Insecticidal protein and use thereof

Abstract

Provided are an insecticidal protein and the use thereof. The insecticidal protein comprises: (a) a protein consisting of an amino acid sequence as shown in any one of SEQ ID NOs: 1-4; or (b) a protein derived from (a), which is obtained by means of substitution, deletion or addition of one or several amino acids in the amino acid sequence of (a) and has an insecticidal activity. The insecticidal protein exhibits an improved toxicity to lepidopteran insects.

Description

Insecticidal proteins and their uses Technical Field

[0001] This invention relates to an insecticidal protein and its uses, particularly to a novel insecticidal protein that exhibits lethal or development-inhibiting activity against lepidopteran insects and its uses. Background Technology

[0002] Lepidoptera insects are a major cause of damage to field crops (such as corn and soybeans), reducing crop yields in affected areas. Lepidoptera pests that negatively impact agriculture include, but are not limited to, the cotton bollworm, the corn borer, and the fall armyworm. In recent years, with the intensification of the global greenhouse effect, both the types and numbers of pests have increased.

[0003] Bacillus thuringiensis (Bt) has historically been used as a primary source of insecticidal proteins because Bt strains have been found to exhibit high toxicity to certain insects. Bt strains are known to produce delta endotoxins (e.g., Cry protein) located within parasporal crystal inclusions at the onset of sporulation and during the stationary growth phase, and are also known to produce secretory insecticidal proteins (e.g., Vip protein). Upon ingestion by susceptible insects, both the delta endotoxins and secretory toxins exert their effects on the surface of midgut epithelial cells, thereby disrupting the cell membrane and leading to cell destruction and death.

[0004] Over the past 70 years, Bt-derived toxin proteins have been used in a variety of agricultural applications to protect important agricultural plants from insect infestations, reduce the need for chemical pesticides, and increase yields. Examples include mechanically spraying microbial preparations containing various Bt strains onto plant surfaces, and using genetic transformation techniques to produce transgenic plants and seeds expressing insecticidal proteins to control crop-related pests.

[0005] The ability to generate insect-resistant plants through the transformation of Bt insecticidal genes has revolutionized modern agriculture and increased the importance and value of insecticidal proteins and their genes. Several Bt proteins have been used in transgenic plants to produce insect resistance, including Cry1Ab, Cry1Ac, Cry1F, Cry2Ab, Cry3Bb, and Vip3A proteins. However, with the widespread application of transgenic crops, insects will evolve resistance to the Bt proteins expressed in transgenic plants under continuous selective pressure. Once such resistance develops and cannot be effectively controlled, it will undoubtedly limit the commercial value of transgenic crop varieties containing Bt proteins. Therefore, the development of novel insecticidal proteins is crucial for insect resistance management. New toxins with improved efficacy and control over a broader spectrum of susceptible insect species will reduce the number of surviving insects that can develop resistant alleles. Furthermore, if two or more insecticidal toxins are toxic to the same pest and have different modes of action when expressed in a single plant, the likelihood of the target insect species developing resistance to any of the toxins will be further reduced. Therefore, it is necessary to develop more new insecticidal proteins with improved or different mechanisms.

[0006] Cry1Da1 protein exhibits excellent insecticidal activity against the fall armyworm, but its activity against other major lepidopteran pests, including the cotton bollworm and the Asian corn borer, is relatively low. Current technology indicates that Cry1Da1 protein has a different insecticidal mechanism of action than the insecticidal proteins Cry1Ac, Cry1Ab, Cry1A.105, Cry1Fa, Cry2Ae, and Cry2Ab2, which are widely used in transgenic crops, by recognizing different midgut receptor proteins in lepidopteran pests. Therefore, the optimization and modification of Cry1Da1 is of great significance. Cry1Da1_7 (derived from CN106852147B) and Cry1Da2 (derived from CN106455513B) are known commercially available insecticidal proteins derived from the modification of the natural Cry1Da1 protein, but their insecticidal spectrum and resistance still cannot meet the crop's requirements for resistance to multiple major lepidopteran pests.

[0007] To meet this need, this invention discloses novel Cry1Da insecticidal proteins that exhibit activity against major target lepidopteran pest species, named GRIC31-GRIC34 respectively.

[0008] No reports have been found regarding the insecticidal spectrum and toxicity of the aforementioned novel insecticidal protein. The novel insecticidal protein disclosed in this invention addresses the ongoing need in the art to engineer other toxic insecticidal proteins to possess improved insecticidal properties, such as increased efficacy against a wider range of target insect pest species and different modes of action. Summary of the Invention

[0009] The purpose of this invention is to provide an insecticidal protein and its uses, wherein the insecticidal protein has improved toxicity against lepidopteran insects such as cotton bollworm or fall armyworm.

[0010] In one aspect, to achieve the above objectives, the present invention provides an insecticidal protein in one embodiment, comprising:

[0011] (a) A protein having the amino acid sequence shown in any one of SEQ ID NO: 1-4; or

[0012] (b) A protein derived from (a) whose amino acid sequence in (a) has been substituted and / or deleted and / or added with one or more amino acids and has insecticidal activity.

