A mutant insecticidal protein Vip3 and its application

Abstract

This invention relates to the field of biotechnology, and more specifically, to a mutant insecticidal protein Vip3 and its applications. This invention obtains a mutant insecticidal protein, Vip3, with reduced toxicity to plant cells and high expression in plant cells through point mutations in the original Vip3 family protein sequences, exhibiting excellent insecticidal effects against a variety of pests.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more specifically, to a mutant insecticidal protein Vip3 and its applications. Background Technology

[0002] Agricultural pests are the most significant factor affecting crop production. With the rapid development of transgenic technology, the ability to produce insect-resistant plants by transforming Bt (Bacillus thuringiensis) insecticidal protein genes has revolutionized modern agriculture and increased the importance and value of insecticidal proteins and their genes. Several Bt proteins have already 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, there is still an urgent need to obtain transgenic plants with high expression levels and good insect resistance. Invention Summary

[0003] To address the aforementioned problems in the prior art, the present invention provides a mutant insecticidal protein Vip3, which comprises an amino acid sequence having the following mutations compared to the amino acid sequence of any Vip3 family protein: the 12th amino acid in the amino acid sequence corresponding to SEQ ID NO:4 is mutated to any other amino acid and / or the 14th amino acid is mutated to any other amino acid.

[0004] In one specific embodiment, the mutant insecticidal protein Vip3 comprises an amino acid sequence having the following mutations compared to the amino acid sequence of any Vip3 family protein: the 12th amino acid in the amino acid sequence corresponding to SEQ ID NO:4 is mutated from alanine to glycine, valine, leucine, isoleucine, methionine, proline, tryptophan, serine, tyrosine, cysteine, phenylalanine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine, or histidine; and / or the 14th amino acid is mutated from proline to alanine, glycine, valine, leucine, isoleucine, methionine, tryptophan, serine, tyrosine, cysteine, phenylalanine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine, or histidine.

[0005] In one specific embodiment, the amino acid sequence of the Vip3 family protein is as shown in SEQ ID NO:4, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28 or SEQ ID NO:30.

[0006] In one specific embodiment, the mutant insecticidal protein Vip3 comprises an amino acid sequence that has the following mutations compared to the amino acid sequence of any Vip3 family protein:

[0007] The amino acid at position 12 in the amino acid sequence corresponding to SEQ ID NO:4 is mutated from alanine to glycine and / or the amino acid at position 14 is mutated from proline to glutamine.

[0008] The amino acid at position 12 in the amino acid sequence corresponding to SEQ ID NO:4 is mutated from alanine to proline and / or the amino acid at position 14 is mutated from proline to aspartic acid.

[0009] The amino acid at position 12 in the amino acid sequence corresponding to SEQ ID NO:4 is mutated from alanine to glycine and / or the amino acid at position 14 is mutated from proline to asparagine.

[0010] The amino acid at position 12 in the amino acid sequence corresponding to SEQ ID NO:4 is mutated from alanine to glycine and / or the amino acid at position 14 is mutated from proline to leucine.

[0011] The amino acid at position 12 in the amino acid sequence corresponding to SEQ ID NO:4 is mutated from alanine to glycine and / or the amino acid at position 14 is mutated from proline to arginine;

[0012] The amino acid at position 12 in the amino acid sequence corresponding to SEQ ID NO:4 is mutated from alanine to tyrosine and / or the amino acid at position 14 is mutated from proline to lysine.

[0013] The amino acid at position 12 in the amino acid sequence corresponding to SEQ ID NO:4 is mutated from alanine to serine and / or the amino acid at position 14 is mutated from proline to valine.

[0014] The amino acid at position 12 in the amino acid sequence corresponding to SEQ ID NO:4 is mutated from alanine to histidine and / or the amino acid at position 14 is mutated from proline to glutamine.

[0015] The amino acid at position 12 in the amino acid sequence corresponding to SEQ ID NO:4 is mutated from alanine to histidine and / or the amino acid at position 14 is mutated from proline to aspartic acid.

[0016] The amino acid at position 12 in the amino acid sequence corresponding to SEQ ID NO:4 is mutated from alanine to valine and / or the amino acid at position 14 is mutated from proline to cysteine.

[0017] The amino acid at position 12 in the amino acid sequence corresponding to SEQ ID NO:4 is mutated from alanine to proline and / or the amino acid at position 14 is mutated from proline to glycine.

[0018] The amino acid at position 12 in the amino acid sequence corresponding to SEQ ID NO:4 is mutated from alanine to tryptophan and / or the amino acid at position 14 is mutated from proline to threonine;

[0019] The amino acid at position 12 in the amino acid sequence corresponding to SEQ ID NO:4 is mutated from alanine to glycine and / or the amino acid at position 14 is mutated from proline to aspartic acid.

[0020] The amino acid at position 12 in the amino acid sequence corresponding to SEQ ID NO:4 is mutated from alanine to phenylalanine and / or the amino acid at position 14 is mutated from proline to isoleucine.

[0021] The amino acid at position 12 in the amino acid sequence corresponding to SEQ ID NO:4 is mutated from alanine to glycine and / or the amino acid at position 14 is mutated from proline to alanine.

[0022] The amino acid at position 12 in the amino acid sequence corresponding to SEQ ID NO:4 is mutated from alanine to phenylalanine and / or the amino acid at position 14 is mutated from proline to histidine.

[0023] The amino acid at position 12 in the amino acid sequence corresponding to SEQ ID NO:4 is mutated from alanine to asparagine and / or the amino acid at position 14 is mutated from proline to glutamine.

