Compositions and methods for controlling coleopteran insects

Abstract

Novel pesticidal polypeptides having activity against coleopteran pests are disclosed. Nucleic acid molecules encoding the novel insecticidal proteins are also provided. Nucleotide sequences encoding the pesticidal polypeptides can be used to transform prokaryotic and eukaryotic organisms to express the pesticidal proteins. Also disclosed are methods of making the insecticidal proteins and methods of using the insecticidal proteins, for example, to confer protection from insect damage in transgenic plants.

Description

[0001] Statement Concerning Electronic Submission of Sequence Listings

[0002] A sequence listing in XML format is provided as an alternative to a paper copy, which is submitted pursuant to 37 C.FR §1.831(a), is named "82735-SamsoniaCRW_Sequence Listing_ST26", is about 239 kilobytes in size, was generated on August 1, 2023, and was submitted via EFS-Web. This sequence listing is hereby incorporated by reference into this specification for its disclosure content. Technical Field

[0003] The present disclosure relates to novel pesticidal proteins having insecticidal activity, nucleic acid molecules encoding these pesticidal proteins and whose expression produces these pesticidal proteins, and compositions and methods for controlling agriculturally relevant pests of crop plants. Background Art

[0004] Insects are a major cause of crop loss. Many commercially valuable plants, including common crops, are susceptible to attack by plant pests, including insects and nematodes, resulting in significant reductions in crop yield and quality. For example, plant pests are a major cause of loss of important agricultural crops worldwide. Insect pests are also a burden to vegetable and fruit growers, to producers of ornamental flowers, and they are a nuisance to home gardeners.

[0005] Multiple species of corn rootworm are considered to be the most destructive corn pests. In the U.S., three important species are the corn rootworm (Diabrotica virgifera virgifera, also known as the western corn rootworm), the long-horned barber's rootworm (D.longicornis barberi, also known as the northern corn rootworm), and the eleven-star rootworm (D.undecimpunctata howardi, also known as the southern corn rootworm). In the U.S. Corn Belt, only the western and northern corn rootworms are considered to be major corn pests. In addition, an important corn rootworm pest in the southern U.S. is the Mexican corn rootworm (Diabrotica virgifera zeae). Corn rootworm larvae cause substantial plant damage by almost exclusively feeding on corn roots. This damage has been shown to increase plant lodging, reduce grain yield and nutritional yield together with changes in the nutrient content of grains. Larval feeding also causes indirect effects on corn by opening access to the root for bacteria and fungal infections that potentially cause root rot and stem rot. Adult corn rootworms are active in corn fields in late summer where they feed on ears, silks, and pollen, thereby interfering with normal pollination.

[0006] Corn rootworms are primarily controlled by intensive applications of chemical pesticides, which are active by inhibiting insect growth, preventing insect feeding or reproduction, or death. Thus, good corn rootworm control can be achieved, but not without some inefficiencies. In some cases, the application of these chemicals can affect other beneficial organisms. In addition, the widespread use of chemical pesticides can lead to the development of resistant insect varieties. Finally, the underground feeding preference of corn rootworm larvae makes it difficult to apply insecticide rescue treatments. Therefore, most insecticide applications are performed preventively at planting, which results in a significant environmental burden. This situation has been partially improved by various farmland management practices, but there is an increasing need for alternative mechanisms of pest control.

[0007] Biological pest control agents, such as Bacillus thuringiensis (Bt) strains expressing insecticidal toxins like delta-endotoxins (Δ-endotoxins; also known as crystalline toxins or Cry proteins), have also been applied to crop plants, producing satisfactory results against insect pests. Delta-endotoxins are proteins contained in a crystalline matrix, known to have insecticidal activity when ingested by plant pests (including insects) of certain orders and species, but are harmless to plants and other non-target organisms. Several natural Cry proteins or engineered Cry proteins from Bacillus thuringiensis have been expressed in transgenic crop plants to control certain lepidopteran and coleopteran insect pests as an alternative or supplement to chemical pesticides. Since 2003, transgenic corn hybrids that control corn rootworm have been commercially available in the United States and express toxins such as Cry3Bb1, Cry34Ab1 / Cry35Ab1, modified Cry3A (mCry3A), or Cry3Ab (eCry3.1Ab).

[0008] Although it has been shown that transgenic plants expressing Cry proteins are very effective, insect pests that are resistant to Cry proteins expressed in certain transgenic plants are now known. Therefore, there is still a need to identify new and effective pest control agents that provide economic benefits to farmers and are environmentally acceptable. Particularly needed are proteins that are toxic to Diabrotica species (a major corn pest) that have different modes of action compared to existing insect control products to mitigate resistance development. In addition, it is desirable to deliver insect control agents through these products that minimize the environmental burden (such as through transgenic plants). Summary of the invention

[0009] The present disclosure provides polypeptides that are insecticidal against at least one coleopteran pest, such as against corn rootworm (WCR, corn rootworm), and uses of such polypeptides and related nucleic acids in compositions and methods, such as in plants or in methods of controlling coleopteran pests.

[0010] Thus, in some aspects, a polypeptide is provided comprising an amino acid sequence having at least 85% identity to SEQ ID NO: 1. In some embodiments, the polypeptide has at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98%) identity to SEQ ID NO: 1. In some embodiments, the polypeptide has at least 99% (e.g., at least 99.5%) identity to SEQ ID NO: 1. Additional aspects of the disclosure include polypeptides comprising SEQ ID NO: 1, any one of SEQ ID NO: 5-23, or any one of SEQ ID NO: 71-117. In some embodiments, the polypeptide has at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5%) identity to SEQ ID NO: 1, and includes at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5%) identity to SEQ ID NO: 1. NO:1 I52, S53, I67, I72, I80, V83, A85, I95, I123, S125, I153, I175, I207, I237, D7, T 9. T11, E27, S28, S29, M30, K31, T32, H33, R34, L35, V37, L38, S39, V83, G89, S94, G97, S 99, Q100, G104, N106, P107, D111, E113, S114, N115, G118, I123, V129, T135, T140, V142, H143, N148, E164, T199 or V241 corresponding to one or more substitution mutations (e.g., conservative amino acid substitutions, alanine substitutions or leucine substitutions). In some embodiments, the one or more substitution mutations are leucine substitutions at one or more positions corresponding to any one or more of I52, S53, I67, I72, I80, V83, A85, I95, I123, S125, I153, I175, I207 or I237 of SEQ ID NO: 1. In some embodiments, the one or more substitution mutations are I52L and I180L, I152L and S53L, or I52L and S53L and I80L.In some embodiments, the one or more substitution mutations are one or more conservative amino acid or alanine substitutions at one or more positions corresponding to any one or more of D7, T9, T11, E27, S28, S29, M30, K31, T32, H33, R34, L35, V37, L38, S39, V83, G89, S94, G97, S99, Q100, G104, N106, P107, D111, E113, S114, N115, G118, 1123, V129, T135, T140, V142, H143, N148, E164, T199, or V241 of SEQ ID NO: 1. In some embodiments, the one or more substitution mutations are one or more alanine substitutions at one or more positions corresponding to any one or more of D7, T9, T11, E27, S28, S29, M30, K31, T32, H33, R34, L35, V37, L38, S39, V83, G89, S94, G97, S99, Q100, G104, N106, P107, D111, E113, S114, N115, G118, 1123, V129, T135, T140, V142, H143, N148, E164, T199, or V241 of SEQ ID NO: 1. In some embodiments, the polypeptide does not comprise a substitution mutation at one or more positions corresponding to one or more of W16, F36, T51L, W105, F122, L127, Y131, and S139 of SEQ ID NO: 1. In some embodiments, a polypeptide having at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity to any one of SEQ ID NO: 24-26 is provided. In some embodiments, the polypeptide comprises any one of SEQ ID NO: 24-26. In some embodiments, the polypeptide is insecticidal against coleopteran pests. In some embodiments, the polypeptide is insecticidal against rootworm pests (e.g., corn rootworms).

[0011] The disclosure also provides polypeptides comprising an amino acid sequence having at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity to SEQ ID NO: 1 and being toxic to coleopteran pests (e.g., rootworm pests such as western corn rootworm (corn rootworm)). The disclosure also encompasses polypeptides comprising an amino acid sequence having at least 90% identity to SEQ ID NO: 1 and being toxic to coleopteran pests (e.g., rootworm pests such as western corn rootworm (corn rootworm)), wherein the polypeptide is derived from a Samsonia bacterium. Additional aspects of the disclosure include polypeptides consisting of the amino acid sequence of SEQ ID NO: 1 or differing from the amino acid sequence of SEQ ID NO: 1 by conservative substitutions, deletions, and / or additions of 1-5 amino acids, wherein the polypeptide is toxic to coleopteran pests (e.g., rootworm pests such as western corn rootworm). In some embodiments, the polypeptide consists of SEQ ID NO: 1, any one of SEQ ID NOs: 5-23, or any one of SEQ ID NOs: 71-117.

[0012] Another aspect of the present disclosure is a nucleic acid molecule comprising a coding sequence encoding a polypeptide of any of the above embodiments. In some embodiments, the coding sequence comprises a nucleotide sequence having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) identity with any one of SEQ ID NO:2-4, 27-45, 49-67 or 118-164 or comprises any one of SEQ ID NO:2-4, 27-45, 49-67 or 118-164. In some embodiments, the coding sequence comprises a nucleotide sequence having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) identity with any one of SEQ ID NO:4 or 49-67 or comprises any one of SEQ ID NO:4 or 49-67. In some embodiments, the coding sequence is codon optimized for expression in plants (e.g., in maize plants). In some embodiments, the coding sequence is operably linked to a heterologous promoter, for example, a plant-expressible heterologous promoter.

[0013] Still other aspects of the present disclosure are vectors comprising any of the nucleic acid molecules and transgenic host cells comprising any of the polypeptides or any of the nucleic acid molecules. In certain embodiments, the transgenic host cell is a plant cell, such as a monocot cell, for example, a maize cell. In certain embodiments, the transgenic host cell is a bacterial cell, such as an Agrobacterium, a Bacillus or an Escherichia coli cell.

[0014] Other aspects of this disclosure are compositions comprising any of the polypeptides, optionally further comprising an agriculturally acceptable carrier. Still other aspects of this disclosure are plants comprising any of the polypeptides or comprising any of the nucleic acid molecules. This disclosure contemplates that these plants are monocots and that these plants are maize plants. Other aspects of this disclosure are seeds of any of the plants and cells of any of the plants.

[0015] The present disclosure also provides a method for producing a transgenic plant, the method comprising the following steps: introducing any of the nucleic acid molecules into a plant cell, selecting a plant cell comprising the nucleic acid molecule; and regenerating a plant from the selected plant cell. A method for producing a transgenic plant with enhanced insecticidal properties is also provided, the method comprising the following steps: sexually crossing a first parent plant with a second parent plant, wherein the first or second parent plant is a polypeptide or nucleic acid molecule comprising the present disclosure, and selecting a first generation progeny plant with enhanced insecticidal properties, wherein the selected progeny plant comprises the nucleic acid molecule. In some embodiments, the method further comprises selfing the first generation progeny plant, thereby producing a plurality of second generation progeny plants; and selecting a plant with enhanced insecticidal properties from the second generation progeny plant, wherein the selected second generation progeny plant comprises a nucleic acid molecule.

[0016] The disclosure also relates to methods for controlling coleopteran pests, comprising delivering any of the polypeptides to the pest or its environment, such as in a plant or composition. In some embodiments, the coleopteran pest is a rootworm pest, such as western corn rootworm (corn rootworm). In some embodiments, the polypeptide is delivered by feeding (e.g., the pest feeds on a plant part comprising the polypeptide). The disclosure also contemplates the use of a sequence of any one of SEQ ID NOs: 1 to 164 in bioinformatics analysis to identify insecticidal proteins and the use of a polypeptide comprising an amino acid sequence of any one of SEQ ID NOs: 1, 5-26, or 71-117 in insect bioassays to identify insecticidal proteins (e.g., insecticidal properties for coleopteran pests, such as rootworm pests, such as western corn rootworm (corn rootworm)).

[0017] Brief description of the sequences in the sequence listing

[0018] SEQ ID NO: 1 is the amino acid sequence of SamsoniaCRW identified in Samsonia erythrinae.

[0019] SEQ ID NO: 2 is the native nucleotide sequence of Samsonia CRW.

[0020] SEQ ID NO: 3 is the E. coli codon-optimized nucleotide sequence of Samsonia CRW.

[0021] SEQ ID NO: 4 is the maize codon-optimized nucleotide sequence of Samsonia CRW.

[0022] SEQ ID NO:5 is the amino acid sequence of SamsoniaCRW T51L.

[0023] SEQ ID NO:6 is the amino acid sequence of SamsoniaCRW I52L

[0024] SEQ ID NO:7 is the amino acid sequence of SamsoniaCRW S53L.

[0025] SEQ ID NO: 8 is the amino acid sequence of SamsoniaCRW I67L.

[0026] SEQ ID NO:9 is the amino acid sequence of SamsoniaCRW I72L

[0027] SEQ ID NO: 10 is the amino acid sequence of SamsoniaCRW I80L.

[0028] SEQ ID NO: 11 is the amino acid sequence of SamsoniaCRW V83L.

[0029] SEQ ID NO: 12 is the amino acid sequence of SamsoniaCRW A85L.

[0030] SEQ ID NO: 13 is the amino acid sequence of SamsoniaCRW I95L.

[0031] SEQ ID NO: 14 is the amino acid sequence of SamsoniaCRW I123L.

[0032] SEQ ID NO: 15 is the amino acid sequence of SamsoniaCRW S125L.

[0033] SEQ ID NO: 16 is the amino acid sequence of SamsoniaCRW I153L.

[0034] SEQ ID NO: 17 is the amino acid sequence of SamsoniaCRW I175L.

[0035] SEQ ID NO: 18 is the amino acid sequence of SamsoniaCRW I207L.

[0036] SEQ ID NO: 19 is the amino acid sequence of SamsoniaCRW I237L.

[0037] SEQ ID NO: 20 is the amino acid sequence of Samsonia CRW 152L / 180L.

[0038] SEQ ID NO:21 is the amino acid sequence of SamsoniaCRW T51L / I52L.

[0039] SEQ ID NO:22 is the amino acid sequence of SamsoniaCRW I52L / S53L.

[0040] SEQ ID NO:23 is the amino acid sequence of SamsoniaCRW T51L / I52L / S53L.

[0041] SEQ ID NO: 24 is the amino acid sequence of MGYP000214679871 (Samsonia CRW ortholog).

[0042] SEQ ID NO: 25 is the amino acid sequence of MGYP000317625865 (SamsoniaCRW ortholog).

[0043] SEQ ID NO: 26 is the amino acid sequence of MGYP000301824029 (SamsoniaCRW ortholog).

[0044] SEQ ID NO: 27 is the E. coli codon-optimized nucleotide sequence of SamsoniaCRW T51L.

[0045] SEQ ID NO: 28 is the E. coli codon-optimized nucleotide sequence of SamsoniaCRW I52L.

[0046] SEQ ID NO: 29 is the E. coli codon-optimized nucleotide sequence of SamsoniaCRW S53L.

[0047] SEQ ID NO: 30 is the E. coli codon-optimized nucleotide sequence of Samsonia CRW I67L.

[0048] SEQ ID NO:31 is the E. coli codon-optimized nucleotide sequence of SamsoniaCRW I72L.

[0049] SEQ ID NO: 32 is the E. coli codon-optimized nucleotide sequence of Samsonia CRW I80L.

[0050] SEQ ID NO: 33 is the E. coli codon-optimized nucleotide sequence of SamsoniaCRW V83L.

[0051] SEQ ID NO:34 is the E. coli codon-optimized nucleotide sequence of SamsoniaCRW A85L.

[0052] SEQ ID NO: 35 is the E. coli codon-optimized nucleotide sequence of SamsoniaCRW I95L.

[0053] SEQ ID NO: 36 is the E. coli codon-optimized nucleotide sequence of SamsoniaCRW I123L.

[0054] SEQ ID NO: 37 is the E. coli codon-optimized nucleotide sequence of SamsoniaCRW S125L.

[0055] SEQ ID NO:38 is the E. coli codon-optimized nucleotide sequence of SamsoniaCRW I153L.

[0056] SEQ ID NO:39 is the codon-optimized nucleotide sequence of E. coli SamsoniaCRW I175L

[0057] SEQ ID NO:40 is the E. coli codon-optimized nucleotide sequence of SamsoniaCRW I207L.

[0058] SEQ ID NO:41 is the E. coli codon-optimized nucleotide sequence of SamsoniaCRW I237L.

[0059] SEQ ID NO:42 is the codon-optimized nucleotide sequence of SamsoniaCRW I52L / I80L in E. coli.

[0060] SEQ ID NO:43 is the codon-optimized nucleotide sequence of SamsoniaCRW T51L / I52L ​​in E. coli.

[0061] SEQ ID NO:44 is the E. coli codon-optimized nucleotide sequence of SamsoniaCRW I52L / S53L.

[0062] SEQ ID NO:45 is the E. coli codon-optimized nucleotide sequence of SamsoniaCRW T51L / I52L / S53L.

[0063] SEQ ID NO:46 is the E. coli codon-optimized nucleotide sequence of MGYP000214679871 (Samsonia CRW ortholog).

[0064] SEQ ID NO:47 is the E. coli codon-optimized nucleotide sequence of MGYP000317625865 (Samsonia CRW ortholog).

[0065] SEQ ID NO:48 is the E. coli codon-optimized nucleotide sequence of MGYP000301824029 (Samsonia CRW ortholog).

[0066] SEQ ID NO:49 is the maize codon-optimized nucleotide sequence of SamsoniaCRW T51L.

[0067] SEQ ID NO:50 is the maize codon-optimized nucleotide sequence of SamsoniaCRW I52L.

[0068] SEQ ID NO:51 is the maize codon-optimized nucleotide sequence of SamsoniaCRW S53L.

[0069] SEQ ID NO:52 is the maize codon-optimized nucleotide sequence of SamsoniaCRW I67L.

[0070] SEQ ID NO:53 is the maize codon-optimized nucleotide sequence of SamsoniaCRW I72L.

[0071] SEQ ID NO:54 is the maize codon-optimized nucleotide sequence of SamsoniaCRW I80L.

[0072] SEQ ID NO:55 is the maize codon-optimized nucleotide sequence of SamsoniaCRW V83L.

[0073] SEQ ID NO:56 is the maize codon-optimized nucleotide sequence of SamsoniaCRW A85L.

[0074] SEQ ID NO:57 is the maize codon-optimized nucleotide sequence of SamsoniaCRW I95L.

[0075] SEQ ID NO:58 is the maize codon-optimized nucleotide sequence of SamsoniaCRW I123L.

[0076] SEQ ID NO:59 is the maize codon-optimized nucleotide sequence of SamsoniaCRW S125L.

[0077] SEQ ID NO: 60 is the maize codon-optimized nucleotide sequence of SamsoniaCRW I153L.

[0078] SEQ ID NO:61 is the maize codon-optimized nucleotide sequence of SamsoniaCRW I175L

[0079] SEQ ID NO: 62 is the maize codon-optimized nucleotide sequence of SamsoniaCRW I207L.

[0080] SEQ ID NO: 63 is the maize codon-optimized nucleotide sequence of SamsoniaCRW I237L.

[0081] SEQ ID NO:64 is the maize codon-optimized nucleotide sequence of SamsoniaCRW I52L / I80L.

[0082] SEQ ID NO:65 is the maize codon-optimized nucleotide sequence of SamsoniaCRW T51L / I52L.

[0083] SEQ ID NO: 66 is the maize codon-optimized nucleotide sequence of SamsoniaCRW I52L / S53L.

[0084] SEQ ID NO:67 is the maize codon-optimized nucleotide sequence of SamsoniaCRW T51L / I52L / S53L.

[0085] SEQ ID NO: 68 is the maize codon-optimized nucleotide sequence of MGYP000214679871 (Samsonia CRW ortholog).

[0086] SEQ ID NO:69 is the maize codon-optimized nucleotide sequence of MGYP000317625865 (Samsonia CRW ortholog).

[0087] SEQ ID NO:70 is the maize codon-optimized nucleotide sequence of MGYP000301824029 (Samsonia CRW ortholog).

[0088] SEQ ID NO:71 is the amino acid sequence of SamsoniaCRW D7A

[0089] SEQ ID NO:72 is the amino acid sequence of SamsoniaCRW T9A

[0090] SEQ ID NO:73 is the amino acid sequence of SamsoniaCRW T11A

[0091] SEQ ID NO:74 is the amino acid sequence of SamsoniaCRW W16A

[0092] SEQ ID NO:75 is the amino acid sequence of SamsoniaCRW E27A

[0093] SEQ ID NO:76 is the amino acid sequence of SamsoniaCRW S28A

[0094] SEQ ID NO:77 is the amino acid sequence of SamsoniaCRW S29A

[0095] SEQ ID NO:78 is the amino acid sequence of SamsoniaCRW M30A

[0096] SEQ ID NO:79 is the amino acid sequence of SamsoniaCRW K31A

[0097] SEQ ID NO:80 is the amino acid sequence of SamsoniaCRW T32A

[0098] SEQ ID NO:81 is the amino acid sequence of SamsoniaCRW H33A

[0099] SEQ ID NO:82 is the amino acid sequence of SamsoniaCRW R34A

[0100] SEQ ID NO:83 is the amino acid sequence of SamsoniaCRW L35A

[0101] SEQ ID NO:84 is the amino acid sequence of SamsoniaCRW F36A

[0102] SEQ ID NO:85 is the amino acid sequence of SamsoniaCRW V37A

[0103] SEQ ID NO:86 is the amino acid sequence of SamsoniaCRW L38A

[0104] SEQ ID NO:87 is the amino acid sequence of SamsoniaCRW S39A

[0105] SEQ ID NO:88 is the amino acid sequence of SamsoniaCRW V83A

[0106] SEQ ID NO:89 is the amino acid sequence of SamsoniaCRW G89A

[0107] SEQ ID NO:90 is the amino acid sequence of SamsoniaCRW S94A

[0108] SEQ ID NO:91 is the amino acid sequence of SamsoniaCRW G97A

[0109] SEQ ID NO:92 is the amino acid sequence of SamsoniaCRW S99A

[0110] SEQ ID NO:93 is the amino acid sequence of SamsoniaCRW Q100A

[0111] SEQ ID NO:94 is the amino acid sequence of SamsoniaCRW G104A

[0112] SEQ ID NO:95 is the amino acid sequence of SamsoniaCRW W105A

[0113] SEQ ID NO:96 is the amino acid sequence of SamsoniaCRW N106A

[0114] SEQ ID NO:97 is the amino acid sequence of SamsoniaCRW P107A

[0115] SEQ ID NO:98 is the amino acid sequence of SamsoniaCRW D111A

[0116] SEQ ID NO:99 is the amino acid sequence of SamsoniaCRW E113A

[0117] SEQ ID NO: 100 is the amino acid sequence of SamsoniaCRW S114A

[0118] SEQ ID NO: 101 is the amino acid sequence of SamsoniaCRW N115A

[0119] SEQ ID NO: 102 is the amino acid sequence of SamsoniaCRW G118A

[0120] SEQ ID NO: 103 is the amino acid sequence of SamsoniaCRW F122A

[0121] SEQ ID NO: 104 is the amino acid sequence of SamsoniaCRW I123A

[0122] SEQ ID NO: 105 is the amino acid sequence of SamsoniaCRW L127A

[0123] SEQ ID NO: 106 is the amino acid sequence of SamsoniaCRW V129A

[0124] SEQ ID NO: 107 is the amino acid sequence of SamsoniaCRW Y131A

[0125] SEQ ID NO: 108 is the amino acid sequence of SamsoniaCRW S139A

[0126] SEQ ID NO: 109 is the amino acid sequence of SamsoniaCRW T135A

[0127] SEQ ID NO: 110 is the amino acid sequence of SamsoniaCRW T140A

[0128] SEQ ID NO: 111 is the amino acid sequence of SamsoniaCRW V142A

[0129] SEQ ID NO: 112 is the amino acid sequence of SamsoniaCRW H143A

[0130] SEQ ID NO: 113 is the amino acid sequence of SamsoniaCRW N148A

[0131] SEQ ID NO: 114 is the amino acid sequence of Samsonia CRW E164A

[0132] SEQ ID NO: 115 is the amino acid sequence of SamsoniaCRW T199A

[0133] SEQ ID NO: 116 is the amino acid sequence of SamsoniaCRW V241A

[0134] SEQ ID NO: 117 is the amino acid sequence of SamsoniaCRW I52L / S53L / I80L

[0135] SEQ ID NO: 118 is the E. coli codon-optimized nucleotide sequence of Samsonia CRW D7A

[0136] SEQ ID NO: 119 is the codon-optimized nucleotide sequence of SamsoniaCRW T9A in E. coli

[0137] SEQ ID NO: 120 is the codon-optimized nucleotide sequence of SamsoniaCRW T11A in E. coli

[0138] SEQ ID NO: 121 is the codon-optimized nucleotide sequence of Samsonia CRW W16A in E. coli

[0139] SEQ ID NO: 122 is the codon-optimized nucleotide sequence of Samsonia CRW E27A in E. coli

[0140] SEQ ID NO: 123 is the codon-optimized nucleotide sequence of Samsonia CRW S28A in E. coli

[0141] SEQ ID NO: 124 is the codon-optimized nucleotide sequence of Samsonia CRW S29A in E. coli

[0142] SEQ ID NO: 125 is the codon-optimized nucleotide sequence of Samsonia CRW M30A in E. coli

[0143] SEQ ID NO: 126 is the codon-optimized nucleotide sequence of SamsoniaCRW K31A in E. coli

[0144] SEQ ID NO: 127 is the codon-optimized nucleotide sequence of SamsoniaCRW T32A in E. coli

[0145] SEQ ID NO: 128 is the codon-optimized nucleotide sequence of SamsoniaCRW H33A in E. coli

[0146] SEQ ID NO: 129 is the codon-optimized nucleotide sequence of Samsonia CRW R34A in E. coli

[0147] SEQ ID NO: 130 is the codon-optimized nucleotide sequence of SamsoniaCRW L35A in E. coli

[0148] SEQ ID NO: 131 is the codon-optimized nucleotide sequence of Samsonia CRW F36A in E. coli

[0149] SEQ ID NO: 132 is the codon-optimized nucleotide sequence of Samsonia CRW V37A in E. coli

[0150] SEQ ID NO: 133 is the codon-optimized nucleotide sequence of SamsoniaCRW L38A in E. coli

[0151] SEQ ID NO: 134 is the codon-optimized nucleotide sequence of SamsoniaCRW S39A in E. coli

