A mutant Cry51Aa protein and its application in pest control

Abstract

This invention relates to a mutant Cry51Aa protein and its uses. The mutant Cry51Aa protein contains at least one amino acid mutation compared to the amino acid sequence shown in SEQ ID NO:2, and has at least 90% sequence identity with SEQ ID NO:2. It exhibits excellent insect-resistant properties, particularly providing protection for the entire plant throughout its growth cycle against thrips and leaf beetles, with no pollution or residue, stable and thorough effects, and is simple, convenient, and economical.

Description

Technical Field

[0001] This invention relates to the field of agricultural biotechnology, and in particular to a mutant Cry51Aa protein and its application in controlling pests, especially thrips or spotted stink bugs. Background Technology

[0002] Cotton and soybeans are important economic and food crops in China, and the annual losses to grain and the economy caused by pests, especially thrips and leafhoppers, are enormous. As agricultural pests, thrips use their specialized mouthparts to scab and suck the leaves, buds, or flowers of their host plants. Affected leaves initially show white spots that later merge, resembling spot diseases on the upper surface, while black frass appears on the underside. Severe infestations cause leaves to become smaller, wrinkled, and even yellow, dry, and wilt, severely impacting photosynthesis and leading to yield reduction. Leafhoppers, as a significant pest of legumes, were previously not given much attention, considered a serious soybean pest only in Japan and South Korea. However, in recent years, due to outbreaks of soybean pod rot, this harmful organism has received increasing attention. Leafhoppers and other piercing-sucking pests cause buds and flowers to wither and fall off when legumes begin to form pods, resulting in unfilled pods or shriveled grains; in severe cases, the entire plant dies, resulting in no harvest. Furthermore, it has been confirmed that leafhoppers and other piercing-sucking pests are one of the main causes of the "unfilled pods" type of soybean pod rot. Therefore, there is an urgent need for effective pest control methods to control the damage to plants caused by thrips or spodumene bugs. Summary of the Invention

[0003] The purpose of this invention is to provide a mutant Cry51Aa protein and its application in controlling pests, especially thrips or spotted bugs.

[0004] The present invention provides a mutant Cry51Aa protein, which contains at least one amino acid mutation compared with the amino acid sequence shown in SEQ ID NO:2, and has at least 90% sequence identity with SEQ ID NO:2.

[0005] In one specific embodiment, the mutant Cry51Aa protein has at least 98% or 99% sequence identity with SEQ ID NO:2.

[0006] In one specific embodiment, the amino acid mutation includes:

[0007] In the amino acid sequence corresponding to SEQ ID NO:2, the 214th amino acid is mutated to any other amino acid; and / or the 216th amino acid is mutated to any other amino acid; and / or the 270th amino acid is mutated to any other amino acid; or

[0008] The amino acid at position 147 in the amino acid sequence corresponding to SEQ ID NO:2 is mutated to any other amino acid; and / or the amino acid at position 149 is mutated to any other amino acid; and / or the amino acid at position 155 is mutated to any other amino acid; and / or the amino acid at position 157 is mutated to any other amino acid.

[0009] In another specific embodiment, the amino acid mutation includes:

[0010] In the amino acid sequence corresponding to SEQ ID NO:2, position 214 is mutated from serine to asparagine; and / or position 216 is mutated from arginine to proline; and / or position 270 is mutated from tryptophan to arginine; or

[0011] In the amino acid sequence corresponding to SEQ ID NO:2, position 147 is mutated from serine to valine; and / or position 149 is mutated from glutamic acid to cysteine; and / or position 155 is mutated from glutamic acid to cysteine; and / or position 157 is mutated from asparagine to aspartic acid.

[0012] In one specific embodiment, the mutant Cry51Aa protein has the amino acid sequence shown in SEQ ID NO:4 or 7.

[0013] The present invention also provides an isolated polynucleotide comprising a nucleic acid sequence selected from the following:

[0014] (1) The nucleic acid sequence encoding the mutant Cry51Aa protein or its complementary sequence;

[0015] (2) The nucleic acid sequence shown in SEQ ID NO: 11, 14, 18 or 19 or its complementary sequence;

[0016] (3) A nucleic acid sequence that hybridizes to the sequence shown in (1) or (2) under stringent conditions; and / or

[0017] (4) A nucleic acid sequence that encodes the same amino acid sequence as the sequence shown in (1) or (2) due to the degeneracy of the genetic code, or its complementary sequence;

[0018] In one specific embodiment, the polynucleotide is a DNA molecule.

[0019] In another specific embodiment, the nucleic acid sequence is optimized for expression in plant cells.