[0024] The amino acid at position 12 in the amino acid sequence corresponding to SEQ ID NO:4 is mutated from alanine to glutamine and / or the amino acid at position 14 is mutated from proline to histidine;

[0025] The amino acid at position 12 in the amino acid sequence corresponding to SEQ ID NO:4 is mutated from alanine to proline and / or the amino acid at position 14 is mutated from proline to methionine.

[0026] The amino acid at position 12 in the amino acid sequence corresponding to SEQ ID NO:4 is mutated from alanine to aspartic acid and / or the amino acid at position 14 is mutated from proline to phenylalanine.

[0027] The amino acid at position 12 in the amino acid sequence corresponding to SEQ ID NO:4 is mutated from alanine to histidine and / or the amino acid at position 14 is mutated from proline to lysine.

[0028] The amino acid at position 12 in the amino acid sequence corresponding to SEQ ID NO:4 is mutated from alanine to phenylalanine and / or the amino acid at position 14 is mutated from proline to methionine.

[0029] The 12th amino acid in the amino acid sequence corresponding to SEQ ID NO:4 is mutated from alanine to glutamic acid;

[0030] The amino acid at position 12 in the amino acid sequence corresponding to SEQ ID NO:4 is mutated from alanine to glycine and / or the amino acid at position 14 is mutated from proline to isoleucine;

[0031] The amino acid at position 12 in the amino acid sequence corresponding to SEQ ID NO:4 is mutated from alanine to aspartic acid and / or the amino acid at position 14 is mutated from proline to glycine.

[0032] The amino acid at position 12 in the amino acid sequence corresponding to SEQ ID NO:4 is mutated from alanine to glutamine and / or the amino acid at position 14 is mutated from proline to valine.

[0033] The amino acid at position 12 in the amino acid sequence corresponding to SEQ ID NO:4 is mutated from alanine to serine and / or the amino acid at position 14 is mutated from proline to glycine.

[0034] The amino acid at position 12 in the amino acid sequence corresponding to SEQ ID NO:4 is mutated from alanine to phenylalanine and / or the amino acid at position 14 is mutated from proline to glycine.

[0035] The amino acid at position 12 in the amino acid sequence corresponding to SEQ ID NO:4 is mutated from alanine to glycine and / or the amino acid at position 14 is mutated from proline to serine.

[0036] The amino acid at position 12 in the amino acid sequence corresponding to SEQ ID NO:4 is mutated from alanine to arginine and / or the amino acid at position 14 is mutated from proline to methionine.

[0037] The 12th amino acid in the amino acid sequence corresponding to SEQ ID NO:4 is mutated from alanine to histidine;

[0038] The 12th amino acid in the amino acid sequence corresponding to SEQ ID NO:4 is alanine mutant threonine;

[0039] The 14th amino acid in the amino acid sequence corresponding to SEQ ID NO:4 is mutated from proline to cysteine;

[0040] The 14th amino acid in the amino acid sequence corresponding to SEQ ID NO:4 is mutated from proline to valine.

[0041] The 14th amino acid in the amino acid sequence corresponding to SEQ ID NO:4 is mutated from proline to alanine.

[0042] The 14th amino acid in the amino acid sequence corresponding to SEQ ID NO:4 is mutated from proline to aspartic acid.

[0043] The 14th amino acid in the amino acid sequence corresponding to SEQ ID NO:4 is mutated from proline to glutamic acid;

[0044] The 14th amino acid in the amino acid sequence corresponding to SEQ ID NO:4 is mutated from proline to phenylalanine;

[0045] The 14th amino acid in the amino acid sequence corresponding to SEQ ID NO:4 is mutated from proline to histidine;

[0046] The 14th amino acid in the amino acid sequence corresponding to SEQ ID NO:4 is mutated from proline to isoleucine; or...

[0047] The 14th amino acid in the amino acid sequence corresponding to SEQ ID NO:4 is mutated from proline to lysine.

[0048] In another specific embodiment, the amino acid sequence of the mutant insecticidal protein Vip3 is as shown in SEQ ID NO:1, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27 or SEQ ID NO:29, SEQ ID NO:41-80.

[0049] The present invention also provides an isolated polynucleotide comprising a nucleic acid sequence encoding the mutant insecticidal protein Vip3 or its complementary sequence.

[0050] In one specific embodiment, the polynucleotide is DNA, RNA, or a hybrid thereof.

[0051] In one specific embodiment, the polynucleotide is single-stranded or double-stranded.

[0052] In one specific embodiment, the polynucleotide has a nucleic acid sequence selected from the following:

[0053] (1) A nucleic acid sequence or its complementary sequence that encodes the amino acid sequence shown in SEQ ID NO: 1, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27 or SEQ ID NO: 29, SEQ ID NO: 41-80;

[0054] (2) The nucleic acid sequence or its complementary sequence shown in any one of SEQ ID NO: 2, SEQ ID NO: 31-40, or SEQ ID NO: 81-120;

[0055] (3) A nucleic acid sequence that hybridizes to the sequence shown in (1) or (2) under stringent conditions; and / or

[0056] (4) A nucleic acid sequence that encodes the same amino acid sequence as the sequence shown in (1) or (2) due to the degeneracy of the genetic code, or its complementary sequence.

[0057] In another specific embodiment, the nucleic acid sequence is optimized for expression in plant cells.

