Insecticidal protein and use thereof

Abstract

Provided are an insecticidal protein and a use thereof. The insecticidal protein comprises: (a) a protein consisting of the amino acid sequence as shown in any one of SEQ ID NOs: 1-7; or (b) a protein derived from (a) by substitution, deletion, or addition of one or more amino acids in the amino acid sequence of (a) while having insecticidal activity. The insecticidal protein has improved toxicity against 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] To meet this need, this invention discloses novel Cry1B insecticidal proteins that exhibit activity against major target lepidopteran pest species, named GRIC24-GRIC30 respectively.

[0007] 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

[0008] 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 the Asian corn borer, cotton bollworm, or fall armyworm.

[0009] In one aspect, to achieve the above objectives, the present invention provides an insecticidal protein in one embodiment, comprising:

[0010] (a) A protein having the amino acid sequence shown in any one of SEQ ID NO: 1-7; or

[0011] (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.

[0012] In another embodiment, the present invention provides an insecticidal gene, comprising:

[0013] (a) The nucleotide sequence encoding the insecticidal protein of claim 1; or

[0014] (b) A nucleotide sequence that hybridizes to the nucleotide sequence defined in (a) under stringent conditions and encodes a protein with insecticidal activity; or

[0015] (c) Having a nucleotide sequence shown in any one of SEQ ID NO:9-15.

[0016] 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.

[0017] 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;

[0018] Preferably, the transgenic host organism includes plants, bacteria, yeast, baculoviruses, nematodes, or algae;

[0019] Furthermore, the bacteria are Bacillus thuringiensis or Escherichia coli.

[0020] 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.

[0021] 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.

[0022] On the other hand, to achieve the above objectives, one embodiment of the present invention provides a method for generating insecticidal proteins, comprising:

[0023] Obtain cells of a transgenic host organism containing the insecticidal gene, or the expression cassette, the DNA construct, or the recombinant vector;

[0024] The cells of the transgenic host organism were cultured under conditions that allowed for the production of insecticidal proteins;

[0025] The insecticidal protein was recovered;

[0026] In a preferred embodiment, the transgenic host organism includes plant cells, animal cells, bacteria, yeast, baculovirus, nematodes, or algae;

[0027] In a more preferred embodiment, the plant is corn, soybean, cotton, rice, or wheat; and the bacteria is Bacillus thuringiensis or Escherichia coli.

[0028] 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.

[0029] In this invention, the expression of the insecticidal proteins GRIC24 to GRIC30 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 protein 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.

[0030] 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.

[0031] In other alternative embodiments, the insecticidal polynucleotide is a dsRNA that inhibits important genes in the target insect.

[0032] 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.

[0033] In a preferred embodiment, the plant is corn, soybean, cotton, rice, or wheat.

[0034] 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.

[0035] In a preferred embodiment, the plant is corn, soybean, cotton, rice, or wheat.

[0036] 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.

[0037] In a preferred embodiment, the pest is a lepidopteran insect.

[0038] In yet another embodiment of the present invention, there is a use of the insecticidal protein to control lepidopteran insects.

[0039] 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.

[0040] 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.

[0041] 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.

[0042] 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 are intended to encompass a broader range of Cry toxins found in various Bt strains, including toxins such as Cry1, Cry2, or Cry3. Sequences of all Bt proteins discovered to date and named by the Bt Protein Nomenclature Committee, along with their respective domains, are available for download from the Bt Protein Database BPPRC (bpprc-db.org).

[0043] A comprehensive consideration of publicly available structural analyses of Bt toxins and reported functions related to specific structures and motifs suggests that specific regions of the toxin are associated with specific functions and discrete steps in the protein's mode of action. For example, many toxins isolated from Bt are typically described as comprising three domains: a seven-helical bundle involved in pore formation, a three-lamellar domain associated with receptor binding, and a β-sandwich motif. Nucleic acid sequences encoding Cry family peptides were obtained, and the characteristics of the target domains and their connections to surrounding domains were analyzed based on considerations of the proposed target domain functions in the toxin's mode of action. Similar domains and closely linked sequence domains were replaced using recombination techniques, and the resulting peptides' pest-killing activity was 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.

