Madeira et al. Nucleic Acids Research, June 2019, 47(W1):W636-W641. As used herein, the term “equivalent program” refers to any sequence comparison program that, when comparing corresponding alignments generated by EMBOSS Needle for any two sequences under discussion, produces alignments with identical nucleotide or amino acid residue matches and identical percentages of sequence identity. In some embodiments, substantially identical nucleic acids or amino acid sequences may perform substantially the same function.

[0095] In some embodiments, the polynucleotides or peptides of this disclosure are “isolated.” The term “isolated” polynucleotide or polypeptide is a polynucleotide or polypeptide that no longer exists in its natural environment. The isolated polynucleotides or polypeptides of this disclosure may exist in a purified form or in a recombinant host, such as in transgenic bacteria or transgenic plants. Thus, in some embodiments, the “isolated” nucleic acid molecule encompasses the nucleic acid molecule if the nucleic acid molecule is contained in the genome of a transgenic plant.

[0096] The term “isolated,” when used with respect to nucleic acid molecules or polynucleotides of this disclosure, refers to a polynucleotide that is identified within its respective source organism and isolated / separated from its chromosomal polynucleotides within its respective source organism. An isolated nucleic acid or polynucleotide is not a nucleic acid because it occurs in its natural context if it actually has a naturally occurring counterpart. In contrast, unisolated nucleic acids are nucleic acids such as DNA and RNA, which are found in the state in which they exist in nature. For example, a given polynucleotide (e.g., a gene) is found on a host cell chromosome near adjacent genes. An isolated nucleic acid molecule may exist in single-stranded or double-stranded form. Alternatively, it may contain both sense and antisense strands (i.e., the nucleic acid molecule may be double-stranded). In some embodiments, the nucleic acid molecules of this disclosure are isolated.

[0097] As used herein, the term “maize” includes all plant varieties that can be bred on Zea mays, including Zea mays and wild maize species. The terms “maize” and “corn” are used interchangeably herein.

[0098] The terms "motif," "consensus sequence," or "signature" refer to short, conserved regions within evolutionarily related protein sequences. Motifs are often highly conserved portions of a domain, but may contain only a portion of the domain or may be located outside the conserved domain (if all amino acids of the motif are outside the defined domain).

[0099] "Natural" or "wild-type" nucleic acids, polynucleotides, nucleotide sequences, polypeptides, or amino acid sequences refer to naturally occurring or endogenous nucleic acids, polynucleotides, nucleotide sequences, polypeptides, or amino acid sequences.

[0100] A “nucleic acid molecule,” “nucleic acid,” or “polynucleotide” (these terms are used interchangeably herein) is a segment of single-stranded, double-stranded, or partially double-stranded DNA or RNA, or a hybrid thereof, that can be isolated or synthesized from any source. In the context of this disclosure, a nucleic acid molecule is typically a segment of DNA. In some embodiments, the nucleic acid molecules of this disclosure are isolated nucleic acid molecules. In some embodiments, the nucleic acid molecules of this disclosure are contained within a vector, a plant, a plant cell, or a bacterial cell. These terms also include references to deoxyribopolynucleotides, ribopolynucleotides, or analogs thereof that have the essential properties of natural ribonucleotides in that they hybridize to substantially the same nucleotides as naturally occurring nucleotides under stringent hybridization conditions and / or enable translation into the same amino acids as naturally occurring nucleotides. A nucleic acid molecule may be a natural or heterologous structure, or a full-length or partial sequence of a regulatory gene. Unless otherwise indicated, this term includes references to a given sequence, its complementary sequence, or sequence. Therefore, DNA or RNA having a modified skeleton for stability or other reasons is a “polynucleotide” as intended herein. Furthermore, to give just two examples, DNA or RNA containing unusual bases such as inosine or modified bases such as tritylated bases is a polynucleotide as used herein. It will be understood that a wide variety of modifications are made to DNA and RNA to serve many useful purposes known to those skilled in the art. The term polynucleotide as used herein encompasses 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 simple and complex cells, in particular.

[0101] "Operatively linked" refers to the binding of a polynucleotide to a single nucleic acid molecule such that the function of one influences the function of the other. For example, a promoter is operatively linked to a coding polynucleotide (i.e., the coding polynucleotide is under the transcriptional control of the promoter) when it can influence the expression of that coding polynucleotide. A coding polynucleotide in sense or antisense orientation can be operatively linked to a regulatory polynucleotide.

[0102] The term "plant" includes references to the entire plant, plant organs, plant tissues (e.g., leaves, stems, roots, etc.), seeds, and plant cells, as well as their offspring. Plant cells as used herein include, but are not limited to, seeds, suspension cultures, embryos, meristematic tissues, callus tissue, leaves, roots, twigs, gametophytes, sporophytes, pollen, and microspores.The plant species that may be used in the methods of this disclosure generally include the following genera: Cucurbita, Rosa, Vitis, Juglans, Fragaria, Lotus, Medicago, Onobrychis, Trifolium, Trigonella, Vigna, Citrus, Linum, Geranium, and Phytolacca americana. Genus Manihot, Daucus, Arabidopsis, Brassica, Raphanus, Sinapis, Atropa, Capsicum, Datura, Hyoscyamus, Lycopersicon, Nicotiana, Solanum, Petunia, Digitalis, and Oriental Honeysuckle. Genus Majorana, Ciahorium, Helianthus, Lactuca, Bromus, Asparagus, Antirrhinum, Heterocallis, Nemesis, Pelargonium, Panieum, Pennisetum, Ranunculus, Senecio, The range of species suitable for transformation techniques is as broad as that of higher plants, including both monocots and dicots, such as species of Salpiglossis, Cucumis, Browaalia, Glycine, Pisum, Phaseolus, Lolium, Oryza, Avena, Hordeum, Secale, Allium, and Triticum.A particularly favored plant is corn.

[0103] A "plant cell" is a structural and physiological unit of a plant, including its protoplast and cell wall. Plant cells may be in the form of an isolated single cell or a cultured cell, or they may be in the form of part of a higher tissue unit, such as a plant tissue, plant organ, or the whole plant.

[0104] "Plant cell cultures" refer to, for example, cultures of plant units such as protoplasts, cells in cell cultures, plant tissues, pollen, pollen tubes, ovules, embryo sacs, zygotes, and cultures of cells in embryos at various stages of development.

[0105] "Plant material" refers to leaves, stems, roots, flowers or flower parts, fruits, pollen, egg cells, zygotes, seeds, cuttings, cell or tissue cultures, or any other part or product of a plant.

[0106] A "plant organ" is a distinct, visible, structured, and differentiated part of a plant, such as a root, stem, leaf, flower bud, or embryo.

[0107] As used herein, “plant material,” “plant part,” or “plant tissue” means plant cells, plant protoplasts, plant cell tissue cultures from which a plant can regenerate, plant callus, plant clumps, and intact plant cells in a plant or plant part, such as embryos, pollen, ovules, seeds, leaves, flowers, branches, fruits, grains, spikes, rachis, barks, stems, roots, root tips, anthers, tubers, rhizomes, etc. Any tissue of a planted or cultured plant is included in the term “plant tissue.”

[0108] As used herein, “plant sample” or “biological sample” refers to either intact or non-intact plant tissue (e.g., ground seeds or plant tissue, shredded plant tissue, freeze-dried tissue). It may also be an extract containing intact or non-intact seeds or plant tissue. The biological sample or extract may be selected from the group consisting of cereals manufactured whole or in part to include corn flour, cornmeal, corn syrup, corn oil, corn starch, and corn by-products.

[0109] "Polynucleotide of the Target" or "Nucleic Acid of the Target" refers to any polynucleotide that, when introduced into an organism, such as a plant, confers to the organism a desired trait, such as insect resistance, disease resistance, herbicide tolerance, antibiotic tolerance, improved nutritional value, improved performance in industrial processes, production of commercially valuable enzymes or metabolites, or altered reproductive capacity.

[0110] It will be understood that the “part” or “fragment” of the polypeptide in this disclosure means an amino acid sequence or nucleic acid sequence of reduced length compared to the reference amino acid sequence or nucleic acid sequence in this disclosure. Such a part or fragment in this disclosure may, as necessary, be included in a larger polypeptide or nucleic acid (e.g., a tagged or fusion protein or expression cassette) in which it is a component. In embodiments, the “part” or “fragment” substantially retains the activity of the full-length protein or nucleic acid, e.g., insecticidal activity (e.g., at least 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, or even 100% activity) or has even higher activity than the full-length protein, e.g., insecticidal activity.

[0111] As used herein, “vegetative propagator” refers to any material used to propagate a plant, preferably a transgenic plant. A vegetative propagator may be a seed, a cutting, or a group of cells derived from a transgenic plant, which can be used to produce a crop of the transgenic plant.

[0112] The terms "protein," "peptide," and "polypeptide" are used synonymously in this specification.

[0113] As used herein, the term “promoter” typically refers to a polynucleotide upstream (5') of the translation start site of a coding sequence, which controls the expression of the coding sequence by providing other factors necessary for recognition and proper transcription by RNA polymerase. For example, a promoter may include a region containing a basal promoter element recognized by RNA polymerase, the 5' untranslated region (UTR) of the coding sequence, and optionally a region containing an intron.

[0114] A "pollen-free promoter" is a promoter that drives little to no detectable gene expression in the pollen of a target plant species. Quantification of the mRNA transcript of the target protein in the pollen can be measured by various methods, including qRT-PCR / RNA-Seq, and the protein can be measured by commonly used ELISA and Western blotting methodologies. In this disclosure, a promoter is considered pollen-free if it drives the expression of the protein in the pollen with a TSP (total soluble protein) of <10 ng / mg.

[0115] As used herein, the term “recombinant” refers to a form of nucleic acid (e.g., DNA or RNA), protein, cell, tissue, or organism that is not normally found in nature and is therefore produced by human intervention. As used herein, “recombinant nucleic acid molecule” refers to a nucleic acid molecule containing a combination of polynucleotides that does not exist together in nature and is the result of human intervention, for example, a nucleic acid molecule containing a combination of at least two polynucleotides that are heterogeneous, or a nucleic acid molecule that is artificially synthesized, for example, a polynucleotide synthesized using an assembled nucleotide sequence and deviating from polynucleotides normally found in nature, or a nucleic acid molecule containing a transgene artificially incorporated into the genomic DNA of a host cell and related adjacent DNA of the host cell's genome. Another example of a recombinant nucleic acid molecule is a DNA molecule obtained from the insertion of a transgene into the genomic DNA of a plant, which may ultimately result in the expression of recombinant RNA or protein molecules in that organism. As used herein, “recombinant plant” refers to a plant that does not normally exist in nature, is the result of human intervention, and contains a transgene or heterogeneous nucleic acid molecule that may be incorporated into its genome. As a result of such genomic alterations, the recombinant plant is distinctly different from the associated wild-type plant. Recombinant bacteria are bacteria not found in nature that contain heterologous nucleic acid molecules. Such bacteria can be produced by transforming bacteria with nucleic acid molecules or by transfer such as plasmid conjugation from one bacterial strain to another, where the plasmid contains nucleic acid molecules.

[0116] The terms “reduce,” “reduced,” “reducing,” “reduction,” “diminish,” and “suppress” (and their grammatical variations), and similar terms, as used herein, refer to, for example, a reduction in the survival, growth, and / or reproduction of a plant pest by bringing it into contact with the plants of this disclosure. This reduction in survival, growth, and / or reproduction may refer to the level observed in the absence of the nucleic acid molecule of this disclosure (e.g., a plant that does not contain this nucleic acid molecule). Accordingly, in embodiments, the terms “reduce,” “reduced,” “is reducing,” “reduce,” “decrease,” and “suppress” (and their grammatical variations), and similar terms, mean a reduction of at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more compared to a plant not in contact with the nucleic acid molecule of this disclosure (e.g., a plant not containing this nucleic acid molecule). In typical embodiments, this reduction results in no detectable survival, growth, and / or reproduction of plant pests, or essentially no detectable survival, growth, and / or reproduction of plant pests (i.e., a small amount, for example, less than about 10%, less than about 5%, or even less than about 1%).

[0117] "Regulatory elements" refer to nucleotide sequences located upstream (5' non-coding sequence), internally, or downstream (3' non-coding sequence) of a coding sequence, and affect the transcription, RNA processing, stability, or translation of the associated coding sequence. Regulatory sequences include enhancers, promoters, translational enhancer sequences, introns, terminators, and polyadenylation signal sequences. They include sequences that may be native and synthetic sequences, as well as combinations of synthetic and native sequences. Regulatory sequences may determine expression levels, spatial and temporal patterns of expression, and, in the case of a subset of promoters, expression under inducing conditions (modulation by external factors such as light, temperature, chemicals, and hormones).

[0118] As used herein, “selectable marker” means a nucleotide sequence that, when expressed, confers a different phenotype to a plant, plant part, and / or plant cell expressing the marker, thereby enabling such transformed plants, plant parts, and / or plant cells to be distinguished from those without the marker. Such a nucleotide sequence may encode either a selectable marker or a screenable marker, depending on whether the marker confers a trait that can be selected by chemical means, for example, by using a selector (e.g., an antibiotic, herbicide, etc.), or whether the marker is simply a trait that can be identified through observation or testing, such as by screening (e.g., an R locus trait).

[0119] The terms “stringent conditions” or “stringent hybridization conditions” refer to conditions under which nucleic acids selectively hybridize to a target sequence to a detectably greater degree than other sequences (e.g., at least twice as much as non-target sequences), and optionally substantially exclude binding to non-target sequences. Stringent conditions are sequence-dependent and will vary under different circumstances. By controlling the stringency of hybridization and / or washing conditions, target sequences that may be up to 100% complementary to a reference nucleotide sequence can be identified. Alternatively, moderate or low stringency conditions may be used to tolerate some mismatch in the sequence so that a lower degree of sequence similarity is detected. For example, those skilled in the art will understand that, in order to function as a primer or probe, nucleic acid sequences only need to be sufficiently complementary to the target sequence to substantially bind to them in order to form a stable double-stranded structure under the conditions employed. Thus, primers or probes can be used under high, moderate, or even low stringency conditions. Similarly, low or moderate stringency conditions may be advantageous for detecting homologous, orthologous, and / or paralogous sequences that have a lower degree of sequence identity than those identified under highly stringent conditions. Typically, stringent conditions are a pH of 7.0–8.3 with a salt concentration of less than about 1.5 M Na ions, typically about 0.01–1.0 M Na ions (or other salts), and a temperature of at least about 30°C for short probes (e.g., 10–50 nucleotides) and at least about 60°C for long probes (e.g., 50 nucleotides). Stringent conditions may also be achieved by adding an stabilizer such as formamide or Denhardt's solution (500 ml of water, 5 g Ficol, 5 g polyvinylpyrrolidone, 5 g bovine serum albumin).Exemplary low-stringency conditions include hybridization in a buffer solution of 30%–35% formamide, 1M NaCl, and 1% SDS (sodium dodecyl sulfate) at 37°C, and washing in 1×–2×SSC (20×SSC = 3.0M NaCl / 0.3M trisodium citrate) at 50°C–55°C. Exemplary moderate-stringency conditions include hybridization in 40%–45% formamide, 1M NaCl, and 1% SDS at 37°C, and washing in 0.5×–1×SSC at 55°C–60°C. Exemplary high-stringency conditions include hybridization in 50% formamide, 1M NaCl, and 1% SDS at 37°C, and washing in 0.1×SSC at 60°C–65°C. A more non-limiting example of high-stringency conditions includes hybridization in 4×SSC, 5×Denhardt solution, 0.1 mg / ml boiled salmon sperm DNA, and 25 mM sodium phosphate at 65°C, and washing in 0.1×SSC and 0.1% SDS at 65°C. Another example of high-stringency hybridization conditions includes hybridization in 7% SDS, 0.5 M NaPO4, and 1 mM EDTA at 50°C, along with washing in 2×SSC and 0.1% SDS at 50°C, or with washing in 1×SSC and 0.1% SDS at 50°C, or with washing in 0.5×SSC and 0.1% SDS at 50°C, or with washing in 0.1×SSC and 0.1% SDS at 50°C, or even with washing in 0.1×SSC and 0.1% SDS at 65°C. Those skilled in the art will understand that specificity is typically a typical function of post-hybridization washing, and that the relevant factors are the ionic strength and temperature of the final washing solution.

[0120] As used herein, “stable transformation” or “stable transformed” means that a nucleic acid is introduced into a cell and integrated into the cell’s genome. Thus, the integrated nucleic acid can be inherited by their offspring, more specifically by multiple successive generations of offspring. As used herein, “genome” includes the nuclear and plastid genomes, and therefore includes the integration of nucleic acids into, for example, the chloroplast genome. As used herein, stable transformation can also refer to a transgene maintained outside of a chromosome, for example, as a microchromosome.