[0020] The present invention also provides a plant genome containing the aforementioned polynucleotides.

[0021] The present invention also provides a vector construct comprising the aforementioned polynucleotide and a homologous or non-homologous promoter operably linked thereto.

[0022] The present invention also provides a host cell comprising the mutant Cry51Aa protein, the polynucleotide, the plant genome, or the vector construct.

[0023] In one specific embodiment, the host cell is a plant cell.

[0024] The present invention provides a method for cultivating transgenic plants with or enhanced insect resistance, including regenerating plants from said plant cells.

[0025] The present invention also provides plants produced by the above method.

[0026] The present invention provides a method for managing pest resistance or controlling pests, comprising contacting the pest with at least the said mutant Cry51Aa protein.

[0027] In one embodiment, the mutant Cry51Aa protein is present in at least one host cell that produces the mutant Cry51Aa protein, and the pest comes into contact with the mutant Cry51Aa protein at least by ingesting the host cell.

[0028] In another specific embodiment, the mutant Cry51Aa protein is present in at least the bacteria or transgenic plants that produce the mutant Cry51Aa protein, and the pest comes into contact with the mutant Cry51Aa protein at least by ingesting the tissues of the bacteria or the transgenic plants. After contact, the growth of the pest is inhibited and / or it leads to death, thereby achieving control over its damage to the plant.

[0029] In one specific embodiment, the mutant Cry51Aa protein in Escherichia coli has the nucleotide sequence shown in SEQ ID NO:11 or 14; the mutant Cry51Aa protein in transgenic plants has the nucleotide sequence shown in SEQ ID NO:18 or 19.

[0030] The present invention also provides the application of the mutant Cry51Aa protein, polynucleotide, plant genome, vector construct, host cell, or the method thereof in improving plant insect resistance, preparing agents with insect resistance effects, or cultivating transgenic plants with or with enhanced insect resistance.

[0031] In one specific embodiment, the plant is preferably Arabidopsis thaliana, soybean, cotton, or cowpea.

[0032] In another specific embodiment, the pest is preferably thrips and / or spotted bugs.

[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 "wild type" refers to nucleic acid molecules or proteins that can be found in nature.

[0036] In this invention, "plant" should be understood as any differentiated multicellular organism capable of photosynthesis, particularly monocotyledonous or dicotyledonous plants. In one specific embodiment, the plant is Arabidopsis thaliana, cotton, soybean, or cowpea.

[0037] In this invention, the term "plant tissue" or "plant part" includes plant cells, protoplasts, plant tissue cultures, plant callus, plant blocks, as well as plant embryos, pollen, ovules, seeds, leaves, stems, flowers, branches, seedlings, fruits, kernels, spikes, roots, root tips, anthers, etc.

[0038] 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.

[0039] 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.

[0040] 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.

[0041] The step preceding the contact step is to plant a plant containing a polynucleotide encoding the Cry51Aa protein.

[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] In this invention, the terms "control" and / or "prevention" refer to pests coming into contact with the Cry51Aa protein at least once, resulting in inhibited growth and / or death of the pests. Further, the pests come into contact with the Cry51Aa protein at least once 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 the consumption and / or generation of products. Furthermore, plants and / or seeds containing the polynucleotide sequence encoding the Cry51Aa 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 Cry51Aa protein 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 Cry51Aa protein) simultaneously and / or asynchronously contains and / or produces Cry51Aa protein and / or another substance that can control pests, then the presence of the other substance neither affects the "control" and / or "prevention" effect of Cry51Aa protein 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 Cry51Aa protein. In the field, the process of pests feeding on plant tissues is usually 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 the Cry51Aa 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 Cry51Aa protein.

[0044] In this invention, the expression of Cry51Aa 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 Cry51Aa protein, and a second plant (the second parent) can be genetically engineered to express Cry-type insecticidal proteins and / or Vip-type insecticidal proteins. Offspring plants expressing all genes introduced from both the first and second parents are obtained through hybridization.

[0045] 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.