[0044] 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. Cry1B.868 protein possesses typical Cry1 protein structural features and has been extensively studied due to its excellent insecticidal activity against lepidopteran insects; however, its protein structure and how it interacts with insect intestinal cell receptors remain unclear.

[0045] The Asian corn borer (Ostrinia furnacalis), commonly known as the corn borer, is an insect belonging to the genus Ostrinia in the family Pyralidae of the order Lepidoptera. It is a major pest in corn production in my country. This insect feeds on corn leaves and bores into the main stem or ears of corn, reducing photosynthesis, affecting nutrient transport, and causing various secondary diseases, leading to reduced corn yield and quality. In recent years, with changes in climate conditions, farming practices, increased corn planting density, and improved fertilizer and water conditions, the damage caused by the Asian corn borer has become increasingly severe.

[0046] The "peach moth (Conogethes punctiferalis)" described in this invention is an insect belonging to the genus Conogethes in the family Pyralidae of the order Lepidoptera. The peach moth is mainly distributed in Korea, Japan, Indonesia, India, and Sri Lanka; in China, it is found in Liaoning, Beijing, Hebei, Jiangsu, Zhejiang, Shandong, and Guizhou provinces. The peach moth primarily damages fruit trees such as peach, pear, apple, plum, and hawthorn, as well as various crops including corn, onions, sunflowers, and castor beans. When damaging corn, it bores into the stems, feeding on leaves, ears, and kernels, causing significant yield reductions.

[0047] The cotton bollworm (Helicover paarmigera), also known as the peach borer or heart borer, is an insect belonging to the genus Helicoverpas 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 regions 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.

[0048] 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.

[0049] 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.

[0050] The "Oriental armyworm (Mythimna separata)" described in this invention is an insect belonging to the genus Mythimna in the family Noctuidae of the order Lepidoptera. It is mainly distributed in Asian countries, as well as Australia and New Zealand; in China, it can be found in all regions except Xinjiang. The Oriental armyworm primarily damages grasses such as wheat, rice, corn, sorghum, and barley.

[0051] The "black cutworm (Agrotis ipsilon)" described in this invention is an insect belonging to the genus Agrotis in the family Noctuidae of the order Lepidoptera. Black cutworms are a global pest with a wide distribution, found on almost every continent, including Asia, Africa, Europe, and the Americas. In China, black cutworms are extremely widespread, covering almost all provinces. They primarily damage cotton, corn, wheat, sorghum, tobacco, potatoes, hemp, beans, vegetables, and various low-growing herbaceous plants. They also harm seedlings of linden, ash, walnut, and Korean pine. Young larvae feed on the leaves and tender stems of seedlings, while older larvae damage the base of the stems and roots, causing gaps in the rows.

[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] The diamondback moth (Plutella xylostella (Linnaeus)) described in this invention is an insect belonging to the genus Plutella in the family Plutellaidae of the order Lepidoptera. The diamondback moth is a global agricultural pest, most severe in tropical and subtropical regions. In North China, it has 4-6 generations per year, 10-11 generations in the middle and lower reaches of the Yangtze River, and 20 generations in Guangdong and Guangxi, often exhibiting multiple generations of damage. In North China, it overwinters as a pupa, while in South China, there is no overwintering phenomenon. The diamondback moth is an oligophagous pest, generally feeding only on cruciferous vegetables. The larvae damage the leaves; early-instar larvae burrow into the leaf tissue and feed on the mesophyll, while slightly larger larvae gnaw on the epidermis and mesophyll, leaving only one side of the epidermis, forming a transparent spot, commonly known as a "window." Third- and fourth-instar larvae create holes and notches in the leaves; in severe cases, the leaves are eaten into a net-like pattern, or the damage is concentrated in the heart of the vegetable, feeding on the growing point and reducing the edible and commercial value of the vegetables. Currently, the diamondback moth is becoming increasingly resistant to pesticides, posing a significant challenge to vegetable production.

