Insecticidal proteins and their use in controlling pests

Abstract

Disclosed are a pesticidal protein and its use in controlling pests. The present application provides the amino acid sequence of the pesticidal protein and the nucleotide sequence encoding the protein, and the pesticidal protein exhibits excellent pesticidal activity against pests such as Thysanoptera, Lepidoptera or Hemiptera.

Description

Technical Field

[0001] This invention relates to the field of molecular biology, specifically providing an insecticidal protein and disclosing its application in pest control. These insecticidal proteins and the nucleic acid sequences encoding them can be used to produce transgenic insect-resistant plants to control pests such as Thysanoptera, Lepidoptera, or Hemiptera. Background Technology

[0002] Agricultural pests are the number one major factor affecting crop production. With the rapid development of transgenic technology, the creation of insect-resistant plants by transforming the Bt (Bacillus thuringiensis) insecticidal protein gene has revolutionized modern agriculture, increasing the importance and value of insecticidal proteins and their genes, and providing farmers with an environmentally friendly alternative to traditional chemical pesticide control methods. Although they have proven to be very commercially successful, these genetically engineered insect-resistant plants are only effective against certain specific insects due to the high selectivity of their insecticidal proteins.

[0003] There are many types of harmful insects, and currently available genetically modified insect-resistant genes mainly target certain species in the Lepidoptera and Coleoptera orders. Many other harmful insect species still lack effective insect-resistant proteins / genes. Therefore, it remains necessary to develop insect-resistant proteins or genes for these pests. Invention Summary

[0004] To develop novel insecticidal proteins with enhanced insecticidal activity, diverse activity spectra, and / or modes of action, such as insecticidal proteins active against various insects in the orders Thysanoptera, Lepidoptera, and Hemiptera, including but not limited to insect pests resistant to existing insecticides, this invention provides the following technical solutions:

[0005] This invention discloses an insecticidal protein (referred to herein as a KIR-associated insecticidal protein), comprising an amino acid sequence having 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%, or at least 100% sequence identity with SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, or SEQ ID NO:24.

[0006] The present invention also provides a polynucleotide comprising a nucleotide sequence selected from the following:

[0007] (a) The nucleotide sequence encoding the above-mentioned insecticidal protein or its complementary sequence;

[0008] (b) The nucleotide sequences shown in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25 or SEQ ID NO:26, or their complementary sequences;

[0009] (c) A nucleotide sequence that hybridizes to the sequence shown in (a) or (b) under stringent conditions; and / or

[0010] (d) A nucleotide sequence that encodes the same amino acid sequence as the sequence shown in (a) or (b) due to the degeneracy of the genetic code, or a complementary sequence thereof.

[0011] The present invention also provides the use of the insecticidal protein or the polynucleotide described herein in the control of pests.

[0012] The present invention also provides an expression cassette comprising the polynucleotide under the regulation of an effectively linked regulatory sequence.

[0013] The present invention also provides a plant transformation vector comprising the aforementioned polynucleotide or the aforementioned expression cassette.

[0014] The present invention also provides a composition for controlling pest infestation, comprising one or more of the insecticidal proteins described above and at least one suitable carrier, excipient or diluent.

[0015] Furthermore, the composition also contains a second insecticidal protein different from the insecticidal protein described above.

[0016] The present invention also provides a method for controlling pest infestation, the method comprising delivering an effective amount of the insecticidal protein or the composition to the pest's growth environment and contacting the pest, causing the pest to die and / or inhibiting its growth.

[0017] The present invention also provides a method for enhancing plant resistance to pests, the method comprising producing an insecticidally effective amount of the insecticidal protein in the plant, or expressing the polynucleotide, the expression cassette, or the plant transformation vector in the plant to produce an insecticidally effective amount of the insecticidal protein, contacting the plant with pests, wherein the plant is unaffected by and / or has reduced damage from the pests.

[0018] The present invention also provides a method for producing offspring seeds resistant to pests, the method comprising:

[0019] a. Planting a first seed containing the polynucleotide or capable of producing the insecticidal protein;

[0020] b. A plant grows from the seed described in step a; and

[0021] c. Harvest the seeds of the offspring from the plant, wherein the harvested seeds contain the polynucleotide or are capable of producing the insecticidal protein.

[0022] The present invention also provides a commercial product derived from a plant or a part thereof, wherein the commercial product comprises the insecticidal protein or the polynucleotide.

[0023] The present invention also provides a method for detecting the presence of the polynucleotide in a plant genomic DNA sample, the method comprising:

[0024] a. Contact the sample with a specific nucleotide probe, wherein the nucleotide probe is sequence homologous to or complementary to the polynucleotide;

[0025] b. subjecting the sample and the nucleotide probe to stringent hybridization conditions; and

[0026] c. Detect the hybridization of the nucleotide probe with the polynucleotide;

[0027] The detection of the hybridization confirmed the presence of the polynucleotide in the plant genomic DNA sample.

[0028] Furthermore, the specific nucleotide probe hybridizes with plant genomic DNA containing the said polynucleotide under strict hybridization conditions, and does not hybridize with other plant genomic DNA not containing the said polynucleotide under the same hybridization conditions.

[0029] The present invention also provides a method for detecting the presence of the insecticidal protein in a commercial product, the method comprising:

[0030] a. Contacting the product with an immunoreactive antibody, said immunoreactive antibody being specific for binding to the insecticidal protein or a fragment thereof; and

[0031] b. Detect the binding of the antibody to the insecticidal protein or a fragment thereof. Invention Details

[0033] The term "transgenic" plant refers to a plant containing heteropolynucleotides. Preferably, the heteropolynucleotides are stably integrated into the genome, allowing the polynucleotides to be passed on to successive generations. Heteropolynucleotides may be integrated into the genome alone or as part of a recombinant expression cassette. "Transgenic" herein refers to any cell, cell line, callus, tissue, plant part, or plant whose genotype has been altered due to the presence of heteronucleotides, including those originally altered transgenic organisms or cells, and those produced from hybridization or asexual reproduction of the initial transgenic organism or cell. As used herein, the term "transgenic" is not intended to include changes to the genome (chromosomal or extrachromosomal) by conventional plant breeding methods (e.g., hybridization) or by naturally occurring events (e.g., self-fertilization, random hybridization, non-recombinant viral infection, non-recombinant bacterial transformation, non-recombinant transposition, or spontaneous mutation). The transgenic plant may be at any stage of growth.

[0034] The control of pest damage to plants is not affected by changes in planting location and / or planting time.

[0035] The term "wildtype" refers to nucleic acid molecules or proteins that can be found in nature. "Wildtype" plants can be plants, plant cells, or plant parts that do not express exogenous resistance genes.

[0036] In this invention, "plant" should be understood as any differentiated multicellular organism capable of photosynthesis, particularly monocotyledonous or dicotyledonous plants. In this invention, the term "plant tissue" or "plant part" includes plant cells, protoplasts, plant tissue cultures, plant callus, plant masses, as well as plant embryos, pollen, ovules, seeds, leaves, stems, flowers, branches, seedlings, fruits, kernels, spikes, roots, root tips, anthers, etc.

[0037] In this invention, "plant cell" should be understood as any cell derived from or found in a plant that is capable of forming, for example, undifferentiated tissues such as callus, differentiated tissues such as embryos, components of a plant, or a seed.