[0046] The term "thrips" as used in this invention refers to the general term for insects belonging to the order Thysanoptera. Adult thrips are tiny, typically only 1-2 mm in length, and are mostly pale yellow, brown, or black in color. They have marginal bristles along the posterior margin of the prothorax, and long, transparent wings surrounded by numerous fine hairs. Eggs are oblong and are usually laid on the underside of leaves, inside flower buds, or in the tissues of young stems. Initially milky white, they gradually turn pale yellow or orange-red as they develop. Nymphs are pale yellow, similar in appearance to adults, but smaller and wingless. There are many species of thrips, common ones including the western flower thrips (Frankliniella occidentalis) (Pergande), the tobacco thrips (Thripsalliorum) (Priesner), the bean thrips (Megalurothrips usitatus), the melon thrips (Thrips flevas) (Schrank), and the rice thrips (Stenchaetothrips biformis) (Bagnall). Western flower thrips is a world-renowned dangerous pest with a wide host range and diverse diet, hosting more than 500 plant species, including various vegetables, flowers, and cash crops such as cotton. Tobacco thrips has more than 30 host plant species and mainly damages vegetables of the Liliaceae, Cucurbitaceae, and Solanaceae families, as well as cotton. Bean thrips mainly damage legumes such as cowpeas, broad beans, and peanuts, and also occasionally damage eggplants, potatoes, and corn. Melon thrips mainly damage cucurbit crops, severely affecting the yield and quality of cucumbers, watermelons, and other cucurbit vegetables. Rice thrips mainly damage rice, with adults and nymphs rasping and sucking sap from rice leaves, causing white or yellowish-brown spots on the leaves, and in severe cases, affecting the normal growth and development of rice.

[0047] Thrips not only directly harm plants but also act as vectors for various plant viruses. For example, the western flower thrips can transmit several important plant viruses, such as tomato spotted wilt virus (TSWV). These viruses often cause more severe damage to crops than the thrips themselves, leading to large-scale yield reductions or even crop failure. Therefore, effectively controlling thrips damage is of great significance for ensuring the stability and safety of agricultural production.

[0048] The spotted pedestris (Fabricius) described in this invention is an insect belonging to the family Riptortusidae in the order Hemiptera. Its 1st to 4th instar nymphs resemble ants, while the 5th instar resembles an adult, except for shorter wings. The adult is entirely yellowish-brown to dark brown; the head is triangular; the pronotum has spines on both sides; the abdomen is constricted at the front, and the smooth yellow markings on the sides of the head and thorax are patchy or absent; the first antennal segment is longer than the second, and the fourth segment is longer than the sum of the second and third segments; the pronotum and pleura have many irregular black granules; the scent gland groove is long, curving forward, almost reaching the anterior margin of the metapleura; the lateral margins of the abdomen are black and yellow; the hind femora have rows of spines, the tibiae are curved, shorter than the femora, and pale in the middle.

[0049] The spotted cricket bug (Spodoptera litura) is distributed in China from Heilongjiang in the north to Taiwan and Hainan in the south, east to the border, and west to Jilin, Liaoning, Shanxi, Gansu, Sichuan, Yunnan, and Tibet. In other countries, it is found in North Korea, Japan, Vietnam, Laos, Thailand, Myanmar, India, Sri Lanka, and Malaysia. The spotted cricket bug has two generations per year, overwintering as adults. Emerging from hibernation in April of the following year when host plants sprout, mating and laying eggs in May and June, with eggs scattered on the undersides of leaves, tender shoots, and young stems. Newly hatched nymphs initially congregate to feed, gradually dispersing during the third instar. Mature nymphs emerge as adults in July and August, mating and laying eggs to reproduce and cause damage. The second generation of adults emerges in October and overwinters in late autumn. The spotted cricket bug prefers various legumes, followed by cotton, hemp, loofah, strawberries, rice, and wheat. Adults must feed on the reproductive organs of plants, such as flowers, for normal development and reproduction.

[0050] The Cry51Aa protein described in this invention is a type of β-open-pore protein. Enzymatic activation within the insect gut, binding to receptors on the insect gut, and the physicochemical environment within the gut are key factors in the function of β-open-pore proteins. Only after β-open-pore proteins are enzymatically cleaved into active fragments and then bind to receptors on the insect intestinal epithelial cell membrane can a particular β-open-pore protein exhibit an insecticidal effect against the pest. Receptor binding requires precise matching; often, a single amino acid difference in either the open-pore protein or the receptor protein can alter the binding to the same receptor. For example, the aerolysin protein, also a type of β-open-pore protein, exhibited a qualitative change in virulence against the CTLL-2 cell line after the R336A mutation (Osusky, Teschk et al., 2008). Similarly, changes in receptors can also lead to alterations in the virulence of the same β-open-pore protein. For example, suppressing the HAVCR1 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 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.

[0052] 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.

[0053] 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.

[0054] In this invention, nucleic acid molecules or fragments thereof hybridize with the Cry51Aa gene of this invention under stringent conditions. Any conventional nucleic acid hybridization or amplification method can be used to identify the presence of the Cry51Aa 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, then 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, then 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, then 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. For a nucleic acid molecule to function as a primer or probe, it only needs to be sufficiently complementary in sequence to form a stable double-stranded structure under the specific solvent and salt concentration used.