[0121] As used herein, gene or trait “stacking” refers to combining a desired gene or trait into a single transgenic plant line. One approach is for plant breeders to stack transgenic traits by crossing parents, each possessing the desired trait, and then identifying offspring that possess both of these desired traits (a so-called “breeding stack”). Another method of gene stacking involves introducing two or more genes simultaneously into the cell nucleus of a plant during transformation. Another method of gene stacking involves re-transforming a transgenic plant with another gene of interest. For example, gene stacking can be used to combine two different insect resistance traits, an insect resistance trait and a disease resistance trait, or a herbicide resistance trait (e.g., Bt11). In addition to the gene of interest, the use of selectable markers is also considered gene stacking.

[0122] "Synthetic" refers to a nucleotide sequence that contains bases or structural features not present in natural sequences. For example, artificial sequences encoding proteins of this disclosure that closely resemble the G+C content and normal codon distribution of dicotyledonous or monocotyledonous plant genes are called synthetic.

[0123] As used herein, the "toxic" nature of the disclosed proteins to pests means that the protein functions as an orally active insecticide to kill the pest, or that the protein can interfere with or deter contact with an insect, or cause growth inhibition in the pest, both of which may or may not cause death of the insect. When the toxic proteins of the disclosed are delivered to an insect, or when an insect comes into orally contact with a toxic protein, the result is typically death of the insect, delayed growth of the insect, or cessation of the insect's feeding of the source that made the toxic protein available to the insect.

[0124] The terms “toxin fragment” and “toxin moiety” are used interchangeably to refer to a fragment or portion of a longer (e.g., full-length) insecticidal protein of the Disclosure, where “toxin fragment” or “toxin moiety” retains insecticidal activity. For example, it is known in the Art that the natural Cry protein, when expressed as a protoxin, is treated at its N-terminus and C-terminus to produce a mature toxin. In embodiments, the “toxin fragment” or “toxin moiety” of the chimeric insecticidal protein of the Disclosure is cleaved at the N-terminus and / or C-terminus. In embodiments, the “toxin fragment” or “toxin moiety” is cleaved at the N-terminus, removing part or all of the N-terminal peptidyl fragment, and optionally containing at least about 400, 425, 450, 475, 500, 510, 520, 530, 540, 550, 560, 570, 580, or 590 consecutive amino acids of the insecticidal protein specifically described herein, or an amino acid sequence substantially equivalent thereto. Therefore, in the embodiment, the "toxin fragment" or "toxin portion" of the insecticidal protein is cleaved at the N-terminus (for example, to remove some or all of the peptidyl fragment), for example, by cleaving one or more amino acids, for example, up to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 or more amino acids at the N-terminus.In embodiments, the "toxin fragment" or "toxin portion" of the insecticidal protein is cleaved at the C-terminus (for example, to remove part or all of the protoxin tail), for example, one amino acid or two or more amino acids, for example, up to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 70, 80, 90, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, and 560 or more C-terminal cleavages of amino acids are performed. In embodiments, the "toxin fragment" or "toxin moiety" includes domains 1 and 2 and a core domain 3. In embodiments, the "toxin fragment" or "toxin moiety" is a mature (i.e., processed) toxin (e.g., Cry toxin).

[0125] "Transformation" is the process of introducing a different nucleic acid into a host cell or organism. In certain embodiments, "transformation" means the stable integration of a DNA molecule into the genome (nucleus or plastid) of the organism of interest. In certain embodiments, introduction into a plant, plant part, and / or plant cell is via bacterial-mediated transformation, particle impact transformation, calcium phosphate-mediated transformation, cyclodextrin-mediated transformation, electroporation, liposome-mediated transformation, nanoparticle-mediated transformation, polymer-mediated transformation, virus-mediated nucleic acid delivery, whisker-mediated nucleic acid delivery, microinjection, sonication, infiltration, polyethylene glycol-mediated transformation, protoplast transformation, or any other electrical, chemical, physical, and / or biological mechanism or combination thereof that results in the introduction of nucleic acids into a plant, plant part, and / or its cells. Procedures for transforming plants are well known and commonplace in the art and are described throughout the literature. Non-limiting examples of methods for plant transformation include bacterial-mediated nucleic acid delivery (e.g., via bacteria from the genus Agrobacterium), viral-mediated nucleic acid delivery, silicon carbide or nucleic acid whisker-mediated nucleic acid delivery, liposome-mediated nucleic acid delivery, microinjection, microparticle impaction, calcium phosphate-mediated transformation, cyclodextrin-mediated transformation, electroporation, nanoparticle-mediated transformation, sonication, infiltration, PEG-mediated nucleic acid uptake, and transformation via any other electrical, chemical, physical (mechanical) and / or biological mechanisms (including any combination thereof) that result in the introduction of nucleic acids into plant cells.General guidelines for various plant transformation methods known in this field include those by Miki et al. ("Procedures for Introducing Foreign DNA into Plants" in Methods in Plant Molecular Biology and Biotechnology, Glick, BRand Thompson, JE, Eds. (CRC Press, Inc., Boca Raton, 1993), pages 67-88) and Rakowoczy-Trojanowska (2002, Cell Mol Biol Lett 7:849-858 (2002)).

[0126] "Transformed" and "transgenic" refer to host organisms, such as bacteria or plants, into which heterologous nucleic acid molecules have been introduced. The nucleic acid molecules can either be stably integrated into the host's genome or exist as extrachromosomal molecules. Such extrachromosomal molecules can self-replicate. It is understood that transformed cells, tissues, or plants include not only the final product of the transformation process but also their transgenic offspring. "Untransformed," "non-transgenic," or "non-recombinant" hosts refer to wild-type organisms, such as bacteria or plants, that do not contain heterologous nucleic acid molecules.

[0127] The term “transgenic plant” includes references to plants into which heterologous nucleic acid molecules have been introduced. Generally, heterologous nucleic acid sequences are stably integrated into the genome so that the nucleic acid sequence is passed on to subsequent generations. Heterologous nucleic acid sequences may be integrated into the genome alone or as part of a recombinant expression cassette. “Transgenic” is used herein to include any cell, cell lineage, callus, tissue, plant part, or plant whose phenotype is modified by the presence of heterologous nucleic acid sequences, including those of the transgenic plant that was initially modified in this way, as well as those produced by sexual mating or asexual reproduction from an early transgenic plant.

[0128] The term "vector" refers to a composition for transporting, delivering, or introducing nucleic acids (or multiple nucleic acids) into a cell. A vector comprises a nucleic acid molecule containing the nucleotide sequence to be transported, delivered, or introduced. Examples of vectors include plasmids, cosmids, phagemids, artificial chromosomes, phages, or viral vectors.

[0129] The term “yield” may refer to bushels per acre of a grain crop at harvest, adjusted for grain moisture (e.g., typically 15% for maize) and for the volume of biomass produced (for forage crops such as alfalfa and multiple crops, the size of the plant roots). Grain moisture is measured in the grain at harvest. The adjusted test weight of the grain is determined by the weight in pounds per bushel, adjusted for grain moisture levels at harvest. Biomass is measured as the weight of harvestable plant material produced. Yield can be influenced by many characteristics, including, but are not limited to, plant height, number of panicles, position of panicles on the plant, number of internodes, rate of split panicle appearance, grain size, efficiency of nodule formation and nitrogen fixation, efficiency of nutrient assimilation, carbon assimilation, plant structure, germination rate, seed vigor, and seedling traits. Yield can also be influenced by germination efficiency (including germination under stress conditions), growth rate (including growth rate under stress conditions), number of ears, number of seeds per ear, seed size, seed composition (starch, oil, protein), and seed filling characteristics. Plant yield can be measured in many ways, including by weight, number of seeds per plant, seed weight, number of seeds per unit area (i.e., seeds or seed weight per acre), bushels per acre, tons per acre, or kilograms per hectare. For example, maize yield may be measured as the production of hulled corn kernels per unit area, for example, bushels per acre, or metric tons per hectare, and is often reported based on adjusted moisture, for example, at 15.5 percent moisture. Furthermore, a corn bushel is defined by Iowa law as weighing 56 pounds, and a useful conversion factor for corn yield is as follows: 100 bushels per acre is equivalent to 6.272 metric tons per hectare. Other measurements of yield are common practice in the art. In certain embodiments of this disclosure, yields may be increased under stressed and / or non-stressed conditions.

[0130] nucleic acid molecule This disclosure provides compositions and methods for controlling harmful pests. In particular, this disclosure provides nucleic acid molecules that, when expressed in cells, confer insecticidal properties to cells, such as insecticidal activity against lepidopteran pests like Spodoptera frugiperda (fall armyworm).

[0131] For different expression cassettes, multiple different constructs were produced to determine the efficacy and agrochemical effects of the expressed proteins. Surprisingly, one vector, SEQ ID NO: 2, when transformed in maize plants, conferred excellent insecticidal properties with little to no adverse effects on the vegetative development or reproductive capacity of the transgenic plants. The expression cassette from the vector is SEQ ID NO: 1.

[0132] A person skilled in the art will recognize that during the insertion of a nucleic acid molecule, such as SEQ ID NO: 1, into a cell, the 5' and / or 3' ends of the inserted molecule may be deleted or reconfigured. Such deletions or reconfigurations do not affect the function of the inserted molecule, and these relatively small changes result in an inserted molecule that can be considered substantially identical to SEQ ID NO: 1. A person skilled in the art will also recognize that nucleic acid molecules, such as those containing SEQ ID NO: 1, may undergo complete or partial reconfiguration or duplication during the insertion event, and that the thereby inserted molecule is a complete or partial reconfiguration or duplication of the starting nucleic acid molecule. A person skilled in the art will recognize that this inserted molecule may still possess the same properties and / or traits as the starting molecule, and that the thereby inserted molecule is substantially identical to SEQ ID NO: 1, and the transformed cells or resulting transformed plants are still desirable.

[0133] Those skilled in the art will recognize that, for a transgene intended for commercial use, such as a nucleic acid molecule containing Sequence ID No. 1, relatively minor modifications to the nucleic acid sequence may be necessary to comply with government regulatory standards. Such modifications should not affect the function of the resulting molecule, and the resulting molecule will be substantially identical to Sequence ID No. 1. Those skilled in the art will recognize that the modified nucleic acid molecule is essentially the same as the starter molecule.

[0134] Therefore, the Disclosure also includes nucleic acid molecules substantially identical to SEQ ID NO: 1, in which certain nucleotides in SEQ ID NO: 1 are deleted, substituted, or rearranged, resulting in a mutated SEQ ID NO: 1, and the functionality of the mutated SEQ ID NO: 1 is the same as that of the starting molecule. Therefore, in some embodiments, the Disclosure includes nucleic acid sequences that are at least 90% identical to SEQ ID NO: 1 (for example, at least 90% identical to SEQ ID NO: 1, at least 91% identical to SEQ ID NO: 1, at least 92% identical to SEQ ID NO: 1, at least 93% identical to SEQ ID NO: 1, at least 94% identical to SEQ ID NO: 1, at least 95% identical to SEQ ID NO: 1, at least 96% identical to SEQ ID NO: 1, at least 97% identical to SEQ ID NO: 1, at least 98% identical to SEQ ID NO: 1, at least 99% identical to SEQ ID NO: 1, or at least 99.5% identical to SEQ ID NO: 1), or the same Complementary bodiesThe present invention provides nucleic acid molecules comprising the following: In some embodiments, the nucleic acid molecule encodes the same protein encoded by SEQ ID NO: 1. In some embodiments, the nucleic acid sequence comprises SEQ ID NO: 1 or any one of 8-31, or any one or more of the variants in Table 3. In some embodiments, the nucleic acid molecule produces a protein that is insecticidal against one or more lepidopteran pests, for example, at least against Spodoptera frugiperda (fall armyworm). In some embodiments, the nucleic acid molecule produces a protein that is insecticidal against at least two (e.g., two, three, or four) of the following species: Spodoptera frugiperda (white fall armyworm), Mythimna separata (sea armyworm), Spodoptera litura (beet armyworm / oriental leaf worm), and Ostrinia furnacalis (Asian corn borer). In some embodiments, the nucleic acid molecule is isolated. In some embodiments, the nucleic acid molecule is present in the plant.

[0135] The disclosed insecticidal proteins encoded by the nucleic acid molecules of this disclosure (e.g., one of SEQ ID NOs. 1 or 8-31, or one or more of the variants in Table 3) have insecticidal activity against lepidopteran pests. In some embodiments, the insecticidal proteins are active against one or more of the following non-limiting examples of lepidopteran pests: species of the genus Spodoptera, e.g., S. frugiperda (fall armyworm), S. littoralis (Egyptian cottonleaf worm), S. ornithogalli (yellow-striped army worm), S. praefica (Western army worm) Yellow-striped army worm, S. eridania (Southern army worm), S. litura (Oriental reef worm), S. cosmioides (Black army worm), S. exempta (African army worm), S. mauritia (Lone army worm), and / or S. exigua (White-striped army worm) (U); Species of the genus Ostrinia, e.g., O. nubilalis (European corn borer) and / or O. furnacalis (Asian corn borer); Species of the genus Plutella, e.g., P. xylostella (diamondback moth); Species of the genus Agrotis, e.g., A. ipsilon (black cutworm), A. segetum (common moth) (Cutworm), A. gladiaria (clayback cutworm), and / or A. orthogonia (pale western cutworm); species of the genus Striacosta, e.g., S. albicosta (western bean cutworm); species of the genus Helicoverpa, e.g., H. zea (American tobacco moth / soybean pod worm), H. punctigera (H.*H. punctigera* (false tobacco worm), and / or *H. armigera* (cotton ball worm); species of the genus *Heliothis*, e.g., *H. virescens* (tobacco bat worm); species of the genus *Diatraea*, e.g., *D. grandiosella* (Southwestern corn borer), and / or *D. saccharalis* (sugarcane borer); species of the genus *Trichoplusia*, e.g. For example, the nettle moth (T. ni) (cabbage looper); species of the genus Sesamia, e.g., S. nonagroides (Mediterranean corn borer), S. inferens (pink stem borer), and / or S. calamistis (pink stem borer); species of the genus Pectinophora, e.g., P. gossypiella (pink ball worm); species of the genus Cochylis, e.g., C. hospes (C. h ospes) (banded sunflower moss); Manduca species, e.g., tobacco hawk moth (M. sexta) (tobacco hornworm) and / or tomato hawk moth (M. quinquemaculata) (tomato hornworm); Elasmopalpus species, e.g., E. lignosellus (sorghum moth); Pseudoprusia species, e.g., P. includens (soybean inchworm); Anticalcium Species of the genus Anticarsia, e.g., A. gemmatalis (velvet bean caterpillar); species of the genus Platipena, e.g., P. scabra (green clover worm); species of the genus Pieris, e.g., P. brassicae (large white butterfly); species of the genus Papaipema, e.g., P. nebris (stoke borer); species of the genus Pseudaletia, e.g., cutworm (P.unipuncta) (common army worm); species of the genus Peridroma, e.g., P. saucia (variegated cutworm); species of the genus Keiferia, e.g., K. lycopersicella (tomato pinworm); species of the genus Artogeia, e.g., A. rapae (imported) cabbageworm; species of the genus Phthorimaea, e.g., P. operculella (potato leafworm); species of the genus Chrysodeixis, e.g., C. includens (soybean looper); species of the genus Feltia, e.g., F. ducens (denzi cutworm); species of the genus Chilo, e.g., C. suppressalis (striped stem borer), C. agamemnon (oriental corn borer), and C. partellus (spotted stalk borer); species of the genus Cnaphalocrocis, e.g., C. medalis (rice leaf folder); Konogete Species of the genus Conogethes, e.g., *C. punctiferalis* (yellow peach moss); species of the genus Mythimna, e.g., *M. separata* (oriental army worm); species of the genus Athetis, e.g., *A. lepigone* (two-spotted army worm); species of the genus Busseola, e.g., B . Fusca (corn stalk borer), Etiella species, e.g., E. zinckenella (pulse pod borer), Leguminivora species, e.g., L. glycinivorella (soybean pod borer); Matsumuraeses species, e.g., M.Species of the genus Omiodes, e.g., O. indicata (soybean leaf folder / bean leaf webworm); species of the genus Rachiplusia, e.g., R. nu (sunflower looper), or any combination of the above. In some embodiments, at least one of the insecticidal proteins encoded by nucleic acid molecules has insecticidal activity against the fall armyworm (Spodoptera frugiperda). In some embodiments, at least one of the insecticidal proteins encoded by nucleic acid molecules has insecticidal activity against at least two (e.g., two, three, or four) of the following species: Spodoptera frugiperda (fall armyworm), Mythimna separata (sea armyworm), Spodoptera litura (beet armyworm / oriental leaf worm), and Ostrinia furnacalis (Asian corn borer). In some embodiments, the insecticidal protein may optionally have insecticidal activity against fall armyworm pests or colonies that are resistant to another insecticide containing another insecticidal protein (e.g., Bt protein). In some embodiments, the insecticidal protein has insecticidal activity against fall armyworm colonies resistant to Vip3A protein (e.g., Vip3Aa, including, but not limited to, maize event MIR162), Cry1F protein (e.g., Cry1Fa, including, but not limited to, maize event TC1507 or DP-4114), Cry1A protein (e.g., Cry1A.105, including, but not limited to, maize event MON89034), or Cry2 protein (e.g., Cry2Ab, including, but not limited to, maize event MON89034).

