Novel insect inhibitory proteins

Abstract

To provide a new class of proteins exhibiting insect inhibitory activity against agriculturally relevant pests of crop plants and seeds.SOLUTION: There is provided a recombinant nucleic acid molecule comprising a heterologous promoter operably linked to a polynucleotide segment encoding a PirA, PirB, and PirB fusion insecticidal protein or an insecticidal fragment thereof comprising a specific amino acid sequence, or comprising an amino acid sequence having at least a certain level of identity, or consisting of a nucleic acid molecule hybridizing to a polynucleotide having a specific nucleotide sequence.SELECTED DRAWING: None

Description

[Technical Field]

[0001] REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 62 / 736,236, filed September 25, 2018. No. 60 / 699,999, filed on Dec. 1, 2007, which provisional application is incorporated herein by reference in its entirety.

[0002] Incorporation of sequence listings On September 24, 2019, a " A file named "MONS465wo-sequence_listing.txt" This file is 456 kilobytes (MS-Windows (registered trademark) ) and electronic filing (using the USPTO EFS-Web Filing System) ), which is incorporated by reference in its entirety into this application.

[0003] FIELD OF THE INVENTION The present invention relates generally to the field of insect-inhibiting proteins. A novel class of proteins is disclosed that exhibits insect-inhibitory activity against pest insects. The proteins shown are useful for combating agriculturally relevant pests of crop plants and seeds, particularly Coleoptera and Lepidoptera. The toxin proteins encoding one or more of the disclosed toxin proteins have insecticidal activity against pest species. Plants, plant parts, and seeds containing the recombinant polynucleotide constructs are provided. [Background technology]

[0004] Agriculture, including corn, soybeans, sugarcane, rice, wheat, vegetables, and cotton, among others Improving yields from critically important plants is becoming increasingly important. In addition to the growing need for agricultural products to feed, clothe and provide energy, Climate-related impacts and pressures from a growing population to use land for non-agricultural practices are driving agricultural These factors are expected to reduce the amount of arable land available for plant biodiversity. The grim prognosis for food security in the absence of significant improvements in biotechnology and agricultural practices is dire. In light of these pressures, environmental impacts in technology, agricultural practices, and pest management have been increasing. Sustainable improvements are needed to expand crop production on the limited amount of arable land available for agriculture. It is an essential tool for

[0005] Insects, especially those within the orders Lepidoptera, Coleoptera, and Hemiptera, are considered the main cause of damage to field crops. This reduces crop yields in areas where it is infested. Intensive application of chemical insecticides has been relied upon as pest control agents in agriculture. In addition to these problems, environmental and human health concerns have stimulated research and development of biopesticides. Research efforts have led to the advanced discovery and use of various insect pathogenic microbial species, including bacteria.

[0006] The possibility of insect pathogenic bacteria, especially those belonging to the genus Bacillus, was discovered, and biological When developed as a pest control agent, it changed the paradigm of biological control. Bacillus thuringiensis (Bt) strains are specific to specific insects. Since its discovery as being highly toxic, it has been used as a source of insecticidal protein. Bt strains exhibit intradeltaic cytoplasmic spores localized within parasporular crystal inclusions at the onset of sporulation and during stationary growth. They are known to produce toxins (e.g., Cry proteins) and secrete insecticidal proteins. When ingested by susceptible insects, they are also known to produce delta-endotoxins. Similarly, the secreted toxin exerts its effect on the surface of the midgut epithelium, destroying the cell membrane and causing cell destruction and death. The genes encoding insecticidal proteins are also found in other Bacillus species and in other strains, e.g. For example, Brevibacillus laterosporus, Lysinibaci llus sphaericus (formerly known as Bacillus sphaericus) known as "Ls"), Paenibacillus popilliae, Phot B, including a diversity of additional bacterial species such as B. orhabdus and B. xenorhabdus It has also been identified in bacterial species other than t.

[0007] Crystalline and secreted soluble insecticidal toxins are highly host specific and are an alternative to chemical insecticides. For example, insecticidal toxin proteins are important agricultural products. To protect plants from insect infestation, reduce the need for chemical pesticide applications and increase yields It has been employed in various agricultural applications. Insecticidal toxin proteins are produced by various bacterial strains containing microorganisms. By mechanical methods such as spraying to distribute the biologics onto the plant surface, and by genotyping. Transgenic plants and seeds expressing insecticidal toxin proteins using transformation techniques - Patents.com is used to control agriculturally relevant pests of crop plants by producing

[0008] The use of transgenic plants expressing insecticidal toxin proteins has been adopted worldwide. For example, in 2012, 26.1 million hectares were covered with transgenic plants expressing Bt toxins. Transgenic crops were planted (James, C., Global Status of Commercialized Biotech / GM Crops:2012.IS AAA.Brief No.44). Global Use of Transgenic Insect-Protected Crops and the limited number of insecticidal toxin proteins used in these crops are currently available. Create selective pressure for existing insect alleles that confer resistance to the insecticidal protein. are.

[0009] The development of resistance in target pests to insecticidal toxin proteins is due to the New methods useful for managing the rise of insect resistance to emerging transgenic crops This creates a continuing need for the discovery and development of forms of insecticidal toxin proteins that improve efficacy. New proteins have been developed that demonstrate control against an even broader spectrum of susceptible insect species The toxin will reduce the number of surviving insects that can develop resistance alleles In addition, two or more transgenic plants that are toxic to the same pest but exhibit different modes of action are also known. The use of a single insecticidal toxin protein in a single plant may limit the likelihood of resistance in a single target insect species. Reduces the potential.

[0010] Therefore, the inventors herein provide a method for the prevention and treatment of pests of target lepidopterans, coleopterans, and hemipterans. Similar toxin proteins, mutant proteins, and exemplary combinations that exhibit insecticidal activity against insect species. Derived from Xenorhabdus and Photorhabdus along with recombinant proteins A family of protein toxins is disclosed. Summary of the Invention

[0011] Disclosed herein are compounds that exhibit inhibitory activity against one or more pests of crop plants. and the PirAB (Photorhabdus insect associated) protein, as denoted herein. It is a group of insecticidal proteins (toxic proteins) that have insect-inhibitory activity called toxins. The irAB protein toxin class proteins can be used alone or in combination with the PirA protein. as fusions with irB cell proteins or in combination with other insecticidal proteins and toxic agents. It can be used in formulations and plants in combination with other compounds currently used in agricultural systems. It provides an alternative to insecticidal proteins and pesticide chemicals.

[0012] In one embodiment, the present application discloses a method for treating an insecticidal protein or a fragment thereof. A composition comprising a heterologous promoter operably linked to a polynucleotide segment that encodes a recombinant nucleic acid molecule, wherein (a) the insecticidal protein is selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, , 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 58, 60, 62, 64, 66, 6 8, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94 , 96, 98, 100, 102, 105, 107, 109, 111, 113, 115, 1 17, 119, 121, 123, 125, 127, 129, 131, 133, 135, 1 37, 139, 141, 143, 145, 147, 149, 151, 153, 155, or 157 amino acid sequence; or (b) said insecticidal protein comprises (i) SEQ ID NO: For Nos. 44, 46, 48, 123, 127, 129, 131, 133, and 145, at least 65% identity; or (ii) to SEQ ID NOs: 109, 121, and 125 or (iii) at least 70% identity to SEQ ID NOs: 12, 18, 24, 36, 4 2, 62, 68, 74, 80, 86, 98, 113, 117, 119, 147, 149, 153, 155, and 157; or (iv) SEQ ID NO: At least 82% identity to sequences 30, 92, 111, 115, and 151; or (v) at least 86% identity to SEQ ID NOs: 6 and 50; or (iv) the sequence (vii) at least 94% identity to SEQ ID NOs: 137 and 141; or 4, 26, and 32; or (viii) SEQ ID NO: At least 98% identity to 2, 28, 34, 102, and 102; or x) at least 99% identity to SEQ ID NO: 135; or (x) SEQ ID NO: 8, 10, 14, 16, 20, 22, 38, 40, 58, 60, 64, 66, 70, 72, 76, 7 8, 82, 84, 88, 90, 94, 96, 100, 105, 107, 139, and 14 3, or (c) the polynucleotide comprises an amino acid sequence having 100% identity to The dot segments are SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 4 9, 51, 52, 53, 54, 55, 56, 57, 59, 61, 63, 65, 67, 69 , 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 104, 106, 108, 110, 112, 114, 11 6, 118, 120, 122, 124, 126, 128, 130, 132, 134, 13 6, 138, 140, 142, 144, 146, 148, 150, 152, 154, 15 hybridizes to a polynucleotide having a 6, or 158 nucleotide sequence; or (d) the recombinant nucleic acid molecule is operably linked to a vector, the vector being a plasmid. Consists of smids, phagemids, bacmids, cosmids, and bacterial or yeast artificial chromosomes. The recombinant nucleic acid molecule is selected from the group consisting of: or a plant cell that is expressed in an insecticidal effective amount of an insecticidal sequence. Produces protein.

[0013] In another embodiment of the present application is a host cell comprising a recombinant nucleic acid molecule of the present application, wherein: The host cell is selected from the group consisting of a bacterial cell and a plant cell. Contemplated host cells include: Agrobacterium, Rhizobium, Bacillus, Breviba cillus, Escherichia, Pseudomonas, Klebsiell a, Pantoea, and Erwinia. In certain embodiments, the Ba The cillus species are Bacillus cereus or Bacillus thur ingiensis, and the Brevibacillus is Brevibacillus s laterosperus, or said Escherichia is Escherichia Contemplated plant host cells include dicotyledonous and monocotyledonous plant cells. Further contemplated plant host cells include alfalfa, banana, and other plant cells. , barley, beans, broccoli, cabbage, brassica, carrots, cassava, rice Sesame, cauliflower, celery, chickpeas, Chinese cabbage, citrus fruits, coconut, coffee, tomato Sorghum, clover, cotton (Gossypium sp.), gourd, cucumber, douglas fir Fir, eggplant, eucalyptus, flax, garlic, grapes, hops, green onions, lettuce, loblolly Pine, millet, melon, nuts, oats, olives, onions, ornamental plants, palms, Grass, peas, peanuts, pepper, pigeon peas, pine, potato, poplar , pumpkin, radiata pine, radish, rapeseed, rice, rhizome, Wheat, safflower, shrubs, sorghum, southern pine, soybeans, spinach, pumpkins (squash), strawberries, sugar beets, sugarcane, sunflowers, sweet corn, sweet Togum, sweet potato, switchgrass, tea, tobacco, tomato, triticale, turfgrass, Examples include squid and wheat plant cells.

[0014] In yet another embodiment, the insecticidal protein is directed against western corn rootworm, western corn borer, Sorghum rootworm, Northern corn rootworm, Mexican corn rootworm, Brachypodium Gill-type corn rootworm, Colorado potato beetle, Diabrotica viriduli Brazilian maize root cuttings consisting of a and Diabrotica speciosa Insect complex, Coleoptera including the Brassicaceae flea beetle, the striped flea beetle, and the Western black flea beetle Active against insects.

[0015] In another embodiment, the insecticidal protein is effective against black armyworm, tobacco budworm, and diamondback moth. , European corn borer, brown armyworm, southern armyworm, soybean looper, Southwestern American Corn borer, Tobacco Budworm, Velvet Bean Canker Tapir, sugarcane borer, corn moth, black armyworm, beetle Armyworm, Helicoverpa armigera (Old World Bollworm), Spodoptera litura, It is active against lepidopteran insects including the pink bollworm.

[0016] In yet another embodiment, the insecticidal protein is effective against the southern green stink bug, the subtropical brown marsh moth, Stink bugs, southern green stink bugs, subtropical brown marmorated stink bugs, red spotted stink bugs, black thorns Green stink bugs, German stink bugs, brown marmorated stink bugs, green stink bugs, brown marmorated stink bugs Active against Hemiptera insect species including the bug, the Western rusty mites, and the rusty mites. Indicates gender.

[0017] Also contemplated in this application are polypeptides encoding insecticidal proteins or fragments thereof. A recombinant nucleic acid molecule comprising a heterologous promoter operably linked to a oligonucleotide segment. a plant containing an insecticidal protein selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 39, 39, , 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 58, 60, 62, 64, 66, 6 8, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94 , 96, 98, 100, 102, 105, 107, 109, 111, 113, 115, 1 17, 119, 121, 123, 125, 127, 129, 131, 133, 135, 1 37, 139, 141, 143, 145, 147, 149, 151, 153, 155, or 157 amino acid sequence; or (b) said insecticidal protein comprises (i) SEQ ID NO: For Nos. 44, 46, 48, 123, 127, 129, 131, 133, and 145, at least 65% identity; or (ii) to SEQ ID NOs: 109, 121, and 125 or (iii) at least 70% identity to SEQ ID NOs: 12, 18, 24, 36, 4 2, 62, 68, 74, 80, 86, 98, 113, 117, 119, 147, 149, 153, 155, and 157; or (iv) sequences At least 82% identity to numbers 30, 92, 111, 115, and 151; if or (v) at least 86% identity to SEQ ID NOs: 6 and 50; or (vi) a sequence (vii) at least 94% identity to SEQ ID NOs. 137 and 141; or (viii) at least 97% identity to SEQ ID NOs. 4, 26, and 32; or At least 98% identity to sequences 2, 28, 34, 102, and 102; or ix) at least 99% identity to SEQ ID NO: 135; or (x) SEQ ID NOs: 8, 10, 14, 16, 20, 22, 38, 40, 58, 60, 64, 66, 70, 72, 76, 7 8, 82, 84, 88, 90, 94, 96, 100, 105, 107, 139, and 14 or (c) the polynucleotide comprises an amino acid sequence having 100% identity to 3. The hybridization segments of SEQ ID NOs: 49, 51, 5 2, 53, 54, 55, 56, 146, 148, 150, 152, 154, 156 or hybridizes to the complement of the nucleotide sequence of 158; or (d) said plant In certain embodiments, the insecticidal protein is present in a detectable amount. The quality is SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 2 6, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 58 , 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 105, 107, 109 , 111, 113, 115, 117, 119, 121, 123, 125, 127, 129 , 131, 133, 135, 137, 139, 141, 143, 145, 147, 149 , 151, 153, 155, or 157. In one embodiment, the plant is a monocot or In another embodiment, the plant is either alfalfa, banana, or dicotyledonous. , barley, beans, broccoli, cabbage, brassica, carrots, cassava, rice Sesame, cauliflower, celery, chickpeas, Chinese cabbage, citrus fruits, coconut, coffee, tomato Sorghum, clover, cotton, gourd, cucumber, Douglas fir, eggplant, eucalyptus, flax, Garlic, grapes, hops, green onions, lettuce, loblolly pine, millet, melon, nuts, Oats, olives, onions, ornamentals, palms, grasses, peas, peanuts , pepper, pigeon pea, pine, potato, poplar, pumpkin ), radiata pine, radish, rapeseed, rice, rhizomes, rye, safflower, shrubs, Sorghum, Southern pine, soybeans, spinach, squash, strawberries, Sugar beet, sugarcane, sunflower, sweet corn, sweet gum, sweet potato, From the group consisting of: switchgrass, tea, tobacco, tomato, triticale, turfgrass, watermelon, and wheat be selected.

[0018] In a further embodiment, a seed comprising the recombinant nucleic acid molecule is disclosed.

[0019] In another embodiment, an insect inhibiting composition comprising the recombinant nucleic acid molecules disclosed in the present application is contemplated. The insect inhibiting composition further comprises at least one protein different from the insecticidal protein. It may contain a nucleotide sequence encoding another pesticide. The insecticide is a group consisting of an insect-inhibiting protein, an insect-inhibiting dsRNA molecule, and an accessory protein. At least one other insecticide in the insect inhibiting composition is selected from Lepidoptera, Coleoptera Insect-inhibiting compositions are active against one or more pest species of the order Hemiptera, or Pestidae. In one embodiment, the other insecticide is Cry1A, Cry1Ab, Cry1Ac, Cry1A.105, Cry1Ae, Cry1B, Cry1C, Cry1C mutant, Cr y1D, Cry1E, Cry1F, Cry1A / F chimera, Cry1G, Cry1H, C ry1I, Cry1J, Cry1K, Cry1L, Cry2A, Cry2Ab, Cry2 Ae, Cry3, Cry3A mutant, Cry3B, Cry4B, Cry6, Cry7, C ry8, Cry9, Cry15, Cry34, Cry35, Cry43A, Cry43B , Cry51Aa1, ET29, ET33, ET34, ET35, ET66, ET70, TIC400, TIC407, TIC417, TIC431, TIC800, TIC80 7, TIC834, TIC853, TIC900, TIC901, TIC1201, TI C1415, TIC2160, TIC3131, TIC836, TIC860, TIC8 67, TIC869, TIC1100, VIP3A, VIP3B, VIP3Ab, AXM I-AXMI-, AXMI-88, AXMI-97, AXMI-102, AXMI-11 2, AXMI-117, AXMI-100, AXMI-115, AXMI-113, and AXMI-005, AXMI134, AXMI-150, AXMI-171, AXMI- 184, AXMI-196, AXMI-204, AXMI-207, AXMI-209, AXMI-205, AXMI-218, AXMI-220, AXMI-221z, AXM I-222z, AXMI-223z, AXMI-224z and AXMI-225z, AX MI-238, AXMI-270, AXMI-279, AXMI-345, AXMI-3 35, AXMI-R1 and its mutants, IP3 and its mutants, DIG-3, DIG-5 , DIG-10, DIG-657 DIG-11, Cry71Aa1, Cry72Aa1 , PHI-4 mutant type, PIP-72 mutant type, PIP-45 mutant type, PIP-64 mutant type, PIP-74 mutant, PIP-75 mutant, PIP-77 mutant, Axmi422, Di g-305, Axmi440, PIP-47 mutant, Axmi281, BT-009, B T-0012, BT-0013, BT-0023, BT0067, BT-0044, BT -0051, BT-0068, BT-0128, DIG-17, DIG-90, DIG- 79, Cry1JP578V, Cry1JPS1, and Cry1 JPS1P578V is selected from the group consisting of:

[0020] Commercial products containing detectable amounts of the recombinant nucleic acid molecules disclosed in this application are contemplated. Such commodity products include commodity corn, corn bagged by grain handlers, Corn flakes, corn cake, corn flour, corn meal, corn syrup, corn Production of oil, corn silage, corn starch, corn cereals, etc., and corresponding cotton products products, e.g., whole cottonseed or processed for feed or food, fiber, paper, biomass Processed cottonseed, cotton oil, lint, seeds and plant parts, and fuel or cotton gin derived from cotton oil Fuel products such as waste-derived pellets and corresponding soybean commodity products, e.g., whole grain Soybean seeds or processed soybean seeds, soybean oil, soybean protein, soybean meal, Soy flour, soy flakes, soy bran, soy milk, soy cheese, soy wine, soy animal feed containing soybeans, paper containing soybeans, cream containing soybeans, soybean biomass, and soybean Fuel products produced using parts of rice and soybean plants, and the corresponding rice, wheat, Detection of sorghum, pigeon peas, peanuts, fruits, melons, and, where applicable, the present application Such products containing possible amounts of such polynucleotides and / or polypeptides Includes juices, concentrates, jams, jellies, marmalades, and other edible forms of the product Examples include plant-based commodity products.

[0021] Also contemplated in this application are species containing the recombinant nucleic acid molecules disclosed in this application. The method comprises the step of growing a seed containing a recombinant nucleic acid molecule as disclosed in the present application, and then culturing the seed to produce offspring. Plant at least one of the following: growing the plant from the seed; and harvesting the seed from the plant. wherein the harvested seeds comprise a recombinant nucleic acid molecule of the present application.

[0022] In another illustrative embodiment, a plant is provided that is resistant to insect infestation, wherein said The plant cells contain (a) SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20; 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 4 8, 50, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80 , 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, or 157. or (b) a recombinant nucleic acid molecule encoding an effective amount of an insecticidal protein; The proteins are (i) SEQ ID NOs: 44, 46, 48, 123, 127, 129, 131, 13 3, and at least 65% identity to 145; or (ii) SEQ ID NO: 109, 121, and 125; or (iii) SEQ ID NO: 1 2, 18, 24, 36, 42, 62, 68, 74, 80, 86, 98, 113, 117, At least 80% identity to 119, 147, 149, 153, 155 and 157 or (iv) at least to SEQ ID NOs: 30, 92, 111, 115, and 151; or (v) at least 86% identical to SEQ ID NOs: 6 and 50; or (vi) at least 94% identity to SEQ ID NOs: 137 and 141; or (vii) at least 97% identity to SEQ ID NOs: 4, 26, and 32; if or (viii) at least 98% of SEQ ID NOs: 2, 28, 34, 102, and 102 % identity; or (ix) at least 99% identity to SEQ ID NO: 135; if <(x) SEQ ID NOs: 8, 10, 14, 16, 20, 22, 38, 40, 58, 60, 64 , 66, 70, 72, 76, 78, 82, 84, 88, 90, 94, 96, 100, 10 5, 107, 139, and 143 have 100% amino acid sequence identity.

[0023] Also disclosed in this application are methods for controlling pests of the Coleoptera or Lepidoptera species. and a method for controlling the infestation of pests of the order Coleoptera or Lepidoptera on plants, especially crop plants. In one embodiment, the method comprises: (a) a sequence selected from SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16 , 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 58, 60, 62, 64, 66, 68, 70, 72, 74, 7 6, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 1 02, 105, 107, 109, 111, 113, 115, 117, 119, 121, 1 23, 125, 127, 129, 131, 133, 135, 137, 139, 141, 1 43, 145, 147, 149, 151, 153, 155, or 157 contacting the pest with an insecticidally effective amount of one or more insecticidal proteins; or b) the insecticidal protein is selected from the group consisting of (i) SEQ ID NOs: 44, 46, 48, 123, 127, 12 at least 65% identity to 9, 131, 133, and 145; or (ii) At least 70% identity to SEQ ID NOs: 109, 121, and 125; or ii) SEQ ID NOs: 12, 18, 24, 36, 42, 62, 68, 74, 80, 86, 98; At least 80% of 113, 117, 119, 147, 149, 153, and 155 identity and 157; or (iv) SEQ ID NOs: 30, 92, 111, 115, and 151 or (v) at least 82% identity to SEQ ID NOs: 6 and 50; or or (vi) at least 9% identity to SEQ ID NOs: 137 and 141; 4% identity; or (vii) at least 97% identity to SEQ ID NOs: 4, 26, and 32. % identity to SEQ ID NOs: 2, 28, 34, 102, and 102; or (viii) to SEQ ID NOs: 2, 28, 34, 102, and 102 or (ix) at least 99% identity to SEQ ID NO: 135. or (x) SEQ ID NOs: 8, 10, 14, 16, 20, 22, 38, 40, 58, 60, 64, 66, 70, 72, 76, 78, 82, 84, 88, 90, 94, 96, 1 Amino acid sequences with 100% identity to 00, 105, 107, 139, and 143 Includes.

[0024] Further provided herein are polypeptides encoding insecticidal proteins or fragments thereof. A method for detecting the presence of a recombinant nucleic acid molecule comprising a nucleotide segment, a) the insecticidal protein is selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18 , 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 7 8, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, containing the amino acid sequence of 145, 147, 149, 151, 153, 155, or 157 or (b) the insecticidal protein is selected from the group consisting of (i) SEQ ID NOs: 44, 46, 48, 123, 1 At least 65% identity to 27, 129, 131, 133, and 145; or (ii) at least 70% identity to SEQ ID NOs: 109, 121, and 125; or (iii) SEQ ID NOs: 12, 18, 24, 36, 42, 62, 68, 74, 80, 8 6, 98, 113, 117, 119, 147, 149, 153, 155 and 157 or (iv) SEQ ID NOs: 30, 92, 111, 115, and 151; or (v) at least 82% identity to SEQ ID NOs: 6 and 50; or (vi) at least 86% identity to SEQ ID NOs: 137 and 141; at least 94% identity; or (vii) at least 94% identity to SEQ ID NOs: 4, 26, and 32. or (viii) SEQ ID NOs: 2, 28, 34, 102, and 1 or (ix) at least 98% identical to SEQ ID NO: 135; 99% identity; or (x) SEQ ID NOs: 8, 10, 14, 16, 20, 22, 38, 4 0, 58, 60, 64, 66, 70, 72, 76, 78, 82, 84, 88, 90, 94 , 96, 100, 105, 107, 139, and 143, which have 100% identity with or (c) the polynucleotide segment comprises the amino acid sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 3 3, 35, 37, 39, 41, 43, 45, 47, 49, 51, 52, 53, 54, 55 , 56, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 10 4, 106, 108, 110, 112, 114, 116, 118, 120, 122, 12 4, 126, 128, 130, 132, 134, 136, 138, 140, 142, 14 4, 146, 148, 150, 152, 154, 156, or 158 nucleotides In one embodiment of the present invention, the method comprises: The pesticidal proteins provided herein are hybridized under stringent hybridization conditions. A genome D from a plant containing a polynucleotide segment encoding a protein or a fragment thereof NA, but under such hybridization conditions, Nucleic acid probes that do not hybridize to genomic DNA from other isogenic plants wherein the probe is selected from the group consisting of SEQ ID NOs: 49, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77 , 53, 54, 55, 56, 146, 148, 150, 152, 154, 156, or 158, or (i) SEQ ID NOs: 44, 46, 48, At least 65% identical to 123, 127, 129, 131, 133, and 145 or (ii) at least 70% similar to SEQ ID NOs: 109, 121, and 125 identity; or (iii) SEQ ID NOs: 12, 18, 24, 36, 42, 62, 68, 74 , 80, 86, 98, 113, 117, 119, 147, 149, 153, 155 and 1 57; or (iv) SEQ ID NOs: 30, 92, 111 (v) at least 82% identity to SEQ ID NOs: 6 and 151; and 50; or (vi) SEQ ID NOs: 137 and 141 or (vii) at least 94% identity to SEQ ID NOs: 4, 26, and 32; or (viii) at least 97% identity to SEQ ID NOs: 2, 28, 34, 10 2, and 102; or (ix) SEQ ID NO: 135 at least 99% identity; or (x) SEQ ID NOs: 8, 10, 14, 16, 20, 22 , 38, 40, 58, 60, 64, 66, 70, 72, 76, 78, 82, 84, 88, 100% identity with 90, 94, 96, 100, 105, 107, 139, and 143 or a sequence encoding an insecticidal protein comprising an amino acid sequence having The method further comprises: (a) subjecting the sample and the probe to stringent hybridization; (b) subjecting the probe to hybridization conditions; and (b) detecting hybridization between the probe and the sample DNA. The method may include:

[0025] Also provided by the present invention is a method for detecting insecticidal proteins or is a method for detecting the presence of a fragment thereof, wherein the insecticidal protein is SEQ ID NO:2 , 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 58, 60, 62, 6 4, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90 , 92, 94, 96, 98, 100, 102, 105, 107, 109, 111, 113 , 115, 117, 119, 121, 123, 125, 127, 129, 131, 133 , 135, 137, 139, 141, 143, 145, 147, 149, 151, 153 , 155, or 157 amino acid sequences; or the insecticidal protein comprises (i ) SEQ ID NOs: 44, 46, 48, 123, 127, 129, 131, 133, and 145 or (ii) SEQ ID NOs: 109, 121, and 12 or (iii) SEQ ID NOs: 12, 18, 24, 36, 42, 62, 68, 74, 80, 86, 98, 113, 117, 119, 147, At least 80% identity to 149, 153, 155, and 157; or (iv ) at least 82% identity to SEQ ID NOs: 30, 92, 111, 115, and 151 or (v) at least 86% identity to SEQ ID NOs: 6 and 50; or (v i) at least 94% identity to SEQ ID NOs: 137 and 141; or (vii) at least 97% identity to SEQ ID NOs: 4, 26, and 32; or (viii) At least 98% identity to SEQ ID NOs: 2, 28, 34, 102, and 102; if or (ix) at least 99% identity to SEQ ID NO: 135; or (x) SEQ ID NO: 8, 10, 14, 16, 20, 22, 38, 40, 58, 60, 64, 66, 70, 72, 7 6, 78, 82, 84, 88, 90, 94, 96, 100, 105, 107, 139, and and 143. In one embodiment, the method The method involves (a) contacting a sample with an immunoreactive antibody and (b) combining the antibody with an insecticidal protein or and detecting binding to the antibody or a fragment thereof, wherein binding indicates the presence of the protein. In some embodiments, the detecting step comprises ELISA or Western blot. .