[0054] 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.

[0055] 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).

[0056] 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).

[0057] In this invention, the term "recombinant" refers to non-naturally occurring DNA, proteins, cells, seeds, or organisms that are induced by genetic engineering and are not normally found in nature. "Recombinant DNA molecule" refers to a DNA molecule that is non-naturally occurring in nature and 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 are not normally found 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.

[0058] 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.

[0059] 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.

[0060] 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.

[0061] Therefore, amino acid sequences that have a certain degree of homology with the amino acid sequences shown in any one of sequences 1-7 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.

[0062] 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.

[0063] 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.

[0064] 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).

[0065] 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.

[0066] 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).

[0067] 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.

[0068] 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.

[0069] 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.

[0070] 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.

[0071] 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.

[0072] 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).

[0073] 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.

[0074] 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.

[0075] This invention provides an insecticidal protein and its use, wherein the insecticidal proteins GRIC24 to GRIC30 can all exhibit insecticidal activity against one or more Lepidoptera insects; compared with Cry1B.868 protein, some insecticidal proteins enhance insecticidal activity against at least one insect or expand at least one insecticidal spectrum.

[0076] The technical solution of the present invention will be further described in detail below through embodiments. Attached Figure Description

[0077] 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:

[0078] Figure 1 shows the effects of three insects feeding on the leaves of transgenic maize plants mGRIC25, mGRIC26, or mGRIC29, respectively, in relation to the insecticidal protein and its uses in this invention. In the figure, FAW represents the fall armyworm (Spodoptera frugiperda), ACB represents the Asian corn borer (Ostrinia furnacalis), and CBW represents the bollworm (Helicoverpa armigera); mGRIC25 represents maize expressing the insecticidal protein GRIC25; mGRIC26 represents maize expressing the insecticidal protein GRIC26; and mGRIC29 represents maize expressing the insecticidal protein GRIC29.

[0079] Figure 2 shows the effects of four insects feeding on the leaves of transgenic soybean plants sGRIC25, sGRIC26, or sGRIC29, respectively, in relation to the insecticidal protein of the present invention and its uses. In this figure, FAW represents the fall armyworm (Spodoptera frugiperda), OAW represents the oriental armyworm (Mythimna separata), BAW represents the beet armyworm (Spodoptera exigua), and CBW represents the cotton bollworm (Helicoverpa armigera); sGRIC25 represents maize expressing the insecticidal protein GRIC25; sGRIC26 represents maize expressing the insecticidal protein GRIC26; and sGRIC29 represents maize expressing the insecticidal protein GRIC29. Detailed Implementation

[0080] The technical solution of the insecticidal protein of the present invention and its use 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.

[0081] First embodiment: Obtaining the insecticidal protein and its coding sequence of the present invention.

[0082] The amino acid sequences of insecticidal proteins GRIC24 to GRIC30 and the optimized nucleic acid sequences were obtained by modifying the Cry1B.868 protein. The relevant sequence information is shown in Table 1.

[0083] 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.

[0084] Table 1. SEQ ID NO of each insecticidal protein

[0085] Second embodiment: Determining the insecticidal activity of prokaryotically expressed insecticidal proteins GRIC24 to GRIC30.