[0038] The genome of a plant, plant tissue, or plant cell as described in this invention refers to any genetic material within a plant, plant tissue, or plant cell, including the nucleus, plastids, and mitochondrial genome.

[0039] The "plant cell" or "plant" mentioned in this invention may include, but is not limited to, dicotyledonous plants or monocotyledonous plants. "Plant cells" or "plants" may also include, but are not limited to, alfalfa, bananas, barley, broccoli, cabbage, rapeseed, carrots, cassava, castor beans, cauliflower, celery, chickpeas, Chinese cabbage, citrus, coconuts, coffee, corn, clover, cotton, gourds, cucumbers, Douglas fir, eggplant, eucalyptus, flax, garlic, grapes, hops, leeks, lettuce, slash pine, millet, melons, nuts, oats, olives, onions, ornamental plants, palms, pasture grasses, peas, peanuts, pepper, pigeon peas, pine trees, potatoes, poplars, pumpkins, radishes, rapeseed, rice, rootstocks, rye, safflower, shrubs, sorghum, southern pine, soybeans, spinach, zucchini, strawberries, sugar beets, sugarcane, sunflowers, sweet corn, sweet potato, switchgrass, tea trees, tobacco, tomatoes, black wheat, turfgrass, watermelons, and wheat. Transgenic plants can be obtained from transgenic seeds by separating a portion from the plant through cutting, breaking, grinding, or other means. The plant portion can be a seed, boll, leaf, flower, stem, root, or any part thereof, or a non-renewable portion of the transgenic plant portion. As described in this invention, a "non-renewable" portion of a transgenic plant portion is a portion that cannot be induced to form a complete plant, or a portion that cannot be induced to form a complete plant capable of sexual and / or asexual reproduction. In some embodiments, the non-renewable portion of the plant portion is a part of the transgenic seed, boll, leaf, flower, stem, or root.

[0040] In this invention, "host organism" should be understood as any single-celled or multi-celled organism into which mutant protein-encoding nucleic acids can be introduced, including, for example, bacteria such as Escherichia coli, fungi such as yeast (e.g., Saccharomyces cerevisiae), molds (e.g., Aspergillus), plant cells, and plants.

[0041] The terms "protein," "polypeptide," and "peptide" are used interchangeably in this invention to refer to polymers of amino acid residues, including polymers in which one or more amino acid residues are chemical analogs of natural amino acid residues. The proteins and polypeptides of this invention can be generated through recombinant synthesis or through chemical synthesis.

[0042] In this invention, "contact" refers to insects and / or pests touching, staying on, and / or feeding on plants, plant organs, plant tissues, or plant cells. The plants, plant organs, plant tissues, or plant cells may express insecticidal proteins within themselves, or they may have insecticidal proteins on their surface and / or have microorganisms that produce insecticidal proteins.

[0043] The step preceding the contact step is to plant a plant containing a polynucleotide encoding the KIR protein.

[0044] In this invention, the terms "control" and / or "prevention" refer to pests coming into contact with the KIR protein at least, resulting in inhibited growth and / or death of the pests after contact. Further, the pests come into contact with the KIR protein at least by ingesting plant tissues, resulting in the inhibition of all or part of the pests' growth and / or death after contact. Inhibition refers to sublethality, meaning it does not cause death but induces some effect on growth, development, behavior, physiology, biochemistry, and tissue aspects, such as slowed and / or stopped growth. Simultaneously, the plant should be morphologically normal and culturable under conventional methods for product consumption and / or generation. Furthermore, plants and / or seeds containing polynucleotide sequences encoding the KIR protein that control pests, under conditions of artificial inoculation with pests and / or natural pest damage, exhibit reduced plant damage compared to non-transgenic wild-type plants, specifically manifested in, but not limited to, improved stem resistance, and / or increased grain weight, and / or increased yield. The "control" and / or "prevention" effect of KIR proteins on pests can exist independently and is not weakened or eliminated by the presence of other substances that can "control" and / or "prevent" pests. Specifically, if any tissue of a transgenic plant (containing a polynucleotide sequence encoding a KIR protein) simultaneously and / or asynchronously contains and / or produces KIR proteins and / or another substance that can control pests, the presence of the other substance neither affects the "control" and / or "prevention" effect of KIR proteins on pests, nor causes the "control" and / or "prevention" effect to be wholly and / or partially achieved by the other substance, and is unrelated to KIR proteins. Typically, in the field, the process of pests feeding on plant tissues is brief and difficult to observe with the naked eye. Therefore, under conditions of artificial inoculation of pests and / or natural pest damage, such as the presence of dead pests in any tissue of a transgenic plant (containing a polynucleotide sequence encoding a KIR protein), and / or pests with inhibited growth remaining on it, and / or reduced plant damage compared to non-transgenic wild-type plants, the method and / or use of the present invention is achieved, namely, the method and / or use of controlling pests by having the pests at least come into contact with the KIR protein.

[0045] "Effective amount" refers to the concentration of an insecticidal protein that inhibits the ability of insects to survive, grow, feed, and / or reproduce through toxic effects, or limits insect-related damage or crop loss. "Effective amount" may or may not mean killing insects, although it is preferred to mean killing insects.

[0046] In this invention, the expression of the KIR protein in a transgenic plant can be accompanied by the expression of one or more Cry-type insecticidal proteins and / or Vip-type insecticidal proteins. The co-expression of more than one insecticidal protein in the same transgenic plant can be achieved through genetic engineering to include and express the desired genes in the plant. Alternatively, one plant (the first parent) can be genetically engineered to express the KIR protein, and a second plant (the second parent) can be genetically engineered to express Cry-type and / or Vip-type insecticidal proteins. Offspring plants expressing all genes introduced from both the first and second parents are obtained through hybridization.

[0047] RNA interference (RNAi) refers to a highly conserved evolutionary phenomenon characterized by the efficient and specific degradation of homologous mRNA induced by double-stranded RNA (dsRNA). Therefore, in this invention, RNAi technology can be used to specifically knock out or shut down the expression of specific genes in target insect pests.

[0048] This disclosure relates to compositions and methods for controlling pests. These methods involve transforming organisms with nucleic acid sequences encoding insecticidal polypeptides (proteins) of the present disclosure. Specifically, the nucleic acid sequences of the embodiments can be used to prepare plants and microorganisms with pest-killing activity. Thus, transformed bacteria, plants, plant cells, plant tissues, and seeds are provided. The compositions are pest-killing nucleic acids and proteins of bacterial species. These nucleic acid sequences can be used to construct expression vectors subsequently transformed into target organisms, as probes for isolating other homologous (or partially homologous) genes, and for producing altered insecticidal polypeptides by methods known in the art, such as local mutagenesis, domain exchange, or DNA shuffling.