[0026] A brief description of arrays SEQ ID NO: 1 is the sequence of Xenopus erythematosus encoding the TIC4771 PirA insecticidal protein. Nucleic acid sequence obtained from rhabdus nematophila strain ISB000002 is.

[0027] SEQ ID NO: 2 is the amino acid sequence of the TIC4771 PirA protein.

[0028] SEQ ID NO: 3 is the sequence of Xenopus erythematosus encoding the TIC4772 PirB insecticidal protein. Nucleic acid sequence obtained from rhabdus nematophila strain ISB000002 is.

[0029] SEQ ID NO: 4 is the amino acid sequence of the TIC4472 PirB protein.

[0030] SEQ ID NO: 5 is the sequence of TIC4771 and TIC4772 operably linked in frame. A nuclear gene encoding a PirAB fusion protein, TIC6880, consisting of the coding sequence of It is an acid sequence.

[0031] SEQ ID NO: 6 is the amino acid sequence of the TIC6880 PirAB fusion protein.

[0032] SEQ ID NO: 7 is the sequence of the Xenopus genus PirA encoding the TIC7575 PirA insecticidal protein. This is the nucleic acid sequence obtained from strain 85823 of Rhabdus ehlersii.

[0033] SEQ ID NO: 8 is the amino acid sequence of the TIC7575 PirA protein.

[0034] SEQ ID NO: 9 is the sequence of Xenopus erythematosus encoding the TIC7576 PirB insecticidal protein. This is the nucleic acid sequence obtained from strain 85823 of Rhabdus ehlersii.

[0035] SEQ ID NO: 10 is the amino acid sequence of the TIC7576 PirB protein.

[0036] SEQ ID NO: 11 is the sequence of TIC7575 and TIC757 operably linked in frame. Encodes TIC9316, a PirAB fusion protein composed of the coding sequence of 6 It is a nucleic acid sequence.

[0037] SEQ ID NO: 12 is the amino acid sequence of the TIC9316 PirAB fusion protein.

[0038] SEQ ID NO: 13 is the Xen encoding sequence of the TIC7660 PirA insecticidal protein. orhabdus cabanillasii strain 85908. do.

[0039] SEQ ID NO: 14 is the amino acid sequence of the TIC7660 PirA protein.

[0040] SEQ ID NO: 15 is the Xen encoding sequence of the TIC7661 PirB insecticidal protein. orhabdus cabanillasii strain 85908. do.

[0041] SEQ ID NO: 16 is the amino acid sequence of the TIC7661 PirB protein.

[0042] SEQ ID NO: 17 is the sequence of TIC7660 and TIC766 operably linked in frame. Encoding a PirAB fusion protein, TIC9317, consisting of the coding sequence of 1 It is a nucleic acid sequence.

[0043] SEQ ID NO: 18 is the amino acid sequence of the TIC9317 PirAB fusion protein.

[0044] SEQ ID NO: 19 is the Xen encoding sequence of the TIC7662 PirA insecticidal protein. orhabdus ehlersii strain 85887.

[0045] SEQ ID NO: 20 is the amino acid sequence of the TIC7662 PirA protein.

[0046] SEQ ID NO: 21 is the Xen encoding sequence of the TIC7663 PirB insecticidal protein. orhabdus ehlersii strain 85887.

[0047] SEQ ID NO: 22 is the amino acid sequence of the TIC7663 PirB protein.

[0048] SEQ ID NO: 23 is the sequence of TIC7662 and TIC766 operably linked in frame. Encodes a PirAB fusion protein, TIC9318, composed of the coding sequence of 3 It is a nucleic acid sequence.

[0049] SEQ ID NO: 24 is the amino acid sequence of the TIC9318 PirAB fusion protein.

[0050] SEQ ID NO: 25 is the Xen encoding sequence of the TIC7664 PirA insecticidal protein orhabdus poinarii strain 86198.

[0051] SEQ ID NO: 26 is the amino acid sequence of the TIC7664 PirA protein.

[0052] SEQ ID NO: 27 is the Xen encoding sequence of the TIC7665 PirB insecticidal protein orhabdus poinarii strain 86198.

[0053] SEQ ID NO: 28 is the amino acid sequence of the TIC7665 PirB protein.

[0054] SEQ ID NO: 29 is the sequence of TIC7664 and TIC766 operably linked in frame. Encoding a PirAB fusion protein, TIC9319, composed of the coding sequence of 5 It is a nucleic acid sequence.

[0055] SEQ ID NO: 30 is the amino acid sequence of the TIC9319 PirAB fusion protein.

[0056] SEQ ID NO: 31 is the Pho encoding sequence of the TIC7666 PirA insecticidal protein. The nucleic acid sequence obtained from strain 86197 of Torrhabdus luminescens do.

[0057] SEQ ID NO: 32 is the amino acid sequence of the TIC7666 PirA protein.

[0058] SEQ ID NO: 33 is a Phot encoding the sequence of the TIC7667 insecticidal PirB protein. orhabdus luminescens strain 86197. .

[0059] SEQ ID NO: 34 is the amino acid sequence of the TIC7667 PirB protein.

[0060] SEQ ID NO: 35 is a sequence encoding TIC7666 and TIC7666 operably linked in frame. Encodes TIC9322, a PirAB fusion protein composed of the coding sequence of 7 It is a nucleic acid sequence.

[0061] SEQ ID NO: 36 is the amino acid sequence of the TIC9322 PirAB fusion protein.

[0062] SEQ ID NO: 37 is the Pho encoding sequence of the TIC7668 PirA insecticidal protein. The nucleic acid sequence obtained from strain 86194 of Torrhabdus luminescens do.

[0063] SEQ ID NO: 38 is the amino acid sequence of the TIC7668 PirA protein.

[0064] SEQ ID NO: 39 is the Pho encoding sequence of the TIC7669 PirB insecticidal protein. The nucleic acid sequence obtained from strain 86194 of Torrhabdus luminescens do.

[0065] SEQ ID NO: 40 is the amino acid sequence of the TIC7669 PirB protein.

[0066] SEQ ID NO: 41 is an inflatable fragment of TIC7668 and TIC7669 operably linked together. A nuclear gene encoding a PirAB fusion protein, TIC9320, consisting of the coding sequence of It is an acid sequence.

[0067] SEQ ID NO: 42 is the amino acid sequence of the TIC9320 PirAB fusion protein.

[0068] SEQ ID NO: 43 is a microRNA encoding the sequence of the TIC7939 insecticidal PirA protein. Nucleic acid sequences obtained from unknown bacterial strains contained within the biome.

[0069] SEQ ID NO: 44 is the amino acid sequence of the TIC7939 PirA protein.

[0070] SEQ ID NO: 45 is the sequence encoding the TIC7940 PirB insecticidal protein. These are nucleic acid sequences obtained from unknown bacterial strains contained within the lobiome.

[0071] SEQ ID NO: 46 is the amino acid sequence of the TIC7940 PirB protein.

[0072] SEQ ID NO: 47 is the sequence of TIC7939 and TIC794 operably linked in frame. Encodes a PirAB fusion protein, TIC9321, consisting of the coding sequence of . It is a nucleic acid sequence.

[0073] SEQ ID NO: 48 is the amino acid sequence of the TIC9321 PirAB fusion protein.

[0074] SEQ ID NO: 49 is a plant cell clone encoding the TIC6880PL PirAB fusion protein A synthetic coding sequence encoding the TIC4771 protein for use in expression in An additional alanine codon has been inserted immediately after the initiating methionine codon of one fragment.

[0075] SEQ ID NO: 50 is the amino acid sequence of the TIC6880PL PirAB fusion protein .

[0076] SEQ ID NO: 51 is a sequence encoding the TIC9316 PirAB fusion protein in plant cells. It is a synthetic coding sequence used for expression.

[0077] SEQ ID NO: 52 is a sequence encoding the TIC9317 PirAB fusion protein in plant cells. It is a synthetic coding sequence used for expression.

[0078] SEQ ID NO: 53 is a gene encoding the TIC9318 PirAB fusion protein in plant cells. It is a synthetic coding sequence used for expression.

[0079] SEQ ID NO: 54 is a gene encoding the TIC9319 PirAB fusion protein in plant cells. It is a synthetic coding sequence used for expression.

[0080] SEQ ID NO: 55 is a gene encoding the TIC9320 PirAB fusion protein in plant cells. It is a synthetic coding sequence used for expression.

[0081] SEQ ID NO: 56 is a gene encoding the TIC9322 PirAB fusion protein in plant cells. It is a synthetic coding sequence used for expression.

[0082] SEQ ID NO: 57 is the She encoding sequence of the TIC10357 insecticidal PirA protein. This is the nucleic acid sequence obtained from the DSS12 strain of Vanella violacea.

[0083] SEQ ID NO: 58 is the amino acid sequence of the TIC10357 PirA protein.

[0084] SEQ ID NO: 59 is the She encoding sequence of the TIC10366 insecticidal PirB protein. This is the nucleic acid sequence obtained from the DSS12 strain of Vanella violacea.

[0085] SEQ ID NO: 60 is the amino acid sequence of the TIC10366 PirB protein.

[0086] SEQ ID NO: 61 is the sequence of TIC10357 and TIC10 operably linked in frame. The PirAB fusion protein TIC10375, which consists of the coding sequence of 366, was encoded. It is a nucleic acid sequence that

[0087] SEQ ID NO: 62 is the amino acid sequence of the TIC10375 PirAB fusion protein.

[0088] SEQ ID NO: 63 is the Pho encoding sequence of the TIC10358 insecticidal PirA protein. laumondii TTO1 strain of Torrhabdus luminescens It is a nucleic acid sequence.

[0089] SEQ ID NO: 64 is the amino acid sequence of the TIC10358 PirA protein.

[0090] SEQ ID NO: 65 is the Pho encoding sequence of the TIC10367 insecticidal PirB protein. laumondii TTO1 strain of Torrhabdus luminescens It is a nucleic acid sequence.

[0091] SEQ ID NO: 66 is the amino acid sequence of the TIC10367 PirB protein.

[0092] SEQ ID NO: 67 is the sequence of TIC10358 and TIC10 operably linked in frame. The PirAB fusion protein TIC10376, which consists of the coding sequence of 367, was encoded. It is a nucleic acid sequence that

[0093] SEQ ID NO: 68 is the amino acid sequence of the TIC10376 PirAB fusion protein .

[0094] SEQ ID NO: 69 is the PirA gene encoding the TIC10360 insecticidal PirA protein sequence. The nucleic acid sequence was obtained from Otorhabdus asymbiotica.

[0095] SEQ ID NO: 70 is the amino acid sequence of the TIC10360 PirA protein.

[0096] SEQ ID NO: 71 is the Pho encoding sequence of the TIC10369 insecticidal PirB protein torhabdus asymbiotica.

[0097] SEQ ID NO: 72 is the amino acid sequence of the TIC10369 PirB protein.

[0098] SEQ ID NO: 73 is the sequence of TIC10360 and TIC10 operably linked in frame. The PirAB fusion protein TIC10377, which consists of the coding sequence of 369, was encoded. It is a nucleic acid sequence that

[0099] SEQ ID NO: 74 is the amino acid sequence of the TIC10377 PirAB fusion protein.

[0100] SEQ ID NO: 75 is the Xen encoding sequence of the TIC10361 insecticidal PirA protein. orhabdus sp. strain NBAIIXenSa04.

[0101] SEQ ID NO: 76 is the amino acid sequence of the TIC10361 PirA protein.

[0102] SEQ ID NO: 77 is the Xen encoding the TIC10370 insecticidal PirB protein sequence. orhabdus sp. strain NBAIIXenSa04.

[0103] SEQ ID NO: 78 is the amino acid sequence of the TIC10370 PirB protein.

[0104] SEQ ID NO: 79 is the sequence of TIC10361 and TIC10 operably linked in frame. The PirAB fusion protein TIC10378, which consists of the coding sequence of 370, was encoded. It is a nucleic acid sequence that

[0105] SEQ ID NO: 80 is the amino acid sequence of the TIC10378 PirAB fusion protein.

[0106] SEQ ID NO: 81 is the Yer encoding the sequence of the TIC10362 insecticidal PirA protein. This is the nucleic acid sequence obtained from the 670-83 strain of S. sinia aldovae.

[0107] SEQ ID NO: 82 is the amino acid sequence of the TIC10362 PirA protein.

[0108] SEQ ID NO: 83 is the Yer encoding sequence of the TIC10371 insecticidal PirB protein. This is the nucleic acid sequence obtained from the 670-83 strain of S. sinia aldovae.

[0109] SEQ ID NO: 84 is the amino acid sequence of the TIC10371 PirB protein.

[0110] SEQ ID NO: 85 is the sequence of TIC10362 and TIC10 operably linked in frame. The PirAB fusion protein TIC10379, which consists of the coding sequence of 371, was encoded. It is a nucleic acid sequence that

[0111] SEQ ID NO: 86 is the amino acid sequence of the TIC10379 PirAB fusion protein.

[0112] SEQ ID NO: 87 is the Xen encoding the TIC10363 insecticidal PirA protein sequence. orhabdus doucetiae strain FRM16.

[0113] SEQ ID NO: 88 is the amino acid sequence of the TIC10363 PirA protein.

[0114] SEQ ID NO: 89 is the X encoding the TIC10372 insecticidal PirB protein sequence. The nucleic acid sequence obtained from the FRM16 strain of Enorhabdus doucetiae .

[0115] SEQ ID NO: 90 is the amino acid sequence of the TIC10372 PirB protein.

[0116] SEQ ID NO: 91 is the sequence of TIC10363 and TIC10 operably linked in frame. The PirAB fusion protein TIC10380, which consists of the coding sequence of 372, was encoded. It is a nucleic acid sequence that

[0117] SEQ ID NO: 92 is the amino acid sequence of the TIC10380 PirAB fusion protein .

[0118] SEQ ID NO: 93 encodes the sequence of the TIC10364 insecticidal PirA protein. norhabdus griffiniae strain BMMCB. .

[0119] SEQ ID NO: 94 is the amino acid sequence of the TIC10364 PirA protein.

[0120] SEQ ID NO: 95 is the X encoding sequence of the TIC10373 insecticidal PirB protein. The nucleic acid sequence obtained from the BMMCB strain of Enorhabdus griffiniae do.

[0121] SEQ ID NO: 96 is the amino acid sequence of the TIC10373 PirB protein.

[0122] SEQ ID NO: 97 is the sequence of TIC10364 and TIC10 operably linked in frame. The PirAB fusion protein TIC10381, which consists of the coding sequence of 364, was encoded. It is a nucleic acid sequence that

[0123] SEQ ID NO: 98 is the amino acid sequence of the TIC10381 PirAB fusion protein.

[0124] SEQ ID NO: 99 is the Xen encoding sequence of the TIC10359 insecticidal PirA protein. orhabdus nematophila.

[0125] SEQ ID NO: 100 is the amino acid sequence of the TIC10359 PirA protein.

[0126] SEQ ID NO: 101 encodes the sequence of the TIC10368 insecticidal PirB cell protein Nucleic acid sequence obtained from Xenorhabdus nematophila.

[0127] SEQ ID NO: 102 is the amino acid sequence of the TIC10368 PirB protein.

[0128] SEQ ID NO: 103 is an OCR sequence consisting of the coding sequences TIC10359 and TIC10368. 1 is a nucleic acid sequence encoding peron.

[0129] SEQ ID NO: 104 encodes the PirA_ABE68878 insecticidal PirA protein sequence Nucleic acid sequences obtained from the Hm strain of Photorhabdus luminescens is.

[0130] SEQ ID NO: 105 is the amino acid sequence of PirA_ABE68878 PirA protein be.

[0131] SEQ ID NO: 106 encodes the PirB_ABE68879 insecticidal PirB protein sequence. Nucleic acid sequences obtained from the Hm strain of Photorhabdus luminescens It is a column.

[0132] SEQ ID NO: 107 is the amino acid sequence of PirB_ABE68879 PirB protein be.

[0133] SEQ ID NO: 108 is a sequence operably linked in-frame to PirA_ABE68878 and and a PirAB fusion protein consisting of the coding sequences of PirB_ABE68879, 1 is a nucleic acid sequence encoding TIC10434.

[0134] SEQ ID NO: 109 is the amino acid sequence of the TIC10434 PirAB fusion protein .

[0135] SEQ ID NO: 110 is the sequence of TIC7575 and TIC76 operably linked in frame. Encoding a PirAB fusion protein, TIC11210, consisting of 65 coding sequences It is a nucleic acid sequence.

[0136] SEQ ID NO: 111 is the amino acid sequence of the TIC11210 PirAB fusion protein .

[0137] SEQ ID NO: 112 is the sequence of TIC7575 and TIC76 operably linked in frame. Encoding a PirAB fusion protein, TIC11211, consisting of 67 coding sequences It is a nucleic acid sequence.

[0138] SEQ ID NO: 113 is the amino acid sequence of the TIC11211 PirAB fusion protein .

[0139] SEQ ID NO: 114 is the sequence of TIC7662 and TIC76 operably linked in frame. Encoding a PirAB fusion protein, TIC11212, consisting of 65 coding sequences It is a nucleic acid sequence.

[0140] SEQ ID NO: 115 is the amino acid sequence of the TIC11212 PirAB fusion protein .

[0141] SEQ ID NO: 116 is the sequence of TIC7575 and TIC76 operably linked in frame. Encoding a PirAB fusion protein, TIC11301, consisting of 61 coding sequences It is a nucleic acid sequence.

[0142] SEQ ID NO: 117 is the amino acid sequence of the TIC11301 PirAB fusion protein .

[0143] SEQ ID NO: 118 is the sequence of TIC7660 and TIC75 operably linked in frame. Encoding TIC11302, a PirAB fusion protein consisting of 76 coding sequences It is a nucleic acid sequence.

[0144] SEQ ID NO: 119 is the amino acid sequence of the TIC11302f PirAB fusion protein do.

[0145] SEQ ID NO: 120 is an in-frame operably linked TIC4771, TIC477 1, and a PirAB fusion protein consisting of the coding sequence of TIC4472, TIC 11440 is a nucleic acid sequence encoding 11440.

[0146] SEQ ID NO: 121 is the amino acid sequence of the TIC11440 PirAB fusion protein do.

[0147] SEQ ID NO: 122 is an in-frame operably linked TIC7575, TIC757 TIC1, a PirAB fusion protein composed of the coding sequences of TIC7576 and TIC1 1441 is a nucleic acid sequence encoding 1441.

[0148] SEQ ID NO: 123 is the amino acid sequence of the TIC11441f PirAB fusion protein be.

[0149] SEQ ID NO: 124 is an in-frame operably linked TIC7575, TIC477 1, and a PirAB fusion protein consisting of the coding sequence of TIC4472, TIC 11442 is a nucleic acid sequence encoding 11442.

[0150] SEQ ID NO: 125 is the amino acid sequence of the TIC11442 PirAB fusion protein .

[0151] SEQ ID NO: 126 is an in-frame operably linked TIC7660, TIC757 5, and a PirAB fusion protein, TIC1, composed of the TIC7576 coding sequence. 1443 is a nucleic acid sequence encoding 1443.

[0152] SEQ ID NO: 127 is the amino acid sequence of the TIC11443 PirAB fusion protein do.

[0153] SEQ ID NO: 128 is the sequence of TIC7660 and TIC7660 operably linked in frame. The PirAB fusion protein TIC11444, which consists of the coding sequence of 576, was encoded. It is a nucleic acid sequence that

[0154] SEQ ID NO: 129 is the amino acid sequence of the TIC11444 PirAB fusion protein .

[0155] SEQ ID NO: 130 is an in-frame operably linked TIC7660, TIC766 2, and a PirAB fusion protein, TIC1, composed of the TIC7663 coding sequence. 1445 is a nucleic acid sequence encoding 1445.

[0156] SEQ ID NO: 131 is the amino acid sequence of the TIC11445 PirAB fusion protein .

[0157] SEQ ID NO: 132 is an in-frame operably linked TIC7662, TIC766 0, and a fusion protein, TIC11446, composed of the TIC7661 coding sequence. It is a nucleic acid sequence encoding

[0158] SEQ ID NO: 133 is the amino acid sequence of the TIC11446 PirAB fusion protein .

[0159] SEQ ID NO: 134 is the Xe encoding sequence of the TIC11505 insecticidal PirB protein. norhabdus nematophila strain MDI-0035777 It is a nucleic acid sequence.

[0160] SEQ ID NO: 135 is the amino acid sequence of the TIC11505 PirB protein.

[0161] SEQ ID NO: 136 is the sequence of TIC10364 and TIC1 operably linked in frame. The PirAB fusion protein TIC11506, which consists of the coding sequence of 1505, was cloned. It is a nucleic acid sequence that is read.

[0162] SEQ ID NO: 137 is the amino acid sequence of the TIC11506 PirAB fusion protein .

[0163] SEQ ID NO: 138 is the Xe encoding sequence of the TIC11510 insecticidal PirB protein. norhabdus bovienii strain MDI-0035808 It is a column.

[0164] SEQ ID NO: 139 is the amino acid sequence of the TIC11510 PirB protein.

[0165] SEQ ID NO: 140 is the sequence of TIC10364 and TIC1 operably linked in frame. The PirAB fusion protein TIC11512, which consists of the coding sequence of 1510, was cloned. It is a nucleic acid sequence that is read.

[0166] SEQ ID NO: 141 is the amino acid sequence of the TIC11512 PirAB fusion protein .

[0167] SEQ ID NO: 142 is the Xe encoding sequence of the TIC11511 insecticidal PirB protein. Nucleic acid sequence obtained from norhabdus nematophila strain AN6 / 1 is.

[0168] SEQ ID NO: 143 is the amino acid sequence of the TIC11511 PirB protein.

[0169] SEQ ID NO: 144 is the sequence of TIC10364 and TIC1 operably linked in frame. The PirAB fusion protein TIC11513, which consists of the coding sequence of 1511, was cloned. It is a nucleic acid sequence that is read.

[0170] SEQ ID NO: 145 is the amino acid sequence of the TIC11513 PirAB fusion protein .

[0171] SEQ ID NO: 146 is a plant antigen encoding the TIC10376PL PirAB fusion protein A synthetic coding sequence used for expression in cells, encoding the TIC10358 protein. An additional alanine codon has been inserted immediately after the initiating methionine codon of the fragment.

[0172] SEQ ID NO: 147 is the amino acid sequence of the TIC10376PL PirAB fusion protein be.

[0173] SEQ ID NO: 148 is a plant sequence encoding the TIC10378PL PirAB fusion protein A synthetic coding sequence used for expression in cells, encoding the TIC10361 protein. An additional alanine codon has been inserted immediately after the initiating methionine codon of the fragment.

[0174] SEQ ID NO: 149 is the amino acid sequence of the TIC10378PL PirAB fusion protein be.

[0175] SEQ ID NO: 150 is a plant sequence encoding the TIC10380PL PirAB fusion protein A synthetic coding sequence used for expression in cells encoding the TIC10363 protein. An additional alanine codon has been inserted immediately after the initiating methionine codon of the loading fragment.

[0176] SEQ ID NO: 151 is the amino acid sequence of the TIC10380PL PirAB fusion protein be.

[0177] SEQ ID NO: 152 is a plant sequence encoding the TIC10381PL PirAB fusion protein A synthetic coding sequence used for expression in cells, encoding the TIC10364 protein. An additional alanine codon has been inserted immediately after the initiating methionine codon of the fragment.

[0178] SEQ ID NO: 153 is the amino acid sequence of the TIC10381PL PirAB fusion protein be.

[0179] SEQ ID NO: 154 is the coding sequence of operably linked TIC7661 and TIC7660. in plant cells encoding the TIC11103 PirAB fusion protein, which is composed of is a synthetic coding sequence used for expression of

[0180] SEQ ID NO: 155 is the amino acid sequence of the TIC11103 PirAB fusion protein .

