Chimeric insecticidal protein and use thereof

Abstract

The present invention relates to a chimeric insecticidal protein and a use thereof. The protein comprises domain I and domain II of Cry1Ac protein and domain III of Cry1Igg protein which are sequentially linked, and exhibits strong weight-inhibitory activity and lethal activity against various lepidopteran pests. The provided chimeric insecticidal protein broadens the insecticidal spectrum of the original protein, and also enhances toxicity against pests, effectively slowing the development of pest resistance in nature, and has good application prospects in agricultural production.

Description

Chimeric insecticidal proteins and their uses Technical Field

[0001] This invention relates to a chimeric insecticidal protein, a nucleic acid molecule encoding the protein, and methods and uses for controlling lepidopteran pests. Background Technology

[0002] Currently, agricultural production faces both biotic stresses (such as diseases and pests) and abiotic stresses (such as drought, cold damage, and salinity damage), leading to weakened crop growth and reduced yields, posing a significant threat to global food security. Among these, pests are one of the main biotic stress factors affecting agricultural and forestry productivity. As the environmental problems caused by the use of chemical pesticides for pest control become increasingly serious, the use of biological pesticides is gradually coming into focus.

[0003] Bacillus thuringiensis (Bt) is a Gram-positive bacterium widely distributed in nature and is also an entomopathogenic bacterium. The biggest difference between Bt and other Bacillus species is that Bt produces crystal proteins accompanying spore formation in the later stages of its growth; these are generally called parasporal crystal proteins (including Cry and Cyt proteins). These proteins are the main or decisive factors in producing insect pathogenicity. Numerous studies have reported the insecticidal activity of various Bt proteins against Lepidoptera, Coleoptera, Diptera, Hymenoptera, and Homoptera, such as Cry1Ab, Cry1Ac, Cry1F, Cry2Ab, and Cry3Bb. The commercial cultivation of Bt transgenic insect-resistant crops, prepared using plant genetic engineering, has become one of the main drivers of significantly improving agricultural productivity. These transgenic insect-resistant plants not only effectively control the occurrence and damage of target pests but also reduce the use of chemical pesticides, providing important guarantees for food and ecological security. However, with the widespread application of genetically modified crops, insects will evolve resistance to the Bt protein expressed in genetically modified plants under continuous selective pressure. If such resistance cannot be effectively controlled, it will limit the commercial value of genetically modified plant varieties containing Bt protein. At the same time, some Bt proteins also have strong cross-lethal activity against non-target organisms. These problems limit the further application of Bt protein.

[0004] One approach to addressing the aforementioned problems is to rationally design functionally improved Bt chimeric proteins (or fusion proteins) based on the domains of Bt proteins. This method involves introducing or replacing domains of heterologous Bt proteins or other types of insecticidal proteins into a single Bt protein, or recombining domains of different Bt proteins, thereby obtaining Bt chimeric proteins with stronger insecticidal activity, a broader insecticidal spectrum, and even the ability to resist pesticide resistance in target pests. Currently, related research mainly focuses on domain recombination between Cry-like Bt proteins with relatively clear structures and functions. For example, Ballester V et al. reported that replacing domain III of Cry1Ab protein with domain III of Cry1C protein significantly increased the toxicity to diamondback moth compared to both Cry1Ab and Cry1C proteins (Applied and Environmental Microbiology, 1999, 65(5): 1900-1903); SHAH JV et al. reported that replacing domain I of Cry9Aa with domain I of Cry1Ac increased the lethal toxicity to bollworm by 4.9 times compared to Cry1Ac (Archives of Microbiology, 2017, 199(7): 1069-1075); DAS A et al. spliced ​​domain I of Cry1Aa with domain II of Cry1Ab and then with domain III of Cry1Ac, and the resulting hybrid had significantly higher insecticidal activity than the original protein (Frontiers in Plant Science, 2017, 8: 1423.

[0005] However, due to differences in structure, function, receptor binding sites, and insecticidal mechanisms among different Cry proteins, the reassembled chimeric proteins often exhibit incorrect structural forms, or fail to produce chimeric proteins that are identical to the parent protein or have enhanced insecticidal activity. Even with extensive analysis, design, screening, and validation, it is not always possible to construct chimeric insecticidal proteins that exhibit improved insecticidal activity compared to the parent protein. Therefore, designing more novel chimeric proteins with enhanced insecticidal activity and a broader insecticidal spectrum to provide more options for insecticidal protein bodies used in agricultural production remains a hot topic and a challenge in this field.

[0006] Summary of the Invention

[0007] To address the shortcomings of existing technologies, this invention provides a chimeric insecticidal protein capable of simultaneously targeting multiple lepidopteran pests. This chimeric insecticidal protein exhibits strong weight-inhibiting and lethal activity against fall armyworm, cotton bollworm, silver-striped armyworm, beet armyworm, oriental armyworm, and Asian corn borer. After transferring the gene encoding this protein into plants, the plants are endowed with strong insecticidal activity, particularly against fall armyworm and cotton bollworm, achieving mortality rates of 61% and 88%, respectively. The protein provided by this invention is a novel chimeric insecticidal protein that, compared to the original insecticidal protein, possesses a broader insecticidal spectrum and higher insecticidal activity, effectively mitigating the development of pest resistance in nature and showing promising application prospects in agricultural production.

[0008] In a first aspect, the present invention provides a chimeric insecticidal protein comprising a first domain (domain I) and a second domain (domain II) of a Cry1Ac protein and a third domain (domain III) of a Cry1Ig protein connected in sequence.

[0009] Furthermore, the chimeric insecticidal protein comprises, in sequence, a first domain (domain I) and a second domain (domain II) of the Cry1Ac protein, a third domain (domain III) of the Cry1Ig protein, and the protoxin tail of the Cry1Ac protein (the protoxin domain or domains IV-VII of the Cry1Ac protein) or a fragment thereof.

[0010] Preferably, the structural domains are connected sequentially from the 5' end (N end) to the 3' end (C end).

[0011] Preferably, the Cry1Ac protein protoxin tail is a complete protoxin tail.

[0012] More preferably, the Cry1Ac protoxin tail fragment is a truncated 3' (C-terminus) segment of the Cry1Ac protoxin tail.

[0013] Preferably, the accession number of the Cry1Ac protein in GenBank is AJU57501.1 or AAA22331.1, and the accession number of the Cry1Ig protein in GenBank is AGU13866.1.

[0014] More preferably, the Cry1Ac protein protoxin tail is a fragment obtained by truncating 6 amino acids from the 3' end (C-terminus) of the protoxin tail (amino acids 616-1182) of the AJU57501.1 protein.

[0015] More preferably, the Cry1Ac protein protoxin tail can also be the protoxin tail (amino acids 618-1178) of the Cry1Ac protein with GenBank accession number AAA22331.1.

[0016] More preferably, the amino acid sequence of the chimeric insecticidal protein includes SEQ ID NO:1.

[0017] More preferably, the amino acid sequence of the chimeric insecticidal protein includes SEQ ID NO:2.

[0018] In another aspect, the present invention also provides a 3' (C-terminus) truncated form of the above-mentioned chimeric insecticidal protein.

[0019] Preferably, the chimeric insecticidal protein comprises the 3' (C-terminus) truncated sequence of SEQ ID NO:2.

[0020] In a second aspect, the present invention provides a nucleic acid molecule encoding the chimeric insecticidal protein described herein.

[0021] Preferably, the sequence of the nucleic acid molecule includes SEQ ID NO:3 or SEQ ID NO:5;

[0022] Preferably, the sequence of the nucleic acid molecule includes SEQ ID NO:4 or SEQ ID NO:6.

[0023] In a third aspect, the present invention provides an expression cassette comprising the nucleic acid molecule described in this application.

[0024] In a fourth aspect, the present invention provides an expression vector comprising the nucleic acid molecule or expression cassette described in the present invention.

[0025] In a fifth aspect, the present invention provides a host cell comprising the nucleic acid molecule, expression cassette, or expression vector described in the present invention.

[0026] Preferably, the host cell described in this invention is selected from plant cells;

[0027] More preferably, the plant cells are selected from monocotyledonous plant cells or dicotyledonous plant cells;

[0028] More preferably, the plant cells are selected from the cells of soybean, corn, Arabidopsis thaliana, cotton, rapeseed, rice, sorghum, wheat, barley, millet, tobacco, sunflower, sugarcane or oats;

[0029] More preferably, the plant cell is a soybean cell.

[0030] Preferably, the host cell described in this invention is selected from bacteria;

[0031] More preferably, the bacteria are selected from Agrobacterium, Bacillus, or Escherichia coli.