[0049] The insecticidal peptides disclosed herein can be used to control or kill target pests, including but not limited to species of Thysanoptera, Lepidoptera, and Hemiptera. These Thysanoptera include, but are not limited to, western flower thrips (Frankliniella occidentalis), flower thrips (Frankliniella intonsa (Trybom)), bean thrips (Megalurothripsusitatus), and tobacco thrips (Thrips alliorum (Priesner)). These Lepidoptera include, but are not limited to, fall armyworm (Spodoptera frugiperda), cotton bollworm (Helicoverpa armigera), beet armyworm (Spodoptera exigua), oriental armyworm (Mythimna seperata (Walker)), beet armyworm (Spodoptera frugiperda), Asian corn borer (Ostrinia furnacalis (Guenée)), corn ear borer (Helicoverpa zeaBoddie), European corn borer (Ostrinia nubilalis), and small sugarcane stem borer (Diatraea). Saccharalis and other species; these hemiptera insects include, but are not limited to, the spotted pedestris (Fabricius), the green mirid bug (Apolygus lucorum Meyer), the pasture mirid bug (Lygus pratensis), the rice green bug (Nezara viridula Linnaeus), and the hero bug (Euschistus heros).

[0050] The KIR protein described in this invention is a type of β-pore-forming protein. Enzymatic activation within the insect gut, specific binding to receptors on the insect gut, and the physicochemical environment within the gut are key factors in the function of β-pore-forming proteins. Only after the β-pore-forming protein is enzymatically cleaved into active fragments and specifically binds to receptors on the insect intestinal epithelial cell membrane can a particular β-pore-forming protein form pores in the intestinal epithelial cell membrane, disrupting the cell membrane and thus exerting an insecticidal effect against the pest. The receptor binding process requires precise matching; often, a difference of even one amino acid in the pore-forming protein or the receptor protein can alter the binding to the same receptor. For example, the aerolysin protein, which belongs to the β-pore-forming protein family, exhibited a qualitative change in virulence against the CTLL-2 cell line after the R336A mutation (Osusky, Teschk et al., 2008). Similarly, changes in the receptor can also lead to changes in the virulence of the same β-pore-forming protein. For example, inhibiting the expression of the HAVCR1 receptor-encoding gene in the MDCK cell line using dsRNA resulted in a hundredfold difference in the cytotoxicity of epsilon-toxin (Ivie, Fennessey et al., 2011). This fully demonstrates that the interaction between β-aporphyrin and enzymes and receptors in insects is complex and unpredictable.

[0051] The present invention covers insecticidal compositions comprising one or more insecticidal proteins that act together as insecticidal proteins and at least one suitable carrier, excipient or diluent.

[0052] The insecticidal composition of this invention further comprises a second insecticidal agent. The second insecticidal agent is a biological agent (second insecticidal protein) or a chemical agent. This biological agent is or is derived from insecticidal proteins of Bacillus thuringiensis, Bacillus cereus, pathogenic bacteria, luminescent bacteria, Bacillus retroflexus, Bacillus spheruliticus, Chromobacterium, Yersinia pestis, Paenibacillus popiliae, or Clostridium species. The biological agent is or is derived from dsRNA, Cry protein, Vip protein, potato glycoprotein, protease, protease inhibitor, urease, α-amylase inhibitor, porogen, lectin, engineered antibody or antibody fragment, or chitinase. The chemical agent is a carbamate, pyrethroid, organophosphate, neonicotinoid, organochloride, nereistoxin, or a combination thereof; or the chemical agent contains an active ingredient selected from the group consisting of: carbofuran, carbamate, methomyl, bifenthrin, heptafluthrin, permethrin, cypermethrin, lambda-cyhalothrin, lambda-cyhalothrin, deltamethrin, chlorpyrifos, oxychloride, dimethoate, ethoprophos, malathion, methyl parathion, phorate, terbufos, terbufos, fipronil, acetamiprid, imidacloprid, thiamethoxam, endosulfan, sulfadiazine, and combinations thereof.

[0053] The polynucleotides and / or nucleotides described in this invention form a complete "gene" that encodes a protein or polypeptide in the desired host cell. Those skilled in the art will readily recognize that the polynucleotides and / or nucleotides of this invention can be placed under the control of regulatory sequences in the target host.

[0054] As is well known to those skilled in the art, DNA typically exists in a double-stranded form. In this arrangement, one strand is complementary to the other, and vice versa. Because DNA replicates in plants, other complementary strands of DNA are produced. Thus, this invention includes the use of the polynucleotides and their complementary strands as exemplified in the sequence listing. The term "coding strand" as commonly used in the art refers to the strand that binds to the antisense strand. To express proteins in vivo, typically one strand of DNA is transcribed into a complementary strand of mRNA, which serves as a template for protein translation. The mRNA is actually transcribed from the "antisense" strand of DNA. The "sense" or "coding" strand has a series of codons (codons are three nucleotides, and reading three at a time produces a specific amino acid) that can be read as an open reading frame (ORF) to form the target protein or peptide. This invention also includes RNA that functions substantially similarly to the DNA of the examples.

[0055] In this invention, nucleic acid molecules or fragments thereof hybridize with the KIR gene of this invention under stringent conditions. Any conventional nucleic acid hybridization or amplification method can be used to identify the presence of the KIR gene of this invention. Nucleic acid molecules or fragments thereof can specifically hybridize with other nucleic acid molecules under certain conditions. In this invention, if two nucleic acid molecules can form antiparallel double-stranded nucleic acid structures, it can be said that these two nucleic acid molecules can specifically hybridize with each other. If two nucleic acid molecules exhibit perfect complementarity, one nucleic acid molecule is called a "complement" of the other nucleic acid molecule. In this invention, when every nucleotide of one nucleic acid molecule is complementary to the corresponding nucleotide of another nucleic acid molecule, the two nucleic acid molecules are said to exhibit "perfect complementarity". If two nucleic acid molecules can hybridize with sufficient stability to anneal and bind to each other under at least conventional "low-string" conditions, the two nucleic acid molecules are called "minimally complementary". Similarly, if two nucleic acid molecules can hybridize with sufficient stability to anneal and bind to each other under conventional "high-string" conditions, the two nucleic acid molecules are said to be "complementary". Deviations from perfect complementarity are permissible, as long as such deviations do not completely prevent the two molecules from forming a double-stranded structure. In order for a nucleic acid molecule to be used as a primer or probe, it is only necessary to ensure that it has sufficient sequence complementarity so that it can form a stable double-stranded structure under the specific solvent and salt concentration used.

[0056] In this invention, the substantially homologous sequence is a nucleic acid molecule that, under highly stringent conditions, can specifically hybridize with the complementary strand of a matching nucleic acid molecule. Suitable stringent conditions that promote DNA hybridization, such as treatment with 6.0× sodium chloride / sodium citrate (SSC) at approximately 45°C followed by washing with 2.0× SSC at 50°C, are well known to those skilled in the art. For example, the salt concentration in the washing step can be selected from approximately 2.0× SSC, 50°C for low-stringent conditions to approximately 0.2× SSC, 50°C for high-stringent conditions. Furthermore, the temperature conditions in the washing step can be increased from approximately 22°C (room temperature) for low-stringent conditions to approximately 65°C for high-stringent conditions. Both the temperature conditions and the salt concentration can be changed, or one can remain constant while the other is changed. Preferably, the stringent conditions described in this invention can be as follows: specific hybridization occurs at 65°C in a 6×SSC, 0.5% SDS solution, followed by washing the membrane once each with 2×SSC, 0.1% SDS and 1×SSC, 0.1% SDS.

[0057] Therefore, sequences possessing insecticidal activity and hybridizing under stringent conditions with SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, or SEQ ID NO:23 of the present invention are included in the present invention. These sequences are at least about 40%-50% homologous, about 60%, 65%, or 70% homologous, and even at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater sequence homology with the sequences of the present invention.