[0058] The present invention also provides an expression vector comprising the aforementioned polynucleotide and an expression regulatory element operatively linked thereto.

[0059] The present invention also provides an expression vector comprising a gene tandem expression cassette expressing the mutant insecticidal proteins Vip3 and Pat as described above.

[0060] In one specific embodiment, the nucleotide sequence of the mutant insecticidal protein Vip3 is shown in any one of SEQ ID NO: 2, SEQ ID NO: 31-40, and SEQ ID NO: 81-120, and the nucleotide sequence of the gene Pat is SEQ ID NO: 6.

[0061] In another specific embodiment, the gene tandem expression frame further includes:

[0062] The promoter CaMV 35S promoter for initiating Pat expression, as shown in SEQ ID NO: 5, and the termination sequence CaMV poly(A)signal for terminating gene expression, as shown in SEQ ID NO: 7;

[0063] The promoter OsUbi2promoter for initiating the expression of the mutant insecticidal protein Vip3, as shown in SEQ ID NO: 8; the chloroplast guide peptide CTP-TS-SSU, as shown in SEQ ID NO: 3; and the terminator T-Ara5, as shown in SEQ ID NO: 9, for terminating the expression of this gene.

[0064] In another specific embodiment, the nucleotide sequence of the expression vector is shown in SEQ ID NO: 10.

[0065] The present invention also provides a host cell containing the aforementioned polynucleotide or the aforementioned expression vector.

[0066] In one specific embodiment, the host cell is a plant cell.

[0067] The present invention also provides a method for cultivating transgenic plants with or enhanced insect resistance, and plants produced by said method, including regenerating plants from said plant cells.

[0068] The present invention also provides the application of the expression vector or the host cell described herein in improving the insect resistance characteristics of plants, preparing agents with insect resistance effects, or cultivating transgenic plants with or with enhanced insect resistance.

[0069] The present invention also provides a method for managing insect resistance or controlling insects, comprising bringing insects into contact with the aforementioned plant at least, wherein the insects, by ingesting plant tissues, come into contact with the mutant insecticidal protein Vip3 at least, and upon contact, the growth of the insects is inhibited and / or death occurs, thereby managing the resistance of the insects or controlling the damage caused by the insects to the plant.

[0070] In one specific embodiment, the plant is corn, cotton, or soybean.

[0071] In one specific implementation, the insect resistance is against Lepidoptera or the insects are Lepidoptera.

[0072] This invention obtains mutant Vip3 family insecticidal proteins with reduced toxicity to plant cells and high expression in plant cells by point mutation of the original Vip3 family protein sequence, which have excellent insecticidal effects on a variety of pests. Invention Details

[0074] Some of the terms used in this specification are defined as follows.

[0075] In this invention, "plant" should be understood as any differentiated multicellular organism capable of photosynthesis, especially monocotyledonous or dicotyledonous plants.

[0076] In this invention, the term "plant tissue" or "plant part" includes plant cells, protoplasts, plant tissue cultures, plant callus, plant blocks, as well as plant embryos, pollen, ovules, seeds, leaves, stems, flowers, branches, seedlings, fruits, kernels, spikes, roots, root tips, anthers, etc.

[0077] In this invention, "plant cell" should be understood as any cell derived from or found in a plant that is capable of forming, for example, undifferentiated tissues such as callus, differentiated tissues such as embryos, components of a plant, or a seed.

[0078] In this invention, "host organism" should be understood as any single-celled or multi-celled organism into which mutant protein-encoding nucleic acids can be introduced, including, for example, bacteria such as Escherichia coli, fungi such as yeast (e.g., Saccharomyces cerevisiae), molds (e.g., Aspergillus), plant cells, and plants.

[0079] The terms "protein," "polypeptide," and "peptide" are used interchangeably in this invention to refer to polymers of amino acid residues, including polymers in which one or more amino acid residues are chemical analogs of natural amino acid residues. The proteins and polypeptides of this invention can be generated through recombinant synthesis or through chemical synthesis.

[0080] The specific amino acid positions (numbers) within the protein described in this invention are determined by comparing the amino acid sequence of the target protein with Vip3Aa using standard sequence alignment tools, such as the Smith-Waterman algorithm or the CLUSTALW2 algorithm. The sequence is considered aligned when the alignment score is the highest. The alignment score can be calculated according to the method described in Wilbur, WJ and Lipman, DJ (1983) Rapid similarity searches of nucleic acid and protein data banks. Proc. Natl. Acad. Sci. USA, 80:726-730. In the ClustalW2 (1.82) algorithm, the default parameters are preferably used: protein gap opening penalty = 10.0; protein gap extension penalty = 0.2; protein matrix = Gonnet; protein / DNA terminal gap = -1; protein / DNA GAPDIST = 4.

[0081] The AlignX program (part of the vectorNTI group) is preferably used with default parameters suitable for multiple alignments (gap opening penalty: 10; gap extension penalty: 0.05). The positions of specific amino acids within the protein of the present invention are determined by aligning the amino acid sequence of the protein to Vip3Aa.

[0082] Amino acid sequence identity can be verified using conventional methods, using data from the National Center for Biotechnology Information (NCBI). www.ncbi.nlm.nih.gov / The BLAST algorithm obtained (Altschul et al., 1990, Mol.Biol. 215: 403-10) is determined using default parameters.