[0032] In a sixth aspect, the present invention provides a plant or plant part comprising the chimeric insecticidal protein, nucleic acid molecule, expression cassette or expression vector described in the present invention.

[0033] Preferably, the plant part is selected from plant cells, plant tissues, plant tissue cultures, and callus cultures;

[0034] Preferably, the plant or plant part is selected from corn, soybean, Arabidopsis thaliana, cotton, rapeseed, rice, sorghum, wheat, barley, millet, tobacco, sunflower, sugarcane or oats.

[0035] In a seventh aspect, this application provides a method for producing transgenic plants, the method comprising:

[0036] 1) Transform the nucleic acid molecules, expression cassettes, or expression vectors described in this application into plant cells;

[0037] 2) Select plant cells containing the nucleic acid molecule, expression cassette, or expression vector;

[0038] 3) Regenerate plants from the selected plant cells.

[0039] Preferably, the plant is selected from monocotyledonous or dicotyledonous plants;

[0040] More preferably, the plant is selected from soybean, corn, Arabidopsis thaliana, cotton, rapeseed, rice, sorghum, wheat, barley, millet, tobacco, sunflower, sugarcane or oats.

[0041] More preferably, the plant is soybean.

[0042] In an eighth aspect, the present invention provides an insecticidal composition comprising the chimeric insecticidal protein described in this application.

[0043] In a ninth aspect, this application provides a method for controlling lepidopteran pests, comprising contacting the lepidopteran pests with the chimeric insecticidal protein, host cell, plant or plant part or insecticidal composition described in this application.

[0044] In a tenth aspect, this application provides a method for controlling lepidopteran pests, comprising introducing the nucleic acid molecule, expression cassette, and expression vector described in this application into a plant, thereby bringing the lepidopteran pests into contact with the plant.

[0045] Preferably, the lepidopteran pests include noctuid moths or pyralid moths;

[0046] More preferably, the lepidopteran pests include fall armyworm, beet armyworm, sugar beet armyworm, silver-striped armyworm, oriental armyworm, cotton bollworm and / or Asian corn borer.

[0047] Preferably, the plant is selected from monocotyledonous or dicotyledonous plants;

[0048] More preferably, the plant is selected from soybean, corn, Arabidopsis thaliana, cotton, rapeseed, rice, sorghum, wheat, barley, millet, tobacco, sunflower, sugarcane or oats.

[0049] More preferably, the plant is soybean.

[0050] In the eleventh aspect, this application provides the use of the chimeric insecticidal protein, nucleic acid molecule, expression cassette, recombinant expression vector, or insecticidal composition in controlling lepidopteran pests.

[0051] Preferably, the method involves contacting lepidopteran pests with the insecticidal protein or insecticidal composition described in this application to control lepidopteran pests; or introducing the nucleic acid molecules, expression cassettes, or recombinant expression vectors described in this application into plants to allow lepidopteran pests to contact the plants and thus control the lepidopteran pests.

[0052] Preferably, the lepidopteran pests include noctuid moths or pyralid moths;

[0053] More preferably, the lepidopteran pests include fall armyworm, beet armyworm, sugar beet armyworm, silver-striped armyworm, oriental armyworm, cotton bollworm and / or Asian corn borer.

[0054] Preferably, the plant is selected from monocotyledonous or dicotyledonous plants;

[0055] More preferably, the plant is selected from soybean, corn, Arabidopsis thaliana, cotton, rapeseed, rice, sorghum, wheat, barley, millet, tobacco, sunflower, sugarcane or oats.

[0056] More preferably, the plant is soybean.

[0057] In a twelfth aspect, this application provides a product, commodity, or processed agricultural product obtained by processing the plant or plant part described in this application or the genetically modified plant, wherein the product is grain, starch, seed oil, syrup, flour, coarse flour, cereal, or protein.

[0058] Preferably, the product contains the chimeric insecticidal protein or nucleic acid molecule described in this application.

[0059] The terms "comprising" and "including" as used in this invention mean "including but not limited to".

[0060] The terms "polypeptide," "peptide," and "protein" used in this invention are interchangeable 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.

[0061] The insecticidal proteins described in this invention include not only specific example sequences, but also insecticidal active feature portions and / or fragments (including those with deletions compared to the full-length protein and / or at the ends), variants, mutants, substitutes (proteins with substituted amino acids), chimeras, and fusion proteins of the specific example proteins. The insecticidal active feature portions and / or fragments, variants, mutants, substitutes, chimeras, and fusion proteins are "equivalent proteins" to the specific example sequence proteins described in this invention, that is, proteins that have the same or substantially the same biological activity against lepidopteran pests as the specific example sequence proteins described in this invention.

[0062] Variations, substitutions, deletions, or additions of the amino acid sequence of insecticidal proteins are conventional techniques in the art. Preferably, these variations refer to amino acid substitutions, deletions, additions, or insertions that do not substantially affect the insecticidal activity of the protein. More preferably, such amino acid changes are: minor property changes, i.e., conserved amino acid substitutions that do not significantly affect the folding and / or activity of the protein; minor deletions, typically about 1-5 amino acid deletions; and minor amino or carboxyl terminal extensions, such as an extension of one methionine residue to the amino terminus.

[0063] Those skilled in the art will recognize that Three-Domain Cry (3D Cry) is the most representative type of Cry protein in the Cry protein family. When target pests ingest this type of Cry protein, the alkaline environment of their intestines digests the Cry protein into Cry protoxin. The N-terminus and C-terminus of the Cry protoxin (protoxin domains or domains IV-VII) are further specifically hydrolyzed and removed by proteases, resulting in an activated structure composed of three domains (Domain I, Domain II, and Domain III). Domain I mainly participates in the binding of intestinal wall proteins and insertion into the cell membrane to form perforations. Domain II is mainly involved in the specific recognition and binding of intestinal receptors of pests. Domain III mainly participates in the binding of midgut receptors and insertion into the cell membrane, and also plays a role in maintaining the stability of the protein structure. The insertion of the activated Cry protein into the cell membrane causes perforation and lysis, thereby causing intestinal damage, impaired digestion and absorption, and ultimately leading to the death of the pest. It is evident that the three domains described above are essential components for the function of this type of Cry insecticidal protein. Specifically, in this invention, both the Cry1Ac protein and the Cry1Ig protein belong to the 3D Cry type among the aforementioned Cry proteins.

[0064] Furthermore, the chimeric insecticidal protein of the present invention may include the N-terminal and / or C-terminal portions of the Cry protein protoxin. The chimeric insecticidal protein of the present invention may also be a protein in which the N-terminus and / or C-terminus of the Cry protein protoxin is truncated to remove part or all of the N-terminal and / or C-terminal fragments. On one hand, the chimeric insecticidal protein is a mature (i.e., processed) protein containing domains I, II, and III. In a specific embodiment, the chimeric insecticidal protein comprises a first domain (domain I) and a second domain (domain II) of the Cry1Ac protein, and a third domain (domain III) of the Cry1Ig protein, connected in sequence. On the other hand, the chimeric insecticidal protein is a protein containing domains I, II, III, and the protoxin tail (protoxin domain or domains IV-VII) or fragments thereof. In a specific embodiment, the chimeric insecticidal protein comprises, in sequence, a first domain (domain I) and a second domain (domain II) of the Cry1Ac protein, a third domain (domain III) of the Cry1Ig protein, and a Cry1Ac protein protoxin tail (the protoxin domain or domains IV-VII of the Cry1Ac protein) or a fragment thereof. The Cry1Ac protein protoxin tail fragment may be a truncated segment of the 3' end (C-terminus) of the Cry1Ac protein protoxin tail.

[0065] In a specific embodiment of the present invention, the chimeric insecticidal protein includes Domain I and Domain II of Cry1Ac protein and Domain III of Cry1Ig protein. More specifically, SEQ ID NO:1, SEQ ID NO:3, and SEQ ID NO:5 of this application all include Domain I and Domain II of Cry1Ac and Domain III of Cry1Ig; SEQ ID NO:2, SEQ ID NO:4, and SEQ ID NO:6 of the present invention include Domain I + Domain II of Cry1Ac protein, Domain III of Cry1Ig, and the protoxin tail of Cry1Ac protein (protoxin domain or Domain IV-VII). As disclosed by Shaoyan Liu et al., there is no significant difference in toxicity when feeding pests with the protoxin of 3D Cry protein and the activated toxin after enzymatic cleavage (PLoS One, 2020, 15(1):e0228159). Based on this, and according to research in this field on the structure and insecticidal mechanism of 3D Cry proteins, those skilled in the art can reasonably expect that any protein containing Domain I, Domain II, and Domain III of the Cry protein will possess insecticidal activity. In other words, the chimeric insecticidal protein and nucleic acid molecule described in this application, which contain the above three domains, can both exert normal insecticidal effects.