[0058] The genes and proteins described in this invention include not only specific example sequences, but also portions and / or fragments (including those with incomplete or terminal deletions compared to the full-length protein), variants, mutants, substitutes (proteins with substituted amino acids), chimeras, and fusion proteins that preserve the insecticidal activity characteristics of the specific example proteins. The term "variant" or "mutation" refers to a nucleotide sequence encoding the same protein or an equivalent protein with insecticidal activity. The term "equivalent protein" refers to a protein having the same or substantially the same insecticidal biological activity as the protein of the claims.

[0059] The “fragment” or “truncated” DNA molecule or protein sequence described in this invention refers to a portion of the original DNA or protein sequence (nucleotide or amino acid) or its artificially modified form (e.g., a sequence suitable for plant expression). The length of the aforementioned sequence may vary, but the length is sufficient to ensure that the (encoded) protein has insecticidal activity.

[0060] Genes can be modified and gene variants can be easily constructed using standard techniques. For example, techniques for creating point mutations are well known in the art. Another example is Gene shuffling, a method described in U.S. Patent No. 5,605,793 that uses DNA reassembly after random breaks to generate additional molecular diversity.

[0061] This invention can derive equivalent proteins and / or genes encoding these equivalent proteins from β-open-pore protein isolates and / or DNA libraries. Various methods exist for obtaining the insecticidal proteins of this invention. For example, antibodies to the insecticidal proteins disclosed and claimed in this invention can be used to identify and isolate other proteins from a protein mixture. In particular, the antibody may be generated from the protein fraction that is most constant and most distinct from other β-open-pore proteins. These antibodies can then be used to specifically identify the characteristically active equivalent proteins by immunoprecipitation, enzyme-linked immunosorbent assay (ELISA), or Western blotting. Antibodies to the proteins, equivalent proteins, or fragments of such proteins disclosed in this invention can be readily prepared using standard procedures in the art. Genes encoding these proteins can then be obtained from microorganisms.

[0062] Due to the degeneracy of the genetic codon, many different DNA sequences can encode the same amino acid sequence. The production of alternative DNA sequences encoding these identical or substantially identical proteins is within the skill level of those skilled in the art. These different DNA sequences are included within the scope of this invention. The term "substantially identical" means a sequence with amino acid substitutions, deletions, additions, or insertions that do not substantially affect insecticidal activity, and also includes fragments that retain insecticidal activity.

[0063] The substitution, deletion, or addition of amino acid sequences in this invention is a conventional technique in the art. Preferably, such amino acid changes are: small property changes, i.e., conserved amino acid substitutions that do not significantly affect protein folding and / or activity; small deletions, typically about 1-30 amino acid deletions; small amino or carboxyl terminus extensions, such as an amino terminus extension of one methionine residue; and small linker peptides, such as about 20-25 residues long.

[0064] Examples of conserved substitutions are those occurring within the following groups of amino acids: basic amino acids (such as arginine, lysine, and histidine), acidic amino acids (such as glutamic acid and aspartic acid), polar amino acids (such as glutamine and asparagine), hydrophobic amino acids (such as leucine, isoleucine, and valine), aromatic amino acids (such as phenylalanine, tryptophan, and tyrosine), and small-molecule amino acids (such as glycine, alanine, serine, threonine, and methionine). Those amino acid substitutions that do not typically alter specific activity are well known in the art and have been described, for example, by N. Neurath and R.L. Hill in *Protein*, published by Academic Press in New York in 1979. The most common interchanges are Ala / Ser, Val / Ile, Asp / Glu, Thu / Ser, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Tyr / Phe, Ala / Pro, Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, Ala / Glu, and Asp / Gly, as well as their opposite interchanges.

[0065] It will be apparent to those skilled in the art that such substitution can occur outside the regions where molecular function is important, and still produce an active polypeptide. For the polypeptides of the present invention, the amino acid residues essential for their activity and therefore selected as unsubstituted can be identified according to methods known in the art, such as site-directed mutagenesis or alanine scanning mutagenesis (see, Cunningham and Wells, 1989, Science 244: 1081-1085). The latter technique involves introducing a mutation at each positively charged residue in the molecule and detecting the insecticidal activity of the resulting mutant molecule, thereby identifying the amino acid residues important for the activity of the molecule. The substrate-enzyme interaction site can also be determined by analyzing its three-dimensional structure, which can be determined by techniques such as nuclear magnetic resonance analysis, crystallography, or photoaffinity labeling (see, for example, de Vos et al., 1992, Science 255: 306-312; Smith et al., 1992, J. Mol. Biol 224: 899-904; Wlodaver et al., 1992, FEBS Letters 309: 59-64).

[0066] In this invention, KIR proteins include, but are not limited to, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, or SEQ ID NO:24, and amino acid sequences having a certain degree of homology with the amino acid sequences shown in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, or SEQ ID NO:24 are also included in this invention. These sequences typically exhibit greater than 78% similarity / identity with the sequences of this invention, preferably greater than 85%, more preferably greater than 90%, even more preferably greater than 95%, and may be greater than 99%. Preferred polynucleotides and proteins of this invention may also be defined according to more specific ranges of similarity and / or identity. For example, sequences of the present invention have 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% similarity.

[0067] The following terms are used to describe sequence relationships between two or more nucleotides or amino acids (peptides, proteins, protein sequences): (a) "reference sequence", (b) "comparison window", (c) sequence "identity", (d) percentage of sequence "identity" and (e) "substantial identity".

[0068] (a) As used herein, a “reference sequence” is a defined sequence used as the basis for sequence comparison. A reference sequence may be a subset or the whole of a specified sequence; for example, a segment of a full-length cDNA or gene sequence, or the complete cDNA or gene sequence.

[0069] (b) As used herein, a “comparison window” refers to a continuous and specified segment of a polynucleotide sequence, wherein the polynucleotide sequence in the comparison window may include additions or deletions (i.e., vacancies) compared to a reference sequence (which does not include additions or deletions) used for the best alignment of the two sequences. Typically, the length of the comparison window is at least 20 consecutive nucleotides, and optionally may be 30, 40, 50, 100 or longer. Those skilled in the art will understand that, due to the presence of gaps in the polynucleotide sequence, a gap penalty is typically introduced and subtracted from the number of matches to avoid high similarity with the reference sequence.

[0070] The alignment methods used for comparing sequences are well known in the art. Therefore, the determination of percentage sequence “identity” between any two sequences can be performed using a mathematical algorithm. A non-limiting example of such a mathematical algorithm is the work of Myers and Miller (1988) in CABIOS. The algorithms in 4:11-17; the local alignment algorithm in Smith et al. (1981) Adv. Appl. Math. [Advances in Applied Mathematics] 2:482; the global alignment algorithm in Needleman and Wunsch (1970) J. Mol. Biol. [Journal of Molecular Biology] 48:443-453; the search-based local alignment method in Pearson and Lipman (1988) Proc. Natl. Acad. Sci. [Proceedings of the National Academy of Sciences] 85:2444-2448; the algorithm in Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences] 872-264; as modified in Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences] 90:5873-5877.