[0013] In another embodiment, the present invention provides an insecticidal gene, comprising:

[0014] (d) The nucleotide sequence encoding the insecticidal protein of claim 1; or

[0015] (e) A nucleotide sequence that hybridizes to the specified nucleotide sequence in (d) under stringent conditions and encodes a protein with insecticidal activity; or

[0016] (f) Having the nucleotide sequence shown in any one of SEQ ID NO:5-8.

[0017] In another embodiment of the present invention, an expression cassette, DNA construct, or recombinant vector is provided, containing the insecticidal gene under the regulation of an effectively linked regulatory sequence.

[0018] In another embodiment, the present invention provides a transgenic host organism comprising the insecticidal gene, or the expression cassette, the DNA construct, or the recombinant vector;

[0019] Preferably, the transgenic host organism includes plants, bacteria, yeast, baculoviruses, nematodes, or algae;

[0020] Furthermore, the bacteria are Bacillus thuringiensis or Escherichia coli.

[0021] In another embodiment of the present invention, an agricultural product or commodity is provided, characterized in that it contains a detectable amount of the insecticidal protein or the insecticidal gene.

[0022] The agricultural products or commodities mentioned are not plant varieties, but can be processed or treated grains, processed foods, feed products or their components, fuel products or their components, etc.

[0023] On the other hand, to achieve the above objectives, one embodiment of the present invention provides a method for generating insecticidal proteins, comprising:

[0024] Obtain cells of a transgenic host organism containing the insecticidal gene, or the expression cassette, the DNA construct, or the recombinant vector;

[0025] The cells of the transgenic host organism were cultured under conditions that allowed for the production of insecticidal proteins;

[0026] The insecticidal protein was recovered;

[0027] In a preferred embodiment, the transgenic host organism includes plant cells, animal cells, bacteria, yeast, baculovirus, nematodes, or algae;

[0028] In a more preferred embodiment, the plant is corn, soybean, cotton, rice, or wheat; and the bacteria is Bacillus thuringiensis or Escherichia coli.

[0029] In another embodiment of the invention, a method for increasing the range of target insects is provided, comprising: expressing the insecticidal protein in a plant together with at least one insecticidal polynucleotide different from the protein.

[0030] In this invention, the expression of the insecticidal proteins GRIC31 to GRIC34 in a transgenic plant can be accompanied by the expression of one or more other insecticidal polynucleotides. The co-expression of more than one insecticidal substance in the same transgenic plant can be achieved through genetic engineering to enable the plant to include and express the desired genes. Furthermore, one plant (the first parent) can be genetically engineered to express the insecticidal proteins of this invention, and a second plant (the second parent) can be genetically engineered to express other insecticidal polynucleotides. Offspring plants expressing all genes introduced from both the first and second parents are obtained through hybridization.

[0031] In some embodiments, the insecticidal polynucleotide may encode Cry-type insecticidal proteins, Vip-type insecticidal proteins, ETX / MTX-type insecticidal proteins, protease inhibitors, lectins, α-amylases, or peroxidases.

[0032] In other alternative embodiments, the insecticidal polynucleotide is a dsRNA that inhibits important genes in the target insect.

[0033] In another embodiment of the present invention, a method for producing insect-resistant plants is provided, comprising: introducing the insecticidal gene, or the expression cassette, the DNA construct, or the recombinant vector into a plant.

[0034] In a preferred embodiment, the plant is corn, soybean, cotton, rice, or wheat.

[0035] In another embodiment of the present invention, a method for protecting plants from insect-induced damage is provided, comprising: introducing the insecticidal gene, or the expression cassette, the DNA construct, or the recombinant vector into the plant, such that the plant produces an amount of insecticidal protein sufficient to protect it from insect infestation.

[0036] In a preferred embodiment, the plant is corn, soybean, cotton, rice, or wheat.

[0037] In another embodiment of the present invention, a method for controlling pests is provided, comprising: contacting the pests with an inhibitory amount of the insecticidal protein or a protein having insecticidal activity encoded by the insecticidal gene.

[0038] In a preferred embodiment, the pest is a lepidopteran insect.

[0039] In yet another embodiment of the present invention, there is a use of the insecticidal protein to control lepidopteran insects.

[0040] The following description and definitions are provided to better define the invention and to guide those skilled in the art in practicing it. Unless otherwise stated, the terminology should be understood according to its conventional usage by those skilled in the art.

[0041] The terms "toxin," "insecticide protein," or "insecticide protein" as used in this invention refer to polypeptides that exhibit pest-killing or insecticidal activity, or improved pest-killing or insecticidal activity. "Bt" or "Bacillus thuringiensis" toxins aim to include a broader range of Cry or Vip toxins found in various Bt strains, including toxins such as Cry1, Cry2, Cry3, or Vip3. Sequences of all Bt proteins discovered to date and named by the Bt Protein Nomenclature Committee are available for download from the Bt Protein Database BPPRC (bpprc-db.org). The three-dimensional structures of an increasing number of Bt proteins have been resolved; based on their overall structural similarity and sequence conservation, it is generally believed that most Bt proteins possess multiple domains, each acting alone or synergistically in insecticidal activity. With the rapid development of bioinformatics, protein engineering can be used to modify existing Bt proteins to obtain new Bt proteins with a wider insecticidal spectrum or enhanced toxicity.