[0181] SEQ ID NO: 156 is the coding sequence of operably linked TIC7663 and TIC7662. in plant cells encoding the TIC11104 PirAB fusion protein, which is composed of is a synthetic coding sequence used for expression of

[0182] SEQ ID NO: 157 is the amino acid sequence of the TIC11104 PirAB fusion protein .

[0183] SEQ ID NO: 158 is a plant cell clone encoding the TIC11302 PirAB fusion protein It is a synthetic coding sequence used for expression in

[0184] SEQ ID NO: 159 was expressed in Escherichia coli and purified as a protein. a synthetic coding sequence encoding a histidine tag operably linked to the coding sequence used in It is a code array.

[0185] SEQ ID NO: 160 is the amino acid sequence of the histidine tag. DETAILED DESCRIPTION OF THE INVENTION

[0186] The problem in the field of agricultural pest control is to develop a method for controlling pests that is effective against target pests and that is specific to the target pest species. It exhibits a broad spectrum of toxicity to plants without causing undesirable agricultural problems. Alternative actions compared to current toxins that can be expressed and commercially used in plants This can be characterized as the need for new toxin proteins that provide the mechanism.

[0187] Disclosed herein are P. cerevisiae strains that provide resistance to Coleoptera, Hemiptera, and Lepidoptera pests. irA proteins TIC4771, TIC7575, TIC7660, TIC7662, TIC7664, TIC7666, TIC7668, TIC7939, TIC10357 , TIC10358, TIC10360, TIC10361, TIC10362, TIC 10363, TIC10364, TIC10359, and PirA_ABE68878( collectively, "PirA proteins"); PirB proteins TIC4772, TIC75 76, TIC7661, TIC7663, TIC7665, TIC7667, TIC76 69, TIC7940, TIC10366, TIC10367, TIC10369, TI C10370, TIC10371, TIC10372, TIC10373, TIC103 68, PirB_ABE68879, TIC11505, TIC11510, and TIC 11511 (collectively, "PirB proteins"); and PirAB fusion proteins , TIC6880, TIC9316, TIC9317, TIC9318, TIC9319 , TIC9322, TIC9320, TIC9321, TIC6880PL, TIC10 375, TIC10376, TIC10377, TIC10378, TIC10379, TIC10380, TIC10381, TIC10434, TIC11210, TIC1 1211, TIC11212, TIC11301, TIC11302, TIC11440 , TIC11441, TIC11442, TIC11443, TIC11444, TIC 11445, TIC11446, TIC11506, TIC11512, TIC1151 3, TIC10376PL, TIC10378PL, TIC10380PL, TIC10 381PL, TIC11103, and TIC11104 (collectively, "PirAB fusion proteins"). This is a novel class of insecticidal proteins exemplified by the PirAB (PirAb) insecticidal proteins.

[0188] Also disclosed are PirAB fusion proteins, TIC6880PL, TIC9 316, TIC9317, TIC9318, TIC9319, TIC9320, TIC9 322, TIC10376PL, TIC10378PL, TIC10380PL, TIC Codes 10381PL, TIC11103, TIC11104, and TIC11302 It is a synthetic coding sequence designed for expression in plant cells. The PirA protein, PirB protein, or PirAB fusion protein a recombinant comprising a promoter operably linked to a coding sequence encoding one or more of It is a nucleic acid molecule.

[0189] In this application, "PirA protein," "PirA protein toxin," and "PirA toxin" "PirA insecticidal protein," "PirA-associated toxin," or "Pi References to "rA-related toxin proteins" etc. are to Coleopteran, Hemiptera and Lepidopteran pests. and sequence comparison of such proteins with any of the PirA proteins. If the amino acid sequence identity is between about 20 and about 100 percent, insecticidal activity is expected. TIC4771 (SEQ ID NO: 2), including any protein that exhibits insecticidal or insect-inhibitory activity; TIC7575 (SEQ ID NO: 8), TIC7660 (SEQ ID NO: 14), TIC7662 (SEQ ID NO: Sequence number 20), TIC7664 (sequence number 26), TIC7666 (sequence number 32), T TIC7668 (SEQ ID NO: 38), TIC7939 (SEQ ID NO: 44), TIC10357( SEQ ID NO: 58), TIC10358 (SEQ ID NO: 64), TIC10360 (SEQ ID NO: 70 ), TIC10361 (SEQ ID NO: 76), TIC10362 (SEQ ID NO: 82), TIC1 TIC10363 (SEQ ID NO: 88), TIC10364 (SEQ ID NO: 94), TIC10359 (SEQ ID NO: Sequence number 100), and insecticidal protein of PirA_ABE68878 (sequence number 105) and the sequences of insecticidal or insect-inhibitory proteins and their insecticidal or insect-inhibitory segments. comprising, consisting of, substantially homologous to, Any novel insecticidal or insect-inhibiting protein similar to or derived therefrom It refers to quality.

[0190] In this application, "PirB protein," "PirB protein toxin," and "PirB toxin" "PirB insecticidal protein," "PirB-associated toxin," or "Pi References to "rB-related toxin proteins" etc. are to Coleopteran, Hemiptera and Lepidopteran pests. and sequence comparison of such proteins with any of the PirB proteins. If the amino acid sequence identity is between about 24 and about 100 percent, insecticidal activity is expected. TIC4772 (SEQ ID NO: 4), including any protein that exhibits insecticidal or insect-inhibitory activity; TIC7576 (SEQ ID NO: 10), TIC7661 (SEQ ID NO: 16), TIC7663 ( SEQ ID NO: 22), TIC7665 (SEQ ID NO: 28), TIC7667 (SEQ ID NO: 34), TIC7669 (SEQ ID NO: 40), TIC7940 (SEQ ID NO: 46), TIC10366 (SEQ ID NO: 60), TIC10367 (SEQ ID NO: 66), TIC10369 (SEQ ID NO: 7 2), TIC10370 (SEQ ID NO: 78), TIC10371 (SEQ ID NO: 84), TIC 10372 (SEQ ID NO: 90), TIC10373 (SEQ ID NO: 96), TIC10368( SEQ ID NO: 102), PirB_ABE68879 (SEQ ID NO: 107), TIC11505 (SEQ ID NO: 135), TIC11510 (SEQ ID NO: 139), and TIC11511 (SEQ ID NO: 140). Sequences of insecticidal or insect-inhibiting proteins in column 143 and their insecticidal properties comprising or consisting of an insect-inhibiting segment or an insect-inhibiting segment or a combination thereof , any novel insecticidal compound substantially homologous thereto, similar thereto, or derived therefrom It refers to a protein or insect-inhibiting protein.

[0191] The term "PirAB fusion protein" is used in this application to refer to a protein that is adjacent to a PirB protein. Used to describe proteins containing both the PirA protein and the adjacent PirA protein. The DNA sequence encoding the B fusion protein, when expressed in cells, produces the PirA protein. The PirB protein is then ligated to form a fusion protein containing both the PirB protein and the PirB protein. a PirA protein operably linked in frame to a coding sequence encoding a protein; The PirAB protein can be expressed in the same bacterial operon as the PirAB gene. It can be composed of PirA protein and PirB protein derived from Instead, the PirA and PirB proteins originate from different bacterial operons. An example in which the PirA protein is adjacent to the PirB protein. Exemplary PirAB fusion proteins are provided in Table 1. Table 1. Exemplary Pir proteins composed of a PirA protein flanked by PirB proteins AB fusion protein and the corresponding PirA and PirB proteins contained therein Plagiarism. TIFF2026010055000001.tif227164

[0192] The term "PirAB fusion protein" is also used in this application to refer to a PirA protein. Used to describe proteins that contain the PirB protein adjacent to the PirA protein. The DNA sequence encoding the PirB fusion protein, when expressed in cells, The PirA protein is then added to generate a fusion protein containing both the PirA protein and the PirA protein. the PirB protein operably linked in frame to a coding sequence encoding a protein The PirB protein can contain a coding sequence encoding the same bacterial operon or Instead, it is composed of PirB and PirA proteins from different operons. In an exemplary embodiment, the PirB protein is adjacent to the PirA protein. The proteins are provided in Table 2. Table 2. Exemplary Pir proteins composed of a PirB protein adjacent to a PirA protein AB fusion protein and the corresponding PirB and PirA proteins contained therein. Plagiarism. TIFF2026010055000002.tif39159

[0193] The term "PirAB fusion protein" is also used in this application to mean a PirAB fusion protein. It is used to describe a protein that contains two adjacent PirA proteins. DNA sequences encoding various PirAB fusion proteins were expressed in cells to produce Pi A fusion protein comprising a PirA protein and another PirA protein and a PirB protein. operably linked to a coding sequence encoding a PirB protein so as to produce engineered coding sequences that encode the same or different PirA proteins The PirA protein may be operably linked to a coding sequence encoding the PirA protein. An exemplary PirAB fusion protein containing two PirA proteins flanking a PirB protein. Proteins are provided in Table 3. Table 3. PirA proteins adjacent to another PirA protein and adjacent to a PirB protein Exemplary PirAB fusion proteins comprised of a PirAB protein and the corresponding PirAB protein contained therein. PirA and PirB proteins. TIFF2026010055000003.tif70165

[0194] The term "PirAB fusion protein" is also used in this application to refer to multiple PirAB fusion proteins adjacent to each other. Describes a protein containing multiple PirA proteins and / or multiple PirB proteins. Multiple PirA and / or PirB proteins are used to The PirA or PirB protein may be a PirA protein or a PirB protein produced by the The fusion protein can be a PirA protein or a PirB protein. The combination of all PirA and / or PirB proteins is specific It can enhance activity against target pest species or increase the range of pest species for which it is active. You may do so.

[0195] The term "segment" or "fragment" is used in this application to refer to a PirA protein, P The complete amino acid sequence describing one of the irB or PirAB proteins used to describe a contiguous amino acid or nucleic acid sequence that is shorter than the Segments or fragments that exhibit insect inhibitory activity are those that combine such segments or fragments with Column numbers 2, 8, 14, 20, 26, 32, 38, 44, 58, 64, 70, 76, 82, 88, 94, 100, or 105; SEQ ID NOs: 4, 10 , 16, 22, 28, 34, 40, 46, 60, 66, 72, 78, 84, 90, 96, 102, 107, 135, 139, or 143; or are SEQ ID NOs: 6, 12, 18, 24, 30, 36, 42, 48, 50, 62, 68, 74, 80, 86, 92, 98, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 137, 141, 145, 147, PirAB fusion proteins described in 149, 151, 153, 155, or 157 or sequence comparison with the corresponding section of the insecticidal protein of a related family member Between about 65 and about 100 percent of the segments or fragments and the corresponding sections of the aligned proteins If a sequence comparison of the identity of the first sequence occurs, it is also disclosed in this application. The PirA, PirB, or PirAB protein described A segment or fragment of one of the PirA protein, PirB protein, or or PirAB protein, at least about 50 contiguous amino acids, at least about 75 contiguous amino acids, at least about 100 contiguous amino acids, At least about 125 contiguous amino acids, at least about 150 contiguous amino acids, At least about 200 contiguous amino acids, at least about 250 contiguous amino acids, At least about 300 contiguous amino acids, at least about 350 contiguous amino acids, At least about 400 contiguous amino acids, at least about 450 contiguous amino acids, at least about 500 contiguous amino acids, at least about 550 contiguous amino acids, or It may comprise at least about 600 contiguous amino acids. One of the irA, PirB, or PirAB proteins The fragment or fragment may exhibit the activity of the base sequence.

[0196] In this application, "active" or "activity", "insecticidal activity" or "insecticidal" or References to the terms "insecticidal activity," "insect-inhibiting," or "insecticidal" refer to an effective amount of Pir A protein, PirB protein, or a PirAB fusion protein. inhibiting (inhibiting growth, feeding, fertility, or viability) certain crops; , inhibit (inhibit growth, feeding, reproduction, or viability), control (Preventing pest infestations and pest feeding activities) These terms refer to the effectiveness of toxic agents such as toxic proteins that cause disease when exposed to pests. An insecticidal amount of toxic tannins that results in a catastrophic condition, mass mortality, reduced fecundity, or stunted growth. These terms are intended to include the results of providing proteins to pests. damage to plants, plant tissues, or plant tissues as a result of providing large amounts of toxic proteins in or on the plant pests that originate from parts of plants, seeds, plant cells, or the particular geography in which plants may grow In general, insecticidal activity is measured by the effects of growth, development, survival rate, feeding, and other factors on the repelling of pests from the target site. Effective by disrupting feeding, mating, or fecundity, or by targeting Lepidoptera, Coleoptera, or This protein, protein of specific target pests, including but not limited to Hemiptera insects. Induced by insect feeding on protein fragments, protein segments or polynucleotides It refers to the ability of a toxic protein to be effective in measurably reducing the adverse effects caused. Proteins can be produced by plants or can be found in plants or in the areas where plants are located. The terms "bioactive," "effective," "effective" or "effective" or any of its equivalents may be used. These variations are also used in this application to describe the effect of the proteins of the invention on target pests. are terms used interchangeably in

[0197] An effective amount of the poison is provided in the bait of the target pest, and when the poison comes into contact with the pest, The toxic agent may be an insecticidal protein or one or more of the toxic agents known in the art. It can be a chemical. Insecticidal or insecticidal chemicals and insecticidal or insecticidal Protein drugs can be used alone or in combination with each other. These include dsRNA molecules that target specific genes for the control of target pests, organochlorine compounds, and compounds, organophosphates, carbamates, pyrethroids, neonicotinoids, and rhynoids Insecticidal or insecticidal protein agents include, but are not limited to, Protein toxins described in the application, as well as Coleoptera, Lepidoptera, Hemiptera, Thysanoptera, or Other proteinaceous poisons include those that target Dipteran pest species.

[0198] "Photorhabdus insect-associated" proteins, or PirAB proteins, It is a two-component toxin that has insecticidal activity against some insects. It has been shown to have lepidopteran activity when injected into the hemocoel of insects. Oral administration of PirAB protein had little or no activity when presented in the diet. (e.g., Yang, et al. (2017), PirAB protein in from Xenorhabdus nematophila HB310 ex hibits a binary toxin with insecticidal activity and cytotoxicity in Galleria me llonella.J.Invertebr.Pathol,148:43-50;Li ,et al.(2014),Photorhabdus luminescens P irAB-fusion protein exhibits both cytoto xicity and insecticidal activity.FEMS Mi crobial.Lett,356:23-31;Wu and Yunhong,(2016 ),Scientific Reports,6,Article number:34 996;doi:10.1038 / srep34996;and Zhang,et a l.(2013),XaxAB-like binary toxin from Ph otorhabdus luminescens exhibits both ins ecticidal activity and cytotoxicity.FEMS Microbiol. Lett. 350:48-56). Oral activity of the PirAB protein has been reported, but these studies were limited to the insect diet. Instead of purified toxin, the bait contained E. coli bacteria expressing the PirAB protein. (e.g., Waterfield, et al. 2005),The Photorhabdus Pir toxins are si milar to a developmentally regulated ins ect protein but show no juvenile hormone esterase activity.FEMS Microbiol.Lett,2 45:47-52 and Blackburn, et al. (2006), Remarka ble susceptibility of the diamondback mo th (Plutella xylostella) to ingestion of Pir toxins from Photorhabdus luminescen s.Entomologia Experimentalis et Applic a, 121:31-37). In stark contrast, the examples herein As reported, PirA protein, PirB protein, and PirAB fusion protein Protein preparations of the plaque were used in insect bait bioassays. Oral activity against ptera pests has been observed and is presented in the examples. B fusion proteins, TIC9316, TIC9317, and TIC9318. Leaf discs derived from plants containing the lepidopteran pests European corn borer and Southwestern American corn borer were used to It was used in an oral insect feeding test demonstrating activity against the wheat borer (SWCB). Expressing TIC10376, TIC10378, TIC10380, and TIC10381 Leaf discs derived from plants containing the compounds showed activity against SWCB. TIC9315 and TIC11302 were stably transformed as described in It showed activity against the western corn rootworm pest in plants.

[0199] References to pests, particularly crop pests, include references to pests of crops, particularly PirA proteins, PirB proteins, proteins, and PirAB proteins, related families of insecticidal proteins, or means the pest controlled by at least one of those segments or pieces is intended.

[0200] As described in the Examples, PirA protein, PirB protein, or P One or more of the irAB proteins are expressed in Coleoptera, Hemiptera, and Neanderthida, including adults, pupae, larvae, and neonates. It exhibits insecticidal activity against pests from Lepidoptera pest species.

[0201] Lepidoptera include armyworms, cutworms, and other insects of the Noctuidae family. Cutworms, loopers, and heliothins, e.g., Fall Armyworm (S podoptera frugiperda), Spodoptera frugiperda a exigua), black armyworm (Spodoptera exempta), bell Mamestra configurata, Southern armyworm (Spo doptera eridania), Tamanaya moth (Agrotis ipsilon) , cabbage looper (Trichoplusia ni), soybean looper (Pseud oplusia includens), Velvet Bean Caterpillar (Antica rsia gemmatalis), green cloverworm (Hypena sca bra), Heliothis virescens, Granular cutwa Agrotis subterranea, cutworm (Pseudaleti a unipuncta), Western root cutter (Agrotis Orthogonia); Borers, casebearers, webworms, cornworms, and cabbage worms of the family Pyralidae Corn borers and skeletonizers, e.g., European corn borer (Ostrinia nubii) lalis), Navel orangeworm (Amyelois transitella) , Corn root webworm (Crambus caliginosellus), Sot Brown webworm (Herpetogramma licarsisalis), sunflower Moth (Homoeosoma electellum), Lesser corn stalk borer ( Elasmopalpus lignosellus); Tortricidae Turnip worms, budworms, seedworms, and fruitworms, such as the codling moth (C ydia pomonella), Grapeberry Moss (Endopiza vitea na), Japanese fruit moth (Grapholita molesta), sunflower bud moss (Suleima helianthana); as well as many other economically important Lepidoptera, e.g., diamondback moth (Plutella xylostella), pink ball moth Pectinophora gossypiella and Lymantria dispar antria dispar) and other Lepidoptera. Examples of pests include the cotton bollworm (Alabama argillacea), Tree leafroller (Archips argyrospila), European leafroller (A Archips rosana) and other Archips species (Chilo suppres salis, Asian rice leafroller, or rice stem borer), rice leafroller (Cnaphal ocrocis medinalis), Corn root webworm (Crambus caliginosellus), Bluegrass webworm (Crambus tet errellus), southwestern corn borer (Diatraea grandiosell) a), Sagar Cane Borer (Diatraea saccharalis), thorny A ballworm (Earias insEarias vittella), Otaba Helicoverpa armigera, Helicoverpa armigera verpa zea, also known as the soybean podworm and cotton bollworm), Heliothis virescens, sod webworm petogramma licarsisalis), Western bean rootworm (Stria costa albicosta), European grapevine moth (Lobesia bot rana), Citrus leaf miner (Phyllocnistis citrella ), large cabbage white (Pieris brassicae), small cabbage white ( Pieris rapae, also known as the imported caterpillar), tobacco root cutter Cluster caterpillar (also known as Spodoptera litura) ), and the tomato leaf moth (Tuta absoluta).

[0202] Among the Coleoptera, the western corn rootworm (Diabrotica vulgaris) is a particularly pest. irgifera, WCR), Agriotes spp., Anthono mus spp., Atomaria linearis, Chaetocnema t. ibialis, Cosmopolites spp., Curculio spp., Dermestes spp., Diabrotica spp., Epilachna spp., Eremnus spp., Leptinotarsa ​​decemlin eata, Lissorhoptrus spp., Melolontha spp., Orycaephilus spp., Otiorhynchus spp., Phly ctinus spp., Popillia spp., Psylliodes spp. ., Rhizopertha spp., Scarabeidae, Sitophilu s spp., Sitotroga spp., Tenebrio spp., Trib olium spp., and Trogoderma spp., northern corn root cutters Diabrotica barberi (NCR), Mexican type corn root cutter Diabrotica virgifera zeae (MCR), Brazilian type Corn rootworm (Diabrotica balteata, BZR), southern type Sorghum rootworm (Diabrotica undecimpunctata howa rdii, SCR), Colorado potato beetle (Leptinotarsa ​​decemline) ata, CPB), Brazilian corn rootworm complex (BCR, Diabroti ca viridula and Diabrotica speciosa), Leaf beetle (Phyllotreta crucifer)Phyllotreta striolata), striped flea beetle (Phyllotreta striolat a) and the Western black flea beetle (Phyllotreta pusilla). (But not limited to these.)

[0203] Hemiptera include stink bugs of the Pentatomidae family, and bugs of the Chinavia genus. Chinavia hilaris, Chinavia margina ta, and Chinavia pensylvanica), Chlorochroa spp. Stink bug (Chlorochroa granulose, Chlorochroa kanei, Chlorochroa ligata, Chlorochroa lin eate, Chlorochroa opuntiae, Chlorochroa pe rsimilis, Chlorochroa rossiana, Chlorochro a sayi, Chlorochroa uhleri, Chlorochroa be lfragii, Chlorochroa faceta, Chlorochroa o sborni, Chlorochroa saucia, and Chlorochroa senilis), Southern green stink bug (Nezara viridula), Ede Stink bugs of the genus Edessa (Edessa meditabunda, Edessa bifi da, and Edessa florida), subtropical brown marmorated stink bug (Euschist us heros), Euschistus genus stink bugs (Euschistus ac uminatus, Euschistus biformis, Euschistus conspersus, Euschistus crenator, Euschistus s egglestoni, Euschistus ictericus, Euschi stus inflatus, Euschistus latimarginatus, Euschistus obscures, Euschistus politus, E uschistus quadrator, Euschistus sevus, Eus chistus strenuous, Euschistus tristigmus, and Euschistus variolarius), brown marmorated stink bug (Halyom orpha halys), red stink bug (Thyanta accerra), T Stink bugs of the genus Thyanta (Thyanta calceata, Thyanta cu) stator, Thyanta pallidovirens, Thyanta per ditor, Thyanta maculate, and Thyanta pseudoc asta), Dichelops genus green stink bug (Dichelops mel acanthus) and other stink bugs (Dichelops avilapiresi, Dichelops bicolor, Dichelops dimidatus, Di chelops furcatus, Dichelops furcifrons, Di chelops lobatus, Dichelops miriamae, Diche lops nigrum, Dichelops peruanus, Dichelops phoenix, and Dichelops saltensis), red spotted stink bug (Piezodorus guildinni) as well as Piezodorus lit uratus; and Plataspidae insects, such as the Taiwan stink bug ( Megacopta cribraria), Western green thrush (Lygus he sperus, and the green mistletoe bug (Lygus lineolaris). Examples include, but are not limited to:

[0204] References in this application to "isolated DNA molecules" or equivalent terms or phrases are The DNA molecule may be present alone or in combination with other compositions, but may not be present in its own right. It is intended to mean something that does not exist in the natural environment. For example, Coding sequences, intron sequences, and untranslated leader sequences naturally found within the DNA of the genome Nucleic acid elements such as promoter sequences, transcription termination sequences, etc., are used to identify the sequence of interest when the element is present in the genome of an organism. A gene is not considered "isolated" as long as it is in the location within the genome in which it is found in nature. However, each of these elements, and the subdivisions of these elements, It is within the scope of this disclosure to the extent that it is not within the genome of an organism and is not the location within the genome in which it is found in nature. Similarly, an insecticidal protein or any of its proteins would be "isolated" within the context of The nucleotide sequence encoding a naturally occurring insecticidal variant of Unless the protein-encoding sequence was not found in the bacterial DNA found in nature, The isolated nucleotide sequence is a naturally occurring insecticidal protein. A synthetic nucleotide sequence encoding the amino acid sequence of a protein is isolated for purposes of this disclosure. For the purposes of this disclosure, any transgenic nucleotide sequence i.e., inserted into the genome of a plant or bacterial cell or present on an extrachromosomal vector. The nucleotide sequence of the DNA present in the plasmid or It may or may not be present in a similar structure used in the genome of a plant or a bacterium. or tissues, progeny, biological samples or commercial products derived from plants or bacteria. A nucleotide sequence is considered to be isolated whether or not it is present in detectable amounts in a product. will be done.

[0205] As further described herein, the PirA protein, TIC4771 (sequence No. 1) and two encoding the PirB cell protein, TIC4772 (SEQ ID NO: 3). The operons containing the open reading frames (ORFs) are set forth in SEQ ID NOs: 2 and 3, respectively. Xenorhabdus ne encoding the protein toxin presented as SEQ ID NO:4 matophila strain ISB000002. The RF was used to clone the DNA sequence, TIC6880 (SEQ ID NO: 5), encoding a PirAB fusion producing a protein, wherein the two coding sequences are operably linked in frame; The TIC6880 PirAB fusion protein presented as SEQ ID NO: 6 was generated. Bioassays using TIC4771 derived from living host cells demonstrated that it inhibits the growth of the Lepidoptera species Tabacum officinalis. Helicoverpa zea (CEW), diamondback moth (Plutella xyl ostella, DEM), European corn borer (Ostrinia nubilalis , ECB), Velvet Bean Caterpillar (Anticarsia gemmatal is, VBC), and southern armyworm (Spodoptera eridania, SA W); Colorado potato beetle (Leptinotarsa ​​decemlineat a, CPB); and the Hemiptera species Lygus lineolaris ( TIC4772 derived from microbial host cells was shown to be active against TPB. The assay showed activity against Lepidoptera species, CEW, DBM, VBC and Hemiptera species, TPB. The PirAB gene derived from microbial host cells was composed of TIC4771 and TIC4772. Bioassays using the fusion protein TIC6880 demonstrated that the lepidopteran species Fall armyworm (Spodoptera frugiperda, FAW), CEW, Southwestern America Diatraea grandiosella, SWCB, DBM, EC B, and VBC, Coleoptera species CPB and Diabrotidae a virgifera, WCR); Hemiptera species, Lygus linnatus eolaris, TPB), Western green turtle (Lygus hesperus, W TP), Southern Green Bug (Nezara viridula, SGB), and Subtropical Brown marmorated stink bug (Euschistus heros, NBSB) and other Diptera species It showed activity against Aedes aegypti (YFM).