[0071] Computer implementations of these mathematical algorithms can be used for sequence comparisons to determine sequence identity. These implementations include, but are not limited to: CLUSTAL from the PC / Gene program (available from Intelligenetics, Mountain View, CA); ALIGN program (version 2.0); and GAP, BESTFIT, BLAST, FASTA, and TFASTA from GCG Wisconsin Genetics software package version 10 (available from Accelrys Inc., 9685 Scranton Road, San Diego, CA, USA). Comparisons using these programs can be performed with default parameters. The following fully describe the CLUSTAL procedure: Higgins et al. (1988) Gene 73: 237-244 (1988); Higgins et al. (1989) CABIOS 5: 151-153; Corpet et al. (1988) Nucleic Acids Res. 16: 10881-90; Huang et al. (1992) CABIOS 8: 155-65; and Pearson et al. (1994) Meth. Mol. Biol. 24: 307-331. The ALIGN procedure is based on the algorithm of Myers and Miller (1988) (ibid.). When comparing amino acid sequences, the ALIGN procedure can use the PAM120 weighted residue table with a nick length penalty of 12 and a nick penalty of 4. The BLAST procedure proposed by Altschul et al. (1990) J. Mol. Biol. [Journal of Molecular Biology] 215:403 is based on the algorithm in Karlin and Altschul (1990) above. A BLAST nucleotide search can be performed using the BLASTN procedure with a score of 100 and a word length of 12 to obtain nucleotide sequences homologous to the nucleotide sequence of the protein encoding the example. A BLAST protein search can be performed using the BLASTX procedure with a score of 50 and a word length of 3 to obtain amino acid sequences homologous to the protein or polypeptide of the example. For gapped alignments for comparative purposes, GappedBLAST (in BLAST 2.0) as described by Altschul et al. (1997) Nucleic Acids Res. [Nucleic Acids Research] 25:3389 can be used. Alternatively, PSI-BLAST (in BLAST 2.0) can be used for iterative searches to detect distant relationships between molecules. See Altschul et al. (1997), ibid.When using BLAST, nick BLAST, or PSI-BLAST, the default parameters for each program can be used (e.g., BLASTN for nucleotide sequences, BLASTX for proteins). See the National Center for Biotechnology Information website, ncbi.hlm.nih.gov. Alignment can also be performed manually via inspection.

[0072] (c) As used herein, “sequence identity” or “identity” in the context of two nucleic acid or polypeptide sequences means the same residues in two sequences when compared at maximum correspondence on a specified comparison window. When using a percentage of sequence identity for a protein, it is recognized that dissimilar residue positions are generally distinguished by conserved amino acid substitutions, where an amino acid residue is substituted by another amino acid residue having similar chemical properties (e.g., charge or hydrophobicity) and therefore does not alter the functional properties of the molecule. When sequences differ in terms of conserved substitutions, the percentage of sequence identity can be adjusted upwards to correct for the conservatism of that substitution. Sequences differing from these conserved substitutions are referred to as having “sequence similarity” or “similarity.” Methods for making such adjustments are well known to those skilled in the art. Typically, this involves scoring conserved substitutions as partial rather than complete mismatches, thereby increasing the percentage of sequence identity. Thus, for example, a score for a conserved substitution is between zero and 1 when the same amino acid scores 1 and a non-conserved substitution scores zero. Scores for conserved substitutions are calculated, for example, as implemented in the program PC / GENE (Edali Genetics, Mountain View, CA).

[0073] (d) As used herein, the sequence “identity” percentage refers to the value determined by comparing two best-aligned sequences within a comparison window, where the polynucleotide sequence portion of the comparison window may contain additions or deletions (i.e., vacancies) compared to a reference sequence (which does not contain additions or deletions) for optimal alignment of the two sequences. This percentage is calculated by determining the number of positions in both sequences where the same nucleic acid base or amino acid residue occurs to generate the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window, and then multiplying the result by 100 to produce the sequence identity percentage.

[0074] (e)(i) The term “substantial identity” for a polynucleotide sequence means that when a polynucleotide is compared with a reference sequence using one of the alignment procedures described above with standard parameters, it includes sequences having at least 70%, 80%, 90%, or 95% or higher sequence identity. Those skilled in the art will recognize that these values ​​can be appropriately adjusted to determine the corresponding identity of proteins encoded by two nucleotide sequences by taking into account codon degeneracy, amino acid similarity, reading frame positioning, etc. For these purposes, substantial identity of the amino acid sequence generally means sequence identity with at least 60%, 70%, 80%, 90%, or 95% or higher sequence identity.

[0075] Another indicator of substantially identical nucleotide sequences is whether the two molecules hybridize with each other under stringent conditions. Typically, stringent conditions are chosen to be about 5°C lower than the Tm of a particular sequence at defined ionic strengths and pH. However, stringent conditions encompass temperatures ranging from about 1°C to about 20°C lower than Tm, depending on the desired level of stringency as limited elsewhere in this document. If nucleic acids that do not hybridize with each other under stringent conditions encode substantially identical polypeptides, then these nucleic acids remain substantially identical. This can occur, for example, when a single copy of a nucleic acid is produced using the maximum codon degeneracy allowed by genetic coding. Another indicator of substantially identical nucleic acid sequences is that the polypeptide encoded by the first nucleic acid is immunely cross-reactive with the polypeptide encoded by the second nucleic acid.

[0076] (e)(ii) In the context of peptides, the term “substantially similar” means that the peptide contains a sequence that has at least 70%, 80%, 85%, 90%, 95%, or higher sequence identity with a reference sequence within a specified comparison window. Optimal alignment for these purposes can be performed using the global alignment algorithm described above by Needleman and Wunsch (1970). One indication that two peptide sequences are substantially identical is that one peptide is immunoreactive with an antibody against the second peptide. Thus, for example, when one peptide is not identical to a second peptide only due to conserved substitutions, the two peptides are substantially identical. “Substantially similar” peptides share the sequence as described above, except that the positions of not entirely identical residues may differ due to conserved amino acid changes. The “expression cassette” as described in this invention refers to a nucleic acid sequence capable of directing the expression of a specific nucleotide sequence in a suitable host cell, comprising a promoter operatively linked to a target nucleotide sequence operatively linked to a termination signal. It also typically contains the sequence required for proper translation of the nucleotide sequence. An expression cassette containing the target nucleotide sequence may have at least one of its components that is heterologous to at least one of its other components. Expression cassettes can also be naturally occurring but obtained in a recombinant form useful for heterologous expression. However, typically, the expression cassette is heterologous relative to the host, meaning that 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 its ancestor through a transformation event. Expression of the nucleotide sequence in the expression cassette can be under the control of a constitutive or inducible promoter, which initiates transcription only when the host cell is exposed to specific external stimuli. In the case of multicellular organisms (such as plants), the promoter can also be tissue- or organ-specific, or developmentally stage-specific.

[0077] Expression cassettes containing the target nucleotide sequence can be chimeric, meaning that at least one of its components is heterologous relative to at least one of its other components. Expression cassettes can also be expression cassettes containing a natural promoter driving their native gene, however, they have been obtained in a recombinant form useful for heterologous expression. This use of the expression cassette makes it not so naturally present in the cell in which it is introduced.

[0078] The regulatory sequences described in this invention include, but are not limited to, promoters, transport peptides, terminators, enhancers, leader sequences, introns, and other regulatory sequences operatively linked to the KIR protein.