[0136] The disclosed insecticidal proteins may also possess insecticidal activity against Coleopteran, Hemipteran, Dipteran, Lygus species, and / or other stinging and sucking insects, such as Orthoptera or Thysanoptera. In some embodiments, the insecticidal protein has activity against one or more of the following non-limiting examples: Coleopteran pests: Diabrotica species, e.g., D. barberi (Northern corn rootworm), D. virgifera virgifera (Western corn rootworm), D. undecimpunctata howardii (Southern corn rootworm), D. balteata (Banded cucumber beetle), D. undecimpunctata D. undecimpunctata (Western Spotted Cucumber Beetle), D. significata (Three-Spotted Leaf Beetle), D. speciosa (Cucumber Beetle), D. virgifera zeae (Mexican Corn Rootworm), D. beniensis, D. cristata, D. curviplustalata, D. dissimilis, D. elegantula, D. emorsitans, D. graminea, D. hispanloe, D. lemniscata, D. linsleyi, D. milleri, D. nummularis, D. occlusal, D. porrecea, D. scutellata, D. tibialis,D. trifasciata and / or D. viridula; species of the genus Leptinotarsa, e.g., L. decemlineata (colored potato beetle); species of the genus Chrysomela, e.g., C. scripta (cottonwood leaf beetle); species of the genus Hypothenemus, e.g., H. hampei (coffee berry baller); Sitophilis Species of the genus *Ilus*, e.g., rice weevil (S. zeamais); species of the genus *Epitrix*, e.g., tobacco flea beetle (E. hirtipennis) and / or E. cucumeris (potato flea beetle); species of the genus *Phyllotreta*, e.g., *P. cruciferae* (oil flea beetle) and / or *P. pusilla* (Western black flea beetle); species of the genus *Anthonomus*, e.g., *A. eugeni* i) Pepper weevil; species of the genus Hemicrepidus, e.g., H. memnonius (wireworm); species of the genus Melanotus, e.g., M. communis (wireworm); species of the genus Ceutorhychus, e.g., C. assimilis (cabbage seedpod weevil); species of the genus Phyllotreta, e.g., P. cruciferae (oilworm) Leaf beetles; species of the genus Aeolus, e.g., A. mellillus (wireworm); species of the genus Aeolus, e.g., A. mancus (wheat wireworm); species of the genus Horistonotus, e.g., H. uhlerii (sand wireworm); species of the genus Sphenophorus, e.g., S. maidis (corn weevil), S. zeae (timothy bill bug),S. parvulus (bluegrass bill bug) and S. callosus (Southern corn bill bug); species of the genus Phyllophaga (whitegrubs); species of the genus Chaetocnema, e.g., C. pulicaria (corn flea beetle); species of the genus Popillia, e.g., P. japonica (Japanese beetle) - ; species of the genus Epilachna, e.g., E. varivestis (Mexican beetle); species of the genus Cerotoma, e.g., C. trifurcate (bean leaf beetle); species of the genus Epicauta, e.g., E. pestifera and E. lemniscata (blister beetle); or any combination of the above. Insects within the order Hemiptera are not limited to,Chinavia hilaris (green stink bug); Anasa tristis De Geer (squash bug); Blissus leucopterus (chinch bug); Corythuca gossypii Fabricius (cotton lace bug); Cyrtopeltis modesta Distant (tomato bug); Dysdercus suturellus Hern ch-Schaffer (cotton stainer); Euschistus servus Say (brown stink bug); E. variolarius Palisot de Beauvois (One-spotted stink bug); Graptosthethus species (complex of seed bugs); Leptoglossus corculus Say (Leaffoot pine seed bug); Lygus lineolaris Palisot de Beauvois (Tarnished plant bug); L. Hesperus Knight (Western turned plant bug); L. pratensis Linnaeus (Common meadow bug); L. rugulipennis Poppius (European turned plant bug); Lygocoris pabulinus Linnaeus (Common green capsid); Nezara viridula Linnaeus (Southern Green Stinkbug); Oebalus pugnax Fabricius (Inesting Stinkbug); Oncopeltus fasciatus Dallas (Large Milkweed Bug); Pseudatomoscelis seriatus Reuter (Cotton Leap Hopper)Calocoris norvegicus Gmelin (Strawberry Bug); Orthops campestris Linnaeus; Plesiocoris rugicollis Fallen (Apple Capsid); Cryptopeltis modestus Distant (Tomato Bug); Cryptopeltis notatus Distant (Suckfly); Spanagonicus albofasciatus Reuter (White Marked Flare Hopper); Diaphnocoris chlorionis Say (Honey Locust Plant Bug); Labopidicola allii Knight) (Onion Plant Bug); Pseudatomoscelis seriatus Reuter (Cotton Flare Hopper); Adelphocoris rapidus Say (Rapid Plant Bug); Poecilocapsus lineatus Fabricius (Fore-lined Plant Bug); Nysius ericae Schilling (False Chinchinch Bug); Nysius raphanus Howard (False Chinchinch Bug); Nezara viridula Examples include Linnaeus (Southern Green Stinkbug); species of the genera Eurygaster; Coreidae; Pyrrhocoridae; Tinidae; Blostomatidae; Reduviidae; and Cimicidae. Insects of the order Diptera, though not limited to these, include species of the genus Liriomyza, for example,Examples include the bean leafminer (L. trifolii) and L. sativae (vegetable leafminer); species of the genus Scrobipalpula, e.g., S. absoluta (tomato moth); species of the genus Delia, e.g., D. platura (seed corn fly), D. brassicae (cabbage fly), and D. radicum (cabbage root fly); species of the genus Psilia, e.g., P. rosae (carrot rust fly); species of the genus Tetanops, e.g., T. myopaeformis (sugar beet root maggot); and any combination of the above. Among the Orthoptera insects, but not limited to, are species of the genus Melanoplus, e.g., M. differentialis (differential grasshopper), M. femurrubrum (red-legged grasshopper), M. bivittatus (two-striped grasshopper); and any combination thereof. Among the Thymoptera insects, but not limited to, are species of the genus Frankliniella, e.g., F. occidentalis (western flower thrips) and F. fusca (tobacco thrips); and species of the genus Thrips, e.g., onion thrips. Examples include the onion thrips (T. tabaci), the southern yellow thrips (T. palmi), and any combination of the above.

[0137] The disclosed insecticidal proteins may also have insecticidal activity against one or more of the following: species of the genus Phyllophaga, Rhopalosiphum maidis, Pratylenchus penetrans, Melanotus cribulosus, Cyclocephala lurida, Limonius californicus, Tetranychus urticae, Haplothrips aculeatus, Tetranychus truncates, Anomala corpulenta, Oedaleus infernalis infernalis), Frankliniella tenuicornis, Tetranychus cinnabarinus, Aiolopus thalassinus tamulus, Trachea tokionis, Laodelphax striatellus, Holotrichia oblita, Dichelops furcatus, Diloboderus abderu, Dalbulus maidis, Astylus variegathus, Scaptocoris castanea castanea), Locusta migratoria manilensis, Agriotes lineatus, Peregrinus maidis, Oscinella frit, Frankliniella 'Williamsii'williamsi), Zyginidia manaliensis, Atherigona soccata, Nicentrites testaceipes, Mylocerus undecimpustulatus, Atherigona naquii, Amsecta albistriga, Plodia interpuctella, Melanotus caudex, Microtermes species, Atherigona oryzae, Tanymecus dilaticollis, Delphacodes kosherii *Leptospermum kuschelli*, *Lepidiota stigma*, *Phyllophaga hellery*, *Tribolium castaneum*, *Pelopidas mathias*, *Oxya chinensis (Thunberg)*, *Stenocranus pacificus*, *Scutigerella immaculata*, *Chrysodeixis chalcites*, species of the genus *Euproctis* (Lymantriidae), species of the genus *Phyllotreata* (undulata), *Reptalus* Panzer), Cyrtacanthacris tartarica Linnaeus, Orgyia postica, Dactylispa lameyi, Patanga succinctaJohanson), species of the genera Tetranychus, species of Calomycterus, Adoretus compressus Weber, and Paratetranychus stickney.

[0138] In some embodiments, the Disclosure provides vectors comprising nucleic acid molecules of the Disclosure. Examples of vectors include plasmids, cosmids, phagemids, artificial chromosomes, phages, or viral vectors. In embodiments, the vector is, for example, a plant vector for use in plant transformation. In embodiments, the vector is, for example, a bacterial vector for use in bacterial transformation. Suitable vectors for plants, bacteria, and other organisms are known in the Art.

[0139] In some embodiments, the nucleic acid molecules or vectors of this disclosure may include sequences encoding other desired traits in addition to insecticidal proteins. Such expression cassettes containing stacked traits may be used to produce plants, plant parts, or plant cells having a desired phenotype with stacked traits (i.e., molecular stacking). Such stacked combinations in plants may also be produced by other methods, including but not limited to crossbreeding plants by any conventional methodology. When stacked by genetically transforming plants, the desired nucleotide sequences can be combined at any time and in any order. For example, a transgenic plant containing one or more desired traits may be used as a target to introduce further traits by subsequent transformation. Additional nucleotide sequences may be introduced simultaneously with the nucleic acid molecules or vectors of this disclosure in a co-transformation protocol. For example, if two nucleotide sequences are introduced, they may be incorporated into separate cassettes (trans) or into the same cassette (cis). Polynucleotide expression may be driven by the same promoter or by different promoters. It is further understood that polynucleotides can be stacked at desired genomic locations using site-specific nucleases or recombinant systems (e.g., FRT / Flp, Cre / Lox, TALE-endonucleases, zinc finger nucleases, CRISPR / Cas, and related technologies). See U.S. Patent Nos. US7214536, US8921332, US8765448, US5527695, US5744336, US5910415, US6110736, US6175058, US6720475, US6455315, US6458594, and U.S. Patent Publication Nos. US2019093090, US2019264218, US2018327785, US2017240911, US2016208272, and US2019062765.

[0140] In some embodiments, the nucleic acid molecules or vectors of this disclosure may include one or more polynucleotides or additional double-stranded RNA (dsRNA) coding sequences for agricultural traits that are particularly beneficial to seed companies, growers, or grain processors. The polypeptide of interest may be any polypeptide encoded by the nucleotide sequence of interest. Non-limiting examples of polypeptides of interest suitable for plant production include those that result in agriculturally important traits such as herbicide resistance (sometimes also referred to as "herbicide tolerance"), virus resistance, bacterial pathogen resistance, insect resistance, nematode resistance, or fungal resistance. See, for example, U.S. Patents No. 5,569,823, No. 5,304,730, No. 5,495,071, No. 6,329,504, and No. 6,337,431. Polypeptides may also increase plant vitality or yield (including traits that allow plants to grow at different temperatures, soil conditions, and levels of sunlight and rainfall), or enable the identification of plants exhibiting desired traits (e.g., selectable markers, seed coat color, etc.). Polypeptides for various purposes, and methods for introducing these polypeptides into plants, are, for example, described in U.S. Patents No. 4,761,373, 4,769,061, 4,810,648, 4,940,835, 4,975,374, 5,013,659, 5,162,602, 5,276,268, and 5,304,730. This is described in U.S. Patent Publication No. 5,495,071, No. 5,554,798, No. 5,561,236, No. 5,569,823, No. 5,767,366, No. 5,879,903, No. 5,928,937, No. 6,084,155, No. 6,329,504, and No. 6,337,431, as well as in U.S. Patent Publication No. 2001 / 0016956.

[0141] Polynucleotides conferring resistance / tolerance to herbicides that inhibit growth or meristematic tissue, such as imidazolinone or sulfonylurea, may also be suitable in some embodiments. Exemplary polynucleotides in this category encode mutant ALS and AHAS enzymes, as described, for example, in U.S. Patent Nos. 5,767,366 and 5,928,937. U.S. Patents Nos. 4,761,373 and 5,013,659 are aimed at plants resistant to various imidazolinone or sulfonamide herbicides. U.S. Patent No. 4,975,374 relates to plant cells and plants containing nucleic acids encoding mutant glutamine synthase (GS) resistant to inhibition by herbicides known to inhibit GS, such as phosphinothricin and methionine sulfoximine. U.S. Patent No. 5,162,602 discloses plants resistant to inhibition by cyclohexanedione and aryloxyphenoxypropanoate herbicides. Resistance is conferred by a modified acetylcoenzyme A carboxylase (ACCase).

[0142] Polypeptides encoded by nucleotide sequences that confer resistance to glyphosate are also suitable for this disclosure. See, for example, U.S. Patent No. 4,940,835 and U.S. Patent No. 4,769,061. U.S. Patent No. 5,554,798 discloses a transgenic glyphosate-resistant maize plant in which resistance is conferred by a modified 5-enolpyruvir-3-phosphosikimic acid (EPSP) synthase gene.

[0143] Phosphono compounds, such as glufosinate ammonium or phosphinotricin, and polynucleotides encoding resistance to pyridinoxy or phenoxypropionic acid, as well as cyclohexones, are also preferred. See European Patent Application No. 0242246. Also see U.S. Patents Nos. 5,879,903, 5,276,268, and 5,561,236.

[0144] Other suitable polynucleotides include those encoding resistance to photosynthesis-inhibiting herbicides, such as triazines and benzonitriles (nitrilases). See, for example, U.S. Patent No. 4,810,648. Additional suitable polynucleotides encoding herbicide resistance include those encoding resistance to 2,2-dichloropropionic acid, cethoxydime, haloxyfop, imidazolinone herbicides, sulfonylurea herbicides, triazolopyrimidine herbicides, s-triazine herbicides, and bromoxynil. Also preferred are polynucleotides that confer resistance to protox enzymes, or enhanced resistance to plant diseases, enhanced tolerance to harmful environmental conditions (abiotic stresses) such as drought, excessive cold, excessive heat, excessive soil salinity, or extreme acidity or alkalinity, as well as modifications in plant structure or development, including changes in developmental timing. For example, see U.S. Patent Publication No. 2001 / 0016956 and U.S. Patent No. 6,084,155.

[0145] Additional suitable polynucleotides include those encoding insecticidal polypeptides. These polypeptides can be produced in amounts sufficient to control pests (i.e., insect control levels). It is understood that the amount of insecticidal polypeptides produced in plants necessary to control insects or other pests may vary depending on the cultivar, pest species, environmental factors, etc. Additional polynucleotides useful for insect or pest resistance include, for example, those encoding toxins identified in the genus Bacillus. Polynucleotides containing nucleotide sequences encoding the Bacillus thuringiensis (Bt)Cry protein from several subspecies have been cloned, and recombinant clones have been found to be toxic to lepidopteran, diptera, and / or coleopteran insect larvae. Examples of such Bt insecticidal proteins include Cry proteins, such as Cry1Aa, Cry1Ab, Cry1Ac, Cry1B, Cry1C, Cry1D, Cry1Ea, Cry1Fa, Cry3A, Cry9A, Cry9B, and Cry9C, as well as vegetative insecticidal proteins, such as Vip1, Vip2, and Vip3. A complete list of Bt-derived proteins can be found on the World Wide Web in the Bacillus thuringiensis Toxin Nomenclature Database managed by the University of Sussex (see also Crickmore et al. (1998) Microbiol. Mol. Biol. Rev. 62:807-813).

[0146] In the embodiment, additional polypeptides include alpha-amylase, peroxidase, cholesterol oxidase, patatin, protease, protease inhibitor, urease, alpha-amylase inhibitor, pore-forming protein, chitinase, lectin, engineered antibody or antibody fragment, and Bacillus cereus. Insecticidal proteins from *Cereus*, *Xenorhabdus* species (such as *X. nematophila* or *X. bovienii*), *Photorhabdus* species (such as *P. luminescens* or *P. asymobiotica*), *Brevibacillus* species (such as *B. laterosporus*), *Lysinibacillus* species (such as *L. sphearicus*), and *Chromobacterium*. These are insecticidal polypeptides derived from non-Bt sources, including but not limited to genus species (such as C. subtsugae or C. piscinae), Yersinia species (such as Y. entomophaga), Paenibacillus species (such as P. propylaea), Clostridium species (such as C. bifermentans), Pseudomonas species (such as P. fluorescens), and lignin.