[0066] In this invention, amino acid sequences that share a certain degree of homology with the amino acid sequence of the chimeric insecticidal protein are included. The amino acid sequence of the chimeric insecticidal protein includes, but is not limited to, amino acid sequences that share a certain degree of homology with the 3' truncated sequence of SEQ ID NO:1 or SEQ ID NO:2 or SEQ ID NO:2. The homology / identity / similarity / identity of these sequences with the sequences of this invention is typically greater than 60%, preferably greater than 75%, more preferably greater than 90%, even more preferably greater than 95%, and can be greater than 99%. Preferred proteins of the invention may also be defined based on more specific ranges of similarity and / or likeness, for example, having 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% similarity to sequences in the examples of the invention.

[0067] As used in this invention, the term "nucleic acid molecule" refers to a polymeric form of nucleotides, which may include both sense and antisense strands of RNA, cDNA, and genomic DNA, as well as synthetic forms and mixed polymers of the foregoing. Nucleotides may refer to ribonucleotides, deoxyribonucleotides, or modified forms of either of these types of nucleotides. As used herein, "nucleic acid molecule" is synonymous with "nucleic acid" and "polynucleotide." Unless otherwise specified, nucleic acid molecules are generally at least 10 bases in length. The term includes both single-stranded and double-stranded forms of DNA. Nucleic acid molecules include dimer (so-called tandem) forms and transcripts of nucleic acid molecules. Nucleic acid molecules may include naturally occurring nucleotides and modified nucleotides linked together by naturally occurring nucleotide linkages and / or non-naturally occurring nucleotide linkages. As will be readily understood by those skilled in the art, nucleic acid molecules may be chemically or biochemically modified, or may contain non-natural or derivatized nucleotide bases.

[0068] Those skilled in the art will recognize that, due to the abundance of genetic codons, many different DNA sequences can encode the same amino acid sequence. Alternative DNA sequences that generate these proteins encoding the same or substantially the same are within the skill level of those skilled in the art, and these different DNA sequences are included within the scope of this invention. The term "substantially the same sequence" refers to sequences with amino acid substitutions, deletions, additions, or insertions that do not substantially affect insecticidal activity, and also includes fragments that retain insecticidal activity.

[0069] As is well known to those skilled in the art, DNA typically exists in a double-stranded form. In this arrangement, one strand is complementary to the other, and vice versa. The replication of DNA in plants produces other complementary strands. The term "coding strand" or "sense strand," as commonly used in the art, refers to the strand that binds to the antisense strand. To express proteins in vivo, typically one strand of DNA is transcribed into a complementary strand of mRNA, which serves as a template for protein translation. The mRNA is actually transcribed from the "antisense strand" of DNA. The "sense" or "coding" strand has a series of codons (codons are three nucleotides, and reading three at a time produces a specific amino acid) that can be read as an open reading frame (ORF) to form the target protein or peptide. This invention also includes RNA and PNA (peptide nucleic acid) that function similarly to the example DNA.

[0070] As used in this invention, the term "encoding" or "coded" refers to a nucleic acid containing the necessary information to guide the translation of a polynucleotide sequence or gene into a specific protein. The information used to encode the protein is detailed using codons. The nucleic acid encoding the protein may contain untranslated sequences (e.g., introns) within the translated region of the nucleic acid, or may lack such inserted untranslated sequences (e.g., in cDNA).

[0071] In this invention, all nucleic acid molecules encoding the chimeric insecticidal protein described herein are within the scope of protection of this invention. In a specific embodiment, the nucleic acid molecule is a nucleic acid molecule encoding a truncated C-terminal (3' end) sequence comprising SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:2. In another specific embodiment, the nucleic acid molecule comprises a truncated SEQ ID NO:3, SEQ ID NO:4, or a truncated SEQ ID NO:4, and a truncated 3' end sequence comprising SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:6.

[0072] Furthermore, sequences possessing insecticidal activity and hybridizing under stringent conditions with nucleic acid molecules encoding chimeric insecticidal proteins as described in this invention are included in this invention. In specific embodiments, sequences hybridizing with 3' truncated sequences comprising SEQ ID NO:3 or SEQ ID NO:4 or SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6 or SEQ ID NO:6 are included in this invention. Any conventional nucleic acid hybridization or amplification method can be used to identify the presence of the chimeric insecticidal protein gene of this invention. Nucleic acid molecules or fragments thereof can specifically hybridize with other nucleic acid molecules under certain conditions. In this invention, if two nucleic acid molecules can form antiparallel double-stranded nucleic acid structures, it can be said that these two nucleic acid molecules can specifically hybridize with each other. If two nucleic acid molecules exhibit perfect complementarity, then one nucleic acid molecule is called a "complement" of the other nucleic acid molecule. In this invention, when every nucleotide of one nucleic acid molecule is complementary to the corresponding nucleotide of another nucleic acid molecule, the two nucleic acid molecules are said to exhibit "perfect complementarity". Two nucleic acid molecules are said to be "minimally complementary" if they can hybridize with sufficient stability to anneal and bind to each other under at least conventional "low-strict" conditions. Similarly, two nucleic acid molecules are said to be "complementary" if they can hybridize with sufficient stability to anneal and bind to each other under conventional "high-strict" conditions. Deviations from perfect complementarity are permissible, as long as such deviations do not completely prevent the two molecules from forming a double-stranded structure. For a nucleic acid molecule to function as a primer or probe, it only needs to be sufficiently complementary in sequence to form a stable double-stranded structure under the specific solvent and salt concentration used.

[0073] In this invention, nucleic acid molecules can specifically hybridize with the complementary strand of a matching nucleic acid molecule under highly stringent conditions. Suitable stringent conditions that promote DNA hybridization, such as treatment with 6.0× sodium chloride / sodium citrate (SSC) at approximately 45°C followed by washing with 2.0× SSC at 50°C, are well known to those skilled in the art. For example, the salt concentration in the washing step can be selected from approximately 2.0× SSC, 50°C for low-stringency conditions to approximately 0.2× SSC, 50°C for high-stringency conditions. Furthermore, the temperature conditions in the washing step can be increased from approximately 22°C (room temperature) for low-stringency conditions to approximately 65°C for high-stringency conditions. Both temperature conditions and salt concentration can be changed, or one can remain constant while the other is changed. Preferably, the stringent conditions of this invention can be specific hybridization with nucleic acid molecules at 65°C in a 6× SSC, 0.5% SDS solution, followed by washing once each with 2× SSC, 0.1% SDS and 1× SSC, 0.1% SDS.

[0074] Furthermore, sequences that are at least about 40%-50% homologous to the nucleic acid sequences of the present invention are also included in the present invention, preferably about 60%, 65% or 70% homologous, or even at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence homology.

[0075] As used in this invention, the terms "similarity," "identity," and "homology," in the context of two nucleic acid or polypeptide sequences, refer to the same residues in the two sequences when compared with maximum correspondence on a specified comparison window. To achieve optimal sequence alignment, the sequence portion in the comparison window may contain additions or deletions (i.e., vacancies) compared to a reference sequence (which contains no additions or deletions). A number of matching positions is generated by determining the number of positions in the sequence where the same nucleotide or amino acid residues appear. This number of matching positions is divided by the total number of positions in the comparison window, and the result is multiplied by 100 to obtain the percentage of sequence identity. This percentage is then calculated.

[0076] In this invention, the term "fragment" or "truncated" nucleic acid or protein sequence refers to a portion of the original nucleic acid or protein sequence or its artificially modified form (e.g., a sequence suitable for plant expression).

[0077] The expression cassette described in this invention refers to a DNA sequence containing all the necessary components that work together to ensure the expression of one or more genes of interest in a host cell, and is a genetic construct with a specific function. The expression cassette includes, but is not limited to, at least one regulatory sequence operatively linked to the nucleic acid molecule that drives its expression. A “regulatory sequence” or “control element” refers to a nucleotide sequence that affects the timing and level / amount of transcription, RNA processing or stability, or the translation of a related coding sequence. Regulatory sequences include, but are not limited to, promoters, transport peptides, terminators, enhancers, leader sequences, introns, marker genes, and other regulatory sequences operatively linked to the nucleic acid molecule. The regulatory sequence may be located upstream and / or downstream of the coding sequence to which it is operatively linked. Furthermore, the regulatory sequence operatively linked to the coding sequence may be located on the relevant complementary strand of the double-stranded nucleic acid molecule.