[0086] 1. Expression of insecticidal proteins GRIC24 to GRIC30 in Escherichia coli

[0087] The optimized nucleic acid sequences SEQ ID NO: 9-16 of the first embodiment were synthesized, and the aforementioned optimized nucleic acid sequences were ligated into the protein expression vector pET28a (Novagen, USA, CAT: 69864-3) according to the specification to obtain recombinant prokaryotic expression vectors. Using a heat shock method known in the art, the recombinant expression vectors containing the optimized nucleic acid sequences (any one of SEQ ID NO: 9-16) of the insecticidal proteins GRIC24 to GRIC30 or the Cry1B.868 protein were transformed into *E. coli* BL21(DE3) competent cells (Transgen, China, CAT: CD501) to express the insecticidal proteins GRIC24 to GRIC30 and Cry1B.868 protein. The expressed proteins were extracted using protein extraction methods known in the art and finally dissolved in CBS solution (Sigma-Aldrich, China, CAT: 3041). The concentration of the dissolved protein solution was estimated using the BSA quantification method.

[0088] 2. Insecticidal activity assay of insecticidal proteins GRIC24 to GRIC30

[0089] The insecticidal activity of insecticidal proteins GRIC24 to GRIC30 was determined using the protein solutions with concentrations determined in Part 1 of the second embodiment and the following nine insect species: cotton bollworm (CBW), fall armyworm (FAW), oriental armyworm (OAW), cutworm (BCW), Asian corn borer (ACB), peach fruit borer (YPM), beet armyworm (TCW), sugar beet armyworm (BAW), and diamondback moth (DBM). Artificial feed was added to petri dishes, with approximately 100 μL of protein solution added per gram of feed to achieve a final concentration of 50–200 μg / g. Protein solutions expressing fluorescent protein GFP served as negative controls, and protein solutions expressing Cry1B.868 protein served as positive controls. For the protein solutions of insecticidal proteins GRIC24 to GRIC30, Cry1B.868 protein, and GFP protein, ten newborn larvae of each insect species were placed in each petri dish. Three to six days after each batch was fed, if the larval mortality rate was higher than 10% or the larvae showed developmental inhibition compared to the negative control, it indicated that the insecticidal function was effective and was marked as "+". If the larval mortality rate was lower than 10% or the larvae did not show developmental inhibition (similar to the negative control), it was defined as ineffective and marked as "-". The experimental results are shown in Table 2.

[0090] Table 2. Experimental results of 9 insect species against insecticidal proteins GRIC24 to GRIC30

[0091] The experimental results in Table 2 show that: (1) Compared with the negative control GFP protein, the insecticidal proteins GRIC24 to GRIC30 can all show insecticidal activity against at least one Lepidoptera insect; (2) Compared with the positive control Cry1B.868 protein, the insecticidal protein GRIC26 has expanded the insect resistance spectrum, that is, it has added resistance against peach borer.

[0092] Third Example: Insecticidal Activity Determination of Insecticidal Proteins GRIC24 to GRIC30 in Maize

[0093] 1. Obtaining transgenic maize plants

[0094] DNA constructs comprising the optimized nucleic acid sequences SEQ ID NO: 9-16 of the first embodiment were constructed sequentially. Each DNA construct comprises two tandem expression cassettes: the first expression cassette consists of the maize ubiquitin 1 gene promoter effectively linked to the optimized nucleic acid sequence of any of the insecticidal proteins GRIC24 to GRIC30 or the Cry1B.868 protein (any one of SEQ ID NO: 9-16), and effectively linked to the terminator of the carmine synthase gene; the second expression cassette consists of the maize ubiquitin 1 gene promoter effectively linked to the phosphinic acid acetyltransferase gene (PAT), and effectively linked to the terminator of the carmine synthase gene (the sequences of the remaining elements can be found in Chinese patent application CN202411218579.9).

[0095] 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 maize embryos to obtain transgenic maize plants. Transgenic maize plants containing optimized nucleic acid sequences of either Cry1B.868 protein or any of the insecticidal proteins GRIC24 to GRIC30, and which were obtained as single copies, were used for testing by TaqMan assay.