[0147] Polypeptides suitable for production in plants include, for example, those that improve or otherwise facilitate the conversion of harvested plants or plant parts into commercially useful products, including increased or altered carbohydrate content or distribution, improved fermentation properties, increased oil content, increased protein content, improved digestibility, and nutritional supplement content, such as increased phytosterol content, increased tocopherol content, increased stanol content, or increased vitamin content. The target polypeptides also include those that result in or contribute to a reduction in unwanted components in the harvested crop, such as phytic acid or glycosphagocytes. “Resulting in” or “contributing” means that the target polypeptide is intended to directly or indirectly contribute to the presence of the target trait (e.g., increasing cellulose degradation by the use of heterologous cellulase enzymes).

[0148] In some embodiments, polypeptides contribute to improved digestibility of food or feed. Xylanase is a hemicellulose-degrading enzyme that improves the breakdown of plant cell walls, leading to better utilization of plant nutrients by animals. This results in improved growth rates and feed conversion. Furthermore, the viscosity of feed containing xylan can be reduced. Heterogeneous production of xylanase in plant cells can also facilitate the conversion of lignocellulose to fermentable sugars in industrial processing.

[0149] Numerous xylanases derived from fungal and bacterial microorganisms have been identified and characterized (see, for example, U.S. Patent No. 5,437,992; Coughlin et al. (1993) “Proceedings of the Second TRICEL Symposium on Trichoderma reesei Cellulases and Other Hydrolases” Espoo; Souminen and Reinikainen, eds. (1993) Foundation for Biotechnical and Industrial Fermentation Research 8:125-135; U.S. Patent Publication No. 2005 / 0208178; and PCT Publication No. WO03 / 16654). In particular, three specific xylanases (XYL-I, XYL-II, and XYL-III) have been identified in Trichoderma reesei (Tenkanen et al. (1992) Enzyme Microb. Technol. 14:566; Torronen et al. (1992) Bio / Technology 10:1461; and Xu et al. (1998) Appl. Microbiol. Biotechnol. 49:718).

[0150] In other embodiments, polypeptides useful to the disclosure may be polysaccharide-degrading enzymes. Plants of the disclosure that produce such enzymes may be useful, for example, for generating fermentation raw materials for bioprocesses. In some embodiments, enzymes useful for fermentation processes include alpha-amylase, protease, pullulanase, isoamylase, cellulase, hemicellulase, xylanase, cyclodextrin tricotransferase, lipase, phytase, laccase, oxidase, esterase, cutinase, granular starch hydrolase, and other glucoamylases.

[0151] Polysaccharide-degrading enzymes include starch-degrading enzymes, such as α-amylase (EC3.2.1.1) and glucuronidase (EC3.2.1.131); exo-1,4-α-D-glucanases, such as amyloglucosidase and glucoamylase (EC3.2.1.3), β-amylase (EC3.2.1.2), α-glucosidase (EC3.2.1.20), and other exo-amylases; and starch debranching enzymes, such as a) isoamylase (EC3.2.1.20). 3.2.1.68) pullulanase (EC3.2.1.41), etc.; b) cellulases, e.g., exo-1,4-3-cellobiohydrolase (EC3.2.1.91), exo-1,3-β-D-glucanase (EC3.2.1.39), β-glucosidase (EC3.2.1.21); c) L-arabinases, e.g., endo-1,5-α-L-arabinase (EC3.2.1.99), α-arabinosidase (EC3.2.1.55), etc.; d ) Galactanases, e.g., endo-1,4-β-D-galactanase (EC3.2.1.89), endo-1,3-β-D-galactanase (EC3.2.1.90), α-galactosidase (EC3.2.1.22), β-galactosidase (EC3.2.1.23), etc.; e) Mannanases, e.g., endo-1,4-β-D-mannanase (EC3.2.1.78), β-mannosidase (EC3.2.1.25), α-mannosidase ( Examples include EC3.2.1.24); f) xylanases, e.g., endo-1,4-β-xylanase (EC3.2.1.8), β-D-xylosidase (EC3.2.1.37), 1,3-β-D-xylanase, etc.; and g) other enzymes, e.g., α-L-fucosidase (EC3.2.1.51), α-L-rhamnosidase (EC3.2.1.40), revanase (EC3.2.1.65), inulanase (EC3.2.1.7), etc. In one embodiment, the α-amylase is Amy797E, a synthetic α-amylase described in whole in U.S. Patent No. 8,093,453, which is incorporated herein by reference in whole.

[0152] Further enzymes that may be used in this disclosure include proteases such as fungal and bacterial proteases. Fungal proteases include, but are not limited to, those obtained from the genera Aspergillus, Trichoderma, Mucor, and Rhizopus, such as A. niger, A. awamori, A. oryzae, and M. miehei. In some embodiments, the polypeptide of this disclosure may be a cellobiohydrolase (CBH) enzyme (EC3.2.1.91). In one embodiment, the cellobiohydrolase enzyme may be CBH1 or CBH2.

[0153] Other enzymes useful in this disclosure include, but are not limited to, hemicellulases, e.g., mannase and arabinofuranosidase (EC3.2.1.55); ligninases; lipases (e.g., EC3.1.1.3), glucose oxidases, pectinases, xylanases, transglucosidases, alpha-1,6-glucosidases (e.g., EC3.2.1.20); esterases, e.g., ferulate esterase (EC3.1.1.73) and acetylxylan esterase (EC3.1.1.72); and cutinases (e.g., EC3.1.1.74).

[0154] Examples of double-stranded RNA molecules useful in this disclosure include, but are not limited to, those that repress target insect genes. Where used herein, the term “gene repression” is intended to mean, collectively, any known method for reducing the level of protein produced as a result of gene transcription to mRNA and subsequent translation of the mRNA. Gene repression is also intended to mean the reduction of protein expression from a gene or coding sequence, including post-transcriptional gene repression and transcriptional repression. Post-transcriptional gene repression refers to a substantial and measurable reduction in the amount of available mRNA available in the cell due to ribosome binding, mediated by homology between all or part of the mRNA transcribed from the targeted gene or coding sequence for repression and the corresponding double-stranded RNA used for repression. The transcribed RNA may be in either a sense orientation that affects so-called co-repression, an antisense orientation that affects so-called antisense repression, or an orientation that produces dsRNA that affects so-called RNA interference (RNAi). Transcriptional repression is mediated by the intracellular presence of the gene repressor dsRNA and the promoter DNA sequence or its Complementary bodiesIt exhibits substantial sequence identity and affects what is called promoter trans-repression. Gene repression may be effective, for example, against native genes associated with traits, providing plants with reduced levels of proteins encoded by the native genes or enhanced or reduced levels of affected metabolites. Gene repression may also be effective against target genes in plant pests that ingest or come into contact with plant material containing gene repressors, specifically those designed to inhibit or suppress the expression of one or more homologous or complementary sequences in the cells of the pests. Such genes targeted for repression may encode essential proteins, and their predicted functions are selected from the group consisting of myogenesis, juvenile hormone formation, juvenile hormone regulation, ion regulation and transport, digestive enzyme synthesis, maintenance of cell membrane potential, amino acid biosynthesis, amino acid degradation, spermatogenesis, pheromone synthesis, pheromone sensing, antenna formation, wing formation, leg formation, development and differentiation, ooogenesis, larval maturation, digestive enzyme formation, hemolymph synthesis, hemolymph maintenance, neurotransmission, cell division, energy metabolism, respiration, and apoptosis.

[0155] Transgenic cells, plants, plant parts In some embodiments, the Disclosure further provides transgenic cells, plants, plant parts, etc., comprising the nucleic acid molecules or vectors of the Disclosure (e.g., one of SEQ ID NOs. 1 or 8-31, or one or more of the variants in Table 3). In some embodiments, the Disclosure provides non-human host cells comprising the nucleic acid molecules or vectors of the Disclosure. Examples of transgenic non-human host cells include, but are not limited to, plant cells (including monocotyledonous plant cells and / or dicotyledonous plant cells), yeast cells, bacterial cells, or insect cells. Therefore, in some embodiments, genera include Bacillus, Brevibacillus, Clostridium, Xenorhabdus, Photorhabdus, Pasteuria, Escherichia, Pseudomonas, Erwinia, Serratia, Klebsiella, Salmonella, Pasteurella, and Xanthomonas. Bacterial cells selected from the genera Streptomyces, Rhizobium, Rhodopseudomonas, Methylophilus, Agrobacterium, Acetobacter, Lactobacillus, Arthrobacter, Azotobacter, Leuconostoc, or Alcaligenes are provided.

[0156] In some embodiments, the transgenic plant cells are dicotyledonous plant cells or monocotyledonous plant cells. In additional embodiments, the dicotyledonous plant cells are soybean cells, sunflower cells, tomato cells, kohl crop cells, cotton cells, sugar beet cells, or tobacco cells. In further embodiments, the monocotyledonous plant cells are barley cells, maize cells, oat cells, rice cells, sorghum cells, sugarcane cells, or wheat cells. In preferred embodiments, the monocotyledonous plant cells are maize cells. In some embodiments, the disclosure provides a plurality of dicotyledonous plant cells or monocotyledonous plant cells (e.g., a plurality of maize cells containing the nucleic acid molecules or vectors of the disclosure). In embodiments, the plurality of cells are juxtaposed to form an apoplast and grown in natural sunlight. In embodiments, the transgenic plant cells cannot regenerate an entire plant.

[0157] In other embodiments of the Disclosure, the nucleic acid molecules of the Disclosure are expressed in higher organisms, such as plants. Such transgenic plants express an effective amount of insecticidal protein encoded by the nucleic acid molecule to control plant pests, such as harmful insects. When insects begin to feed on such transgenic plants, they ingest the expressed insecticidal protein. This prevents the insects from biting further into the plant tissue, or it may even harm or kill the insects. In some embodiments, the nucleic acid molecules of the Disclosure are stably incorporated into the genome of a plant. In other embodiments, the nucleic acid molecules of the Disclosure are contained in a non-pathogenic self-replicating virus.

[0158] In some embodiments, the transgenic plant is insecticidal against at least Spodoptera frugiperda (the fall armyworm). In some embodiments, the transgenic plant is insecticidal against at least two (e.g., two, three, or four) of the following: Spodoptera frugiperda (the fall armyworm), Mythimna separata (the armyworm), Spodoptera litura (the beet armyworm), and Ostrinia furnacalis (the Asian corn borer). In some embodiments, the transgenic plants have enhanced insecticidal properties, for example, against Spodoptera frugiperda (fall armyworm), compared to a control plant that does not contain the nucleic acid molecule.

[0159] In some embodiments of the present disclosure, transgenic plant cells comprising the nucleic acid molecules of the present disclosure are cells of plant parts, plant organs, or plant cultures (as described herein, respectively), including but not limited to root, leaf, seed, flower, fruit, pollen cell, organ, or plant culture, or callus cells or cultures.

[0160] Transgenic plants or plant cells transformed in accordance with this disclosure may be monocots or dicots or plant cells, including corn, soybeans, rice, wheat, barley, rye, oats, sorghum, millet, sunflower, safflower, sugar beet, cotton, sugarcane, rapeseed, alfalfa, tobacco, peanuts, sweet potatoes, beans, peas, chicory, lettuce, cabbage, cauliflower, broccoli, turnips, carrots, eggplants, cucumbers, radishes, and bell peppers. Examples of plants in this disclosure include, but are not limited to, vegetables such as potatoes, tomatoes, asparagus, onions, garlic, melons, peppers, celery, squash, pumpkins, and zucchinis; fruits such as apples, pears, quince, plums, cherries, peaches, nectarines, apricots, strawberries, grapes, raspberries, blackberries, pineapples, avocados, papayas, mangoes, and bananas; horticultural plants such as those of the genus Arabidopsis; and woody plants such as conifers and deciduous trees. Preferably, the plants of this disclosure are crop plants such as corn, sorghum, wheat, sunflowers, tomatoes, Brassicaceae plants, peppers, potatoes, cotton, rice, soybeans, sugar beets, sugarcane, tobacco, barley, and rapeseed.

[0161] Once a desired nucleic acid molecule is transformed into a specific plant species, it can be propagated within that species using any appropriate technique, including traditional breeding techniques, or transferred to other varieties of the same species, particularly commercial varieties.

[0162] The insecticidal proteins encoded by the nucleic acid molecules of this disclosure can function as insect control agents in plant parts, plant cells, plant organs, seeds, harvested products, processed products, or extracts. In other words, the insecticidal proteins can continue to perform the insecticidal function they had in the transgenic plant. The nucleic acid molecules can function to express the insecticidal proteins. Instead of expressing the insecticidal proteins of this disclosure, in some embodiments, the nucleic acid molecules can function to identify the transgenic plant parts, plant cells, plant organs, seeds, harvested products, processed products, or extracts of this disclosure that contain this nucleic acid molecule.

[0163] In embodiments, the transgenic plant, plant part, plant cell, plant organ, or seed of the Disclosure is semi-conjugated to the nucleic acid molecule of the Disclosure. In embodiments, the transgenic plant, plant part, plant cell, plant organ, or seed is homozygous to the nucleic acid molecule of the Disclosure.

[0164] Additional embodiments of the Disclosure include harvested products produced from the transgenic plants or parts thereof of the Disclosure, and processed products produced from the harvested products. The harvested products may be the whole plant or any plant part as described herein. In some embodiments, non-limiting examples of harvested products include seeds, fruits, flowers or parts thereof (e.g., anthers, stigmas, etc.), leaves, stems, etc. In other embodiments, processed products may include, but are not limited to, grain flour, edible flour, oil, starch, syrup, cereal, and similar products produced from the harvested seeds or other plant parts of the Disclosure, wherein such seeds or other plant parts include nucleic acid molecules of the Disclosure.

[0165] In other embodiments, the Disclosure provides extracts from transgenic seeds or transgenic plants of the Disclosure, the extracts containing nucleic acid molecules of the Disclosure. Extracts from plants or plant parts can be prepared according to procedures well known in the art (see de la Torre et al., Food, Agric. Environ. 2(1):84-89 (2004); Guidet, Nucleic Acids Res. 22(9):1772-1773 (1994); Lipton et al., Food Agric. Immun. 12:153-164 (2000)). Such extracts may be used, for example, in methods for detecting the presence of nucleic acid molecules of the Disclosure.

[0166] In some embodiments, transgenic plants, plant parts, plant cells, plant organs, seeds, harvested products, processed products, or extracts have increased insecticidal activity against one or more pests (e.g., lepidopteran pests) compared to preferred controls that do not contain the nucleic acid molecules of the Disclosure. In some embodiments, transgenic plants, plant parts, plant cells, plant organs, seeds, harvested products, processed products, or extracts have increased insecticidal activity against at least Spodoptera frugiperda (fall armyworm). In some embodiments, transgenic plants, plant parts, plant cells, plant organs, seeds, harvested products, processed products, or extracts have increased insecticidal activity against at least two (e.g., two, three, or four) of the following species: Spodoptera frugiperda (fall armyworm), Mythimna separata (sea armyworm), Spodoptera litura (beet armyworm / oriental leaf worm), and Ostrinia furnacalis (Asian corn borer).

[0167] Plant transformation and breeding Procedures for transforming plants are well-known and commonplace in the art and are described throughout the literature. Non-exclusive examples of methods for transforming plants include transformation via bacterial-mediated nucleic acid delivery (e.g., via Agrobacterium), viral-mediated nucleic acid delivery, silicon carbide or nucleic acid whisker-mediated nucleic acid delivery, liposome-mediated nucleic acid delivery, microinjection, particulate gun method, calcium phosphate-mediated transformation, cyclodextrin-mediated transformation, electroporation, nanoparticle-mediated transformation, sonication, infiltration, PEG-mediated nucleic acid uptake, and any other electrical, chemical, physical (mechanical), or biological mechanisms (including any combination thereof) that result in the introduction of nucleic acid molecules into plant cells. General guidelines for various plant transformation methods known in this field include those by Miki et al. ("Procedures for Introducing Foreign DNA into Plants" in Methods in Plant Molecular Biology and Biotechnology, Glick, BRand Thompson, JE, Eds. (CRC Press, Inc., Boca Raton, 1993), pages 67-88) and Rakowoczy-Trojanowska (Cell. Mol. Biol. Lett. 7:849-858 (2002)).