[0055] 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.

[0056] Therefore, sequences that have insecticidal activity and hybridize with SEQ ID NO: 11, 14, 18 or 19 of the present invention under stringent conditions are included in the present invention. These sequences are at least about 40%-50% homologous, about 60%, 65% or 70% homologous, or 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.

[0057] 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.

[0058] The “fragment” or “truncated” DNA molecule or protein sequence referred to in this invention refers to a portion of the original DNA or protein sequence (nucleotide or amino acid) involved 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 insect-resistant activity.

[0059] 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 U.S. Patent No. 5,605,793, which describes a method for generating additional molecular diversity by reassembling DNA after random breaks. Fragments of full-length genes can be produced using commercially available restriction endonucleases, and exonucleases can be used according to standard procedures. For example, enzymes such as Bal31 or site-directed mutagenesis can be used to systematically remove nucleotides from the ends of these genes. Genes encoding active fragments can also be obtained using various restriction endonucleases. Active fragments of these insect-resistant proteins can be obtained directly using proteases.

[0060] 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.

[0061] Due to the abundance of genetic codons, 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.

[0062] 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.

[0063] 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.

[0064] It will be apparent to those skilled in the art that such substitution can occur outside the region 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).

[0065] In this invention, the Cry51Aa protein includes, but is not limited to, SEQ ID NO:4 or 7, and amino acid sequences having a certain degree of homology with the amino acid sequences shown in SEQ ID NO:4 or 7 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 can also be defined according to more specific ranges of similarity and / or identity. For example, sequences exhibiting 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% similarity and / or identity with the sequences exemplified in this invention.

[0066] 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 Cry51Aa protein.

[0067] 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.

[0068] 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 a sequence encoding a chloroplast transport peptide to target the chloroplast, or using the 'KDEL' reserved sequence to target the endoplasmic reticulum, or using the CTPP of the barley plant lectin gene to target the vacuoles.

[0069] 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.

[0070] 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).

[0071] 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.

[0072] 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.

[0073] 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).

[0074] In this invention, "insecticide" or "insect-resistant" refers to insecticides that are toxic to crop pests, thereby achieving "control" and / or "prevention" of crop pests. Preferably, "insecticide" or "insect-resistant" means killing crop pests. More specifically, the target insects are thrips pests (such as western flower thrips, tobacco thrips, bean thrips, flower thrips, corn yellow thrips, rice thrips, and rice thrips, etc.) or spotted stink bugs.

[0075] In this invention, the Cry51Aa protein is toxic to pests. The plants used in this invention, particularly soybeans, cotton, and cowpeas, contain exogenous DNA in their genomes. This exogenous DNA contains a nucleotide sequence encoding the Cry51Aa 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 the consumption and / or generation of the product. Furthermore, this plant essentially eliminates the need for chemical or biological pesticides (specifically, pesticides targeting pests targeted by the Cry51Aa protein).

[0076] The expression level of insecticidal protein (β-opening protein) 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 and specifically detecting the amount of insecticidal protein produced.

[0077] 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 thrips or spotted stink bugs.

[0078] In this invention, the Cry51Aa protein may have the amino acid sequence shown in SEQ ID NO:4 or 7 of the sequence listing. In addition to the coding region of the Cry51Aa protein, it may also contain other elements, such as proteins encoding selective markers.

[0079] Furthermore, the expression cassette containing the nucleotide sequence encoding the Cry51Aa 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), bromoxynil 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.

[0080] In this invention, exogenous DNA is introduced into plants, such as introducing the gene encoding the Cry51Aa protein, its expression cassette, or a recombinant vector into plant cells. Conventional transformation methods include, but are not limited to, Agrobacterium-mediated transformation, microemission bombardment, direct DNA uptake into protoplasts, electroporation, or whisker-silicon-mediated DNA introduction.

[0081] The beneficial effects of this invention are as follows: the Cry51Aa protein produces excellent insect-resistant properties, especially it can protect the entire plant throughout its growth period to prevent damage from thrips and spodumene bugs, and it is pollution-free, residue-free, stable and thorough in effect, and simple, convenient and economical. Attached Figure Description

[0082] Figure 1 This is an SDS-PAGE gel image of the purified Cry51Aa-M3 protein, which has insecticidal protein properties according to the present invention. Lane 1: bacterial culture after induction; Lane 2: cell lysis precipitate; Lane 3: cell lysis supernatant; Lane 4: Ni column purification flow-through; Lane 5: 50 mM imidazole washing; Lane 6: target protein elution.