[0083] Those skilled in the art will also understand that the structure of a protein can be altered without adversely affecting its activity and function. For example, one or more conserved amino acid substitutions can be introduced into the amino acid sequence of a protein without adversely affecting the activity and / or three-dimensional conformation of the protein molecule. Examples and implementations of conserved amino acid substitutions are familiar to those skilled in the art. Specifically, an amino acid residue can be substituted with another amino acid residue belonging to the same group as the site to be substituted, i.e., a nonpolar amino acid residue can replace another nonpolar amino acid residue, a polar uncharged amino acid residue can replace another polar uncharged amino acid residue, a basic amino acid residue can replace another basic amino acid residue, and an acidic amino acid residue can replace another acidic amino acid residue. Conservative substitutions in which an amino acid is replaced by another amino acid belonging to the same group fall within the scope of this invention, provided that the substitution does not impair the biological activity of the protein.

[0084] Therefore, in addition to the mutations described above, the mutant proteins of the present invention may also contain one or more other mutations, such as conserved substitutions, in their amino acid sequences. Furthermore, the present invention also covers mutant proteins containing one or more other non-conserved substitutions, provided that such non-conserved substitutions do not significantly affect the desired function and biological activity of the proteins of the present invention.

[0085] As is well known in the art, one or more amino acid residues can be deleted from the N and / or C-terminus of a protein while retaining its functional activity. Therefore, in another aspect, the present invention also relates to fragments of mutant proteins that have one or more amino acid residues deleted from their N and / or C-terminus while retaining their desired functional activity; these are also within the scope of the present invention and are referred to as bioactive fragments. In the present invention, a "bioactive fragment" refers to a portion of the mutant protein of the present invention that retains the biological activity of the mutant protein of the present invention. For example, a bioactive fragment of a mutant protein may be a portion of the protein in which one or more (e.g., 1-50, 1-25, 1-10, or 1-5, e.g., 1, 2, 3, 4, or 5) amino acid residues are deleted from the N and / or C-terminus, but which still retains the biological activity of the full-length protein.

[0086] The terms "Vip3 family proteins", "Vip3 family genes", and "Vip3" refer to the classification of Vip (vegetative insecticidal protein) proteins based on amino acid sequence homology, including genes such as vip3A, vip3B, and vip3C.

[0087] In one specific embodiment, the amino acid sequence of the Vip3 family protein is as shown in SEQ ID NO:4, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28 or SEQ ID NO:30.

[0088] The term "mutation" refers to a single amino acid variation in a polypeptide and / or at least a single nucleotide variation in a nucleic acid sequence relative to the normal sequence, wild-type sequence, or reference sequence.

[0089] The terms “polynucleotide,” “nucleic acid,” “nucleic acid molecule,” or “nucleic acid sequence” are used interchangeably to refer to oligonucleotides, nucleotides, or polynucleotides and fragments or portions thereof, which may be single-stranded or double-stranded, and indicate sense or antisense strands. Nucleic acids include DNA, RNA, or hybrids thereof, and may have natural or synthetic origins. For example, nucleic acids may include mRNA or cDNA. Nucleic acids may include nucleic acids that have been amplified (e.g., using polymerase chain reaction). The nucleotide name “R” indicates a purine, such as guanine or adenine; “Y” indicates a pyrimidine, such as cytosine or thymine (or uracil if it is RNA); “M” indicates adenine or cytosine; “K” indicates guanine or thymine; and “W” indicates adenine or thymine.

[0090] The term "isolated," when referring to nucleic acids, means a nucleic acid that is separate from the substantial portion of the genome in which it is naturally present and / or substantially separated from other cellular components that naturally accompany it. For example, any nucleic acid that has been synthesized (e.g., by sequential base condensation) is considered isolated. Similarly, recombinantly expressed nucleic acids, cloned nucleic acids, nucleic acids produced by primer extension reactions (e.g., PCR), or other nucleic acids excised from the genome are also considered isolated.

[0091] Those skilled in the art will readily understand that, due to the degeneracy of the genetic code, a variety of different nucleic acid sequences can encode the amino acid sequences disclosed herein. Generating other nucleic acid sequences encoding the same protein is within the capabilities of those skilled in the art; therefore, this invention covers nucleic acid sequences encoding the same amino acid sequence due to the degeneracy of the genetic code. For example, to achieve high expression of a heterologous gene in a target host organism such as a plant, the gene can be optimized using codons preferred by the host organism to improve its expression.

[0092] The term "transgenic" plant refers to a plant containing heteropolynucleotides. Preferably, the heteropolynucleotides are stably integrated into the genome, allowing the polynucleotides to be passed on to successive generations. Heteropolynucleotides may be integrated into the genome alone or as part of a recombinant expression cassette. "Transgenic" as used herein refers to any cell, cell line, callus, tissue, plant part, or plant whose genotype has been altered due to the presence of heteronucleotides, including those originally altered transgenic organisms or cells, and those produced from hybridization or asexual reproduction of the initial transgenic organism or cell. As used herein, the term "transgenic" is not intended to include changes to the genome (chromosomal or extrachromosomal) by conventional plant breeding methods (e.g., hybridization) or by naturally occurring events (e.g., autofertilization, random hybridization, non-recombinant viral infection, non-recombinant bacterial transformation, non-recombinant transposition, or spontaneous mutation).

[0093] Based on the methods commonly used in this industry, the herbicide resistance Pat gene and Vip3 family genes can be introduced into plants and transgenic operations can be performed through appropriate plant transformation expression vectors.