[0152] SEQ ID NO: 135 is the codon-optimized nucleotide sequence of SamsoniaCRW V83A in E. coli

[0153] SEQ ID NO: 136 is the codon-optimized nucleotide sequence of Samsonia CRW G89A in E. coli

[0154] SEQ ID NO: 137 is the codon-optimized nucleotide sequence of Samsonia CRW S94A in E. coli

[0155] SEQ ID NO: 138 is the codon-optimized nucleotide sequence of Samsonia CRW G97A in E. coli

[0156] SEQ ID NO: 139 is the codon-optimized nucleotide sequence of Samsonia CRW S99A in E. coli

[0157] SEQ ID NO: 140 is the codon-optimized nucleotide sequence of Samsonia CRW Q100A in E. coli

[0158] SEQ ID NO: 141 is the codon-optimized nucleotide sequence of Samsonia CRW G104A in E. coli

[0159] SEQ ID NO: 142 is the codon-optimized nucleotide sequence of Samsonia CRW W105A in E. coli

[0160] SEQ ID NO: 143 is the codon-optimized nucleotide sequence of Samsonia CRW N106A in E. coli

[0161] SEQ ID NO: 144 is the codon-optimized nucleotide sequence of Samsonia CRW P107A in E. coli

[0162] SEQ ID NO: 145 is the codon-optimized nucleotide sequence of Samsonia CRW D111A in E. coli

[0163] SEQ ID NO: 146 is the codon-optimized nucleotide sequence of Samsonia CRW E113A in E. coli

[0164] SEQ ID NO: 147 is the codon-optimized nucleotide sequence of Samsonia CRW S114A in E. coli

[0165] SEQ ID NO: 148 is the codon-optimized nucleotide sequence of SamsoniaCRW N115A in E. coli

[0166] SEQ ID NO: 149 is the codon-optimized nucleotide sequence of SamsoniaCRW G118A in E. coli

[0167] SEQ ID NO: 150 is the codon-optimized nucleotide sequence of Samsonia CRW F122A in E. coli

[0168] SEQ ID NO: 151 is the codon-optimized nucleotide sequence of Samsonia CRW I123A in E. coli

[0169] SEQ ID NO: 152 is the codon-optimized nucleotide sequence of Samsonia CRW L127A in E. coli

[0170] SEQ ID NO: 153 is the codon-optimized nucleotide sequence of SamsoniaCRW V129A in E. coli

[0171] SEQ ID NO: 154 is the codon-optimized nucleotide sequence of Samsonia CRW Y131A in E. coli

[0172] SEQ ID NO: 155 is the codon-optimized nucleotide sequence of Samsonia CRW S139A in E. coli

[0173] SEQ ID NO: 156 is the codon-optimized nucleotide sequence of SamsoniaCRW T135A in E. coli

[0174] SEQ ID NO: 157 is the codon-optimized nucleotide sequence of SamsoniaCRW T140A in E. coli

[0175] SEQ ID NO: 158 is the codon-optimized nucleotide sequence of Samsonia CRW V142A in E. coli

[0176] SEQ ID NO: 159 is the E. coli codon-optimized nucleotide sequence of Samsonia CRW H143A

[0177] SEQ ID NO: 160 is the codon-optimized nucleotide sequence of Samsonia CRW N148A in E. coli

[0178] SEQ ID NO: 161 is the codon-optimized nucleotide sequence of Samsonia CRW E164A in E. coli

[0179] SEQ ID NO: 162 is the codon-optimized nucleotide sequence of SamsoniaCRW T199A in E. coli

[0180] SEQ ID NO: 163 is the codon-optimized nucleotide sequence of Samsonia CRW V241A in E. coli

[0181] SEQ ID NO: 164 is the codon-optimized nucleotide sequence of E. coli SamsoniaCRW I52L / S53L / I80L DETAILED DESCRIPTION

[0182] This specification is not intended to be an inventory of all the different ways in which the present disclosure can be implemented or an inventory of all the features that can be added to the present disclosure. For example, features described with respect to one embodiment can be incorporated into other embodiments, and features described with respect to a particular embodiment can be deleted from that embodiment. Therefore, the present disclosure contemplates that in some embodiments, any feature or combination of features set forth herein may be excluded or omitted. In addition, in view of the present disclosure, numerous changes and additions to the various embodiments suggested herein are apparent to those skilled in the art, and this does not depart from the present disclosure. Therefore, the following description is intended to illustrate some special embodiments of the present disclosure, and does not exhaustively narrate all its permutations, combinations, and variations.

[0183] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. The terms used in the description disclosed herein are for the purpose of describing specific embodiments only and are not intended to limit this disclosure.

[0184] All publications, patent applications, patents, and other references cited herein are incorporated by reference in their entirety for the teachings of the pertinent sentence and / or paragraph to which the reference is addressed.

[0185] The nucleotide sequences provided herein are presented from left to right in the 5' to 3' direction and are represented using the standard code representing nucleotide bases, as set forth in 37 CFR §§1.821-1.825 and World Intellectual Property Organization (WIPO) Standard ST.25, e.g., adenine (A), cytosine (C), thymine (T), and guanine (G).

[0186] Amino acids are also indicated using WIPO Standard ST.25, for example: alanine (Ala; A), arginine (Arg; R), asparagine (Asn; N), aspartic acid (Asp; D), cysteine ​​(Cys; C), glutamine (Gln; Q), glutamic acid (Glu; E), glycine (Gly; G), histidine (His; H), isoleucine (Ile; 1), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y), and valine (Val; V).

[0187] Unless the context indicates otherwise, it is expressly contemplated that the various features of the disclosure described herein may be used in any combination. Moreover, the disclosure also contemplates that in some embodiments of the disclosure, any feature or combination of features stated herein may be excluded or omitted. For example, if the specification states that a composition comprises components A, B, and C, it is expressly contemplated that any one or combination of A, B, or C may be omitted and abandoned, singly or in any combination.

[0188] definition

[0189] For the sake of clarity, certain terms used in this specification are defined and presented below:

[0190] As used herein and in the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a plant" is a reference to one or more plants and includes equivalents thereof known to those skilled in the art, and so forth.

[0191] As used herein, the term "or" also includes "and / or" unless the context clearly indicates otherwise.

[0192] The term "about" is used herein to mean approximately, roughly, approximately, or around. When the term "about" is used in conjunction with a numerical range, it defines the range by extending the boundaries above and below the stated values. In general, the term "about" is used herein to define a numerical value to a variation of 20%, preferably 10% above and below (higher or lower) above and below the stated value. With respect to temperature, the term "about" means ±1°C, preferably ±0.5°C. When the term "about" is used in the context of the present disclosure (e.g., in combination with a temperature or molecular weight value), the exact value (i.e., no "about") is preferred.

[0193] Unless the context indicates otherwise, as used herein, phrases such as "between about X and Y," "between about X and about Y," "from X to Y," and "from about X to about Y" (and similar phrases) should be interpreted as including X and Y. Units, prefixes, and symbols may be represented in their SI recognized form. Unless otherwise indicated, nucleic acids are written from left to right in 5' to 3' orientation; amino acid sequences are written from left to right in N-terminal to C-terminal orientation, respectively. Amino acids may be referred to herein by either their commonly known three letter symbols or by the single-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Similarly, nucleotides may be referred to by their generally accepted single-letter codes.

[0194] The "activity" of the insecticidal proteins of the present disclosure means that the insecticidal proteins act as an orally active insect control agent, have toxic effects (e.g., inhibit the ability of insect pests to survive, grow and / or reproduce), and / or are able to interfere with or prevent insect feeding, which may or may not cause the death of the insect. When the insecticidal proteins of the present disclosure are delivered to insects, this result is typically the death of the insect, or the insect does not feed on a source to which the insecticidal protein is available to the insect. "Pesticide" is defined as a toxic biological activity that is able to control pests (such as insects, nematodes, fungi, bacteria or viruses), preferably by killing or destroying them. "Insecticidal" is defined as a toxic biological activity that is able to control insects, preferably by killing them. "Pesticide" is an agent with pesticidal activity. "Insecticide" is an agent with insecticidal activity.

[0195] A "coding sequence" is a nucleic acid sequence that is transcribed into RNA (eg, mRNA, rRNA, tRNA, snRNA, sense RNA, or antisense RNA) and then preferably translated in an organism to produce a protein.

[0196] As used herein, "codon optimized" sequence means the following nucleotide sequence, wherein codons are selected to reflect the specific codon preference that a host cell or organism may have. This is typically accomplished in such a manner that the amino acid sequence of a polypeptide encoded by the nucleotide sequence to be optimized is maintained. In certain embodiments, the DNA sequence of a recombinant DNA construct includes a sequence that has been codon optimized for the cell (e.g., animal cell, plant cell, or fungal cell) to be expressed therein for the construct. For example, a construct to be expressed in a plant cell can have its entire or partial sequence (e.g., a first gene suppression element or a gene expression element) codon optimized for expression in a plant. See, e.g., U.S. Patent No. 6,121,014, which is incorporated herein by reference. In an embodiment, the polynucleotides of the present disclosure are codon optimized for expression in a plant cell (e.g., a dicotyledonous plant cell or a monocotyledonous plant cell) or a bacterial cell.

[0197] "Controlling" insects means inhibiting the ability of insect pests to survive, grow, feed, and / or reproduce through toxic effects, and / or limiting crop plant damage or loss associated with insects, and / or protecting the yield potential of crops when grown in the presence of insect pests. "Controlling" insects may or may not mean killing insects, although it preferably means killing insects. In some embodiments of the present disclosure, "controlling" insects means killing insects.

[0198] The terms "comprises", "comprising", "includes", "including", "having" and their variations mean "including but not limited to". These terms indicate the presence of stated features, integers, steps, operations, elements, or components but do not exclude the presence or addition of one or more other features, integers, steps, operations, elements, components, or groups thereof. The term "consisting of" means "including and limited to".

[0199] As used herein, the transitional phrase "consisting essentially of (and grammatical variations) means that the scope of the claim is to be interpreted as encompassing the specified materials or steps recited in the claim and those that do not materially alter one or more of the basic and novel characteristics of the claimed disclosure. Thus, the term "consisting essentially of" when used in the claims of the present disclosure is not intended to be interpreted as equivalent to "comprising."

[0200] In the context of the present disclosure, "corresponding to or corresponds to" means that when the amino acid sequence of a reference sequence is aligned with a second amino acid sequence (e.g., a variant sequence or a homologous sequence) that is different from the reference sequence, the amino acids "corresponding to" certain enumerated positions in the second amino acid sequence are those that are aligned with these positions in the reference amino acid sequence, but not necessarily at these exact numerical positions relative to the specific reference amino acid sequence of the present disclosure.

[0201] "Delivering" or "delivering" a composition or insecticidal protein means that the composition or insecticidal protein contacts an insect, which promotes oral ingestion of the composition or insecticidal protein, thereby producing a toxic effect and control on the insect. The composition or insecticidal protein can be delivered in many recognized ways, for example, by a transgenic plant expressing the insecticidal protein, one or more formulated protein compositions, one or more sprayable protein compositions, a bait matrix, or any other toxin delivery system recognized in the art.

[0202] The term "domain" refers to a group of amino acids that are conserved at specific positions along the alignment of the sequences of evolutionarily related proteins. Although amino acids at other positions may differ between homologs, highly conserved amino acids at specific positions indicate amino acids that are likely to be essential in the structure, stability or function of the protein. They are identified by their high conservation in the aligned sequences of a family of protein homologs and can be used as identifiers to determine whether any polypeptide in question belongs to a previously identified group of polypeptides.

[0203] The "engineered" protein of the present disclosure refers to a protein having a different sequence at at least one amino acid position compared to at least one corresponding parent protein. The engineered protein can be a mutant protein, which comprises, for example, one or more modifications relative to the parent protein, such as deletions, additions and / or substitutions of one or more amino acid positions. The engineered protein can be a chimeric protein and comprises, for example, one or more exchanged or shuffled domains or fragments from at least two parent proteins.

[0204] By "effective insect controlling amount" is meant a concentration of an insecticidal protein that inhibits the ability of insects to survive, grow, feed and / or reproduce, or limits insect-related damage or crop plant losses, through toxic effects. An "effective insect controlling amount" may or may not mean killing insects, although it preferably means killing insects. A transgenic plant with "enhanced insecticidal properties" is a plant that expresses one or more proteins in an effective insect controlling amount, so that in some embodiments, the plant is insecticidal to an expanded range of insect species relative to an untransformed plant of the same species. This expanded range of insect species includes insect plant pests, such as coleopteran insect pests, such as the corn rootworm (western corn rootworm).

[0205] The term "event" refers to the original transformant and / or the progeny of the transformant comprising heterologous DNA. The term "event" also refers to the progeny produced by sexual outcrossing (outcross) between the transformant and another maize strain. Even after repeated backcrossing with the recurrent parent, the insertion DNA and flanking DNA from the transformed parent are present in the same chromosome position of the hybrid progeny. The term "event" also refers to the DNA from the original transformant comprising the DNA inserted and the flanking genomic sequence next to the DNA inserted, and it will be expected to be transferred to a progeny produced by sexual hybridization of a parental strain (such as the original transformant and the progeny produced by self-fertilization) comprising the DNA inserted and a parental strain not containing the DNA inserted. Typically, the transformation of plant tissue produces multiple events, and each of the above-mentioned events represents that the DNA construct is inserted into the different positions in the genome of the plant cell.

[0206] As used herein, "expression cassette" means a nucleic acid sequence capable of directing the expression of a specific nucleotide sequence in an appropriate host cell, comprising a promoter operably connected to a target nucleotide sequence, which is operably connected to a termination signal. It also typically comprises a sequence required for the appropriate translation of the nucleotide sequence. The expression cassette comprising the target nucleotide sequence may have at least one of its components, which is heterologous relative to at least one of its other components. The expression cassette may also be a naturally occurring expression cassette obtained in a recombinant form for heterologous expression. However, typically, the expression cassette is heterologous relative to the host, i.e., the specific nucleic acid sequence of the expression cassette is not naturally present in the host cell, and must have been introduced into the host cell or the ancestor of the host cell by a transformation event. The expression of the nucleotide sequence in the expression cassette may be under the control of a constitutive promoter or an inducible promoter, which initiates transcription only when the host cell is exposed to some specific external stimuli. In the case of multicellular organisms (such as plants), the promoter may also be specific for a specific tissue, or an organ, or a developmental stage.

[0207] The expression cassette comprising the nucleotide sequence of interest may be chimeric, meaning that at least one of its components is heterologous relative to at least one of its other components. The expression cassette may also be an expression cassette comprising a native promoter driving its native gene; however, it has been obtained in a recombinant form that can be used for heterologous expression. Such use of an expression cassette renders it not so naturally present in the cell into which it is introduced.

[0208] The expression cassette can also optionally include a transcription and / or translation termination region (i.e., termination region) that plays a role in plants. A variety of transcription terminators are available for use in the expression cassette and are responsible for transcription termination and correct mRNA polyadenylation when exceeding the heterologous nucleotide sequence of interest. The termination region can be natural for the transcription initiation region, can be natural for the nucleotide sequence of interest that is operably connected, can be natural for the plant host, or can be derived from another source (i.e., foreign or heterologous for promoter, nucleotide sequence of interest, plant host, or any combination thereof). Suitable transcription terminators include, but are not limited to, CAMV 35S terminator, tml terminator, nopaline synthase terminator, and / or pea rbcs E9 terminator. These terminators can be used in both monocotyledons and dicotyledons. In addition, the natural transcription terminator of the coding sequence can be used. Any terminator known to play a role in plants that can be used can be used in the context of this disclosure.

[0209] When used with reference to polynucleotides (such as genes, ORFs or parts thereof, or transgenics of plants), the term "expression" refers to the process by which the genetic information encoded in a gene is converted into RNA (e.g., mRNA, rRNA, tRNA or snRNA) by the "transcription" of the gene (i.e., via the enzymatic action of an RNA polymerase), and, where applicable (e.g., if the gene encodes a protein), into a protein by the "translation" of the mRNA. Gene expression can be regulated at many stages of the method. For example, in the case of an antisense construct or a dsRNA construct, expression may refer only to the transcription of the antisense RNA or only to the transcription of the dsRNA, respectively. In an embodiment, "expression" refers to the transcription and stable accumulation of a sense (mRNA) or functional RNA. "Expression" may also refer to the production of a protein.

[0210] "Gene" is a limited region located within a genome and comprising a coding nucleic acid sequence, and typically also comprises other major regulatory nucleic acids responsible for controlling the expression (i.e., transcription and translation) of the coding portion. A gene may also comprise other 5' and 3' untranslated sequences and termination sequences. Other possible elements are, for example, introns. As found in nature, the regulatory nucleic acid sequence of a gene may not be operably connected to the associated nucleic acid sequence under normal circumstances, and therefore will not be a mosaic gene.

[0211] The term "heterologous" when used in reference to a gene or polynucleotide or polypeptide means that the gene or polynucleotide or polypeptide is not in its natural environment or contains a portion of it that is present in a non-natural environment (i.e., has been artificially altered). For example, a heterologous gene may include a polynucleotide introduced from one species into another species. A heterologous gene may also include a polynucleotide that is native to an organism that has been altered in some way (e.g., by mutation; addition in multiple copies; attachment to a non-natural promoter or enhancer polynucleotide, etc.). A heterologous gene may further include a plant gene polynucleotide that includes a cDNA form of a plant gene; the cDNA may be expressed in a sense orientation (to produce mRNA) or an antisense orientation (to produce an antisense RNA transcript that is complementary to the mRNA transcript). In one aspect of the present disclosure, heterologous genes are distinguished from endogenous plant genes in that heterologous gene polynucleotides are typically joined to polynucleotides containing regulatory elements such as promoters, and these polynucleotides are not found to be naturally associated with genes for proteins encoded by heterologous genes or with plant gene polynucleotides in chromosomes, or these polynucleotides are associated with parts of chromosomes not found in nature (e.g., genes expressed in loci that do not normally express genes). In addition, "heterologous" polynucleotides refer to polynucleotides that are not naturally associated with host cells into which the polynucleotides are introduced, including multiple copies of non-naturally occurring polynucleotides. Heterologous nucleic acid sequences or nucleic acid molecules can include chimeric sequences, such as chimeric expression cassettes, in which promoters and coding regions are derived from multi-source organisms. The promoter sequence can be a constitutive promoter sequence, a tissue-specific promoter sequence, a chemically inducible promoter sequence, a wound-inducible promoter sequence, a stress-inducible promoter sequence, or a developmental stage-specific promoter sequence.

[0212] A "homologous" nucleic acid sequence is a nucleic acid sequence naturally associated with a host cell into which it is introduced.

[0213] As used herein, the terms "increase, increasing, increased," "enhance, enhanced, enhancing and enhancement" and similar terms describe an increase in the control of plant pests, such as by contacting the plant with a polypeptide of the disclosure (such as, for example, by transgenic expression or by topical application methods). Increased control can refer to the level of control of plant pests in the absence of the polypeptide of the disclosure (e.g., a plant that is not transgenic expressing the polypeptide or not topically treated with the polypeptide). Thus, in embodiments, the terms "increase, increasing, increased," "enhance, enhanced, enhancing and enhancement," and similar terms can indicate an increase of at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 125%, 150%, 200%, 300%, 400%, 500% or more as compared to a suitable control (e.g., a plant, plant part, plant cell not contacted with the polypeptide of the disclosure).

[0214] The terms "identity" or "identical" in the context of two nucleic acid or amino acid sequences refers to the percentage of identical nucleotides or amino acids in the linear polynucleotide or amino acid sequence of a reference ("query") sequence (or its complementary strand) when the two sequences are fully aligned, such as compared to a test ("subject") sequence. Unless otherwise indicated, sequence identity as used herein refers to the value obtained by using the Needleman and Wunsch algorithm ((1970) J. Mol. Biol. 48:443-453) implemented in the EMBOSS Needle alignment tool, using the default matrix file EBLOSUM62 (for proteins) and default parameters (gap open=10, gap extension=0.5, terminal gap penalty=false, terminal gap open=10, terminal gap extension=0.5) or DNAfull (for nucleic acids) and default parameters (gap open=10, gap extension=0.5, terminal gap penalty=false, terminal gap open=10, terminal gap extension=0.5); or any equivalent program thereof. EMBOSS Needle is available, for example, from EMBL-EBI, for example at the following website: ebi.ac.uk / Tools / psa / emboss_needle / and as described in the following publication: "The EMBL-EBI search and sequence analysis tools APIs in 2019." Madeira et al. Nucleic Acids Research, June 2019, 47(W1): W636-W641. As used herein, the term "equivalent program" refers to any sequence comparison program that generates an alignment with identical nucleotide or amino acid residue matches and identical sequence identity percentages for any two sequences in question when compared to the corresponding alignment generated by EMBOSS Needle. In some embodiments, substantially identical nucleic acid or amino acid sequences can perform substantially the same function.

[0215] As used herein, "insecticide" is defined as a toxic biological activity that is capable of controlling insect pests, optionally but preferably by killing them.

[0216] In some embodiments, the polynucleotides or polypeptides of the present disclosure are "isolated". The term "isolated" polynucleotide or polypeptide is a polynucleotide or polypeptide that is no longer present in its natural environment. The isolated polynucleotides or polypeptides of the present disclosure may exist in a purified form, or may exist in a recombinant host, such as a transgenic bacterium or a transgenic plant. Thus, for example, the requirement for an "isolated" polynucleotide or polypeptide encompasses a nucleic acid molecule when the nucleic acid molecule is contained within a transgenic plant genome.

[0217] When used in the context of nucleic acid molecules or polynucleotides disclosed herein, the term "isolated" refers to polynucleotides identified and separated / separated in the context of chromosome polynucleotides in the corresponding source organism. If the isolated nucleic acid or polynucleotides do have naturally occurring counterparts, the isolated nucleic acid or polynucleotides are not nucleic acids present in their natural environment. In contrast, non-isolated nucleic acids are nucleic acids (such as DNA and RNA), which are found under the state of natural existence. For example, given polynucleotides (e.g., genes) are found on the host cell chromosome near adjacent genes. Isolated nucleic acid molecules can exist in single-stranded or double-stranded forms. Alternatively, it can include sense strands and antisense strands (i.e., nucleic acid molecules can be double-stranded). In certain embodiments, nucleic acid molecules disclosed herein are isolated.

[0218] The term "motif" or "consensus sequence" or "signature" refers to a short conserved region in the sequence of evolutionarily related proteins. A motif is often a highly conserved portion of a domain, but may also include only a portion of a domain, or be located outside a conserved domain (if all of the amino acids of the motif are located outside the defined domain).

[0219] A "native" or "wild-type" nucleic acid, polynucleotide, nucleotide sequence, polypeptide or amino acid sequence refers to a naturally occurring or endogenous nucleic acid, polynucleotide, nucleotide sequence, polypeptide or amino acid sequence.

[0220] "Nucleic acid molecule" or "nucleic acid" is a segment of single-stranded, double-stranded or partially double-stranded DNA or RNA, or a hybrid thereof, which can be isolated or synthesized from any source. In the context of the present disclosure, a nucleic acid molecule is typically a segment of DNA. In some embodiments, the nucleic acid molecule of the present disclosure is an isolated nucleic acid molecule. In some embodiments, the nucleic acid molecule of the present disclosure is contained in a vector, a plant, a plant cell, or a bacterial cell. These terms also include deoxyribopolynucleotides, ribopolynucleotides, or analogs thereof having the necessary properties of natural ribonucleotides, which have the necessary properties because they hybridize substantially the same nucleotide sequence compared to naturally occurring nucleotides under stringent hybridization conditions, and / or allow translation into one or more identical amino acids compared to one or more naturally occurring nucleotides. Nucleic acid molecules can be full-length sequences or subsequences of natural or heterologous structural genes or regulatory genes. Unless otherwise indicated, the term includes reference to a specified sequence and its complementary sequence. Therefore, DNA or RNA to which the backbone has been modified for stability or other reasons is a "polynucleotide" as the term is meant herein. In addition, to name just two examples, DNA or RNA containing rare bases (such as inosine) or modified bases (such as tritylated bases) are polynucleotides as the term is used herein. It will be recognized that a wide variety of modifications have been made to DNA and RNA, which serve many useful purposes known to those skilled in the art. The term polynucleotide as used herein encompasses these chemically modified, enzymatically modified, or metabolically modified forms of polynucleotides, as well as chemical forms of DNA and RNA that are unique to viruses and cells (especially including simple cells as well as complex cells).

[0221] The terms "nucleic acid," "nucleic acid molecule," and "polynucleotide" are used interchangeably herein.

[0222] "Operably linked" refers to the association of polynucleotides on a single nucleic acid molecule such that the function of one affects the function of the other. For example, a promoter is operably linked to a coding polynucleotide when the promoter is capable of affecting the expression of the coding polynucleotide (i.e., the coding polynucleotide is under the transcriptional control of the promoter). A coding polynucleotide in either the sense or antisense orientation can be operably linked to a regulatory polynucleotide.

[0223] As used herein, "pesticide," "insecticide," and the like refer to the ability of a protein of the disclosure to control pests or the amount of one or more proteins of the disclosure that can control pests.

[0224] The term "plant" includes whole plants, plant organs, plant tissues (e.g., leaves, stems, roots, etc.), seeds, and plant cells and progeny thereof. As used herein, plant cells include, but are not limited to, cells in seeds, suspension cultures, embryos, meristem regions, calli, leaves, roots, branches, gametophytes, sporophytes, pollen, and microspores. Plant species that can be used for the present disclosure method are generally as extensive as higher plant species suitable for transformation techniques, including both monocots and dicots, including species from the following genera: Cucurbita, Rosa, Vitis, Juglans, Fragaria, Nelumbo, Medicago, Sainfoin, Clover, Trigonella, Vigna, Citrus, Flax, Geranium, Cassava, Daucus, Arabidopsis, Brassica, Raphanus, Brassica, Belladonna, Capsicum, Datura, Scopolamine, Tomato, Nicotiana, Solanum, Petunia, Digitalis The preferred plant is sorghum, ...

[0225] A "plant cell" is a structural and physiological unit of a plant, comprising a protoplast and a cell wall. A plant cell may be in the form of an isolated single cell or a cultured cell, or as part of a higher organization unit such as, for example, a plant tissue, a plant organ, or a whole plant.

[0226] "Plant cell culture" means a culture of plant units such as, for example, protoplasts, cell culture cells, cells in plant tissues, pollen, pollen tubes, ovules, embryo sacs, fertilized eggs, and embryos at various stages of development.

[0227] "Plant material" refers to leaves, stems, roots, flowers or flower parts, fruits, pollen, egg cells, zygotes, seeds, cuttings, cell or tissue cultures, or any other part or product of a plant.