[0206] PirA protein TIC7575 (SEQ ID NO: 7) and PirB protein TIC75 An operon containing two ORFs encoding 76 (SEQ ID NO: 9), respectively, is shown in SEQ ID NO: Xenorhabd encoding the protein toxin presented as SEQ ID NO: 8 and SEQ ID NO: 10 Two ORFs were found in DNA from strain 85823 of C. ehlersii. The DNA sequence, TIC9316 (SEQ ID NO: 11), was used to identify the PirAB fusion protein encoding The two coding sequences are operably linked in frame to produce a protein. The TIC9316 PirAB fusion protein shown as column number 12 was generated. Bioassays using TIC7575 and TIC7576 derived from living host cells were However, TIC7575 and TIC Bioassay using PirAB fusion protein TIC9316 composed of 7576 The species of Lepidoptera are SWCB, Agrotis ipsilon (BCW), SAW, Heliothis virescens (TBW), ECB, and VBC, Coleoptera species, CPB, and Hemiptera species, TPB, WTP, SGB, and NBSB. It showed activity against

[0207] PirA protein TIC7660 (SEQ ID NO: 13) and PirB protein TIC7 The operon containing two ORFs encoding 661 (SEQ ID NO: 15) is Xenorhabdin, which encodes the protein toxin presented as sequence number 14 and sequence number 16 was found in DNA obtained from strain 85908 of A. abdus cabanillasii Two ORFs were used to construct the DNA sequence, TIC9317 (SEQ ID NO: 17), encoding P An irAB fusion protein is generated, in which the two coding sequences are engineered in-frame. TIC9317 PirAB fusion protein linked to and presented as SEQ ID NO: 18 Bioassay using TIC7660 and TIC7661 derived from microbial host cells The assay showed no activity against the insects used in the assay. The PirAB fusion protein TIC9317, which is composed of TIC660 and TIC7661, was used. The bioassays performed were for Lepidoptera species, SWCB, ECB, and VBC, for Coleoptera species, CPB, and It showed activity against WCR, Hemiptera species, TPB, WTP, and SGB.

[0208] PirA protein TIC7662 (SEQ ID NO: 19) and PirB protein TIC76 An operon containing two ORFs encoding 63 (SEQ ID NO: 21), respectively, has the sequence Xenorha encoding the protein toxin presented as SEQ ID NO: 20 and SEQ ID NO: 22 Two O The RF was used to identify the DNA sequence, PirAB fusion protein encoding TIC9318 (SEQ ID NO: 23). A fusion protein is produced, wherein the two coding sequences are operably linked in frame. , to generate the TIC9318 PirAB fusion protein presented as SEQ ID NO: 24 Bioassays using TIC7662 and TIC7663 derived from microbial host cells showed However, TIC7662 and T Bioassay using PirAB fusion protein TIC9318 composed of IC7663 The lepidopteran species, SWCB, BCW, TBW, ECB, and VBC, and the coleopteran species, CP B and WCR, and Hemiptera species, TPB, WTP, SGB, and NBSB. did.

[0209] PirA protein TIC7664 (SEQ ID NO: 25) and PirB protein TIC7 The operon containing two ORFs encoding 665 (SEQ ID NO: 27) is Xenorhabdin, which encodes the protein toxin presented as SEQ ID NO: 26 and SEQ ID NO: 28 It was found in DNA obtained from strain 86198 of B. abdus poinarii. The ORF was used to identify the DNA sequence, PirAB encoding TIC9319 (SEQ ID NO: 29). A fusion protein is created, in which the two coding sequences are operably linked in frame. The TIC9319 PirAB fusion protein was generated and presented as SEQ ID NO: 30. Bioassays using microbial host cell-derived TIC7664 demonstrated that CPB of Coleoptera species Bioassays using TIC7665 derived from microbial host cells showed activity against It showed activity against TBW of Pterygota species. Bioassays using the PirAB fusion protein TIC9319 from Lepidoptera species, SW CB, BCW, ECB, and VBC, Coleoptera, CPB, and Hemiptera, TPB, WTP , and SGB.

[0210] PirA protein TIC7666 (SEQ ID NO: 31) and PirB protein TIC7 The operon containing two ORFs encoding 667 (SEQ ID NO: 33) is Photorhizin encoding the protein toxin shown as sequence number 32 and sequence number 34 It was found in DNA obtained from strain 86197 of B. abdus luminescens. Two ORFs were used to construct the DNA sequence, Pi encoding TIC9322 (SEQ ID NO: 35). A rAB fusion protein was generated, in which the two coding sequences were operable in frame. The TIC9322 PirAB fusion protein, presented as SEQ ID NO: 36, was Bioassays using TIC7666 derived from microbial host cells showed that The insects tested showed no activity against the insects used. Bioassays showed activity against Lepidoptera species, SWCB. Bioassay using PirAB fusion protein TIC9322 composed of 7667 I: for Lepidoptera species, SWCB and VBC, Coleoptera species, CPB, and Hemiptera species, TPB It showed activity.

[0211] PirA protein TIC7668 (SEQ ID NO: 37) and PirB protein TIC76 An operon containing two ORFs encoding 69 (SEQ ID NO: 39), respectively, has the sequence Photorh encoding the protein toxin presented as SEQ ID NO: 38 and SEQ ID NO: 40 It was found in DNA obtained from strain 86194 of B. abdus luminescens. Two ORFs are used to encode the DNA sequence, TIC9320 (SEQ ID NO: 41). An AB fusion protein is created, in which the two coding sequences are operably linked in frame. The resulting TIC9320 PirAB fusion protein was ligated and presented as SEQ ID NO: 42. Bioassay using TIC7668 and TIC7669 derived from microbial host cells However, TIC766 showed no activity against the insects used in the assay. 8 and TIC7669 using the PirAB fusion protein TIC9320. The ioassay was performed for Lepidoptera species, SWCB, ECB, and VBC, and for Coleoptera species, CPB and WC. R and showed activity against Hemiptera species, TPB, SGB, and NBSB.

[0212] PirA protein TIC7939 (SEQ ID NO: 43) and PirB protein TIC79 An operon containing two ORFs encoding 40 (SEQ ID NO: 45), respectively, has the sequence Microbial toxins encoding the protein toxins presented as SEQ ID NO: 44 and SEQ ID NO: 46 It was discovered in DNA from an unknown bacterial species contained within the genome. , DNA sequence, PirAB fusion protein encoding TIC9321 (SEQ ID NO: 47) wherein the two coding sequences are operably linked in frame to generate SEQ ID NO:4 The TIC9321 PirAB fusion protein, presented as 8, was generated.

[0213] PirA protein TIC10357 (SEQ ID NO: 57) and PirB protein TIC1 The operon containing two ORFs encoding 0366 (SEQ ID NO: 59) is Shewa encoding protein toxins presented as SEQ ID NO: 58 and SEQ ID NO: 60 It was discovered in DNA obtained from Nella violacea. The DNA sequence, TIC10375 (SEQ ID NO: 61), is encoded using the A PirAB fusion protein is generated, in which the two coding sequences are engineered in frame. TIC10375 PirAB fusion protein linked to the nucleotide sequence shown in SEQ ID NO: 62 Protein was produced.

[0214] PirA protein TIC10358 (SEQ ID NO: 63) and PirB protein TIC The operon contains two ORFs encoding 10367 (SEQ ID NO: 65), respectively. , Phot encoding the protein toxins presented as SEQ ID NO: 64 and SEQ ID NO: 66 orhabdus luminescens laumondii TTO1 strain Using two ORFs, the DNA sequence, TIC10376 (SEQ ID NO: No. 67), where the two coding sequences are TIC10376 operably linked in frame and presented as SEQ ID NO: 68 PirAB fusion proteins were produced using TIC10358 and TIC10358 derived from microbial host cells. Bioassays using C10367 showed no activity against the insects used in the assay. However, the PirAB fusion protein consisting of TIC10358 and TIC10367 Bioassays using the fusion protein TIC10376 were performed on Lepidoptera species, SWCB and Coleoptera. Order species, northern corn root cutter (Diabrotica barberi, NCR) and It showed activity against HIV-1 and WCR.

[0215] PirA protein TIC10360 (SEQ ID NO: 69) and PirB protein TIC The operon contains two ORFs encoding 10369 (SEQ ID NO: 71), respectively. , Phot encoding the protein toxin presented as SEQ ID NO: 70 and SEQ ID NO: 72 orhabdus asymbiotica. The RF was used to identify the DNA sequence, TIC10377 (SEQ ID NO: 73), encoding PirAB A fusion protein is created, in which the two coding sequences are operably linked in frame. and generate the TIC10377 PirAB fusion protein presented as SEQ ID NO: 74. did.

[0216] PirA protein TIC10361 (SEQ ID NO: 75) and PirB cell protein T The operon containing the two ORFs encoding IC10370 (SEQ ID NO: 77) is Xen encoding protein toxins presented as SEQ ID NO: 76 and SEQ ID NO: 78, respectively. orhabdus spp. Two ORFs were used to encode the DNA sequence TIC10378 (SEQ ID NO: 79). A PirAB fusion protein was generated, in which the two coding sequences were engineered in frame. TIC10378 PirAB fusion protein operably linked to the TIC10378 PirAB fusion protein and presented as SEQ ID NO: 80 Protein was produced.

[0217] PirA protein TIC10362 (SEQ ID NO: 81) and PirB protein TIC The operon contains two ORFs encoding 10371 (SEQ ID NO: 83), respectively. , Yers encoding protein toxins presented as SEQ ID NO: 82 and SEQ ID NO: 84 was found in DNA obtained from strain 670-83 of I. aldovae. The PirAB fusion protein encoding DNA sequence TIC10379 (SEQ ID NO: 85) was used to The two coding sequences are operably linked in frame to produce a fusion protein having sequence no. The TIC10379 PirAB fusion protein, presented as no. 86, was generated.

[0218] PirA protein TIC10363 (SEQ ID NO: 87) and PirB protein TIC The operon contains two ORFs encoding 10372 (SEQ ID NO: 89), respectively. Xenor encoding the protein toxins presented as SEQ ID NO: 88 and SEQ ID NO: 90 It was found in DNA obtained from the FRM16 strain of Habdus doucetiae. Two ORFs were used to construct the Pir encoding DNA sequence TIC10380 (SEQ ID NO: 91). An AB fusion protein is created, in which the two coding sequences are operably linked in frame. The TIC10380 PirAB fusion protein was ligated and presented as SEQ ID NO: 92. Bio-based bioassays using TIC10363 and TIC10372 derived from microbial host cells were performed. The assay showed no activity against the insects used in the assay. PirAB fusion protein TIC1038 composed of 10363 and TIC10372 Bioassays using 0 were performed on Lepidoptera species, FAW, Coleoptera species, NCR and WCR, and Hemiptera. It showed activity against the genus NBSB.

[0219] PirA protein TIC10364 (SEQ ID NO: 93) and PirB protein TIC The operon contains two ORFs encoding 10373 (SEQ ID NO: 95), respectively. , Xeno, encoding the protein toxins presented as SEQ ID NO: 94 and SEQ ID NO: 96 Found in DNA obtained from the BMMCB strain of rhabdus griffiniae Two ORFs were used to construct the P encoding DNA sequence TIC10381 (SEQ ID NO: 97). An irAB fusion protein is generated, in which the two coding sequences are engineered in-frame. TIC10381 PirAB fusion protein linked to and presented as SEQ ID NO: 98 The microbial host cell-derived TIC10364 and TIC10373 were used to generate the microbial protein. The bioassay showed no activity against the insects used in the assay. PirAB fusion protein TIC10 composed of IC10364 and TIC10373 Bioassays using 378 were performed on Coleoptera, NCR and WCR, and Hemiptera species, NBSB. It showed activity against

[0220] PirA protein TIC10359 (SEQ ID NO: 99) and PirB protein TIC The operon containing two ORFs encoding 10368 (SEQ ID NO: 101) is and X encoding the protein toxin presented as SEQ ID NO: 100 and SEQ ID NO: 102. It was discovered in DNA obtained from enorhabdus nematophila. The operon sequence containing both C10359 and TIC10368 is presented as SEQ ID NO: 103 It has been done.

[0221] PirA protein PirA_ABE68878 (SEQ ID NO: 104) and PirB protein Contains two ORFs encoding the protein PirB_ABE68879 (SEQ ID NO: 106) The operons encoding the proteins are presented as SEQ ID NO: 105 and SEQ ID NO: 107, respectively. A protein derived from the Hm strain of Photorhabdus luminescens encoding a phosphotoxin Using two ORFs, the DNA sequence TIC10434 (sequence A PirAB fusion protein encoding the PirAB gene (SEQ ID NO: 108) was generated, in which two coding sequences were The sequences are operably linked in frame and presented as SEQ ID NO: 109 in TIC103 78 PirAB fusion proteins were generated. PirA_ABE68878 and PirB_ A microbial host cell-derived PirAB fusion protein TI, consisting of ABE68879 Bioassays using C10434 showed activity against Coleoptera species, NCR and WCR. did.

[0222] Contains the ORF of the PirB protein encoding TIC11505 (SEQ ID NO: 134) The operon encoding the protein toxin Xenopus is presented as SEQ ID NO: 135. It was discovered in the rhabdus nematophila strain MDI-0035777. The coding sequence of the PirAB fusion protein TIC11056 (SEQ ID NO: 136) is TIC10364 coding sequence operably linked in frame to the 11505 coding sequence TIC11506 PirAB fusion protein, presented as SEQ ID NO: 137, containing the sequence Protein was produced.

[0223] Contains the ORF of the PirB protein encoding TIC11510 (SEQ ID NO: 138) The operon encoding the protein toxin Xenopus is presented as SEQ ID NO: 139. It was discovered in the rhabdus nematophila strain MDI-0035777. The coding sequence of the PirAB fusion protein TIC11512 (SEQ ID NO: 140) is TIC10364 coding sequence operably linked in frame to the 11505 coding sequence TIC11056 PirAB fusion protein, presented as SEQ ID NO: 141, containing the sequence Protein was produced.

[0224] Contains the ORF of the PirB protein encoding TIC11511 (SEQ ID NO: 142) The operon encoding the protein toxin Xenopus is presented as SEQ ID NO: 143. It was discovered in the rhabdus nematophila strain MDI-0035777. The coding sequence of the PirAB fusion protein TIC11513 (SEQ ID NO: 144) is TIC10364 coding sequence operably linked in frame to the 11513 coding sequence TIC11056 PirAB fusion protein, presented as SEQ ID NO: 145, containing the sequence Protein was produced.

[0225] PirAB fusion proteins TIC11210, TIC11211, and TIC1130 1 are PirA protein TIC7575 and PirB protein TIC766, respectively. 5, TIC7667, and TIC7661. PirAB fusion proteins TI C11212 is a PirA protein TIC7662 and a PirB protein TIC76 The PirAB fusion protein TIC11302 containing 65 was synthesized by the PirA protein TIC7 660 and PirB protein TIC7576. PirAB fusion protein TIC 11210 and TIC11211 for Lepidoptera species, SWCB and Hemiptera species, NBSB The PirAB fusion proteins TIC11301 and TIC11302 showed activity. Lepidoptera species, SWCB, ECB, and VBC; Coleoptera species, WCR; and Hemiptera species, NBSB and WTP.

[0226] The PirAB fusion proteins, TIC11103 and TIC11104, The PirAB fusion protein TIC1 contained the PirB protein, which is consistent with the protein 1103 from PirB protein TIC7661 and PirA protein TIC7660 The PirAB fusion protein TTIC11104 is composed of the PirB protein TIC 7663 and the PirA protein TIC7662.

[0227] The PirAB fusion protein TIC11140 is a fusion protein of the PirA protein TIC4771 and P It consists of a copy of the irB protein TIC4472. PirAB fusion protein TI C11141 is the PirA protein TIC7575 and the PirB protein TIC7576 The PirAB fusion protein TIC11142 consists of a copy of the PirA protein. It is composed of proteins TIC7575 and TIC4771 and PirB protein TIC4772. The PirAB fusion protein TIC11443 is synthesized from the PirA protein TIC766 It is composed of PirA and TIC7575 and the PirB protein TIC7576. B fusion protein TIC11444 is a fusion protein of PirA proteins TIC7575 and TIC7 PirAB fusion protein TIC7661 is composed of 660 and PirB protein TIC7661. Protein TIC11445 is a PirA protein TIC7660 and TIC7662 and a Pir The PirAB fusion protein TIC114 is composed of the B protein TIC7663 and the PirAB fusion protein TIC114. 46 is a PirA protein TIC7662 and TIC7660 and a PirB protein T TIC11442, derived from bacterial host cells, is effective against Hemiptera pest species, TIC11444, derived from bacterial host cells, showed activity against Lepidoptera species, SW It showed activity against CB and Hemiptera species, and NBSB.

[0228] As described in the Examples, the PirAB fusion protein TIC6880PL (sequence No. 49), TIC9316 (SEQ ID NO: 51), TIC9317 (SEQ ID NO: 53), TI C9318 (SEQ ID NO: 55), TIC9320 (SEQ ID NO: 57), TIC9322 (SEQ ID NO: No. 59), TIC10376PL (SEQ ID NO: 146), TIC10378PL (SEQ ID NO: No. 148), TIC10380PL (SEQ ID NO: 150), TIC10381PL (SEQ ID NO: No. 152), TIC11103 (SEQ ID NO: 154), TIC11104 (SEQ ID NO: 156 ), and a synthetic DNA sequence encoding TIC11302 (SEQ ID NO: 158) were expressed in plant cells. TIC9316, TIC9317, and TIC9318 were designed for the expression of Maize plants transformed with the binary transformation plasmid constructs expressing It showed activity against the European corn borer and the southwestern corn borer.

[0229] For expression in plant cells, the PirAB fusion protein is expressed in the cytosol. or can be expressed to target various organelles in plant cells. For example, targeting a protein to the chloroplast can The level of protein expressed in transgenic plants can be increased while preventing the development of phenotypes. Targeting may also result in increased pest resistance in transgenic events. The targeting or transport peptide may target the nucleus, mitochondria, Within the cell, including the endoplasmic reticulum (ER), chloroplasts, apoplast, peroxisomes, and plasma membrane A short (3-70 amino acid long) peptide chain that directs protein transport to a specific region Some target peptides are translocated by signal peptidases after the protein is transported. The protein is cleaved from the chloroplast. To target the protein to the chloroplast, approximately 40-50 amino acids are added. The chloroplast transit peptide is a chloroplast transit peptide. See Patent Nos. 5,188,642 and 5,728,925. The protein localized in the chloroplast is expressed as a precursor from a nuclear gene and encodes a chloroplast transit peptide (C TP) to the chloroplast. Examples of such isolated chloroplast proteins Contains ribulose-1,5-bisphosphate carboxylase, ferredoxin, and ferroxin. Synthon oxidoreductase, light-harvesting complex protein I and protein II, thioredoxin phenanthrene F, enolpyruvylshikimate phosphate synthase (EPSPS), and U.S. Pat. The small subunit (SSU) of the transport peptide described in Patent No. 7,193,133 Non-chloroplast proteins include, but are not limited to, heterologous CT proteins. It may be targeted to the chloroplast by using a protein fusion with P, CTP is sufficient to target proteins to chloroplasts in vivo and in vitro. For example, EPSPS of Arabidopsis thaliana CTP(CTP2)(Klee et al.,Mol.Gen.210:437-44 2, 1987) or Petunia hybrida EPSPS CT P(CTP4)(della-Cioppa et al.,Proc.Natl.Ac Ad. Sci. USA, 83:6873-6877, 1986). The incorporation of a suitable chloroplast transit peptide allows the heterologous E to enter the chloroplasts in transgenic plants. It has been shown to target PSPS protein sequences (U.S. Patent No. 5,627 ,061; EP 5,633,435; and EP 5,312,910; and EP0 See EP 218571; EP 189707; EP 508909; and EP 924299. To target one of the PirAB fusion proteins to the chloroplast, The sequence encoding the transit peptide was inserted into Pi, which is designed for optimal expression in plant cells. operably linked and linked to a synthetic coding sequence encoding one of the rAB fusion proteins. Place it in the 5' position in frame.

[0230] Associated with PirA protein, PirB protein, and PirAB fusion protein Additional toxin protein sequences include PirA protein, PirB protein, and PirA B. Using the naturally occurring amino acid sequence of the fusion protein, differences at the amino acid sequence level were identified. Incorporating the novel amino acid sequence variants and making appropriate changes to the recombinant nucleic acid sequence encoding the variants. It is contemplated that the method can be produced by performing the method.

[0231] The disclosure further provides PirA proteins, PirB proteins, and PirAB fusion proteins. Improved variants of proteins can be engineered using various gene editing methods known in the art. It is intended that the gene can be manipulated in plants by using the gene for genome editing. Such technologies include ZFN (zinc finger nucleases), meganucleases, T ALEN (activator-like effector nuclease), and CRISPR (cluster CRISPR (Cat 3D Recombinant Regularly Interspaced Short Palindromic Repeats) / Cas These genome editing methods include, but are not limited to, the following: , the transformed toxin protein coding sequence in the plant cell is changed to a different toxin coding sequence. Specifically, new protein amino acid sequences can be obtained through these methods. Alternatively, one or more codons within the toxin coding sequence may be altered to manipulate the Substituting or deleting fragments within a sequence or inserting additional DNA fragments into the coding sequence The new coding sequence is then used to engineer a new toxin coding sequence that is highly active against pests or It is possible to encode toxin proteins with new properties, such as specificity or spectrum. provides activity against pest species that have developed resistance to the original insect toxin protein The gene-edited toxin coding sequence can be prepared by methods known in the art. Plant cells containing the virulence factor can be used to generate whole plants expressing new toxin proteins. do.

[0232] Similar to PirA protein, PirB protein, and PirAB fusion protein Proteins can be identified using various computer-based algorithms known in the art. For example, PirA protein can be identified by comparing it with other proteins. Amino acids of proteins associated with proteins, PirB proteins, and PirAB fusion proteins Acid sequence identity is calculated using the following default parameters: weight matrix: blosum, gap Gap opening penalty: 10.0, Gap widening penalty: 0.05, Hydrophilic gap: C with on, hydrophilic residues: GPSNDQERK, residue-specific gap penalty: on can be analyzed using lustal W sequence comparison (Thompson, et al. l.,(1994)Nucleic Acids Research,22:4673- 4680). The percent amino acid identity is further multiplied by 100% (amino acid identity / target Other sequence comparison algorithms are also known in the art. available in the field and results similar to those obtained using Clustal W sequence comparisons to provide.

[0233] Proteins that exhibit insect-inhibitory activity against insect species of the orders Lepidoptera, Coleoptera, or Hemiptera are Sequence comparison of a query protein such as TIC7939 with the subject protein Between protein and protein, approximately 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72% ,73%,74%,75%,76%,77%,78%,79%,80%,81%,82% ,83%,84%,85%,86%,87%,88%,89%,90%,91%,92% , 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% of amino acid sequences identity (or any fraction of this range) along the length of the query protein or if the query shows at least 65% to about 100% amino acid identity; The sequence of the protein is compared with that of TIC7664 or TIC7666. Approximately 97%, 98%, 99%, or 100% amino acid identity (or at least 97% along the length of the query protein that is any fraction of this range of ratios ~100% amino acid identity; or TI with such a query protein The sequence comparison of C4771 between the query protein and the subject protein was approximately 98% and 99%. , a query with 100% amino acid sequence identity (or any portion of this range of percentages) show at least 98% to approximately 100% amino acid identity along the length of the protein; or or such a query protein and TIC7575, TIC7660, TIC7662 , TIC7668, TIC10357, TIC10358, TIC10360, TIC1 0361, TIC10362, TIC10364, TIC10359, or PirA_ Sequence comparison with ABE68878 was 100% between the query protein and the subject protein. When identity is shown, it is intended to be with respect to the PirA protein.

[0234] Proteins that exhibit insect-inhibitory activity against insect species of the orders Lepidoptera, Coleoptera, or Hemiptera are Sequence comparison of a query protein such as TIC7940 with the subject protein Between protein and protein, approximately 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72% ,73%,74%,75%,76%,77%,78%,79%,80%,81%,82% ,83%,84%,85%,86%,87%,88%,89%,90%,91%,92% , 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% of amino acid sequences identity (or any fraction of this range) along the length of the query protein or if the query shows at least 65% to about 100% amino acid identity; Sequence comparison of the query protein with TIC4772 reveals a significant difference between the query protein and the subject protein. Approximately 97%, 98%, 99%, 100% amino acid identity (or any percentage within this range) At least 97% to approximately 100% of the amino acids along the length of the query protein are If it shows identity; or if it shows identity with such query protein, TIC7665, TIC76 67 or TIC10368 sequence comparison between the query protein and the subject protein and about 98%, 99%, 100% amino acid sequence identity between the two or any of the percentages within this range. At least 98% to about 100% amino acid identity along the length of the query protein (part or if the query protein and TIC7576, TIC766 1, TIC7663, TIC7669, TIC10366, TIC10367, TIC1 0369, TIC10370, TIC10371, TIC10372, TIC10373 , PirB_ABE68879, TIC11510, or TIC11511 Comparison shows 100% amino acid sequence identity between the query protein and the subject protein. When used herein, it is intended to refer to the PirB protein.