[0094] Using any appropriate promoter, including vectors, is a common practice in the plant transgenic industry. For example, commonly used promoters in plant transgenics include, but are not limited to, the SP6 promoter, T7 promoter, T3 promoter, PM promoter, maize ubiquitin promoter, cauliflower mosaic virus (CaMV) 35S promoter, alpha-linolenic acid synthase (NOS) promoter, Scrophularia mosaic virus 35S promoter, sugarcane stalk virus promoter, bamboo mottle virus promoter, light-induced ribulose-1,5-ketocarboxylase (ssRUBISCO small subunit) promoter, rice cytoplasmic triose phosphate isomerase (TPI) promoter, Arabidopsis thaliana adenine transphosphoribosylase (APRT) promoter, octopine synthase promoter, and BCB (copper-binding protein) promoter.

[0095] Plant transgenic vectors include polyadenylated signal sequences that can induce 3'-terminal polyadenylation. Examples include, but are not limited to, the NOS 3'-terminal derivative of the Agrobacterium tumefaciens alpha-lipoic acid synthase gene, the 3'-terminal derivative of the octopine synthase gene of Agrobacterium tumefaciens, the 3'-terminus of the tomato or potato protease resistance I or II gene, the CaMVPoly A signal sequence, the 3'-terminus of the rice α-amylase gene, and the 3'-terminus of the betaine gene.

[0096] Vectors also include coding genes that can be selectively labeled as reporter molecules. Examples of selective labeling include, but are not limited to, antibiotic (e.g., neomycin, carbenicillin, kanamycin, spectinomycin, hygromycin, bleomycin, chloramphenicol, etc.) or herbicide resistant (glyphosate, glufosinate, glufosinate, etc.) genes.

[0097] Vector transformation methods include Agrobacterium-mediated transformation, electroporation, microparticle bombardment, and polyethylene glycol-medium absorption to introduce recombinant plasmids into plants.

[0098] In this invention, plant transformation receptors include plant cells (including suspension cultured cells), protoplasts, callus tissue, hypocotyls, seeds, cotyledons, buds, and mature plants.

[0099] The scope of transgenic plants includes not only contemporary plants from which genes have been introduced, but also their clones and offspring (T1, T2, or subsequent generations). The scope of this invention also includes all mutants and variants of the aforementioned transgenic plants that exhibit characteristics of the primary transgenic plant after hybridization and fusion. The scope of this invention also includes parts of a plant, such as seeds, flowers, stems, fruits, leaves, roots, tubers, or rhizomes, derived from a plant that has been genetically modified in advance using the methods mentioned in this invention, or its offspring, and which must consist at least of a portion of genetically modified cells.

[0100] In this invention, "insecticide" or "insect-resistant" refers to a substance that is toxic to crop pests, thereby achieving "control" and / or "prevention" of crop pests. Preferably, "insecticide" or "insect-resistant" refers to killing crop pests. These pests include Lepidoptera, such as the corn borer and / or fall armyworm.

[0101] The "inhibition of insect growth" as described in this invention refers to sublethal, meaning that it is not lethal but can cause certain effects on growth, development, behavior, physiology, biochemistry, and tissue aspects, such as slowed and / or stopped growth and development. Meanwhile, the plant should be morphologically normal and can be cultured using conventional methods for the consumption and / or generation of products.

[0102] This invention can be embodied in many different forms, and the methods of implementation are not limited to those described herein. The embodiments described herein are provided to achieve thorough and complete effects, and those skilled in the art will fully understand the scope of the invention. The same reference numerals refer to the same elements throughout this invention.

[0103] The terminology used herein is for the purpose of describing particular embodiments and is not intended to be limiting. Unless otherwise expressly stated herein, the terms “a,” “an,” and “the” as used in the foregoing include their plural forms. The terms “comprises” and / or “comprising,” or “includes” and / or “including” as used herein specifically refer to the presence of the features, factors, and / or ingredients described herein, without excluding the presence and addition of one or more other features, factors, and ingredients. The term “and / or” as used above includes all or one of the items in the list of combinations.

[0104] This invention has been described in detail through a series of embodiments, but the invention is not limited to the disclosed embodiments. Any variations, substitutions, or replacements that fall within the scope of this invention, not described herein, may be modified according to public needs. Attached Figure Description

[0105] Figure 1 This is a schematic diagram of the pQYI0187 carrier.

[0106] Figure 2 Comparison of plant cytotoxicity of callus tissue from transgenic maize QYI186 (transformed into MIR162 Vip3Aa, top) and QYI187 (transformed into Vip3Aa-K1, bottom) 4 weeks after callus differentiation.

[0107] Figure 3 For comparison of the plant toxicity of the remaining Vip3Aa protein mutants.

[0108] Figure 4 Mortality rates of fall armyworm larvae at different dilutions of freeze-dried leaf powder from QYI187 and QYI186 transgenic maize. The left figure shows QYI187 diluted 50 times, and the right figure shows QYI186 diluted 4 times.

[0109] Figure 5 Representative experimental results of feeding cotton bollworms with leaves of non-GMO and GMO soybeans. The left image shows a leaf of non-GMO wild-type recipient soybean, and the right image shows a leaf of Vip3Aa-K1 transgenic soybean.

[0110] Sequence Description

[0111]

[0112]

[0113]

[0114] Detailed Implementation

[0115] The following embodiments are provided to provide those skilled in the art with a complete disclosure and description of how to prepare and use the invention, and these embodiments are not intended to limit the scope of the invention as viewed by the inventors, nor are they intended to represent or imply that the experiments described below are all or only the experiments performed. Those skilled in the art will understand that many variations and / or modifications can be made to the invention shown in specific aspects without departing from the spirit or scope broadly described herein. Therefore, this document is to be considered illustrative rather than restrictive in all aspects.