[0228] A "plant organ" is a distinct and distinct structured and differentiated part of a plant, such as a root, stem, leaf, flower bud or embryo.

[0229] As used herein, "plant material", "plant part" or "plant tissue" refers to plant cells, plant protoplasts, plant cell tissue cultures from which plants can be regenerated, plant calli, plant clumps, and intact plant cells in plants or plant parts such as embryos, pollen, ovules, seeds, leaves, flowers, branches, fruits, grains, ears, cobs, husks, stems, roots, root tips, anthers, tubers, rhizomes, etc. Any plant tissue in plants or in culture is included in the term "plant tissue".

[0230] As used herein, "plant tissue" means a group of plant cells organized into structural and functional units. Any plant tissue in a plant or in culture is included. The term includes, but is not limited to, whole plants, plant organs, plant seeds, tissue cultures, and any group of plant cells organized into structural and / or functional units. The use of this term in conjunction with any specific type of plant tissue as listed above or otherwise covered by this definition or alone is not intended to exclude any other type of plant tissue.

[0231] As used herein, "plant sample" or "biological sample" refers to plant tissue, intact or incomplete (e.g., ground seeds or plant tissue, chopped plant tissue, freeze-dried tissue). It can also be an extract containing intact or incomplete seeds or plant tissue. The biological sample or extract can be selected from the group consisting of corn flour, corn meal, corn syrup, corn oil, corn starch, and manufactured cereal foods containing corn by-products in whole or in part.

[0232] "Polynucleotide of interest" or "nucleic acid of interest" refers to any polynucleotide that, when transferred into an organism (e.g., a plant), confers a desired characteristic to the organism, such as insect resistance, disease resistance, herbicide tolerance, antibiotic resistance, improved nutritional value, improved performance in an industrial process, production of commercially valuable enzymes or metabolites, altered reproductive capacity, etc.

[0233] A "portion" or "fragment" of a polypeptide disclosed herein will be understood to mean an amino acid sequence or nucleic acid sequence of reduced length relative to a reference amino acid sequence or nucleic acid sequence disclosed herein. According to the disclosure, such a portion or fragment may be included in a larger polypeptide or nucleic acid of which it is a component (e.g., a tagged or fusion protein or expression cassette), where appropriate. In embodiments, a "portion" or "fragment" substantially retains activity such as insecticidal activity (e.g., the insecticidal activity of the full-length protein or nucleic acid (e.g., at least 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95% or even 100% of the activity), or has higher activity such as insecticidal activity than the full-length protein.

[0234] The terms "protein," "peptide," and "polypeptide" are used interchangeably herein.

[0235] As used herein, the term "promoter" refers to a polynucleotide generally located upstream (5') of the translation start site of a coding sequence, which controls the expression of the coding sequence by providing recognition of RNA polymerase and other factors required for correct transcription. For example, a promoter may contain a region comprising a basic promoter element recognized by RNA polymerase, a region containing the 5' untranslated region (UTR) of the coding sequence, and optionally an intron.

[0236] As used herein, the term "recombination" refers to the following form of nucleic acid (e.g., DNA or RNA) or protein or organism, which is not usually found in nature and is produced by human intervention for this reason. As used herein, "recombinant nucleic acid molecule" is a nucleic acid molecule comprising a combination of polynucleotides, which do not coexist naturally and are the result of human intervention, for example, a nucleic acid molecule composed of a combination of at least two polynucleotides heterologous to each other, or an artificially synthesized (e.g., a polynucleotide synthesized using an assembled nucleotide sequence) and comprising a nucleic acid molecule of a polynucleotide different from a polynucleotide usually present in nature, or a nucleic acid molecule comprising a transgenic artificially incorporated into the genomic DNA of a host cell and the host cell genome-related flanking DNA. Another example of a recombinant nucleic acid molecule is a DNA molecule produced by inserting a transgenic into the genomic DNA of a plant, which can ultimately lead to the expression of a recombinant RNA / or protein molecule in the organism. As used herein, "recombinant plant" is a plant that is not usually present in nature, is the result of human intervention, and contains a transgenic or heterologous nucleic acid molecule that can be incorporated into its genome. Due to such genome changes, recombinant plants are significantly different from related wild-type plants. "Recombinant" bacteria are bacteria not found in nature that contain a heterologous nucleic acid molecule. Such bacteria can be produced by transforming the bacteria with the nucleic acid molecule, or by conjugatively transferring a plasmid from one bacterial strain to another bacterial strain, whereby the plasmid contains the nucleic acid molecule.

[0237] As used herein, the terms "reduce," "reduced," "reducing," "reduction," "diminish," and "suppress" (and grammatical variations thereof) and similar terms refer to a reduction in the survival, growth and / or reproduction of a plant pest, such as by contacting the plant with a polypeptide of the disclosure (such as, for example, by transgenic expression or by topical application methods). This reduction in survival, growth and / or reproduction can be referenced to levels observed in the absence of a polypeptide of the disclosure (e.g., a plant that is not transgenic expressing the polypeptide or not topically treated with the polypeptide). Thus, in embodiments, the terms "reduce," "reduced," "reducing," "reduction," "diminish," and "suppress" (and grammatical variations thereof) and similar terms mean a reduction of at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more compared to a plant not contacted with a polypeptide of the disclosure (e.g., a plant not transgenic expressing the polypeptide or not topically treated with the polypeptide). In representative embodiments, the reduction results in no or substantially no (i.e., an insignificant amount, e.g., less than about 10%, less than about 5%, or even less than about 1%) detectable survival, growth, and / or reproduction of a plant pest.

[0238] "Regulatory elements" refer to nucleotide sequences that are located upstream (5' non-coding sequences), within or downstream (3' non-coding sequences) of a coding sequence and affect the transcription, RNA processing or stability, or translation of the associated coding sequence. Regulatory sequences include enhancers, promoters, translation enhancer sequences, introns, terminators, and polyadenylation signal sequences. They include natural sequences and synthetic sequences, as well as sequences that may be a combination of synthetic and natural sequences. Regulatory sequences can determine expression levels, spatial and temporal patterns of expression, and, for a subset of promoters, expression under inductive conditions (regulated by external factors such as light, temperature, chemicals, and hormones).

[0239] As used herein, "selectable marker" means a nucleotide sequence that, when expressed, confers a different phenotype to a plant, plant part, and / or plant cell expressing the marker, and thus allows such transformed plants, plant parts, and / or plant cells to be distinguished from those that do not have the marker. Such a nucleotide sequence can encode a selectable or screenable marker, depending on whether the marker confers a trait that can be selected for chemically, such as by using a selective agent (e.g., an antibiotic, herbicide, or the like), or whether the marker is simply a trait that one can identify by observation or testing, such as by screening (e.g., R gene traits).

[0240] As used herein, "stable transformation" or "stably transformed" means that a nucleic acid is introduced into a cell and integrated into the cell genome. Thus, the integrated nucleic acid is capable of being inherited by its progeny, more particularly, by multiple successive generations of progeny. As used herein, "genome" also includes nuclear and plasmid genomes, and thus includes integration of the nucleic acid into, for example, a chloroplast genome. As used herein, stable transformation may also refer to a transgene that is maintained extrachromosomally, for example, as a mini-chromosome.

[0241] "Synthetic" refers to a nucleotide sequence that contains bases or one or more structural features that are not present in the natural sequence. For example, an artificial sequence encoding a protein of the present disclosure that is more similar to the G+C content and normal codon distribution of a dicot or monocot gene is described as synthetic.

[0242] As used herein, a protein of the present disclosure that is "toxic" to an insect pest means that the protein acts as an orally active insect control agent to kill the insect pest, or that the protein is capable of disrupting or preventing insect feeding, or causing growth inhibition of the insect pest, both of which may or may not cause insect death. When a toxic protein of the present disclosure is delivered to an insect or an insect comes into oral contact with a toxic protein, the result is typically the death of the insect, or a slowing of the growth of the insect, or the cessation of feeding on a source of the toxic protein available to the insect.

[0243] The terms "toxin fragment" and "toxin portion" are used interchangeably herein and refer to fragments or portions of longer (e.g., full-length) insecticidal proteins of the present disclosure, wherein the "toxin fragment" or "toxin portion" retains insecticidal activity. For example, it is known in the art that native Cry proteins are expressed as protoxins, which are processed at the N-terminus and C-terminus to produce mature toxins. In embodiments, the "toxin fragment" or "toxin portion" of the chimeric insecticidal protein of the present disclosure is truncated at the N-terminus and / or C-terminus. In embodiments, the "toxin fragment" or "toxin portion" is truncated at the N-terminus to remove part or all of the N-terminal peptidyl fragment, and optionally comprises at least about 400, 425, 450, 475, 500, 510, 520, 530, 540, 550, 560, 570, 580 or 590 consecutive amino acids of the insecticidal protein specifically described herein, or an amino acid sequence substantially identical thereto. Thus, in embodiments, a "toxin fragment" or "toxin portion" of an insecticidal protein is truncated at the N-terminus (e.g., to omit part or all of the peptidyl fragment), e.g., an N-terminal truncation of one amino acid or more than one amino acid, e.g., up to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60 or more amino acids. In embodiments, a "toxin fragment" or "toxin portion" of an insecticidal protein is truncated at the C-terminus (e.g., to omit part or all of the protoxin tail), e.g., a C-terminal truncation of one amino acid or more than one amino acid, e.g., up to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 70, 80, 90, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 560 or more amino acids C-terminal truncation. In an embodiment, the "toxin fragment" or "toxin portion" comprises domains 1 and 2, and core domain 3.In embodiments, the "toxin fragment" or "toxin portion" is a mature (ie, processed) toxin (eg, a Cry toxin).

[0244] "Transformation" is a method for introducing heterologous nucleic acids into a host cell or organism. In specific embodiments, "transformation" means that a DNA molecule is stably integrated into the genome (nucleus or plastid) of a target organism. In some specific embodiments, introduction into plants, plant parts and / or plant cells is via: bacterial-mediated transformation, particle bombardment transformation, calcium phosphate-mediated transformation, cyclodextrin-mediated transformation, electroporation, liposome-mediated transformation, nanoparticle-mediated transformation, polymer-mediated transformation, virus-mediated nucleic acid delivery, whisker-mediated nucleic acid delivery, microinjection, ultrasonic treatment, infiltration, polyethylene glycol-mediated transformation, protoplast transformation, or any other electrical, chemical, physical and / or biological mechanism that results in the introduction of nucleic acids into plants, plant parts and / or their cells, or a combination thereof. Procedures for transforming plants are well known in the art and conventional and are generally described in the literature. Non-limiting examples of methods for plant transformation include transformation via bacteria-mediated nucleic acid delivery (e.g., via bacteria from the genus Agrobacterium), virus-mediated nucleic acid delivery, silicon carbide or nucleic acid whisker-mediated nucleic acid delivery, liposome-mediated nucleic acid delivery, microinjection, particle bombardment, calcium phosphate-mediated transformation, cyclodextrin-mediated transformation, electroporation, nanoparticle-mediated transformation, sonication, infiltration, PEG-mediated nucleic acid uptake, and any other electrical, chemical, physical (mechanical), and / or biological mechanism that results in the introduction of nucleic acids into plant cells, including any combination thereof. General guides to the various plant transformation methods known in the art include Miki et al. ("Procedures for Introducing Foreign DNA into Plants," in Methods in Plant Molecular Biology and Biotechnology, Glick, BR and Thompson, JE, eds. (CRC Press, Inc., Boca Raton, 1993), pp. 67-88) and Rakowoczy-Trojanowska (2002, Cell Mol Biol Lett 7:849-858 (2002)).

[0245] "Transformed" and "transgenic" refer to a host organism (e.g., bacteria or plant) into which a heterologous nucleic acid molecule has been introduced. The nucleic acid molecule can be stably integrated into the host's genome, or the nucleic acid molecule can also exist as an extrachromosomal molecule. Such extrachromosomal molecules can replicate autonomously. Transformed cells, tissues or plants should be understood to encompass not only the end product of the transformation process, but also its transgenic progeny. "Non-transformed," "non-transgenic," or "non-recombinant" hosts refer to wild-type organisms, such as bacteria or plants, that do not contain heterologous nucleic acid molecules.

[0246] The term "transgenic plant" includes plants into which heterologous nucleic acid molecules have been introduced. Typically, the heterologous nucleic acid sequence is stably integrated into the genome so that the nucleic acid sequence is passed on to successive generations. The heterologous nucleic acid sequence can be integrated into the genome alone or as part of a recombinant expression cassette. "Transgenic" is used herein to include any cell, cell line, callus, tissue, plant part or plant whose genotype has been altered by the presence of a heterologous nucleic acid sequence, including those transgenics that were originally so altered and those transgenics that were generated by sexual crosses or asexual propagation from the original transgenic.

[0247] The term "vector" refers to a composition used to transfer, deliver or introduce one or more nucleic acids into a cell. A vector comprises a nucleic acid molecule containing one or more nucleotide sequences to be transferred, delivered or introduced. Exemplary vectors include plasmids, cosmids, phagemids, artificial chromosomes, phages or viral vectors. Insecticide proteins, peptides, nucleic acid molecules

[0248] The present disclosure provides novel insecticidal proteins that are active against Coleoptera, such as Diabrotica barberi (Western Corn Rootworm; WCR), Diabrotica barberi (Northern Corn Rootworm; NCR), and / or Diabrotica undecimpunctata howardi (Southern Corn Rootworm; SCR), and / or other Diabrotica species, including Diabrotica virgifera zeae (Mexican Corn Rootworm), and / or other Coleopteran insect pests, such as Colorado Potato Beetle. The present disclosure also relates to nucleic acids, the expression of which produces the insecticidal proteins of the present disclosure, and to methods of making and using these insecticidal proteins to control insect pests. In embodiments, expression of these nucleic acids produces insecticidal proteins that can be used to control Coleopteran insects, such as Western, Northern, and / or Southern Corn Rootworm, particularly when expressed in transgenic plants, such as transgenic corn plants.

[0249] In some embodiments, the disclosure provides a polypeptide comprising an amino acid sequence having at least 85% sequence identity to any one of SEQ ID NOs: 1, 5-23, or 71-117 (at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8 or at least 99.9% sequence identity).

[0250] In some embodiments, the polypeptide comprises SEQ ID NO:1.

[0251] In another embodiment, the polypeptide comprises any one of SEQ ID NOs: 5-23.

[0252] In another embodiment, the polypeptide comprises any one of SEQ ID NOs:71-117.

[0253] In some embodiments, the polypeptide is toxic to coleopteran pests (e.g., rootworm pests, such as western corn rootworm). In some embodiments, the polypeptide is derived from a specific bacterium, e.g., Samsonia bacteria. In further embodiments, a polypeptide is provided, which consists of the amino acid sequence of any one of SEQ ID NO: 1, 5-23 or 71-117, or differs from the amino acid sequence of SEQ ID NO: 1, 5-23 or 71-117 by substitution (e.g., conservative amino acid substitution), deletion and / or addition of 1-10 amino acids (e.g., 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2 or 1). A "conservative amino acid substitution" is a substitution in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains have been defined in the art. These families include: amino acids with basic side chains (e.g., lysine, arginine, histidine); amino acids with acidic side chains (e.g., aspartic acid, glutamic acid); amino acids with polar, negatively charged residues and their amides (e.g., aspartic acid, asparagine, glutamic acid, glutamine); amino acids with uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine); amino acids with small aliphatic nonpolar or slightly polar residues (e.g., alanine, serine, threonine, proline); , glycine); amino acids with nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan); amino acids with large aliphatic nonpolar residues (e.g., methionine, leucine, isoleucine, valine, cystine); amino acids with β-branched side chains (e.g., threonine, valine, isoleucine); amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine); amino acids with large aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan).In some embodiments, the polypeptide has at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5%) identity to SEQ ID NO: 1 and includes I52, S53, I67, I72, I80, V83, A85, I95, I123, S125, I153, I175, I207, I237, D7, T9, T11, E27, S28, S29, M30, K31, T32, H33, R34, L35, V37, L38, S39, V83, G89, S94, G97, S 99, Q100, G104, N106, P107, D111, E113, S114, N115, G118, I123, V129, T135, T140, V142, H143, N148, E164, T199 or V241 corresponding to one or more substitution mutations (e.g., conservative amino acid substitutions, alanine substitutions or leucine substitutions). In some embodiments, the one or more substitution mutations are leucine substitutions at one or more positions corresponding to any one or more of I52, S53, I67, I72, I80, V83, A85, I95, I123, S125, I153, I175, I207 or I237 of SEQ ID NO: 1. In some embodiments, the one or more substitution mutations are I52L and I180L, I152L and S53L, or I52L and S53L and I80L. In some embodiments, the one or more substitution mutations are one or more conservative amino acid or alanine substitutions at one or more positions corresponding to any one or more of D7, T9, T11, E27, S28, S29, M30, K31, T32, H33, R34, L35, V37, L38, S39, V83, G89, S94, G97, S99, Q100, G104, N106, P107, D111, E113, S114, N115, G118, I123, V129, T135, T140, V142, H143, N148, E164, T199, or V241 of SEQ ID NO: 1.In some embodiments, the one or more substitution mutations are one or more alanine substitutions at one or more positions corresponding to any one or more of D7, T9, T11, E27, S28, S29, M30, K31, T32, H33, R34, L35, V37, L38, S39, V83, G89, S94, G97, S99, Q100, G104, N106, P107, D111, E113, S114, N115, G118, 1123, V129, T135, T140, V142, H143, N148, E164, T199, or V241 of SEQ ID NO: 1. In embodiments, the polypeptide is toxic to coleopteran pests (e.g., Diabrotica pests, such as western corn rootworm). In another embodiment, the present disclosure provides a polypeptide consisting of the amino acid sequence of any one of SEQ ID NO: 1, 5-23 or 71-117.

[0254] Certain sequences disclosed herein are orthologs of each other and have certain identity percentages to each other. Those identity percentages are disclosed in Table 1.

[0255] Dietary exposure is the main way that humans can be exposed to insecticidal proteins expressed in transgenic plants. Mammalian acute oral toxicity and protein digestibility are the endpoints of EPA's human health risk assessment. Further scientific evidence for the safety of insecticidal proteins is that they have been shown to degrade rapidly in vitro using simulated gastric fluid. For example, the results of seven in vitro assays with representative Cry1, Cry2 and Cry3 proteins established that these proteins are generally rapidly degraded within 30 seconds. These results support the broader conclusion that members of these groups of Cry proteins (with significant amino acid sequence identity) may degrade rapidly after human ingestion. Similar tests are performed on each transgenic protein expressed in plants. Another consideration is whether insecticidal proteins may cause allergic reactions. The confirmed rapid degradation of transgenic insecticidal proteins in vitro should minimize the possibility of such occurrence. In contrast, food allergens are usually retained in in vitro gastrointestinal models, while common food proteins without allergic history are rapidly degraded in simulated gastric fluid (Metcalfe et al. 1996).

[0256] The simulated gastric fluid (SGF) assay measures the in vitro digestibility of the test protein under strictly controlled conditions representing the upper digestive tract of mammals. For example, at 37°C over a period of one hour, the test Cry protein produced by bacteria (at a concentration of 0.5-5 mg / ml) is exposed to pepsin (from porcine gastric mucosa, dissolved in 2 mg / ml NaCl, pH 1.2) at a ratio of 10 units of pepsin activity / μg test protein. Samples are taken out at 1, 2, 5, 10, 30 and 60 minutes and immediately quenched by adding a preheated (95°C-2 minutes) stop buffer (65% 0.5M sodium bicarbonate pH 11, 35% Tricine loading buffer) to inactivate pepsin immediately and return to heat for 5 minutes. Once the assay is completed, time point samples and controls (only test protein, only pepsin) are checked on 10%-20% Tris-Tricine gels (peptides visible as low as 1 kDa) by SDS-PAGE to track the kinetics and level of digestion by pepsin. If significant polypeptide fragments of the test protein or proteins herein are visible at, for example, the 5 and / or 10 minute time points, then it is not digestible or not fully digestible by the SGF assay and can be scored qualitatively as "no" or "indigestible". If the test protein and any significant polypeptide fragments are visible at, for example, the 5 minute time point, then it can be digested by the SGF assay and can be scored qualitatively as "yes" or "digestible".

[0257] Therefore, in some embodiments, the disclosed insecticidal proteins can be modified to improve the properties of the protein, such as its insecticidal activity, its solubility, its stability and / or its digestibility. For example, the disclosed insecticidal proteins can additionally include introduced protease cleavage sites and / or cysteine ​​substitutions. The introduced protease cleavage sites and / or cysteine ​​substitutions are not naturally occurring, but are introduced into the polypeptide sequence as substitution mutations or as insertion or deletion mutations or some combination thereof. The introduced protease cleavage site can be introduced by inserting at least one leucine residue into the polypeptide sequence. In some embodiments, the polypeptide comprises a cysteine ​​substitution, for example, replacing cysteine ​​with another amino acid such as alanine or leucine. The introduced mutation can make the polypeptide unstable, thereby allowing the protease to approach the cleavage site, which was previously inaccessible due to the tight and / or stable folding of the protein or due to steric hindrance. The introduced protease cleavage site can be a mutation introduced in the polypeptide sequence, which is recognized as a site for proteolytic cleavage by a protease such as chymotrypsin, trypsin or pepsin.

[0258] In certain embodiments, the protease cleavage site introduced can change the existing protease cleavage site so that it is recognized by different proteases. Protease cleavage sites for chymotrypsin, trypsin and pepsin are well known in the art. Chymotrypsin preferentially cuts peptide amide bonds, wherein the carboxyl side (P1 position) of the amide bond is a large hydrophobic amino acid (tyrosine, tryptophan and phenylalanine). Trypsin mainly cuts the peptide chain at the carboxyl side of the amino acid lysine or arginine, unless any one of them is followed by proline. Pepsin is most effective in cutting the peptide bond between hydrophobic amino acids and preferably aromatic amino acids (such as phenylalanine, tryptophan, tyrosine and leucine). These cleavage sites are preferential cleavage sites, do not include all cleavage sites identified by chymotrypsin, trypsin or pepsin, and do not include all cleavage sites of all proteases.

[0259] Cysteine ​​in proteins is usually covalently bonded to other cysteine ​​residues to form disulfide bonds. Disulfide bonds play an important role in the folding and stability of some proteins. Compared with the wild-type polypeptide, the variant polypeptide may have a changed or less stable tertiary structure. For example, the introduced mutation can make the three-dimensional folding of the polypeptide "loose", so that the protease cleavage site that was previously inaccessible (and therefore not cut) can be approached by the protease. This causes the introduced mutation to introduce a protease cleavage site that does not exist in the unmodified polypeptide. In a preferred embodiment, the mutation does not change or does not significantly change the activity or insecticidal activity of the polypeptide against coleopteran pests.

[0260] Each of the one or more insecticidal proteins disclosed has insecticidal activity against lepidopteran pests. In some embodiments, the one or more insecticidal proteins are active against one or more of the following coleopteran pests: Diabrotica spp., such as Diabrotica barbatae (northern corn rootworm), Diabrotica zeae (western corn rootworm), D. undecimpunctata howardii (southern corn rootworm), D. balteata (banded cucumber beetle), D. undecimpunctata undecimpunctata (western spotted cucumber beetle), D. significata (3-spotted leaf beetle), D. speciosa (cucurbit beetle), D. speciosa (cucurbit beetle). beetle), Mexican corn rootworm (Mexican corn rootworm), Beni rootworm (D. beiensis), Christa rootworm (D. cristata), Cove rootworm (D. curviplustalata), two-spotted rootworm (D. dissimilis), elegant rootworm (D. elegantula), Emer rootworm (D. emorsitans), Graminea rootworm (D. graminea), Hispanolae rootworm (D. hispanolae), Lemini rootworm (D. lemniscata), D. linsleyi, D. milleri, D. nummularis, D. occlusal, D. porracea, D. scutellata, D. tibialis, D. trifasciata and / or D. viridula; species of the genus Leptinotarsa spp., such as L. decemlineata (Colorado potato beetle); Chrysomela spp., such as C. scripta (cottonwood leaf beetle); Hypothenemus spp., such as H.hampei (coffee berry borer); Sitophilus spp., such as S. zeamais (maize weevil); Epitrix spp., such as E. hirtipennis (tobacco flea beetle). (tobacco flea beetle) and / or E. cucumeris (potato flea beetle); Phyllotreta spp., such as P. cruciferae (crucifer flea beetle) and / or P. pusilla (western black flea beetle); Anthonomus spp., such as A. Eugenii (pepper weevil); Hemicrepidus spp., such as H. memnonius (wireworms); Melanotus spp., such as M. communis (wireworms); Ceutorhychus spp. spp., such as C. assimilis (cabbage seed podweevil); Phyllotreta spp., such as P. cruciferae (crucifer flea beetle); Aeolus spp., such as A. mellillus (wireworm); Aeolus spp., such as A. mancus (wheat wireworm); Horistonotus spp., such as H. uhlerii (sand wireworm); Sphenophorus spp., such as S. maidis (maize billbug), S. zeae (timothy billworm); billbug), S. parvulus (bluegrass billbug), and S. callosus (southern corn billbug); Phyllophaga spp. (white grub); Chaetocnema spp., such as C. pulicaria (corn flea beetle); Popillia spp.), such as P. japonica (Japanese beetle); Epilaxna spp., such as E. varivestis (Mexican bean beetle); Cerotoma spp., such as C. trifurcate (Bean leaf beetle); Epicauta spp., such as E. pestifera and E. lemniscata (Blister beetles); Holotrichia spp., such as H. diomphalia Bates (Northeast larger black chafer); or any combination of the foregoing.

[0261] In some embodiments, the one or more insecticidal proteins have insecticidal activity against Diabrotica virgata (western corn rootworm) and Diabrotica barnetta (northern corn rootworm).

[0262] In some embodiments, the one or more insecticidal proteins have insecticidal activity against an insect pest or colony that is resistant to another insecticide, including another insecticidal protein such as, for example, a Bt protein. In some embodiments, the one or more insecticidal proteins have insecticidal activity against a pest that is resistant to: a Cry3A protein (e.g., mCry3A, including but not limited to maize event MIR604 or MZIR098 or eCry3.1Ab, including but not limited to maize event 5307 or MZIR098), a Cry3B protein (e.g., Cry3Bb1, including but not limited to maize event MON87411 or MON88017), a Cry34 / 35 protein (e.g., Cry34 and Cry35, including but not limited to maize event 59122 and DP-4114), or an IPD072 protein (e.g., IPD072Aa, including but not limited to maize event DP-023211-2).