[0083] Figure 2-1 Comparison of the toxicity of Cry51Aa-WT, Cry51Aa-M2, and Cry51Aa-M3 proteins to western flower thrips.

[0084] Figure 2-2 Comparison of the toxicity of TIC807_M13, Cry51Aa-M4, Cry51Aa-M5 and Cry51Aa-M6 proteins to western flower thrips.

[0085] Figure 2-3 Comparison of the toxicity of TIC807_M13, Cry51Aa-M3 and Cry51Aa-M6 proteins to tobacco thrips.

[0086] Figure 3-1 Comparison of the developmental rates of Cry51Aa-WT, Cry51Aa-M2, and Cry51Aa-M3 proteins on western flower thrips.

[0087] Figure 3-2 Comparison of the developmental rates of western flower thrips by TIC807_M13, Cry51Aa-M4, Cry51Aa-M5 and Cry51Aa-M6 proteins.

[0088] Figure 3-3 Comparison of the developmental rates of TIC807_M13, Cry51Aa-M3, and Cry51Aa-M6 proteins on tobacco thrips.

[0089] Figure 4 Comparison of the toxicity of Cry51Aa-WT, Cry51Aa-M2, and Cry51Aa-M3 proteins to the spotted rimetrope bug.

[0090] Figure 5-1 To detect the toxic effects of different concentrations of Cry51Aa-M6 protein on western flower thrips.

[0091] Figure 5-2 To detect the toxic effects of different concentrations of Cry51Aa-M6 protein on tobacco thrips.

[0092] Figure 6 This is a comparison of feeding behavior of western flower thrips on Cry51Aa-M3 transgenic Arabidopsis thaliana T1 generation (experimental material number 11484) and non-transgenic Arabidopsis thaliana (COL).

[0093] Figure 7 This is a comparison of the feeding behavior of western flower thrips on Cry51Aa-M3 transgenic soybean generation T0 (experimental material number event-1-3) and non-transgenic soybean (CK).

[0094] Figure 8 This is a comparison of feeding behavior of western flower thrips on Cry51Aa-M6 transgenic cotton T0 generation (experimental material number Ghevent-1-3) and non-transgenic cotton (CK). Detailed Implementation

[0095] The following specific embodiments further illustrate the technical solution for the use of the insecticidal protein of the present invention.

[0096]

[0097] Example 1: Gene Synthesis

[0098] To improve plant resistance to pests, optimized nucleotide sequences of Cry51Aa wild-type and its mutants in Escherichia coli and Arabidopsis thaliana were synthesized (SEQ ID NO: 8-19).

[0099] Example 2: Construction of recombinant expression vector and acquisition of Cry51Aa protein

[0100] Taking Cry51Aa-M3 as an example, the synthesized E. coli codon optimized Cry51Aa-M3 nucleotide sequence (SEQ ID NO: 11) was ligated into the protein expression vector pET15b (Novagen, USA), with the gene insertion sites being NdeI and BamHI, to obtain the recombinant expression vector pET15b-Cry51Aa-M3.

[0101] Following the method described above for constructing the recombinant expression vector pET15b-Cry51Aa-M3, vectors pET15b-Cry51Aa-WT, pET15b-TIC807_M13, pET15b-Cry51Aa-M2, pET15b-Cry51Aa-M4, pET15b-Cry51Aa-M5, and pET15b-Cry51Aa-M6 were constructed, respectively.

[0102] The above expression vector was transformed into E. coli, and the corresponding Cry51Aa protein (SEQ ID NO:1-SEQ ID NO:7) was obtained by Ni-NTA affinity chromatography. The purity of the final obtained protein was over 90%. Representative results are shown in [link to relevant documentation]. Figure 1 .

[0103] Example 3: Identification of the insecticidal effect of feeding Cry51Aa protein on thrips and spotted bugs.

[0104] The Cry51Aa-WT and Cry51Aa mutant proteins obtained in Example 2 were used to test their insecticidal effects on western flower thrips. Seven protein solutions were diluted to different concentration gradients, and equal volumes of the dilutions were mixed into the feed. Cry51Aa-WT, Cry51Aa-M2, and Cry51Aa-M3 were set to final concentration gradients of 250, 500, and 750 μg / ml, respectively; TIC807_M13, Cry51Aa-M4, Cry51Aa-M5, and Cry51Aa-M6 were set to a final concentration of 500 μg / ml. An equal volume of buffer solution was mixed with the feed as a control (CK). Each treatment had six replicates, with ten nymphs per replicate. The mortality rate of the test insects was assessed daily, and a new feed containing the protein dilutions was introduced on day 3. The mortality rate of the test insects was assessed on day 6. Figure 2-1 and 2-2 ) and development rate ( Figure 3-1 and 3-2 Statistical analysis was performed separately.