[0116] Example 1: Construction of a maize transgenic vector

[0117] Based on the Vip3Aa sequence information listed on the Bt gene nomenclature website (http: / / www.lifesci.sussex.ac.uk / home / Neil_Crickmore / Bt / vip.html), the Vip3Aa protein sequence (GenBank: ABG20429.1, its amino acid sequence is shown in SEQ ID NO: 4), which performed well in transgenic maize mir162, was selected. After protein structure prediction, the 12th position of its amino acid sequence was mutated from Ala to Gly, and the 14th position was mutated from Pro to Gln, named Vip3Aa-K1, its amino acid sequence is shown in SEQ ID NO: 1. The maize codon was optimized for this amino acid sequence, and the corresponding encoding nucleotide sequence is shown in SEQ ID NO: 2, which includes 2367 nucleotides and encodes 789 amino acids. The nucleotide sequence was synthesized by GenScript Biotech.

[0118] During the artificial synthesis of the Vip3Aa-K1 gene sequence, the chloroplast localization peptide CTP-TS-SSU was simultaneously synthesized upstream of the ATG gene. Its nucleotide sequence is shown in SEQ ID NO: 3. The artificially synthesized CTP-TS-SSU-Vip3Aa-K1 gene fragment was constructed downstream of the Ubiquitin2 promoter and upstream of the T-Ara5 terminator in rice to obtain the OsUbi2 promoter-driven Vip3Aa-K1 gene expression cassette. The Vip3Aa-K1 gene expression cassette was then inserted into a vector containing the Pat gene using homologous recombination seamless cloning to obtain an expression cassette containing the insect-resistant gene Vip3Aa-K1 and the glufosinate-tolerant gene Pat. Finally, the two expression cassettes were linked to the LB and RB of the pCAMBIA1300 backbone using homologous recombination to construct the vector pQYI0187. Figure 1 ).

[0119] The vector pQYI0186 was constructed according to the above method. The difference between pQYI0186 and pQYI0187 is that Vip3Aa-K1 is replaced with MIR162 Vip3Aa.

[0120] Ten representative Vip3 family protein sequences (SEQ ID NO: 12, 14, 16, 18, 20, 22, 24, 26, 28, 30) were selected. The second amino acid position of SEQ ID NO: 11 (corresponding to the 14th position in the amino acid sequence shown in SEQ ID NO: 4) was mutated from Pro to Gln. The 12th position of the remaining nine sequences was mutated from Ala to Gly, and the 14th position was mutated from Pro to Gln. The resulting amino acid sequences are shown in SEQ ID NO: 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, respectively. The above proteins and the mutant proteins were used to construct vectors pQYI0011-pQYI0030, respectively, using the same method as vector pQYI0187.

[0121] Additionally, site-directed saturation mutations were generated at amino acids 12 and / or 14 of the sequence in SEQ ID NO: 4, either individually or simultaneously. Besides SEQ ID NO: 1, this method generated a total of 398 new amino acid sequences. Following the construction method of vector pQYI0186, these 398 amino acid sequences were optimized using maize codons to construct two-point mutation and single-point mutation vectors for transformation of maize embryo callus tissue.

[0122] Example 2: Comparison of Cytotoxicity in Transgenic Plants

[0123] Transgenic vectors pQYI0187 and pQYI0186 were transformed into maize callus tissue using Agrobacterium-mediated transformation. Transformants QYI187 and QYI186 were obtained after screening and culture. During the genetic transformation process, the emergence rates of intermediate materials from the QYI187 and QYI186 transformants were compared.

[0124] Table 1. Positive transformation seedling status of QYI187 and QYI186

[0125] Insect-resistant gene transformants Transformed embryos (number) Positive transformation seedlings (strains) QYI187 1000 630 QYI186 1000 60

[0126] The results showed that callus growth of the QYI186 transformant was severely inhibited and damaged, with only 60 positive transformants out of 1000 transformed embryos. In contrast, callus growth of the QYI187 transformant was good, with 630 positive transformants out of 1000 transformed embryos. Figure 2 See Table 1. This indicates that the QYI187(Vip3Aa-K1) transgenic material significantly reduced the cytotoxicity of recipient plants.

[0127] Similarly, the transgenic vector pQYI0011-pQYI0030 was transformed into maize callus tissue using Agrobacterium-mediated transformation, and the transformants QYI11-QYI30 were obtained after screening and culture. During the genetic transformation process, the emergence of intermediate materials from the maize QYI11-QYI30 transformants was compared. The results showed that the callus growth of the unmutated QYI12 and QYI14 transformants was severely inhibited and damaged, with very few positive transformant seedlings (58 and 75 plants respectively) out of 1000 transformed immature embryos. In contrast, the callus growth of the QYI11 and QYI13 transformants containing the mutant protein was good, with 614 and 571 positive transformant seedlings (614 and 571 plants respectively) out of 1000 transformed immature embryos. This indicates that the transgenic materials containing the mutant proteins, QYI11 (Vip3Aa truncated protein double mutant) and QYI13 (Vip3Aa19 double mutant), showed significantly reduced cytotoxicity to the recipient plants. Similarly, other transgenic materials with mutant Vip3 family proteins (QYI15, QYI17, QYI19, QYI21, QYI23, QYI25, QYI27 and QYI29) also showed significantly reduced plant cytotoxicity.