[0168] For Agrobacterium-mediated transformation, binary vectors or vectors carrying at least one T-DNA border sequence are generally preferred, while for direct gene transfer (e.g., by a particulate gun), any vector is preferred, and linear DNA containing only the construct of interest can be used. In the case of direct gene transfer, transformation with a single DNA species or co-transformation can be used (Schocher et al., Biotechnology 4:1093-1096 (1986)). For both direct gene transfer and Agrobacterium-mediated gene transfer, transformation is usually (but not always) carried out using a selectable marker (e.g., phosphomannose isomerase) that may be a positive choice providing resistance to antibiotics (e.g., kanamycin, hygromycin, or methotrexate) or herbicides (e.g., glyphosate or glufosinate). However, the selection of the selectable marker is not important to this disclosure.

[0169] Agrobacterium-mediated transformation is a commonly used method for transforming plants due to its high transformation efficiency and versatility across many different species. Agrobacterium-mediated transformation typically involves introducing a binary vector carrying the foreign DNA of the target into a suitable Agrobacterium strain, which carries the vir gene either on a coexisting Ti plasmid or on a chromosome within the host Agrobacterium strain. Complementary bodiesThis may depend on (Uknes et al. (1993) Plant Cell 5:159-169). The introduction of recombinant binary vectors into Agrobacterium strains can be achieved by a tripearing mating procedure using Escherichia coli, which is a helper Escherichia coli strain carrying a plasmid capable of recruiting the recombinant binary vector to the target Agrobacterium strain. Alternatively, recombinant binary vectors can be introduced into Agrobacterium strains by nucleic acid transformation ((Hoefgen & Willmitzer (1988) Nucleic Acids Res. 16:9877).

[0170] Dicotyledonous and monocotyledonous plants can be transformed using the genus Agrobacterium. Methods for Agrobacterium-mediated transformation of rice include well-known methods for rice transformation, such as those described in any of the following: European Patent Application No. EP1198985, Aldemita and Hodges (Planta 199:612-617, 1996); Chan et al. (Plant Mol Biol 22(3):491-506, 1993); Hiei et al. (Plant J 6(2):271-282, 1994) (their disclosures are incorporated herein by reference as if they were fully described). For maize transformation, the methods described are those found in either Ishida et al. (Nat. Biotechnol 14(6):745-50, 1996) or Frame et al. (Plant Physiol 129(1):13-22, 2002) (these disclosures are incorporated herein by reference as if they were fully described). These methods are further described, for example, in B. Jenes et al., Techniques for Gene Transfer, in: Transgenic Plants, Vol. 1, Engineering and Utilization, eds. SDKung and R. Wu, Academic Press (1993) 128-143 and Potrykus Annu. Rev. Plant Physiol. Plant Molec. Biol. 42 (1991) 205-225). The nucleic acid or construct to be expressed is preferably cloned into a vector, which is suitable for transforming Agrobacterium tumefaciens, for example, pBin19 (Bevan et al., Nucl. Acids Res. 12 (1984) 8711).Agrobacteria transformed with such vectors can then be used in known methods for the transformation of plants, such as model plants like Arabidopsis, or crop plants such as tobacco, by immersing wounded or chopped leaves in an Agrobacteria solution and then culturing them in a suitable medium. Plant transformation using Agrobacterium tumefaciens is known, for example, from Hagen and Willmitzer in Nucl. Acid Res. (1988) 16, 9877, or in particular from FF White, Vectors for Gene Transfer in Higher Plants; in Transgenic Plants, Vol. 1, Engineering and Utilization, eds. SDKung and R. Wu, Academic Press, 1993, pp. 15-38.

[0171] Soybean plant material can be suitably transformed, and fertile plants have been regenerated by many methods well known to those skilled in the art. Examples of soybean transformation methods can be found in U.S. Patent No. 5,024,944; Finer and McMullen (1991) In Vitro Cell Dev. Biol. 27P:175-182; McCabe et al. (1988) Bio / technology 6:923-926; Khalafalla et al. (2006) African J. of Biotechnology 5:1594-1599; U.S. Patent No. 7,001,754; Hinchee et al. (1988) Bio / Technology 6:915-922; U.S. Patent No. 7,002,058; U.S. Patent Application Publication No. 20040034889; U.S. Patent Application Publication No. 20080229447; and Paz et al. (2006) Plant Cell Report 25:206-213.

[0172] Transgenic plants can be generated using different transformation methods with previously described binary vectors containing selectable marker genes. For example, the vector may be used to transform immature seed targets as described in order to directly generate transgenic HPPD plants using HPPD inhibitors such as mesotrione as the selector (see, e.g., U.S. Patent Application Publication 20080229447). Optionally, other herbicide resistance genes may be present in polynucleotides together with other sequences that provide additional means of selection / identification of transformed tissues, including known genes that provide resistance to, for example, kanamycin, hygromycin, phosphinotricin, porphenacil, or glyphosate. For example, different binary vectors containing PAT or EPSPS selectable marker genes are transformed using Agrobacterium-mediated transformation and glufosinate or glyphosate selection as described (see, e.g., U.S. Patent Application Publication 20080229447).

[0173] Plant transformation with recombinant Agrobacterium typically involves co-culturing Agrobacterium with explants from plants, followed by methods well known in the art. The transformed tissue is regenerated on a selective medium carrying antibiotic or herbicide resistance markers across a binary plasmid T-DNA border.

[0174] As previously discussed, another method for transforming plants, plant parts, and plant cells involves propelling inactive or biologically active particles into plant tissues and cells. See, for example, U.S. Patent Nos. 4,945,050, 5,036,006, and 5,100,792. Generally, this method involves propelling inactive or biologically active particles into plant cells under conditions that allow them to penetrate the outer surface of the cell and be incorporated into its interior. When inactive particles are used, the vector can be introduced into the cell by coating the particles with a vector containing the nucleic acid of interest. Alternatively, the cell can be surrounded by the vector so that the vector is carried into the cell following the particles. Biologically active particles (e.g., dried yeast cells, dried bacteria, or bacteriophages, each containing one or more nucleic acids to be introduced) can also be propelled into plant tissues.

[0175] In other embodiments, the nucleic acid molecules of this disclosure can be directly transformed into a plastid genome. Plastidic transformation techniques are extensively described in U.S. Patent Nos. 5,451,513, 5,545,817, and 5,545,818, PCT application WO95 / 16783, and McBride et al. (1994) Proc. Nati. Acad. Sci. USA 91,7301-7305.

[0176] Methods for selecting transformed transgenic plants, plant cells, or plant tissue cultures are commonplace in the art and can be employed in the methods of the Disclosure provided herein. For example, the nucleic acid molecules or vectors of the Disclosure may also include expression cassettes containing nucleotide sequences for selectable markers that can be used to select transformed plants, plant parts, or plant cells.

[0177] Examples of selectable markers include, but are not limited to, nucleotide sequences encoding neo or nptII to confine resistance to kanamycin G418 (Potrykus et al. (1985) Mol. Gen. Genet. 199:183-188); nucleotide sequences encoding bar to confine resistance to phosphinotricin; nucleotide sequences encoding modified 5-enolpyruvirshikimic acid-3-phosphate (EPSP) synthase to confine resistance to glyphosate (Hinchee et al. (1988) Biotech. 6:915-922); and nucleotide sequences encoding nitrilases such as bxn from Klebsiella ozaenae to confine resistance to bromoxyn (Stalker et al. (1988) Science). 242:419-423); Nucleotide sequences encoding modified acetolactate synthase (ALS) that confer resistance to imidazolinone, sulfonylurea, or other ALS inhibitors (European Patent Application No. 154204); Nucleotide sequences encoding methotrexate-resistant dihydrofolate reductase (DHFR) (Thillet et Examples include nucleotide sequences encoding darapon dehalogenase that confer resistance to darapon (1988) J. Biol. Chem. 263:12500-12508); nucleotide sequences encoding mannose-6-phosphate isomerase (also known as phosphomannose isomerase (PMI)) that confer the ability to metabolize mannose (U.S. Patent Nos. 5,767,378 and 5,994,629); nucleotide sequences encoding modified anthranilate synthase that confer resistance to 5-methyltryptophan; or nucleotide sequences encoding hph that confer resistance to hygromycin. Those skilled in the art can select suitable selectable markers for use in the expression cassettes of this disclosure.

[0178] Additional selectable markers include, but are not limited to, nucleotide sequences encoding β-glucuronidase or uidA (GUS), which encodes an enzyme known to have various chromogenic substrates; R-locus nucleotide sequences encoding products that regulate the production of anthocyanin pigments (red) in plant tissues (Dellaporta et al., “Molecular cloning of the maize R-nj allele by transposon-tagging with Ac” 263-282 In: Chromosome Structure and Function: Impact of New Concepts, 18th Stadler Genetics Symposium (Gustafson & Appels eds., Plenum Press 1988)); nucleotide sequences encoding β-lactamase, an enzyme known to have various chromogenic substrates (e.g., PADAC, chromogenic cephalosporins) (Sutcliffe (1978) Proc. Natl. Acad. Sci. USA 75:3737-3741); and nucleotide sequences encoding xylE, which encodes catechol dioxygenase (Zukowsky et al. (1983) Proc. Natl. Acad. Sci. USA 80:1101-1105); a nucleotide sequence encoding tyrosinase, an enzyme that can oxidize tyrosine to DOPA and dopaquinone, which then condense to form melanin (Katz et al. (1983) J. Gen. Microbiol. 129:2703-2714); a nucleotide sequence encoding β-galactosidase, an enzyme that contains a chromogenic substrate; a nucleotide sequence encoding luciferase (lux), which enables bioluminescence detection (Ow et al. (1986) Science 234:856-859); a nucleotide sequence encoding aequorin, which can be used in calcium-sensitive bioluminescence (Prasher et al. (1985) Biochem. Biophys. Res. Comm. 126:1259-1268); or green fluorescent protein (Niedz et al.Examples include nucleotide sequences encoding other fluorescent proteins (e.g., dsRed or mCherry, as described in (1995) Plant Cell Reports 14:403-406). Those skilled in the art can select suitable markers for use in the expression cassettes of this disclosure.

[0179] Furthermore, as is well known in the art, intact transgenic plants can be regenerated from transformed plant cells, plant tissue cultures, or cultured protoplasts using any of the various known techniques. Regeneration of plants from plant cells, plant tissue cultures, or cultured protoplasts is described, for example, in Evans et al. (Handbook of Plant Cell Cultures, Vol. 1, MacMilan Publishing Co., New York (1983)); and Vasil IR (ed.) (Cell Culture and Somatic Cell Genetics of Plants, Acad. Press, Orlando, Vol. I (1984), and Vol. II (1986)).

[0180] Furthermore, the genetic characteristics incorporated into the transgenic seeds and plants, plant parts, or plant cells of the present disclosure can be inherited by sexual reproduction or vegetative growth, and thus maintained and propagated in offspring plants. Generally, maintenance and propagation utilize known agricultural methods developed to suit specific purposes such as harvesting, sowing, or cultivation.

[0181] Therefore, the nucleic acid molecules of this disclosure can be introduced into plants, plant parts, or plant cells by any method known in the art as described above. Thus, there is no particular method to rely on for introducing nucleic acid molecules into plants, but rather any method that enables the nucleic acid molecules to be stably incorporated into the plant genome can be used. When two or more polynucleotides are introduced, each polynucleotide can be assembled as part of a single nucleic acid molecule or as a separate nucleic acid molecule, and can be located on the same or different nucleic acid molecules. Thus, polynucleotides can be introduced into target cells in a single transformation event, in separate transformation events, or, for example, in plants as part of a breeding protocol.

[0182] Once a desired nucleic acid molecule is transformed into a specific plant species, it can be propagated within that species or transferred to other varieties of the same species, particularly commercial varieties, using traditional breeding techniques.

[0183] In some embodiments, the transgenic plants, plant parts, plant cells, plant organs, seeds, harvested products, processed products, or extracts of the Disclosure may contain one or more other nucleic acids intended to provide one or more input traits (e.g., insect resistance, herbicide resistance, fungal resistance, viral resistance, stress tolerance, disease resistance, male sterility, stem strength, etc.) and / or output traits (e.g., increased yield, modified starch, improved oil profile, balanced amino acids, high lysine or methionine, improved digestibility, improved fiber quality, drought resistance, etc.). In some embodiments, the transgenic plants of the Disclosure may be bred together with another transgenic plant containing one or more other nucleic acids of interest.

[0184] In some embodiments, one or more other nucleic acids of interest encode one or more second pest control agents, such as Bacillus thuringiensis (Bt) insecticidal proteins and / or non-Bt insecticides, including but not limited to Xenorhabdus species insecticidal proteins, Photorhabdus species insecticidal proteins, Brevibacillus laterosporus insecticidal proteins, Bacillus sphaericus insecticidal proteins, protease inhibitors (both serine and cysteine ​​types), lectins, alpha-amylase, peroxidase, cholesterol oxidase, or double-stranded RNA (dsRNA) molecules. In additional embodiments, the second pest control agent may be one or more of several Bacillus thuringiensis insecticidal proteins, including but not limited to Cry protein, vegetative growth-stage insecticidal protein (VIP), and insecticidal chimeras of any of the aforementioned insecticidal proteins. In some embodiments, the second pest control agent may be non-proteinogenic and may be an interfering RNA molecule, such as dsRNA.

[0185] In some embodiments, the second pest control agent comprises one or more insecticidal proteins or dsRNAs present in any of the following events: Bt11 event (see U.S. Patent No. 6114608), MIR604 event (see U.S. Patent No. 8884102), MIR162 event (see U.S. Patent No. 8232456), 5307 event (see U.S. Patent No. 10428393), MZIR098 event (see U.S. Patent Application No. 20200190533). ), TC1507 event (see U.S. Patent No. US7288643), DAS-59122-7 event (see U.S. Patent No. US7323556), MON810 event (see U.S. Patent No. US6713259), MON863 event (see U.S. Patent No. US7705216), MON89034 event (see U.S. Patent No. US8062840), MON88017 event (see U.S. Patent No. US9556492), DP-4114 event (see U.S. Patent No. US97257 See Specification No. 72), MON87411 event (see U.S. Patent No. US9441240), DP-032218-9 event (see U.S. Patent Application No. US2015361447), DP-033121-3 event (see U.S. Patent Application No. US2015361446), DP-023211-2 event (PCT Publication No. WO2019209700), MON95379 event (U.S. Patent Application No. US2020032289), DBN9936 event (see PCT Publication No. WO2016173361) (see), DBN9501 event (see PCT publication WO20207125), GH5112E-117C event (see PCT publication WO17 / 088480), LP007-1 (see Chinese Patent Application No. CN112852801), LP007-2 (see Chinese Patent Application No. CN112831584), LP007-3 (see Chinese Patent Application No. CN112877454), LP007-4 (see Chinese Patent Application No. CN112831585), LP007-5 (see Chinese Patent Application No. CN113151534),LP007-6 (Chinese Patent Application No. CN113151533), LP007-7 (Chinese Patent Application No. CN112852991), LP007-8 (Chinese Patent Application No. CN113980958), Ruifeng8, ND207, or Ruifeng125 events (see Chinese Patent Application No. CN105017391). In some embodiments, the second pest control agent includes one or more of the following events: Bt11 event (see U.S. Patent No. US6114608), MIR604 event (see U.S. Patent No. US8884102), MIR162 event (see U.S. Patent No. 8232456), 5307 event (see U.S. Patent No. US10428393), MZIR098 event (see U.S. Patent Application No. US202 See U.S. Patent No. 00190533), TC1507 event (see U.S. Patent No. 7288643), DAS-59122-7 event (see U.S. Patent No. 7323556), MON810 event (see U.S. Patent No. 6713259), MON863 event (see U.S. Patent No. 7705216), MON89034 event (see U.S. Patent No. 8062840) i) MON88017 event (see U.S. Patent No. US9556492), DP-4114 event (see U.S. Patent No. US9725772), MON87411 event (see U.S. Patent No. US9441240), DP-032218-9 event (see U.S. Patent Application No. US2015361447), DP-033121-3 event (see U.S. Patent Application No. US2015361446) (Tai), DP-023211-2 case (see PCT Publication WO2019209700), MON95379 case (see US Patent Application No. US2020032289), DBN9936 case (see PCT Publication WO2016173361), DBN9501 case (see PCT Publication WO20207125), GH5112E-117C case (see PCT Publication WO17 / 088480),LP007-1 (see Chinese Patent Application No. CN112852801), LP007-2 (see Chinese Patent Application No. CN112831584), LP007-3 (see Chinese Patent Application No. CN112877454), LP007-4 (see Chinese Patent Application No. CN112831585), LP007-5 (see Chinese Patent Application No. CN113151534), LP007-6 (see Chinese Patent Application No. CN113151533), LP007-7 (see Chinese Patent Application No. CN112852991), LP007-8 (see Chinese Patent Application No. CN113980958), Ruifeng8, ND207, or Ruifeng125 (see Chinese Patent Application No. CN105017391). ,

[0186] In the embodiment, the second pest control agent may be derived from a source other than B. thuringiensis. For example, the second pest control agent may be alpha-amylase, peroxidase, cholesterol oxidase, patatin, protease, protease inhibitor, urease, alpha-amylase inhibitor, pore-forming protein, chitinase, lectin, engineered antibody or antibody fragment, Bacillus cereus Insecticidal proteins from *Cereus*, *Xenorhabdus* species (such as *X. nematophila* or *X. bovienii*), *Photorhabdus* species (such as *P. luminescens* or *P. asymobiotica*), *Brevibacillus* species (such as *B. laterosporus*), *Lysinibacillus* species (such as *L. sphearicus*), and *Chromobacterium*. These may include insecticidal proteins from species of the genus Chromobacterium (such as C. subtsugae or C. piscinae), species of Yersinia (such as Y. entomophaga), species of Paenibacillus (such as P. propylaea), species of Clostridium (such as C. bifermentans), species of Pseudomonas (such as P. fluorescens), and lignin. In other embodiments, the second agent may be at least one insecticidal protein derived from an insecticidal toxin complex (Tc) from the genera Photorhabdus, Xenorhabus, Serratia, or Yersinia.In other embodiments, the insecticidal protein may be an ADP-ribosyltransferase derived from an insecticidal bacterium, such as a species of the genus Photorhabdus. In other embodiments, the insecticidal protein may be a VIP protein such as VIP1 and / or VIP2 from B. cereus. In yet another embodiment, the insecticidal protein may be a binary toxin derived from an insecticidal bacterium, such as ISP1A and ISP2A from B. laterosporus, or BinA and BinB from L. sphaericus. In yet another embodiment, the insecticidal protein may be a modified version of any of the aforementioned insecticidal proteins, or a hybrid or chimera thereof.