[0263] Each of the one or more insecticidal proteins disclosed may also have insecticidal activity against Lepidoptera, Hemiptera, Diptera, Lygus species, and / or other piercing-sucking insects (e.g., Orthoptera or Thysanoptera). In some embodiments, each of the one or more insecticidal proteins disclosed is active against one or more of the following Lepidoptera pests: Spodoptera spp., such as S. frugiperda (Fall Armyworm), S. littoralis (Egyptian Cotton Leafworm), S. ornithogalli (yellowstriped Armyworm), S. praefica (western yellowstriped Armyworm), S. eridania (southern Armyworm), S. litura (Common Cutworm / Oriental Leafworm), S. cosmioides (black Armyworm), S. exempta (African Armyworm), S. mauritia (lawnworm), S. armyworm and / or S. exigua (beet armyworm); Ostrinia spp., such as O. nubilalis (European corn borer) and / or O. furnacalis (Asian corn borer); Plutella spp., such as P. xylostella (diamondback moth); Agrotis spp., such as A. ipsilon (black cutworm), A. segetum (common cutworm), A. gladiaria (claybacked cutworm) and / or A. orthogonia (pale western cutworm); Striacosta spp., such as S. albicosta (western bean cutworm);zea (corn earworm / soybean podworm), H. punctigera (native budworm) and / or H. armigera (cotton bollworm); Heliothis spp., such as H. virescens (tobacco budworm); Diatraea spp., such as D. grandiosella (southwestern corn borer) and / or D. saccharalis (sugarcane borer); Trichoplusia spp., such as T. ni (cabbage looper); Sesamia spp., such as H. virescens (tobacco budworm); spp., such as S. nonagroides, Mediterranean corn borer, S. inferens, Pink stem borer and / or S. calamistis, pink stem borer; Pectinophora spp., such as P. gossypiella, pink bollworm; Cochylis spp., such as C. hospes, banded sunflower moth; Manduca spp., such as M. sexta, tobacco hornworm and / or M. quinquemaculata, tomato hornworm; Elasmopalpus spp., such as M. sexta, tobacco hornworm and / or M. quinquemaculata, tomato hornworm; spp., such as E. Lignosellus (lesser cornstalk borer); Pseudoplusia spp., such as P. includens (soybean looper); Anticarsia spp., such as A. gemmatalis (velvet bean caterpillar);Species of the genus Plathypena, such as P.scabra (green cloverworm); species of the genus Pieris, such as P.brassicae (cabbage butterfly); species of the genus Papaipema, such as P.nebris (stalk borer); species of the genus Pseudaletia, such as P.unipuncta (common armyworm); species of the genus Peridroma, such as P.saucia (variegated cutworm); species of the genus Keiferia, such as K.lycopersicella (tomato borer); pinworm); Artogeia spp., such as A. rapae (imported cabbageworm); Phthorimaea spp., such as P. operculella (potato tuberworm); Chrysodeixis spp., such as C. includens (soybean looper); Feltia spp., such as F. ducens (dingy cutworm); Chilo spp., such as C. suppressalis (striped stem borer), C. Agamemnon (oral corn borer), C. venosatus (spotted borer and spotted stalk borer (C. partellus); Cnaphalocrocis spp., such as rice leaf folder (C. medinalis, rice leaf folder); Conogethes spp., such as yellow peach moth (C. punctiferalis); Mythimna spp., such as Oriental armyworm (M. separata);Athetis spp., such as A. lepigone, Two-spotted armyworm; Busseola spp., such as B. fusca, maize stalk borer; Etiella spp., such as E. zinckenella, pulse pod borer; Leguminivora spp., such as L. glycinivorella, soybean podborer; Matsumuraeses spp., such as M. phaseoli, adzuki podworm; Omiodes spp., such as Etiella zinckenella, pulse pod borer; Leguminivora spp., such as L. glycinivorella, soybean podborer; Matsumuraeses spp., such as M. phaseoli, adzuki podworm; spp.), such as O. indicata, Soybean leaf folder / Bean-leaf webworm; Rachiplusia spp., such as R. nu, Sunflower Looper; Maruca spp., such as M. Testulalis Geyer, Bean pod borer; Monolepta spp., such as M. hieroglyphica, Double-spotted leaf beetle; or any combination of the foregoing. In some embodiments, the one or more insecticidal proteins are active against one or more of the following Hemiptera pests: Chinavia hilaris (green stink bug); Anasa tristis De Geer (squash bug); bug); Blissus leucopterus (chinch bug); Corythuca gossypii Fabricius (cotton lace bug); Cyrtopeltismodesta Distant (tomato bug); Dysdercus suturellus Hernch-Schaffer (cottonstainer); Euschistus servus Say (brown stink bug);one-spotted stink bug (E. variolarius Palisot de Beauvois); Graptostethus spp. (complex of seed bugs); Leptoglossus corculus Say (leaf-footed pine seed bug); Lygus lineolaris Palisot de Beauvois (tarnished plant bug); L. Hesperus Knight (Western tarnished plant bug); L. pratensis Linnaeus (common meadow bug); L. rugulipennis Poppius (European tarnished plant bug); Lygocoris pabulinus Linnaeus (common green capsid; Nezara viridula Linnaeus (southern green bug); Oebalus pugnax Fabricius (rice stink bug); Oncopeltus fasciatus Dallas (large milkweed bug); Pseudatomoscelis seriatus Reuter (cotton fleahopper); Calocoris norvegicus Gmelin (strawberry bug); Orthops campestris Linnaeus (wilderness bug); Plesiocoris rugicollis Fallen (apple capsid); Cyrtopeltis modestus Distant (tomato bug); Cyrtopeltis notatus Distant (suckfly); Spanagonicus albofasciatus Reuter (whitemarked fleahopper); Diaphnocoris chlorionis Say, honeylocust plant bug;Labopidicola allii Knight (onion plant bug); Pseudatomoscelis seriatus Reuter (cotton fleahopper); Adelphocoris rapidus Say (rapid plant bug); Poecilocapsus lineatus Fabricius (four-lined plant bug); Nysius ericae Schilling (false chinchbug); Nysius raphanus Howard (false chinch bug); Nezara viridula Linnaeus (southern green bug); Eurygaster spp.; Coreidae spp.; Pyrrhocoridae spp.; Tinidae spp.); Blostomatidae spp.; Reduviidae spp. and Cimicidae spp.; or a combination of any of the foregoing. In some embodiments, the one or more insecticidal proteins are active against one or more of the following Diptera pests: Liriomyza spp., such as L. trifolii (leafminer) and L. sativae (vegetable leafminer); Scrobipalpula spp., such as S. absoluta (tomato leafminer); Delia spp., such as D. platura (seedcorn maggot), D. brassicae (cabbage maggot), and D. radicum (cabbage root fly); Psilia spp., such as P. rosae (carrot rust fly); Tetanops spp., such as D. platura (seedcorn maggot), D. brassicae (cabbage maggot), and D. radicum (cabbage root fly); spp.), such as T. myopaeformis (sugarbeet root maggot);In some embodiments, the one or more insecticidal proteins are active against one or more of the following Orthoptera pests: Melanoplus spp., such as M. differentialis, Differentialgrasshopper, M. femurrubrum, Redlegged grasshopper, M. bivittatus, Twostriped grasshopper; or any combination of the foregoing. In some embodiments, the one or more insecticidal proteins are active against one or more of the following Thysanoptera pests: Frankliniella spp., such as F. occidentalis (western flower thrips) and F. fusca (tobacco thrips); and Thrips spp., such as T. tabaci (onion thrips), T. palmi, melon thrips; or any combination of the foregoing. ;

[0264] The one or more insecticidal proteins disclosed may also have insecticidal activity against any one or more of the following: Phyllophaga spp., Rhopalosiphum maidis, Pratylenchus penetrans, Melanotus cribulosus, Cyclocephalalurida, Limonius californicus, Tetranychus urticae, Haplothrips aculeatus, Tetranychus truncates, Anomalacorpulenta, Oedaleus infernalis, Frankliniella tenuicornis, Tetranychus cinnabarinus, Aiolopus thalassinus tamulus, Trachea spp. tokionis), Laodelphax striatellus, Holotrichia oblita, Dichelops furcatus, Diloboderus abderu, Dalbulus maidis, Astylus variegathus, Scaptocoris castanea, Locusta migratoria manilensis, Agriotes lineatus, Peregrinus maidis, Oscinella frit, Frankliniella williamsi, Zyginidia manaliensis, Atherigona soccata, Nicentrites testaceipes, Myllocerus undecimpustulatus, Atherigona naquii, Amsecta albistriga, Plodia interpuctella, Melanotus serratus, caudex), Microtermes spp.), Atherigona oryzae, Tanymecus dilaticollis, Delphacodes kuschelli, Lepidiota stigma, Phyllophaga hellery, Tribolium castaneum, Pelopidas mathias, Oxya chinensis, Thunberg, Stenocranus pacificus, Scutigerella immaculata, Chrysodeixis chalcites, Euproctis sp. (Lysimidae), Phyllotreata spp. (undulata), Reptalus panzer, Cyrtacanthacris tartarica Linnaeus, Orgyia postica, Dactylispa lameyi, Patanga succincta Johanson, Tetranychus spp., Calomycterus sp., Adoretus compressus Weber, and Paratetranychus stickney.

[0265] The disclosed insecticidal proteins may also be active against nematodes. As used herein, the term "nematode" encompasses any organism currently known or later identified that is classified as an organism of the phylum Nematoda of the animal kingdom, including, but not limited to, nematodes in the class Adenophora (including, for example, Rhizophora, Isopharyngea, Monodonta, Lanceolata, Trichocephala, Trachelata, Muspa, Lactropharyngea, Chromatidae, Taeniformes, Catenonemata, and Monogynae) and / or the class Tubulonemata (including, for example, Rhabditis, Strongyloides, Ascaridae, Spirulina, Camelidae, Digastricales, Sphaeroidea, and Sphaeroidea).

[0266] Nematodes include, but are not limited to, parasitic nematodes, such as root-knot nematodes, cyst nematodes, and / or decay nematodes. Exemplary genera of nematodes according to the present disclosure include, but are not limited to: Root-knot nematodes (root-knot nematodes), Heterodera (cyst nematodes), Globodera (cyst nematodes), Penetrate nematodes (piercing nematodes), Renoides (kidney-shaped kidney-shaped nematodes), Pratylenchus (rot nematodes), Aphelenchus (leaf nematodes), Heliconematodes (helical nematodes), Nematodes (lance nematodes), Pseudo-burrs (short and thick root nematodes), Long-nematodes, Pearl Nematodes (pseudo-root-knot nematodes), Subanguina, Stinging Nematodes (thorn nematodes), Microcircle Nematodes (microcircle nematodes), Angiospermum (angiosperm nematodes), Ditylenchus (dendrum nematodes), Conynematode (conynematodes), Hemicyclenematode (hemicycle nematodes), Sheathnematode (sheath nematodes), Subroot Nematode (subroot nematodes), Hypsoperine, Ditylenchus (large stem nematodes), Melinius, Punctodera, Quinisulcius, Shield Nematode (shield nematodes), Xiphinema (dagger nematodes), Dwarf Nematode (dwarf nematodes), Penelopenetes (piercing nematodes), Bursaphelenchus (roundworms), and any combination thereof.

[0267] Exemplary plant parasitic nematodes according to the present disclosure include, but are not limited to: Belonolaimus gracilis, Belonolaimus longicaudatus, Bursaphelenchus xylophilus, pine wood nematode, Criconemoides ornata, Ditylenchus destructor, potato rot nematode, Ditylenchus dipsaci, stem and bulb nematode, Globodera pallida, potato cyst nematode, Globodera rostochiensis (golden nematode), Heterodera glycines, soybean cyst nematode, Heteroderaschachtii, sugar beet cyst nematode. nematode); Heterodera zeae, corn cyst nematode; Heterodera avenae,cereal cyst nematode), Heterodera carotae, Heterodera trifolii, Hoplolaimus columbus, Hoplolaimus galeatus, Hoplolaimusmagnistylus, Longidorus breviannulatus, Meloidogynearenaria, Meloidogyne chitwoodi, Meloidogyne hapla, Meloidogyne incognita, Meloidogyne javanica, Mesocriconema xenoplax, Nacobbus aberrans, Naccobus dorsalis, Paratrichodorus christiei, Paratrichodorus minor), Pratylenchus brachyurus, Pratylenchus crenatus, Pratylenchus hexincisus, Pratylenchus negletus, Pratylenchus penetrans, Pratylenchus projectus, Pratylenchus scribneri, Pratylenchus tenuicaudatus, Pratylenchus thornei, Pratylenchus zeae, Punctodera chaccoensis, Quinisulciusacutus, Radopholus similis, Rotylenchulus reniformis, Tylenchorhynchus dubius), Tylenchulus semipenetrans, Siphinema americanum, X. Mediterraneum, and any combination of the foregoing.

[0268] The present disclosure also encompasses antibodies that specifically bind to the insecticidal proteins of the present disclosure. The antibodies may optionally be monoclonal antibodies or polyclonal antisera. Such antibodies can be produced using standard immunological techniques that produce polyclonal antisera, and if desired, antibody-producing cells of immortalized immune hosts are used as monoclonal antibody production sources. Techniques for producing antibodies to any substance of interest are well known, for example, as described in Harlow and Lane (1988. Antibodies a laboratory manual. [Antibodies: Laboratory Manual] p. 726. Cold Spring Harbor Laboratory [Cold Spring Harbor Laboratory]) and as described in Goding (Monoclonal Antibodies: Principles & practice. [Monoclonal Antibodies: Principles & Practice] 1986. Academic Press, Inc. [Academic Press Company], Orlando, Florida). The present disclosure also encompasses insecticidal proteins that cross-react with antibodies, particularly monoclonal antibodies, to produce one or more of the insecticidal proteins of the present disclosure.

[0269] Antibodies according to the disclosure can be used, for example, in immunoassays to determine the amount or presence of the insecticidal proteins or antigen-related polypeptides of the disclosure, for example, in biological samples. Such assays are also useful in quality control production of compositions containing one or more of the insecticidal proteins or antigen-related polypeptides of the disclosure. In addition, these antibodies can be used to evaluate the efficacy of recombinant production of one or more of the proteins or antigen-related polypeptides of the disclosure, and to screen expression libraries to determine the presence of nucleotide sequences encoding one or more of the proteins or antigen-related polypeptides of the disclosure. Antibodies can further be used as affinity ligands for purification or separation of any one or more of the proteins or antigen-related polypeptides of the disclosure.

[0270] In an embodiment, a nucleic acid molecule encoding a polypeptide disclosed herein is provided. In some embodiments, these nucleic acid molecules are codon optimized, for example, as further described herein. In some embodiments, the coding sequence is at least 80% identical to any one of SEQ ID NO: 2-4, 27-45, 49-67 or 118-163 (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8 or at least 99.9% identical). In other embodiments, the coding sequence comprises or consists of the sequence of any one of SEQ ID NOs: 2-4, 27-45, 49-67, or 118-163. In some embodiments, the coding sequence comprises a degenerate nucleotide sequence encoding the same amino acid sequence of any one of SEQ ID NOs: 2-4, 27-45, or 49-67.

[0271] Expression cassettes and vectors

[0272] In some aspects, the disclosure provides expression cassettes and vectors encoding insecticidal proteins of the disclosure. In some embodiments, the coding sequence comprises a synthetic nucleotide sequence for expression in a plant (e.g., a transgenic monocot host or a transgenic dicot host, such as a corn or soybean plant) through codon optimization. In an embodiment, the nucleotide coding sequence is partially or completely synthesized. In a representative embodiment, the nucleotide sequence of the disclosure is modified and / or optimized for expression in a transgenic plant (e.g., corn or soybean). For example, although in many cases, genes from microbial organisms can be expressed at high levels in plants without modification, low expression in transgenic plants may be due to microbial nucleotide sequences, which have codons that are not preferred in plants. It is known in the art that living organisms have specific codon usage preferences, and the codons of these nucleotide sequences described in the disclosure can be changed to conform to plant preferences while maintaining the amino acids encoded therein. In addition, it is known in the art that high expression in plants (e.g., corn plants) can be achieved by coding sequences having a GC content of at least about 35%, or at least about 45%, or at least about 50%, or at least about 60%. Microbial nucleotide sequences with low GC content may not express well in plants. Although some nucleotide sequences can be fully expressed in both monocot and dicot species, the sequences can be modified to cater to the codon preferences and GC content preferences specific to monocot or dicot plants, as these preferences have been shown to be different (Murray et al. Nucl. Acids Res. 17: 477-498 (1989)). In addition, in some embodiments, the nucleotide sequence is modified to remove abnormal splicing sites that may cause message truncation. Such modifications to the nucleotide sequences can be made using the methods described, for example, in US Patent Nos. 5,625,136, 5,500,365, and 6,013,523, using well-known site-directed mutagenesis, PCR, and synthetic gene construction techniques.

[0273] In certain embodiments, the disclosure provides synthetic coding sequences or polynucleotides prepared according to the program disclosed in U.S. Patent No. 5,625,136. In this program, maize preferred codons, i.e., the single codons that most frequently encode the amino acid in maize, are used. The maize preferred codons for specific amino acids can be derived from, for example, known gene sequences derived from maize. For example, maize codons for 28 genes from maize plants are used to find in the following documents: Murray et al., Nucleic Acids Research [nucleic acid research] 17: 477-498 (1989). It should be recognized that codons are optimized for expression in a plant species and will also act on other plant species, but codons are optimized for expression in plant species that may not be at the same level. In this way, these nucleotide sequences can be optimized for expression in any plant. It should be recognized that all or any part of the nucleotide sequence can be optimized or synthetic. That is to say, polynucleotides can include nucleotide sequences as part of the native sequence and part of the codon optimized sequence.

[0274] In representative embodiments, the polynucleotides of the present disclosure are isolated polynucleotides. In embodiments, the polynucleotides of the present disclosure are recombinant polynucleotides.

[0275] In some embodiments, a heterologous promoter is operably linked to a nucleic acid comprising, consisting essentially of, or consisting of a coding sequence encoding a protein of the disclosure that is toxic to coleopteran pests. For example, a promoter may include a constitutive, inducible, temporally regulated, developmentally regulated, chemically regulated, tissue-preferred, and / or tissue-specific promoter. In particular aspects, a promoter suitable for use in the disclosure is a promoter capable of initiating transcription of a nucleotide sequence in a plant cell, such as in a monocot (e.g., maize or rice) or a dicot (e.g., soybean, cotton) cell.

[0276] In an embodiment, the heterologous promoter is a plant expressible promoter (e.g., a monocot expressible or dicot expressible promoter). For example, but not limited to, the plant expressible promoter is selected from the group consisting of ubiquitin, tuberose yellow virus, corn TrpA, OsMADS 6, maize H3 histone, bacteriophage T3 gene 9 5'UTR, maize sucrose synthase 1, maize alcohol dehydrogenase 1, maize light harvesting complex, maize heat shock protein, maize mtl, pea small subunit RuBP carboxylase, rice actin, rice cyclophilin, Ti plasmid mannopine synthase, Ti plasmid nopaline synthase, petunia chalcone isomerase, legume glycine-rich protein 1, potato patatin, lectin, CaMV 35S, and S-E9 small subunit RuBP carboxylase promoter.

[0277] Although many promoters from dicots have been shown to be operable in monocots and vice versa, in an embodiment, a dicot promoter is selected for expression in dicots, and a monocot promoter is selected for expression in monocots. However, there is no limitation on the origin of the selected promoters; it is sufficient as long as they can effectively drive the expression of the nucleotide sequence in the desired cell.

[0278] The selection of promoter can depend on the time and space of expression and needs to change, and also depends on the host cell to be transformed and changes.Therefore, for example, the expression of the nucleotide sequence of the present disclosure can be in any plant and / or plant part (for example, in leaf, in stalk or stem, in spike, in inflorescence (for example, spike, panicle, cob etc.), in root, seed and / or seedling etc.). For example, in the case where it is desired to express in specific tissues or organs, tissue-specific or tissue-preferred promoters (for example, root-specific / preferred promoters) can be used. For example, when it is not desired to express in specific tissues or organs, tissue-free promoters can be used.In an embodiment, a "pollen-free" promoter is provided, which causes low or no detectable gene expression in the pollen of a target plant species.By contrast, in the case where it is desired to express in response to stimulation, a promoter that can be induced by stimulation or chemicals can be used.In the case where it is desired to express continuously with a relatively constant level in all cells of a plant, a constitutive promoter can be selected.

[0279] Promoters suitable for use in the present disclosure include, but are not limited to, those that constitutively drive the expression of a nucleotide sequence, those that drive expression upon induction, and those that drive expression in a tissue or development-specific manner. These different types of promoters are known in the art.

[0280] Suitable constitutive promoters include, for example, the CaMV 35S promoter (Odell et al., Nature 313:810-812, 1985); the Arabidopsis thaliana At6669 promoter (see PCT Publication No. WO 04081173A2); maize Ubi 1 (Christensen et al., Plant Mol. Biol. 18:675-689, 1992); rice actin (McElroy et al., Plant Cell 2:163-171, 1990); pEMU (Last et al., Theor. Appl. Genet. 81:581-588, 1991); CaMV 19S (Nilsson et al., Physiol. Plant 100:456-462, 1997); GOS2 (de =Pater et al., Plant J. Nov;2(6):837-44, 1992); ubiquitin (Christensen et al., Plant Mol. Biol. 18:675-689, 1992); rice cyclophilin (Bucholz et al., Plant Mol Biol. 25(5):837-43, 1994); maize H3 histone (Lepetit et al., Mol. Gen. Genet. 231:276-285, 1992); actin 2 (An et al., Plant J. 10(1);107-121, 1996), the constitutive root apical CT2 promoter (see PCT Application No. IL / 2005 / 000627) and the synthetic super MAS (Ni et al., The Plant Journal [Plant Journal] 7:661-76, 1995). Other constitutive promoters include those described in the following references: U.S. Patent Nos. 5,659,026, 5,608,149; 5,608,144; 5,604,121; 5,569,597; 5,466,785; 5,399,680; 5,268,463; and 5,608,142.

[0281] Tissue-specific or tissue-preferred promoters useful for expressing the polypeptides of the present disclosure in plants (optionally maize) include those that express directly in roots, pith, leaves, or pollen. Suitable tissue-specific promoters include, but are not limited to, leaf-specific promoters (as described, for example, by Yamamoto et al., Plant J. [Plant Journal] 12:255-265, 1997; Kwon et al., Plant Physiol. [Plant Physiology] 105:357-67, 1994; Yamamoto et al., Plant Cell Physiol. [Plant Cell Physiology] 35:773-778, 1994; Gotor et al., Plant J. [Plant Journal] 3:509-18, 1993; Orozco et al., Plant Mol. Biol. [Plant Molecular Biology] 23:1129-1138, 1993; and Matsuoka et al., Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences of the United States of America] 90:9586-9590, 1993), seed-preferred promoters (e.g., from seed-specific genes; Simon et al., Plant Mol. Biol. [Plant Molecular Biology] 5.191, 1985; Scofield et al., J. Biol. Chem. [Journal of Biological Chemistry] 262:12202, 1987; Baszczynski et al., Plant Mol. Biol. [Plant Molecular Biology] 14:633, 1990), Brazil nut albumin (Pearson et al., Plant Mol. Biol. [Plant Molecular Biology] 18:235-245, 1992), legumin (Ellis et al., Plant Mol. Mol. Biol. [Plant Molecular Biology] 10: 203-214, 1988), glutenin (Takaiwa et al., Mol. Gen. Genet. [Molecular Genetics and General Genetics] 208: 15-22, 1986; Takaiwa et al., FEBS Letts. [FEBS Letters] 221: 43-47, 1987), zein (Matzke et al., Plant Mol Biol. [Plant Molecular Biology], 143: 323-32 1990), napA (Stalberg et al., Planta [Plant] 199: 515-519, 1996), wheat SPA (Albanietal et al., Plant Cell [Plant Cell], 9: 171-184, 1997), sunflower oil protein (Cummins et al., Plant Mol. Biol. [Plant Molecular Biology ...9: 171-184, 1997), sunflower oil protein (Cummins et al.[Plant Mol Biol] 19:873-876, 1992), endosperm-specific promoters (e.g., wheat LMW and HMW, glutelin-1 (Mol Gen Genet [Molecular Genetics and General Genetics] 216:81-90, 1989; NAR 17:461-2)), wheat a, b and g alcohol-soluble proteins (EMB03:1409-15, 1984), barley ltrl promoter, barley B1, C, D alcohol-soluble proteins (Theor Appl Gen [Theoretical and Applied Genetics] 98:1253-62, 1999; Plant J [Plant Journal] 4:343-55, 1993; Mol Gen Genet [Molecular Genetics and General Genetics] 250:750-60, 1996), barley DOF (Mena et al., The Plant Journal [Plant Journal], 116 (1): 53-62, 1998), Biz2 (EP99106056.7), synthetic promoter (Vicente-Carbajosa et al., Plant J [Plant Journal]. 13: 629-640, 1998), rice prolamin NRP33, rice globulin Glb-1 (Wu et al., Plant Cell Physiology [Plant Cell Physiology] 39 (8) 885-889, 1998), rice α-globulin REB / OHP-1 (Nakase et al., Plant Mol. Biol. [Plant Molecular Biology] 33: 513-S22, 1997), rice ADP-glucose PP (Trans Res [Transformation Research] 6: 157-68, 1997), maize ESR gene family (Plant J [Plant Journal] 12:235-46, 1997), sorghum gamma-kafirin (Plant Mol. Biol [Plant Molecular Biology] 32:1029-35, 1996)], embryo-specific promoters (e.g., rice OSH1; Sato et al., Proc. Nat. Acad. Sci. USA [Proceedings of the National Academy of Sciences of the United States of America] 93:8117-8122), KNOX (Postma-Haarsma et al., Plant Mol. Biol. [Plant Molecular Biology] 39:257-71, 1999), rice oleosomal protein (Wu et al., J. Biochem. [Biological Chemistry], 123:386, 1998)], flower-specific promoters (e.g., AtPRP4, chalone synthase (chsA) (Van der Meer et al., Plant Mol.Biol.[Plant Molecular Biology]15,95-109,1990), LAT52 (Twell et al., Mol.Gen Genet.[Molecular Genetics and General Genetics] 217: 240-245; 1989), apetala-3), and promoters specific to plant reproductive tissues (e.g., OsMADS promoter; U.S. Patent Publication No. 2007 / 0006344). .

[0282] Examples of promoters suitable for preferred expression in green tissues include many promoters that regulate genes involved in photosynthesis, and many of these have been cloned from both monocots and dicots. One such promoter is the maize PEPC promoter (Hudspeth and Grula, Plant Molec. Biol. [Plant Molecular Biology] 12: 579-589 (1989)) derived from the phosphoenol carboxylase gene. Another promoter for root-specific expression is the promoter described by de Framond (FEBS 290: 103-106 (1991) or U.S. Patent number 5,466,785). Another promoter useful in this disclosure is the stem-specific promoter described in U.S. Patent number 5,625,136, which drives the expression of the maize trpA gene naturally.