[0042] Cry1 proteins (such as Cry1Aa and Cry1Ba) exhibit insecticidal activity against lepidopteran pests. Cry1 proteins can be divided into seven domains, the first three of which are associated with insect-specific toxicity. Domain I, one of the first three domains, contains the first third of the active toxin fragment and has been shown to be indispensable for pore formation; domains II and III are both related to receptor binding and insect specificity. Cry1Da1_7 and Cry1Da2 proteins possess typical Cry1 protein structural features and have been extensively studied due to their excellent insecticidal activity against lepidopteran insects; however, the interaction between their protein structure and insect intestinal cell receptors remains unclear.

[0043] The "modification" described in this invention is based on the structural regularities of protein molecules and their relationship with biological functions, and involves modifying existing proteins through gene modification or gene synthesis. Modification methods for Bt proteins include, but are not limited to: obtaining proteins with higher insecticidal activity using site-directed mutagenesis, and obtaining chimeric proteins with broadened insecticidal spectrum and / or enhanced insecticidal activity through domain exchange.

[0044] The "point mutation" described in this invention includes adding one or more amino acids to the N-terminus and / or C-terminus of a natural protein; deleting or adding one or more amino acids at one or more sites in a natural protein; or modifying or replacing one or more amino acids at one or more sites in a natural protein.

[0045] In this invention, a "domain" refers to a group of amino acids conserved at a specific position along the sequence alignment of evolution-related proteins. While amino acids at other positions may differ between homologues, highly conserved amino acids at specific positions indicate that they are likely essential for the protein's structure, stability, or function. Identified by their high conservation in sequence alignments within protein homologue families, they can be used as identifiers to determine whether any polypeptide under discussion belongs to a previously identified polypeptide group.

[0046] A comprehensive consideration of publicly available structural analyses of Bt toxins and reported functions related to specific structures and motifs indicates that specific regions of the toxin are associated with specific functions and discrete steps in the protein's mode of action. Nucleic acid sequences encoding Cry family peptides were obtained. Based on considerations of the target domain function proposed in the toxin's mode of action, the characteristics of the target domain and its connection features with surrounding domains were analyzed. Similar domains and closely linked sequence domains were replaced using recombination techniques. The resulting peptides' pest-killing activity was then determined. A series of recombinants can be generated and placed in various background sequences to produce novel insecticidal proteins with enhanced or altered pest-killing activities.

[0047] The term "activity" or "insectic activity" as used in this invention refers to the efficacy of a toxic agent (such as an insecticidal protein) in inhibiting (inhibiting growth, feeding, fertility, or viability), suppressing (inhibiting growth, feeding, fertility, or viability), controlling (controlling pest infestation, controlling the feeding activity of pests containing an effective dose of the insecticidal protein on a particular crop), or killing (causing disease, death, or reduced fertility) pests. These terms include providing an effective dose of the insecticidal protein to a pest, exposing the pest to the insecticidal protein to cause disease, death, reduced fertility, or stunted development. These terms also include repelling pests from the plant, plant tissue, plant parts, seeds, plant cells, or a particular geographical location from which the plant may grow by providing an effective dose of the insecticidal protein in or on the plant. Generally, insecticidal activity refers to the effectiveness of an insecticidal protein in inhibiting the growth, development, survival, feeding behavior, mating behavior, and reproductive capacity of a specific target pest (including, but not limited to, lepidopteran insects), or the ability to produce any measurable reduction in adverse effects caused by insects feeding on the protein, protein fragments, or polynucleotides. Insecticidal proteins may be produced by plants or applied to plants or the environment in which they are located. The terms "bioactivity," "effectiveness," or variations thereof used herein are also interchangeable in describing the effects of the insecticidal proteins of this invention on target insects.

[0048] Recombinant DNA molecules or DNA constructs containing insecticidal protein-coding sequences may further include DNA regions encoding one or more toxic agents. These DNA regions may be configured to co-express or be expressed with DNA sequences encoding chimeric insecticidal proteins, proteins distinct from insecticidal proteins, insect repressive dsRNA molecules, or helper proteins. Helper proteins include, but are not limited to, cofactors, enzymes, binding chaperones, or other agents that function to enhance the effects of insect inhibitors, such as by assisting their expression, influencing their stability in plants, optimizing oligomer free energy, enhancing their toxicity, and increasing their activity spectrum. Helper proteins may, for example, promote the absorption of one or more insect inhibitors or enhance the toxic effects of the toxic agents.

[0049] The cotton bollworm (Helicoverpa armigera), also known as the peach borer or heart borer, is an insect belonging to the genus Helicoverpa in the family Noctuidae of the order Lepidoptera. It is distributed throughout Europe, Asia, Africa, and Australia from 50°N to 50°S latitude, and is found in all cotton-growing areas of China. Adults are nocturnal, spending the day in shady places such as the undersides of leaves and flower petals among plants, becoming active in the evening. It is omnivorous, feeding on a wide variety of hosts, damaging not only cotton but also various cultivated crops such as corn, wheat, and sorghum, as well as many wild plants. Since the 1990s, the cotton bollworm has caused outbreaks of damage in cotton-growing areas north of the Huai River and in the Yellow River basin of China, becoming a focus of attention for agricultural production and scientific research in China.