[0078] As used in this invention, the term "expression" refers to a process by which the coding information of nucleic acid transcription units (including, for example, genomic DNA or cDNA) is translated into operational, non-operational, or structural parts of a cell, typically involving protein synthesis. Gene expression can be influenced by external signals, such as exposure of cells, tissues, or organisms to agents that increase or decrease gene expression. Gene expression can also be regulated at any point in the pathway from DNA to RNA to protein. Regulation of gene expression occurs, for example, by controlling the action on transcription, translation, RNA transport and processing, degradation of intermediate molecules (such as mRNA), or by activation, inactivation, compartmentalization, or degradation of specific protein molecules after their production, or a combination thereof. Gene expression can be measured at the RNA or protein level by any method known in the art, including but not limited to Northern blotting, RT-PCR, Western blotting, or in vitro, in situ, or in vivo protein activity assays.

[0079] In this invention, "linkage" and "operably linked" refer to the connection of nucleic acid sequences, which enables one sequence to provide the function required for the linked sequences. Methods of "linking" nucleic acid sequences include, but are not limited to, in-frame linking and fusion linking. In-frame linking refers to arranging two or more different DNA fragments or biological elements in a specific order and linking them to the same expression cassette or recombinant vector using recombinant DNA technology. In-frame linking allows multiple genes to be expressed simultaneously in the same cell to construct biological systems capable of expressing multiple genes or having complex functions. Fusion linking refers to linking two or more different biological elements or protein sequences together using molecular biology techniques (e.g., linker sequences) to form a new fusion protein or fusion gene.

[0080] In this invention, "operably linked" 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, "operably linked" means that the promoter is linked to the sequence in a manner that allows for efficient translation of the resulting transcript. If the link between the promoter and the coding sequence is a transcript fusion and the desired expression of the encoded protein is desired, such a link is created such that the first translation start codon in the resulting transcript is the start codon of the coding sequence. If the link between the promoter and the coding sequence is a translation fusion and the desired expression of the encoded protein is desired, such a link is created such that the first translation start codon contained in the 5' untranslated sequence is linked to the promoter, and the linking method ensures 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 "operably linked" include, but are not limited to: sequences that provide gene expression functions, i.e., gene expression elements (e.g., 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 functions (i.e., T-DNA boundary sequences, site-specific recombinase recognition sites, and integrase recognition sites); sequences that provide selective functions (i.e., antibiotic resistance markers and biosynthetic genes); sequences that provide scoring marker functions; 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 functions (i.e., bacterial origin of replication, autonomous replication sequences, and centromere sequences).

[0081] The promoters described in this invention are plant-expressible promoters, which ensure the expression of the target gene or nucleic acid linked to them within plant cells. Plant-expressible promoters can be constitutive promoters, examples of which include, but are not limited to, the 35S promoter derived from cauliflower mosaic virus, the Arabidopsis Ubi10 promoter, the maize Ubi promoter, and the promoter of the rice GOS2 gene. Plant-expressible promoters can also be tissue-specific promoters, meaning that the expression level of the coding sequence guided by this promoter is higher in some plant tissues, such as green tissues, than in other plant tissues (this can be determined through conventional RNA assays), such as the PEP carboxylase promoter. Plant-expressible promoters can also be wound-induced promoters. Wound-induced promoters, or promoters that guide wound-induced expression patterns, refer to promoters that significantly increase the expression of the coding sequence under their regulation compared to normal growth conditions when plants experience mechanical or insect-induced trauma. Examples of trauma-inducible promoters include, but are not limited to, promoters of the protease repressor genes (pin I and pin II) of potato and tomato and the maize protease repressor gene (MPI).

[0082] The transport peptide described in this invention, also known as a secretory signal sequence or guide sequence, guides transgenic products to specific organelles or cell compartments. For the receptor protein, the transport peptide can be heterologous. For example, it can target chloroplasts using a sequence encoding a chloroplast transport peptide, or target the endoplasmic reticulum using a 'KDEL' reserved sequence, or target vacuoles using the CTPP of the barley plant lectin gene.

[0083] The leader sequences described in this invention include, but are not limited to: small RNA virus leader sequences, such as the EMCV leader sequence (5' untranslated region of encephalomyocarditis virus); potato Y virus group leader sequences, such as the MDMV (maize dwarf mosaic virus) leader sequence; human immunoglobulin heavy chain binding protein (BiP); untranslated leader sequence of alfalfa mosaic virus capsid protein mRNA (AMV RNA4); and tobacco mosaic virus (TMV) leader sequences.

[0084] The enhancers described in this invention include, but are not limited to, enhancers for cauliflower mosaic virus (CaMV), enhancers for scrophularia mosaic virus (FMV), enhancers for carnation weathering ring virus (CERV), enhancers for cassava vein mosaic virus (CsVMV), enhancers for four o'clock mosaic virus (MMV), enhancers for night-blooming jasmine yellow leaf curl virus (CmYLCV), enhancers for cotton leaf curl virus (CLCuMV), enhancers for dayflower yellow mottle virus (CoYMV), and enhancers for peanut chlorotic streak mosaic virus (PCLSV).

[0085] For monocotyledonous plants, the introns described in this invention include, but are not limited to: maize hsp70 intron, maize ubiquitin intron, Adh intron 1, sucrose synthase intron, or rice Act1 intron. For dicotyledonous plants, the introns described in this invention include, but are not limited to: CAT-1 intron, pKANNIBAL intron, PIV2 intron, and "super ubiquitin" intron.

[0086] The terminator described in this invention can be a suitable polyadenylation signal sequence that functions in plants, including but not limited to polyadenylation signal sequences derived from the Agrobacterium tumefaciens carmine synthase (NOS) gene, polyadenylation signal sequences derived from the protease inhibitor II (pin II) gene, polyadenylation signal sequences derived from the pea ssRUBISCO E9 gene, and polyadenylation signal sequences derived from the α-tubulin gene.

[0087] As used in this invention, a "vector" refers to a nucleic acid molecule introduced into a cell, for example, to produce a transformed cell. A vector may contain a nucleic acid sequence, such as an origin of replication, that allows it to replicate within a host cell. Examples of vectors include, but are not limited to, plasmids, phages, or viruses carrying exogenous DNA into the cell. A vector may also include one or more genes, antisense molecules, and / or selectable marker genes, as well as other genetic elements known in the art. A vector can transduce, transform, or infect cells, thereby causing the cells to express the nucleic acid molecule and / or the protein encoded by the vector. Optionally, a vector may include substances that facilitate the entry of the nucleic acid molecule into the cell (e.g., liposomes, protein coatings, etc.).

[0088] The host cells described in this invention include, but are not limited to, plant cells or bacterial cells. "Bacterial cells" or "bacteria" may include, but are not limited to, Agrobacterium, Bacillus, Escherichia coli, Salmonella, Pseudomonas, or Rhizobium cells. Alternatively, the host cells described in this invention may be non-plant cells or non-animal cells.

[0089] In this invention, "plant" refers to the whole plant, including all plants and plant populations, such as desired and unwanted wild plants or crop plants (including naturally occurring crop plants). Crop plants can be plants obtained through conventional breeding and optimization methods or through biotechnology and recombination methods, or a combination of these methods, including transgenic plants.

[0090] The term "plant part" as used in this invention includes plant cells, plant organs, plant protoplasts, plant cell tissue cultures from which plants can regenerate, plant callus, plant clumps, and intact plant cells in a plant or plant part. Examples of plant parts include embryos, endosperm, pollen, ovules, seeds, leaves, flowers, branches, fruits, stems, roots, root tips, anthers, etc. It should be understood that parts of transgenic plants within the scope of this invention include, but are not limited to, plant cells, protoplasts, tissues, callus, embryos, endosperm, and flowers, stems, fruits, leaves, and roots, all of which are derived from transgenic plants or their progeny that have been previously transformed with the DNA molecules of this invention and are therefore at least partially composed of transgenic cells. Alternatively, a plant part can be a plant cell.

[0091] On one hand, plant cells / plant parts are either non-regenerative cells / non-regenerative parts or regenerative cells / non-regenerative parts. Non-regenerative cells refer to cells / parts that cannot be regenerated into a whole plant through induction or in vitro culture. Non-regenerative cells can be in the plant or plant part (e.g., leaf, seed, flower, stem, or root) of the present invention. Non-regenerative cells can be cells in a seed or the seed coat of said seed. Mature plant organs (including mature leaves, mature stems, or mature roots) contain at least one non-regenerative cell. Non-regenerative plant cells can be structurally incomplete living plant cells, such as plant cells without a nucleus. Specifically, plant cells are sieve tube cells without a nucleus (mature sieve tube cells). On one hand, plant cells are somatic cells; on the other hand, plant cells are reproductive cells, such as ovules or cells that are part of pollen. In one aspect, pollen cells are vegetative (non-reproductive) cells or sperm cells.