[0128] In addition, the intermediate materials of the transformants of the two-point mutation and single-point mutation vectors constructed in Example 1 were also compared. The results showed that the number of positive seedlings exceeded QYI186, and the cytotoxicity of the mutants to plants was reduced. Specifically, when the amino acid mutated at positions 12 and / or 14 of Vip3Aa (SEQ ID NO: 4) to 12P / 14D, 12G / 14N, 12G / 14L, 12G / 14R, 12Y / 14K, 12S / 14V, 12H / 14Q, 12H / 14D, 12V / 14C, 12P / 14G, 12W / 14T, 12G / 14D, 12F / 14I, 12G / 14A, 14V, 12F / 14H, 14C, 12N / 14Q, 12Q / 14H, 12P / 14M, 12D / 14F, 12 When H / 14K, 12F / 14M, 12E, 12G / 14I, 12D / 14G, 12Q / 14V, 12S / 14G, 12F / 14G, 12G / 14S, 12R / 14M, 12H, 12T, 14A, 14D, 14E, 14F, 14H, 14I, and 14K were used, a relatively large number of positive plantlets were obtained (338–626 positive transformed plantlets per 1000 transformed embryos), and the intermediate transformants grew normally on the screening culture dishes, indicating a significant reduction in cytotoxicity to the recipient plants. Representative plant toxicity comparison images are shown below. Figure 3 .

[0129] Example 3: Detection of protein content and insecticidal activity in transgenic maize leaves

[0130] Protein expression levels were measured, and during the T2 generation V7-V8 stage of transgenic maize, the average expression level of Vip3Aa-K1 in the leaves of transgenic maize QYI187 reached 130 μg / g (fresh weight of leaves), while the average expression level of MIR162Vip3Aa in the leaves of transgenic maize QYI186 was 11 μg / g (fresh weight of leaves). Furthermore, tests revealed that the expression levels of the corresponding mutant proteins in other transgenic maize varieties were also significantly higher than those in the corresponding transgenic maize varieties containing the original proteins.

[0131] Upper leaves were taken from test maize plants of QYI186 and QYI187 T2 generation V7-V8 leaf stage with consistent growth. These leaves were placed in sealed bags with corresponding labels, retrieved to the laboratory, and then cut into 2cm pieces. 2 The larvae were ground in liquid nitrogen in separate mortars of varying sizes, and the freeze-dried leaf powder was diluted with 4× and 50× dilution factors, respectively. When the prepared feed temperature dropped to 45℃, the freeze-dried leaf powder was added in proportion and stirred thoroughly. After complete cooling, feed cakes of uniform shape and weight were made using a fixed membrane device and placed in a bioassay apparatus. One second-instar larva of the fall armyworm was placed in each apparatus, with 10 replicates. The experiment was conducted in a rearing room at 27±1℃, RH 75%, and L:D = 16h:8h. Larval mortality was assessed in each apparatus after 7 days.

[0132] Second-instar larvae of the fall armyworm from 10 replicate experiments were collected into one experimental setup. The results are as follows: Figure 4 As shown, the insecticidal protein concentrations of QYI187 diluted 50 times and QYI186 diluted 4 times were 2.6 μg / g (fresh feed weight) and 2.75 μg / g (fresh feed weight), respectively. The concentrations of Vip3Aa-K1 and Vip3Aa contained in them were similar, and the mortality rate of second-instar fall armyworm larvae was 100% in both cases.

[0133] In addition, the median lethal concentration (LC50) of each original protein and each mutant protein against fall armyworm was determined using the feed surface method. Freshly prepared artificial feed was poured into beakers and immersed in hot water. Using a manual continuous dispenser, the feed was dispensed into 24-well cell culture plates, with 1 mL of feed dispensed into each well (1.6 cm in diameter). After solidification, the resulting surface area was 2 cm². 2The mutant protein to be tested was serially diluted with Na2CO3 / NaHCO3 buffer (pH=10) to four concentrations. Using a manual continuous dispenser, 50 μl of the diluted solution was dispensed into each well and shaken to ensure complete protein coverage of the feed surface. After sample addition, the 24-well cell culture plates were dried in a clean bench. Once the protein had fully penetrated the feed surface, second-instar larvae of the fall armyworm were inoculated. One larva was inoculated into each well, the plates were capped and sealed, and placed under conditions of 25–27℃, 65–70% relative humidity, and a light / D ratio of 16h / 8h. A control group containing 50 μl of buffer was used. The experiment was repeated twice. After 7 days, the mortality rate of the test insects was observed, and the corresponding median lethal concentration (LC50) was calculated. The results showed that the LC50 value of the mutant protein in this application was not significantly different from that of its corresponding Vip3 original proteins, maintaining or even improving the insecticidal effect against the fall armyworm. Representative data are shown in Table 2.

[0134] Table 2 shows the median lethal concentration (LC50) of the Vip3 mutant protein against the fall armyworm.

[0135]

[0136]

[0137] Example 4: Detection of Insect Resistance in Transgenic Soybean Leaves

[0138] Following the same method as in Example 1, a soybean transgenic vector containing Vip3Aa-K1 was constructed, transformed into a soybean recipient, and transgenic soybeans were obtained. Upper leaves from soybean plants of uniform growth stage were taken, placed in sealed bags with corresponding labeling, and retrieved to the laboratory. The leaf material was then cut into 2cm pieces. 2 The larvae were placed in bioassay apparatus, with three second-instar larvae introduced into each apparatus, and ten replicates were set up. The experiment was conducted in an insect rearing room at 27±1℃, RH 75%, and L:D = 16h:8h. After 6 days, the mortality rate and leaf feeding of the larvae in each apparatus were investigated.