[0050] The fall armyworm (Spodoptera frugiperda), described in this invention, is an insect belonging to the genus Spodoptera in the family Noctuidae of the order Lepidoptera. While native to tropical and subtropical regions of the Americas, the fall armyworm has invaded Central and Southeast Asia due to increased international trade and its powerful flight capabilities. It is an omnivorous pest that parasitizes plants, feeding primarily on corn, cotton, sorghum, and rice, but also on alfalfa, barley, buckwheat, oats, millet, peanuts, ryegrass, sugar beets, Sudan grass, soybeans, tobacco, tomatoes, potatoes, onions, and wheat. The fall armyworm is a major agricultural pest under global warning by the Food and Agriculture Organization of the United Nations. In 2017, it was ranked among the "Top Ten Plant Pests in the World" by the International Centre for Agricultural and Biosciences. In 2018, it caused over $3 billion in economic losses in Africa, and in January 2019, it entered China and spread rapidly. In 2020, it was listed as a Class I crop disease and pest in China, ranking first on the list.

[0051] The "beet armyworm (Spodoptera exigua)" described in this invention is an insect belonging to the genus Spodoptera in the family Noctuidae of the order Lepidoptera. The beet armyworm is a global agricultural pest, widely distributed in temperate, subtropical, and tropical regions worldwide. In China, its distribution is extremely widespread, covering almost every province. The beet armyworm is an omnivorous insect with a very wide range of pests, known to damage over 170 plant species, including vegetables, fruits, grain crops, cash crops, and flowers. When damaging corn, it mainly feeds on leaves, ears, and kernels, affecting yield; when damaging soybeans, it mainly feeds on leaves.

[0052] The "spodoptera litura" (Tabacco cutworm) described in this invention is an insect belonging to the genus Spodoptera in the family Noctuidae of the order Lepidoptera. Spodoptera litura is reported to be distributed in parts of Asia, Africa, Oceania, South America, and Europe; in China, its distribution is widespread, covering almost the entire country. Spodoptera litura is an omnivorous insect that damages nearly 100 families and over 300 species of plants, including apples, pears, corn, wheat, rice, sorghum, soybeans, and cotton. When damaging corn, it mainly feeds on leaves, and in severe cases, on the husks and kernels of the ear; when damaging soybeans, it mainly feeds on leaves, and in severe cases, on the flower buds and pods, affecting fruit setting and yield.

[0053] 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).

[0054] The “recombinant” nucleic acid (or DNA) molecule described in this invention refers to a nucleic acid sequence (or DNA) in a recombinant bacterial or plant host cell. In some embodiments, the “isolated” or “recombinant” nucleic acid does not contain sequences that are naturally located on the flanking side of the nucleic acid in the genomic DNA of the organism from which the nucleic acid is derived (i.e., sequences located at the 5’ and 3’ ends of the nucleic acid).

[0055] In this invention, the term "recombinant" refers to non-naturally occurring DNA, proteins, cells, seeds, or organisms that are induced by genetic engineering and do not normally exist in nature. A recombinant DNA molecule is a DNA molecule that occurs non-naturally in nature and is produced by human intervention, such as a DNA molecule composed of at least two heterologous DNA molecules. Genetic engineering refers to the creation of non-natural DNA, proteins, or organisms that do not normally exist in nature and require human intervention. Genetic engineering can be used to conceive and create engineered DNA, proteins, or organisms using one or more of the following biotechnologies: molecular biology, protein biochemistry, bacterial transformation, and plant transformation.

[0056] 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.

[0057] Examples of conservative 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. 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.

[0058] 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 essential amino acid residues for their activity, and therefore the selected unsubstituted residues, can be identified according to methods known in the art, such as site-directed mutagenesis or alanine scanning mutagenesis. 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 that molecule. The substrate-enzyme interaction sites can also be determined by analysis of their three-dimensional structure, which can be determined by techniques such as nuclear magnetic resonance analysis, crystallography, or photoaffinity labeling.

[0059] Therefore, amino acid sequences that have a certain degree of homology with the amino acid sequences shown in any one of sequences 1-4 of this invention are also included in this invention. These sequences have at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence identity with the sequences of this invention. Generally, "sequence identity" refers to the degree to which two best-aligned DNA or amino acid sequences remain unchanged across the entire alignment window of components (e.g., nucleotides or amino acids). The "identity score" of the aligned segments of the test sequence and the reference sequence is calculated by dividing the number of common components shared by the two aligned sequences by the total number of components in the reference sequence segment (i.e., the complete reference sequence or a smaller defined portion of the reference sequence). The "identity %" is the identity score multiplied by 100. For example, the BLAST algorithm, available from the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov), is used to determine this using default parameters.

[0060] The "optimized nucleic acid sequence" described in this invention refers to a nucleic acid that, under appropriate conditions, can be optimized to increase expression in a host organism. Therefore, when the host organism is bacteria or a plant, the synthesized nucleic acid can utilize bacterial or plant-preferred codons to improve expression.