[0235] Proteins that exhibit insect-inhibitory activity against insect species of the orders Lepidoptera, Coleoptera, or Hemiptera are Query proteins such as TIC9321, TIC11411, TIC11443, and T Sequence comparison with IC11444, TIC11445, TIC11446, and TIC11513 The correlation between the query protein and the subject protein was approximately 65%, 66%, 67%, 68%, and 6 9%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 7 9%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 8 9%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 9 9%, 100% amino acid sequence identity (or any portion of this range). At least 65% to approximately 100% amino acid identity along the length of the protein or such query protein TIC10434, TIC11440, or Sequence comparison with TIC11442 was approximately 70% between the query protein and the subject protein. ,71%,72%,73%,74%,75%,76%,77%,78%,79%,80% ,81%,82%,83%,84%,85%,86%,87%,88%,89%,90% ,91%,92%,93%,94%,95%,96%,97%,98%,99%,100 % amino acid sequence identity (or any portion of this range) with the query protein exhibits at least 70% to about 100% amino acid identity along the length of the Query proteins such as TIC9316, TIC9317, TIC9318, and TIC9 322, TIC9320, TIC10375, TIC10376, TIC10377, T IC10378, TIC10379, TIC10381, TIC11211, TIC11 301, TIC11302, TIC10376PL, TIC10378PL, TIC10 Sequence comparison with 381PL, TIC11103, or TIC11104 was performed on the query protein. Approximately 80%, 81%, 82%, 83%, 84%, 85%, and 90% of the chromatin was found to be chromatic aberrations between the chromatin and the subject protein. 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% amino acid sequence identity (or ratios within this range) at least 80% to approximately 100% along the length of the query protein (any portion of the or the amino acid identity of such query protein TIC9319, T IC10380, TIC11210, TIC11212, or TIC10380PL The sequence comparison between the query protein and the subject protein was approximately 82%, 83%, and 84%. ,85%,86%,87%,88%,89%,90%,93%,94%,95%,96% , 97%, 98%, 99%, 100% amino acid sequence identity (or any percentage within this range) At least 82% to approximately 100% amino acid sequence along the length of the query protein (part of the intended sequence) or TIC6880 or TIC6880 of such query protein. The sequence comparison with IC6880PL was approximately 86% between the query protein and the subject protein. ,87%,88%,89%,90%,91%,92%,93%,94%,95%,96% , 97%, 98%, 99%, 100% amino acid sequence identity (or any percentage within this range) At least 86% to approximately 100% amino acid sequence along the length of the query protein (part of the intended sequence) or TIC11506 or T of such query protein The sequence comparison with IC11512 was approximately 94% between the query protein and the subject protein; 95%, 96%, 97%, 98%, 99%, 100% amino acid sequence identity (or at least 94% to approximately 100% along the length of the query protein (any portion of the ratio in the range When 100% amino acid identity is shown, it is intended to refer to a PirAB fusion protein. can be.

[0236] Exemplary PirA proteins include TIC4771, TIC7575, TIC7660, T IC7662, TIC7664, TIC7666, TIC7668, TIC7939, T IC10357, TIC10358, TIC10360, TIC10361, TIC10 362, TIC10363, TIC10364, TIC10359, and PirA_AB E68878 were aligned to each other using the Clustal W algorithm. As reported, the amino acid sequence identity percentage for each of the full-length proteins A matrix of cent pairs was created. TIFF2026010055000004.tif138165TIFF2026010055000005.tif138166

[0237] Exemplary PirB proteins include TIC4772, TIC7576, TIC7661, T IC7663, TIC7665, TIC7667, TIC7669, and TIC7940 were aligned with each other using the Clustal W algorithm. To do this, we create a pairwise matrix of percent amino acid sequence identity for full-length proteins. Successful. TIFF2026010055000006.tif155168TIFF2026010055000007.tif150165

[0238] Exemplary PirAB fusion proteins TIC6880, TIC9316, TIC9317 , TIC9318, TIC9319, TIC9322, TIC9320, TIC9321 ,TIC6880PL,TIC10375,TIC10376,TIC10377,TI C10378, TIC10379, TIC10380, TIC10381, TIC104 34, TIC11210, TIC11211, TIC11212, TIC11301, T IC11302, TIC11440, TIC11441, TIC11442, TIC11 443, TIC11444, TIC11445, TIC11446, TIC11506, TIC11512, TIC11513, TIC10376PL, TIC10378PL, TIC10380PL, TIC10381PL, TIC11103, and TIC1110 4 were aligned with each other using the Clustal W algorithm. Tables 8, 9, 10, and 11 As reported in

[2012] , the amino acid sequence identity percentages for each of the full-length proteins were A pairwise matrix of cents was created. TIFF2026010055000008.tif252167TIFF2026010055000009.tif252168TIFF2026010055000010.tif24816 8TIFF2026010055000011.tif35167TIFF2026010055000012.tif249169TIFF2026010055000013.tif36165

[0239] In addition to the percent identity, the PirA protein, the PirB protein, and the PirA B fusion proteins are differentiated by their primary structure (conserved amino acid motifs) and length (Pir PirA is about 133 to about 141 amino acids, and PirB is about 414 to about 428 amino acids. amino acids, and about 549 to about 566 amino acids for the PirAB fusion protein) The PirA and PirB proteins can be related by their specificity and other characteristics. The properties of the protein and the PirAB fusion protein are reported in Table 12. TIFF2026010055000014.tif250167TIFF2026010055000015.tif251167TIFF2026010055000016.tif109164

[0240] As further described in the Examples of this application, a PirAB fusion protein encoding The sequence of the recombinant nucleic acid molecule is designed for use in plants. Exemplary recombinant nucleic acid molecule sequences that have been optimized for plants are set forth in SEQ ID NOs: 49, 51, 52, 53, 54, 55, 56, 146, 148, 150, 152, 154, 156, and 15 It is presented as 8.

[0241] Expression cassettes and vectors containing the sequences of these recombinant nucleic acid molecules are well known in the art. Constructed and transformed into corn, soybean, cotton or other plants according to known transformation methods and techniques. For example, Agrobacterium-mediated Transformation was performed according to the methods described in U.S. Patent Application Publication Nos. 2009 / 0138985A1 (soybean), 2008 / 0280361A1 (soybean), 2009 / 0142837A1 (corn), 20 08 / 0282432 (cotton), 2008 / 025667 (cotton), 2003 / 0110 531 (wheat), 2001 / 0042257A1 (sugar beet), U.S. Pat. No. 5,750 No. 7,026,528 (wheat), and No. 6,365 , No. 807 (rice), and Arencibia, et al., (1998), Tran sgenic Res.7:213-222 (sugarcane), and all of The transformed cells contain the PirAB fusion Proteins TIC6880PL, TIC9316, TIC9317, TIC9318, T Transgenic plants expressing TIC9319, TIC9320, or TIC9322 were regenerated. To examine the insecticidal activity, the transformed plants were cultured as described in the Examples. Bioassays were performed in the presence of lepidopteran pest larvae using plant leaf discs obtained from the To examine the insecticidal activity against coleopteran pests, Transformed plants of the R0 and F1 generations are used in rootworm assays. To investigate the insecticidal activity against the pods of transformed plants, either from tissue removed or tissue remaining in the plant, Either panicles or leaves are used in the assay.

[0242] As an alternative to traditional transformation methods, DNA such as transgenes, expression cassette(s), etc. The A sequence is integrated into a specific site or region in the genome of a plant or plant cell via site-specific integration. may be inserted or integrated into the locus. The NA construct(s) and molecule(s) are inserted into the genome of the plant or plant cell. a donor containing at least one transgene, expression cassette, or other DNA sequence for Such donor templates for site-specific integration may also contain template sequences. The insert may further comprise an insertion sequence (i.e., a sequence to be inserted into the plant genome, a transgene, a cassette, The recombinant DNA construct of the present disclosure may comprise one or two homology arms flanking the homologous sequence (e.g., a nucleotide sequence). The entity(ies) may further comprise a site-specific nuclease and / or a gene encoding a gene that performs site-specific integration. The expression cassette(s) may also include an expression cassette(s) encoding any relevant proteins for the These nuclease expression cassette(s) are the same molecule or vector as the donor template. It may be present in a vector (cis) or in another molecule or vector (trans). Cleave the NA to create a double-strand break (DSB) or nick at the desired genomic site or locus Various proteins (or protein complexes and / or guide RNAs) that produce Several methods for site-specific integration are known in the art, including As understood in the art, repairing a DSB or nick introduced by a nuclease enzyme is During the repair process, the donor template DNA is integrated into the genome at the site of the DSB or nick. The insertion event occurs via non-homologous end joining (NHEJ). However, the presence of homology arms(s) in the donor template may facilitate homologous recombination. The method may facilitate the introduction and targeting of insertion sequences into the plant genome during the repair process via Examples of site-specific nucleases that may be used include zinc finger nucleases. , engineered or native meganucleases, TALE endonucleases, and RNA-guided endonucleases (e.g., Cas9 or Cpf1) are included. Methods using A-derived site-specific nucleases (e.g., Cas9 or Cpf1) In some cases, the recombinant DNA construct(s) also contain a nucleic acid sequence that is inserted into the desired site within the plant genome. The nucleic acid sequence may also include a sequence encoding one or more guide RNAs for directing the enzyme.

[0243] PirA protein, PirB protein, or PirAB fusion protein, or Recombinant nucleic acid molecule compositions encoding relevant insecticidal proteins are contemplated. For example, P irA protein, PirB protein, or PirAB fusion protein, or related The insecticidal protein is a polynucleotide molecule comprising an ORF encoding the protein. , e.g., promoters and other regulatory elements necessary for expression in the system in which the construct is intended. Expressed in a recombinant DNA construct operably linked to a gene expression element such as a Non-limiting examples include PirAB fusion proteins, TIC6880PL, T IC9316, TIC9317, TIC9318, TIC9319, TIC9320, T IC9322, TIC10376PL, TIC10378PL, TIC10380PL, TIC10381PL, TIC11103, TIC11104, and TIC11302 or related family members encoding sequences for the expression of proteins in plants. a promoter functional in plants operably linked to an insecticidal protein, or PirA; PirA protein such as _ABE68878 or PirB protein TIC4 772, TIC7576, TIC7661, TIC7663, TIC7665, TIC7 667, TIC7669, TIC7940, TIC10366, TIC10367, TI C10369, TIC10370, TIC10371, TIC10372, TIC103 73, TIC10368, PirB_ABE68879, TIC11505, TIC11 510 and TIC11511; or PirAB fusion protein, TIC6880, T IC9316, TIC9317, TIC9318, TIC9319, TIC9322, T IC9320, TIC9321, TIC6880PL, TIC10375, TIC103 76, TIC10377, TIC10378, TIC10379, TIC10380, T IC10381, TIC10434, TIC11210, TIC11211, TIC11 212, TIC11301, TIC11302, TIC11440, TIC11441, TIC11442, TIC11443, TIC11444, TIC11445, TIC1 1446, TIC11506, TIC11512, TIC11513, TIC10376 PL, TIC10378PL, TIC10380PL, TIC10381PL, TIC1 1103, and TIC11104; or Bt bacteria or other Bacillus species operably to encode sequences for the expression of the protein in the relevant insecticidal protein The Bt promoter is functionally linked to enhancers, introns, and non- The translation leader, the encoded protein immobilization tag (HIS tag), the translocation peptide (i.e. plastid transit peptides, signal peptides), polypeptides for post-translational modification enzymes These include, but are not limited to, nucleotide sequences, ribosome binding sites, and RNAi target sites. Other elements include PirA protein, PirA_ABE68878, PirB protein, TIC4772, TIC7576, TIC7661, TIC7663, TIC7665, TIC7667, TIC7669, TIC7940, TIC10366, TIC1036 7, TIC10369, TIC10370, TIC10371, TIC10372, TI C10373, TIC10368, PirB_ABE68879, TIC11505, T IC11510 and TIC11511, or the PirAB fusion protein, TIC68 80, TIC9316, TIC9317, TIC9318, TIC9319, TIC93 22, TIC9320, TIC9321, TIC6880PL, TIC10375, TI C10376, TIC10377, TIC10378, TIC10379, TIC103 80, TIC10381, TIC10434, TIC11210, TIC11211, T IC11212, TIC11301, TIC11302, TIC11440, TIC11 441, TIC11442, TIC11443, TIC11444, TIC11445, TIC11446, TIC11506, TIC11512, TIC11513, TIC1 0376PL, TIC10378PL, TIC10380PL, TIC10381PL, TIC11103 and TIC11104 or related insecticidal protein coding sequences They can be operably linked.

[0244] Exemplary recombinant polynucleotide molecules provided herein include, for example, those having SEQ ID NO: No. 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 1 4, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, Sequence number 38, sequence number 40, sequence number 42, sequence number 44, sequence number 46, sequence number 48 , SEQ ID NO: 50, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:74, SEQ ID NO:76, SEQ ID NO: No. 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 92, SEQ ID NO: 94, SEQ ID NO: 96, SEQ ID NO: 98, SEQ ID NO: 1 00, SEQ ID NO: 102, SEQ ID NO: 105, SEQ ID NO: 107, SEQ ID NO: 109, SEQ ID NO: 1 11, SEQ ID NO: 113, SEQ ID NO: 115, SEQ ID NO: 117, SEQ ID NO: 119, SEQ ID NO: 1 21, SEQ ID NO: 123, SEQ ID NO: 125, SEQ ID NO: 127, SEQ ID NO: 129, SEQ ID NO: 1 31, SEQ ID NO: 133, SEQ ID NO: 135, SEQ ID NO: 137, SEQ ID NO: 139, SEQ ID NO: 1 41, SEQ ID NO: 143, SEQ ID NO: 145, SEQ ID NO: 147, SEQ ID NO: 149, SEQ ID NO: 1 51, SEQ ID NO: 153, SEQ ID NO: 155, and SEQ ID NO: 157. SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, Sequence number 5, sequence number 7, sequence number 9, sequence number 11, sequence number 13, sequence number 15, sequence No. 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 3 9, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, Sequence number 57, sequence number 59, sequence number 61, sequence number 63, sequence number 65, sequence number 67 , SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:89, SEQ ID NO: No. 91, SEQ ID NO: 93, SEQ ID NO: 95, SEQ ID NO: 97, SEQ ID NO: 99, SEQ ID NO: 101 , SEQ ID NO: 104, SEQ ID NO: 106, SEQ ID NO: 108, SEQ ID NO: 110, SEQ ID NO: 112 , SEQ ID NO: 114, SEQ ID NO: 116, SEQ ID NO: 118, SEQ ID NO: 120, SEQ ID NO: 122 , SEQ ID NO: 124, SEQ ID NO: 126, SEQ ID NO: 128, SEQ ID NO: 130, SEQ ID NO: 132 , SEQ ID NO: 134, SEQ ID NO: 136, SEQ ID NO: 138, SEQ ID NO: 140, SEQ ID NO: 142 , SEQ ID NO: 144, SEQ ID NO: 146, SEQ ID NO: 148, SEQ ID NO: 150, SEQ ID NO: 152 , SEQ ID NO: 154, SEQ ID NO: 156, and SEQ ID NO: 158. Examples of heterologous promoters include, but are not limited to, heterologous promoters operably linked to the promoter. The motors TIC6880PL, TIC9316, and TIC93 also target plastids. 17, TIC9318, TIC9319, TIC9320, TIC9322, TIC10 376PL, TIC10378PL, TIC10380PL, TIC10381PL, T TIC11103, TIC11104, and TIC11302, or non-targeted TIC 6880PL, TIC9316, TIC9317, TIC9318, TIC9319, T IC9320, TIC9322, TIC10376PL, TIC10378PL, TIC 10380PL, TIC10381PL, TIC11103, TIC11104 and TI engineered into a synthetic DNA coding sequence encoding C11302 or a related insecticidal protein Recombinant proteins encoding the proteins disclosed herein may also be linked to Codons in nucleic acid molecules may be substituted with synonymous codons (known in the art as silent substitutions). can be replaced by

[0245] As used herein, the term "recombinant" refers to a gene that is not normally found in nature and is "Recombinant" refers to non-naturally occurring DNA, proteins, or organisms created through human intervention. "DNA molecules" are DNA molecules that would not exist together in nature and are the result of human intervention. For example, a DNA molecule containing a transgene and a At least two DNA fragments heterologous to each other, such as the plant genomic DNA flanking the transgene. A DNA molecule composed of a combination of A molecules is a recombinant DNA molecule.

[0246] As used herein, the term "heterologous" refers to the nature of such combinations. refers to a combination of two or more DNA molecules that is not found in the genome. For example, The offspring may be from different species and / or the two DNA molecules may contain different genes, e.g. For example, different genes from the same species or the same gene from different species Therefore, regulatory elements may be used in combinations where such combinations are not normally found in nature. That is, when the transcribable DNA molecule does not naturally occur operably linked to regulatory elements. , heterologous with respect to the operably linked transcribable DNA molecule.

[0247] PirA protein, PirB protein, or PirAB fusion protein, or The recombinant DNA construct containing the coding sequence for the relevant insecticidal protein may further comprise the PirA tag. Protein, PirB protein, or PirAB fusion protein, or related insecticide DNA sequences encoding functional proteins, insect-inhibitory dsRNA molecules, or accessory proteins One or more insect inhibitors can be expressed or configured to be co-expressed in the sequence. Accessory proteins can include regions of DNA that encode cofactors, enzymes, and binding proteins. The partner, or for example, aids its expression and influences its stability in plants, Optimizing the free energy for merization, enhancing its toxicity, and improving its activity spectrum. Other agents that function to aid in the effectiveness of insect inhibitors by increasing the The accessory protein may be, for example, a protein that is involved in the uptake of one or more insect inhibitors. The toxic agent may facilitate uptake or may enhance the toxic effect of the toxic agent.

[0248] so that all proteins or dsRNA molecules are expressed from one promoter , or so that each protein or dsRNA molecule is under the control of a separate promoter or any combination thereof. The PirA protein, PirB protein, or PirAB fusion protein of the present invention can be used. The protein and related proteins include PirA protein, PirB protein, or The PirAB fusion protein, or one or more related proteins, may be expressed in the selected expression system. Optionally, a common nucleotide sequence containing other open reading frames and promoters may be used. The gene can be expressed from a multigene expression system in which the gene is expressed from a sequence segment. For example, Bacterial multigene expression systems utilize a single promoter to express multiple genes from within a single operon. Expression of multiple / tandem open reading frames (i.e., polycistronic expression) In another example, a plant multi-gene expression system can drive different genes. multiply linked vectors expressing a target protein or other agent such as one or more dsRNA molecules. It is possible to use an expression cassette that is not specifically designed.

[0249] PirA protein, PirB protein, or PirAB fusion protein, or Recombinant nucleic acid molecules or compositions containing sequences encoding related family member proteins Recombinant DNA constructs can be vectors, such as plasmids, baculoviruses, synthetic chromosomes, virions, cosmids, phagemids, phages, or viral vectors to deliver the virus to host cells Such vectors can be used to deliver the PirA protein, Pir B protein, or PirAB fusion protein, or related proteins in the host cell Stable or transient expression of the coding sequence or the encoded polypeptide Subsequent expression can be achieved by inducing a gene encoding a protein in a host cell. The introduced exogenous recombinant polynucleotide or recombinant DNA construct is referred to herein as It is called a "transgene."

[0250] PirA protein, PirB protein, or PirAB fusion protein, or Recombinant polynucleotides expressing any one or more of the sequences encoding the relevant proteins transgenic bacteria, transgenic plant cells, transgenic plants containing Plants and parts of transgenic plants. The term "bacterial cell" or "bacteria" , Agrobacterium, Bacillus, Escherichia, Salm May contain cells of Onella, Pseudomonas, or Rhizobium The term "plant cell" or "plant" refers to a dicotyledonous plant. The cells may include, but are not limited to, plant cells or monocotyledonous plant cells. Plants and plant cells include alfalfa, banana, barley, beans, broccoli, and cabbage. Beets, brassicas, carrots, cassava, castor beans, cauliflower, celery, chickpeas Rice, Chinese cabbage, citrus fruits, coconut, coffee, corn, clover, cotton, melon , cucumber, Douglas fir, eggplant, eucalyptus, flax, garlic, grapes, hops, leeks, Lettuce, loblolly pine, millet, melon, nuts, oats, olives, onions, Ornamental plants, palms, grasses, peas, peanuts, pepper, pigeon peas, pine, Potato, poplar, pumpkin, radiata pine, radish, nata Neem, rice, rhizome, rye, safflower, shrub, sorghum, southern pine, soybean, bay leaf Spinach, squash, strawberries, sugar beets, sugarcane, sunflowers, sweetcorn Sweet corn, sweet gum, sweet potato, switchgrass, tea, tobacco, tomatoes, lamb Plant cells or plants include, but are not limited to, wheat, turfgrass, watermelon, and wheat. In another embodiment, the transgenic plants regenerated from the transgenic plant cells are In certain embodiments, transgenic plants and parts of transgenic plants are provided. Transgenic plants are those that have been cut, broken, crushed, or otherwise separated from the plant. In certain embodiments, the plant can be obtained from transgenic seeds by A part of a plant is a seed, capsule, leaf, flower, stem, root, or any part thereof, or a tiger It can be a non-regenerative part of a transgenic plant. When transgenic plants are grown, some "non-regenerable" parts of the plants form whole plants. or whole plants capable of sexual and / or asexual reproduction. In certain embodiments, a non-regenerable part of a plant is a part that cannot be induced to regenerate. The portion may be a part of a seed, capsule, leaf, flower, stem, or root of a transgenic plant.

[0251] an insect, Coleoptera or Lepidoptera or Hemiptera inhibiting amount of TIC6880PL, TIC9 316, TIC9317, TIC9318, TIC9319, TIC9320, TIC9 322, TIC10376PL, TIC10378PL, TIC10380PL, TIC 10381PL, TIC11103, TIC11104, or TIC11302 or Methods for producing transgenic plants containing the relevant proteins are provided. The plant may be any of TIC6880PL, TIC9316, TIC9 317, TIC9318, TIC9319, TIC9320, TIC9322, TIC1 0376PL, TIC10378PL, TIC10380PL, TIC10381PL, TIC11103, TIC11104, or TIC11302, or related proteins and introducing a recombinant polynucleotide encoding the compound into a plant cell, and and selecting a plant derived from said plant cell that expresses an amount of the protein that inhibits the growth of the genus Pseudomonas or Hemiptera. Plants can be produced by regeneration, seeds, pollen, or meristematic tissue. can be derived from plant cells by conventional transformation techniques. This method is known in the art.

[0252] The processed product contains detectable amounts of TIC6880PL, TIC9316, TIC9317, and T IC9318, TIC9319, TIC9320, TIC9322, TIC10376P L, TIC10378PL, TIC10380PL, TIC10381PL, TIC11 103, TIC11104, or TIC11302, or related proteins, Processed plant products or any characteristic parts thereof containing insect-inhibiting segments or fragments. Also disclosed in the present application. In certain embodiments, the processed product comprises a plant part, Plant biomass, oil, meal, sugar, animal feed, wheat flour, flakes, bran, lint In certain embodiments, the processed product is selected from the group consisting of hulls, processed seeds, and seeds. Plant products are derived from transgenic plants or transgenic plants. It may include goods or other products of commerce derived from parts of an object, and the goods or other products , TIC6880PL, TIC9316, TIC9317, TIC9318, TIC93 19, TIC9320, TIC9322, TIC10376PL, TIC10378PL , TIC10380PL, TIC10381PL, TIC11103, TIC11104 or TIC11302; or encoding or containing a characteristic portion of a related protein by detecting nucleotide segments or expressed RNA or proteins containing It can be traced through commerce.

[0253] TIC6880PL, TIC9316, TIC9317, TIC9318, TIC93 19, TIC9320, TIC9322, TIC10376PL, TIC10378PL , TIC10380PL, TIC10381PL, TIC11103, TIC11104 Plants expressing TIC11302, or related proteins, have been shown to inhibit the growth of other toxic proteins. expressing proteins and / or conferring herbicide tolerance genes, yield or stress tolerance traits, etc. Transgenic events that express other transgenic traits, such as genes that contribute to They can be crossed by mating with or by crossing with the Such traits can be combined in a single vector.

[0254] As further described in the Examples, TIC6880PL, TIC9316, TIC 9317, TIC9318, TIC9319, TIC9320, TIC9322, TIC 10376PL, TIC10378PL, TIC10380PL, TIC10381PL The sequences encoding TIC11103, TIC11104, and TIC11302 are These synthetic or artificial nucleotide sequences are designed for use in Expression cassettes and expression vectors containing sequences can be prepared using methods known in the art for transformation. According to the methods and techniques, the vectors are constructed and introduced into plant cells of corn, cotton, and soybean. The transformed cells are TIC6880PL, TIC9316, and TIC931 7, TIC9318, TIC9319, TIC9320, TIC9322, TIC103 76PL, TIC10378PL, TIC10380PL, TIC10381PL, TI It was observed that the cells expressed TIC11103, TIC11104, and TIC11302. To examine the insecticidal activity, the transformants were cultured on Lepidoptera, Coleoptera, and Hemiptera. Bioassays are performed in the presence of pests.

[0255] As further described in the Examples, TIC6880PL, TIC9316, TIC 9317, TIC9318, TIC9319, TIC9320, TIC9322, TIC 10376PL, TIC10378PL, TIC10380PL, TIC10381PL , TIC11103, TIC11104, or TIC11302 or related tampering sequences encoding proteins and having a substantial percentage of identity to these proteins The sequences can be analyzed by techniques known in the art, such as polymerase chain reaction (PCR), thermal amplification, and hybridization. For example, TIC6880PL, TIC93 16, TIC9317, TIC9318, TIC9319, TIC9320, TIC93 22, TIC10376PL, TIC10378PL, TIC10380PL, TIC1 0381PL, TIC11103, TIC11104, or TIC11302 protein The protein or related proteins can be used to raise antibodies that specifically bind to the related proteins. To select and find other closely related protein members that can be used. It can be used to

[0256] In addition, TIC6880PL, TIC9316, TIC9317, TIC9318, T IC9319, TIC9320, TIC9322, TIC10376PL, TIC103 78PL, TIC10380PL, TIC10381PL, TIC11103, TIC1 Nucleotides encoding the 1104 or TIC11302 protein or related proteins The nucleic acid sequences were used as probes and primers for screening and thermocycling. Identify other members of the class using linear or isothermal amplification and hybridization techniques. For example, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53 , SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 146, SEQ ID NO: 148, SEQ ID NO: SEQ ID NO: 150, SEQ ID NO: 152, SEQ ID NO: 154, SEQ ID NO: 156, and SEQ ID NO: 158 Oligonucleotides derived from the sequences described above may be used to identify nucleotides derived from commercial products. TIC6880PL, TIC9316, TIC9317 in deoxyribonucleic acid samples TIC9318, TIC9319, TIC9320, TIC9322, TIC10376 PL, TIC10378PL, TIC10380PL, TIC10381PL, TIC1 1103, TIC11104, or TIC11302 protein or related proteins The presence or absence of a transgene can be determined using oligonucleotides. Given the sensitivity of certain nucleic acid detection methods, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 52, Sequence number 53, sequence number 54, sequence number 55, sequence number 56, sequence number 146, sequence number 1 48, SEQ ID NO: 150, SEQ ID NO: 152, SEQ ID NO: 154, SEQ ID NO: 156 and SEQ ID NO: 158 to prepare commercial products using oligonucleotides derived from the sequences described in Only some of the sequences are SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, and SEQ ID NO: 54. , SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 146, SEQ ID NO: 148, SEQ ID NO: 150, A vector containing sequence number 152, sequence number 154, sequence number 156, or sequence number 158 TIC68 in commercial products derived from pooled sources derived from transgenic plants 80PL, TIC9316, TIC9317, TIC9318, TIC9319, TIC 9320, TIC9322, TIC10376PL, TIC10378PL, TIC10 380PL, TIC10381PL, TIC11103, TIC11104, or TI It is expected that the transgene of C11302 can be detected. Using the code, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54 , SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 146, SEQ ID NO: 148, SEQ ID NO: 150, Nucleotides in sequence number 152, SEQ ID NO: 154, SEQ ID NO: 156, or SEQ ID NO: 158 It is further recognized that mutations in the gene sequence can be introduced. The "allergenic" oligonucleotides are used to induce the growth of various insect inhibitors in transgenic plant host cells. TIC6880PL, TIC9316, and TIC9317, which show anti-cancer activity or diverse expression. TIC9318, TIC9319, TIC9320, TIC9322, TIC10376 PL, TIC10378PL, TIC10380PL, TIC10381PL, TIC1 1103, TIC11104, or TIC11302, or related amino acid sequence variants It is useful for identifying variants.