[0079] The promoters mentioned are plant-expressible promoters, meaning promoters that ensure the expression of the coding sequence linked to them within plant cells. Plant-expressible promoters can be constitutive promoters. Examples of promoters guiding constitutive expression in plants include, but are not limited to, the 35S promoter derived from cauliflower mosaic virus, the maize Ubi promoter, and the promoter of the rice GOS2 gene. Alternatively, plant-expressible promoters can be tissue-specific promoters, meaning that the promoter guides the expression level of the coding sequence in some plant tissues, such as green tissues, to be higher than in other plant tissues (which can be determined by conventional RNA assays), such as the PEP carboxylase promoter. Alternatively, plant-expressible promoters can be wound-induced promoters. Wound-induced promoters, or promoters that guide wound-induced expression patterns, refer to promoters that significantly increase the expression of the coding sequence under their regulation compared to normal growth conditions when plants experience mechanical or insect-induced trauma. Examples of trauma-inducible promoters include, but are not limited to, promoters of the protease repressor genes (pin I and pin II) in potatoes and tomatoes and the protease repressor gene (MPI) in maize.

[0080] The transport peptide (also known as a secretory signal sequence or guide sequence) guides the transgenic product to a specific organelle or cell compartment. The transport peptide can be heterologous to the receptor protein, for example, using the CTP sequence encoding chloroplast transport peptide to target chloroplasts, or using the 'KDEL' reserved sequence to target the endoplasmic reticulum, or using the CTPP of the barley plant lectin gene to target vacuoles.

[0081] The leader sequence includes, but is not limited to, small RNA virus leader sequences, such as the EMCV leader sequence (5' untranslated region of encephalomyocarditis virus); potato Y virus group leader sequences, such as the MDMV (maize dwarf mosaic virus) leader sequence; human immunoglobulin heavy chain binding protein (BiP); untranslated leader sequence of alfalfa mosaic virus capsid protein mRNA (AMVRNA4); and tobacco mosaic virus (TMV) leader sequence.

[0082] The enhancers include, but are not limited to, enhancers for cauliflower mosaic virus (CaMV), enhancers for scrophularia mosaic virus (FMV), enhancers for carnation weathering ring virus (CERV), enhancers for cassava vein mosaic virus (CsVMV), enhancers for four o'clock mosaic virus (MMV), enhancers for night-blooming jasmine yellow leaf curl virus (CmYLCV), enhancers for cotton leaf curl virus (CLCuMV), enhancers for dayflower yellow mottle virus (CoYMV), and enhancers for peanut chlorotic streak mosaic virus (PCLSV).

[0083] For monocotyledonous plant applications, the introns include, but are not limited to, the maize hsp70 intron, the maize ubiquitin intron, the Adh intron 1, the sucrose synthase intron, or the rice Act1 intron. For dicotyledonous plant applications, the introns include, but are not limited to, the CAT-1 intron, the pKANNIBAL intron, the PIV2 intron, and the "super ubiquitin" intron.

[0084] The terminator can be a suitable polyadenylation signal sequence that functions in plants, including but not limited to polyadenylation signal sequences derived from the Agrobacterium tumefaciens carmine synthase (NOS) gene, polyadenylation signal sequences derived from the protease inhibitor II (pin II) gene, polyadenylation signal sequences derived from the pea ssRUBISCO E9 gene, and polyadenylation signal sequences derived from the α-tubulin gene.

[0085] In this invention, "effective linkage" refers to the connection of nucleic acid sequences such that one sequence provides the function required for the linked sequences. In this invention, "effective linkage" can refer to linking a promoter to a sequence of interest, such that the transcription of the sequence of interest is controlled and regulated by the promoter. When the sequence of interest encodes a protein and its expression is desired, "effective linkage" means that the promoter is linked to the sequence in a manner that allows for efficient translation of the resulting transcript. If the linkage between the promoter and the coding sequence is a transcript fusion and the desired expression of the encoded protein is desired, such a linkage is created such that the first translation start codon in the resulting transcript is the start codon of the coding sequence. Alternatively, if the linkage between the promoter and the coding sequence is a translational fusion and the desired expression of the encoded protein is desired, such a linkage is created such that the first translation start codon contained in the 5' untranslated sequence is linked to the promoter, and the linkage is such that the relationship between the resulting translation product and the open reading frame encoding the desired protein conforms to the reading frame. Nucleic acid sequences that can be "effectively linked" include, but are not limited to: sequences that provide gene expression function (i.e., gene expression elements, such as promoters, 5' untranslated regions, introns, protein-coding regions, 3' untranslated regions, polyadenylation sites, and / or transcription terminators); sequences that provide DNA transfer and / or integration function (i.e., T-DNA boundary sequences, site-specific recombinase recognition sites, and integrase recognition sites); sequences that provide selective function (i.e., antibiotic resistance markers and biosynthetic genes); sequences that provide scoreable marker function; sequences that assist in sequence manipulation in vitro or in vivo (i.e., multiple adapter sequences and site-specific recombination sequences); and sequences that provide replication function (i.e., bacterial origin of replication, autonomous replication sequences, and centromere sequences).

[0086] In this invention, the KIR protein is toxic to pests. The plants used in this invention, particularly Arabidopsis thaliana, cotton, and soybean, contain exogenous DNA in their genomes. This exogenous DNA contains a nucleotide sequence encoding the KIR protein. Pests come into contact with this protein by ingesting plant tissues, and upon contact, their growth is inhibited and / or they die. Inhibition refers to lethality or sublethality. Simultaneously, the plants should be morphologically normal and can be cultured using conventional methods for product consumption and / or generation. Furthermore, this plant essentially eliminates the need for chemical or biological pesticides (these pesticides are only those targeting pests targeted by the KIR protein, excluding pesticides targeting non-target pests).

[0087] The expression level of insecticidal protein (β-openpoprotein) in plant material can be detected by a variety of methods described in the art, such as quantifying the mRNA encoding insecticidal protein produced in the tissue by applying specific primers, or directly using specific antibodies to detect the amount of insecticidal protein produced.

[0088] Different experiments can be used to determine the insecticidal effect of β-pore-opening protein in plants. The target insects in this invention are mainly Hemiptera pests such as the spotted stink bug, green mirid bug, and rice green stink bug; Lepidoptera pests such as the fall armyworm, corn borer, cotton bollworm, and armyworm; and Thysanoptera pests such as thrips.

[0089] In this invention, the KIR protein may have the amino acid sequence shown in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, or SEQ ID NO:24 in the sequence listing. In addition to the coding region of the KIR protein, it may also contain other elements, such as proteins encoding selective markers.

[0090] Furthermore, the expression cassette containing the nucleotide sequence encoding the KIR protein of the present invention can also be expressed in plants along with at least one protein encoding a herbicide resistance gene, including but not limited to glufosinate resistance genes (such as bar genes, pat genes), benzyladenine resistance genes (such as pmph genes), glyphosate resistance genes (such as EPSPS genes), bromooxynil resistance genes, sulfonylurea resistance genes, herbicide resistance genes, ammonia nitrile resistance genes, or glutamine synthetase inhibitor resistance genes (such as PPT), thereby obtaining transgenic plants that have both high insecticidal activity and herbicide resistance.