[0105] The insecticidal effects of the selected TIC807_M13, Cry51Aa-M3, and Cry51Aa-M6 proteins obtained in Example 2 on tobacco thrips were tested. The final concentration for each protein was set at 500 μg / ml, and each protein was mixed into a fixed mass of artificial feed. An equal volume of buffer solution was mixed with the feed as a control (CK). Each treatment had 6 replicates, with 10 nymphs per replicate. The mortality rate of the test insects was monitored daily, and a new feed containing the protein dilution was introduced on day 3. The mortality rate of the test insects was assessed on day 6. Figure 2-3 ) and development rate ( Figure 3-3 Statistical analysis was performed separately.

[0106] The Cry51Aa-WT, Cry51Aa-M2, and Cry51Aa-M3 proteins obtained in Example 2 were used to test their insecticidal effects on the spotted bug. The three proteins were diluted to different concentration gradients, and equal volumes of the dilutions were mixed with fixed masses of artificial feed, resulting in final concentrations of 12.5, 25, 50, and 100 μg / g, respectively. An equal volume of buffer solution was mixed with the feed as a control (CK). Each treatment had five replicates, with 15 nymphs per replicate. The mortality rate of the test insects was investigated on days 3 and 6, with a fresh feed containing the protein dilutions introduced on day 3. The mortality rate of the test insects was statistically analyzed on day 6. Figure 4 ).

[0107] The results showed that, compared with the control group and other Cry51Aa strains, feeding with Cry51Aa-M3 and Cry51Aa-M6 proteins significantly increased the mortality rate of both thrips species and had a significant inhibitory effect on nymph development. At the same time, Cry51Aa-M3 protein also showed good resistance activity against the spotted bug.

[0108] Example 4: Determination of the lethal median concentration of Cry51Aa-M6 protein in thrips after feeding

[0109] The Cry51Aa-M6 protein obtained in Example 2 was used to conduct lethal concentration tests on western flower thrips and tobacco thrips. The protein was diluted to different concentration gradients, and equal volumes of the diluents were mixed with fixed masses of artificial feed. The final protein concentrations for testing western flower thrips were 50, 100, 150, 200, 250, 300, and 350 μg / ml, and the final protein concentrations for testing tobacco thrips were 6.25, 12.5, 25, 50, 100, 200, and 400 μg / ml. An equal volume of buffer solution was mixed with the feed as a control. Each treatment had six replicates, with ten nymphs per replicate. Mortality rates were investigated on days 3 and 6, with the feed containing the protein diluent replaced on day 3. Mortality and development rates were statistically analyzed on day 6. The LC50 of Cry51Aa-M6 against western flower thrips was 172.5 μg / ml, and against tobacco thrips was 25.7 μg / ml. Mortality rates at different concentrations are shown in [reference needed]. Figure 5-1 and 5-2 .

[0110] Example 5: Resistance test of transgenic Arabidopsis thaliana to second instar nymphs of western flower thrips.

[0111] 1. Constructing and transforming Arabidopsis transgenic expression vectors

[0112] Based on the pCAMBIA1301 vector, transgenic vectors P1-P5 containing Cry51Aa-WT (SEQ ID NO:15), TIC807_M13 (SEQ ID NO:16), Cry51Aa1-M2 (SEQ ID NO:17), Cry51Aa1-M3 (SEQ ID NO:18), and Cry51Aa2-M6 (SEQ ID NO:19) were constructed using conventional methods and successfully transformed into Arabidopsis thaliana. Individual transgenic Arabidopsis plants were transplanted into a bioassay device, and after 15 days, insect resistance tests were conducted on transgenic Arabidopsis plants with uniform growth.

[0113] 2. Insect resistance test of T1 generation transgenic Arabidopsis thaliana

[0114] Fifty healthy, identical second-instar nymphs of western flower thrips were inoculated onto each Arabidopsis plant. Observations continued until day 15, during which the number of surviving adults was recorded, and leaf feeding was documented via photographs. Non-transgenic Arabidopsis (COL) was used as a control group, and univariate analysis was employed to compare the differences in survival rates among different treatments.