[0061] Suitable methods for transforming host plant cells include any method known in the art for introducing DNA into cells (e.g., stably integrating a recombinant DNA construct into a plant chromosome). Exemplary and widely used methods for introducing recombinant DNA constructs into plants include the Agrobacterium transformation system, well known to those skilled in the art. Another exemplary method for introducing recombinant DNA constructs into plants is by inserting the recombinant DNA construct into the plant genome at a predetermined site using a site-directed integration approach. Site-directed integration can be achieved by any method known in the art, such as using zinc finger nucleases, engineered or naturally occurring large-scale nucleases, TALE endonucleases, or RNA-directed endonucleases (e.g., the CRISPR / Cas9 system). Transgenic plants can be regenerated from transformed plant cells using plant cell culture methods. Methods for regenerating plants are also well known in the art. For example, Ti plasmid vectors have been used for delivering exogenous DNA, as well as direct DNA uptake, liposomes, electroporation, microinjection, and microelectromagnetism. A transgenic plant homozygous for the transgene (i.e., two copies of the transgene's alleles) can be obtained by self-pollinating (self-crossing) a transgenic plant containing a single transgene allele with itself (such as an F0 plant) to produce F1 seeds. One-quarter of the resulting F1 seeds are homozygous for the transgene. Plants growing from germinating F1 seeds can be tested for conjugation using SNP assays, DNA sequencing, or thermoamplification assays that allow for differences between heterozygotes and homozygotes; this is called a conjugation assay.

[0062] In this invention, the term "DNA construct" refers to a recombinant DNA molecule containing two or more heterologous DNA sequences. DNA constructs can be used for transgenic expression and can be contained in vectors and plasmids. DNA constructs can be used in vectors for transformation purposes, i.e., introducing heterologous DNA into host cells to produce transgenic cells and plants, and can also be contained in plasmid DNA or genomic DNA of transgenic cells, plants, or parts thereof, or seeds. A "plant transformation vector" typically contains a plasmid vector containing cis-acting sequences required for T-DNA transfer (e.g., left and right boundaries), a selective marker engineered to be expressed in plant cells, and a target gene. This plasmid vector also contains sequences required for bacterial replication, with the cis-acting sequences arranged in a manner that allows efficient transfer to and expression in plant cells. For example, the selective marker gene and the pest-killing gene are located between the left and right boundaries. As understood in the art, the “plant transformation vector” contains virulence functions (Vir gene) that allow infection of plant cells by Agrobacterium, as well as DNA transfer via boundary sequence cleavage and Vir-mediated DNA transfer (Hellens and Mullineaux, Trends in Plant Science, (2000) 5:446-451).

[0063] 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 and plastid genome and the mitochondrial genome.

[0064] Transgenic plants may contain a superposition of one or more insecticidal polynucleotides of the present invention with one or more additional polynucleotides, resulting in the production or inhibition of multiple polypeptide sequences. Transgenic plants containing multiple superpositions of polynucleotide sequences can be obtained by conventional breeding methods or by genetic engineering methods. These methods include, but are not limited to: breeding individual lines each containing the desired polynucleotide, transforming a transgenic plant containing the insecticidal gene of the present invention with subsequent gene transformation, and co-transforming the gene into a single plant cell. The transgenic plants, progeny, seeds, plant cells, and plant parts of the present invention may also contain one or more additional traits. Additional traits can be introduced by hybridizing a transgenic plant containing a nucleic acid sequence encoding the insecticidal protein described in the present invention with another plant containing one or more additional traits. The term "trait" refers to a phenotype obtained from a specific sequence or sequence set. The term "hybridization" refers to breeding two individual plants to produce progeny plants. The two plants can thus be hybridized to produce progeny containing the desired trait from each parent. The term "progeny" refers to any generation of offspring of the parent plants, and the transgenic progeny contains a nucleic acid sequence encoding the insecticidal protein described in the present invention inherited from at least one parent plant. Additional traits can also be introduced by co-transformation of a DNA construct containing the additional trait with a DNA construct containing a nucleic acid sequence encoding the insecticidal protein of the present invention (all DNA constructs being present as part of the same vector used for plant transformation), or by inserting the additional trait into a transgenic plant containing the DNA construct of the present invention (containing a nucleic acid sequence encoding the insecticidal protein of the present invention) or vice versa (e.g., by plant transformation, or by gene editing in transgenic plants or plant cells). These additional traits include, but are not limited to: resistance to diseases, insects, and herbicides; tolerance to heat and drought; shortened crop maturity time; improved industrial processing (e.g., for converting starch or biomass into fermentable sugars); and improved agronomic qualities (e.g., high oil and high protein content).

[0065] The regulatory sequences selected in the vector of this invention are well known to those skilled in the art. These regulatory sequences include, but are not limited to, promoters (e.g., CaMV 35S promoter, maize Ubi promoter, rice GOS2 gene promoter, etc.), transport peptides (e.g., chloroplast-targeting transport peptides, etc.), terminators (e.g., polyadenylation signal sequences of NOS gene, pinII gene, E9 gene, etc.), enhancers (e.g., CaMV enhancer, FMV enhancer, MMV enhancer, etc.), leader sequences (e.g., TMV leader sequence, MDMV leader sequence, potato virus Y group leader sequence, etc.), introns (e.g., maize hsp70 intron, maize ubiquitin intron, CAT-1 intron, etc.), and other regulatory sequences effectively linked to the insecticidal gene.