[0187] In some embodiments, one or more other nucleic acids of interest encode one or more herbicide resistance agents, for example, PAT (phosphinotricin N-acetyltransferase), AAD-1 (allyloxyalkanoate dioxygenase 1), EPSPS (5-enolpyruvulshikimate-3-phosphate synthase), or an inhibitor of protoporphyrinogen oxidase (PPO, see, for example, U.S. Patent Application No. US2019185873). In some embodiments, the herbicide resistant agent includes one or more of the following events: GA21 (see PCT Publication WO98 / 44140), NK603 (see U.S. Patent No. US6825400), DAS40278 (see PCT Publication WO2011 / 022469), DBN9858 (see PCT Publication WO2016173508), MON87429 (see PCT Publication WO19 / 152316), LW2-2 (see Chinese Patent Application No. CN113278721), and T25 (see USDA / APHIS Petition 94-357-01 for Determination of Nonregulated Status for Glufosinate Resistant Corn Transformation Events T14 and T25, June 1995).

[0188] In some embodiments, one or more other nucleic acids of interest encode one or more enzymes, for example, alpha-amylase. In some embodiments, the enzyme comprises 3272 events (see U.S. Patent No. 7635799).

[0189] In some embodiments, one or more other nucleic acids of interest include one or more of the following: MZDT09Y (see, for example, U.S. Patent No. 9121033), LY038 (see, for example, U.S. Patent No. 7157281), BT176 (Koziel et al. (1993) Biotechnology 11:194-200), and DP202216-6 (see, for example, U.S. Patent Application No. U.S. 2019320607).

[0190] Transgenic plants or seeds containing the nucleic acid molecules of this disclosure may also be treated with insecticides or insecticidal seed coatings, for example, as described in U.S. Patent No. 5,849,320 and No. 5,876,739. In some embodiments, both the insecticides or insecticidal seed coatings of this disclosure and the transgenic plants or seeds are active against the same target insect, for example, lepidopteran pests (e.g., fall armyworm). Accordingly, in some embodiments, a method is provided for enhancing control of a lepidopteran insect population, comprising providing the transgenic plants or seeds of this disclosure and applying the plants or seeds to the insecticide or insecticidal seed coating.

[0191] Even if the insecticide or insecticide seed coating is active against different insects, the insecticide or insecticide seed coating is useful for expanding the scope of insect control by applying an insecticide or insecticide seed coating active against Coleopteran insects to the transgenic seeds of the Disclosure (in some embodiments, which are active against Lepidopteran insects), and the coated transgenic seeds produce control against both Lepidopteran and Coleopteran pests.

[0192] Methods for using nucleic acid molecules and transgenic plants In some embodiments, the Disclosure also provides methods for producing and using nucleic acid molecules of the Disclosure, as well as related compositions such as cells and plants containing nucleic acid molecules, and uses thereof.

[0193] In some embodiments, the methods of the present disclosure provide control of at least one lepidopteran pest, including, but not limited to, one or more of the following: species of the genus Spodoptera, e.g., S. frugiperda (fall armyworm), S. littoralis (Egyptian cottonleaf worm), S. ornithogalli (yellow-striped army worm), S. praefica (Western yellow-striped army worm) ), S. eridania (Southern Army Worm), S. litura (Beet Armyworm / Oriental Leaf Worm), S. cosmioides (Black Army Worm), S. exempta (African Army Worm), S. mauritia (Lone Army Worm), and / or S. exigua (White-spotted Armyworm); species of the genus Ostrinia, for example, O. nubilalis (O. nubilalis). O. alis (European corn borer), and / or O. furnacalis (Asian corn borer); species of the genus Plutella, e.g., P. xylostella; species of the genus Agrotis, e.g., A. ipsilon (black cutworm), A. segetum (common cutworm), A. gladiaria (claybacked cutworm), and / or A. orthogoria (orthogo nia) (Pale Western Cutworm); species of the genus Striacosta, e.g., S. albicosta (Western Bean Cutworm); species of the genus Helicoverpa, e.g., H. zea (American Tobacco Bud / Soybean Pod Worm), H. punctigera (False American Tobacco Bud), and / or H. armigera (Cotton Ball Worm); species of the genus Heliothis, e.g., H. wirense (H.virescens) (tobacco batworm); species of the genus Diatraea, e.g., D. grandiosella (Southwestern corn borer) and / or D. saccharalis (sugarcane borer); species of the genus Trichoplusia, e.g., T. ni (cabbage looper); species of the genus Sesamia, e.g., S. nonagroides (Mediterranean corn borer), Rice armyworm (S. inferens) (pink stem borer), and / or S. calamistis (pink stem borer); species of the genus Pectinophora, e.g., cotton earworm (P. gossypiella) (pink ball worm); species of the genus Cochylis, e.g., C. hospes (banded sunflower moss); species of the genus Manduca, e.g., tobacco hawk moth (M. sexta) (tobacco horn worm), and / or Tomato hawk moth (M. quinquemaculata) (tomato hornworm); species of the genus Elasmopalpus, e.g., E. lignosellus (sorghum moth); species of the genus Pseudoplusia, e.g., P. includens (soybean inchworm); species of the genus Anticarsia, e.g., A. gemmatalis (velvet bean caterpillar); Platipena (Pl Species of the genus *Athypena*, e.g., *P. scabra* (green clover worm); species of the genus *Pieris*, e.g., *P. brassicae* (large white butterfly); species of the genus *Papaipema*, e.g., *P. nebris* (stoke borer); species of the genus *Pseudaletia*, e.g., cutworm (*P. unicpuncta*) (common army worm); species of the genus *Peridroma*, e.g., false caterpillar (*P.saucia) (variegated cutworm); species of the genus Keiferia, e.g., K. lycopersicella (tomato pinworm); species of the genus Artogeia, e.g., A. rapae (imported cabbageworm); species of the genus Phthorimaea, e.g., P. operculella (potato moth); species of the genus Chrysodeixis, e.g., C. includens (soybean looper); species of the genus Feltia, e.g., F. ducens (denzi cutworm); species of the genus Chilo, e.g., C. su C. suppressalis (striped stem borer), C. agamemnon (oriental cone borer), and C. partellus (spotted stalk borer); species of the genus Cnaphalocrocis, e.g., C. medinalis (rice leaf folder); species of the genus Conogethes, e.g., peach leaf borer. Caterpillar moth (C. punctiferalis) (yellow peach moss); species of the genus Mythimna, e.g., armyworm (M. separata) (Oriental army worm); species of the genus Athetis, e.g., black armyworm (A. lepigone) (two-spotted army worm); species of the genus Busseola, e.g., B. fusca (corn stalk borer), Etiella (Eti Species of the genus E. zinckenella (pulsepod borer), for example, species of the genus Leguminivora, for example, species of L. glycinivorella (soybean pod borer); species of the genus Matsumuraeses, for example, species of M. phaseoli (adzuki bean pod borer); species of the genus Omiodes, for example, species of O. indicata (O.(e.g., two, three, or four) of the following species: *Rachiplusia indicata* (soybean leaf folder / bean leaf webworm); *Rachiplusia* species, e.g., *Rachiplusia nu* (sunflower looper); or any combination thereof. In some embodiments, the lepidopteran pest is at least *S. frugiperda* (white fall armyworm); in some embodiments, the lepidopteran pest is at least two (e.g., two, three, or four) of *Spodoptera frugiperda* (white fall armyworm), *Mythimna separata* (sea armyworm), *Spodoptera litura* (beet armyworm / oriental leaf worm), and *Ostrinia furnacalis* (Asian corn borer).

[0194] In some embodiments, the method provides control of fall armyworm pests or colonies resistant to other insecticidal proteins, such as Vip3A protein (e.g., Vip3Aa, including, but not limited to, maize event MIR162), Cry1F protein (e.g., Cry1Fa, including, but not limited to, maize event TC1507 or DP-4114), Cry1A protein (e.g., Cry1A.105, including, but not limited to, maize event MON89034), and / or Cry2 protein (e.g., Cry2Ab, including, but not limited to, maize event MON89034).

[0195] In further embodiments, a method is provided for controlling lepidopteran pests, the method comprising delivering a plant or plant part containing an effective amount of the nucleic acid molecule of the Disclosure to the pest. For effectiveness, the insecticidal protein expressed by the nucleic acid molecule of the Disclosure is ingested orally by the pest. In some embodiments, the insecticidal protein is delivered to the pest in a transgenic plant, where the pest ingests (ingests) one or more parts of the transgenic plant, thereby ingesting the insecticidal protein expressed in the transgenic plant.

[0196] Methods for producing transgenic plants with enhanced insecticidal properties are also included. In a typical embodiment, the method involves introducing the nucleic acid of the Disclosure into a plant, which is then expressed in the plant to produce an insecticidal protein, thereby conferring enhanced insecticidal properties to the plant.

[0197] In some embodiments, a method for introducing the nucleic acid molecules of the Disclosure into a plant comprises first transforming plant cells with the nucleic acid molecules of the Disclosure, and then regenerating a transgenic plant therefrom, the transgenic plant comprising the nucleic acid molecules of the Disclosure. In some embodiments, the method comprises introducing the nucleic acids of the Disclosure into a plant, tissue culture, or plant cell to obtain a transgenic plant, transgenic tissue culture, or transgenic cell having enhanced insecticidal properties, and growing the transgenic plant or regenerating a transgenic plant from the transgenic tissue culture or transgenic plant cell, thereby producing a transgenic plant having enhanced insecticidal properties.

[0198] Alternatively or additionally, the introduction step may include crossing a first plant containing the nucleic acid molecule of the Disclosure with a second plant (e.g., a plant different from the first plant, e.g., a plant that does not contain the nucleic acid molecule of the Disclosure) to optionally produce offspring plants containing the nucleic acid molecule of the Disclosure. Thus, the transgenic plant includes the plant that is the direct result of the transformation event, and its offspring (of any generation) containing the nucleic acid molecule of the Disclosure.

[0199] This disclosure further provides a method for identifying transgenic plants of the Disclosure, the method comprising detecting the presence of nucleic acid molecules of the Disclosure in a plant (or plant cells, plant parts, etc. derived therefrom) and thereby identifying the plant as a transgenic plant of the Disclosure based on the presence of nucleic acid molecules of the Disclosure.

[0200] Some embodiments further provide a method for producing transgenic plants having increased resistance to at least one pest (e.g., at least one lepidopteran pest), the method comprising planting seeds containing nucleic acid molecules or vectors of the Disclosure, and growing transgenic plants from the seeds, the transgenic plants containing nucleic acids of the Disclosure.

[0201] The method for producing transgenic plants described herein optionally includes a further step of harvesting seeds from the transgenic plants, the seeds containing the nucleic acids of the Disclosure. Optionally, the seeds produce further transgenic plants containing the nucleic acid molecules of the Disclosure.

[0202] This disclosure further provides plant parts, plant cells, plant organs, plant cultures, seeds, plant extracts, harvested products and processed products of transgenic plants produced by the methods of this disclosure.

[0203] In a further embodiment, the Disclosure also provides a method for producing seeds, the method comprising providing a transgenic plant containing the nucleic acid molecules of the Disclosure, and harvesting seeds from the transgenic plant, the seeds containing the nucleic acid molecules of the Disclosure. Optionally, the seeds produce further transgenic plants containing the nucleic acid molecules of the Disclosure. In a typical embodiment, the step of providing a transgenic plant includes planting seeds that produce transgenic plants.

[0204] A method for producing hybrid plant seeds is further provided, comprising crossing a first inbred plant, which is a transgenic plant containing the nucleic acid molecules of the Disclosure, with a different inbred plant (e.g., an inbred plant that does not contain the nucleic acid molecules of the Disclosure), and causing the hybrid seeds to form. Optionally, the method further comprises harvesting the hybrid seeds. In some embodiments, the hybrid seeds contain the nucleic acid molecules of the Disclosure. In some embodiments, the hybrid seeds produce a transgenic plant containing the nucleic acid molecules of the Disclosure.

[0205] In some embodiments, the Disclosure provides a method for producing commercial plant products, the method comprising using transgenic plants containing nucleic acid molecules of the Disclosure to produce such commercial plant products from them. Examples of commercial plant products include grains, starches, seed oils, syrups, grain flours, edible flours, starches, cereals, proteins, and the like. Methods for producing such commercial plant products are well known in the art.

[0206] In some embodiments, the Disclosure provides a method for detecting the presence of nucleic acid molecules in a sample, the method comprising: (a) contacting the sample with a pair of primers that produce an amplicon for diagnosing the nucleic acid molecule when used in a nucleic acid amplification reaction with DNA containing a nucleic acid molecule of any of the embodiments described herein or any other embodiment described herein (e.g., one of SEQ ID NOs: 1 or 8-31, or one or more of the variants in Table 3); (b) carrying out the nucleic acid amplification reaction to produce an amplicon; and (c) detecting the amplicon. In some embodiments, the primer pair is a first primer and a second primer, the first primer comprising at least 10 (e.g., at least 10, at least 15, or at least 20) complementary to one of SEQ ID NOs: 1 or 8-31, or one or more of the variants in Table 3; and the second primer comprising at least 10 consecutive nucleotides complementary to the reverse complementary sequence of one of SEQ ID NOs: 1 or 8-31, or one or more of the variants in Table 3. In some embodiments, the first and second primers are 10–50, 10–40, 10–30, or 10–20 nucleotides long. In some embodiments, the sample is a sample obtained from a maize plant part or cell.

[0207] In some embodiments, the Disclosure provides a method for detecting the presence of a nucleic acid molecule in a sample, the method comprising: (a) hybridizing the sample with DNA containing a nucleic acid molecule of any of the embodiments described above or any other embodiment described herein (e.g., one of SEQ ID NOs: 1 or 8-31, or one or more of the variants in Table 3), and contacting it with a probe that does not hybridize with DNA of a control maize plant that does not contain this nucleic acid molecule under high-stringent conditions; (b) subjecting the sample and the probe to high-stringent hybridization conditions; and (c) detecting the hybridization of the probe to the nucleic acid molecule. In some embodiments, the probe contains at least 10 (e.g., at least 10, at least 15, or at least 20) consecutive nucleotides complementary to one of SEQ ID NOs: 1 or 8-31, or one or more of the variants in Table 3, or their reverse complementary sequence. In some embodiments, the probe is 10-50, 10-40, 10-30, or 10-20 nucleotides long. In some embodiments, the sample is a sample obtained from a maize plant part or cell.