[0283] In addition, promoters that function in plastids can be used. Non-limiting examples of such promoters include the phage T3 gene 9 5'UTR and other promoters disclosed in U.S. Patent No. 7,579,516. Other promoters suitable for use in the present disclosure include, but are not limited to, the S-E9 small subunit RuBP carboxylase promoter and the Kunitz trypsin inhibitor gene promoter (Kti3).

[0284] In certain embodiments, inducible promoters can be used. Therefore, for example, chemically regulated promoters can be used to regulate gene expression in plants by using exogenous chemical regulators. The expression of the nucleotide sequence of the present disclosure is regulated by the promoter that is chemically regulated so that the polypeptide of the present disclosure can be synthesized only when the crop plants are treated with inducing chemicals. Depending on the purpose, when applying chemicals to induce gene expression, the promoter can be a chemically inducible promoter, or when applying chemicals to suppress gene expression, the promoter can be a chemically repressible promoter. Such examples of chemically induced technology for genetic expression are described in detail in open application EP 0 332 104 and U.S. Patent number 5,614,395.

[0285] Chemically inducible promoters are known in the art and include, but are not limited to, the maize In2-2 promoter (which is activated by benzenesulfonamide herbicide safeners), the maize GST promoter (which is activated by hydrophobic electrophilic compounds used as pre-emergence herbicides), the tobacco PR-1a promoter (which is activated by salicylic acid) (e.g., the PR1a system), steroid-responsive promoters (see, e.g., Schena et al. (1991) Proc. Natl. Acad. Sci. USA 88, 10421-10425 and McNellis et al. (1998) Plant Genetically Modified Organisms (EPTEMBER 2002-03) and the promotor promoter (see, e.g., Schena et al. (1991) Proc. Natl. Acad. Sci. USA 88, 10421-10425 and McNellis et al. (1998) Plant Genetically Modified Organisms (EPTEMBER 2002-03)). J. [Plant Journal] 14, 247-257), tetracycline-inducible promoters and tetracycline-repressible promoters (see, for example, Gatz et al. (1991) Mol. Gen. Genet. [Molecular Genetics and General Genetics] 227, 229-237 and U.S. Pat. Nos. 5,814,618 and 5,789,156), Lac repressor system promoters, copper-inducible system promoters, salicylic acid-inducible system promoters (e.g., PR1a system), glucocorticoid-inducible promoters (Aoyama et al. (1997) Plant J. [Plant Journal] 11: 605-612) and ecdysone-inducible system promoters.

[0286] Other non-limiting examples of inducible promoters include ABA-inducible and cell swelling-inducible promoters, the auxin binding protein gene promoter (Schwob et al. (1993) Plant J. 4:423-432), the UDP-glucose flavonoid glycosyltransferase promoter (Ralston et al. (1988) Genetics 119:185-197), the MPI proteinase inhibitor promoter (Cordero et al. (1994) Plant J. 6:141-150), and the glyceraldehyde-3-phosphate dehydrogenase promoter (Kohler et al. (1995) Plant J. 10:114-115). Mol. Biol. [Plant Molecular Biology] 29: 1293-1298; Martinez et al. (1989) J. Mol. Biol. [Journal of Molecular Biology] 208: 551-565; and Quigley et al. (1989) J. Mol. Evol. [Journal of Molecular Evolution] 29: 412-421). Also included are benzenesulfonamide-inducible (U.S. Pat. No. 5,364,780) and ethanol-inducible (International Patent Application Publication Nos. WO 97 / 06269 and WO 97 / 06268) systems and glutathione S-transferase promoters. Likewise, any of the inducible promoters described in Gatz (1996) Current Opinion Biotechnol. 7:168-172 and Gatz (1997) Annu. Rev. Plant Physiol. Plant Mol. Biol. 48:89-108 may be used. Other chemically inducible promoters suitable for directing expression of the disclosed nucleotide sequences in plants are disclosed in U.S. Pat. No. 5,614,395. Chemical induction of gene expression is also described in detail in EP 0 332 104 (issued to Ciba-Geigy) and U.S. Pat. No. 5,614,395.

[0287] Another type of promoter useful in the present disclosure is a wound-inducible promoter. Examples of such promoters include those described by Stanford et al. Mol. Gen. Genet. [Molecular Genetics and General Genetics] 215: 200-208 (1989), Xu et al. Plant Molec. Biol. [Plant Molecular Biology] 22: 573-588 (1993), Logemann et al. Plant Cell [Plant Cell] 1: 151-158 (1989), Rohrmeier and Lehle, Plant Molec. Biol. [Plant Molecular Biology] 22: 783-792 (1993), Firek et al. Plant Molec. Biol. [Plant Molecular Biology] 22: 129-142 (1993), and Warner et al. Plant J. [Plant Journal] 3: 191-201 (1993).

[0288] In some embodiments, a recombinant vector is provided, which comprises a nucleic acid molecule or an expression cassette of the present disclosure. Certain vectors used in the transformation of plants and other organisms are known in the art. In other embodiments, non-limiting examples of vectors include plasmids, cosmids, phagemids, artificial chromosomes, phages, or viral vectors. In an embodiment, the vector is a plant vector, such as for plant transformation. In an embodiment, the vector is a bacterial vector, such as for bacterial transformation. Vectors suitable for plants, bacteria, and other organisms are known in the art.

[0289] Some embodiments relate to expression cassettes designed to express the polynucleotides and nucleic acid molecules of the present disclosure. In some embodiments, the expression cassette comprises a nucleic acid molecule having at least one control sequence operably linked to a nucleotide sequence of interest (e.g., a nucleotide sequence encoding an insecticidal protein of the present disclosure). In this manner, for example, a plant promoter operably linked to a nucleotide sequence to be expressed can be provided in an expression cassette for expression in a plant, plant part, or plant cell.

[0290] The expression cassette comprising the polynucleotide of interest may be chimeric, meaning that at least one of its components is heterologous relative to at least another of its other components. An expression cassette may also be a naturally occurring expression cassette that has been obtained in a recombinant form suitable for heterologous expression. Typically, however, the expression cassette is heterologous relative to the host, i.e., the specific nucleic acid sequence of the expression cassette is not naturally present in the host cell and must have been introduced into the host cell or an ancestor of the host cell by a transformation event.

[0291] In addition to the promoter operably linked to the nucleotide sequence of the present disclosure, the expression cassette of the present disclosure may also include other regulatory sequences. Regulatory sequences include, but are not limited to, enhancers, introns, translation leader sequences, termination signals, and polyadenylation signal sequences.

[0292] In certain embodiments, the expression cassette may also include polynucleotides encoding other desired traits in addition to the disclosed protein. Such an expression cassette comprising superimposed traits can be used to produce a desired plant, plant part or plant cell having a phenotype with superimposed traits (i.e., molecular superposition). Such a superimposed combination in a plant may also be produced by other methods, including but not limited to hybrid breeding plants by any conventional methodology. If superimposed by genetic transformation of these plants, the target nucleotide sequence may be combined at any time and in any order. For example, a transgenic plant comprising one or more desired traits may be used as a target to introduce other traits by subsequent conversion. Additional nucleotide sequences may be introduced simultaneously with the nucleotide sequence, nucleic acid molecule, nucleic acid construct or composition of the present disclosure provided by any combination of expression cassettes in a co-transformation scheme. For example, if two nucleotide sequences are to be introduced, they may be merged in a separate box (trans) or merged in the same box (cis). The expression of polynucleotides may be driven by the same promoter or by different promoters. It is also recognized that site-specific nucleases or recombination systems (e.g., FRT / Flp, Cre / Lox, TALE-endonucleases, zinc finger nucleases, CRISPR / Cas and related technologies) can be used to stack polynucleotides at a desired genomic location. See U.S. Patent Nos. 7,214,536, 8,921,332, 8,765,448, 5,527,695, 5,744,336, 5,910,415, 6,110,736, 6,175,058, 6,720,475, 6,455,315, 6,458,594 and U.S. Patent Publication Nos. 2019093090, 2019264218, 2018327785, 2017240911, 2016208272, 2019062765.

[0293] The expression cassette can also include additional coding sequences for one or more polypeptides of interest or double-stranded RNA molecules (dsRNA) for agronomic traits, the main beneficiaries of which are seed companies, growers or grain processors. The polypeptide of interest can be any polypeptide encoded by a nucleotide sequence of interest. Non-limiting examples of polypeptides of interest suitable for production in plants include those that produce agronomically important traits, such as herbicide resistance (sometimes also referred to as "herbicide tolerance"), virus resistance, bacterial pathogen resistance, insect resistance, nematode resistance, or fungal resistance. See, e.g., U.S. Patent Nos. 5,569,823; 5,304,730; 5,495,071; 6,329,504; and 6,337,431. The polypeptide can also be a trait that increases plant vigor or yield (including traits that allow plants to grow at different temperatures, soil conditions, and sunlight and precipitation levels), or a trait that allows plants exhibiting the trait of interest to be identified (e.g., selective markers, seed coat color, etc.). Various polypeptides of interest and methods of introducing these polypeptides into plants are described, for example, in U.S. Pat. Nos. 4,761,373; 4,769,061; 4,810,648; 4,940,835; 4,975,374; 5,013,659; 5,162,602; 5,276,268; 5,304,730; 5,495,071; 5,554,798; 5,561,236; 5,569,823; 5,767,366; 5,879,903; 5,928,937; 6,084,155; 6,329,504 and 6,337,431; and in U.S. Patent Publication No. 2001 / 0016956.

[0294] Polynucleotides that confer resistance / tolerance to herbicides (such as imidazolinones or sulfonylureas) that inhibit growing points or meristems may also be applicable in some embodiments. For example, exemplary polynucleotides in this classification number for mutant ALS and AHAS enzymes are described in U.S. Patents 5,767,366 and 5,928,937. U.S. Patents 4,761,373 and 5,013,659 relate to plants resistant to different imidazolinones or sulfonylurea herbicides. U.S. Patent 4,975,374 relates to plant cells and plants containing nucleic acids encoding mutant glutamine synthetase (GS), which are resistant to the inhibitory effects of known herbicides that inhibit GS (e.g., phosphinothricin and methionine sulfoximine). U.S. Patent 5,162,602 discloses plants resistant to the inhibitory effects of cyclohexanedione and aryloxyphenoxypropionic acid herbicides. This resistance is conferred by altered acetyl-CoA carboxylase (ACCase).

[0295] Polypeptides encoded by nucleotide sequences that confer resistance to glyphosate are also suitable for use in the present disclosure. See, for example, U.S. Pat. No. 4,940,835 and U.S. Pat. No. 4,769,061. U.S. Pat. No. 5,554,798 discloses transgenic maize plants resistant to glyphosate, the resistance being conferred by an altered 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase gene.

[0296] Polynucleotides encoding resistance to phosphoryl compounds such as glufosinate or phosphinothricin, and pyridinyloxypropionic acid or phenoxypropionic acid and cyclohexanone are also suitable. See, European Patent Application No. 0 242 246. See also U.S. Patent Nos. 5,879,903, 5,276,268 and 5,561,236.

[0297] Other suitable polynucleotides include those encoding resistance to herbicides that inhibit photosynthesis, such as triazines and benzocyanate (nitrilase), see U.S. Patent No. 4,810,648. Other suitable polynucleotides encoding for herbicide resistance include those encoding resistance to 2,2-dichloropropionic acid, sethoxydim, haloxyfop-butyl, imidazolinone herbicides, sulfonylurea herbicides, triazolopyrimidine herbicides, s-triazine herbicides and bromoxynil. Also suitable are polynucleotides that confer resistance to the original enzyme, or polynucleotides that provide enhanced resistance to plant diseases; enhanced tolerance to adverse environmental conditions (abiotic stress), including but not limited to drought, cold, overheating or soil salinity is too high or extremely acidic or alkaline; and changes in plant structure or development, including changes in development time. See, for example, U.S. Patent Publication No. 2001 / 0016956 and U.S. Patent No. 6,084,155.

[0298] Other suitable polynucleotides include those encoding pest-killing (e.g., insect-killing) polypeptides. These polypeptides can be produced in an amount sufficient to control, for example, insect pests (i.e., insect control amounts). It should be recognized that the production of pest-killing polypeptides necessary for controlling insects or other harmful organisms in plants can vary, depending on the type of cultivated variety, harmful organisms, environmental factors, etc. Polynucleotides that can be used for other insect or harmful organism resistance include, for example, those encoding toxins identified in Bacillus (Bacillus) organisms. Polynucleotides comprising nucleotide sequences encoding Bacillus thuringiensis (Bt) Cry proteins from several subspecies have been cloned, and it has been found that these recombinant clones are toxic to Lepidoptera, Diptera, and / or Coleopteran insect larvae. Examples of such Bt insecticidal proteins include Cry proteins such as Cry1Aa, Cry1Ab, Cry1Ac, Cry1B, Cry1C, Cry1D, Cry1Ea, Cry1Fa, Cry3A, Cry9A, Cry9B, Cry9C, etc., as well as vegetative insecticidal proteins such as Vip1, Vip2, Vip3, etc. A complete list of Bt-derived proteins can be found on the World Wide Web in the Bacillus thuringiensis Toxin Nomenclature Database maintained by the University of Sussex (see also Crickmore et al. (1998) Microbiol. Mol. Biol. Rev. 62:807-813).

[0299] In an embodiment, the additional polypeptide is an insecticidal polypeptide derived from a non-Bt source, including but not limited to: alpha amylase, peroxidase, cholesterol oxidase, potato glycoprotein, protease, protease inhibitor, urease, alpha-amylase inhibitor, pore-forming protein, chitinase, lectin, engineered antibody or antibody fragment, Bacillus cereus insecticidal protein, Xenorhabdus species (such as X. nematophila or Xenorhabdus bovienii) insecticidal protein, Photorhabdus species (such as P. luminescens or P. asymobiotica) insecticidal protein, Brevibacillus species (such as B. laterosporous) insecticidal protein, Lysinibacillus species (such as Lysinibacillus spp.) (such as spherical lysine Bacillus (L. Sphearicus) insecticidal proteins, Chromobacterium species (such as C. subtsugae or C. piscinae) insecticidal proteins, Yersinia species (such as Yersinia entomophage (Y. entomophaga)) insecticidal proteins, Paenibacillus species (such as P. propylaea) insecticidal proteins, Clostridium species (such as C. bifermentans) insecticidal proteins, Pseudomonas species (Pseudomonas spp.) (such as Pseudomonas fluorescens) insecticidal proteins and lignin.

[0300] Polypeptides suitable for production in plants further include those that improve or otherwise aid in the conversion of harvested plants or plant parts into commercially useful products, including, for example, increased or altered carbohydrate content or distribution, improved fermentation characteristics, increased oil content, increased protein content, improved digestibility, and increased nutrient content (e.g., increased phytosterol content, increased tocopherol content, increased stanol content, or increased vitamin content). Target polypeptides also include, for example, those that result in or contribute to a reduction in the level of undesirable components (e.g., phytic acid, or enzymes that degrade sugars) in the harvested crop. "Resulting in" or "contributing to" means that the target polypeptide can directly or indirectly contribute to the presence of the target trait (e.g., by increasing cellulose degradation through the use of a heterologous cellulase).

[0301] In certain embodiments, the polypeptide contributes to the improved digestibility of food or feed. Xylanases are hemicellulolytic enzymes that improve the decomposition of plant cell walls, which results in animals making better use of these plant nutrients. This results in improved growth rates and feed conversion. Similarly, the viscosity of feeds containing xylanases can be reduced. Heterogeneous production of xylanases in plant cells can also promote the conversion of lignocellulose into fermentable sugars in industrial processing.

[0302] Many xylanases from fungal and bacterial microorganisms have been identified and characterized (see, e.g., U.S. Pat. No. 5,437,992; Coughlin et al. (1993) "Proceedings of the Second TRICEL Symposium on Trichoderma reesei Cellulases and Other Hydrolases" Espoo; Souminen and Reinikainen, eds. (1993) Foundation for Biotechnical and Industrial Fermentation Research 8:125-135; U.S. Pat. Publication No. 2005 / 0208178; and PCT Publication No. WO 03 / 16654). In particular, three specific xylanases (XYL-I, XYL-II and XYL-III) have been identified in Trichoderma reesei (Tenkanen et al. (1992) Enzyme Microb. Technol. 14:566; Torronen et al. (1992) Bio / Technology 10:1461; and Xu et al. (1998) Appl. Microbiol. Biotechnol. 49:718).

[0303] In other embodiments, the polypeptide useful for the present disclosure can be a polysaccharide degrading enzyme. Plants of the present disclosure that produce such enzymes can be useful for producing fermentation raw materials such as for bioprocessing. In some embodiments, enzymes that can be used in fermentation processes include alpha amylases, proteases, pullulanases, isoamylases, cellulases, hemicellulases, xylanases, cyclodextrin glycosyltransferases, lipases, phytases, laccases, oxidases, esterases, cutinases, granular starch hydrolases and other glucoamylases.

[0304] Polysaccharide degrading enzymes include: starch degrading enzymes such as α-amylase (EC 3.2.1.1), glucuronidase (EC 3.2.1.131); exo-1,4-α-D-glucanases such as amyloglucosidase and glucoamylase (EC 3.2.1.3), β-amylase (EC 3.2.1.2), α-glucosidase (EC 3.2.1.20) and other exoamylases; starch debranching enzymes such as a) isoamylase (EC 3.2.1.68), pullulanase (EC 3.2.1.41), etc.; b) cellulases such as exo-1,4-3-cellobiohydrolase (EC 3.2.1.91), exo-1,3-β-D-glucanase (EC 3.2.1.39), β-glucosidase (EC 3.2.1.21); c) L-arabinase (arabinase), such as endo-1,5-α-L-arabinase (EC 3.2.1.99), α-arabinosidase (EC 3.2.1.55), etc.; d) galactanase, such as endo-1,4-β-D-galactanase (EC 3.2.1.89), endo-1,3-β-D-galactanase (EC 3.2.1.90), α-galactosidase (EC 3.2.1.22), β-galactosidase (EC 3.2.1.23), etc.; e) mannanase, such as endo-1,4-β-D-mannanase (EC 3.2.1.78), β-mannosidase (EC 3.2.1.25), α-mannosidase (EC 3.2.1.24), etc.; f) xylanase, such as endo-1,4-β-xylanase (EC 3.2.1.8), β-D-xylosidase (EC 3.2.1.37), 1,3-β-D-xylanase, etc.; and g) other enzymes such as α-L-fucosidase (EC 3.2.1.51), α-L-rhamnosidase (EC 3.2.1.40), levanase (EC3.2.1.65), inulinase (EC 3.2.1.7), etc. In one embodiment, the α-amylase is the synthetic α-amylase Amy797E described in U.S. Pat. No. 8,093,453 (incorporated herein by reference in its entirety).

[0305] Other enzymes that can be used together with the present disclosure include proteases, such as fungal and bacterial proteases. Fungal proteases include but are not limited to those obtained from Aspergillus, Trichoderma, Mucor and Rhizopus, such as A. niger, A. awamori, A. oryzae and M. miehei. In certain embodiments, the polypeptide of the present disclosure can be a cellobiohydrolase (CBH) (EC3.2.1.91). In one embodiment, the cellobiohydrolase can be CBH1 or CBH2.

[0306] Other enzymes useful in the present disclosure include, but are not limited to, hemicellulases, such as mannanases and arabinofuranosidases (EC 3.2.1.55); ligninases; lipases (e.g., EC 3.1.1.3), glucose oxidases, pectinases, xylanases, transglucosidases, α1,6 glucosidases (e.g., EC 3.2.1.20); esterases, such as ferulic acid esterases (EC 3.1.1.73) and acetylxylan esterases (EC 3.1.1.72); and cutinases (e.g., EC 3.1.1.74).

[0307] The double-stranded RNA molecules useful for the present disclosure include but are not limited to those that inhibit target insect genes. As used herein, the term "gene inhibition" is intended to refer to any well-known method for reducing the level of protein produced as a result of gene transcription into mRNA and the subsequent translation of the mRNA when considered together. Gene inhibition is also intended to mean reducing the expression of protein from a gene or coding sequence, including post-transcriptional gene inhibition and transcriptional inhibition. Post-transcriptional gene inhibition is mediated by homology between all or a portion of the mRNA transcribed from the gene or coding sequence of the targeted inhibition and the corresponding double-stranded RNA for inhibition, and refers to the substantial and measurable reduction of the amount of available mRNA available for use by ribosomes in cells. The transcribed RNA can be in the sense direction and play a role, which is called co-suppression, in the antisense direction and play a role, which is called antisense inhibition, or in both directions to produce dsRNA and play a role, which is called RNA interference (RNAi). Transcriptional inhibition is mediated by the presence of dsRNA that acts as a gene inhibitor in the cell and exhibits substantial sequence identity with the promoter DNA sequence or its complement, which is called promoter trans inhibition. Gene suppression can be effective for natural plant genes associated with traits, for example, to provide plants with reduced levels of proteins encoded by the natural genes or with enhanced or reduced levels of affected metabolites. Gene suppression can also be effective for target genes in plant pests, which can ingest or contact plant materials containing gene suppressors that are specifically designed to suppress or inhibit the expression of one or more homologous or complementary sequences in the cells of the pest. Such genes targeted for suppression can encode essential proteins whose predicted functions are selected from the group consisting of: muscle formation, juvenile hormone formation, juvenile hormone regulation, ion regulation and transport, digestive enzyme synthesis, maintenance of cell membrane potential, amino acid biosynthesis, amino acid degradation, sperm formation, pheromone synthesis, pheromone sensing, antenna formation, wing formation, leg formation, development and differentiation, egg formation, larval maturation, digestive enzyme formation, hemolymph synthesis, hemolymph maintenance, neurotransmission, cell division, energy metabolism, respiration and apoptosis.

[0308] Genetically modified cells, plants, plant parts, seeds

[0309] In some aspects, the disclosure further provides transgenic cells, plants, plant parts and seeds comprising the insecticidal proteins or nucleic acids of the disclosure. In some embodiments, the disclosure provides non-human host cells, which include polynucleotides, nucleic acid molecules, expression cassettes, vectors or polypeptides of the disclosure. Transgenic non-human host cells may include but are not limited to plant cells (including monocotyledonous plant cells and / or dicotyledonous plant cells), yeast cells, bacterial cells or insect cells. Therefore, in some embodiments, a bacterial cell selected from the following genus is provided: Bacillus, Brevibacillus, Clostridium, Xenorhabdus, Photobacterium, Pasteurella, Escherichia, Pseudomonas, Erwinia, Serratia, Klebsiella, Salmonella, Pasteurella, Xanthomonas, Streptomyces, Rhizobium, Rhodopseudomonas, Methylophilus, Agrobacterium, Acetobacter, Lactobacillus, Arthrobacter, Azotobacter, Leuconostoc or Alcaligenes. Thus, for example, as a biological insect control agent, the disclosed insecticidal proteins can be produced by expressing a polynucleotide encoding the same in a bacterial cell. For example, in some embodiments, a Bacillus thuringiensis cell comprising a polynucleotide encoding an insecticidal protein of the present disclosure is provided.

[0310] In some embodiments, the transgenic plant cell is a dicotyledon cell or a monocotyledon cell. In other embodiments, the dicotyledon cell is a soybean cell, a sunflower cell, a tomato cell, a rape cell, a cotton cell, a beet cell, or a tobacco cell. In further embodiments, the monocotyledon cell is a barley cell, a maize cell, an oat cell, a rice cell, a sorghum cell, a sugarcane cell, or a wheat cell. In some embodiments, the disclosure provides a variety of dicotyledon cells or monocotyledon cells comprising polynucleotides expressing the disclosed insecticidal proteins. In embodiments, a plurality of cells are juxtaposed to form apoplasts and grown in natural light. In embodiments, the transgenic plant cell cannot regenerate a whole plant.

[0311] In other embodiments of the present disclosure, the insecticidal proteins of the present disclosure are expressed in higher organisms (e.g., plants). Such transgenic plants express effective amounts of insecticidal proteins to control plant pests, such as insect pests. When insects begin to feed on such transgenic plants, they ingest the expressed insecticidal proteins. This can prevent insects from further feeding on plant tissues or can even injure or kill insects. In some embodiments, the disclosed polynucleotides are inserted into expression cassettes, which are then stably integrated into the genome of the plant. In other embodiments, the polynucleotides are contained in non-pathogenic self-replicating viruses.

[0312] In some embodiments of the present disclosure, the transgenic plant cell comprising the nucleic acid molecule or polypeptide of the present disclosure is a cell of a plant part, plant organ or plant culture (each as described herein), including but not limited to roots, leaves, seeds, flowers, fruits, pollen cells, organs or plant cultures, etc., or callus cells or cultures.

[0313] Transgenic plants or plant cells transformed according to the present disclosure can be monocotyledonous or dicotyledonous plants or plant cells, and include, but are not limited to, corn (maize), soybean, rice, wheat, barley, rye, oats, sorghum, millet, sunflower, safflower, sugar beets, cotton, sugarcane, rapeseed, alfalfa, tobacco, peanuts, vegetables (including sweet potatoes, beans, peas, chicory, lettuce, cabbage, cauliflower, broccoli, turnips, carrots, eggplant, cucumber, radish, spinach, potato, tomato, asparagus, onion, garlic, melons, peppers, celery, pumpkin, zucchini, green squash), fruits (including apples, pears, quinces, plums, cherries, peaches, nectarines, apricots, strawberries, grapes, raspberries, blackberries, pineapples, avocados, papayas, mangoes, bananas) and specialty plants such as Arabidopsis, as well as woody plants such as coniferous and deciduous trees. Preferably, the plant of the present disclosure is a crop plant, such as maize, sorghum, wheat, sunflower, tomato, crucifer, pepper, potato, cotton, rice, soybean, sugar beet, sugar cane, tobacco, barley, rapeseed, etc.

[0314] Once a desired polynucleotide has been transformed into a particular plant species, it may be propagated within that species or transferred into other varieties of the same species, particularly including commercial varieties, using any suitable technique, including traditional breeding techniques.

[0315] The disclosed insecticidal proteins can act as insect control agents in plant parts, plant cells, plant organs, seeds, harvested products, processed products or extracts, etc. In other words, in some embodiments, the insecticidal protein can continue to perform the insecticidal function it has in transgenic plants. Nucleic acids can serve to express the insecticidal protein. As an alternative to encoding the insecticidal protein disclosed herein, the nucleic acid can be used to identify the transgenic plant parts, plant cells, plant organs, seeds, harvested products, processed products or extracts disclosed herein.