[0092] This invention can be applied to a variety of plants, including but not limited to alfalfa, green beans, cauliflower, cabbage, carrots, celery, cotton, cucumber, eggplant, lettuce, melon, peas, pepper, zucchini, radish, rapeseed, spinach, soybean, pumpkin, tomato, Arabidopsis thaliana, or watermelon; and monocotyledonous plants including but not limited to corn, rice, sorghum, wheat, barley, rye, millet, sugarcane, oats, or turfgrass.

[0093] As used in this invention, the terms "transformation" or "transduction" refer to the transfer of one or more nucleic acid molecules into a cell. A cell is "transformed" when nucleic acid molecules are stably replicated by the cell through incorporation into the cell's genome or through appendage replication. The term "transformation" encompasses all techniques that can introduce nucleic acid molecules into such cells. Examples include, but are not limited to: transfection with viral vectors; transformation with plasmid vectors; electroporation (Fromm et al., (1986), Nature 319:791-3); liposome transfection (Felgner et al., (1987), Proc. Natl. Acad. Sci. USA 84:7413-7); microinjection (Mueller et al., (1978), Cell 15:579-85); Agrobacterium-mediated transfer (Fraley et al., (1983), Proc. Natl. Acad. Sci. USA 80:4803-7); direct DNA uptake; and particle bombardment (Klein et al., (1987), Nature 327:70).

[0094] The transformation and the scheme for introducing nucleic acid molecules into plants described in this invention vary depending on the type of plant or plant cell being transformed, i.e., monocotyledonous or dicotyledonous plants. Suitable methods for introducing nucleic acid molecules into plant cells and subsequently inserting them into the plant genome include, but are not limited to, Agrobacterium-mediated transformation, microemission bombardment, direct uptake of nucleic acid molecules (DNA) into protoplasts, electroporation, or whisker-silica-mediated DNA introduction. The transformed cells can be grown into plants in a conventional manner. These plants are cultured and pollinated with the same or different transformants to produce hybrids that express the desired identified phenotypic trait. Two or more generations can be cultured to ensure stable maintenance and genetic inheritance of the desired phenotypic trait, and then seeds that guarantee the expression of the desired phenotypic trait are harvested.

[0095] The term "insecticide" or "insect-resistant" as used in this invention refers to substances that are toxic to crop pests, such as inhibiting or suppressing the growth, weight, feeding, reproductive capacity, or survival of pests, controlling their invasion and feeding activities on crops, or killing, causing disease, death, or reducing reproduction of pests, thereby achieving "control" and / or "prevention" of crop pests.

[0096] In this invention, "contact" refers to touching, staying and / or ingesting, specifically insects and / or pests touching, staying and / or ingesting plants, plant organs, plant tissues, plant cells or microbial cells. The plants, plant organs, plant tissues or plant cells may express insecticidal proteins within themselves, or they may have insecticidal proteins on their surface and / or have microorganisms that produce insecticidal proteins.

[0097] The term "control" and / or "prevention" as used in this invention refers to the contact between pests and the chimeric protein described in this invention, or a host cell, plant, plant part expressing the chimeric protein, or the insecticidal composition described in this application, resulting in the inhibition of pest growth and / or death upon contact. The insecticidal protein can be produced within the plant or can be applied to the plant or its environment.

[0098] Furthermore, pests come into contact with the chimeric insecticidal protein by ingesting plant tissues, resulting in the inhibition of growth and / or death of all or part of the pests upon contact. Inhibition refers to sublethality, meaning that it does not cause death but can induce some effect on growth, development, behavior, physiology, biochemistry, and tissue aspects, such as slowed and / or stopped growth and development. Simultaneously, the plant should be morphologically normal and culturable under conventional methods for the consumption and / or generation of products. In addition, plants and / or seeds containing the nucleotide sequence of the chimeric protein that control lepidopteran pests, under conditions of artificial inoculation and natural pest damage, exhibit reduced plant damage compared to non-transgenic wild-type plants, specifically manifested in, but not limited to, improved leaf resistance, and / or increased grain weight, and / or increased yield. The "control" and / or "prevention" effect of chimeric proteins on pests can exist independently. Specifically, if any tissue of a transgenic plant (containing a nucleotide sequence encoding a chimeric protein) simultaneously and / or asynchronously contains and / or produces a chimeric protein and / or another substance that controls pests, the presence of the other substance does not result in the "control" and / or "prevention" effect being entirely and / or partially achieved by the other substance, and is unrelated to the chimeric protein. Typically, in the field, the process of pests feeding on plant tissues is brief and difficult to observe with the naked eye. Therefore, under conditions of natural pest damage caused by artificial inoculation, the method and / or use of the present invention is achieved when any tissue of the transgenic plant (containing a nucleotide sequence encoding a chimeric protein) contains dead pests, and / or pests whose growth is inhibited, and / or exhibits reduced plant damage compared to non-transgenic wild-type plants. This is the method and / or use of controlling lepidopteran pests through contact with the chimeric protein.

[0099] In this invention, the chimeric insecticidal protein can be prepared by recombinant bacteria. Specifically, it can be prepared through steps such as transformation, expression, centrifugation, disruption, purification, filtration, and concentration. The chimeric insecticidal protein, after formulation, can be used for various applications, including the preparation of insecticidal products. The aforementioned chimeric insecticidal protein or product may further contain an agriculturally acceptable carrier, such as powder, spray, emulsion, colloidal suspension, or aqueous solution. On the other hand, the chimeric insecticidal protein or its prepared product / composition, or the plant or plant part expressing the chimeric insecticidal protein, may further contain at least one other insecticide, such as an insecticidal protein or insect-inhibiting dsRNA that is different from the chimeric insecticidal protein but also exhibits insecticidal / inhibitory activity against pests. In other embodiments, the chimeric insecticidal protein or its prepared product / composition may further contain an insecticide different from the pests inhibited by the chimeric insecticidal protein of this invention, thereby broadening the insecticidal range of the product / composition.

[0100] The "commodities", "products", and "processed agricultural products" referred to in this invention mean any composition or product made of materials consisting of plants, seeds, plant cells or plant parts containing the chimeric protein of this invention or nucleic acid molecules encoding the chimeric protein, including but not limited to food, feed, coarse flour, starch, flour, oil, crushed or whole grains or seeds, protein concentrates, protein isolates or biomass.

[0101] This invention provides a chimeric insecticidal protein capable of simultaneously targeting multiple lepidopteran pests. The protein comprises domains I and II of the Cry1Ac protein and domain III of the Cry1Ig protein, linked sequentially. Cry1Ac is a commonly used insecticidal protein in the art, but it has no lethal activity against the fall armyworm and does not affect larval growth, nor does it significantly affect the growth and development of the beet armyworm. The toxicity of Cry1Ig against various pests has not been disclosed in the art. This invention is the first to recombine the domains of the Cry1Ac protein and the Cry1Ig protein, obtaining a novel chimeric insecticidal protein. This protein simultaneously exhibits strong weight-inhibiting and lethal activity against the fall armyworm, cotton bollworm, silver-striped armyworm, beet armyworm, oriental armyworm, and Asian corn borer. After transferring the gene encoding this protein into plants, the plants are endowed with strong insecticidal activity, especially against the fall armyworm and cotton bollworm, with mortality rates reaching 61% and 88%, respectively. Compared to the original insect-resistant protein, the chimeric insecticidal protein provided by this invention has a broader insect-resistant spectrum and higher insecticidal activity, which can effectively slow down the occurrence of pest resistance in nature and has good application prospects in agricultural production.

[0102] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0103] Figure 1 is a flowchart of the construction process of the recombinant expression vector DBN21004-P containing the chimeric insecticidal protein gene of the present invention;

[0104] Figure 2 is a flowchart of the construction process of the recombinant cloning vector DBN21004-T containing the chimeric insecticidal protein gene of the present invention;

[0105] Figure 3 is a flowchart of the construction process of the recombinant expression vector DBN21004-B containing the chimeric insecticidal protein gene of the present invention. Detailed Implementation

[0106] The technical solution of the chimeric insecticidal protein of the present invention and its application is further illustrated below through specific embodiments.

[0107] Example 1: Obtaining the chimeric protein DBN21004 and its truncated form

[0108] The chimeric insecticidal protein of this invention is obtained by replacing Domain 3 of the Cry1Ac protein with Domain 3 of the Cry1Ig protein. It sequentially comprises Domain I and Domain II of the Cry1Ac protein, Domain III of the Cry1Ig protein, and the protoxin tail of the Cry1Ac protein (the protoxin domain or Domains IV-VII of the Cry1Ac protein), and is named DBN21004. Its amino acid sequence is shown in SEQ ID NO:2, and its nucleotide sequence is shown in SEQ ID NO:4. The accession number of the Cry1Ac protein in GenBank is AJU57501.1, and the accession number of the Cry1Ig protein is AGU13866.1. The Cry1Ac protein protoxin tail used in this invention is a fragment obtained by truncating 6 amino acids from the 3' end of the protoxin tail (amino acids 616-1182) of the AJU57501.1 protein. It is also the complete protoxin tail (amino acids 618-1178) of the Cry1Ac protein with GenBank accession number AAA22331.1.