[0257] Homologs of the nucleotide sequences, e.g., those disclosed in this application, may be hybridized under hybridization conditions. by nucleotide sequences that hybridize to each or any of the sequences The encoded insecticidal protein is also an embodiment of the present invention. and detecting a first nucleotide sequence that hybridizes to a nucleotide sequence. wherein the first nucleotide sequence (or its reverse complement) is an insecticidal protein or or an insecticidal fragment thereof, and a second nucleic acid encoding the same under stringent hybridization conditions. In such cases, the second nucleotide sequence hybridizes to the sequence of the second nucleotide. Under stringent hybridization conditions, SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: No. 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: No. 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 4 1, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, Sequence number 59, sequence number 61, sequence number 63, sequence number 65, sequence number 67, sequence number 69 , SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO: 91, SEQ ID NO: No. 93, SEQ ID NO: 95, SEQ ID NO: 97, SEQ ID NO: 99, SEQ ID NO: 101, SEQ ID NO: 10 4, SEQ ID NO: 106, SEQ ID NO: 108, SEQ ID NO: 110, SEQ ID NO: 112, SEQ ID NO: 11 4, SEQ ID NO: 116, SEQ ID NO: 118, SEQ ID NO: 120, SEQ ID NO: 122, SEQ ID NO: 12 4, SEQ ID NO: 126, SEQ ID NO: 128, SEQ ID NO: 130, SEQ ID NO: 132, SEQ ID NO: 13 4, SEQ ID NO: 136, SEQ ID NO: 138, SEQ ID NO: 140, SEQ ID NO: 142, SEQ ID NO: 14 4, SEQ ID NO: 146, SEQ ID NO: 148, SEQ ID NO: 150, SEQ ID NO: 152, SEQ ID NO: 15 4, SEQ ID NO: 156, and SEQ ID NO: 158. Nucleotide coding sequences can be hybridized to each other under suitable hybridization conditions. The proteins encoded by these nucleotide sequences are cross-reacts with antisera raised against any one of the proteins as defined herein. Such stringent hybridization conditions are at least as stringent as hybridization at 42°C. Formation of a denatured ... followed by 30 washes in 0.5x SSC, 0.1% SDS at 65°C. Washes at higher temperatures are more stringent. Hybridization conditions are generally suitable, for example, at 68°C followed by 0.1% SDS. This comprises a wash at 68°C in 2x SSC.

[0258] Those skilled in the art will appreciate that due to the redundancy of the genetic code, many other sequences are known to encode the PirA protein, Pir Encoding protein B, or a protein related to the PirAB fusion protein and their sequences are consistent with their ability to differentiate either in Bacillus strains or plant cells. To the extent that many such redundant coding sequences function to express insecticidal proteins, Under these conditions, the column does not bind to PirA protein, PirB protein, or PirAB fusion protein. Native Xenorhabdus or Photorha encoding synthase proteins It is an embodiment of the present invention that recognizes, of course, that the nucleotide sequence does not hybridize to the bdus sequence. It will be recognized that the present application provides a PirA protein, a PirB protein, or Sequences encoding PirAB fusion proteins or related proteins and These and other methods known to those skilled in the art can be used to identify sequences having a substantial percentage of identity. The use of identification methods is contemplated.

[0259] TIC6880PL, TIC9316, TIC9317, TIC9318, TIC93 19, TIC9320, TIC9322, TIC10376PL, TIC10378PL , TIC10380PL, TIC10381PL, TIC11103, TIC11104 , or TIC11302 protein or related proteins in insects, especially Lepidoptera, A method for controlling infestation of crops by Coleoptera or Hemiptera is also disclosed in the present application. Such methods include administering to an insect, Coleoptera, or Lepidoptera or Hemiptera inhibiting amount of TIC6880 PL, TIC9316, TIC9317, TIC9318, TIC9319, TIC93 20, TIC9322, TIC10376PL, TIC10378PL, TIC1038 0PL, TIC10381PL, TIC11103, TIC11104, or TIC1 1302, or growing a plant containing a related toxin protein. In certain embodiments, such methods further comprise: (i) TIC6880PL, TIC 9316, TIC9317, TIC9318, TIC9319, TIC9320, TIC 9322, TIC10376PL, TIC10378PL, TIC10380PL, TI C10381PL, TIC11103, TIC11104, or TIC11302 Tan Any composition containing or encoding a protein or related toxin protein may be administered to a plant or is applied to seeds that give rise to plants; and (ii) TIC6880PL, TIC93 16, TIC9317, TIC9318, TIC9319, TIC9320, TIC93 22, TIC10376PL, TIC10378PL, TIC10380PL, TIC1 0381PL, TIC11103, TIC11104, or TIC11302 protein or a polynucleotide encoding a toxin protein associated with the plant. and transforming a plant cell to cause the plant to grow. Generally TIC6880PL, TIC9316, TIC9317, TIC9318, TIC 9319, TIC9320, TIC9322, TIC10376PL, TIC10378 PL, TIC10380PL, TIC10381PL, TIC11103, TIC111 04, or TIC11302 protein, or a related toxin protein, in a composition provided, provided in microorganisms, provided in transgenic plants, or Lepidoptera It is contemplated that the compound may be capable of imparting insect inhibitory activity against Coleoptera or Hemiptera insects. can be.

[0260] In certain embodiments, the PirA protein, the PirB protein, or the PirAB fusion protein The recombinant nucleic acid molecule of the fusion protein or related toxin protein is then subjected to conditions suitable for expression. PirA protein, PirB protein, or PirAB fusion protein or related Recombinant Bacillus or Bacillus sp. transformed to express one of the toxin proteins associated with or any other recombinant bacterial cell. Such compositions are the insecticidal active ingredient. Drying, lyophilization, homogenization, extraction, filtration, centrifugation, sedimentation, or other processes for the preparation of recombinant cell cultures such as Such processes can be carried out by Bacillus or are cell extracts, cell suspensions, cell homogenates, cell lysates, and cells of other insect pathogenic bacteria. The supernatant, cell filtrate, or cell pellet may be obtained. By obtaining the polypeptide from a recombinant cell, a composition comprising the recombinant polypeptide can be prepared from a bacterial cell, a cellular The fungal spores and parasporular inclusions may be used in agricultural insect control spray products or bait The compounds can be formulated for a variety of uses, including as insect inhibitors in oocyte assays. do.

[0261] In one embodiment, to reduce the likelihood of resistance development, the PirA protein, PirB protein, or an insect inhibitor containing a PirAB fusion protein or one of the related proteins The insecticidal composition further exhibits insect-inhibiting activity against the same Lepidoptera, Coleoptera, or Hemiptera insect species. As indicated, PirA protein, PirB protein, or PirAB fusion protein, or at least one additional polypeptide known to those skilled in the art that is different from the related toxin protein. Possible additional polypeptides for such compositions include: Examples include insect-inhibitory proteins and insect-inhibitory dsRNA molecules. An example of the use of such ribonucleotide sequences for the purpose of The method is described in Patent Publication 2006 / 0021087 A1.

[0262] Such additional polypeptides for the control of lepidopteran pests include, for example, Cry1A ( Patent No. 5,880,275), Cry1Ab, Cry1Ac, Cry1A.105, Cry1Ae, Cry1B (U.S. Patent Publication No. 10 / 525,318), Cry1C (U.S. Patent No. 6,033,874), Cry1D, Cry1Da and their mutants, Cry 1E, Cry1F, and Cry1A / F chimeras (U.S. Patent No. 7,070,982; Nos. 6,962,705; and 6,713,063), e.g., TIC836, TI C860, TIC867, TIC869, and TIC1100 (International Publication No. WO201 6 / 061391(A2)), TIC2160 (International Publication WO2016 / 06139 2(A2)), such as, but not limited to, Cry1G, Cry1H, Cry 1I, Cry1J, Cry1K, Cry1L, Cry1-type chimera, Cry2A, Cry 2Ab (U.S. Patent No. 7,064,249), Cry2Ae, Cry4B, Cry6, Cr y7, Cry8, Cry9, Cry15, Cry43A, Cry43B, Cry51Aa 1, ET66, TIC400, TIC800, TIC834, TIC1415, Vip3 A, VIP3Ab, VIP3B, AXMI-001, AXMI-002, AXMI-03 0, AXMI-035, AND AXMI-045 (U.S. Patent Publication No. 2013-01178 84 A1), AXMI-52, AXMI-58, AXMI-88, AXMI-97, A XMI-102, AXMI-112, AXMI-117, AXMI-100 (U.S. patent Open2013-0310543 A1), AXMI-115, AXMI-113, AXMI -005 (US Patent Publication No. 2013-0104259 A1), AXMI-134 (US Patent Publication No. 2013-0167264 A1), AXMI-150 (US Patent Publication No. 2010- 0160231 A1), AXMI-184 (U.S. Patent Publication No. 2010-0004176 A1), AXMI-196, AXMI-204, AXMI-207, AXMI-209( U.S. Patent Publication No. 2011-0030096 A1), AXMI-218, AXMI-220 (US Patent Publication 2014-0245491 A1), AXMI-221z, AXMI-2 22z, AXMI-223z, AXMI-224z, AXMI-225z (US Patent Publication 2014-0196175 A1), AXMI-238 (U.S. Patent Publication 2014-003 3363 A1), AXMI-270 (U.S. Patent Publication 2014-0223598 A1) , AXMI-345 (U.S. Patent Publication 2014-0373195 A1), AXMI-33 5 (International Patent Publication WO2013 / 134523(A2)), DIG-3 (U.S. Patent Publication 2 013-0219570 A1), DIG-5 (U.S. Patent Publication 2010-0317569 A1), DIG-11 (US Patent Publication 2010-0319093 A1), AfIP- 1A and their derivatives (U.S. Patent Publication No. 2014-0033361 A1), AfIP- 1B and their derivatives (U.S. Patent Publication No. 2014-0033361 A1), PIP-1 APIP-1B (US Patent Publication 2014-0007292 A1), PSEEN3174 (U.S. Patent Publication No. 2014-0007292 A1), AECFG-592740 (U.S. Patent Publication No. 2014-0007292 A1), Pput_1063 (US Patent Publication No. 2014 -0007292 A1), DIG-657 (International Application Publication WO2015 / 195594 (A2)), Pput_1064 (US Patent Publication 2014-0007292 A1), G S-135 and its derivatives (US Patent Publication 2012-0233726 A1), GS 153 and their derivatives (U.S. Patent Publication No. 2012-0192310 A1), GS15 4 and their derivatives (U.S. Patent Publication No. 2012-0192310 A1), GS155 and and their derivatives (U.S. Patent Publication No. 2012-0192310A1), U.S. Patent Publication No. 201 SEQ ID NO: 2 and derivatives thereof as described in US Pat. No. 2-0167259 A1, SEQ ID NO: 2 and their derivatives as described in Patent Publication 2012-0047606 A1 and derivatives of sequence numbers as described in U.S. Patent Publication No. 2011-0154536 A1. No. 2 and their derivatives, and U.S. Patent Publication No. 2011-0112013 A1. SEQ ID NO: 2 and derivatives thereof, U.S. Patent Publication No. 2010-0192256 A1 SEQ ID NOS: 2 and 4 and derivatives thereof, as described in U.S. Patent Publication No. 2010- SEQ ID NO: 2 and derivatives thereof as described in U.S. Pat. No. 0077507 A1. SEQ ID NO: 2 and their derivatives as described in Publication 2010-0077508 A1 Conductor, SEQ ID NO: 2 as described in U.S. Patent Publication 2009-0313721 A1 and derivatives thereof, as described in U.S. Patent Publication No. 2010-0269221 A1 SEQ ID NO: 2 or 4 and their derivatives, as described in U.S. Patent No. 7,772,465 (B2) SEQ ID NO: 2 and derivatives thereof as described in WO2014 / 008054 A CF161_0085 and derivatives thereof as described in U.S. Patent Publication No. US 2003 / 0129994, 2008-0172762 A1, US2011-0055968 A1, and US20 Lepidopteran toxic proteins and their Derivatives: SEQ ID NO: 2 and derivatives thereof as described in US 7510878 (B2) Conductors, SEQ ID NO: 2 as described in US Pat. No. 7,812,129 (B1) and their Derivatives of Cry71Aa1 and Cry72Aa1 (US Patent Publication US2016-023 0187 A1), Axmi422 (US Patent Publication US2016-0201082 A1 ), Axmi440 (US Patent Publication US2016-0185830 A1), Axmi2 81 (US Patent Publication 2016-0177332 A1), BT-0044, BT-005 1, BT-0068, BT-0128 and their mutants (WO2016-094159 A1), BT-009, BT-0012, BT-0013, BT-0023, BT00 67 and their mutants (WO2016-094165 A1), Cry1JP578V , Cry1JPS1, Cry1 JPS1P578V(WO2016-061208 A 1); and the like, but are not limited to the group consisting of insect inhibitory proteins. may be selected.

[0263] Such additional polypeptides for controlling coleopteran pests include, for example, Cry3Bb (U.S. Patent No. 6,501,009), Cry1C mutant, Cry3A mutant, Cry3 , Cry3B, Cry34 / 35, 5307, AXMI134 (US Patent Publication 2013- 0167264 A1)AXMI-184 (US Patent Publication No. 2010-0004176 A 1), AXMI-205 (US Patent Publication 2014-0298538 A1), AXMI- 207 (US Patent Publication 2013-0303440 A1), AXMI-218, AXMI -220 (US Patent Publication 20140245491A1), AXMI-221z, AXMI -223z (US Patent Publication 2014-0196175 A1), AXMI-279 (US Patent Publication No. 2014-0223599 A1), AXMI-R1 and its mutant forms (U.S. Patent Published 2010-0197592 A1, TIC407, TIC417, TIC431, T IC807, TIC853, TIC901, TIC1201, TIC3131, DIG- 10 (U.S. Patent Publication No. 2010-0319092 A1), eHIP (U.S. Patent Application Publication No. No. 2010 / 0017914), IP3 and its variants (U.S. Patent Publication No. 2012-02 10462A1), v-Hexatoxin-Hv1a (US Patent Application Publication No. 2014-0 366227 A1), PHI-4 mutant (U.S. Patent Application Publication No. 2016-0281105 A1), PIP-72 mutant (WO2016-144688 A1), PIP-45 mutant Variants, PIP-64 mutants, PIP-74 mutants, PIP-75 mutants, and PIP-7 7 mutant (WO2016-144686 A1), DIG-305 (WO2016109 214 A1), PIP-47 mutant (U.S. Patent Publication 2016-0186204 A1) , DIG-17, DIG-90, DIG-79(WO2016-057123 A1), DIG-303 (WO2016-070079 A1); etc., but these The protein may be selected from the group consisting of, but not limited to, insect inhibitor proteins.

[0264] Such additional polypeptides for controlling hemipteran pests include TIC1415 (US Patent Publication 2013-0097735 A1), TIC807 (U.S. Patent No. 860993 6), TIC852 and TIC853 (U.S. Patent Publication 2010-0064394 A1) , TIC834 and its variants (U.S. Patent Publication No. 2013-0269060 A1), AX MI-036 (U.S. Patent Publication No. 2010-0137216 A1), and AXMI-171 (U.S. Patent Publication 2013-0055469 A1), Cry64Ba and Cry64Ca (Liu,et al(2018),Cry64Ba and Cry64Ca,Two ETX / MTX2-Type Bacillus thuringiensis In secticidal Protein Active against Hemipt eran Pests.Applied and Environmental Mic robiology,84(3):1-11) It may be selected from the group consisting of proteins active against Hemiptera.

[0265] In other embodiments, such compositions / formulations may further comprise other insect-inhibiting proteins of the present invention. The spectrum of insect inhibition obtained by the inhibitory activity against insects that are not inhibited by the substance is At least one additional polypeptide to be expanded, e.g., Thysanopterans The polypeptide may comprise an additional polypeptide that exhibits insect-inhibitory activity against the

[0266] The potential for insects to develop resistance to certain pesticides is well documented in the art. One insect resistance management strategy involves the use of two different insect inhibitors that operate via different modes of action. The next step is to use transgenic crops that express insecticides. Insects that are resistant to one can be controlled by the other insect inhibitor. Pest resistance management strategies are used to protect plants that are not protected against the targeted Coleopteran or Lepidopteran pest species. The aim is to provide refuge for such unprotected plants by using a specific Examples of this are described in U.S. Pat. No. 6,551,962, which is incorporated by reference in its entirety. It is being done.

[0267] Seed treatment, spray-on, drip-on, or wipe-on formulations Pests that are also controlled by the proteins disclosed herein when used in conjunction with proteins Other embodiments, such as topically applied insecticide chemistries designed to control insects, include: It can be applied directly to the soil (soil drench) and produces the proteins disclosed herein. or can be applied to growing plants expressing one or more of the disclosed proteins. and (c) formulating the compound to be applied to seeds containing one or more transgenes encoding the above. Such formulations for use in seed treatment are available from a variety of sources known in the art. Such formulations can be applied with adhesives and tackifiers as disclosed. The formulation may contain insecticides that are synergistic in their mode of action with the protein being treated. Insecticides act through various modes of action to be controlled by the disclosed proteins. or such insecticides contain the PirA protein, PirB protein, PirAB fusion protein, or related insecticidal protein Within the broader host range or plant pest species that are not effectively controlled by It acts to control pests.

[0268] The aforementioned compositions / formulations may further be used in the form of baits, powders, dusts, pellets, granules, sprays, emulsions, Colloidal suspension, aqueous solution, Bacillus spore / crystal preparation, seed treatment, 1 or more a recombinant plant cell / plant tissue / seed / plant transformed to express a protein of or agriculturally acceptable organisms such as bacteria transformed to express one or more proteins. The recombinant polypeptide may further comprise a carrier that contains an insect-inhibiting or insecticidal activity inherent therein. Depending on the level of inhibition and the level of formulation applied to the plant or dietary assay, the composition / The formulations may contain various amounts of recombinant polypeptide by weight, for example, from 0.0001% to 0.0 The composition may contain 0.1% to 0.01% to 1% to 99% by weight of recombinant polypeptide. can. [Example]

[0269] In view of the foregoing, one skilled in the art would be able to readily implement the disclosed method without departing from the spirit and scope of the present invention. Modifications can be made in any specific manner and still obtain the same or similar results. It should be fully understood that the present invention can be applied to the Specific structural and functional details should not be construed as limiting. It is understood that the entire disclosure of each cited reference is incorporated within the disclosure of this application. should be.

[0270] Example 1 Identification of PirA and PirB proteins and construction of PirAB fusion protein In this example, the insecticidal PirA proteins TIC4771, TIC7575, and TIC 7660, TIC7662, TIC7664, TIC7666, TIC7668, TIC 7939, TIC10357, TIC10358, TIC10360, TIC10361 , TIC10362, TIC10363, TIC10364, TIC10359, and P irA_ABE68878 (collectively "PirA protein"), PirB protein T IC4772, TIC7576, TIC7661, TIC7663, TIC7665, T IC7667, TIC7669, TIC7940, TIC10366, TIC10367 , TIC10369, TIC10370, TIC10371, TIC10372, TIC 10373, TIC10368, PirB_ABE68879, TIC11505, TI The discovery of C11510 and TIC11511 (collectively "PirB proteins") and PirAB fusion proteins, TIC6880, TIC9316, TIC9317, T IC9318, TIC9319, TIC9322, TIC9320, TIC9321, T IC6880PL, TIC10375, TIC10376, TIC10377, TIC1 0378, TIC10379, TIC10380, TIC10381, TIC10434 , TIC11210, TIC11211, TIC11212, TIC11301, TIC 11302, TIC11440, TIC11441, TIC11442, TIC1144 3, TIC11444, TIC11445, TIC11446, TIC11506, TI C11512, TIC11513, TIC10376PL, TIC10378PL, TI C10380PL, TIC10381PL, TIC11103, and TIC11104( In summary, we will explain the production of the PIsAB protein.

[0271] PirAB insecticidal proteins of Photorabdus and Xenorabdus Sequences to be loaded are identified, synthesized, cloned, and sequenced from proprietary collections and public sequence information. The bacterial operon was sequenced and examined in insect bioassays. The operons were identified from the species PirA and PirB. Insecticidal PirA proteins, TIC4771, TIC7575, and TIC7 660, TIC7662, TIC7664, TIC7666, TIC7668, TIC7 939, TIC10357, TIC10358, TIC10360, TIC10361, TIC10362, TIC10363, TIC10364, TIC10359, and Pi rA_ABE68878; and PirB proteins, TIC4772 and TIC757 6, TIC7661, TIC7663, TIC7665, TIC7667, TIC766 9, TIC7940, TIC10366, TIC10367, TIC10369, TIC 10370, TIC10371, TIC10372, TIC10373, TIC1036 8, PirB_ABE68879, TIC11505, TIC11510, and TIC1 1511 is a list of Photorabdus and Xenorabdus species listed in Table 13. Proteins TIC7939 and TIC7940 were isolated from the operon was identified from microbiome samples, and the bacterial species from which it originated is still unknown. TIFF2026010055000017.tif249164TIFF2026010055000018.tif59162

[0272] PirA and PirB insecticidal proteins of Photorabdus and Xenorabdus Protein-encoding sequences were identified, synthesized, and cloned from proprietary collections and public sequence information. The bacterial operon was identified and expressed. Sequencing each of the 13 listed Photorabdus and Xenorabdus species Polymerase chain reaction (PCR) primers were designed based on the contigs derived from The full-length coding sequence amplicons for each protein toxin were generated using the respective amplicons listed in Table 13. Each amplicon was generated using total DNA isolated from the species. Bacillus subtilis operably linked to a Bt-expressible promoter was prepared using methods known in the art. It was cloned into an expression vector of B. thuringiensis (Bt).

[0273] Fusion proteins comprising PirA and PirB proteins are known in the art. The coding sequence encoding the PirAB fusion protein was generated using known methods. It contains the coding sequences for the PirA and PirB proteins operably linked together, When expressed in cells, it contains the PirA and PirB proteins adjacent to each other. The protein was produced. It consists of the PirA protein adjacent to the PirB protein. The PirAB fusion proteins listed in Table 1 are The protein is composed of the PirA and PirB proteins from the same bacterial operon, It is composed of PirA and PirB proteins derived from different bacterial operons. The PirAB fusion protein is composed of the PirB protein adjacent to the PirA protein. The PirAB fusion proteins are listed in Table 2. The PirAB fusion proteins in Table 2 are from the same bacterial operon It is composed of PirA and PirB proteins derived from another PirA protein. It consists of a PirA protein adjacent to a PirB protein, which in turn is adjacent to a PirB protein. The PirAB fusion proteins listed in Table 3 are The PirA protein components of the protein may consist of overlapping or different PirA proteins. It can be quality.

[0274] Example 2 PirA protein, PirB protein, and PirAB fusion protein are Demonstrates activity against Lepidoptera, Coleoptera, and Hemiptera in assays This example demonstrates the efficacy of PirA proteins against various species of Lepidoptera, Coleoptera, and Hemiptera. Explain the inhibitory activity exhibited by the PirB protein and the PirAB fusion protein. do.

[0275] PirA protein, PirB protein, and PirAB fusion protein are Bt and Expressed in E. coli and toxic to various species of Lepidoptera, Coleoptera, Hemiptera, and Diptera Each toxin preparation was assayed against the lepidopteran pest species Spo doptera frugiperda, FAW, tobacco budworm (Helicov erpa zea, (CEW), also known as the soybean podworm and cotton bollworm ), Southwestern corn borer (Diatraea grandiosella, SWCB) , diamondback moth (Plutella xylostella, DBM), European corn borer (O strinia nubilalis, ECB), Velvet Bean Caterpillar (An ticarsia gemmatalis, VBC), Agrotis i psilon, BCW), southern armyworm (Spodoptera eridania, SAW), soybean looper (Pseudoplusia includes, SBL), and Heliothis virescens (TBW); Coleoptera pests Species: Colorado potato beetle (Leptinotarsa ​​decemlineata, CPB) , Northern corn rootworm (Diabrotica barberi, NCR), Southern Corn rootworm (Diabrotica undecimpunctata ho wardii, SCR), and western corn rootworm (Diabrotica vi rgifera, WCR); Hemiptera species, southern green stink bug (Nezara viridu) la, SG), Subtropical brown marmorated stink bug (Euschistus heros, NBSB ), Green Mire Bug (Lygus lineolaris, TPB), and Western Green Mirid turtle (Lygus hesperus, WTP); and dipteran species, Aedes aegypti Assayed against maca (Aedes aegypti, YFM).