[0316] In embodiments, the transgenic plants, plant parts, plant cells, plant organs or seeds of the present disclosure are hemizygous for the polynucleotides or expression cassettes of the present disclosure, for example, hybrid corn plants in which only one parental plant contains the polynucleotides or expression cassettes of the present disclosure. In embodiments, the transgenic plants, plant parts, plant cells, plant organs or seeds of the present disclosure are homozygous for the polynucleotides or expression cassettes of the present disclosure.

[0317] Other embodiments of the present disclosure include harvest products produced from the transgenic plants of the present disclosure or parts thereof and processed products produced from the harvest products. The harvest products can be whole plants or any plant parts as described herein. Therefore, in some embodiments, non-limiting examples of harvest products include seeds, fruits, flowers or parts thereof (e.g., anthers, stigmas, etc.), leaves, stems, etc. In other embodiments, processed products include but are not limited to flour, meal, oil, starch, cereals, etc. produced from the seeds or other plant parts of the present disclosure of the harvest, wherein the seeds or other plant parts comprise nucleic acid molecules / polynucleotides / nucleotide sequences of the present disclosure.

[0318] In other embodiments, the disclosure provides an extract from a transgenic seed or transgenic plant of the disclosure, wherein the extract comprises a nucleic acid molecule, polynucleotide, nucleotide sequence or insecticidal protein of the disclosure. Extracts from plants or plant parts can be prepared according to procedures well known in the art (see de la Torre et al., Food, Agric. Environ. [Food Agriculture and Environment] 2 (1): 84-89 (2004); Guidet, Nucleic Acids Res. [Nucleic Acids Research] 22 (9): 1772-1773 (1994); Lipton et al., Food Agric. Immun. [Food and Agricultural Immunology] 12: 153-164 (2000)). Such extracts can be used, for example, in methods for detecting the presence of insecticidal proteins or polynucleotides of the disclosure.

[0319] In embodiments, the transgenic plant, plant part, plant cell, plant organ, seed, harvested product, processed product, or extract has increased insecticidal activity against one or more insect pests (e.g., coleopteran pests such as western corn rootworm) as compared to a suitable control that does not comprise a nucleic acid encoding an insecticidal protein of the disclosure.

[0320] Plant transformation

[0321] Non-limiting examples of methods for plant transformation include transformation via bacteria-mediated nucleic acid delivery (e.g., via Agrobacterium), virus-mediated nucleic acid delivery, silicon carbide or nucleic acid whisker-mediated nucleic acid delivery, liposome-mediated nucleic acid delivery, microinjection, particle bombardment, calcium phosphate-mediated transformation, cyclodextrin-mediated transformation, electroporation, nanoparticle-mediated transformation, sonication, infiltration, PEG-mediated nucleic acid uptake, and any other electrical, chemical, physical (mechanical), or biological mechanism that results in the introduction of nucleic acids into plant cells, including any combination thereof. General guides to the various plant transformation methods known in the art include Miki et al. ("Procedures for Introducing Foreign DNA into Plants," in Methods in Plant Molecular Biology and Biotechnology, Glick, BR and Thompson, JE, eds. (CRC Press, Inc., Boca Raton, 1993), pp. 67-88) and Rakowoczy-Trojanowska (Cell. Mol. Biol. Lett. 7:849-858 (2002)).

[0322] For Agrobacterium-mediated transformation, binary vectors or vectors carrying at least one T-DNA border sequence are usually suitable, and for direct gene transfer (e.g., particle bombardment, etc.), any vector is suitable, and a linear DNA containing only the purpose structure can be used. In the case of direct gene transfer, conversion or co-transformation with a single DNA species can be used (Schocher et al., Biotechnology [biotechnology] 4: 1093-1096 (1986)). For both direct gene transfer and Agrobacterium-mediated transfer, conversion is usually (but not necessarily) carried out with the following selective marker, which can be a positive selection (e.g., phosphomannose isomerase), providing resistance to antibiotics (e.g., kanamycin, hygromycin or methotrexate) or herbicides (e.g., glyphosate or glufosinate). However, the selection of selective markers is not crucial for this disclosure.

[0323] Agrobacterium-mediated transformation is a commonly used method for transforming plants due to its high transformation efficiency and its wide utility with many different species. Agrobacterium-mediated transformation typically involves transferring a binary vector carrying the foreign DNA of interest to a suitable Agrobacterium strain, which may depend on the complement of vir genes carried by the host Agrobacterium strain either on the co-resident Ti plasmid or chromosomally (Uknes et al., 1993, Plant Cell 5:159-169). Transfer of the recombinant binary vector to Agrobacterium can be achieved using Escherichia coli carrying the recombinant binary vector and a helper E. coli strain (the helper strain carrying a plasmid capable of mobilizing the recombinant binary vector into the target Agrobacterium strain) by a triparental mating procedure. Alternatively, the recombinant binary vector can be transferred into Agrobacterium by nucleic acid transformation ( and Willmitzer, (1988) Nucleic Acids Res. 16:9877).

[0324] Agrobacterium can be used to transform dicotyledons as well as monocotyledons. Methods for Agrobacterium-mediated rice transformation include well-known rice transformation methods, such as those described in any of the following documents: European Patent Application EP1198985 A1, Aldemita and Hodges (Planta [Plant] 199: 612-617, 1996); Chan et al. (Plant Mol Biol [Plant Molecular Biology] 22 (3): 491-506, 1993), Hiei et al. (Plant J [Plant Journal] 6 (2): 271-282, 1994), the disclosures of which are incorporated herein by reference to the same extent as if fully set forth. In the case of maize transformation, preferred methods are as described in Ishida et al. (Nat. Biotechnol [Nature Biotechnology] 14 (6): 745-50, 1996) or Frame et al. (Plant Physiol [Plant Physiology] 129 (1): 13-22, 2002), the disclosures of which are incorporated herein by reference to the same extent as if fully set forth. The methods are further described by examples in the following literature: B. Jenes et al., Techniques for Gene Transfer [Gene Transfer Technology], in: Transgenic Plants [Transgenic Plants], Vol. 1, Engineering and Utilization [Engineering and Utilization], ed. S. D. Kung and R. Wu, Academic Press [Academic Press of the United States] (1993) 128-143 and Potrykus Annu. Rev. Plant Physiol. Plant Molec. Biol. [Annual Review of Plant Physiology and Plant Molecular Biology] 42 (1991) 205-225. The nucleic acid or construct to be expressed is preferably cloned into a vector suitable for transforming Agrobacterium tumefaciens, for example pBin19 (Bevan et al., Nucl. Acids Res. [Nucleic Acids Research] 12 (1984) 8711). Agrobacterium transformed with such a vector can then be used in a known manner to transform plants, such as plants used as models like Arabidopsis thaliana or crop plants such as tobacco plants, for example by immersing the crushed or chopped leaves in an Agrobacterium solution and then culturing them in a suitable culture medium.The transformation of plants by Agrobacterium tumefaciens is described, for example, by Hagen and Willmitzer, Nucl. Acid Res. (1988) 16, 9877, or, inter alia, by FF White, Vectors for Gene Transfer in Higher Plants; described in Transgenic Plants, Vol. 1, Engineering and Utilization, ed. SD Kung and R. Wu, Academic Press, 1993, pp. 15-38.

[0325] Soybean plant material can be appropriately transformed and regenerated into fertile plants by various methods well known to those of ordinary skill in the art. For example, morphologically normal transgenic soybean plants of fertility can be obtained by the following: 1) somatic embryonic tissue is produced from, for example, immature cotyledons, hypocotyls or other suitable tissues; 2) transformation is carried out by particle bombardment or infection with Agrobacterium; and 3) regeneration of plants. In one example, as described in U.S. Patent No. 5,024,944, cotyledon tissue is excised from immature embryos of soybeans, hypocotyls are preferably removed, and cultured in a medium containing hormones to form somatic embryonic plant material. The material is transformed using, for example, direct DNA methods, DNA-coated microprojectile bombardment or infection with Agrobacterium, cultured on a suitable selection medium, and optionally regenerated into fertile transgenic soybean plants in the continuous presence of a selection agent. The selection agent can be an antibiotic such as kanamycin, hygromycin or a herbicide (such as phosphinothricin or glyphosate), or alternatively, the selection can be based on the expression of a visible marker gene (such as GUS). Alternatively, the target tissue for transformation includes meristems other than somaclonal embryonic tissue or optionally flowers or flower-forming tissue. Other examples of soybean transformation can be found, for example, by physical DNA delivery methods such as particle bombardment (Finer and McMullen (1991) In Vitro Cell Dev. Biol. [In vitro cell and developmental biology] 27P: 175-182; McCabe et al. (1988) Bio / technology [biotechnology] 6: 923-926), whisker method (Khalafalla et al. (2006) African J. of Biotechnology [African Journal of Biotechnology] 5:1594-1599), aerosol beam injection (U.S. Patent No. 7,001,754), or through Agrobacterium-mediated delivery methods (Hinchee et al. (1988) Bio / Technology [Bio / Technology] 6:915-922; U.S. Patent No. 7,002,058; U.S. Patent Application Publication No. 20040034889; U.S. Patent Application Publication No. 20080229447; Paz et al. (2006) Plant Cell Report [Plant Cell Communication] 25:206-213).

[0326] Can use different transformation methods, produce soybean transgenic plant with the above-mentioned binary vector containing selective marker gene.For example, as described (referring to for example U.S. Patent Application Publication No. 20080229447), use carrier to transform immature seed target, thereby directly use HPPD inhibitor (for example mesotrione) as selection agent to produce transgenic HPPD soybean plant.Optionally, other herbicide tolerance genes can be present in polynucleotide beside other sequences, and these other sequences provide the other means of the tissue of selection / differentiation transformation, comprise for example provide the known gene of resistance to kanamycin, hygromycin, phosphinothricin, fluazifop-butyl or glyphosate.For example, different binary vectors containing PAT or EPSPS selective marker gene are transformed in immature soybean seed target, thereby as described use Agrobacterium-mediated transformation and phosphinothricin or glyphosate selection produce pesticide and herbicide tolerance plant (referring to for example, U.S. Patent Application Publication No. 20080229447).

[0327] Plant transformation by recombinant Agrobacterium typically involves co-cultivation of Agrobacterium with explants from plants and follows methods well known in the art. Transformed tissues are regenerated on selection media carrying antibiotic or herbicide resistance markers located between the binary plasmid T-DNA borders.

[0328] As previously discussed, another method for transforming plants, plant parts and plant cells involves advancing inert or biologically active particles on plant tissues and cells. See, for example, U.S. Patent Nos. 4,945,050; 5,036,006 and 5,100,792. Typically, this method involves advancing inert or biologically active particles at plant cells under conditions effective to penetrate the outer surface of the cell and provide incorporation into its interior. When inert particles are utilized, the vector can be introduced into the cell by coating the particles with a vector containing the target nucleic acid. Alternatively, one or more cells can be surrounded by the vector so that the vector is carried into the cell by the excitation of the particle. Biologically active particles (e.g., dry yeast cells, dry bacteria or bacteriophages, each containing one or more nucleic acids intended to be introduced) can also be advanced into plant tissues.

[0329] In other embodiments, the polynucleotides of the present disclosure can be directly transformed into the plastid genome. Plastid transformation techniques are extensively described in U.S. Patent Nos. 5,451,513, 5,545,817 and 5,545,818, in PCT Application No. WO 95 / 16783, and in McBride et al. (1994) Proc. Nati. Acad. Sci. USA [Proceedings of the National Academy of Sciences of the United States] 91, 7301-7305.

[0330] Methods for selecting transformed transgenic plants, plant cells or plant tissue cultures can be used in the methods of the disclosure provided herein. For example, the recombinant vectors of the disclosure can also include an expression cassette comprising a nucleotide sequence for a selective marker, which can be used to select transformed plants, plant parts or plant cells.

[0331] Examples of selectable markers include, but are not limited to, nucleotide sequences encoding neo or nptII, which confer resistance to kanamycin, G418, and the like (Potrykus et al. (1985) Mol. Gen. Genet. 199:183-188); nucleotide sequences encoding bar, which confer resistance to phosphinothricin; nucleotide sequences encoding altered 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase, which confer resistance to glyphosate (Hinchee et al. (1988) Biotech. 6:915-922); nucleotide sequences encoding nitrilase such as bxn from Bacillus foetida, which confer resistance to bromoxynil (Stalker et al. (1988) Science 242:419-423); nucleotide sequences encoding altered acetolactone; Synthase (ALS), which confers resistance to imidazolinone, sulfonylurea or other ALS inhibiting chemical agents (European Patent Application No. 154204); a nucleotide sequence encoding a methotrexate-resistant dihydrofolate reductase (DHFR) (Thillet et al. (1988) J. Biol. Chem. [Journal of Biological Chemistry] 263: 12500-12508); a nucleotide sequence encoding a dalapon dehalogenase, which confers resistance to dalapon; a nucleotide sequence encoding a mannose-6-phosphate isomerase (also known as phosphomannose isomerase (PMI)), which confers the ability to metabolize mannose (U.S. Patent Nos. 5,767,378 and 5,994,629); a nucleotide sequence encoding an altered anthranilate synthase, which confers resistance to 5-methyltryptophan; or a nucleotide sequence encoding hph, which confers resistance to hygromycin. Those skilled in the art will be able to select suitable selectable markers for use in the expression cassettes of the present disclosure.

[0332] Additional selectable markers include, but are not limited to, nucleotide sequences encoding beta-glucuronidase or uidA (GUS) encoding an enzyme for which a variety of chromogenic substrates are known; R locus nucleotide sequences encoding products that regulate the production of anthocyanin pigments (red) in plant tissues (Dellaporta et al., "Molecular cloning of the maize R-nj allele by transposon-tagging with Ac," 263-282 In: Chromosome Structure and Function: Impact of New Concepts, 18th Stadler Genetics Symposium (Gustafson and Appels, eds., Plenum Press [Plum Press] 1988)); a nucleotide sequence encoding β-lactamase, an enzyme known for a variety of chromogenic substrates (e.g., PADAC, a chromogenic cephalosporin) (Sutcliffe (1978) Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences of the United States of America] 75:3737-3741); a nucleotide sequence encoding xylE, which encodes a catechol dioxygenase (Zukowsky et al. (1983) Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences of the United States of America] 80:1101-1105); a nucleotide sequence encoding tyrosinase, an enzyme capable of oxidizing tyrosine to DOPA and dopaquinone, which in turn condense to form melanin (K atz et al. (1983) J. Gen. Microbiol. 129:2703-2714); a nucleotide sequence encoding β-galactosidase, an enzyme in the presence of a chromogenic substrate; a nucleotide sequence encoding luciferase (lux) that allows bioluminescent detection (Ow et al. (1986) Science 234:856-859); a nucleotide sequence encoding aequorin that can be used for calcium-sensitive bioluminescent detection (Prasher et al. (1985) Biochem. Biophys. Res. Comm. 126:1259-1268); or a nucleotide sequence encoding green fluorescent protein (Niedz et al. (1995) Plant Cell Reports 14:403-406) or other fluorescent proteins, such as dsRed or mCherry.Those skilled in the art will be able to select appropriate selectable markers for use in the expression cassettes of the disclosure.

[0333] In addition, as is well known in the art, complete transgenic plants can be regenerated from transformed plant cells, plant tissue cultures or cultured protoplasts using any of a variety of known techniques. Plant regeneration from plant cells, plant tissue cultures or cultured protoplasts is described in, for example, Evans et al. (Handbook of Plant Cell Cultures, Vol. 1, MacMilan Publishing Co., New York (1983)); and Vasil IR (ed.) (Cell Culture and Somatic Cell Genetics of Plants, Acad. Press, Orlando, Vol. I (1984) and Vol. II (1986)).

[0334] In addition, the genetic characteristics engineered into the transgenic seeds and plants, plant parts or plant cells of the present disclosure described above can be transmitted by sexual reproduction or vegetative growth, and thus can be maintained and propagated in progeny plants. Typically, maintenance and propagation utilize known agricultural methods developed to be suitable for specific purposes (such as harvesting, sowing or farming).

[0335] Therefore, polynucleotide can be introduced into this plant, plant part or vegetable cell by any method well known in the art (as mentioned above).Therefore, do not rely on the ad hoc method for introducing one or more polynucleotide into plant, on the contrary can use any method that allows these one or more polynucleotide to be stably integrated into the genome of this plant.In the case of waiting to introduce more than one polynucleotide, these corresponding polynucleotide can be assembled as a part of single nucleic acid molecule or as separated nucleic acid molecule, and can be located on the same or different nucleic acid molecule.Therefore, can be in single transformation event, in separated transformation event or for example as a part of breeding scheme in plant, these polynucleotide are introduced into target cell.

[0336] Once a desired polynucleotide has been transformed into a particular plant species, it can be propagated within that species or transferred into other varieties of the same species, particularly including commercial varieties, using conventional breeding techniques.

[0337] Insecticide composition

[0338] In some embodiments, an insecticidal composition is provided, which comprises an insecticidal protein of the present disclosure in an agriculturally acceptable carrier. As used herein, an "agriculturally acceptable carrier" may include a natural or synthetic organic or inorganic material that is combined with an active protein to promote its application to a plant or its part or in a plant or its part. Examples of agriculturally acceptable carriers include, but are not limited to, powders, dusts, pills, granules, sprays, emulsions, colloids, and solutions. Agriculturally acceptable carriers further include, but are not limited to, inert components, dispersants, surfactants, adjuvants, tackifiers, adhesives, adhesives, or combinations thereof that can be used in agricultural formulations. Such compositions can be applied in any manner that pesticidal proteins or other pest control agents are contacted with these pests. Therefore, these compositions can be applied to the surface of plants or plant parts, including seeds, leaves, flowers, stems, tubers, roots, etc. In other embodiments, the plant producing the insecticidal protein of the present disclosure in the plant is an agriculturally acceptable carrier of the expressed insecticidal protein, and the combination of the plant and the protein is an insecticidal composition.

[0339] In further embodiments, the insecticidal composition comprises bacterial cells or transgenic bacterial cells of the present disclosure, wherein the bacterial cells or transgenic bacterial cells produce insecticidal proteins of the present disclosure. Such insecticidal compositions can be prepared by dehydrating, freeze-drying, homogenizing, extracting, filtering, centrifuging, settling or concentrating cultures of Bacillus thuringiensis (Bt), including transgenic Bt cultures. In embodiments, the compositions of the present disclosure may comprise at least about 1%, at least about 5%, at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 97% or at least 99% of the polypeptides of the present disclosure by weight. In further embodiments, the composition comprises insecticidal proteins of the present disclosure from about 1% to about 99% by weight.

[0340] The disclosed insecticidal proteins can be used in combination with other pest control agents to increase the pest target spectrum and / or for preventing or managing insect resistance. In addition, the disclosed insecticidal proteins are used in combination with insecticides (which have different modes of action or target different receptors in the insect gut) to have specific utility for preventing and / or managing insect resistance.

[0341] Thus, in some embodiments, a composition for controlling one or more plant pests (e.g., insect pests, such as lepidopteran insect pests, coleopteran insect pests, hemipteran insect pests, and / or dipteran insect pests) is provided, wherein the composition comprises a first pest control agent (which is an insecticidal protein disclosed) and at least a second pest control agent (which is different from the first pest control agent). In other embodiments, the composition is a formulation for topical application to a plant. In still other embodiments, the composition is a transgenic plant. In further embodiments, the composition is a combination of formulations topically applied to transgenic plants. In some embodiments, when the transgenic plant comprises a second pest control agent, the formulation comprises a first pest control agent (which is an insecticidal protein disclosed). In other embodiments, when the transgenic plant comprises a first pest control agent (which is an insecticidal protein disclosed herein), the formulation comprises a second pest control agent.

[0342] In some embodiments, the second pest control agent can be one or more of the following: a chemical pesticide (such as an insecticide), a Bacillus thuringiensis (Bt) insecticidal protein, and / or a non-Bt insecticidal agent, including but not limited to Xenorhabdus insecticidal protein, Photorhabdus insecticidal protein, Brevibacillus laterosporus insecticidal protein, Bacillus sphaericus insecticidal protein, protease inhibitors (both serine and cysteine ​​types), lectins, alpha-amylases, peroxidases, cholesterol oxidases, or double-stranded RNA (dsRNA) molecules.

[0343] In other embodiments, the second pest control agent is one or more chemical pesticides, which are optionally seed coatings. Non-limiting examples of chemical pesticides include pyrethroids, carbamates, neonicotinoids, neuronal sodium channel blockers, insecticidal macrolides, gamma-aminobutyric acid (GABA) antagonists, insecticidal ureas, and juvenile hormone mimetics. In other embodiments, the chemical pesticide is one or more of abamectin, acephate, acetamiprid, amidoflumet (S-1955), avermectin, azadirachtin, methyl azinphos-methyl, bifenthrin, binfenazate, buprofezin, carbofuran, chlorfenapyr, chlorpyrifos, methyl chlorpyrifos, chromafenazate, clothianidin, cyfluthrin, beta-cyfluthrin, cyhalothrin, lambda-cyhalothrin, cypermethrin, cyromazine, deltamethrin, cyfluthrin, diazinon, diflubenzuron, dimethoate, benzylpezil, emamectin, endosulfan, cypermethrin, ethiprole, fenothicarb, cypermethrin ... b), fenoxycarb, cypermethrin, fenpyroximate, cypermethrin, fipronil, flonicamid, flucythrinate, t-fluvalinate, cypermethrin, flufenacet (UR-50701), flufenoxuron, flufenoxam, chlorfenapyr, hexaflumuron, imidacloprid, indoxacarb, isoflavone, chlorfenapyr, malathion, metaldehyde, methamidophos, methidathion, methomyl, methoprene, methoxychlor, monocrotophos, methoxyfenozide, nithiazin, bispyribac, polyflumuron (XDE-007), oxamyl, parathion, methyl parathion, permethrin, phorate, phosalone, phosmet, phosphamidon, pirimicarb, profenofos, pymetrozine, pyridalyl, pyriproxyfen, rotenone, spinosad, spiromesifin (BSN 2060), thiopromide, fenbufen, tefluthrin, terbufos, chlorpyrifos, thiomethrin, thiomethoxam, thiodicarb, thiosultap-sodium, tralomethrin, trichlorfon and triflumuron, aldicarb, oxamyl, fenamiphos, amitraz, cypermethrin, ethyl cypermethrin, tricyclam, dicofol, cypermethrin, fenbutatin, cypermethrin, fenpyrad, cypermethrin, and cypermethrin. In still other embodiments, the chemical pesticide is selected from one or more of cypermethrin, tri-cyhalothrin, cyfluthrin and beta-cyfluthrin, esfenvalerate, cypermethrin, tralomethrin, fenthiocarb, methomyl, oxamyl, thiodicarb, clothianidin, imidacloprid, thiacloprid, indoxacarb, spinosad, abamectin, avermectin, avermectin, endosulfan, ethiprole, fipronil, flufenoxuron, triflumuron, dimethoate, pyriproxyfen, pymetrozine, and amitraz.

[0344] In other embodiments, the second pest control agent can be one or more of any number of Bacillus thuringiensis insecticidal proteins, including but not limited to Cry proteins, vegetative insecticidal proteins (VIP), and insecticidal chimeras of any of the foregoing insecticidal proteins. In other embodiments, the second pest control agent is a Cry protein selected from the following: Cry1Aa, Cry1Ab, Cry1Ac, Cry1Ad, Cry1Ae, Cry1Af, Cry1Ag, Cry1Ah, Cry1Ai, Cry1Aj, Cry1Ba, Cry1Bb, Cry1Bc, Cry1Bd, Cry1Be, Cry1Bf, Cry1Bg, Cry1Bh, Cry1Bi, Cry1Ca, Cry1Cb, Cry1Da, Cry1Db, Cry1Dc, Cry1Dd, Cry1Ea, Cry1Eb, Cry1Fa, Cry1E y1Fb, Cry1Ga, Cry1Gb, Cry1Gc, Cry1Ha, Cry1Hb, Cry1Hc, Cry1Ia, Cry1Ib, Cry1Ic, Cry1Id, Cry1Ie, Cry1If, Cry1Ig, Cry1Ja, Cry1Jb, Cry1 Jc, Cry1Jd, Cry1Ka, Cry1La, Cry1Ma, Cry1Na, Cry1Nb, Cry2Aa, Cry2Ab, Cry2Ac, Cry2Ad, Cry2Ae, Cry2Af, Cry2Ag, Cry2Ah, Cry2Ai, Cry2Aj , Cry2Ak, Cry2Al, Cry2Ba, Cry3Aa, Cry3Ba, Cry3Bb, Cry3Ca, Cry4Aa, Cry4Ba, Cry4Ca, Cry4Cb, Cry4Cc, Cry5Aa, Cry5Ab, Cry5Ac, Cry5Ad, C ry5Ba, Cry5Ca, Cry5Da, Cry5Ea, Cry6Aa, Cry6Ba, Cry7Aa, Cry7Ab, Cry7Ac, Cry7Ba, Cry7Bb, Cry7Ca, Cry7Cb, Cry7Da, Cry7Ea, Cry7Fa, Cry 7Fb, Cry7Ga, Cry7Gb, Cry7Gc, Cry7Gd, Cry7Ha, Cry7Ia, Cry7Ja, Cry7Ka, Cry7Kb, Cry7La, Cry8Aa, Cry8Ab, Cry8Ac, Cry8Ad, Cry8Ba, Cry8B b. Cry8Bc, Cry8Ca, Cry8Da, Cry8Db, Cry8Ea, Cry8Fa, Cry8Ga, Cry8Ha, Cry8Ia, Cry8Ib, Cry8Ja, Cry8Ka, Cry8Kb, Cry8La, Cry8Ma, Cry8Na,Cry8Pa、Cry8Qa、Cry8Ra、Cry8Sa、Cry8Ta、Cry9Aa、Cry9Ba、Cry9Bb、Cry9Ca、Cry9Da、Cry9Db、Cry9Dc、Cry9Ea、Cry9Eb、Cry9Ec、Cry9Ed、Cry9Ee、Cry9Fa、Cry9Ga、Cry10Aa、Cry11Aa、Cry11Ba、Cry11Bb、Cry12Aa、Cry13Aa、Cry14Aa、Cry14Ab、Cry15Aa、Cry16Aa、Cry17Aa、Cry18Aa、Cry18Ba、Cry18Ca、Cry19Aa、Cry19Ba、Cry19Ca、Cry20Aa、Cry20Ba、Cry21Aa、Cry21Ba、Cry21Ca、Cry21Da、Cry21Ea、Cry21Fa、Cry21Ga、Cry21Ha、Cry22Aa、Cry22Ab、Cry22Ba、Cry22Bb、Cry23Aa、Cry24Aa、Cry24Ba、Cry24Ca、Cry25Aa、Cry26Aa、Cry27Aa、Cry28Aa、Cry29Aa、Cry29Ba、Cry30Aa、Cry30Ba、Cry30Ca、Cry30Da、Cry30Db、Cry30Ea、Cry30Fa、Cry30Ga、Cry31Aa、Cry31Ab、Cry31Ac、Cry31Ad、Cry32Aa、Cry32Ab、Cry32Ba、Cry32Ca、Cry32Cb、Cry32Da、Cry32Ea、Cry32Eb、Cry32Fa、Cry32Ga、Cry32Ha、Cry32Hb、Cry32Ia、Cry32Ja、Cry32Ka、Cry32La、Cry32Ma、Cry32Mb、Cry32Na、Cry32Oa、Cry32Pa、Cry32Qa、Cry32Ra、Cry32Sa、Cry32Ta、Cry32Ua、Cry33Aa、Cry34Aa、Cry34Ab、Cry34Ac、Cry34Ba、Cry35Aa、Cry35Ab、Cry35Ac、Cry35Ba、Cry36Aa、Cry37Aa、Cry38Aa、Cry39Aa、Cry40Aa、Cry40Ba、Cry40Ca、Cry40Da、Cry41Aa、Cry41Ab、Cry41Ba、Cry42Aa、Cry43Aa、Cry43Ba、Cry43Ca、Cry43Cb、Cry43Cc、Cry44Aa、Cry45Aa、Cry46Aa、Cry46Ab, Cry47Aa, Cry48Aa, Cry48Ab, Cry49Aa, Cry49Ab, Cry50Aa, Cry50Ba, Cry51Aa, Cry52Aa, Cry5 2Ba, Cry53Aa, Cry53Ab, Cry54Aa, Cry54Ab, Cry54Ba, Cry55Aa, Cry56Aa, Cry57Aa, Cry57Ab, Cry58Aa, Cry59Aa, Cry59Ba, Cry60Aa, Cry60Ba, Cry61Aa, Cry62Aa, Cry63Aa, Cry64Aa, Cry65Aa, Cry66Aa, Cry67Aa, Cry68Aa, Cry69Aa, Cry69Ab, Cry70Aa, Cry70Ba, Cry70Bb, Cry71Aa, Cry72Aa, Cry73Aa, or any combination thereof. In some embodiments, the second pest control agent comprises the Cry1Ab protein in the Bt11 event (see U.S. Patent No. US 6,114,608), the Cry3A055 protein in the MIR604 event (see U.S. Patent No. US 8884102), the eCry3.1Ab protein in the 5307 event (see U.S. Patent No. US10428393), and / or the mCry3A protein in the MZI098 event (see U.S. Patent Application No. US20200190533). In some embodiments, the second pest control agent comprises the Bt11 event (see U.S. Patent No. US 6,114,608), the MIR604 event (see U.S. Patent No. US 8884102), the 5307 event (see U.S. Patent No. US 10428393), and / or the MZI098 event (see U.S. Patent Application No. US20200190533).