[0115] The results showed that western flower thrips consumed significantly less food from Arabidopsis thaliana transgenic with Cry51Aa-M3 than the control group (e.g., Figure 6 As shown in the figure, the survival rate of adult western flower thrips that fed on Arabidopsis thaliana transgenic with Cry51Aa-M3 was significantly lower than that of the control group, indicating that transgenic with Cry51Aa-M3 endows Arabidopsis thaliana with superior resistance to western flower thrips.

[0116] Example 6: Resistance test of detached leaves of transgenic soybean to second instar nymphs of western flower thrips

[0117] 1. Construct and transform soybean transgenic expression vectors.

[0118] Based on the pCAMBIA1301 vector, the transgenic vector P6-P9 containing the nucleotide sequences of Cry51Aa-WT (SEQ ID NO:15), TIC807_M13 (SEQ ID NO:16), Cry51Aa-M3 (SEQ ID NO:18), and Cry51Aa-M6 (SEQ ID NO:19) was constructed using conventional methods and successfully transformed into soybean to express the corresponding proteins SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, and SEQ ID NO:7.

[0119] 2. Insect resistance testing of T0 generation genetically modified soybeans

[0120] Cut pieces of absorbent cotton and filter paper and place them at the bottom of the test box, soaking them until the surface is dry. Select healthy, disease-free leaves, taking one compound leaf with its petiole from each plant, wrapping it in moistened absorbent cotton, and sealing it tightly with plastic wrap before placing it in the box. Select healthy, uniformly growing second-instar nymphs, placing 30 nymphs in each box. Investigate the survival and development of the test insects after 7 days.

[0121] Using non-transgenic soybean receptor (CK) and transgenic soybean P6 as control groups, univariate analysis was used to compare the differences in survival rates among different treatments.

[0122] The results showed that western flower thrips consumed significantly less of Cry51Aa-M3 genetically modified soybeans than non-GMO soybeans (e.g., ...). Figure 7 As shown in the figure, the number of western flower thrips that fed on Cry51Aa-M3 soybeans and their development were significantly lower than those of the control group. Although the western flower thrips that fed on Cry51Aa-WT soybeans also showed inhibited development, the inhibition rate was significantly lower than that of soybeans that fed on Cry51Aa-M3. This indicates that the introduction of Cry51Aa-M3 conferred superior resistance to western flower thrips on soybean plants.

[0123] Example 7: Resistance test of detached leaves of transgenic cotton to second instar nymphs of western flower thrips.

[0124] 1. Construct and transform cotton transgenic expression vectors

[0125] Based on the pCAMBIA1301 vector, transgenic vectors P10 and P11 containing the nucleotide sequences TIC807_M13 (SEQ ID NO: 16) and Cry51Aa-M6 (SEQ ID NO: 19) were constructed using conventional methods and successfully transformed into cotton, expressing proteins SEQ ID NO: 2 and SEQ ID NO: 7, respectively.

[0126] 2. Insect resistance testing of T0 generation genetically modified cotton

[0127] Cut pieces of absorbent cotton and filter paper and place them at the bottom of the test box, soaking them until the surface is dry. Select healthy, disease-free leaves, taking one leaf with its petiole from each plant, wrapping it in moistened absorbent cotton, and sealing it tightly with film. Place it in the box for later use. Select healthy, uniformly growing second-instar nymphs of western flower thrips, placing 30 nymphs in each box. Investigate the survival and development of the test insects after 7 days.

[0128] Using non-transgenic cotton receptor (CK) and transgenic cotton P10 as control groups, univariate analysis was used to compare the differences in survival rates among different treatments.

[0129] The results showed that western flower thrips consumed significantly less of transgenic Cry51Aa-M6 cotton than non-transgenic cotton (e.g., ...). Figure 8 As shown in the figure, the number of western flower thrips that fed on Cry51Aa-M6 cotton and their development were significantly lower than those of the control group. Although western flower thrips that fed on TIC807_M13 cotton also showed inhibited development, the inhibition rate was significantly lower than that of cotton that fed on Cry51Aa-M6. This indicates that the introduction of Cry51Aa-M6 endowed cotton plants with superior resistance to western flower thrips.

[0130] The results showed that the transgenic soybeans and cotton exhibited excellent resistance to thrips. Furthermore, numerous tests revealed that introducing the gene described in this invention into other plants also produced excellent insect-resistant characteristics, demonstrating significant industrial value.

[0131] 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. A mutant Cry51Aa protein, characterized in that, The mutant Cry51Aa protein contains at least one amino acid mutation compared to the amino acid sequence shown in SEQ ID NO:2, and has at least 90% sequence identity with SEQ ID NO:

2.

2. The mutant Cry51Aa protein according to claim 1, characterized in that, The mutant Cry51Aa protein has at least 98% or 99% sequence identity with SEQ ID NO:

2.