[0109] The nucleotide sequence of the DBN21004 protein (SEQ ID NO:4) was synthesized by GenScript. The nucleotide sequence of the DBN21004 protein was ligated into the pET-28a plasmid (Novagen, USA, CAT:69864-3) using BamHI and HindIII restriction enzyme sites to obtain the recombinant cloning vector DBN21004-P. The construction process is shown in Figure 1. In the figure, f1 origin represents the replication origin site of bacteriophage f1; Kan represents the kanamycin resistance gene; T7 represents the T7 RNA polymerase promoter; and DBN21004 represents the nucleotide sequence of the DBN21004 protein.

[0110] The recombinant cloning vector DBN21004-P was transformed into *E. coli* BL21(DE3) competent cells using a heat shock method. The heat shock conditions were as follows: 50 μL of *E. coli* BL21(DE3) competent cells, 10 μL of plasmid DNA, incubated at 42°C for 30 s; then cultured at 37°C with shaking at 100 rpm for 1 h; the cultured product was then plated onto LB agar plates (1% tryptone, 1% NaCl, 0.5% yeast extract, 1.5% agar) containing 50 mg / L kanamycin and cultured at 37°C for 12 h. Single colonies were picked and inoculated into 5 mL of LB liquid medium (1% tryptone, 1% NaCl, 0.5% yeast extract, pH adjusted to 7.5 with NaOH), and kanamycin was added to a final concentration of 50 mg / L. The mixture was then incubated on a shaker at 37 °C and 220 rpm for 16 h. Plasmids were extracted using the AxyPrep plasmid DNA extraction kit (CORNING, China, CAT: AP-MN-P-50).

[0111] The obtained plasmid was verified by BamHI and HindIII restriction enzyme digestion and sequencing. The results showed that the target nucleotide sequence inserted in the recombinant cloning vector DBN21004-P was exactly SEQ ID NO:4, and the recombinant cloning vector DBN21004-P was constructed correctly.

[0112] The DBN21004 protein was expressed and purified in vitro according to the following steps:

[0113] 1) Pick the above-mentioned Escherichia coli BL21(DE3) positive monoclonal colonies transformed with the recombinant cloning vector DBN21004-P, inoculate them into 3-5 mL of LB liquid medium containing kanamycin at a final concentration of 50 mg / L, and incubate them on a shaker at 37°C and 220 rpm for 16 h.

[0114] 2) Transfer the bacterial culture to 2×YT medium (1.6% tryptone, 0.5% NaCl, 1% yeast extract) at a ratio of 1:10 and incubate on a shaker at 37°C and 220 rpm for 1 h;

[0115] 3) When the OD of the culture medium 600 When the concentration is 0.6-0.8, add IPTG (final concentration 0.5mM) and incubate on a shaker at 37℃ and 220rpm for 6h to induce expression;

[0116] 4) After induction, centrifuge the bacterial culture at 7000 rpm for 5 min to collect the bacterial cells, discard the supernatant, and resuspend the bacterial cells with an appropriate amount of PBS;

[0117] 5) The bacterial cells were sonicated to obtain a broken bacterial solution. The broken bacterial solution was centrifuged at 7000 rpm for 5 min to obtain soluble and insoluble components. The insoluble components were resuspended in PBS buffer.

[0118] 6) Take appropriate amounts of soluble and insoluble components separately and perform SDS-PAGE analysis;

[0119] 7) Use the AKTA rapid purification system to purify soluble components using a HisTrap HP nickel column, and then use a HiTrap Desalting column to desalt and purify the protein to obtain purified DBN21004 protein. Refer to the AKTA operation manual for operation steps.

[0120] 8) Take an appropriate amount of desalted and purified DBN21004 protein sample and perform SDS-PAGE detection;

[0121] 9) Calculate the protein concentration in the desalted protein solution based on the BSA standard curve;

[0122] 10) The purified DBN21004 protein was stored at -20℃ for later use.

[0123] As previously mentioned, this invention also provides a 3' (C-terminus) truncated form of the DBN21004 protein, namely, an activated protein composed of Domain I and Domain II of Cry1Ac and Domain III of Cry1Ig, named DBN21004-B1, whose amino acid sequence is shown in SEQ ID NO:1 and nucleotide sequence is shown in SEQ ID NO:3. Following the in vitro expression and purification steps of the DBN21004 protein described above, the synthesized DBN21004-B1 nucleotide sequence was also ligated into the pET-28a plasmid, and the DBN21004-B1 protein was successfully expressed and purified.

[0124] Example 2: Insect resistance test of DBN21004 protein and its truncated form

[0125] The insecticidal effect of the DBN21004 protein prepared in Example 1 was tested. The 21004 protein was mixed with the feed of fall armyworm, Asian corn borer, beet armyworm, sugar beet armyworm, silver-striped armyworm, oriental armyworm, and cotton bollworm, respectively. The final concentration of the concentrated DBN21004 protein in the feed of fall armyworm and cotton bollworm was 50 ug / g, and the final concentration in the feed of Asian corn borer, beet armyworm, sugar beet armyworm, silver-striped armyworm, and oriental armyworm was 100 ug / g.

[0126] After the protein and feed were mixed evenly, the mixture was placed in a 5cm diameter petri dish. Healthy, unfed, newly hatched larvae of each insect species were selected as test insects, with 10 larvae introduced into each dish. The dishes were placed at 26±1℃ with a photoperiod (L / D) of 16 / 8 until the third day of the experiment. Four replicates were set up for each insect species, with a negative control consisting of feed supplemented only with sterile water. The experiment was repeated twice.

[0127] After treating each insect for 3 days according to the above steps, the insect weight inhibition rate and mortality rate were calculated. The average insect weight inhibition rate was calculated as follows: (Average insect weight of the negative control group - Average insect weight of the treated group) / Average insect weight of the negative control group × 100% (or 100% if all test insects died). The mortality rate was calculated as: (Number of dead insects / Total number of insects treated) × 100%. The corrected mortality rate was calculated as: (Average mortality rate of the treated group - Average mortality rate of the control group) / (1 - Average mortality rate of the control group) × 100%. The results are shown in Tables 1 and 2.

[0128] Table 1. Average insect weight inhibition rate of protein-fed insects

[0129] Table 2 Corrected mortality rate of protein-fed insects

[0130] As shown in Table 1, the DBN21004 protein exhibits strong weight-inhibiting effects against fall armyworm, Asian corn borer, beet armyworm, silver-striped armyworm, oriental armyworm, and cotton bollworm. It is particularly effective against silver-striped armyworm and Asian corn borer, causing complete mortality in both species. The weight-inhibiting rate against fall armyworm is also as high as 98.12%. Furthermore, Table 2 shows that the DBN21004 protein possesses certain lethal activity against these species. The mortality rate against silver-striped armyworm and Asian corn borer is 100%, against oriental armyworm is 37%, and against fall armyworm is 10%. As can be seen, compared with the original protein, the chimeric insecticidal protein DBN21004 of the present invention not only exhibits good weight-inhibiting and insecticidal effects against fall armyworm and beet armyworm, but also demonstrates good insecticidal activity against a variety of lepidopteran pests simultaneously. It is a chimeric insecticidal protein with promising applications in agricultural production. Similarly, mixing the DBN21004-B1 protein of the present invention (amino acid sequence as shown in SEQ ID NO:1) with the feed of various insects and testing yielded similar weight-inhibiting and insecticidal effects.

[0131] Example 3: Verification of the effect of DBN21004 protein and its truncated form on transgenic soybean plants.

[0132] 3.1 Constructing a recombinant cloning vector containing the DBN21004 gene and its truncated form

[0133] To ensure successful expression of the DBN21004 gene and its truncated variant in soybean, its nucleotide sequence was optimized. The nucleotide sequence of DBN21004 is shown in SEQ ID NO:6, and the nucleotide sequence of the truncated variant DBN21004-B1 is shown in SEQ ID NO:5.