[0345] In further embodiments, the second pest control agent is one or more Vip3 vegetative insecticidal proteins. Some structural features that identify proteins as Vip3-like proteins include: 1) a size of about 80-88 kDa, which is processed by insect or trypsin proteolysis to an approximately 62-66 kDa toxic core (Lee et al. 2003. Appl. Environ. Microbiol. [Applied Environmental Microbiology] 69: 4648-4657); and 2) a highly conserved N-terminal secretion signal that is not naturally processed during secretion in Bacillus thuringiensis. Non-limiting examples of members of the Vip3 class and their corresponding GenBank accession numbers, U.S. patents, or patent publication numbers are Vip3Aa1 (AAC37036), Vip3Aa2 (AAC37037), Vip3Aa3 (U.S. Patent No. 6,137,033), Vip3Aa4 (AAR81079), Vip3Aa5 (AAR81080), Vip3Aa6 (AAR81081), Vip3Aa7 (AAK95326), Vip3Aa8 (AAK97481), Vip3Aa9 (CAA76665), Vip3Aa10 (AAN60738), Vip3Aa11 (AAR36859), Vip3Aa12 (AAM22456), Vip3Aa13 (AAL69542), Vip3Aa14 (AAQ12340), Vip3Aa15 (AAP51131), Vip3Aa16 (AAW65132), Vip3Aa17 (U.S. Patent No. 6,603,063), Vip3Aa18(AAX49395), Vip3Aa19(DQ241674), Vip3Aa19(DQ539887), Vip3Aa20(DQ5 39888), Vip3Aa21(ABD84410), Vip3Aa22(AAY41427), Vip3Aa23(AAY41428), Vip3Aa24(BI 880913), Vip3Aa25 (EF608501), Vip3Aa26 (EU294496), Vip3Aa27 (EU332167), Vip3Aa28 (FJ494817), Vip3Aa29 (FJ62 6674), Vip3Aa30 (FJ626675), Vip3Aa31 (FJ626676), Vip3Aa32 (FJ626677), Vip3Aa33 (GU073128), Vip3Aa34 (GU07312 9), Vip3Aa35(GU733921), Vip3Aa36(GU951510), Vip3Aa37(HM132041), Vip3Aa38(HM117632), Vip3Aa39(HM117631), Vip3Aa40(HM132042), Vip3Aa41(HM132043), Vip3Aa42(HQ587048), Vip3Aa43(HQ594534), Vip3Aa44(HQ650163), Vip 3Ab1 (AAR40284), Vip3Ab2 (AAY88247), Vip3Ac1 (U.S. Patent Application Publication 20040128716), Vip3Ad1 (U.S. Patent Application Publication 20040128716), Vip3Ad2 (CAI43276), Vip3Ae1 (CAI43277), Vip3Af1 (U.S. Patent No. 7,378,493), Vip3Af2 (ADN08753), Vip3Af3 (HM117634), Vip3Ag1 ( ADN08758), Vip3Ag2 (FJ556803), Vip3Ag3 (HM117633), Vip3Ag4 (HQ414237), Vip3Ag5 (HQ542193), Vip3Ah1 (DQ832323), Vip3Ba1 (AAV70653), Vip3Ba2 (HM117635), Vip3Bb1 (U.S. Pat. No. 7,378,493), Vip3Bb2 (AB030520), and Vip3Bb3 (ADI48120). In an embodiment, the Vip3 protein is Vip3Aa (U.S. Pat. No. 6,137,033), for example, as represented by corn event MIR162 (U.S. Pat. No. 8,232,456; U.S. Patent No. 8,455,720; and U.S. Patent No. 8,618,272). In some embodiments, the second pest control agent comprises event MIR162 (U.S. Patent No. 8,232,456; U.S. Patent No. 8,455,720; and U.S. Patent No. 8,618,272).

[0346] In some embodiments, the second pest control agent comprises any one or more insecticidal proteins or dsRNAs present in any of the following events: Bt11 event (see U.S. Pat. No. 6,114,608), MIR604 event (see U.S. Pat. No. 8,884,102), MIR162 event (see U.S. Pat. No. 8,232,456), 5307 event (see U.S. Pat. No. 10,428,393), MZIR098 event (see U.S. Patent Application No. 20200190533), TC1507 event (see U.S. Pat. No. 7,288,643), DAS-59122-7 event (see U.S. Pat. No. 7,323,556), MON810 event (see U.S. Pat. No. 6,713,259), MON863 event (see U.S. Pat. No. 7,705,216), MON89034 event (see U.S. Pat. No. 8,062,840), MON88017 event (see U.S. Pat. No. 8,062,840), 9556492), DP-4114 event (see U.S. Patent No. US9725772), MON87411 event (see U.S. Patent No. US 9441240), DP-032218-9 event (see U.S. Patent Application No. US2015361447), DP-033121-3 event (see U.S. Patent Application No. US2015361446), DP-023211-2 event (see PCT Publication No. WO 2019209700), MON95379 event (see U.S. Patent Application No. US2020032289), DBN9936 event (see PCT Publication No. WO 2016173361), DBN9501 event (see PCT Publication No. WO 20207125), GH5112E-117C event (see PCT Publication No. WO 17 / 088480), LP007-1 event (see Chinese patent application number CN 112852801), LP007-2 event (see Chinese patent application number CN 112831584), LP007-3 event (see Chinese patent application number CN 112877454), LP007-4 event (see Chinese patent application number CN112831585), LP007-5 event (see Chinese patent application number CN 113151534), LP007-6 event (see Chinese patent application number CN 113151533), LP007-7 event (see Chinese patent application number CN 112852991), LP007-8 event (see CN 113980958), Ruifeng 8,ND207 or Ruifeng125 event (see Chinese patent application number CN105017391), KJ1172 event (see Chinese patent application number CN 1164109774), LD02 event (see Chinese patent application number CN 115820630 A), LD05 event (see Chinese patent application number CN 116287384), LG11 event (see Chinese patent application numbers CN 116200529 A and CN 115725571 A), DBN9235 event (see Chinese patent application number CN 116219063), DBN9508 event (see Chinese patent number CN 109971880), DBN9888 event (see PCT publication number WO 16 / 173540), DBN9978 event (see PCT publication number WO 16 / 173362), DBN9953 event (see Chinese patent number CN 104878092), DBN9927 event (see PCT publication No. WO 16 / 173360), LP026-1 event (see Chinese application No. CN 116144672A), LP026-2 event (see Chinese application No. CN 116144818 A), LP026-3 event (see Chinese application No. CN 116144671 A), LP026-4 event (see Chinese application No. CN 116144817 A), LP026-5 event (see Chinese application No. CN 116200519A), 2A-7 event (see Chinese patent application No. CN 112280743 A), CA09328 event (see Chinese patent application No. CN 112126707 A), ZM8-143 event (see Chinese patent application No. CN 108018286), ZM1-027 event (see Chinese patent application number CN 108018368 A), DP-915635-4 event (see PCT publication number WO 21 / 247204), DP-910521-2 event (see PCT publication number WO 23 / 091888), DAS-01131-3 event (see PCT publication number WO 23 / 091884), MON95275 event (see PCT publication number WO 21 / 216571) and EH913 event (see PCT publication number WO 21 / 087586).

[0347] In embodiments, the second pest control agent can be derived from a source other than Bacillus thuringiensis. For example, the second pest control agent can be an alpha amylase, a peroxidase, a cholesterol oxidase, a potato glycoprotein, a protease, a protease inhibitor, a urease, an alpha-amylase inhibitor, a pore-forming protein, a chitinase, a lectin, an engineered antibody or antibody fragment, a Bacillus cereus insecticidal protein, a Xenorhabdus species (such as X. nematophila or Xenorhabdus bovienii) insecticidal protein, a Photobacterium species (such as P. luminescens or P. asymobiotica) insecticidal protein, a Brevibacillus species (such as B. laterosporous) insecticidal protein, a Lysinibacillus species (Lysinibacillus spp.) (such as L. Sphearicus) insecticidal proteins, Chromobacterium species (such as C. subtsugae or C. piscinae) insecticidal proteins, Yersinia species (such as Yersinia entomophage (Y.entomophaga)) insecticidal proteins, Paenibacillus species (such as P. propylaea) insecticidal proteins, Clostridium species (such as C. bifermentans) insecticidal proteins, Pseudomonas species (Pseudomonas spp.) (such as Pseudomonas fluorescens) insecticidal proteins and lignin. In other embodiments, the second agent can be at least one insecticidal protein derived from an insecticidal toxin complex (Tc) from Photorhabdus, Xenorhabdus, Serratia or Yersinia. In other embodiments, the insecticidal protein can be an ADP-ribosyltransferase derived from insecticidal bacteria such as Photorhabdus species. In other embodiments, the insecticidal protein may be a VIP protein, such as VIP1 and / or VIP2 from Bacillus cereus. In still other embodiments, the insecticidal protein may be a binary toxin derived from insecticidal bacteria, such as ISP1A and ISP2A from Bacillus laterosporus or BinA and BinB from Bacillus sphaericus. In still other embodiments, the insecticidal protein may be engineered or may be a hybrid or chimera of any of the aforementioned insecticidal proteins.

[0348] Other examples of the second pest control agent include DIG-657 (US Patent Publication 2015366211); PtIP-96 (US Patent Publication 2017233440); PIP-72 (US Patent Publication US2016366891); PIP-83 (US Patent Publication 2016347799); PIP-50 (US Patent Publication 2017166921); IPD73 (US Patent Publication 2019119334); IPD090 (US Patent Publication 2019136258); IPD80 (US Patent Publication 2019256563); IPD078, IPD084, IPD086, IPD087, IPD089 (US Patent Publication 2020055906); IPD093 (International Application Publication WO2018111551); IPD059 (International Application Publication WO 2018232072); IPD113 (International Application Publication WO2019178042); IPD121 (International Application Publication WO 2018208882); IPD110 (International Application Publication WO2019178038); IPD103 (International Application Publication WO 2019125717); IPD092; IPD095; IPD097; IPD099; IPD100, IPD105; IPD106; IPD107; IPD111; IPD112 (International Application Publication WO 2020055885); IPD102 (International Application Publication WO2020076958) Cry1B.868 and Cry1Da_7 (U.S. Patent Publication 2020-032289); TIC107 (U.S. Patent 8049071); Cry2Ab and Cry1A.105 (U.S. Patent 10584391); Cry1F, Cry34Ab1, Cry35Ab1 (U.S. Patent 10407688); TIC6757, TIC7472, TIC7473, TIC6757 (U.S. Patent Publication 2017058294); TIC3668, TIC3669, TIC3670, TIC4076, TIC4078, TIC 4260, TIC4346, TIC4826, TIC4861, TIC4862, TIC4863, TIC-3668 (U.S. Patent Publication 2016319302); TIC7040, TIC7042, TIC7381, TIC7382, TIC7383, TIC7386, TIC7388, TIC7389 (U.S. Patent Publication 2018291395); TIC7941 (U.S. Patent Publication 2020229445) TIC836, TIC860, TIC867, TIC868, TIC869, and TIC1100 (International Application Publication WO 2016061391), TIC2160 (International Application Publication WO 2016061392), ET66, TIC400, TIC800, TIC834, TIC1415, AXMI-001, AXMI-002, AXMI-030, AXMI-035, ANDAXMI-045 (US Patent Publication 20130117884), AXMI-52, AXMI-58, AXMI-88, AXMI-97, AXMI-102, AXMI-112, AXMI-117, AXMI-100 (US Patent Publication 201-0310543), AXMI-115, AXMI-113, AXMI-005 (US Patent Publication 20130104259), AXMI-134 (US Patent Publication 20130167264), AXMI-150 (U.S. Patent Publication 20100160231), AXMI-184 (U.S. Patent Publication 20100004176), AXMI-196, AXMI-204, AXMI-207, AXMI-209 (U.S. Patent Publication 2011-0030096), AXMI-218, AXMI-220 (U.S. Patent Publication 20140245491), AXMI-221z, AXMI-222z, AXMI-223z, AXMI-224z, AXMI-225z (U.S. Patent Publication 20140196175), AXMI-238 (US Patent Publication 20140033363), AXMI-270 (US Patent Publication 20140223598), AXMI-345 (US Patent Publication 20140373195), AXMI-335 (International Application Publication WO2013134523), DIG-3 (US Patent Publication 20130219570), DIG-5 (US Patent Publication 20100317569), DIG-11 (US Patent Publication 20100319 093), AfIP-1A (US Patent Publication 20140033361), AfIP-1B (US Patent Publication 20140033361), PIP-1APIP-1B (US Patent Publication 20140007292), PSEEN3174 (US Patent Publication 20140007292), AECFG-592740 (US Patent Publication 20140007292), Pput_1063 (US Patent Publication 20140007292), DIG-657 (International Application Publication WO 2015195594), Pput_1064 (US Patent Publication 20140007292), GS-135 (US Patent Publication 20120233726), GS153 (US Patent Publication 20120192310), GS154 (US Patent Publication 20120192310), GS155 (US Patent Publication 20120192310), DIG-911 and DIG-180 (US Patent Publication No. 20150264940); etc.

[0349] In some embodiments, the second pesticidal agent can be non-protein (e.g., interfering RNA molecules, such as dsRNA), which can be transgenic expressed or applied as part of a composition (e.g., using a local method). Interfering RNA molecules typically include at least one RNA fragment for a target gene, a spacer sequence, and a second RNA fragment complementary to the first RNA fragment, thereby forming a double-stranded RNA structure. When an organism recognizes double-stranded RNA (dsRNA) molecules and hydrolyzes them, RNA interference (RNAi) occurs. The resulting hydrolyzate is a small RNA fragment of about 19-24 nucleotides in length, which is referred to as small interfering RNA (siRNA). These siRNAs then diffuse or are carried throughout the organism, including across the cell membrane, where they hybridize with mRNA (or other RNA) and cause the hydrolysis of RNA. Interfering RNA is recognized by the RNA interference silencing complex (RISC), in which the effector strand (or "guide strand") of RNA is located. This guide strand serves as a recognition and destruction template for duplex sequences. Each time siRNA hybridizes with its complementary RNA target, this process is repeated, effectively preventing those mRNAs from being translated, and thus "silencing" the expression of specific genes transcribed therein. Interfering RNA is known in the art to be used for insect control (see, for example, WO 2013 / 192256, which is incorporated herein by reference). Interfering RNA designed for insect control produces non-natural double-stranded RNA that utilizes the natural RNAi pathway in insects to trigger the downregulation of target genes, which may lead to cessation of feeding and / or growth and may cause death of insect pests. Interfering RNA molecules can confer insect resistance to target pests identical to the disclosed proteins or can target different pests. Target insect plant pests can be ingested by chewing, sucking or piercing. Interfering RNA is known in the art to be used for insect control. In an embodiment, dsRNAs useful for insect control are described in U.S. Patent Publications 20190185526, 2018020028, or 20190177736. In an embodiment, the dsRNA useful for insect control is described in U.S. Pat. Nos. 9,238,8223, 9,340,797, or 8,946,510. In an embodiment, the dsRNA useful for insect control is described in U.S. Patent Publication Nos. 20200172922, 20110054007, 20140275208, 20160230185, or 20160230186. In other embodiments, the interfering RNA can confer resistance to non-insect plant pests such as nematode pests or viral pests.

[0350] In still further embodiments, a first insect control agent (which is a disclosed insecticidal protein) and a second pest control agent are co-expressed in a transgenic plant. Co-expression of more than one pesticidal component in the same transgenic plant can be achieved by genetically engineering the plant to contain and express a nucleic acid sequence encoding an insect control agent. For example, co-expression of more than one pesticidal agent in the same transgenic plant can be achieved by preparing a single recombinant vector (which contains a coding sequence for more than one pesticidal agent in a "molecular stack") and genetically engineering the plant to contain and express all the pesticidal agents in the transgenic plant. Such molecular stacking can also be made using mini-chromosomes, as described in, for example, U.S. Patent 7,235,716. Alternatively, a plant (parent 1) can be genetically engineered for expression of a disclosed insecticidal protein. A second plant (parent 2) can be genetically engineered for expression of a second pest control agent. By hybridizing parent 1 with parent 2, a progeny plant expressing an insect control agent from both parent 1 and parent 2 is obtained.

[0351] In other embodiments, the present disclosure provides stacked transgenic plants resistant to infestation by plant pests, the plants comprising a nucleic acid (e.g., DNA) sequence encoding a dsRNA for suppressing an essential gene in a target pest and a nucleic acid (e.g., DNA) sequence encoding a disclosed insecticidal protein that exhibits insecticidal activity against the target pest.

[0352] Transgenic plants or seeds containing and / or expressing the disclosed proteins may also be treated with insecticides or insecticidal seed coatings, as described in U.S. Pat. Nos. 5,849,320 and 5,876,739. In embodiments, where the insecticides or insecticidal seed coatings disclosed herein and the transgenic plants or seeds are active against the same target insects, such as coleopteran pests (e.g., western corn rootworms), the combination (i) is useful in methods for further enhancing the activity of the compositions disclosed herein against the target insects and / or (ii) in methods for preventing resistance to the compositions disclosed herein by providing yet another mechanism of action against the target insects. Thus, in embodiments, methods for enhancing the control of coleopteran insect populations are provided, the methods comprising providing transgenic plants or seeds disclosed herein and applying the insecticides or insecticidal seed coatings disclosed herein to the plants or seeds.

[0353] Even where an insecticide or insecticidal seed coating is active against different insects, the insecticide or insecticidal seed coating is useful for expanding the spectrum of insect control, for example by adding an insecticide or insecticidal seed coating active against coleopteran insects to transgenic seeds of the disclosure (which in some embodiments are active against Lepidoptera insects), the resulting coated transgenic seeds control both Lepidoptera and Coleopteran insect pests.

[0354] Methods of making and using insecticidal proteins, nucleic acids and transgenic plants

[0355] In addition to providing compositions, the present disclosure also provides methods for producing and using the insecticidal proteins of the present disclosure. In some embodiments, the production method includes culturing a transgenic non-human host cell comprising a polynucleotide, expression cassette or vector expressing the described insecticidal protein under conditions where the host cell produces an insecticidal protein that is toxic to coleopteran pests. In some embodiments, the transgenic non-human host cell is a plant cell. In some other embodiments, the plant cell is a maize cell. In other embodiments, the conditions for plant cell growth include natural sunlight. In other embodiments, the transgenic non-human host cell is a bacterial cell. In still other embodiments, the transgenic non-human host cell is a yeast cell.

[0356] In some embodiments, the methods of the disclosure provide control of at least one coleopteran pest, including but not limited to one or more of the following: Diabrotica barberi (Northern Corn Rootworm), Diabrotica zea (Western Corn Rootworm), Diabrotica eleven-spotted (Southern Corn Rootworm), D. balteata (banded cucumber beetle), D. undecimpunctata undecimpunctata (western spotted cucumber beetle), D. significata (3-spotted leaf beetle), D. speciosa (chrysanthemum beetle), D. speciosa (chrysanthemum spp. beetle), Mexican corn rootworm (Mexican corn rootworm), Beni rootworm (D. beiensis), Christa rootworm (D. cristata), Cowe rootworm (D. curviplustalata), two-spotted rootworm (D. dissimilis), elegant rootworm (D. elegantula), Emorsitans, Graminea rootworm (D. graminea), Hispanolae rootworm (D. hispanolae), Lemini Root beetle (D.lemniscata), ochre-legged root beetle (D.linsleyi), miller's root beetle (D.milleri), coin-shaped root beetle (D.nummularis), fan-leaved root beetle (D.occlusal), pra root beetle (D.porracea), snail root beetle (D.scutellata), shin root beetle (D.tibialis), three-line root beetle (D.trifasciata) and micro-green root beetle (D.viridula); and any combination thereof.Other non-limiting examples of coleopteran insect pests include Chrysomela spp., such as Leptinotarsa ​​decemlineata (Colorado potato beetle); Chrysomela spp., such as C. scripta (cottonwood leaf beetle); Hypothenemus spp., such as H. hampei (coffee berry borer); Sitophilus spp., such as S. zeamais (maize weevil); Epitrix spp., such as E. hirtipennis (tobacco flea beetle). (tobacco flea beetle) and E. cucumeris (potato flea beetle); Phyllotreta spp., such as P. cruciferae (crucifer flea beetle) and P. pusilla (western black flea beetle); Anthonomus spp., such as A. Eugenii (pepper weevil); Hemicrepidus spp., such as H. memnonius (wireworms); Melanotus spp., such as M. communis (wireworms); Ceutorhychus spp. spp., such as C. assimilis (cabbage seedpod weevil); Aeolus spp., such as A. mellillus (wireworm); Aeolus spp., such as A. mancus (wheat wireworm); Horistonotus spp., such as H. uhlerii (sand wireworm); Sphenophorus spp., such as S. maidis (maize billbug), S. zeae (timothy billbug), S. parvulus (bluegrass billbug). billbug) and S. callosus (southern cornbillbug); Phyllophaga spp. (white grub); Chaetocnema spp., such as C. pulicaria (corn flea beetle); Popillia spp., such as P. japonensis (Japanese popillia).japonica) (Japanese beetle); Epilaxna spp., such as E. varivestis (Mexican bean beetle); Cerotoma spp., such as C. trifurcate, Bean leaf beetle; Epicauta spp., such as E. pestifera and E. lemniscata (Blister beetles); and any combination of the foregoing. In some embodiments, the insecticidal protein has insecticidal activity against a western corn rootworm colony that is resistant to one or more insecticidal proteins present in a commercial product (e.g., mCry3a, eCry3.1Ab, Cry3Bb1, Cry34 / 35, IPD072Aa, or IPD079Ea). .

[0357] Also contemplated are methods of producing transgenic plants that are resistant to insects (e.g., to coleopteran insects). In some embodiments, the method comprises: introducing into a plant a polynucleotide, expression cassette, or vector comprising a nucleotide sequence encoding a disclosed insecticidal protein, wherein the nucleotide sequence is expressed in the plant to produce the disclosed insecticidal protein, thereby conferring resistance to insect pests on the plant, and producing an insect-resistant transgenic plant (e.g., as compared to a suitable control plant, such as a plant that does not comprise the disclosed polynucleotide, expression cassette, or vector and / or does not express the disclosed insecticidal polypeptide).

[0358] In some embodiments, the pest-resistant transgenic plant is resistant to an insect pest selected from the group consisting of: corn rootworm (Western corn rootworm; WCR), barnett's rootworm (Northern corn rootworm; NCR) and / or eleven-spotted rootworm (Southern corn rootworm; SCR) and / or other rootworm species including Mexican corn rootworm (Mexican corn rootworm).

[0359] In embodiments, methods of introducing a disclosed polynucleotide, expression cassette or vector into a plant comprise first transforming a plant cell with the polynucleotide, expression cassette or vector and regenerating a transgenic plant therefrom, wherein the transgenic plant comprises the polynucleotide, expression cassette or vector and expresses the disclosed chimeric insecticidal protein of the present disclosure.

[0360] Alternatively or additionally, the introducing step can include crossing a first plant comprising the polynucleotide, expression cassette or vector with a second plant (e.g., a plant different from the first plant, such as a plant that does not comprise the polynucleotide, expression cassette or vector), and optionally, producing a progeny plant comprising the polynucleotide, expression cassette or vector and expressing the disclosed insecticidal protein, thereby resulting in increased resistance to at least one insect pest. Thus, transgenic plants encompass plants and their progeny (of any generation) that are the direct result of a transformation event, the plants and their progeny comprising the polynucleotide, expression cassette or vector and optionally expressing the insecticidal protein, thereby resulting in increased resistance to at least one insect pest. Once the desired nucleic acid molecule has been transformed into a particular plant species, it can be propagated in that species or transferred to other varieties of the same species, particularly including commercial varieties, using conventional breeding techniques.