[0091] In this invention, exogenous DNA is introduced into plants, such as introducing the gene encoding the KIR protein, expression cassette, or recombinant vector into plant cells. Conventional transformation methods include, but are not limited to, Agrobacterium-mediated transformation, microparticle emission bombardment (i.e., gene gun), direct DNA uptake into protoplasts, electroporation, or glass fiber-mediated DNA introduction.

[0092] The beneficial effects of this invention are as follows: the KIR protein produces excellent insect-resistant properties, especially it can protect plants throughout their entire growth period and the entire plant to prevent damage from Thysanoptera, Lepidoptera and / or Hemiptera pests, and it is pollution-free, residue-free, stable and thorough, simple, convenient and economical.

[0093] Sequence Description

[0094] SEQ ID NO: 1 is the codon-optimized nucleotide sequence of E. coli KIR-01.

[0095] SEQ ID NO: 2 is the amino acid sequence of KIR-01.

[0096] SEQ ID NO: 3 is the codon-optimized nucleotide sequence of E. coli KIR-02.

[0097] SEQ ID NO: 4 is the amino acid sequence of KIR-02.

[0098] SEQ ID NO: 5 is the codon-optimized nucleotide sequence of E. coli KIR-03.

[0099] SEQ ID NO: 6 is the amino acid sequence of KIR-03.

[0100] SEQ ID NO: 7 is the codon-optimized nucleotide sequence of E. coli KIR-04.

[0101] SEQ ID NO: 8 is the amino acid sequence of KIR-04.

[0102] SEQ ID NO: 9 is the codon-optimized nucleotide sequence of E. coli KIR-05.

[0103] SEQ ID NO: 10 is the amino acid sequence of KIR-05.

[0104] SEQ ID NO: 11 is the codon-optimized nucleotide sequence of E. coli KIR-06.

[0105] SEQ ID NO: 12 is the amino acid sequence of KIR-06.

[0106] SEQ ID NO: 13 is the codon-optimized nucleotide sequence of E. coli KIR-07.

[0107] SEQ ID NO: 14 is the amino acid sequence of KIR-07.

[0108] SEQ ID NO: 15 is the codon-optimized nucleotide sequence of E. coli KIR-08.

[0109] SEQ ID NO: 16 is the amino acid sequence of KIR-08.

[0110] SEQ ID NO: 17 is the codon-optimized nucleotide sequence of E. coli KIR-09.

[0111] SEQ ID NO: 18 is the amino acid sequence of KIR-09.

[0112] SEQ ID NO: 19 is the codon-optimized nucleotide sequence of KIR-10 for E. coli.

[0113] SEQ ID NO: 20 is the amino acid sequence of KIR-10.

[0114] SEQ ID NO: 21 is the codon-optimized nucleotide sequence of E. coli KIR-11.

[0115] SEQ ID NO: 22 is the amino acid sequence of KIR-11.

[0116] SEQ ID NO: 23 is the codon-optimized nucleotide sequence of E. coli KIR-12.

[0117] SEQ ID NO: 24 is the amino acid sequence of KIR-12.

[0118] SEQ ID NO: 25 is the soybean codon-optimized nucleotide sequence of KIR-11.

[0119] SEQ ID NO: 26 is the soybean codon-optimized nucleotide sequence of KIR-12. Detailed Implementation

[0120] The following specific embodiments further illustrate the technical solution for the use of the insecticidal protein of the present invention.

[0121] Example 1: Obtaining Insecticidal Protein

[0122] Twelve insecticidal protein sequences were obtained through screening using bioinformatics methods. Their amino acid sequences are SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, and SEQ ID NO:24, respectively, and named KIR-01-KIR-12. Codon-optimized nucleotide sequences for *E. coli* were obtained through gene synthesis (including compatible 5′ and 3′ restriction sites for downstream cloning into pET15b). These nucleotide sequences are SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, and SEQ ID NO:23.

[0123] The above 12 gene fragments were used to construct recombinant expression vectors, which were transformed into Escherichia coli. Positive clones were screened and confirmed by sequencing before protein expression was performed. The expressed protein was purified by Ni-NTA affinity chromatography and stored at -80 °C for later use.

[0124] Example 2: Identification of insect resistance effect against Thysanoptera by feeding KIR protein

[0125] The KIR protein obtained in Example 1 was used to test its insecticidal effect on western flower thrips. Twelve protein solutions were diluted to the same concentration, and equal volumes of the diluted solutions were mixed into artificial feed for western flower thrips, with a final concentration of 300 μg / ml. An equal volume of buffer solution was mixed with the feed as a control (CK). Each treatment group had six replicates, with ten nymphs per replicate. The mortality rate of the test insects was monitored daily, and on day 3, the feed containing fresh protein diluted solution was replaced. On day 6, the mortality rate or inhibition rate (number of deaths and number of nymphs) was scored. Some results are shown in Tables 1-2, where "++++" indicates an average mortality rate or inhibition rate ≥90%, "+++" indicates an average mortality rate or inhibition rate of 61%-90%, "++" indicates an average mortality rate or inhibition rate of 41%-60%, "+" indicates an average mortality rate or inhibition rate of 21%-40%, and "-" indicates no significant difference in average mortality rate or inhibition rate compared to the CK.

[0126] Table 1. Representative insecticidal effects of KIR protein against western flower thrips (inhibition rate of western flower thrips)

[0127]

[0128] Table 2. Representative insecticidal effects of KIR protein against western flower thrips (mortality rate of western flower thrips)

[0129]

[0130] Example 3: Identification of insect resistance effect against Hemiptera by feeding KIR protein

[0131] The KIR protein obtained in Example 2 was used to test its insect resistance against the spotted leaf bug, green mirid bug, and rice green bug. Twelve proteins were diluted to the same concentration, and equal volumes of the diluted solutions were mixed with a fixed mass of artificial feed for each bug, resulting in a final concentration of 100 μg / g. An equal volume of buffer solution was mixed with the feed as the control (CK). Each treatment had five replicates, with 15 nymphs per replicate. Mortality rates were assessed on days 3 and 6, with fresh feed containing the protein diluted solutions provided on day 3. On day 6, bioassays were performed to assess mortality and growth retardation (partial results are shown in Table 3), using the same scoring criteria as in Example 2.

[0132] Table 3. Representative insecticidal effects of KIR protein against the spotted leaf bug.

[0133]

[0134] Example 4: Identification of insect resistance in Lepidoptera by feeding with KIR protein

[0135] The insecticidal effects of the KIR protein obtained in Example 1 on the fall armyworm susceptible population (SS), Cry1F resistant population (Cry1F-R), Vip3Aa resistant population (Vip3A-R), and population resistant to both Cry1F and Vip3Aa (Cry1F-R xVip3A-R) were tested. The insecticidal activity of the KIR protein against the fall armyworm was determined using the feed surface method. Freshly prepared artificial feed was poured into beakers and immersed in hot water. Using a manual continuous dispenser, the feed was dispensed into the wells of a 24-well cell culture plate, with 1 mL of feed dispensed into each well (1.6 cm in diameter). After the feed solidified, the resulting surface area was 2 cm². 2 The protein to be tested was diluted with Na₂CO₃ / NaHCO₃ buffer (pH=10). Using a manual continuous dispenser, 100 μl was dispensed into each well of a 24-well plate and shaken well to ensure the protein solution completely covers the feed surface, achieving a final concentration of 20 μg / cm³. 2After the 24-well cell culture plates were loaded with samples, they were dried in a clean bench until the protein had completely penetrated the surface of the feed or there was no floating water on the surface. Then, second-instar larvae of *Gnaphalium affine* were inoculated. One larva was inoculated into each well, the plates were capped and tied tightly to prevent escape, and the plates were placed under conditions of 25–27°C, 65–70% relative humidity, and 16 h / 8 h light / d illumination. 50 μl of buffer solution was added as a control. The experiment was repeated 3 times. After 7 days, the mortality rate and growth retardation were assessed using bioassays (partial results are shown in Table 4). The scoring criteria were the same as in Example 2.