[0134] The synthesized DBN21004 nucleotide sequence (SEQ ID NO:6) was ligated into the cloning vector pGEM-T (Promega, Madison, USA, CAT: A3600) via the SpeI and KasI restriction sites. The procedure was performed according to the Promega pGEM-T vector instructions, resulting in the recombinant cloning vector DBN21004-T. The construction process is shown in Figure 2. In the figure, Amp represents the ampicillin resistance gene; f1ori represents the origin of replication of bacteriophage f1; LacZ is the LacZ start codon; SP6 is the SP6 RNA polymerase promoter; T7 is the T7 RNA polymerase promoter; DBN21004 is the DBN21004 nucleotide sequence; and MCS is the multiple cloning site.

[0135] Following the procedures outlined in Example 1, the recombinant cloning vector DBN21004-T was transformed into *E. coli* T1 competent cells (Transgen, Beijing, China, CAT: CD501) using the heat shock method, and plasmids were extracted. Enzyme digestion and sequencing confirmed that the nucleotide sequence inserted into the recombinant cloning vector DBN21004-T was indeed the DBN21004 nucleotide sequence, indicating that the recombinant cloning vector DBN21004-T was constructed correctly.

[0136] Similarly, following the steps above, the synthesized DBN21004-B1 nucleotide sequence (SEQ ID NO:5) was ligated into the cloning vector pGEM-T via the restriction enzyme sites SpeI and KasI to obtain the correctly constructed recombinant cloning vector DBN21004-B1-T.

[0137] 3.2 Constructing a recombinant expression vector containing the DBN21004 gene and its truncated form

[0138] The recombinant cloning vector DBN21004-T and expression vector DBNBC-01 (vector backbone: pCAMBIA2301, which can be provided by the CAMBIA organization) were digested with restriction endonucleases SpeI and KasI, respectively. The excised DBN21004 nucleotide sequence fragment was inserted between the SpeI and KasI sites of the expression vector DBNBC-01 to construct the recombinant expression vector DBN21004-B. The construction process is shown in Figure 3. Among them, Spec: spectinomycin gene (SEQ ID NO:7); RB: right border; e35S: e35S enhancer (SEQ ID NO:8); prBnUBi11C: rapeseed Ubiquitin11 gene promoter (SEQ ID NO:9); DBN21004: DBN21004 nucleotide sequence (SEQ ID NO:6); tAtuORF23: Arabidopsis ORF23 terminator (SEQ ID NO:10); prBrCBP: rapeseed eukaryotic elongation factor gene 1α (Tsf1) promoter (SEQ ID NO:11); spAtCTP2: Arabidopsis chloroplast transport peptide (SEQ ID NO:12); EPSPS: 5-enolpyruvate shikimic acid-3-phosphate synthase gene (SEQ ID NO:13); tPsE9: pea RbcS gene terminator (SEQ ID NO:14); LB: left border).

[0139] Similarly, following the steps above, the recombinant cloning vector DBN21004-B1-T and the expression vector DBNBC-01 were digested with restriction endonucleases SpeI and KasI, respectively. The nucleotide sequence of DBN21004-B1 (SEQ ID NO:5) was ligated to the expression vector DBNBC-01 to obtain the correctly constructed recombinant cloning vector DBN21004-B1-B.

[0140] Recombinant expression vectors DBN21004-B and DBN21004-B1-B were transformed into *E. coli* T1 competent cells using the heat shock method. The heat shock conditions were as follows: 50 μL of *E. coli* T1 competent cells, 10 μL of recombinant expression vector DBN21004-B or DBN21004-B1-B, incubated at 42°C for 30 seconds; then cultured at 37°C with shaking at 100 rpm for 1 hour. The cultured bacterial suspension was then plated onto LB agar plates containing 50 mg / L spectinomycin (10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCl, 15 g / L agar, pH adjusted to 7.5 with NaOH) and incubated at 37°C for 12 hours. White monoclonal colonies were then picked and inoculated onto LB liquid medium (10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCl, 15 g / L agar, pH adjusted to 7.5 with NaOH). 10 g / L spectinomycin (50 mg / L, pH adjusted to 7.5 with NaOH) was added and cultured overnight at 37°C. Plasmids were extracted following the procedures in Example 1, and enzyme digestion and sequencing were performed for identification. The results showed that the recombinant expression vectors DBN21004-B and DBN21004-B1-B were constructed correctly.

[0141] 3.3 Transformation of Agrobacterium with recombinant expression vector

[0142] The correctly constructed recombinant expression vectors DBN21004-B and DBN21004-B1-B were transformed into Agrobacterium LBA4404 (Invitrgen, Chicago, USA, CAT: 18313-015) using liquid nitrogen. The transformation conditions were as follows: 100 μL Agrobacterium LBA4404, 3 μL recombinant expression vector DBN21004-B, placed in liquid nitrogen for 10 minutes, followed by a 37°C water bath for 10 minutes; the transformed Agrobacterium LBA4404 was then inoculated... The culture medium was incubated in LB liquid medium at 28°C and 200 rpm for 2 hours. The culture was then spread onto LB solid plates containing 50 mg / L rifampicin and 100 mg / L spectinomycin until positive single clones grew. Single clones were picked, cultured, and their plasmids were extracted. The recombinant expression vectors DBN21004-B and DBN21004-B1-B were digested with restriction endonucleases for verification. The results showed that the structures of the recombinant expression vectors DBN21004-B and DBN21004-B1-B were completely correct.

[0143] 3.4 Obtaining transgenic soybean plants

[0144] Following the conventional Agrobacterium infection method, cotyledonary node tissues of the aseptically cultured soybean variety Zhonghuang 13 were co-cultured with Agrobacterium that had been successfully transformed with the recombinant expression vectors DBN21004-B and DBN21004-B1-B. The specific steps are as follows:

[0145] Mature soybean seeds were germinated in soybean germination medium (B5 salt 3.1 g / L, B5 vitamin, sucrose 20 g / L, agar 8 g / L, pH 5.6). Seeds were inoculated onto the germination medium and cultured under the following conditions: temperature 25 ± 1℃; photoperiod (light / dark) 16 / 8 h. After 4-6 days of germination, fresh, green, swollen, sterile soybean seedlings were harvested from the cotyledonary nodes. The hypocotyl was removed 3-4 mm below the cotyledonary node, and the cotyledons were longitudinally cut open, removing the terminal bud, lateral buds, and seed roots. The cotyledonary nodes were wounded with the back of a scalpel, and Agrobacterium suspension was applied to the wounded cotyledonary node tissue. Agrobacterium can transfer the DBN21004 and DBN21004-B1 nucleotide sequences to the wounded cotyledonary node tissue (Step 1: Infection Step). In this step, the cotyledonary node tissue was preferably immersed in Agrobacterium suspension (OD). 660=0.5-0.8, inoculated in infection medium (MS salt 2.15 g / L, vitamin B5, sucrose 20 g / L, glucose 10 g / L, acetylsuccinone (AS) 40 mg / L, 2-morpholinoethanesulfonic acid (MES) 4 g / L, zeatin (ZT) 2 mg / L, pH 5.3). Cotyledonary tissue is co-cultured with Agrobacterium for a period (3 days) (Step 2: Co-culture step). Preferably, after the infection step, the cotyledonary tissue is cultured on solid medium (MS salt 4.3 g / L, vitamin B5, sucrose 20 g / L, glucose 10 g / L, MES 4 g / L, ZT 2 mg / L, agar 8 g / L, pH 5.6). After this co-culture phase, a selective "recovery" step can be performed. In the "recovery" step, the recovery medium (B5 salt 3.1 g / L, B5 vitamin, MES 1 g / L, sucrose 30 g / L, ZT 2 mg / L, agar 8 g / L, cephalosporin 150 mg / L, glutamate 100 mg / L, aspartic acid 100 mg / L, pH 5.6) contains at least one known antibiotic (cephalosporin) that inhibits the growth of Agrobacterium, without the addition of a selector for plant transformants (Step 3: Recovery Step). Preferably, the cotyledonary node regenerated tissue blocks are cultured on a solid medium containing antibiotics but without a selector to eliminate Agrobacterium and provide a recovery period for infected cells. Next, the cotyledonary node regenerated tissue blocks are cultured on a medium containing a selector (glyphosate) and the growing transformed callus is selected (Step 4: Selection Step). Preferably, the cotyledonary regenerated tissue blocks are cultured on a selective solid medium containing a selector (B5 salt 3.1 g / L, B5 vitamin, MES 1 g / L, sucrose 30 g / L, 6-benzyladenine (6-BAP) 1 mg / L, agar 8 g / L, cephalosporin 150 mg / L, glutamate 100 mg / L, aspartic acid 100 mg / L, glyphosate 6 mg / L, pH 5.6), leading to selective growth of the transformed cells. The transformed cells then regenerate into plants (step 5: regeneration step). Preferably, the cotyledonary regenerated tissue blocks grown on the selective medium are cultured on solid media (B5 differentiation medium and B5 rooting medium) to regenerate plants.