[0139] Experimental results showed that non-transgenic soybean leaves were severely damaged by bollworm larvae, and the larvae were growing well; while Vip3Aa-K1 transgenic soybean leaves were largely undamaged, and the larvae that did feed on them all died. This indicates that the Vip3Aa-K1 transgenic soybean plants exhibit a better resistance phenotype to bollworm larvae. Figure 5 As shown.

[0140] Meanwhile, numerous tests have revealed that the transgenic plants containing Vip3Aa-K1 protein described in this invention (including but not limited to corn, cotton, soybeans, etc.) also exhibit similar insect-resistant effects against other lepidopteran pests of the genera *Gnaphalium*, *Striacosta*, *Gnaphalium*, *Gnaphalium*, *Gnaphalium*, and *Elasmopalpus*. Furthermore, other mutated Vip3 family proteins also demonstrate excellent insecticidal effects against a variety of pests and exhibit low toxicity to plant cells.

[0141] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.

Claims

1. A mutant insecticidal protein Vip3A, the amino acid sequence of which is based on the amino acid sequence of Vip3A family proteins by the following mutations: at the 12th amino acid position corresponding to the amino acid sequence shown in SEQ ID NO: 4, alanine is mutated to glycine and at the 14th amino acid position, proline is mutated to glutamine.

2. The mutant insecticidal protein Vip3A according to claim 1, characterized in that, The amino acid sequences of the Vip3A family proteins are shown in SEQ ID NO:4, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:24, SEQ ID NO:26 or SEQ ID NO:

28.

3. The mutant insecticidal protein Vip3A according to claim 1 or 2, wherein the amino acid sequence is shown in SEQ ID NO:1, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:23, SEQ ID NO:25 or SEQ ID NO:

27.

4. An isolated polynucleotide, said polynucleotide being a nucleic acid sequence encoding the mutant insecticidal protein Vip3A of any one of claims 1-3 or its complementary sequence.

5. The polynucleotide according to claim 4, characterized in that, The polynucleotides mentioned are DNA or RNA.

6. The polynucleotide according to claim 4 or 5, characterized in that, The polynucleotides are single-stranded or double-stranded.

7. The polynucleotide according to claim 4 or 5, characterized in that, It has a nucleic acid sequence selected from the following: (1) A nucleic acid sequence or its complementary sequence that encodes the amino acid sequence shown in SEQ ID NO: 1, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 23, SEQ ID NO: 25 or SEQ ID NO: 27; (2) The nucleic acid sequence or its complementary sequence shown in any one of SEQ ID NO: 2, SEQ ID NO: 31-35, 37-39; and / or (3) A nucleic acid sequence that encodes the same amino acid sequence as the sequence shown in (1) or (2) due to the degeneracy of the genetic code, or its complementary sequence.

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

9. An expression vector comprising the polynucleotide of any one of claims 4-8 and an expression regulatory element operatively linked thereto.

10. An expression vector comprising a tandem expression cassette for expressing the mutant insecticidal protein Vip3A as described in any one of claims 1-3 and the glufosinate resistance Pat.

11. The expression vector according to claim 10, wherein the nucleotide sequence of the gene encoding the mutant insecticidal protein Vip3A is as shown in any one of SEQ ID NO: 2 and SEQ ID NO: 31-40, and the nucleotide sequence of the gene encoding Pat is SEQ ID NO:

6.

12. The expression vector according to claim 10 or 11, wherein the gene tandem expression cassette further comprises: The nucleotide sequence of the promoter CaMV 35S promoter that initiates Pat expression as shown in SEQ ID NO: 5, and the nucleotide sequence of the termination sequence CaMV poly(A) signal that terminates gene expression as shown in SEQ ID NO:

7. The promoter OsUbi2promoter for initiating the expression of the mutant insecticidal protein Vip3A, as shown in SEQ ID NO: 8; the chloroplast guide peptide CTP-TS-SSU, as shown in SEQ ID NO: 3; and the terminator T-Ara5, as shown in SEQ ID NO: 9, for terminating the expression of this gene.

13. The expression vector according to claim 12, wherein the nucleotide sequence is shown in SEQ ID NO:

10.

14. A host cell comprising the polynucleotide of any one of claims 4-8 or the expression vector of any one of claims 9-13.

15. A method for cultivating transgenic plants with or enhanced insect resistance, comprising regenerating a plant from plant cells containing a polynucleotide as described in any one of claims 4-8 or an expression vector as described in any one of claims 9-13, wherein the insect is a lepidopteran insect and the plant is corn or soybean.

16. The application of the expression vector as described in any one of claims 9-13 or the host cell as described in claim 14 in improving the insect resistance of plants, preparing agents with insect resistance effects, or cultivating transgenic plants with or with enhanced insect resistance, wherein the insect is a lepidopteran insect and the plant is corn or soybean.

17. A method for controlling insects, characterized in that, The method includes contacting an insect with a plant containing at least the polynucleotide of any one of claims 4-8 or the expression vector of any one of claims 9-13, wherein the insect contacts the mutant insecticidal protein Vip3A at least by ingesting the plant tissue, and the insect's growth is inhibited and / or it dies after contact, thereby controlling the insect's damage to the plant, wherein the insect is a lepidopteran insect and the plant is corn or soybean.

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