[0096] 2. Determination of insect resistance in transgenic maize plants

[0097] The insect resistance effect was determined by taking maize plants with optimized nucleic acid sequences of insecticidal protein GRIC24, GRIC30, Cry1B.868, and wild-type maize plants, as well as three insects: fall armyworm (FAW), bollworm (CBW), and Asian corn borer (ACB). Fresh leaves at the V3-V4 stage were selected, washed, and cut into strips of 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 petri 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 the larvae were investigated. Compared with larvae fed on mCK, the following criteria were defined: a larval mortality rate of 75% or higher was defined as highly significant and indicated as "++++"; a larval mortality rate between 50% and 75% was defined as significantly effective and indicated as "+++"; a larval mortality rate between 25% and 50% was defined as effective and indicated as "++"; a larval mortality rate between 10% and 25% or larval developmental inhibition was defined as slightly effective and indicated as "+"; and a larval mortality rate below 10% or no larval developmental inhibition (similar to mCK) was defined as ineffective and indicated as "-". The experimental results are shown in Table 3 and Figure 1.

[0098] Table 3. Experimental results of three insect species on transgenic maize plants expressing insecticidal proteins GRIC24 to GRIC30.

[0099] The experimental results in Table 3 and Figure 1 show that the insecticidal activity of the insecticidal proteins GRIC24 to GRIC30 is stable in transgenic maize plants and there is no significant difference from the experimental results of prokaryotic expressed proteins. In particular, mGRIC26 has improved the insecticidal activity against Asian corn borer compared with m1B.868, which indicates that this activity is sufficient to have an adverse effect on the growth of the target lepidopteran pest and thus control it in the field.

[0100] Fourth Example: Insecticidal Activity Determination of Insecticidal Proteins GRIC24 to GRIC30 in Soybeans

[0101] 1. Obtaining transgenic soybean plants

[0102] The plant transformation vectors obtained in Part 1 of the third embodiment were used to transform wild-type soybean seeds using Agrobacterium-mediated transformation, a method known in the art, to obtain transgenic soybean plants. Transgenic soybean plants containing optimized nucleic acid sequences of either Cry1B.868 protein or any of the insecticidal proteins GRIC24 to GRIC30, and which were single copies, were obtained by TaqMan analysis for testing.

[0103] 2. Insect resistance test of transgenic soybean plants

[0104] Following the method for detecting insect resistance using corn leaves in Part 2 of the third embodiment, soybean plants with optimized nucleic acid sequences of insecticidal protein GRIC24 to GRIC30 (sGRIC24 to sGRIC30), soybean plants with optimized nucleic acid sequences of Cry1B.868 protein (s1B.868), wild-type soybean plants (sCK), and the following four insects—fall armyworm (FAW), oriental armyworm (OAW), beet armyworm (BAW), and cotton bollworm (CBW)—were used to determine the insect resistance effect. The experimental results are shown in Table 4 and Figure 2.

[0105] Table 4. Experimental results of four insect species on transgenic soybean plants expressing insecticidal proteins GRIC24 to GRIC30.

[0106] The experimental results in Table 4 and Figure 2 show that the insecticidal activity of the insecticidal proteins GRIC24 to GRIC30 is stable in transgenic soybean plants and there is no significant difference from the experimental results of prokaryotic expressed proteins. In particular, sGRIC26 and sGRIC29 have non-weakened insecticidal activity against fall armyworm compared with s1B.868, indicating that this activity is sufficient to have an adverse effect on the growth of the target lepidopteran pest and thus control it in the field.

[0107] In summary, this invention discloses for the first time insecticidal proteins GRIC24 to GRIC30, which can not only be stably expressed in plants, but also exhibit activity against at least one type of lepidopteran insect, thus having adverse effects on the growth and development of a wider range of target lepidopteran insects and enabling their control in the field.

[0108] 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: 1-7; 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: (a) The nucleotide sequence encoding the insecticidal protein of claim 1; or (b) A nucleotide sequence that hybridizes to the nucleotide sequence defined in (a) under stringent conditions and encodes a protein with insecticidal activity; or (c) Having a nucleotide sequence shown in any of SEQ ID NO:9-15.

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 a plant 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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