3. The mutant Cry51Aa protein according to claim 1 or 2, wherein the amino acid mutation comprises: In the amino acid sequence corresponding to SEQ ID NO:2, the 214th amino acid is mutated to any other amino acid; and / or the 216th amino acid is mutated to any other amino acid; and / or the 270th amino acid is mutated to any other amino acid; or The amino acid at position 147 in the amino acid sequence corresponding to SEQ ID NO:2 is mutated to any other amino acid; and / or the amino acid at position 149 is mutated to any other amino acid; and / or the amino acid at position 155 is mutated to any other amino acid; and / or the amino acid at position 157 is mutated to any other amino acid.

4. The mutant Cry51Aa protein according to claim 3, wherein the amino acid mutation comprises: In the amino acid sequence corresponding to SEQ ID NO:2, position 214 is mutated from serine to asparagine; and / or position 216 is mutated from arginine to proline; and / or position 270 is mutated from tryptophan to arginine; or In the amino acid sequence corresponding to SEQ ID NO:2, position 147 is mutated from serine to valine; and / or position 149 is mutated from glutamic acid to cysteine; and / or position 155 is mutated from glutamic acid to cysteine; and / or position 157 is mutated from asparagine to aspartic acid.

5. The mutant Cry51Aa protein according to any one of claims 1-4, wherein the amino acid sequence is shown in SEQ ID NO:4 or 7.

6. An isolated polynucleotide comprising a nucleic acid sequence selected from the following: (1) A nucleic acid sequence or its complementary sequence encoding the mutant Cry51Aa protein as described in any one of claims 1-5; (2) The nucleic acid sequence shown in SEQ ID NO: 11, 14, 18 or 19 or its complementary sequence; (3) A nucleic acid sequence that hybridizes to the sequence shown in (1) or (2) under stringent conditions; and / or (4) A nucleic acid sequence that encodes the same amino acid sequence as the sequence shown in (1) or (2) due to the degeneracy of the genetic code, or its complementary sequence; Preferably, the polynucleotide is a DNA molecule.

7. The polynucleotide of claim 6, wherein the nucleic acid sequence is optimized for expression in plant cells.

8. A plant genome comprising the polynucleotides of any one of claims 6-7.

9. A vector construct comprising the polynucleotide of any one of claims 6-7 and a homologous or non-homologous promoter operably linked thereto.

10. A host cell comprising the mutant Cry51Aa protein of any one of claims 1-5, the polynucleotide of claim 6 or 7, the plant genome of claim 8, or the vector construct of claim 9; preferably, the host cell is a plant cell.

11. A method for cultivating transgenic plants with or enhanced insect resistance, and plants produced by said method, including regenerating plants from the plant cells of claim 10.

12. A method for managing pest resistance or controlling pests, characterized in that, This includes contacting the pest with at least the mutant Cry51Aa protein as described in any one of claims 1-5; Preferably, the mutant Cry51Aa protein is present in at least the host cells that produce the mutant Cry51Aa protein, and the pest comes into contact with the mutant Cry51Aa protein at least by ingesting the host cells; More preferably, the mutant Cry51Aa protein is present in at least the bacteria or transgenic plants that produce the mutant Cry51Aa protein, and the pest comes into contact with the mutant Cry51Aa protein at least by ingesting the tissues of the bacteria or the transgenic plants. After contact, the growth of the pest is inhibited and / or it leads to death, thereby achieving control over its damage to the plant.

13. The method according to claim 12, characterized in that, The mutant Cry51Aa protein in Escherichia coli has the nucleotide sequence shown in SEQ ID NO:11 or 14; the mutant Cry51Aa protein in transgenic plants has the nucleotide sequence shown in SEQ ID NO:18 or 19.

14. The application of the mutant Cry51Aa protein according to any one of claims 1-5, the polynucleotide according to claim 6 or 7, the plant genome according to claim 8, the vector construct according to claim 9, the host cell according to claim 10, or the method according to any one of claims 11-13 in improving plant insect resistance, preparing agents with insect resistance effects, or cultivating transgenic plants with or with enhanced insect resistance.

15. The polynucleotide according to claim 7, the plant genome according to claim 8, the host cell according to claim 10, the method according to any one of claims 11-13, the plant according to claim 11, or the application according to claim 14, characterized in that, The preferred plants are Arabidopsis thaliana, soybean, cotton, or cowpea.

16. The method according to claim 12 or 13, or the application according to claim 14, characterized in that, The preferred pests are thrips and / or spotted bugs.

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