[0136] Table 4. Representative insecticidal effects of KIR protein against fall armyworm.

[0137]

[0138] Example 5: Lepidopteran activity of insecticidal proteins in transgenic soybean plants

[0139] A soybean recombinant expression vector containing the optimized nucleotide sequences of soybean codons KIR-11 and KIR-12 (SEQ ID NO: 25 and SEQ ID NO: 26, respectively) was constructed. These insecticidal proteins are expressed under the regulation of the Arabidopsis ubiquitin promoter AtUBQ10. Using cotyledonary nodes as explants, the recombinant plant expression vector was transformed into the soybean recipient variety Williams82 via Agrobacterium-mediated transformation. After screening and identification, transgenic soybean plants infused with KIR-11 and KIR-12 insecticidal proteins were obtained.

[0140] The obtained transgenic soybean plants were planted in a greenhouse. Once the plants reached stage V2 (second compound leaf unfolding), plants with consistent growth were selected, and the outer half of each leaf (approximately 2 cm in length) was cut and placed in a detection device. Ten first-instar fall armyworm larvae were inoculated onto each leaf. Leaves from transgenic negative (participating in the transformation process but failing to successfully transfer the target gene) soybean plants and wild-type soybean plants were used as control groups, and tests were conducted under the same suitable environmental conditions. Resistance levels were evaluated on day 4. The results showed that, compared to transgenic negative soybean plants and wild-type soybean plants, the KIR-11 and KIR-12 insecticidal proteins conferred a significantly enhanced level of resistance to lepidopteran pests (especially the fall armyworm) in soybean plants.

[0141] In addition, the KIR resistance protein described in this application was transferred into Arabidopsis thaliana and cotton plant recipients via Agrobacterium-mediated transformation to obtain transgenic plants. These transgenic plants, or parts thereof, were then fed to different Lepidoptera, Thysanoptera, and Hemiptera insects, and the insects' feeding behavior and growth were observed. The results showed that the obtained transgenic plants possess excellent insect resistance characteristics and have good industrial value.

[0142] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.

Claims

1. An insecticidal protein comprising: An amino acid sequence having 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%, or at least 100% sequence identity with SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, or SEQ ID NO:

24.

2. A polynucleotide comprising a nucleotide sequence selected from the following: (a) The nucleotide sequence encoding the insecticidal protein of claim 1 or its complementary sequence; (b) The nucleotide sequences shown in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25 or SEQ ID NO:26, or their complementary sequences; (c) A nucleotide sequence that hybridizes to the sequence shown in (a) or (b) under stringent conditions; and / or (d) A nucleotide sequence that encodes the same amino acid sequence as the sequence shown in (a) or (b) due to the degeneracy of the genetic code, or a complementary sequence thereof.

3. The use of the insecticidal protein as described in claim 1 or the polynucleotide as described in claim 2 in the control of pests; preferably, the pests are selected from Thysanoptera, Lepidoptera, and Hemiptera insects; more preferably, the Thysanoptera insects are selected from western flower thrips (Frankliniella occidentalis), flower thrips (Frankliniella intonsa (Trybom)), bean thrips (Megalurothrips usitatus), and tobacco thrips (Thrips alliorum (Priesner)); the Lepidoptera insects are selected from fall armyworm (Spodoptera frugiperda), cotton bollworm (Helicoverpa armigera), beet armyworm (Spodoptera exigua), oriental armyworm (Mythimna seperata (Walker)), beet armyworm (Spodoptera litura), Asian corn borer (Ostrinia furnacalis (Guenée)), corn ear borer (Helicoverpa zea Boddie), and European corn borer (Ostrinia The species *Nubilalis* and *Diatraea Saccharalis* were selected from the order Hemiptera. Other insects included were *Riptortus pedestris (Fabricius)*, *Apolygus lucorum Meyer*, *Lygus pratensis*, *Nezara viridula Linnaeus*, and *Euschistus heros*.

4. An expression cassette comprising the polynucleotide of claim 2 under the regulation of an effectively linked regulatory sequence.

5. A plant transformation vector comprising the polynucleotide of claim 2 or the expression cassette of claim 4.

6. A composition for controlling pest infestation, comprising one or more insecticidal proteins as described in claim 1 and at least one suitable carrier, excipient or diluent.

7. The composition according to claim 6 further comprises a second insecticidal protein different from the insecticidal protein according to claim 1.

8. A method for controlling pest infestation, the method comprising delivering an effective amount of the insecticidal protein of claim 1 or the composition of claim 6 or 7 to the pest's growth environment and contacting it with the pest, causing the pest to die and / or inhibiting its growth.

9. A method for enhancing plant resistance to pests, the method comprising producing an insecticidally effective amount of the insecticidal protein of claim 1 in the plant, or expressing the polynucleotide of claim 2, the expression cassette of claim 4, or the plant transformation vector of claim 5 in the plant to produce an insecticidally effective amount of the insecticidal protein, contacting the plant with a pest, wherein the plant is unaffected by and / or has reduced damage from the pest.

10. A method for producing offspring seeds resistant to pests, the method comprising: a. Planting a first seed containing the polynucleotide of claim 2 or capable of producing the insecticidal protein of claim 1; b. A plant grows from the seed described in step a; and c. Harvesting the offspring seeds from the plant, wherein the harvested seeds contain the polynucleotide of claim 2 or are capable of producing the insecticidal protein of claim 1.

11. The method according to claim 9 or 10, wherein the plant is Arabidopsis thaliana, cotton, or soybean.

12. A commercial product derived from a plant or a part thereof, wherein the commercial product comprises the insecticidal protein of claim 1 or the polynucleotide of claim 2.

13. A method for detecting the presence of the polynucleotide as described in claim 2 in a plant genomic DNA sample, the method comprising: a. Contact the sample with a specific nucleotide probe, wherein the nucleotide probe is sequence homologous to or complementary to the polynucleotide of claim 2; b. subjecting the sample and the nucleotide probe to stringent hybridization conditions; and c. Detect the hybridization of the nucleotide probe with the polynucleotide; The detection of the hybridization confirmed the presence of the polynucleotide as described in claim 2 in the plant genomic DNA sample.

14. The method of claim 13, wherein the specific nucleotide probe hybridizes with plant genomic DNA comprising the polynucleotide of claim 2 under strict hybridization conditions and does not hybridize with other plant genomic DNA not comprising the polynucleotide of claim 2 under the hybridization conditions.

15. A method for detecting the presence of the insecticidal protein as described in claim 1 in a commodity product, the method comprising: a. Contact the product with an immunoreactive antibody that is specific for binding to the insecticidal protein or a fragment thereof; as well as b. Detect the binding of the antibody to the insecticidal protein or a fragment thereof.

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