[0276] Expressing PirA protein, PirB protein, and PirAB fusion protein The transformed Bt and E. coli were grown and the spores or solubilized proteins were assayed. The mass mortality and growth inhibition were caused by the PirA and PirB proteins. The growth and development of insects on diets containing one or more of the PirAB fusion proteins was compared with that of untreated controls. The insects were assessed by comparing them with the control diets. The activity of the proteins in the bioassay was observed. are presented in Tables 14 (Lepidoptera) and 15 (Coleoptera, Hemiptera, Diptera), with "+" indicates activity, empty cells indicate no activity was observed, and "NT" indicates that the toxin was not indicates that the product has not been assayed against the specified pest. TIFF2026010055000019.tif249167TIFF2026010055000020.tif116164TIFF2026010055000021.tif249168TIFF2026010055000022.tif122153

[0277] As can be seen in Tables 14 and 15, in most cases, the PirA toxin protein and Pi The rB toxin protein alone did not exhibit insecticidal activity. Fusion proteins containing PirA and PirB proteins are useful for the detection of lepidoptera, coleoptera, It showed broad-spectrum activity against Hemiptera and Diptera pest species. Some of the proteins and PirB proteins were orally active when tested in their individual capacities. For example, the PirA protein TIC4771 is expressed in Lepidoptera species CEW, DBM, ECB, and VBC. It showed activity against PirB, SAW, Coleoptera species, CPB, and Hemiptera species, TPB. Protein TIC4772 is effective against Lepidoptera species, CEW, DBM, VBC, SAW, and Hemiptera species. , and showed activity against TPB. TIC4771 protein and TIC4772 protein Combining this protein with the PirAB fusion protein TIC6880 showed almost complete anti-lepidopteran activity. Most of the activity was retained (CEW, DBM, ECB, and VBC). Although the activity was lost, activity was observed against two additional insect species, FAW and SWCB. C6880 also compares individual TIC4471 and TI for Coleopteran and Hemiptera pest species. TIC6880 showed additional activity compared to C4772. For Hemiptera, TIC68 retains its activity against mites and adds activity against WCR. 80 retained activity against TPB and was observed against SGB, NBSB, and WTP. TIC6880 also exhibited activity not seen with TIC4771 or TIC4772. It also showed activity against Diptera species and YFM, which were not previously detected.

[0278] The PirA and PirB proteins, TIC7575 and TIC7576, While each showed no insect-inhibitory activity against the insects assayed, the corresponding Pi The rAB fusion protein, TIC9316, was found to be effective against Lepidoptera species, SWCB, ECB, VBC, TIC9316 showed activity against BCW, SAW, and TBW. It also showed activity against B and Hemiptera species, SGB, NBSB, TPB, and WTP.

[0279] The PirA and PirB proteins, TIC7660 and TIC7661, However, the corresponding PirAB fusion protein, T IC9317 is for Lepidoptera species, SWCB, ECB, and VBC, Coleoptera species, CPB, and WC. R, as well as the Hemiptera species SGB, TPB, and WTP.

[0280] PirA and PirB proteins, TIC7662 and TIC7663 However, the corresponding PirAB fusion protein, T IC9318 is for Lepidoptera species, SWCB, ECB, VBC, BCW, and TBW, Coleoptera species , CPB and WCR, and for Hemiptera species, SGB, NBSB, TPB, and WTP. It showed activity.

[0281] The PirA protein TIC7664 showed activity against Coleoptera and CPB. Protein TIC7665 showed activity against lepidopteran species, TBW. The corresponding PirA The B fusion protein, TIC9319, was identified in Lepidoptera species, SWCB, ECB, VBC, and For BCW, Coleoptera species, CPB and WCR, and Hemiptera species, SGB, TPB, and WTP. and showed activity.

[0282] The PirA protein TIC7666 did not exhibit insect-inhibitory activity. TIC7667 showed activity against Lepidoptera species, SWCB. The protein TIC9322 is expressed in Lepidoptera species, FAW, CEW, SWCB and VBC, elytra It showed activity against Pterygota species, CPB, and Hemiptera species, TPB.

[0283] PirA and PirB proteins, TIC7668 and TIC7669 The corresponding PirAB fusion protein, TIC9320, showed no activity against Lepidoptera. Order species, SWCB, ECB, and VBC, Coleoptera species, CPB, and Hemiptera species, SGB, NB It showed activity against SB and TPB.

[0284] The PirAB fusion protein TIC9321 exhibits activity against the coleopteran pest CPB. Ta.

[0285] PirA protein TIC10357, PirB protein TIC10366, and The corresponding PirAB fusion protein, TIC10375, inhibits the limited number of lepidopterans assayed. It showed no activity on the eyes.

[0286] PirA protein TIC10358 and PirB protein TIC10367 None of these showed activity against the insect species assayed. The AB fusion protein TIC10376 is effective against lepidopteran pest species, SWCB and coleopteran pests. It showed activity against the species, NCR and WCR.

[0287] PirA protein TIC10360 and PirB protein TIC10369 are None of them showed activity against the limited number of lepidopteran pest species assayed.

[0288] PirA protein TIC10361 and PirB protein TIC10370 are Each showed no activity against the limited number of lepidopteran pest species assayed. While the corresponding fusion protein, TIC10378, inhibits lepidopteran pest species, SWCB, It showed activity against the coleopteran pest species NCR and WCR, and the hemipteran pest species NBSB.

[0289] PirA protein TIC10362 and PirB protein TIC10371 were upregulated. It showed no activity against the limited number of lepidopteran pest species tested.

[0290] PirA protein TIC10363 is active against the limited number of pest species assayed. The PirB protein TIC10372 showed no activity against the lepidopteran insect species SWCB. The corresponding fusion protein, TIC10380, was active against lepidopteran pest species, It retains activity against SWCB, Coleoptera pest species, NCR and WCR, and Hemiptera pest species, NB. Activity against SB was added.

[0291] PirA protein TIC10364 and PirB protein TIC10373 were upregulated. The corresponding fusion protein T showed no activity against the limited number of pest species tested. IC10381 is effective against the Coleoptera pest species NCR and WCR, and the Hemiptera pest species NBSB. showed activity.

[0292] The PirAB fusion protein, TIC10434, was shown to inhibit the growth of coleopteran pest species, NCR and WC. It showed activity against R.

[0293] The PirAB fusion protein TIC11103 is active against the lepidopteran pest species SWCB. showed.

[0294] PirAB fusion protein TIC1104 showed activity against Hemiptera species, NBSB .

[0295] The PirAB fusion protein TIC11210 inhibits the growth of lepidopteran pest species, SWCB and BCW, and It showed activity against the hemipteran pest species NBSB.

[0296] The PirAB fusion protein TIC11211 is effective against lepidopteran pest species, SWCB and hemipteran species. It showed activity against NBSB.

[0297] The PirAB fusion protein TIC11212 is active against the lepidopteran pest species SWCB. showed.

[0298] The PirAB fusion protein TIC11301 was used to treat lepidopteran pest species, SWCB, ECB, and and VBC, Coleopteran pest species, NCR and WCR, and Hemiptera pest species, NBSB and WTP It showed activity against

[0299] The PirAB fusion protein TIC11302 was used to identify lepidopteran pest species, SWCB, ECB, and and VBC, coleopteran pest species, WCR, and hemipteran pest species, NBS and WTP. showed sexuality.

[0300] Example 3 A mixture of PirA, PirB, and a mixture of PirAB fusion proteins were used in insect bioassays. It exhibits activity against Lepidoptera, Coleoptera, and Hemiptera in the stingray. This example shows the PirA proteins TIC7575 and TIC7660 at various concentrations. By mixing with PirB proteins TIC7576 and TIC7661, Similarly, the PirAB fusion proteins TIC9316 and TIC9317; TIC9316 and TIC C11301; and TIC9317 and TIC11302 were mixed. The inhibitory activity of

[0301] Mixtures of PirA and PirB proteins at various concentrations, and PirAB fusion proteins The mixture of proteins was presented in insect diets and the lepidopteran pest species BCW, SWC, and VBC were Activity against Coleoptera pest species, WCR and NCR, and Hemiptera pest species, NBSB The mixtures contained various concentrations of toxin proteins. Table 16 below shows the results of each The insect species for which activity of the mixtures was observed are indicated. TIFF2026010055000023.tif251169TIFF2026010055000024.tif29162

[0302] As can be seen in Table 16, PirA proteins TIC7575 and TIC7660 and P A mixture of irB proteins TIC7576 and TIC7661 was prepared from the corresponding fusion proteins. The PirAB fusion protein provided similar activity to the proteins TIC9316 and TIC9317. Quality TIC9316 and TIC9317; TIC9316 and TIC11301; TIC931 The mixture of 7 and TIC11302 showed similar activity to either or both of the fusion proteins. Ta.

[0303] Example 4 PirAB fusion proteins TIC6880PL and TIC931 for expression in plant cells 6, TIC9317, TIC9318, TIC9319, TIC9320, TIC932 2, TIC10376PL, TIC10378PL, TIC10380PL, TIC10 381PL, encoding TIC11103, TIC11104, and TIC11302 Design of synthetic coding sequences PirAB fusion proteins TIC6880PL, TIC9316, and TIC in plants 9317, TIC9318, TIC9319, TIC9320, TIC9322, TIC 10376PL, TIC10378PL, TIC10380PL, TIC10381PL For use in the expression of TIC11103, TIC11104, and TIC11302 Synthetic or artificial coding sequences were constructed. These synthetic coding sequences were used to generate binary plant traits. The PirAB fusion vector was cloned into a transformation vector and used to transform plant cells. Polyadenylation of ATTTA and A / T-rich plants while maintaining the amino acid sequence of the protein Avoiding certain unrealistic problem sequences, such as silylating sequences, as described in U.S. Pat. No. 5,500,366 The synthetic nucleic acid sequences were synthesized according to the methods generally described in [5]. Protein TIC6880PL, TIC9316, TIC9317, TIC9318, TI C9319, TIC9320, TIC9322, TIC10376PL, TIC1037 8PL, TIC10380PL, TIC10381PL, and TIC11302 The code sequences are presented in Table 17. TIC6880PL, TIC10376PL, TIC10 Coding sequences encoding 378PL, TIC10380PL, and TIC10381PL The PirAB fusion protein coding sequence is the starting sequence of the corresponding PirA coding sequence. It contained an additional alanine codon immediately following the thionine residue. TIFF2026010055000025.tif124167

[0304] Synthetic coding sequences and corresponding protein sequences of TIC11103 and TIC11104 are presented in Table 18 below. TIFF2026010055000026.tif43168

[0305] Example 5 Expression cassettes for the expression of PirAB fusion proteins in plant cells Various plant expression cassettes were designed with the sequences described in Table 17. The cassettes are useful for transient expression in plant protoplasts or for transformation of plant cells. A typical expression cassette is designed with respect to the final location of the protein within the plant cell. For plastid-targeted proteins, synthetic TIC6880PL, TIC9316 , TIC9317, TIC9318, TIC9319, TIC9320, TIC9322 , TIC10376PL, TIC10378PL, TIC10380PL, TIC103 81PL, TIC11103, TIC11104, and TIC11302 insecticidal proteins The protein coding sequence is engineered in frame with the coding sequence for a chloroplast-targeting signal peptide. The resulting plant transformation vector is operably linked 5' to a leader. operably linked 5' to an intron (or optionally no intron), Targeted and non-targeted TIC6880PL, TIC9316, and TIC9317 , TIC9318, TIC9319, TIC9320, TIC9322, TIC1037 6PL, TIC10378PL, TIC10380PL, TIC10381PL, TIC Synthetic codes encoding TIC11103, TIC11104, and TIC11302 proteins a construct in which the nucleotide sequence is operably linked 5' to a nucleotide sequence in the 3'UTR, which is then operably linked 5' to a 3'UTR a first transgene cassette for expression of an insecticidal protein comprising a functional promoter; A second step for selecting transformed plant cells using lyphosate or antibiotic selection All of the above-described elements include, for example, a restriction enzyme Expression cassettes containing nucleotides such as nuclease sites or ligation-independent cloning sites They are often arranged contiguously with additional sequences provided for construction of the target.

[0306] Example 6 TIC9316, TIC9317, TIC9318, TIC10376, TIC1037 Transgenic tomato plants expressing 8, TIC10380, and TIC10381 Rokosi plants show activity against lepidopteran pest species This example demonstrates the expression of a lepidopteran pest gene in transgenic maize plants. PirAB fusion proteins TIC9316, TIC9317, and TIC9318 when assayed with Inhibitory activity of IC9318, TIC10378, TIC10380, and TIC10381 Explain.

[0307] TIC9316, TIC9317, TIC9318, TIC10376, TIC103 Transgenes engineered to express 78, TIC10380, or TIC10381 The binary plant transformation vector containing the progeny cassette is transformed using methods known in the art. The plant transformation vector was operably linked 5' to a leader and operably linked 5' to the intron, TIC9316, TIC9317, TIC93 18, TIC10376, TIC10378, TIC10380, or TIC1038 operably linked 5' to a synthetic coding sequence encoding 1 and 5' to the 3'UTR TIC9316 and TIC9317 contain plant-expressible promoters linked to the TIC9318, TIC10376, TIC10378, TIC10380, or TI C10381 A first transgene cassette for expressing an insecticidal protein and a glyphosate and a second transgene cassette for selecting transformed plant cells using a transgene. The resulting vector was used for Agrobacterium-mediated transformation. Maize plants were stably transformed using the same method as previously described in the art. The plants were induced to form plants by known methods. The assay was performed similarly to that described in U.S. Pat. No. 8,344,207. Non-transformed maize plants were used to obtain tissues to be used as negative controls. Multiple transformation events from each binary vector were analyzed in lepidopteran pest species, FAW, CEW. , SWCB, ECB, and BCW.

[0308] Some transformed events expressing TIC9316 were expressed against SWCB and ECB. Similarly, TIC9317-expressing transforming events showed good to moderate inhibitory activity. TIC9318 also showed good to moderate inhibitory activity against SWCB and ECB. The resulting transformation events showed good to excellent inhibitory activity against SWCB and ECB. TIC10376, TIC10378, TIC10380, and TIC10381 expressing The resulting transformation events showed good to moderate activity against SWCB.

[0309] Example 7 Coleoptera corn root cuttings when expressed in stably transformed maize plants Assay of the activity of PirAB fusion proteins against insect pests This example demonstrates the effectiveness of TIC6880 against various coleopteran species that feed on maize roots. PL, TIC9316, TIC9317, TIC9318, TIC9319, TIC93 20, TIC9322, TIC10376PL, TIC10378PL, TIC1038 0PL, TIC10381PL, TIC11103, TIC11104, or TIC1 The inhibitory activity of 1302 is described.

[0310] Plastid-targeted and non-targeted TIC6880PL, TIC9316, and TIC9 317, TIC9318, TIC9319, TIC9320, TIC9322, TIC1 0376PL, TIC10378PL, TIC10380PL, TIC10381PL, to express both TIC11103, TIC11104, or TIC11302. Binary plant transformation vectors containing the designed transgene cassettes are described in the art. They were cloned using known methods and contain the sequences shown in Tables 17 and 18. The resulting vector can be used to transform corn plants using methods known in the art. A single T-DNA insertion event is selected and propagated. Insecticidal activity is Coleopteran pests, NCR, SCR, and and assayed against WCR.

[0311] Stably transformed plants from R0 were assayed for coleopteran resistance, as well as F Generate 1 progeny. Select multiple single copy events from each binary vector transformation. A portion of the events resulting from the transformation of the binary vector will be used in the R0 coleopteran assay. While another portion of the events is used to generate F1 progeny for further testing.

[0312] Transplant the R0 assay plants into 8-inch pots. Plants are then inoculated with either WCR, NCR, or S CR eggs are inoculated. The eggs are incubated for approximately 10 days before inoculation, and hatching occurs 4 days after inoculation. This ensures that enough larvae survive and are able to attack the corn roots. Transformed plants are inoculated at approximately the V2 to V3 stage. Grow for approximately 28 days. Remove the plants from the pots, carefully wash the roots, and remove all the soil. Damage to roots is assessed using a damage rating scale of 1 to 5 as presented in Table 19. Comparisons are also made to negative controls to ensure the assay is performing properly. Use multiple R0 events for transformation of nary vectors in coleopteran assays. Root damage scores were calculated by the PirAB fusion proteins tested against the coleopteran pests tested. Indicates the resistance imparted. TIFF2026010055000027.tif46142

[0313] The fraction of R0 stably transformed events resulting from transformation of each binary vector are used to produce F1 progeny. R0 stably transformed plants are self-fertilized. The F1 seeds are planted. Heterozygous plants are then grown in a manner consistent with the techniques of the present invention. Identified by known molecular methods in the field and assayed for coleopteran pests, as well as toxins Heterozygous F1 offspring from each event were used for ELISA protein expression measurements. One part of the offspring will be used in insect assays, while another part will measure the expression of toxin proteins. Used for.

[0314] Eggs from WCR, NCR, or SCR were incubated for approximately 10 days so that they hatched within 4 days of inoculation. For WCR, approximately 2,000 eggs are inoculated into each pot. For NCR, fewer eggs may be used due to the low availability of this species. Plants are inoculated at approximately the V2-V3 stage. Plants are grown for approximately 28 days after inoculation. Remove the plant from the pot and carefully wash the roots, removing all soil. The damage is assessed using a damage rating scale of 0 to 3 as presented in Table 20. A low root damage score indicates a high TIC score. 6880PL, TIC9316, TIC9317, TIC9318, TIC9319, T IC9320, TIC9322, TIC10376PL, TIC10378PL, TIC 10380PL, TIC10381PL, TIC11103, TIC11104, or 1 shows the resistance to coleopteran pests conferred by TIC11302. TIFF2026010055000028.tif60164

[0315] Example 8 Lepidoptera when expressed in stably transformed maize, soybean, or cotton plants Assay of activity of PirAB fusion proteins against pests. This example demonstrates the use of stably transformed transgenic mice expressing one of the PirAB fusion proteins. Activity against various lepidopteran pest species feeding on tissue from corn, soybean, or cotton plants The sex assay is described.

[0316] Expressing both plastid-targeted and non-targeted forms of the PirAB fusion protein Binary plant transformation vectors containing transgene cassettes designed to The coding sequences presented in Tables 17 and 18 were cloned using methods known in the art. Includes.

[0317] Maize, soybean, or cotton are transformed using Agrobacterium-mediated The transformants are transformed with the binary transformation vectors described above using the recombinant method. The transformed cells are induced to form plants by methods known in the art. Bioassays using plant leaf discs are described in U.S. Patent No. 8,344,207. The method is carried out in the same manner as described in the literature. Alternatively, cotton plants are used to obtain tissues to be used as negative controls. Multiple transformation events were observed in BCW, CEW, DBM, ECB, FAW, SAW, and SBL. To lepidopteran pests such as, but not limited to, SWCB, TBW, and VBC These insects exhibit stunted growth and / or mass mortality in insect bioassays. The worms have been determined to be susceptible to the PirAB fusion proteins tested.

[0318] Example 9 P against flea beetle pests when stably expressed in transformed canola plants Assay of activity of irAB fusion proteins This example demonstrates the use of transgenic canonical cells expressing one of the PirAB fusion proteins. When mice were fed whole canola plants or tissue derived from transgenic canola plants, Assays for activity against various species of flea beetles are described.

[0319] Expressing both plastid-targeted and non-targeted forms of the PirAB fusion protein Binary plant transformation vectors containing transgene cassettes designed to The coding sequences presented in Tables 17 and 18 were cloned using methods known in the art. Includes.

[0320] The resulting binary transformation vector is used to transfect cells using methods known in the art. Stably transform Nora plant cells. The transformed cells are induced to form plants. Bioassays using plant leaf discs are conducted on field-collected flea plants. This is performed in a manner similar to that described in U.S. Pat. No. 8,344,207 using a mosquito. Non-transformed canola plants were used to obtain tissue for use as a negative control. Multiple transformation events from binary vectors have been reported in, for example, the Brassicaceae flea beetle (Ph yllotreta cruciferae), striped flea beetle (Phyllotre ta striolata), Western black flea beetle (Phyllotreta pusi Evaluated against Coleopteran flea beetle pests such as, but not limited to, As the flea beetles continue to feed, the mortality of the flea beetles is assessed daily. The discs were changed every 2-3 days for 12 days to provide fresh food for the flea beetles. This allows for the use of suitable materials and reduces the effects of proteolysis in the sample.

[0321] Alternatively, transformed canola plants can be planted in fields where flea beetle infestations exist. To prevent flea beetles from emerging from the soil and escaping the experimental plot, Canola leaf damage assessment can be used to determine which plants are more susceptible to damage. It can be determined whether the plants have experienced less damage and have demonstrated resistance to flea beetles.

[0322] Example 10 PirAB fusion proteins against hemipteran pests in stably transformed soybean plants Assay of protein activity This example uses a strain of Escherichia coli stably transformed to express one of the PirAB fusion proteins. This example describes an assay for activity against hemipteran pests in selected soybean plants.

[0323] Both plastid-targeted and non-targeted forms of one PirAB fusion protein were Binary plant transformation vectors containing transgene cassettes designed to express , cloned using methods known in the art and the codes presented in Tables 17 and 18. The soybean plants are transformed using the binary plant transformation vector. Transformed soybean plant cells are induced to form whole plants. Assays for activity against hemipteran pests are performed using a variety of methods depending on the species of hemipteran pest and the preferred target tissue of that pest. For example, hemipteran pest species of stink bugs typically attack soybean plants. They feed on developing seeds and pods. To assay their activity against stink bugs, R5 stage Pods of soybeans were cultured from transgenic soybean plants expressing one of the PirAB fusion proteins. Harvest covered Petri dishes or large multi-well plates containing a layer of agar or moist paper Place 2nd instar stink bug larvae in a Petri dish or large container to provide a humid feeding environment. Place the feeding tube in a multi-well plate. Cover the feeding tube with a cover that provides oxygen exchange while preventing drying. Allow the stink bug larvae to feed for several days. The results showed that the stink bug larvae that feed on pods derived from non-transformed soybean plants were different from those of the stink bug larvae that feed on pods derived from non-transformed soybean plants. compare.

[0324] Alternatively, activity assays can be performed in whole stably transformed plants. Transgenic plants expressing one of the PirAB fusion proteins were grown in a culture chamber or greenhouse. At the R5 stage, the plants are placed on a breathable plastic "pollination" sheet (Vi Made by Lutis and Company Inc., Frankfort, IL The sheet sleeve is secured to the soil surface using Velcro® ties. Each plant is attached to the trunk just above the surface of the plant. A specific number of second-instar stink bug larvae are present on each plant. The insects are released into individual cages through small slits in the side of the cage, and the cages are closed to prevent the insects from escaping. The larvae are allowed to feed on the soybean pods for several days to a week or more. Observations are used to determine measures of stunting and mortality. At the end of the feeding period, live The larvae and dead larvae are collected. The plants are cut off under the cage and transferred to the laboratory where each plant is Before opening the cage, shake the plant vigorously to ensure all insects are removed. Then, open the bottom of the cage and remove all plant material. Remove the material and place it on a black sheet. The insects can be collected using a vacuum or other means. The number of insects and their developmental stages are recorded for each plant. Also, the number of dead larvae is recorded. These measurements are taken from negative control, untransformed plants. The measured value is compared with the measured value.

[0325] Stunted development or mass mortality of stink bug larvae is associated with increased mortality compared with untransformed controls. Any significant difference when compared to the α-amino acid is interpreted as an indication of toxicity.

[0326] Example 11 PirAB fusion tags against hemipteran pests in stably transformed maize plants Protein activity assay This example uses a strain of Escherichia coli stably transformed to express one of the PirAB fusion proteins. This example describes an assay for activity against hemipteran pests in selected corn plants.

[0327] Both plastid-targeted and non-targeted forms of one PirAB fusion protein were Binary plant transformation vectors containing transgene cassettes designed to express , cloned using methods known in the art and the codes presented in Tables 17 and 18. The maize plants are transformed using binary plant transformation vectors. The transformed corn plant cells are induced to form whole plants. Assays for activity against lepidopteran pests are based on the hemipteran pest species and their preferred target tissues. This is done using a variety of techniques depending on the species. For example, hemipteran pest species of stink bugs are usually They feed on young corn plants in late or early summer, resulting in holes in the leaves and severe In late summer, the stink bugs usually feed on the grain ear itself, directly picking the kernels. Destroy it.

[0328] One method to assay activity against stink bugs is to measure P Derived from stably transformed maize plants expressing one of the irAB fusion proteins. The method involves exposing stink bug larvae to the leaf discs that develop. The large maltodextrins were then placed in a pod with leaf discs derived from stably transformed maize plants. The cells were placed on a Chiwell plate and allowed to feed for several days. Stunting and mortality were measured and transformation was confirmed. The results are compared with those of stink bug larvae that had fed on untreated corn leaf discs.

[0329] Alternatively, whole transformed plants can be used to assay activity against stink bugs. Stably transformed maize expressing one of the PirAB fusion proteins can be used. Koshi plants were enclosed in cages in a manner similar to that described for soybean plants in Example 4. Second-instar larvae are introduced onto V3 stage corn plants and allowed to feed for several days to a week. After the prescribed feeding period, live and dead larvae are collected. The set values ​​are compared to untransformed control plants.

[0330] Stably transformed corn ears were used to assay activity against stink bugs. To do this, we can use a similar approach to analyzing V3 stage plants. Stably transformed maize plants expressing one of the PirAB fusion proteins The developing ears of corn allow free exchange of air while preventing the escape of stink bug larvae. The encapsulated ears are then covered with sheets of material that allow the larvae to grow. They infest and feed on developing grains for several days to a week. Measures of stunting and mortality. compared to panicles of untransformed control plants.

[0331] Example 12 PirAB fusion proteins against hemipteran pests in stably transformed cotton plants Assay of quality activity This example uses a strain of Escherichia coli stably transformed to express one of the PirAB fusion proteins. This describes an assay for activity against hemipteran pests on selected cotton plants.