[0361] The present disclosure further provides a method for identifying the transgenic plant of the present disclosure, which method comprises detecting the presence of the polynucleotide, expression cassette, vector or insecticidal protein of the present disclosure in a plant (or a plant cell, plant part, etc. derived therefrom), thereby identifying the plant as a transgenic plant of the present disclosure based on the presence of the polynucleotide, expression cassette, vector or insecticidal protein of the present disclosure.

[0362] Embodiments further provide methods of producing a transgenic plant with increased resistance to at least one insect pest (e.g., at least one lepidopteran pest), the method comprising: planting a seed comprising a polynucleotide, expression cassette, or vector of the disclosure, and growing a transgenic plant from the seed, wherein the transgenic plant comprises the polynucleotide, expression cassette, or vector and produces an insecticidal protein.

[0363] In embodiments, transgenic plants produced by the methods of the present disclosure comprise polynucleotides, expression cassettes or vectors of the present disclosure. In embodiments, transgenic plants produced by the methods of the present disclosure comprise insecticidal proteins of the present disclosure, and optionally have increased resistance to at least one insect pest.

[0364] The method of producing a transgenic plant described herein optionally comprises a further step of harvesting seeds from the transgenic plant, wherein the seeds comprise the polynucleotide, expression cassette or vector and produce an insecticidal protein. Optionally, the seeds produce another transgenic plant comprising the polynucleotide, expression cassette or vector and producing an insecticidal protein, thereby having increased resistance to at least one insect pest.

[0365] The disclosure further provides plant parts, plant cells, plant organs, plant cultures, seeds, plant extracts, harvested products, and processed products of transgenic plants produced by the methods of the disclosure.

[0366] As an additional aspect, the disclosure also provides a method for producing seeds, the method comprising: providing a transgenic plant comprising a disclosed polynucleotide, expression cassette or vector, and harvesting seeds from the transgenic plant, wherein the seeds comprise the polynucleotide, expression cassette, vector and produce an insecticidal protein. Optionally, the seeds produce another transgenic plant, the transgenic plant comprising the polynucleotide, expression cassette or vector and producing an insecticidal protein, thereby having increased resistance to at least one insect pest. In a representative embodiment, the step of providing the transgenic plant comprises planting the seeds that produce the transgenic plant.

[0367] Further provided is a method of producing hybrid plant seeds, the method comprising: hybridizing a first inbred plant (which is a transgenic plant comprising a polynucleotide, expression cassette or vector of the present disclosure and optionally expressing an insecticidal protein of the present disclosure) with a different inbred plant (e.g., an inbred plant that does not comprise a polynucleotide, expression cassette or vector of the present disclosure), and allowing hybrid seeds to form. Optionally, the method further comprises harvesting the hybrid seeds. In embodiments, the hybrid seeds comprise a polynucleotide, expression cassette or vector of the present disclosure, and in embodiments may further comprise an insecticidal protein of the present disclosure and have increased resistance to insect pests. In embodiments, the hybrid seeds produce transgenic plants comprising a polynucleotide, expression cassette or vector of the present disclosure, expressing an insecticidal protein of the present disclosure, and having increased resistance to at least one insect pest.

[0368] In a further embodiment, a method of controlling a coleopteran pest is provided, the method comprising delivering an effective amount of a disclosed insecticidal protein to the pest or an environment containing such a pest. To be effective, the insecticidal protein is first orally ingested by the insect. However, the insecticidal protein can be delivered to the insect in many ways. The way of delivering the protein orally to the insect includes, but is not limited to (1) providing the protein in a transgenic plant, wherein the insect feeds (ingests) one or more parts of the transgenic plant, thereby ingesting the polypeptide expressed in the transgenic plant; (2) providing the protein in one or more formulated protein compositions that can be applied to or incorporated into, for example, an insect growth medium; (3) providing the protein in one or more protein compositions that can be applied to the surface of a plant part (e.g., sprayed onto the surface of a plant part), and then ingested by the insect when the insect feeds on one or more sprayed plant parts; (4) bait matrix; or (5) any other protein delivery system recognized in the art. Therefore, any method of oral delivery to insects can be used to deliver the disclosed insecticidal protein of the present disclosure. In some specific embodiments, the disclosed insecticidal protein is orally delivered to the insect, wherein the insect ingests one or more parts of the transgenic plant.

[0369] In other embodiments, the disclosed insecticidal proteins are delivered orally to insects, wherein the insects ingest one or more parts of the plant covered or partially covered by the composition comprising the insecticidal protein. Any method known to those skilled in the art for applying compounds, compositions, formulations, etc. to plant surfaces can be used to deliver the composition of the present disclosure to the plant surface. Some non-limiting examples of delivery to or contacting plants or parts thereof include spraying, dusting, sprinkling, dispersing, misting, atomizing, broadcasting, soaking, soil injection, soil incorporation, saturation (e.g., root, soil treatment), impregnation, pouring, coating, leaf or stem infiltration, side application or seed treatment, etc. and combinations thereof. These and other procedures for contacting plants or parts thereof with one or more compounds, one or more compositions, or one or more formulations are well known to those skilled in the art.

[0370] In some embodiments, the disclosed nucleotide and polypeptide sequences can be used for bioinformatics analysis to identify additional insecticidal toxins (both nucleotide sequences and proteins encoded by nucleic acids). In embodiments, such identification of additional toxins can be based on percentage identity (e.g., using BLAST or similar algorithms). In other embodiments, the identification of additional toxins can be accomplished using conserved protein domains or epitopes (e.g., Hmmer, psi-BLAST, or hhsuite). In some embodiments, bioinformatics assays include performing sequence identity comparisons and selecting one or more candidate insecticidal toxins, which have sequence identity above a specific threshold (e.g., at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or higher identity) relative to the disclosed nucleotide or polypeptide sequences of the present disclosure. In some embodiments, bioinformatics assays include performing domain or epitope conservation analysis and selecting one or more candidate insecticidal toxins, which have at least one conserved domain or epitope relative to the disclosed nucleotide or polypeptide sequences of the present disclosure.

[0371] In an embodiment, the determination of the insecticidal activity of the disclosed insecticidal protein can be accomplished by insect bioassays. Insect bioassay methods are well known in the art and can be performed "in vitro" or "in plant body". In an in vitro bioassay, after being produced in a cell-based system (e.g., a recombinant bacterial strain, e.g., Escherichia coli, Bacillus thuringiensis Cry-), the disclosed protein is delivered to the desired insect species. The clarified lysate containing the disclosed protein produced in these recombinant bacterial strains can be orally fed to insects. Alternatively, purified protein can be prepared and orally fed to insects. In some embodiments, before insect infestation, the clarified lysate or purified protein is covered on an artificial feed. In other embodiments, before insect infestation, the clarified lysate or purified protein is mixed or incorporated into an artificial feed. In a bioassay in plant body, transgenic plants expressing the disclosed protein are used to deliver toxins to the desired insect species. In an embodiment, the sampled tissue is orally fed to insects. Non-limiting examples of sampled tissues include leaves, roots, pollen, silk, and stems. In some embodiments, plant tissue is mixed or incorporated into the artificial diet prior to insect infestation. In embodiments, the insects evaluated are L1 instar insects or larvae. In other embodiments, the insects evaluated are late larval stages, i.e., L2, L3, L4, or L5 instar insects.

[0372] Examples

[0373] Embodiments of the present invention can be better understood by reference to the following detailed examples. The foregoing and following embodiments of the present invention and the description of various embodiments are not intended to limit the claims, but are illustrative thereof. Therefore, it should be understood that the claims are not intended to be limited to the specific details of these examples. It should be understood by those skilled in the art that other embodiments of the present invention can be practiced without departing from the spirit and scope of the present disclosure, and the scope of the present disclosure is defined by the appended claims. Recombinant DNA and molecular cloning techniques recognized in the art can be found in the following references: for example, J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 4th ed., Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press (2012); TJ Silhavy, ML Berman and LW Enquist, Experiments with Gene Fusions, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1984) and Ausubel, FM et al., Current Protocols in Molecular Biology, New York, John Wiley and Sons Inc., (1988), Reiter et al., Methods in Arabidopsis Research, World Scientific Press (1992), and Schultz et al., Plant Molecular Biology Manual, Kluwer Academic Press (1996). Publishers[Kluwer Academic Publishers](1998).

[0374] Example 1: Identification of proteins with insecticidal activity against western corn rootworm

[0375] The insecticidal protein SamsoniaCRW was identified from the bacterium Samsonia erythrinae (accession number WP_132452716). Other related insecticidal proteins MGYP000214679871, MGYP000317625865 and MGYP000301824029 were identified from the metagenomic sequencing database. The identified proteins are orthologs and have a percentage of identity with each other, ranging from about 17%-70% identity (Table 1, percentage of identity calculated using Blosum62). The genes of the optimized version of Escherichia coli were synthesized and cloned into the pET29a vector. The resulting construct was transformed into Escherichia coli BL21*(DE3) and protein expression was performed overnight in Luria-Bertani broth at 18°C ​​with IPTG induction. The soluble fraction of lysate was prepared from these cultures by using a French pressure cell followed by centrifugation of the entire lysate at 20,000 xg for thirty minutes. The supernatant (soluble fraction) was then tested for bioactivity against western corn rootworm (WCR; Diabrotica zeae).

[0376] Table 1: Percent identity of identified bacterial orthologs (peptides)

[0377]

[0378] Use the dietary incorporation method to carry out bioactivity assay.In brief, the artificial insect diet (Bioserv company, Frenchtown, New Jersey (NJ)) of Escherichia coli BL21* (DE3) lysate and isovolumetric heating is mixed in 1.5mL centrifuge tubes and then applied to a small petri dish.After the meal-sample mixture is cooled and solidified, 12 WCR larvae are added to each plate.The plate is sealed and maintained under environmental laboratory conditions with respect to temperature, illumination and relative humidity.The lysate from the Escherichia coli BL21* (DE3) culture carrying an empty pET29a carrier is used as a negative control.Mortality was assessed on the 3rd day or the 4th day and the 6th day.For this form and all subsequent forms, "-" means no death, "+" means the mortality rate of 1%-24%, "++" means the mortality rate of 25%-49%, "+++" means the mortality rate of 50%-74%, and "++++" means the mortality rate of 75%-100%. For this and all subsequent tables showing insecticidal activity against CRW, the abbreviations in the "Remarks" column are as follows: s = small larvae, sm = small / medium larvae, m = medium larvae, mb = medium / large larvae, b = large larvae, vb = very large larvae. For this and all subsequent tables showing insecticidal activity of the identified proteins or their variants, "SEQ ID NO." refers to the amino acid sequence of the protein. As shown in Table 2, lysates from cultures expressing proteins identified from SamsoniaCRW showed strong bioactivity against WCR. SamsoniaCRW was also tested against Fall Armyworm (Spodoptera frugiperda) and had little insecticidal effect against this pest, indicating that the insecticidal effect against CRW is specific, which is a desirable effect.

[0379] Lysates from cultures expressing the identified orthologous proteins also showed biological activity against WCR as shown in Tables 3 and 4. Confirmatory bioassays using additional lysates expressing these proteins were performed and produced similar results.

[0380] Table 2: Insecticidal activity of Samsonia CRW against western corn rootworm (WCR) at various lysate dilutions

[0381]

[0382] Table 3: Insecticidal activity of lysates of MgNify orthologs against WCR

[0383]

[0384] Table 4: Insecticidal activity of lysates of MgNify orthologs against WCR

[0385]

[0386] Example 2: Samsonia CRW with insecticidal activity against northern corn rootworm

[0387] The efficacy of SamsoniaCRW lysate against northern corn rootworm (NCR) larvae was also tested in a dietary incorporation assay performed essentially as described in Example 1. Lysate from a culture of E. coli BL21*(DE3) carrying an empty pET29a vector was used as a negative control. As shown in Table 5, SamsoniaCRW exhibited insecticidal activity against NCR.

[0388] Table 5: Insecticidal activity of Samsonia CRW against northern corn rootworm (NCR) at various lysate dilutions

[0389]

[0390] Example 3: Purified SamsoniaCRW protein is insecticidal against WCR

[0391] The SamsoniaCRW protein was purified to further characterize its insecticidal properties. An N-terminal hexahistidine-tagged construct was generated for SamsoniaCRW for protein production. One liter of E. coli BL21*(DE3) cells carrying pET29a-6His-SamsoniaCRW were grown in LB medium at 37°C. When the OD reached 0.8-1.0, IPTG (1 mM) was added to the culture, and the culture was then moved to 18°C ​​for 18 hours. The cell pellet was harvested and resuspended in 20 mM Tris (pH 8.0) (buffer A) containing 10% glycerol, 0.5 M NaCl and 5 mM β-mercaptoethanol. The cells were lysed using a French cell press; the lysate was then spun at 100 k xg in an ultracentrifuge.

[0392] After centrifugation, the supernatant of pET29a-6His-SamsoniaCRW was filtered and then applied to a 5mL HisTrap FPLC column balanced in buffer A. The His-tagged protein was eluted from the column using a linear gradient of buffer B (buffer B is buffer A containing 0.25M imidazole). The purity of the fractions was analyzed by SDS-PAGE. The purest fractions were combined and then dialyzed into 1×PBS. The protein was then concentrated and then stored at -80°C. The protein concentration was determined by the BCA protein assay. A series of concentrations of pure protein were then tested for 12 WCR larvae using a dietary incorporation method essentially as described in Example 1. As shown in Table 6, the protein showed concentration-dependent biological activity against WCR within the concentration range tested.

[0393] Table 6: Dose response of purified SamsoniaCRW protein against WCR

[0394]

[0395] Example 4: SamsoniaCRW variants with insecticidal activity against WCR

[0396] Mutations were introduced into SamsoniaCRW and the variants were assayed for protein stability and insecticidal activity. Insecticidal activity was determined using a dietary incorporation assay essentially as described in Example 1, using 12 WCR larvae per experimental assay. The results are shown in Tables 7-9. The SEQ ID NOs correspond to the amino acid sequences of the variants. Most of the variants tested showed insecticidal activity against WCR.

[0397] Table 7: Insecticidal activity of lysates of SamsoniaCRW variants against WCR

[0398]

[0399]

[0400] Table 8: Insecticidal activity of lysates of SamsoniaCRW variants against WCR

[0401]

[0402] Table 9: Insecticidal activity of purified SamsoniaCRW triple mutants against WCR

[0403]

[0404] Example 5: Simulated gastric fluid test on Samsonia CRW variant E. coli lysate preparation

[0405] The bacterial lysate in 50 mM potassium phosphate (pH 7.0), 50 mM sodium chloride was diluted to 3 mg / mL (total protein concentration) for digestibility analysis. The digestion reaction was initiated at 37°C by adding 15 μL of lysate to 285 μL of simulated gastric fluid [10 units of pepsin / μg protein, or approximately 1579 units of pepsin / mL in G-Con solution (2 mg / mL sodium chloride, pH 1.2)]. At 5 or 10 minutes, 100 μL of the lysate-SGF reaction solution was removed and the reaction was terminated by adding it to 100 μL of preheated (95°C) stop solution, which consisted of 65% Tricine loading buffer (Bio-rad 2x Tricine loading buffer w / 10% β-mercaptoethanol) and 35% 500 mM sodium bicarbonate (pH 11.0). Zero time point (T0) was generated by adding 5 μL of test lysate to 100 μL stop solution and 95 μL simulated gastric fluid preheated (95°C). All samples were heated at 95°C for 5 minutes and then stored on ice until SDS-PAGE analysis. Before standard protein gel electrophoresis, 30 microliters of each reaction solution were loaded onto a 10%-20% Tris-tricine peptide gel. Immediately after electrophoresis, the Tris-tricine gel was fixed for 20 minutes with a 40% methanol: 10% acetic acid mixture. The gel was then stained with GelCode blue protein stain at room temperature for 1 hour. After 1 hour, the polyacrylamide gel was decolorized with distilled water for at least 12 hours. The results are qualitatively shown in the "SGF-T5 pepsin digestibility" column. "No" (N) means that the complete SamsoniaCRW protein variant can be detected by GelCode Blue protein staining after gel electrophoresis, which indicates that the protein cannot be completely digested in the SGF assay. "Yes" (Y) indicates that the intact SamsoniaCRW protein variant was not detectable, indicating that the SamsoniaCRW protein variant was digestible in the SGF assay.

[0406] Table 10: SGF digestibility of Samsonia CRW variants

[0407]

[0408]

[0409] Example 6: Example 3: Purified SamsoniaCRWI52L / I80L protein is insecticidal against WCR

[0410] The bacterial lysate double mutant SamsoniaCRW I52L / I80L (SEQ ID NO: 20) was identified as having improved digestibility in the SGF assay and retained its insecticidal activity against WCR in the dietary incorporation bioassay. SamsoniaCRW I52L / I80L was purified to further characterize its insecticidal properties. Purification was performed essentially as described in Example 3 and then performed in the insect bioassay. A range of concentrations of the pure protein were then tested against 12 WCR larvae using a dietary incorporation method essentially as described in Example 1. As shown in Table 11, the protein showed concentration-dependent bioactivity against WCR over the concentration range tested.

[0411] Table 11: Dose response of purified SamsoniaCRW I52L / I80L protein against WCR

[0412]

[0413]

[0414] Example 7: Maize transformation

[0415] Transformation of immature maize embryos was performed essentially as described in Negrotto et al. (Plant Cell Reports (2000) 19:798-803). Briefly, Agrobacterium strain LBA4404 (pSB1) containing an expression vector expressing the insecticidal protein disclosed in Example 1 was grown on YEP (yeast extract (5 g / L), peptone (10 g / L), NaCl (5 g / L), 15 g / l agar, pH 6.8) solid medium at 28°C for 2-4 days. Approximately 0.8×10 9 Agrobacterium cells were suspended in LS-inf medium supplemented with 100 μM As. The bacteria were pre-induced in this medium for about 30-60 minutes.

[0416] The immature embryos from inbred maize lines are excised from the ear of 8-12 days age in liquid LS-inf+100 μM As. The embryos are rinsed once with fresh infection culture medium. Then add Agrobacterium solution, and these embryos are vortexed for 30 seconds and allowed to settle for 5 minutes with bacteria. Then these embryo scutels are transferred upwards to the LSA culture medium, and cultivated two to three days in the dark. Subsequently, embryos between about 20 and 25 of every petri plate are transferred to the LSDc culture medium supplemented with cefotaxime (250mg / l) and silver nitrate (1.6mg / l), and cultivated 10 days in the dark at about 28 ℃.

[0417] The immature embryos that produce embryonic callus are transferred to the LSD1M0.5S culture medium. On this culture medium, the selection that continues about 6 weeks is carried out, and the subculture step is carried out in about 3 weeks. The callus that is surviving is transferred to the Reg1 culture medium that is supplemented with mannose. After cultivating (16 hours of illumination / 8 hours of dark scheme) in illumination, then the green tissue is transferred to the Reg2 culture medium that does not have growth regulator, and hatches about 1-2 weeks. Plantlet is transferred to the Magenta GA-7 box (Magenta Corp, Chicago Ill., Illinois) containing Rcg3 culture medium, and they are grown in illumination. After about 2-3 weeks, the selective marker gene of plant and the existence of disclosed insecticide gene are tested by PCR. The positive plant that measures from PCR is transferred to greenhouse for further evaluation.

[0418] Example 8: Expression activity of SamsoniaCRW and orthologous proteins in maize plants

[0419] Leaf and root tissue samples from each event were tested for the presence of proteins disclosed in Examples 1-6 by ELISA (ng / mg total soluble protein (TSP)) or mass spectrometry. Events expressing the disclosed proteins provided protection against western corn rootworm in whole plant bioassays.

[0420] Example 9: Alanine scanning site-directed mutagenesis of Samsonia CRW

[0421] Mutations were introduced along the entire length of SamsoniaCRW using alanine scanning site-directed mutagenesis to identify residues that may be associated with receptor binding and insecticidal activity. Approximately 50 variants were produced and expressed in bacteria using the method of Example 1. The bacteria were lysed, and the insecticidal activity of bacterial lysates containing mutant variants or purified proteins was determined using a dietary incorporation assay essentially as described in Example 1. The bioassay results of the alanine scanning mutants are shown in Tables 12-17. As shown in the table, changing certain residues in the protein sequence has a negative impact on the CRW biological activity of SamsoniaCRW. These residues include W16A, F36A, W105A, F122A, L127A, Y131A and S139A.

[0422] Table 12: Insecticidal activity of lysates of SamsoniaCRW alanine scanning variants against WCR

[0423]

[0424] Table 13: Insecticidal activity of lysates of SamsoniaCRW alanine scanning variants against WCR

[0425]

[0426] Table 14: Insecticidal activity of lysates of SamsoniaCRW alanine scanning variants against WCR

[0427]

[0428] Table 15: Insecticidal activity of lysates of SamsoniaCRW alanine scanning variants against WCR

[0429]

[0430] Table 16: Insecticidal activity of lysates of SamsoniaCRW alanine scanning variants against WCR

[0431]

[0432] Table 17: Insecticidal activity of lysates of SamsoniaCRW alanine scanning variants against WCR

[0433]

Claims

1. A polypeptide comprising an amino acid sequence having at least 85% identity to SEQ ID NO:

1.

2. The polypeptide of claim 1, wherein the amino acid sequence is at least 90% identical to SEQ ID NO:

1.

3. The polypeptide of claim 1, wherein the amino acid sequence is at least 99% identical to SEQ ID NO:

1.

4. The method of claim 1, wherein the polypeptide is contained in One or more substitution mutations at one or more positions corresponding to I52, S53, I67, I72, I80, V83, A85, I95, I123, S125, I153, I175, I207, I237, D7, T9, T11, E27, S28, S29, M30, K31, T32, H33, R34, L35, V37, L38, S39, V83, G89, S94, G97, S99, Q100, G104, N106, P107, D111, E113, S114, N115, G118, I123, V129, T135, T140, V142, H143, N148, E164, T199 or V241 of NO:

1.

5. The polypeptide of claim 4, wherein the amino acid sequence comprises any one of SEQ ID NO: 1, 5-23 or 71-117.

6. A polypeptide comprising an amino acid sequence at least 90% identical to any one of SEQ ID NOs: 24 to 26.

7. The polypeptide of claim 6, wherein the amino acid sequence comprises any one of SEQ ID NOs: 24 to 26.

8. A nucleic acid molecule comprising a coding sequence encoding the polypeptide according to any one of claims 1 to 7.

9. The nucleic acid molecule of claim 8, wherein the coding sequence comprises a nucleotide sequence that is at least 80% identical to any one of SEQ ID NOs: 2-4, 27-45, 49-67, or 118-164 or comprises any one of SEQ ID NOs: 2-4, 27-45, 49-67, or 118-164.

10. The nucleic acid molecule of claim 8, wherein the coding sequence comprises a nucleotide sequence that is at least 80% identical to any one of SEQ ID NOs: 4 or 49-67 or comprises any one of SEQ ID NOs: 4 or 49-67.

11. The nucleic acid molecule of any one of claims 8 to 10, wherein the coding sequence is codon optimized for expression in plants.

12. The nucleic acid molecule of any one of claims 8 to 11, wherein the coding sequence is operably linked to a heterologous promoter.

13. The nucleic acid molecule of claim 12, wherein the heterologous promoter is a plant-expressible promoter.

14. A vector comprising the nucleic acid according to any one of claims 8 to 13.

15. A transgenic host cell comprising the polypeptide of any one of claims 1 to 7, or the nucleic acid of any one of claims 8 to 13.

16. The transgenic host cell of claim 15, wherein the transgenic host cell is a plant cell.

17. The transgenic host cell of claim 16, wherein the plant cell is a monocotyledonous plant cell.

18. The transgenic host cell of claim 15, wherein the plant cell is a maize cell.

19. The transgenic host cell of claim 18, wherein the transgenic host cell is a bacterial cell.

20. The transgenic host cell of claim 19, wherein the bacterial cell is an Agrobacterium, Bacillus or Escherichia coli cell.

21. A composition comprising the polypeptide of any one of claims 1 to 7.

22. The composition of claim 21, further comprising an agriculturally acceptable carrier.

23. A plant comprising the polypeptide of any one of claims 1 to 7, or the nucleic acid molecule of any one of claims 8 to 13.

24. The plant of claim 23, wherein the plant is a monocot.

25. The plant of claim 24, wherein the plant is a maize plant.

26. A seed of the plant as claimed in any one of claims 23 to 25.

27. A cell of the plant of any one of claims 23 to 25.

28. A method for producing a transgenic plant, the method comprising: a. introducing a nucleic acid molecule as claimed in any one of claims 8 to 13 into a plant cell; b. selecting a plant cell comprising the nucleic acid molecule; and c. Regenerating plants from the selected plant cells.

29. A method for producing a transgenic plant having enhanced insecticidal properties, the method comprising the steps of: a. sexually crossing a first parent plant with a second parent plant, wherein the first or second parent plant is a plant as described in any one of claims 23 to 25; and b. selecting a first generation progeny plant having enhanced insecticidal properties, wherein the selected progeny plant comprises the nucleic acid molecule.

30. The method of claim 29, further comprising the steps of: a. selfing said first generation progeny plants to produce a plurality of second generation progeny plants; and b. selecting plants having enhanced insecticidal properties from said second generation progeny plants, wherein said selected second generation progeny plants comprise said nucleic acid molecule.

31. A method of controlling a coleopteran pest, the method comprising delivering to the pest or its environment a polypeptide as claimed in any one of claims 1 to 7.

32. The method of claim 31, wherein the polypeptide is delivered by ingestion.

33. The method of claim 32, wherein said feeding comprises said pest feeding on a plant part comprising said polypeptide.

34. Use of the sequence of any one of SEQ ID NOs: 1 to 164 in bioinformatics analysis to identify insecticidal proteins.

35. Use of a polypeptide comprising the amino acid sequence of any one of SEQ ID NOs: 1, 5-26 or 71-117 for identifying insecticidal proteins in insect bioassays.

36. A polypeptide comprising an amino acid sequence at least 90% identical to SEQ ID NO: 1 and being toxic to western corn rootworm.

37. A polypeptide comprising an amino acid sequence having at least 90% identity to SEQ ID NO: 1 and being toxic to western corn rootworm, wherein the polypeptide is derived from the bacterium Samsonia.

38. A polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 or differing from the amino acid sequence of SEQ ID NO: 1 by conservative substitutions, deletions and / or additions of 1-5 amino acids, wherein the polypeptide is toxic to western corn rootworm.

39. A polypeptide consisting of the amino acid sequence of any one of SEQ ID NO: 1, 5-26, or 71-117.

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