[0146] Specifically, the selected resistant tissue blocks were transferred to the B5 differentiation medium (B5 salt 3.1 g / L, B5 vitamin, MES 1 g / L, sucrose 30 g / L, ZT 1 mg / L, agar 8 g / L, cephalosporin 150 mg / L, glutamate 50 mg / L, aspartic acid 50 mg / L, gibberellin 1 mg / L, auxin 1 mg / L, glyphosate 6 mg / L, pH 5.6) and cultured for differentiation at 25°C. The differentiated seedlings were then transferred to the B5 rooting medium (B5 salt 3.1 g / L, B5 vitamin, MES 1 g / L, sucrose 30 g / L, agar 8 g / L, cephalosporin 150 mg / L, indole-3-butyric acid (IBA) 1 mg / L) and cultured at 25°C until they reached approximately 10 cm in height. They were then transferred to a greenhouse for further cultivation until fruit set. In the greenhouse, the plants were cultured at 26°C for 16 hours each day, followed by 8 hours at 20°C.

[0147] Approximately 100 mg of leaves from soybean plants transformed with the DBN21004 and DBN21004-B1 genes were collected as samples. Genomic DNA was extracted using Qiagen's DNeasy Plant Maxi Kit (specific method referred to its product instructions). The copy number of the EPSPS gene was determined by TaqMan probe-based quantitative real-time PCR to identify the copy numbers of the DBN21004 and DBN21004-B1 nucleotide sequences. Wild-type soybean plants were used as controls. The experiment was performed in triplicate, and the average value was used. The primer sequences for quantitative real-time PCR are shown in SEQ ID NO:15 and SEQ ID NO:16, and the probe sequence is shown in SEQ ID NO:17.

[0148] The PCR reaction system is as follows:

[0149] The 50× primer / probe mixture contained 45 μL of each primer at a concentration of 1 mM, 50 μL of the probe at a concentration of 100 μM, and 860 μL of 1×TE buffer, and was stored in amber tubes at 4°C. The PCR reaction conditions were: 95°C for 5 min; 95°C for 30 s; 60°C for 1 min, for a total of 40 cycles.

[0150] Data analysis using SDS2.3 (Applied Biosystems) software showed that the EPSPS gene was integrated into the soybean plant chromosome in a single-copy form, indicating that the DBN21004 and DBN21004-B1 nucleotide sequences were also integrated into the soybean plant chromosome in a single-copy form.

[0151] 3.5 Insect Resistance Testing of Soybean Materials

[0152] Leaves from soybean plants inoculated with the DBN21004 and DBN21004-B1 genes were taken and cut into 1cm × 2cm strips. These strips were then laid flat in petri dishes lined with moisturizing filter paper. Ten newly hatched larvae of fall armyworm and bollworm (incubated no more than 24 hours) were placed in each dish. The petri dishes were then sealed tightly and placed in a bioassay box lined with moisturizing gauze. The bioassay box was then placed in a bioassay chamber at a temperature of 24±2℃, a photoperiod (L / D) of 16 / 8, and a humidity of 70-80%. Considering the vulnerability of newly hatched larvae and their susceptibility to mechanical damage, the petri dishes were kept undisturbed as much as possible on the day of inoculation and the first day after inoculation. From the second day after inoculation, the soybean leaves were replaced every two days until the end of the experiment on the sixth day. The experiment was repeated three times. Using leaves from wild-type soybean plants as a control, the average mortality rate of fall armyworm and cotton bollworm was tested. Mortality rate = number of dead insects / total number of infested insects × 100%. The experimental results are shown in Table 3.

[0153] Table 3. Average mortality rate of transgenic soybean plants inoculated with fall armyworm and bollworm.

[0154] As shown in Table 3, the chimeric insecticidal protein of this invention not only exerts insecticidal activity in vitro, but also confers corresponding insecticidal properties to transgenic plants when expressed. Soybeans transformed with the DBN21004 gene exhibited significant lethal activity against fall armyworm and bollworm, with a mortality rate of 35% for fall armyworm and 39% for bollworm. Compared to the DBN21004 gene, soybean plants transformed with the DBN21004-B1 gene showed even better insecticidal effects, with a mortality rate of 61% for fall armyworm and 88% for bollworm. Therefore, compared to the original protein, the chimeric protein of this invention not only has a broader insecticidal spectrum but also significantly improved insecticidal activity. Furthermore, the substitution of Domain3 alters the insecticidal mechanism of the Cry1Ac protein, enabling it to combat pests that also contain Cry1Ac protein, making it a novel insecticidal protein with great application potential.

[0155] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.

Claims

1. A chimeric insecticidal protein, characterized in that, The chimeric insecticidal protein comprises, in sequence, domain I and domain II of the Cry1Ac protein and domain III of the Cry1Ig protein.

2. The chimeric insecticidal protein according to claim 1, characterized in that, The chimeric insecticidal protein comprises, in sequence, domains I and II of the Cry1Ac protein, domain III of the Cry1Ig protein, and the protoxin tail or a fragment thereof of the Cry1Ac protein.

3. The chimeric insecticidal protein according to claim 1 or 2, characterized in that, The amino acid sequence of the chimeric insecticidal protein includes SEQ ID NO:1 or SEQ ID NO:

2.

4. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the chimeric insecticidal protein according to any one of claims 1-3.

5. The nucleic acid molecule according to claim 4, characterized in that, The sequence of the nucleic acid molecule includes SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:

6.

6. An expression box, characterized in that, The expression cassette comprises the nucleic acid molecule as described in any one of claims 4 or 5.

7. An expression carrier, characterized in that, The expression vector comprises the nucleic acid molecule of claim 4 or 5 or the expression cassette of claim 6.

8. A host cell, characterized in that, The host cell comprises the chimeric protein of any one of claims 1-3, the nucleic acid molecule of any one of claims 4-5, the expression cassette of claim 6, or the expression vector of claim 7.

9. A plant or plant part, characterized in that, The plant or plant part comprises the chimeric insecticidal protein of any one of claims 1-3, the nucleic acid molecule of any one of claims 4-5, the expression cassette of claim 6, or the expression vector of claim 7.

10. A method for preparing transgenic plants, the method comprising: 1) Transform the nucleic acid molecule of claims 4-5, the expression cassette of claim 6, or the expression vector of claim 7 into plant cells; 2) Select plant cells containing the nucleic acid molecule, expression cassette, or expression vector; 3) Regenerate plants from the selected plant cells.

11. An insecticidal composition, characterized in that, The insecticidal composition comprises the chimeric insecticidal protein as described in any one of claims 1-3.

12. A method for controlling lepidopteran pests, characterized in that, This includes contacting lepidopteran pests with the chimeric insecticidal protein of any one of claims 1-3, the host cell of claim 8, the plant or plant part of claim 9, or the insecticidal composition of claim 11, or introducing the nucleic acid molecule of any one of claims 4-5, the expression cassette of claim 6, or the expression vector of claim 7 into a plant, thereby causing the lepidopteran pests to contact the plant.

13. The method for controlling lepidopteran pests according to claim 12, characterized in that, The Lepidoptera pests include Noctuidae pests or Pyralidae pests. Preferably, the lepidopteran pests include fall armyworm, beet armyworm, sugar beet armyworm, silver-striped armyworm, oriental armyworm, cotton bollworm and / or Asian corn borer.

14. Use of the chimeric insecticidal protein of claims 1-3, the nucleic acid molecule of claims 4-5, the expression cassette of claim 6, the expression vector of claim 7, or the insecticidal composition of claim 11 in the control of lepidopteran pests.

15. The use according to claim 14, characterized in that, This includes controlling lepidopteran pests by contacting them with the insecticidal protein of any one of claims 1-3 or the insecticidal composition of claim 11, or by introducing the nucleic acid molecule of claims 4-5, the expression cassette of claim 6, or the recombinant expression vector of claim 7 into plants, thereby causing lepidopteran pests to contact the plants and thus controlling the lepidopteran pests. Preferably, the lepidopteran pests include noctuid moths or pyralid moths; More preferably, the lepidopteran pests include fall armyworm, beet armyworm, sugar beet armyworm, silver-striped armyworm, oriental armyworm, cotton bollworm and / or Asian corn borer.

16. A product characterized in that, The product is obtained from the plant or plant part described in claim 9, or from the transgenic plant obtained by the method for preparing transgenic plants described in claim 10, and the product is grain, starch, seed oil, syrup, flour, coarse flour, cereal or protein. Preferably, the product comprises the chimeric insecticidal protein of claims 1-3 or the nucleic acid molecule of claims 4-5.

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