[0332] Both plastid-targeted and non-targeted forms of one PirAB fusion protein were Binary plant transformation vectors containing transgene cassettes designed to express , cloned using methods known in the art and the codes presented in Tables 17 and 18. The cotton plants are transformed using the binary plant transformation vector. The transformed cotton plant cells are induced to form whole plants. Assays for activity may be performed using a variety of techniques depending on the species of hemipteran pest and the preferred target tissue of that pest. For example, stink bugs are usually seed-eating insects, so Damage to cotton capsules is a major concern. They primarily bore holes into the capsules and eat the seeds. Their feeding activity is concentrated on the outside of the large capsule where feeding occurs. The insects can produce dark spots about 1 / 16 inch in diameter on the surface of the cotton near the feeding site. It reduces hair production and can soil the down. Due to their size, adults and 4th and 5th instars The larvae are the most likely to cause damage to the capsules, so it is recommended to catch the insects in the early larval stages. The hemipteran pest species of the genus Lygus primarily attack squares and young capsules. Larvae are the more voracious feeders and tend to cause the most serious damage. When feeding on squares, Lygus targets developing anthers, resulting in the squares These squares often shrink and fall off the plant. The fruit may contain pollen, unfertilized seeds, and anthers that fail to form empty locules. When feeding on fruit, Lygus targets the developing seeds, forming small black spots on the outside of the capsule. It causes blemished spots.

[0333] Assaying the activity of PirAB fusion proteins in stably transformed cotton plants One way to do this is to use squares in insect bioassays. TIC6880PL, TIC9316, TIC9317, TIC9318, TIC931 9, TIC9320, TIC9322, TIC10376PL, TIC10378PL, TIC10380PL, TIC10381PL, TIC11103, TIC11104, or recovered from transgenic cotton plants expressing TIC11302. Each square can be placed in a tri-plate or placed in a well of a large well plate. Young neonate Lygus or stink bug larvae can be placed in a Petri dish or large well. The animals were placed on plates and allowed to feed for a specified period of time. Stunting and mortality were measured over the time course of feeding. Squares from untransformed cotton plants were used in the assay. Compare with a control.

[0334] Alternatively, activity assays can be performed on whole transformed cotton plants. For example, one of the PirAB fusion proteins was expressed to assay for Lygus species. R1 seeds from the resulting plants are sown in 10-inch pots. Cotton plants, preferably from the same variety as the transformed plants, are used as negative controls. The plants are grown under a photoperiod of 16 hours light at 32 degrees Celsius and 8 hours dark at 23 degrees Celsius, and They are maintained in an environmental chamber with a light intensity between 800 and 900 micro-einsteins. After 40 to 45 days, place individual plants on a breathable plastic "pollination" sheet ( Made by Vilutis and Company Inc., Frankfort, IL The sheet sleeve is attached to the soil surface using Velcro® ties. Sexually mature male and female Lygu from laboratory rearing. Two adult (6-day-old) Lygus lineolaris or Lygus hesperus Place a pair of 14 ml round-bottom plastic test tubes (Bacton Dickson Labware, Franklin Lakes, NJ) and used for each plant. Adult insects are released into individual cages through small slits in the side of the cage, and the cage is closed to prevent the insects from escaping. The insects are allowed to mate and the plants are kept in the cage for 21 days.

[0335] After 21 days, the plants were cut off under the cage and transferred to the laboratory, where the insects on each plant were collected. Before opening the cage, shake the plant vigorously to ensure that all insects are removed from the feeding site. Then open the bottom of the cage and remove all the plant material. The plants are then thoroughly inspected and placed on a black sheet. The insects are then collected using a vacuum extractor. The number of insects collected and their developmental stage are recorded for each plant. Lygus maturity: total number of insects based on larvae and adults up to 3rd, 4th, and 5th instars are divided into several groups.

[0336] Expressing one of the PirAB fusion proteins to assay against stink bug species R1 seeds from the plants were sown in pots as described above and propagated to produce ca. Non-transformed cotton plants are also used as negative controls. Moth larvae are used to infest the plants and are allowed to feed on the squares and capsules for several days or weeks. The caged plants were harvested as described above, and the harvested stink bugs were then placed in a Mortality is also examined and scored, as is the maturity of the recorded larvae. These scores are compared to negative control plants.

[0337] Example 13 TIC9318 and TIC11 when expressed in stably transformed maize plants 302 demonstrates activity against western corn rootworm pests This example demonstrates the development of corn roots in stably transformed corn plants. TIC9318 against cutworms (Diabrotica virgifera, WCR) and the inhibitory activity of TIC11302.

[0338] Maize plants were grown for expression of either TIC9318 or TIC11302. The binary plants were transformed with a binary plant transformation construct containing an expression cassette for Transformation vectors were designed to express TIC9318 and TIC11302. The vectors contained transgene cassettes and were cloned using methods known in the art. The transformation vector is operably linked 5' to a leader and operably linked 5' to an intron. operably linked synthetic coding sequences encoding TIC9318 or TIC11302 operably linked 5' to the 3'UTR and operably linked 5' to the 3'UTR. Expression of TIC9318 or TIC11302 insecticidal proteins containing suitable promoters and a first transgene cassette for selecting transformed plant cells using glyphosate. The resulting vector was used to transfect Agroba. Maize plants were stably transformed using the .gamma.-mediated transformation method. The transformed cells can be propagated to form plants by methods known in the art. was led to.

[0339] Testing TIC11302 for WCR resistance using R0 stably transformed plants The F1 progeny were generated in the same manner. Single copy events were selected. A proportion of events resulting from each binary vector transformation were in R0. Used in WCR assays.

[0340] The R0 assay plants were transplanted into 8-inch pots. Approximately 2% of the WCR-derived plants were added to the plants. ,000 eggs were inoculated. The eggs were then soaked for approximately 10 minutes before inoculation to ensure that a sufficient number of larvae survived. The bacteria were incubated for 4 days and hatched 4 days after inoculation, allowing them to attack the maize roots. Approximately 2,000 WCR eggs were inoculated into each pot. The plants were inoculated at the V2 to V3 stage. The plants were grown for approximately 28 days after inoculation. The plants were then removed and the roots were carefully washed to remove all soil. Damage to the roots was measured as shown in Table 17 of Example 17. The damage was assessed using a 1-5 damage rating scale as presented in

[19] . Comparisons with negative controls were also performed to ensure that each TIC11302 binary Multiple R0 events for vector transformation were used in the WCR assay.

[0341] R0 resulting from transformation of TIC9318 and TIC11302 binary vectors A portion of the stably transformed events were used to generate F1 progeny. The transformed plants were self-fertilized to generate F1 progeny. F1 seeds were sown in 8-inch pots. Heterozygous plants were identified via molecular methods known in the art and submitted to WCR. WCR eggs were inoculated into the stable R0 as described above. The root damage was as described for the transformed events. The injury was assessed using a 0-3 injury rating scale as presented in the 20. A negative control was compared to ensure that the root damage rating (RDR) of each construct was 0.01. ) are presented below in Table 21, where "NT" indicates not tested. TIFF2026010055000029.tif66166

[0342] As can be seen in Table 21 above, both TIC9318 and TIC11302 were negative. Western corn rootworm (Diabrotica virgifol) when compared to controls era virgifera).

[0343] All of the compositions disclosed and claimed herein may be prepared without undue experimentation in light of the present disclosure. The compositions of the present invention may be prepared and carried out without the use of the above-described illustrative embodiments. Although described in terms of embodiments, no deviation from the true concept, spirit or scope of the invention exists. The compositions described herein are not intended to be limiting unless variations, changes, modifications and alterations are applied. It will be apparent to one skilled in the art that other methods may be used. More specifically, methods that achieve the same or similar results may be used. While certain agents are both chemically and physiologically related, the present invention provides a method for treating a variety of conditions, including the development of steroid hormones, including steroid hormones, and the like. It will be apparent that the agents listed above may be substituted. All such similar substitutes and modifications are intended to fall within the spirit and spirit of the invention as defined by the appended claims. It is considered to be within the scope and concept.

[0344] All publications and published patents are hereby incorporated by reference as if each individual publication or patent application were incorporated by reference. This document is incorporated by reference to the same extent as if it were specifically and individually indicated to be incorporated. INCORPORATED INTO THE SPECIFICATION.

Claims

1. The polynucleotide segment encoding the insecticidal protein or insecticidal fragment thereof is manipulated.

1. A recombinant nucleic acid molecule comprising a heterologous promoter operably linked to a. The insecticidal protein is selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18 、２０、２２、２４、２６、２８、３０、３２、３４、３６、３８、４０、４２、４４、 ４６、４８、５０、５８、６０、６２、６４、６６、６８、７０、７２、７４、７６、７ ８、８０、８２、８４、８６、８８、９０、９２、９４、９６、９８、１００、１０２、 １０５、１０７、１０９、１１１、１１３、１１５、１１７、１１９、１２１、１２３、 １２５、１２７、１２９、１３１、１３３、１３５、１３７、１３９、１４１、１４３、 145, 147, 149, 151, 153, 155, or 157 amino acid sequences. or b. The insecticidal protein is i. SEQ ID NOs: 44, 46, 48, 123, 127, 129, 131, 133, and 145 at least 65% identical to; or ii. at least 70% identity to SEQ ID NOs: 109, 121, and 125 ;or iii. SEQ ID NOs: 12, 18, 24, 36, 42, 62, 68, 74, 80, 86, 98 , 113, 117, 119, 147, 149, 153, 155, and 157 at least 80% identity; or iv. at least 8 sequences for SEQ ID NOs: 30, 92, 111, 115, and 151 2% identity; or v. at least 86% identity to SEQ ID NOs: 6 and 50; or vi. at least 94% identity to SEQ ID NOs: 137 and 141; or vii. at least 97% identity to SEQ ID NOs: 4, 26, and 32; or viii. At least 98% identity to SEQ ID NOs: 2, 28, 34, 102, and 102 Oneness; or ix. at least 99% identity to SEQ ID NO: 135; or x. SEQ ID NOs: 8, 10, 14, 16, 20, 22, 38, 40, 58, 60, 64, 66 、７０、７２、７６、７８、８２、８４、８８、９０、９４、９６、１００、１０５、１ comprising an amino acid sequence having 100% identity to 07, 139, and 143; and teeth c. the polynucleotide segment is hybridized under stringent hybridization conditions , SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 2 ７、２９、３１、３３、３５、３７、３９、４１、４３、４５、４７、４９、５１、５２ 、５３、５４、５５、５６、５７、５９、６１、６３、６５、６７、６９、７１、７３、 ７５、７７、７９、８１、８３、８５、８７、８９、９１、９３、９５、９７、９９、１ ０１、１０３、１０４、１０６、１０８、１１０、１１２、１１４、１１６、１１８、１ ２０、１２２、１２４、１２６、１２８、１３０、１３２、１３４、１３６、１３８、１ 40, 142, 144, 146, 148, 150, 152, 154, 156, or 1 the recombinant DNA fragment hybridizes to a polynucleotide having a nucleotide sequence of 58. Nucleic acid molecule.

2. a. the recombinant nucleic acid molecule is a sequence that functions to express the insecticidal protein in a plant; Contains columns, or b. The recombinant nucleic acid molecule is expressed in a plant cell to produce an insecticidally effective amount of the insecticidal protein. to occur, or c. the recombinant nucleic acid molecule is operably linked to a vector, the vector being a plasmid from the group consisting of phagemids, bacmids, cosmids, and bacterial or yeast artificial chromosomes. The recombinant nucleic acid molecule of claim 1, selected from the group consisting of:

3. is defined as being present within a host cell, said host cell being selected from the group consisting of bacterial cells and plant cells. The recombinant nucleic acid molecule of claim 1, selected from the group consisting of:

4. The bacterial host cell is selected from the group consisting of Agrobacterium, Rhizobium, Bacillus subtilis, lus, Brevibacillus, Escherichia, Pseudomona s, Klebsiella, Pantoea, and Erwinia.

4. The recombinant nucleic acid molecule of claim 3, which is derived from a genus of bacteria.

5. The Bacillus species is Bacillus cereus or Bacillus thuringiensis, and the Brevibacillus is Breviba cillus laterosperus, or the Escherichia The recombinant nucleic acid molecule of claim 4, which is Escherichia coli.

6. 4. The recombinant nucleic acid molecule of claim 3, wherein the plant cell is a dicotyledonous or monocotyledonous plant. 。

7. The plant host cell may be alfalfa, banana, barley, bean, broccoli, cabbage, Beets, brassicas, carrots, cassava, castor beans, cauliflower, celery, chickpeas Rice, Chinese cabbage, citrus fruits, coconut, coffee, corn, clover, cotton, melon , cucumber, Douglas fir, eggplant, eucalyptus, flax, garlic, grapes, hops, leeks, Lettuce, loblolly pine, millet, melon, nuts, oats, olives, onions, Ornamental plants, palms, grasses, peas, peanuts, pepper, pigeon peas, pine, Potato, poplar, pumpkin, radish, rapeseed , rice, rhizome, rye, safflower, shrub, sorghum, southern pine, soybean, and holly Grass, squash, strawberry, sugar beet, sugarcane, sunflower, sweet potato Corn, sweetgum, sweet potato, switchgrass, tea, tobacco, tomato, rye 7. The method of claim 6, wherein the plant cell is selected from the group consisting of wheat, turfgrass, watermelon, and triticale. Recombinant nucleic acid molecules.

8. 10. The recombinant nucleus of claim 1, wherein the insecticidal protein is active against Coleoptera insects. acid molecule.

9. The insects are Western corn rootworm, Southern corn rootworm, Northern corn rootworm, Corn rootworm, Mexican corn rootworm, Brazilian corn rootworm, Colo Rada beetles, Diabrotica viridula and Diabrotica sp. Brazilian corn rootworm complex consisting of Brassicaceae flea beetles, The recombinant nucleic acid molecule of claim 8, which is a striped flea beetle or a western black flea beetle. 。

10. 10. The recombinant insecticidal protein of claim 1, wherein the insecticidal protein is active against insect species of the order Lepidoptera. Nucleic acid molecules.

11. The insects include the common cutworm moth, the tobacco moth, the diamondback moth, the European corn borer, and the rice moth. Armyworm, Southern armyworm, Soybean looper, Southwestern corn borer, Helicoverpa armigera (To bacco budworm), velvet bean caterpillar, sugarcane borer, Lesser cornstalk borer, black armyworm, beet armyworm, tobacco bollworm (Old World Bollworm), Spodoptera litura, or Pink Bollworm The recombinant nucleic acid molecule of claim 10,

12. 10. The recombinant insecticidal protein of claim 1, wherein the insecticidal protein is active against Hemiptera insect species. Nucleic acid molecules.

13. The insects are the southern green stink bug, the subtropical brown marmorated stink bug, the red spotted stink bug, the black spotted stink bug, Brown-winged stink bugs, brown marmorated stink bugs, green stink bugs, brown marmorated stink bugs 13. The insect according to claim 12, which is a stink bug, a western rusty stink bug, or a rusty stink bug. A recombinant nucleic acid molecule of.

14. A plant or part thereof comprising the recombinant nucleic acid molecule of claim 1.

15. 15. The plant or any of its parts according to claim 14, wherein the plant is a monocotyledonous or dicotyledonous plant. Department.

16. The plant may be alfalfa, banana, barley, bean, broccoli, cabbage, Lassika, carrots, cassava, castor beans, cauliflower, celery, chickpeas, haku Rhino, citrus fruits, coconut, coffee, corn, clover, cotton, gourd, cucumber berries, Douglas fir, eggplant, eucalyptus, flax, garlic, grapes, hops, chives, lettuce, Loblolly pine, millet, melon, nuts, oats, olives, onions, ornamentals palms, grass, peas, peanuts, pepper, pigeon peas, pine, ja Potato, poplar, pumpkin, radiata pine, radish, rapeseed, Rice, rhizome, rye, safflower, shrubs, sorghum, southern pine, soybean, spinach Saw, squash, strawberry, sugar beet, sugarcane, sunflower, sweet Corn, sweetgum, sweet potato, switchgrass, tea, tobacco, tomato, rye 15. The plant of claim 14, selected from the group consisting of barley, turfgrass, watermelon, and wheat. Some of it.

17. 15. A seed of the plant of claim 14, wherein the seed comprises the recombinant nucleic acid molecule.

18. 10. An insect inhibitor composition comprising the recombinant nucleic acid molecule of claim 1.

19. Furthermore, a nucleic acid encoding at least one other insecticide different from the insecticidal protein is also included.

20. The insect inhibitor composition of claim 18, comprising a nucleotide sequence.

20. The at least one other insecticide is selected from the group consisting of an insect-inhibiting protein, an insect-inhibiting dsRNA molecule, 20. The insect inhibitor composition of claim 19, wherein the insect inhibitor composition is selected from the group consisting of: and an accessory protein.

21. The at least one other insecticide is effective against one or more pest species of the order Lepidoptera, Coleoptera, or Hemiptera.

20. The insect inhibiting composition of claim 19, which exhibits activity against

22. The at least one other insecticide is Cry1A, Cry1Ab, Cry1Ac, Cr y1A.105, Cry1Ae, Cry1B, Cry1C, Cry1C mutant, Cry1 D, Cry1E, Cry1F, Cry1A / F chimera, Cry1G, Cry1H, Cry 1I, Cry1J, Cry1K, Cry1L, Cry2A, Cry2Ab, Cry2Ae , Cry3, Cry3A mutant, Cry3B, Cry4B, Cry6, Cry7, Cry 8, Cry9, Cry15, Cry34, Cry35, Cry43A, Cry43B, C ry51Aa1, ET29, ET33, ET34, ET35, ET66, ET70, TI C400, TIC407, TIC417, TIC431, TIC800, TIC807, TIC834, TIC853, TIC900, TIC901, TIC1201, TIC1 415, TIC2160, TIC3131, TIC836, TIC860, TIC867 , TIC869, TIC1100, VIP3A, VIP3B, VIP3Ab, AXMI- AXMI-, AXMI-88, AXMI-97, AXMI-102, AXMI-112, AXMI-117, AXMI-100, AXMI-115, AXMI-113, and AX MI-005, AXMI134, AXMI-150, AXMI-171, AXMI-18 4, AXMI-196, AXMI-204, AXMI-207, AXMI-209, AX MI-205, AXMI-218, AXMI-220, AXMI-221z, AXMI- 222z, AXMI-223z, AXMI-224z and AXMI-225z, AXM I-238, AXMI-270, AXMI-279, AXMI-345, AXMI-33 5, AXMI-R1 and its mutants, IP3 and its mutants, DIG-3, DIG-5, DIG-10, DIG-657 DIG-11, Cry71Aa1, Cry72Aa1, PHI-4 mutant, PIP-72 mutant, PIP-45 mutant, PIP-64 mutant, P IP-74 mutant, PIP-75 mutant, PIP-77 mutant, Axmi422, Dig -305, Axmi440, PIP-47 mutant, Axmi281, BT-009, BT -0012, BT-0013, BT-0023, BT0067, BT-0044, BT- 0051, BT-0068, BT-0128, DIG-17, DIG-90, DIG-7 9, Cry1JP578V, Cry1JPS1, and Cry1 JPS1P578V 22. The insect inhibitor composition of claim 21, wherein the protein is selected from the group consisting of:

23. 19. The method of claim 18, further defined as including a plant cell expressing said recombinant nucleic acid molecule. The insect inhibiting composition.

24. A commercial product containing a detectable amount of said recombinant nucleic acid molecule or the killer gene encoded thereby.

15. A commercial product produced from the plant or part thereof of claim 14, comprising an insect protein. thing.

25. Corn, corn flakes, and corn cakes packed in bags by grain handlers , corn flour, corn meal, corn syrup, corn oil, corn silage, corn starch, corn cereals, etc., whole or processed cottonseed, cotton oil, lint, animal feed or from seeds and plant parts, fibers, paper, biomass, and cotton oil processed for food use Fuel or fuel products such as pellets derived from cotton gin waste, whole soybean seeds or processed Soybean seeds, soybean oil, soybean protein, soybean meal, soybean flour, soybean flavor soybean milk, soybean cheese, soybean wine, animal feed containing soybeans, paper containing soybeans, cream containing soybeans, soybean biomass, and soybean plants and 25. The commodity product of claim 24, wherein the commodity product is selected from the group consisting of fuel products produced using a portion of the commodity product. thing.

26. 1. A method for producing seeds, comprising: a. planting at least a first seed according to claim 17; b. Growing a plant from the seed; c. harvesting seeds from said plant, wherein said harvested seeds contain said recombinant nucleic acid. The method further comprises:

27. 10. A method for producing a plant resistant to insect infestation, wherein the cells of the plant contain the recombinant nucleic acid molecule of claim 1. A plant.

28. A method for controlling pests or infestations of Coleoptera, Lepidoptera, or Hemiptera species, 、 SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26 、２８、３０、３２、３４、３６、３８、４０、４２、４４、４６、４８、５０、５８、 ６０、６２、６４、６６、６８、７０、７２、７４、７６、７８、８０、８２、８４、８ ６、８８、９０、９２、９４、９６、９８、１００、１０２、１０５、１０７、１０９、 １１１、１１３、１１５、１１７、１１９、１２１、１２３、１２５、１２７、１２９、 １３１、１３３、１３５、１３７、１３９、１４１、１４３、１４５、１４７、１４９、 an insecticidally effective amount of an insecticidal agent as described in 151, 153, 155, or 157; contacting said pest with the protein; b. or i. SEQ ID NOs: 44, 46, 48, 123, 127, 129, 131, 133, and 145 at least 65% identical to; or ii. at least 70% identity to SEQ ID NOs: 109, 121, and 125 ;or iii. SEQ ID NOs: 12, 18, 24, 36, 42, 62, 68, 74, 80, 86, 98 , 113, 117, 119, 147, 149, 153, 155, and 157 at least 80% identity; or iv. at least 8 sequences for SEQ ID NOs: 30, 92, 111, 115, and 151 2% identity; or v. at least 86% identity to SEQ ID NOs: 6 and 50; or vi. at least 94% identity to SEQ ID NOs: 137 and 141; or vii. at least 97% identity to SEQ ID NOs: 4, 26, and 32; or viii. At least 98% identity to SEQ ID NOs: 2, 28, 34, 102, and 102 Oneness; or ix. at least 99% identity to SEQ ID NO: 135; or x. SEQ ID NOs: 8, 10, 14, 16, 20, 22, 38, 40, 58, 60, 64, 66 、７０、７２、７６、７８、８２、８４、８８、９０、９４、９６、１００、１０５、１ 100% identity to 07, 139, and 143. contacting said pest with an effective amount of one or more insecticidal proteins at

29. A method for detecting the presence of the recombinant nucleic acid molecule of claim 1 in a sample containing plant genomic DNA. It is a law, a. A plant containing the DNA molecule of claim 1 under stringent hybridization conditions. hybridizes to genomic DNA from the organism, and under such hybridization conditions A hybridization method using genomic DNA derived from another isogenic plant that does not contain the recombinant nucleic acid molecule of claim 1. contacting the sample with an unbridled nucleic acid probe, wherein the probe SEQ ID NOs: 49, 51, 52, 53, 54, 55, 56, 146, 148, 150, 152 154, 156, or 158, or a sequence as defined in claim 1. the nucleic acid molecule described above, or i. SEQ ID NOs: 44, 46, 48, 123, 127, 129, 131, 133, and 145 at least 65% identical to; or ii. at least 70% identity to SEQ ID NOs: 109, 121, and 125 ;or iii. SEQ ID NOs: 12, 18, 24, 36, 42, 62, 68, 74, 80, 86, 98 , 113, 117, 119, 147, 149, 153, 155, and 157 at least 80% identity; or iv. at least 8 sequences for SEQ ID NOs: 30, 92, 111, 115, and 151 2% identity; or v. at least 86% identity to SEQ ID NOs: 6 and 50; or vi. at least 94% identity to SEQ ID NOs: 137 and 141; or vii. at least 97% identity to SEQ ID NOs: 4, 26, and 32; or viii. At least 98% identity to SEQ ID NOs: 2, 28, 34, 102, and 102 Oneness; or ix. at least 99% identity to SEQ ID NO: 135; or x. SEQ ID NOs: 8, 10, 14, 16, 20, 22, 38, 40, 58, 60, 64, 66 、７０、７２、７６、７８、８２、８４、８８、９０、９４、９６、１００、１０５、１ Insecticidal compounds containing amino acid sequences with 100% identity to 07, 139, and 143 a protein-coding sequence; b. subjecting the sample and probe to stringent hybridization conditions; c. detecting hybridization of the probe with the DNA of the sample. How to write.

30. A method for detecting the presence of an insecticidal protein or a fragment thereof in a protein-containing sample. The insecticidal protein is selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 1 ８、２０、２２、２４、２６、２８、３０、３２、３４、３６、３８、４０、４２、４４ 、４６、４８、５０、５８、６０、６２、６４、６６、６８、７０、７２、７４、７６、 ７８、８０、８２、８４、８６、８８、９０、９２、９４、９６、９８、１００、１０２ 、１０５、１０７、１０９、１１１、１１３、１１５、１１７、１１９、１２１、１２３ 、１２５、１２７、１２９、１３１、１３３、１３５、１３７、１３９、１４１、１４３ , 145, 147, 149, 151, 153, 155, or 157 amino acid sequences. or the insecticidal protein is i. SEQ ID NOs: 44, 46, 48, 123, 127, 129, 131, 133, and 145 at least 65% identical to; or ii. at least 70% identity to SEQ ID NOs: 109, 121, and 125 ;or iii. SEQ ID NOs: 12, 18, 24, 36, 42, 62, 68, 74, 80, 86, 98 , 113, 117, 119, 147, 149, 153, 155, and 157 at least 80% identity; or iv. at least 8 sequences for SEQ ID NOs: 30, 92, 111, 115, and 151 2% identity; or v. at least 86% identity to SEQ ID NOs: 6 and 50; or vi. at least 94% identity to SEQ ID NOs: 137 and 141; or vii. at least 97% identity to SEQ ID NOs: 4, 26, and 32; or viii. At least 98% identity to SEQ ID NOs: 2, 28, 34, 102, and 102 Oneness; or ix. at least 99% identity to SEQ ID NO: 135; or x. SEQ ID NOs: 8, 10, 14, 16, 20, 22, 38, 40, 58, 60, 64, 66 、７０、７２、７６、７８、８２、８４、８８、９０、９４、９６、１００、１０５、１ comprising an amino acid sequence having 100% identity to 07, 139, and 143; The method comprises: xi. contacting the sample with an immunoreactive antibody; xii. detecting the binding of said antibody to said insecticidal protein or fragment thereof. wherein binding indicates the presence of said protein.

31. 31. The method of claim 30, wherein the detecting step comprises ELISA or Western blot. method.

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