[0066] In this invention, the term "plant part" or "plant tissue" includes plant cells, plant protoplasts, plant cell tissue cultures from which plants can regenerate, plant callus, plant clusters, and complete plant cells in a plant or in parts of a plant such as embryos, pollen, ovules, seeds, leaves, flowers, branches, fruits, kernels, spikes, rachis, outer shells, stems, roots, root tips, anthers, etc.

[0067] In this invention, the term "plant propagule" includes, but is not limited to, plant sexual propagules and plant asexual propagules. The plant sexual propagule includes, but is not limited to, plant seeds; the plant asexual propagule refers to a plant's vegetative organ or a specific tissue that can produce new plants under in vitro conditions; the vegetative organ or specific tissue includes, but is not limited to, roots, stems, and leaves. For example, plants that use roots as asexual propagules include strawberries and sweet potatoes; plants that use stems as asexual propagules include sugarcane and potatoes (tuber); and plants that use leaves as asexual propagules include aloe vera and begonias.

[0068] The insecticidal protein of this invention can be applied to a variety of plants. The dicotyledonous plants include, but are not limited to, alfalfa, beans, cauliflower, cabbage, carrots, celery, cotton, cucumber, eggplant, lettuce, melon, peas, pepper, zucchini, radish, rapeseed, spinach, soybean, pumpkin, tomato, Arabidopsis thaliana, peanut, or watermelon. Preferably, the dicotyledonous plants refer to cucumber, soybean, Arabidopsis thaliana, tobacco, cotton, peanut, or rapeseed. The monocotyledonous plants include, but are not limited to, corn, rice, sorghum, wheat, barley, rye, millet, sugarcane, oats, or turfgrass. Preferably, the monocotyledonous plants refer to corn, rice, sorghum, wheat, barley, millet, sugarcane, or oats.

[0069] The terms "polypeptide," "peptide," "protein," and "protein protein" are used interchangeably in this invention and refer to polymers of amino acid residues. One or more amino acid residues in the polymer are an artificial chemical analog of a corresponding naturally occurring amino acid, and a naturally occurring amino acid polymer. The polypeptides of this invention can be generated by recombinant synthesis or by chemical synthesis.

[0070] In this invention, the term "transgenic" refers to a DNA molecule that has been incorporated into the genome of an organism due to human intervention (e.g., plant transformation methods).

[0071] The articles “an” and “a kind” used in this invention refer to one or more kinds (i.e., at least one). For example, “an element” means one or more elements.

[0072] The term “comprising” or variations thereof, such as “containing,” “including,” or “including,” means to include the said element, integer, or step, or a group of elements, integers, or steps, but does not exclude any other element, integer, or step, or a group of elements, integers, or steps.

[0073] This invention provides an insecticidal protein and its use, wherein the insecticidal proteins GRIC31 to GRIC34 can all exhibit insecticidal activity against one or more lepidopteran insects; compared with Cry1Da1_7 and Cry1Da2 proteins, some insecticidal proteins have enhanced insecticidal activity against at least one insect.

[0074] The technical solution of the present invention will be further described in detail below through embodiments. Attached Figure Description

[0075] The accompanying drawings, which form part of this specification, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an undue limitation of the invention. In the drawings:

[0076] Figure 1 shows the effects of four insects feeding on transgenic soybean plants sGRIC31 to sGRIC34, representing the insecticidal protein and its uses in this invention. CBW represents the cotton bollworm (Helicoverpa armigera), FAW represents the fall armyworm (Spodoptera frugiperda), TCW represents the beet armyworm (Spodoptera litura), and BAW represents the beet armyworm (Spodoptera exigua). sGRIC31 represents soybean expressing the insecticidal protein GRIC31; sGRIC32 represents soybean expressing the insecticidal protein GRIC32; sGRIC33 represents soybean expressing the insecticidal protein GRIC33; and sGRIC34 represents soybean expressing the insecticidal protein GRIC34.

[0077] Figure 2 shows the effects of two insects feeding on transgenic maize plants mGRIC31 or mGRIC33, respectively, in relation to the insecticidal protein and its uses in this invention; where CBW represents the cotton bollworm (Helicoverpa armigera) and FAW represents the fall armyworm (Spodoptera frugiperda); mGRIC31 represents maize expressing the insecticidal protein GRIC31; and mGRIC33 represents maize expressing the insecticidal protein GRIC33. Detailed Implementation

[0078] The technical solution of the insecticidal protein of the present invention and its uses is further illustrated below through specific embodiments. The methods and operations described in the following embodiments are exemplary and should not be construed as limiting.

[0079] First embodiment: Obtaining the insecticidal protein and its coding sequence of the present invention.