[0208] In some embodiments, the Disclosure provides a pair of polynucleotide primers in a sample for producing an amplicon useful for diagnosing the presence of nucleic acid molecules in the sample, comprising a first polynucleotide primer and a second polynucleotide primer that function together in the presence of nucleic acid molecules of any of the embodiments described above or any other embodiments described herein (e.g., including any one of SEQ ID NOs: 1 or 8-31, or any one or more of the variants in Table 3). In some embodiments, the sample is a sample obtained from a maize plant part or cells. In some embodiments, the first polynucleotide primer comprises at least 10 consecutive nucleotides complementary to any one of SEQ ID NOs: 1 or 8-31, or any one or more of the variants in Table 3, and the second polynucleotide primer comprises at least 10 consecutive nucleotides (e.g., at least 10, at least 15, or at least 20) complementary to the reverse complementary sequence of any one of SEQ ID NOs: 1 or 8-31, or any one or more of the variants in Table 3. In some embodiments, the first and second primers are 10-50, 10-40, 10-30, or 10-20 nucleotides long.

[0209] In some embodiments, the Disclosure provides a kit for detecting nucleic acid molecules of any of the embodiments described above or any other embodiments described herein (e.g., including one of SEQ ID NOs: 1 or 8-31, or one or more of the variants in Table 3), the kit comprising at least one nucleic acid molecule of a sequence of nucleotides long enough to function as a primer or probe in a nucleic acid detection method, the nucleic acid molecule being useful in diagnosing the presence of the nucleic acid molecule in amplification of a target nucleic acid sequence in a sample or hybridization to a target nucleic acid sequence, followed by detection of an amplicon or detection of hybridization to a target sequence. In some embodiments, the at least one nucleic acid molecule comprises at least 10 (e.g., at least 10, at least 15, or at least 20) sequenced nucleotides complementary to one of SEQ ID NOs: 1 or 8-31, or one or more of the variants in Table 3. In some embodiments, at least one nucleic acid molecule comprises a pair of primers, the first polynucleotide primer comprising at least 10 (e.g., at least 10, at least 15, or at least 20) consecutive nucleotides complementary to one of SEQ ID NOs. 1 or 8-31, or one or more of the variants in Table 3, and the second polynucleotide primer comprising at least 10 (e.g., at least 10, at least 15, or at least 20) consecutive nucleotides complementary to one of SEQ ID NOs. 1 or 8-31, or one or more of the reverse complementary sequences of the variants in Table 3. In some embodiments, the first and second primers are 10-50, 10-40, 10-30, or 10-20 nucleotides long. In some embodiments, at least one nucleic acid molecule comprises a probe comprising at least 10 consecutive nucleotides complementary to one of SEQ ID NOs. 1 or 8-31, or one or more of the variants in Table 3, or their reverse complementary sequences. In some embodiments, the probe is 10–50, 10–40, 10–30, or 10–20 nucleotides long. The kits of this disclosure optionally also include reagents and / or instructions for carrying out the detections described herein.

[0210] In some embodiments, the present disclosure provides methods for modifying nucleic acid molecules of the present disclosure, for example, in cells or plants. In some embodiments, the modification is a deletion, insertion (e.g., of a heterologous nucleic acid sequence), substitution, duplication, or inversion, or a combination thereof. In some embodiments, the modification includes the deletion of part or all of a selectable marker coding sequence present in the nucleic acid molecule, for example, a PMI or EPSPS coding sequence. In some embodiments, the modification is introduced using a nuclease such as a CRISPR-Cas nuclease, a zinc finger nuclease, a meganuclease, a TAL effector nuclease (TALEN), or a combination thereof.

[0211] In some embodiments, modification is carried out in host cells or plants of the Disclosure, e.g., maize cells or maize plants, to produce modified transgenic cells or modified transgenic plants. In some embodiments, modification is carried out by expressing a nuclease in host cells or plants (e.g., by transforming host cells or plants with an expression cassette encoding the nuclease, or by crossing a plant with another plant containing an expression cassette, etc.). In some embodiments, modification is carried out by directly introducing the nuclease into host cells or plants using reagents that move the nuclease into the host cells or plants, e.g., through physical methods, e.g., bioistics / microparticle guns, protoplast transfection, nanoparticle-mediated delivery, aerosol beam injection, or whisker-mediated delivery. In some embodiments, the method further includes producing plants from modified transgenic host cells to produce modified transgenic plants. In some embodiments, the method further includes self-pollinating the modified transgenic plant or crossing it with another plant for at least one generation (e.g., one, two, three, four or more generations) to produce modified transgenic offspring plants. In some embodiments, the disclosure provides, for example, modified transgenic cells, modified transgenic plants, or modified transgenic offspring plants produced by the method herein.

[0212] In certain embodiments, nucleic acid modifications are affected by a (modified) zinc finger nuclease (ZFN) system. The ZFN system uses an artificial restriction enzyme produced by fusing a zinc finger DNA-binding domain to a DNA-cleaving domain, which can be manipulated to target a desired DNA sequence. Non-limiting examples of methods using ZFNs can be found, for example, in U.S. Patent Nos. 6,534,261; 6,607,882; 6,746,838; 6,794,136; 6,824,978; 6,866,997; 6,933,113; and 6,979,539.

[0213] In certain embodiments, nucleic acid modifications are affected by meganucleases, which are endodeoxyribonucleases characterized by large recognition sites (double-stranded DNA sequences of 12–40 base pairs). Non-limiting examples of methods using meganucleases can be found in U.S. Patents No. 8,163,514; No. 8,133,697; No. 8,021,867; No. 8,119,361; No. 8,119,381; No. 8,124,369; and No. 8,129,134.

[0214] In certain embodiments, nucleic acid modifications are affected by a CRISPR / Cas complex or system. In certain embodiments, the CRISPR / Cas system or complex is a class 2 CRISPR / Cas system. In certain embodiments, the CRISPR / Cas system or complex is a type II, type V, or type VI CRISPR / Cas system or complex. While the CRISPR / Cas system does not require the production of customized proteins to target specific sequences, rather, the Cas nuclease can be programmed by an RNA guide (gRNA) to recognize a specific nucleic acid target; in other words, the Cas nuclease can be recruited to the desired specific nucleic acid target locus using the short RNA guide.

[0215] Generally, as used herein, CRISPR / Cas or the CRISPR system refers collectively to factors involved in the expression of or induction of the activity of CRISPR-related ("Cas") nucleases, including one or more sequences encoding the Cas gene, as well as tracr (trans-activated CRISPR) sequences (e.g., tracrRNA or active partial tracrRNA), tracr-mate sequences ("direct repeats" and, with respect to the endogenous CRISPR system, tracrRNA-processed partial direct repeats), guide sequences (also referred to as "spacers" with respect to the endogenous CRISPR system), or "RNA" as used herein (e.g., RNA for guiding Cas, such as Cas9, e.g., CRISPR RNA, and, where applicable, trans-activated (tracr)RNA, or single guide RNA (sgRNA) (chimeric RNA)), or other sequences derived from the CRISPR locus, and transcripts. Generally, the CRISPR system is characterized by factors that promote the formation of CRISPR complexes at the site of a target sequence (also referred to as protospacers in the case of the endogenous CRISPR system). In relation to CRISPR complex formation, a "target cell" refers to a sequence whose guide sequence is designed to be complementary, and in this case, hybridization between the target sequence and the guide sequence promotes the formation of the CRISPR complex.

[0216] In certain embodiments, the gRNA is a chimeric guide RNA or a single guide RNA (sgRNA). In certain embodiments, the gRNA comprises a guide sequence and a tracr mate sequence (or direct repeat). In certain embodiments, the gRNA comprises a guide sequence, a tracr mate sequence (or direct repeat), and a tracr sequence. In certain embodiments, the CRISPR / Cas system or complex described herein does not contain a tracr sequence and / or does not depend on the presence of a tracr sequence (for example, when the Cas nuclease is Cas12a).

[0217] The CRISPR-Cas nuclease may be any such nuclease known in the art, such as Cas9, Cas12a, Cas12b, Cas12i, Cas13a (formerly also known as C2c2), C2c3, Cas13b, or any modified version of any of the above. CRISPR-Cas nucleases are well-known in the field (see, for example, Dong et al. Efficient Targeted Mutagenesis Mediated by CRISPR-Cas12a Ribonucleoprotein Complexes in Maize. Front. Genome Ed. (2021), vol.3, article 670529; Wei et al. TALEN or Cas9-Rapid, Efficient and Specific Choices for Genome Modifications. J. of Genetics and Genomics (2013), vol.40, pp.281-289; Sedeek et al. Plant Genome Engineering for Targeted Improvement of Crop Traits. Frontiers in Plant Science (2019), vol.10, article 114; and Zhang et al. Applications and potential of genome editing in crop improvement. Genome Biology (2018), vol.19, article 210). [Examples]

[0218] Example 1: Synthesized structure Binary vector constructs were constructed containing transcriptional enhancers, promoters, transit peptides, and terminators, as well as different combinations of variants of these gene elements that drive the expression of eCry1Gb.1Ig variants. These gene elements were synthesized and ligated into binary vectors via restriction enzyme-based cloning. All promoters used were moderate or strong constitutive or viral promoters. Versions of the eCry1Gb.1Ig gene with different codon selections were created to test desired expression levels and efficacy. Table 1 shows the constructed constructs and lists the gene elements along with their respective coding sequences (CDS). Table 2 describes each of the gene elements named in Table 1.

[0219] [Table 1]

[0220] [Table 2] TIFF0007911013000003.tif217167

[0221] Example 2: Agrobacterium-mediated transformation using phosphomannose isomerase (PMI) selection Each of the binary vector constructs was used to create a maize transgenic event. Transformation of maize (Zea mays) to produce genetically modified maize was achieved using immature embryos via Agrobacterium tumefaciens-mediated transformation, as described by Zhong et al. (2018) (Advances in Agrobacterium-mediated Maize Transformation. In: Lagrimini L. (eds) Maize. Methods in Molecular Biology, vol 1676. Humana Press, New York, NY). The A. tumefaciens strain LBA4404 (recA-), containing the disarmed pTi plasmid pAL4404 and the helper plasmid pVGW7, was used for maize transformation. Detailed information on the pAL4404 and pVGW7 plasmids is described by Hoekema et al. (Nature. (1983) 303:179-189), Ishida et al. (Nat Biotechnol (1996) 14:745-750), and Imayama et al. (U.S. Patent No. 10266835). The A. tumefaciens strain LBA4404 (recA-) containing the individual binary vectors was prepared as described by Li et al. (Plant Physiol (2003) 133:736-47). For maize transformation, immature embryos were collected approximately 9 days after pollination from the inbred maize line NP2222 grown in a greenhouse and used as explants (Zhong et al., 2018). Immature embryos were isolated, inoculated with Agrobacterium, and co-cultured with Agrobacterium using the bulk extraction method described in Zhong et al. (2018).By using this method, gene elements within the left and right boundary sequences of the transformed plasmid were efficiently introduced and integrated into the genome of plant cells, while gene elements outside these boundary sequences were not introduced.

[0222] Transformed tissues and putative transgenic events were regenerated and rooted as previously described (Zhong et al., 2018), using mannose-selective media for events containing phosphomannose isomerase (PMI) selectable markers (Negrotto et al., (2000) Plant Cell Rep. 19:789-803), or using 2 mM N-(phosphonomethyl)-glycine (TouchDown®) herbicide as a selective agent for events containing a modified version of the 5-enolpyruvirshikimic acid-3-phosphate synthase (EPSPS) enzyme.

[0223] The regenerated seedlings were tested for the presence of target genes and plant-selectable marker genes (PMI or EPSPS) by real-time TAQMAN® PCR analysis developed by Ingham et al. (Biotechniques 31(1):132-4,136-40,2001). Plants positive for target genes and selectable markers (also called events) were moved to a greenhouse for further propagation. In one plant transformed with binary vector 24795 (SEQ ID NO: 2), the expression cassette (SEQ ID NO: 1) was found to contain a silent mutation in the coding sequence of cPMI-15 (SEQ ID NO: 7), creating a slightly modified expression cassette sequence (SEQ ID NO: 8) in the plant. Further sequencing revealed additional mutations as shown in Table 3 (see also SEQ ID NOs: 9-31). The plants from which sequencing results were obtained did not appear to have any significant adverse effects on efficacy compared to the pool of other plants containing SEQ ID NO: 1.

[0224] [Table 3] TIFF0007911013000005.tif225165

[0225] Example 3: Quantitative ELISA for the detection of plasma proteins Detection of different plasma proteins was performed using two monoclonal antibodies produced against each protein. Samples were collected from leaves exhibiting transgenic events and extracted in phosphate-buffered saline (PBS) (PBST) pH 7.3 containing 0.05% Tween-20. Total soluble protein (TSP) of the extract was measured using the Pierce BCA Protein Assay (Thermo Scientific, Rockford, IL). High-binding polystyrene plates (Nunc Maxisorp #430341) were coated overnight at 4°C with 1 μg / ml of specific monoclonal antibody (MAb) in 25 mM borate, 75 mM NaCl, pH 8.5. The plates were washed five times with PBST. Samples or standards in ELISA diluent (PBST containing 1% bovine serum albumin) were added to the plates (100 μl / well), incubated at room temperature (RT) for 1 hour with shaking, and washed five times. HRP-labeled secondary MAb, diluted 1 / 10,000 in ELISA diluent, was added to the plate (100 μl / well), incubated at ambient temperature for 1 hour with shaking, and washed as described above. The substrate tetramethylbenzidine (SurModics, Eden Prairie, MN) was added (100 μl / well), and color development was allowed at room temperature for 15-30 minutes with shaking. The reaction was stopped using 1N HCl (100 μl / well). Absorbance at 450 nm was measured using a microplate reader (BioTek Powerwave XS2, Winooski, VT). The standard curve was plotted as concentration versus absorbance using a 4-parameter curve fit. To normalize the extraction efficiency, the concentration of each analyte was divided by the total soluble protein (TSP) concentration.

[0226] [Table 4]

[0227] Constructs 24530, 24534, and 25628 produced only events with very low or no plasma protein expression, even when the plasma protein sequence was paired with a promoter expected to be moderately or strongly expressive.

[0228] Example 4: Testing greenhouse effectiveness 279 transgenic maize events from construct 24795 were confirmed to have single-copy tDNA insertion and plasma protein expression via ELISA analysis as described in Example 3. From this population, 45 transgenic maize events from construct 24795, as well as transgenic maize events from other constructs listed in Table 4, were selected for bioassay testing. The selected events corresponded to a wide range of eCry1Gb.1Ig expression, including a mixture of low, moderate, and high expression levels. Bioassay samples consisting of a cut leaf bioassay, in which a portion of a leaf was excised from a plant, were placed in a petri dish with a filter pad moistened with sterile water and parasitized approximately 10 neonatal fall armyworm (Spodoptera frugiperda) larvae. The assays were incubated at ambient laboratory temperature and scored 5 days after parasitism. Each sample was scored for leaf defense percentage (scale 1-5) and insect mortality (scale 1-3). Events achieving a leaf defense percentage of 1 or 2 (i.e., less than 5% damage to the excised leaf disc) and 100% neonatal larval mortality were considered effective and used as a benchmark for construct performance. Bioassay data for the 45 tested events were extrapolated to events with similar trait gene expression, resulting in a total of 24,795 events across 65 constructs that met the effectiveness and expression criteria, which were then further characterized. Events from constructs 23,698, 24,530, 24,534, and 25,628 did not meet the effectiveness and expression criteria, and these constructs were not further developed.

[0229] Example 5: Field efficacy test Twenty-four transgenic maize events from construct 24795 were tested in a field cycle in Argentina. Each event was planted in three replicates in a single row. Leaf surface ratings for fall armyworm (Spodoptera frugiperda) were assessed from eight plants in each row. Leaf surface damage was evaluated using a Davis scale from 0 to 9 (Davis, FM & Williams, WP1992. Visual rating scales for screening whorl-stage corn for resistance to fall armyworm. Mississippi Agricultural & Forestry Experiment Station, Technical Bulletin 186, Mississippi State University, MS39762, USA). Of the 24 events from the above construct, 14 demonstrated acceptable efficacy against fall armyworm. Another aspect of the present invention may be as follows: [1] A nucleic acid molecule comprising a nucleic acid sequence that is at least 99% identical to SEQ ID NO: 1, or its complement, wherein the nucleic acid sequence encodes a polypeptide comprising the sequence of SEQ ID NO: 4. [2] The nucleic acid molecule according to [1], wherein the nucleic acid sequence includes sequence number 3. [3] The nucleic acid molecule according to [1], wherein the nucleic acid sequence includes one of sequence numbers 1 or 8 to 31. [4] The nucleic acid molecule described in [1] above, wherein the nucleic acid molecule is isolated. [5] A recombinant nucleic acid vector comprising the nucleic acid molecule described in any one of the above items [1] to [3]. [6] A transgenic host cell containing the nucleic acid molecule described in any one of the above items [1] to [3]. [7] The transgenic host cell according to [6], wherein the cell is a bacterial cell or a plant cell. [8] The transgenic host cell according to [7], wherein the cell is a bacterial cell, and the bacterial cell is a cell of Escherichia coli, Bacillus thuringiensis, Bacillus subtilis, Bacillus megaterium, Bacillus cereus, Agrobacterium species, or Pseudomonas species. [9] The transgenic host cell according to [7], wherein the cell is a plant cell, and the plant cell is a cell of maize, sorghum, wheat, sunflower, tomato, cruciferous plant, oat, grass, pasture grass, pepper, potato, cotton, rice, soybean, sugarcane, sugar beet, tobacco, barley, or rapeseed.