[0080] The amino acid sequences of insecticidal proteins GRIC31 to GRIC34 and the optimized nucleic acid sequences were obtained by modifying Cry1Da1_7 and Cry1Da2 proteins. The relevant sequence information is shown in Table 1.

[0081] As is known to those skilled in the art, different codons can encode the same amino acid due to the degeneracy of the genetic codon. Therefore, the nucleotide sequences in Table 1 are exemplary and not limiting.

[0082] Table 1. SEQ ID NO of each insecticidal protein

[0083] Second embodiment: Insecticidal activity determination of insecticidal proteins GRIC31 to GRIC34 in soybeans.

[0084] 1. Obtaining transgenic soybean plants

[0085] DNA constructs comprising the optimized nucleic acid sequences SEQ ID NO:5-10 of the first embodiment were constructed sequentially. Each DNA construct comprises two tandem expression cassettes: the first expression cassette consists of an optimized nucleic acid sequence of any of the insecticidal proteins GRIC31 to GRIC34, the Cry1Da1_7 protein, or the Cry1Da2 protein (any one of SEQ ID NO:5-10) effectively linked to the promoter of the cauliflower mosaic virus; the second expression cassette consists of an optimized nucleic acid sequence of the cauliflower mosaic virus promoter effectively linked to the phosphoserin acetyltransferase gene (PAT) and effectively linked to the terminator of the PAT gene (the cauliflower mosaic virus promoter sequence can be found in Chinese patent application CN202411783526.1, and the sequences of the phosphoserin acetyltransferase gene and the terminator of the PAT gene can be found in Chinese patent application CN 202510474169.9).

[0086] Each of the above DNA constructs was ligated into a plant transformation vector. Using the Agrobacterium-mediated transformation method known in the art, each of the above plant transformation vectors was transformed into wild-type soybean seeds to obtain transgenic soybean plants. Transgenic soybean plants containing optimized nucleic acid sequences of any one of the insecticidal proteins from GRIC31 to GRIC34, the Cry1Da1_7 protein, or the Cry1Da2 protein, and which were single copies, were obtained by TaqMan assay for testing.

[0087] 2. Determination of insect resistance in transgenic soybean plants

[0088] Soybean plants with optimized nucleic acid sequences of insecticidal protein GRIC31 to GRIC34 (sGRIC31 to sGRIC34), soybean plants with optimized nucleic acid sequences of Cry1Da1_7 protein (s1Da1_7), soybean plants with optimized nucleic acid sequences of Cry1Da2 protein (s1Da2), wild-type soybean plants (sCK), and the following four insects—cotton bollworm (CBW), fall armyworm (FAW), beet armyworm (TCW), and beet armyworm (BAW)—were used to determine their insect resistance. Fresh leaves at stages V3-V4 were selected, washed, and cut into strips approximately 1cm × 4cm. One to four leaf strips were placed on moisturizing filter paper at the bottom of a round plastic petri dish. Ten newly hatched larvae of the pests to be tested were placed in each dish. After 3 days of incubation at 25-28℃, 70-80% relative humidity, and a photoperiod (light / dark) of 16:8, the mortality rate and larval development of each insect species were investigated. The corrected lethality rate for each insect species was calculated, with three replicates for each treatment. The corrected lethality rate was calculated as follows: (Lethality rate of each insect species after feeding on sGRIC31 to sGRIC34 - Lethality rate of sCK) / (1 - Lethality rate of sCK) × 100%. The mean corrected lethality rate was calculated using the corrected lethality rate, and the standard deviation was used to show the degree of fluctuation in the results of different replicates of each insect species feeding on sGRIC31 to sGRIC34. The formula for calculating the standard deviation is well known to those skilled in the art. Compared with larvae fed on sCK, the following criteria were defined as follows: highly significant efficacy (denoted as "++++") with an average corrected mortality rate of 75% or higher; significant efficacy (denoted as "+++") with an average corrected mortality rate between 50% and 75%; effective efficacy (denoted as "++") with an average corrected mortality rate between 25% and 50%; slightly effective efficacy (denoted as "+") with an average corrected mortality rate between 10% and 25% or with only developmental inhibition in larvae; and ineffective efficacy (denoted as "-") with an average corrected mortality rate below 10% or with no developmental inhibition in larvae (similar to sCK). The experimental results are shown in Table 2 and Figure 1.

[0089] Table 2. Mean corrected lethality and resistance rating of four insect species against transgenic soybean plants expressing insecticidal proteins GRIC31 to GRIC34.

[0090] The experimental results in Table 2 show that: (1) compared with the negative control sCK, insecticidal proteins GRIC31 to GRIC34 all exhibited insecticidal activity against at least two lepidopteran insects; (2) compared with the positive control s1Da1_7, sGRIC33 and sGRIC34 enhanced insecticidal activity against beet armyworm; (3) compared with the positive control s1Da2, sGRIC31 to sGRIC34 all enhanced insecticidal activity against cotton bollworm; sGRIC31, sGRIC33, and sGRIC34 enhanced insecticidal activity against fall armyworm; and sGRIC33 and sGRIC34 enhanced insecticidal activity against beet armyworm. This activity of insecticidal proteins GRIC31 to GRIC34 is sufficient to adversely affect the growth of the target lepidopteran pests, thereby controlling them in the field.