[10] The transgenic host cell according to [9], wherein the plant cell is a maize cell.

[11] A transgenic plant comprising a nucleic acid molecule as described in any one of the above items [1] to [3].

[12] The transgenic plant described in

[11] , wherein the plant is a monocotyledonous plant.

[13] The transgenic plant described in

[11] , wherein the plant is a dicotyledonous plant.

[14] The transgenic plant described in

[11] , wherein the plant is selected from the group consisting of corn, sorghum, wheat, sunflower, tomato, cruciferous plants, oats, grass, pasture grass, pepper, potato, cotton, rice, soybean, sugarcane, sugar beet, tobacco, barley, or rapeseed.

[15] The entire transgenic maize plant containing the nucleic acid molecule described in [3] above.

[16] Offspring of any generation of the plant described in

[15] , wherein the offspring contains the nucleic acid molecule.

[17] A vegetative propagator of the plant described in

[15] , wherein the vegetative propagator contains the nucleic acid molecule.

[18] A plant part of the plant described in

[15] , wherein the plant part contains the nucleic acid molecule.

[19] The plant part described in

[18] , wherein the plant part is a seed.

[20] A method for producing a transgenic plant having enhanced insecticidal properties, comprising introducing a nucleic acid molecule described in any one of the above items [1] to [3] into a plant to produce a transgenic plant, wherein the nucleic acid molecule expresses a protein in an effective amount for insect control.

[21] A method for producing transgenic plants having enhanced insecticidal properties, a) A step of providing a nucleic acid molecule according to any one of the above items [1] to [3], b) A step of introducing the nucleic acid molecule of step (a) into a plant, tissue culture, or plant cell to obtain a transformed plant, transformed tissue culture, or transformed cell having enhanced insecticidal properties, c) A step of growing the transformed plant or regenerating a transformed plant from the transformed tissue culture or transformed plant cells to produce a transgenic plant having enhanced insecticidal properties; Methods that include...

[22] A method for producing transgenic seeds, a) A step of obtaining a fertile transgenic plant as described in any one of the above items

[11] to

[15] , b) A step of growing the plant under appropriate conditions to produce transgenic seeds, Methods that include...

[23] A method for producing offspring of any generation of a fertile transgenic plant having enhanced insecticidal properties, a) A step of obtaining a fertile transgenic plant having enhanced insecticidal properties containing a nucleic acid molecule as described in any one of the above items [1] to [3], b) A step of collecting transgenic seeds from the transgenic plant, c) The step of planting the collected transgenic seeds, d) A step of growing the offspring transgenic plants from the seeds, Includes, A method wherein the offspring have enhanced insecticidal properties compared to non-transformed plants.

[24] A method for producing transgenic plants having enhanced insecticidal properties, comprising the step of sexually crossing a first parent plant with a second parent plant, wherein the first or second parent plant is a plant described in any one of the above

[11] to

[15] , and producing a first generation of offspring plants containing the nucleic acid molecule.

[25] A method for producing transgenic plants having enhanced insecticidal properties, a) A step of sexually crossing a first parent plant with a second parent plant, wherein the first or second parent plant is a plant described in any one of items

[11] to

[15] above, b) A step of selecting first-generation offspring plants having enhanced insecticidal properties, wherein the selected offspring plants contain the nucleic acid molecule, Methods that include...

[26] a) A step of self-pollinating the first generation of offspring plants to produce multiple second generation of offspring plants, b) A step of selecting a plant having enhanced insecticidal properties from the second generation of offspring plants, wherein the selected second generation of offspring plants contains the nucleic acid molecule, The method according to

[25] , further comprising:

[27] A method for controlling lepidopteran pests, comprising causing the pests to ingest a plant or plant part containing a nucleic acid molecule as described in any one of the above items [1] to [3].

[28] The method according to

[27] , wherein the lepidopteran pest is the fall armyworm (Spodoptera frugiperda).

[29] A method for producing a commercial plant product, wherein the method comprises using a plant described in any one of the above paragraphs

[11] to

[15] and producing the commercial plant product therefrom.

[30] The method according to

[29] , wherein the commodities plant product is grain, starch, seed oil, syrup, grain flour, edible flour, starch, cereal, or protein.

[31] A method for detecting the presence of nucleic acid molecules in a sample, wherein the method is (a) When the sample is used in a nucleic acid amplification reaction with DNA containing a nucleic acid molecule as described in any one of items [1] to [3] above, it is brought into contact with a pair of primers that produce an amplicon useful for diagnosing the nucleic acid molecule, (b) Carrying out a nucleic acid amplification reaction to produce the amplicon, (c) A method comprising detecting the amplicon.

[32] A method for detecting the presence of nucleic acid molecules in a sample, (a) The sample is hybridized with DNA containing the nucleic acid molecule described in any one of the above items [1] to [3] under high stringency conditions, and the sample is brought into contact with a probe that does not hybridize with the DNA of a control maize plant that does not contain the nucleic acid molecule under high stringency conditions. (b) Subjecting the sample and probe to high stringency hybridization conditions, (c) A method comprising detecting the hybridization of the probe to the nucleic acid molecule.

[33] A pair of polynucleotide primers comprising a first polynucleotide primer and a second polynucleotide primer that function together in the presence of a nucleic acid molecule described in any one of the above items [1] to [3] in a sample, and that produce an amplicon useful for diagnosing the presence of the nucleic acid molecule in the sample.

[34] The pair of polynucleotide primers according to

[33] , wherein the first polynucleotide primer comprises at least 10 consecutive nucleotides complementary to any one of SEQ ID NOs: 1 or 8-31, and the second polynucleotide primer comprises at least 10 consecutive nucleotides complementary to the reverse complementary sequence of any one of SEQ ID NOs: 1 or 8-31.

[35] A kit for detecting a nucleic acid molecule as described in any one of the preceding paragraphs [1] to [3], wherein the kit comprises at least one nucleic acid molecule of consecutive nucleotides of sufficient length to function as a primer or probe in a nucleic acid detection method, and is useful in diagnosing the presence of the nucleic acid molecule when amplifying or hybridizing a target nucleic acid sequence in a sample, and subsequently detecting an amplicon or the hybridization to the target sequence.

[36] The kit according to

[35] , wherein the at least one nucleic acid molecule comprises at least 10 consecutive nucleotides complementary to any one of SEQ ID NOs. 1 or 8-31.

[37] A method comprising introducing modifications to nucleic acid molecules present in any one of the transgenic host cells described in [6] to

[10] above, or in any one of the transgenic plants described in

[11] to

[15] above, thereby producing a modified transgenic host cell or a modified transgenic plant.

[38] The method according to

[37] , wherein the modification is a deletion, insertion, substitution, duplication, inversion, or combination thereof.

[39] The method according to

[38] , wherein the modification comprises the deletion of part or all of a selectable marker coding sequence present in the nucleic acid molecule.

[40] The method according to any one of the claims

[37] to

[39] , wherein the modification is introduced using a nuclease, homologous recombination, or a combination thereof.

[41] The method according to

[40] , wherein the nuclease is a CRISPR-Cas nuclease.

[42] The method according to any one of the claims

[37] to

[41] , further comprising producing a plant from the modified transgenic host cell, self-pollinating the plant, or crossing it with another plant to produce a modified transgenic offspring plant.

[43] The method according to any one of the claims

[37] to

[41] , further comprising self-pollinating the modified transgenic plant or crossing it with another plant to produce a modified transgenic offspring plant.

[44] The method according to

[42] or

[43] , further comprising self-pollinating or outcrossing the modified transgenic offspring plants for at least one additional generation.

Claims

1. A nucleic acid molecule comprising a nucleic acid sequence that is at least 99% identical to SEQ ID NO: 1, or a complement thereof, wherein the nucleic acid sequence encodes a polypeptide comprising the sequence of SEQ ID NO:

4.

2. The nucleic acid molecule according to claim 1, wherein the nucleic acid sequence includes sequence number 3.

3. The nucleic acid molecule according to claim 1, wherein the nucleic acid sequence includes one of sequence numbers 1 or 8 to 31.

4. The nucleic acid molecule according to claim 1, wherein the nucleic acid molecule is isolated.

5. A recombinant nucleic acid vector comprising the nucleic acid molecule described in any one of claims 1 to 3.

6. A transgenic host cell comprising the nucleic acid molecule described in any one of claims 1 to 3.

7. The transgenic host cell according to claim 6, wherein the cell is a bacterial cell or a plant cell.

8. The transgenic host cell according to claim 7, wherein the cell is a bacterial cell, and the bacterial cell is a cell of Escherichia coli, Bacillus thuringiensis, Bacillus subtilis, Bacillus megatherium, Bacillus cereus, Agrobacterium species, or Pseudomonas species.

9. The transgenic host cell according to claim 7, wherein the cell is a plant cell, and the plant cell is a cell of corn, sorghum, wheat, sunflower, tomato, cruciferous plant, oat, grass, pasture grass, pepper, potato, cotton, rice, soybean, sugarcane, sugar beet, tobacco, barley, or rapeseed.

10. The transgenic host cell according to claim 9, wherein the plant cell is a maize cell.

11. A transgenic plant comprising the nucleic acid molecule described in claim 1.

12. The transgenic plant according to claim 11, wherein the plant is a monocotyledonous plant.

13. The transgenic plant according to claim 11, wherein the plant is a dicotyledonous plant.

14. The transgenic plant according to claim 11, wherein the plant is selected from the group consisting of corn, sorghum, wheat, sunflower, tomato, cruciferous plants, oats, grass, pasture grass, pepper, potato, cotton, rice, soybean, sugarcane, sugar beet, tobacco, barley, and rapeseed.

15. A whole transgenic maize plant comprising the nucleic acid molecule described in claim 3.

16. A descendant of any generation of the plant according to claim 15, wherein the descendant contains the nucleic acid molecule.

17. A vegetative propagator of a plant according to claim 15, wherein the vegetative propagator contains the nucleic acid molecule.

18. A plant part of a plant according to claim 15, wherein the plant part contains the nucleic acid molecule.

19. The plant part according to claim 18, wherein the plant part is a seed.

20. A method for producing a transgenic plant having enhanced insecticidal properties, comprising introducing a nucleic acid molecule according to any one of claims 1 to 3 into a plant to produce a transgenic plant, wherein the nucleic acid molecule expresses a protein in an effective amount for insect control.

21. A method for producing transgenic plants with enhanced insecticidal properties, a) A step of providing a nucleic acid molecule according to any one of claims 1 to 3, b) A step of introducing the nucleic acid molecule of step (a) into a plant, tissue culture, or plant cell to obtain a transformed plant, transformed tissue culture, or transformed cell having enhanced insecticidal properties, c) A step of growing the transformed plant or regenerating a transformed plant from the transformed tissue culture or transformed plant cells to produce a transgenic plant having enhanced insecticidal properties; Methods that include...

22. A method for producing transgenic seeds, a) A step of obtaining a fertile transgenic plant according to any one of claims 11 to 15, b) A step of growing the plant under appropriate conditions to produce transgenic seeds, Methods that include...

23. A method for producing offspring of any generation of a fertile transgenic plant having enhanced insecticidal properties, a) A step of obtaining a fertile transgenic plant having enhanced insecticidal properties containing a nucleic acid molecule according to any one of claims 1 to 3, b) A step of collecting transgenic seeds from the transgenic plant, c) The step of planting the collected transgenic seeds, d) A step of growing the offspring transgenic plants from the seeds, Includes, A method wherein the offspring have enhanced insecticidal properties compared to non-transformed plants.

24. A method for producing transgenic plants having enhanced insecticidal properties, comprising the step of sexually crossing a first parent plant with a second parent plant, wherein the first or second parent plant is a plant according to any one of claims 11 to 15, and producing a first generation of offspring plants containing the nucleic acid molecule.

25. A method for producing transgenic plants with enhanced insecticidal properties, a) A step of sexually crossbreeding a first parent plant with a second parent plant, wherein the first or second parent plant is a plant described in any one of claims 11 to 15, b) A step of selecting first-generation offspring plants having enhanced insecticidal properties, wherein the selected offspring plants contain the nucleic acid molecule, Methods that include...

26. a) A step of self-pollinating the first generation of offspring plants to produce multiple second generation of offspring plants, b) A step of selecting a plant having enhanced insecticidal properties from the second generation of offspring plants, wherein the selected second generation of offspring plants contains the nucleic acid molecule, The method according to claim 25, further comprising:

27. A method for controlling lepidopteran pests, comprising causing the pests to ingest a plant or plant part containing a nucleic acid molecule as described in any one of claims 1 to 3.

28. The method according to claim 27, wherein the lepidopteran pest is the fall armyworm Spodoptera frugiperda.

29. A method for producing a commercial plant product, the method comprising using a plant described in any one of claims 11 to 15 and producing the commercial plant product therefrom.

30. The method according to claim 29, wherein the commercial plant product is grain, starch, seed oil, syrup, grain flour, edible flour, starch, cereal, or protein.

31. A method for detecting the presence of nucleic acid molecules in a sample, wherein the method is (a) When the sample is used in a nucleic acid amplification reaction with DNA containing the nucleic acid molecule described in any one of claims 1 to 3, it is brought into contact with a pair of primers that produce an amplicon useful for diagnosing the nucleic acid molecule, (b) Performing a nucleic acid amplification reaction to produce the amplicon, (c) A method comprising detecting the amplicon.

32. A method for detecting the presence of nucleic acid molecules in a sample, (a) The sample is hybridized with DNA containing the nucleic acid molecule described in any one of claims 1 to 3 under high stringency conditions, and is brought into contact with a probe that does not hybridize with the DNA of a control maize plant that does not contain the nucleic acid molecule under high stringency conditions, (b) Subjecting the sample and probe to high stringency hybridization conditions, (c) A method comprising detecting the hybridization of the probe to the nucleic acid molecule.

33. A pair of polynucleotide primers comprising a first polynucleotide primer and a second polynucleotide primer that function together in the presence of a nucleic acid molecule described in any one of claims 1 to 3 in a sample to produce an amplicon useful for diagnosing the presence of the nucleic acid molecule in the sample.

34. A pair of polynucleotide primers according to claim 33, wherein the first polynucleotide primer comprises at least 10 consecutive nucleotides complementary to any one of SEQ ID NOs: 1 or 8 to 31, and the second polynucleotide primer comprises at least 10 consecutive nucleotides complementary to the reverse complementary sequence of any one of SEQ ID NOs: 1 or 8 to 31.

35. A kit for detecting a nucleic acid molecule according to any one of claims 1 to 3, wherein the kit comprises at least one nucleic acid molecule of consecutive nucleotides of sufficient length to function as a primer or probe in a nucleic acid detection method, and is useful in diagnosing the presence of the nucleic acid molecule when amplifying or hybridizing a target nucleic acid sequence in a sample, and subsequently detecting an amplicon or the hybridization to the target sequence.

36. The kit according to claim 35, wherein the at least one nucleic acid molecule comprises at least 10 consecutive nucleotides complementary to any one of SEQ ID NOs: 1 or 8 to 31.

37. A method comprising introducing modifications into nucleic acid molecules present in a transgenic host cell according to claim 6, or in a transgenic plant according to any one of claims 11 to 15, thereby producing a modified transgenic host cell or a modified transgenic plant.

38. The method according to claim 37, wherein the modification is a deletion, insertion, substitution, duplication, or inversion, or a combination thereof.

39. The method according to claim 38, wherein the modification includes the deletion of part or all of a selectable marker coding sequence present in the nucleic acid molecule.

40. The method according to claim 37, wherein the modification is introduced using a nuclease, homologous recombination, or a combination thereof.

41. The method according to claim 40, wherein the nuclease is CRISPR-Cas nuclease.

42. The method according to claim 37, further comprising producing a plant from the modified transgenic host cell, self-pollinating the plant, or crossing it with another plant to produce a modified transgenic offspring plant.

43. The method according to claim 37, further comprising self-pollinating the modified transgenic plant or crossbreeding it with another plant to produce modified transgenic offspring plants.

44. The method according to claim 42, further comprising self-pollinating or outcrossing the modified transgenic offspring plants for at least one additional generation.

45. The method according to claim 43, further comprising self-pollinating or outcrossing the modified transgenic offspring plants for at least one additional generation.

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