[0091] Third Example: Insecticidal Activity Determination of Insecticidal Proteins GRIC31 to GRIC34 in Maize

[0092] 1. Obtaining transgenic maize plants

[0093] The plant transformation vectors obtained in Part 1 of the second embodiment were transformed into wild-type maize embryos using Agrobacterium-mediated transformation, a method known in the art, to obtain transgenic maize plants. Transgenic maize plants containing optimized nucleic acid sequences of the GRIC31 protein, GRIC33 protein, Cry1Da1_7 protein, or Cry1Da2 protein, respectively, and which were single copies, were obtained by TaqMan assay for testing.

[0094] 2. Determination of insect resistance in transgenic maize plants

[0095] Following the method for detecting insect resistance using soybean leaves in Part 2 of the second embodiment, corn plants with optimized nucleic acid sequences of insecticidal protein GRIC31 (mGRIC31), GRIC33, Cry1Da1_7, and Cry1Da2 were subjected to insect resistance testing. Wild-type corn plants (mCK) and two insects, namely bollworm (CBW) and fall armyworm (FAW), were also tested. The results are shown in Table 3 and Figure 2.

[0096] Table 3. Average corrected lethality and resistance rating of two insect species against transgenic maize plants expressing insecticidal proteins GRIC31 and GRIC33.

[0097] The experimental results in Table 3 show that the insecticidal activity of the insecticidal proteins GRIC31 and GRIC33 is stable in transgenic maize plants and there is no significant difference from the experimental results of transgenic soybean plants. This indicates that this activity can be stable in different crops and is sufficient to have an adverse effect on the growth of target lepidopteran pests, thereby controlling them in the field.

[0098] In summary, this invention discloses for the first time the insecticidal proteins GRIC31 to GRIC34, which can not only be stably expressed in plants, but also exhibit activity against at least two lepidopteran insects, thus having a negative effect on the growth and development of a wider range of target lepidopteran insects and enabling their control in the field.

[0099] 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, characterized in that, include: (a) A protein having the amino acid sequence shown in any one of SEQ ID NO: 1-4; or (b) A protein derived from (a) whose amino acid sequence in (a) has been substituted and / or deleted and / or added with one or more amino acids and has insecticidal activity.

2. An insecticidal gene, characterized in that, include: (d) The nucleotide sequence encoding the insecticidal protein of claim 1; or (e) A nucleotide sequence that hybridizes to the nucleotide sequence specified in (d) under stringent conditions and encodes a protein with insecticidal activity; or (f) Having a nucleotide sequence shown in any of SEQ ID NO:5-8.

3. An expression cassette, DNA construct or recombinant vector, characterized in that, The insecticidal gene of claim 2 is contained under the regulation of a effectively linked regulatory sequence.

4. A transgenic host organism comprising the insecticidal gene of claim 2, or the expression cassette of claim 3, the DNA construct, or the recombinant vector; Preferably, the transgenic host organism includes plants, bacteria, yeast, baculoviruses, nematodes, or algae; more preferably, the bacteria are Bacillus thuringiensis or Escherichia coli.

5. A method of producing an insecticidal protein, characterized by, include: Obtain cells of a transgenic host organism containing the insecticidal gene of claim 2, or the expression cassette of claim 3, the DNA construct, or the recombinant vector; The cells of the transgenic host organism were cultured under conditions that allowed for the production of insecticidal proteins; The insecticidal protein was recovered; Preferably, the transgenic host organism includes plant cells, animal cells, bacteria, yeast, baculovirus, nematodes, or algae; Preferably, the plant is corn, soybean, cotton, rice or wheat; and the bacteria is Bacillus thuringiensis or Escherichia coli.

6. A method for increasing the range of target insects, characterized in that, include: The insecticidal protein of claim 1 is expressed in plants together with at least one insecticidal polynucleotide different from the insecticidal protein of claim 1; Preferably, the insecticidal polynucleotide encodes Cry-type insecticidal proteins, Vip-type insecticidal proteins, ETX / MTX-type insecticidal proteins, protease inhibitors, lectins, α-amylases, or peroxidases. Optionally, the insecticidal polynucleotide is a dsRNA that inhibits important genes in the target insect.

7. A method of producing a pest-resistant plant, comprising, include: Introduce the insecticidal gene of claim 2, or the expression cassette of claim 3, the DNA construct, or the recombinant vector into a plant; Preferably, the plant is corn, soybean, cotton, rice or wheat.

8. A method for protecting a plant from damage caused by insects, characterized in that, include: Introducing the insecticidal gene of claim 2, or the expression cassette of claim 3, the DNA construct, or the recombinant vector into a plant, so that the plant produces a sufficient amount of insecticidal protein to protect it from insect damage; Preferably, the plant is corn, soybean, cotton, rice or wheat.

9. A method of controlling pests, characterized by, include: The pest is brought into contact with an inhibitory amount of the insecticidal protein of claim 1 or a protein with insecticidal activity encoded by the insecticidal gene of claim 2; Preferably, the pest is a lepidopteran insect.

10. Use of the insecticidal protein of claim 1 to control lepidopteran insects.

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