Mutant insecticidal protein Vip3Aa and application thereof

Abstract

The invention relates to the technical field of biology, in particular to a mutant insecticidal protein Vip3Aa and application thereof. The mutant insecticidal protein Vip3Aa comprises an amino acid sequence with the following mutations compared with an amino acid sequence as shown in SEQ ID NO: 1: alanine mutates into leucine, methionine, threonine, valine or tyrosine at the 12th amino acid in the amino acid sequence as shown in SEQ ID NO: 1; and the 14th amino acid is mutated into glycine, isoleucine, serine, histidine or methionine from proline. According to the invention, the Vip3Aa protein sequence is subjected to point mutation to obtain the mutant insecticidal protein Vip3Aa which has reduced toxicity to plant cells and can be highly expressed in the plant cells, and the mutant insecticidal protein Vip3Aa has an excellent insecticidal effect on various pests.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and more specifically, to a mutant insecticidal protein Vip3Aa and its application. Background Art

[0002] Pest damage is an important factor affecting crop production. An important goal of transgenic crop research is insect resistance. Among them, the most widely planted insect-resistant crops are those transgenic with the insecticidal protein gene of Bacillus thuringiensis (Bt). The insecticidal protein (Vegetative Insecticidal Protein3, abbreviated as Vip3) produced during the vegetative period of Bacillus thuringiensis is a tetramer composed of 5 domains. Its N-terminal mainly controls the stability of the structure, and the C-terminal is the potential specific receptor binding domain, with a total of 14 pattern samples and more than 110 proteins. Due to its different insecticidal mechanism from Cry protein and strong insecticidal activity against Spodoptera frugiperda, Vip3 protein is favored for its good complementarity in the development of transgenic insect-resistant crops. The main one used in the current market is Vip3Aa protein. However, Vip3Aa has certain toxicity to plant cells, resulting in difficulty in obtaining transformants with high expression levels during genetic transformation, and negative impacts on the pollen viability of transgenic maize. There is an urgent need to obtain an insecticidal protein with high expression levels in plants, good insecticidal effects, and no side effects. Brief Description of the Invention

[0004] To solve the above problems existing in the prior art, the present invention provides a mutant insecticidal protein Vip3Aa, which comprises an amino acid sequence having the following mutations compared with the amino acid sequence shown in SEQ ID NO: 1: the amino acid at position 12 corresponding to the amino acid sequence shown in SEQ ID NO: 1 is mutated from alanine to leucine, methionine, threonine, valine or tyrosine; and the amino acid at position 14 is mutated from proline to glycine, isoleucine, serine, histidine or methionine.

[0005] In a specific embodiment, the mutant insecticidal protein Vip3Aa

[0006] the amino acid at position 12 corresponding to the amino acid sequence shown in SEQ ID NO: 1 is mutated from alanine to leucine and the amino acid at position 14 is mutated from proline to glycine;

[0007] The 12th amino acid in the amino acid sequence corresponding to SEQ ID NO:1 is mutated from alanine to methionine and the 14th amino acid is mutated from proline to isoleucine;

[0008] The 12th amino acid in the amino acid sequence corresponding to SEQ ID NO:1 is mutated from alanine to threonine and the 14th amino acid is mutated from proline to serine;

[0009] The 12th amino acid in the amino acid sequence corresponding to SEQ ID NO:1 is mutated from alanine to valine and the 14th amino acid is mutated from proline to histidine; or,

[0010] The 12th amino acid in the amino acid sequence corresponding to SEQ ID NO:1 is mutated from alanine to tyrosine and the 14th amino acid is mutated from proline to methionine.

[0011] In another specific embodiment, the amino acid sequence of the mutant insecticidal protein Vip3Aa is as shown in SEQ ID NO:2-6.

[0012] The present invention also provides an isolated polynucleotide, which contains a nucleic acid sequence encoding the mutant insecticidal protein Vip3Aa or its complementary sequence.

[0013] In one specific embodiment, the polynucleotide is DNA, RNA or a hybrid thereof.

[0014] In one specific embodiment, the polynucleotide is single-stranded or double-stranded.

[0015] In one specific embodiment, the polynucleotide has a nucleic acid sequence selected from the following:

[0016] (1) A nucleic acid sequence encoding the amino acid sequence shown in any one of SEQ ID NO:2-6 or its complementary sequence;

[0017] (2) A nucleic acid sequence shown in any one of SEQ ID NO:7-11 or its complementary sequence;

[0018] (3) A nucleic acid sequence that hybridizes with the sequence shown in (1) or (2) under stringent conditions; and / or

[0019] (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.

[0020] In another specific embodiment, the nucleic acid sequence is optimized for expression in plant cells.

[0021] The present invention also provides an expression vector, which contains the said polynucleotide and an expression regulatory element operably linked thereto.

[0022] The present invention also provides an expression vector, which contains a tandem expression cassette expressing genes such as the said mutant insecticidal proteins Vip3Aa and Pat.

[0023] In a specific embodiment, the nucleotide sequence of the gene mutant insecticidal protein Vip3Aa is shown as any one of SEQ ID NO: 7-11, and the nucleotide sequence of the gene Pat is SEQ ID NO: 12.

[0024] In another specific embodiment, the said tandem expression cassette further contains:

[0025] The promoter CaMV 35S promoter for initiating the expression of Pat with a nucleotide sequence shown as SEQ ID NO: 13, and the termination sequence CaMV poly(A) signal for terminating the expression of this gene with a nucleotide sequence shown as SEQ ID NO: 14;

[0026] The promoter OsUbi2 promoter for initiating the expression of the mutant insecticidal protein Vip3Aa with a nucleotide sequence shown as SEQ ID NO: 15, the chloroplast targeting peptide CTP-TS-SSU with a nucleotide sequence shown as SEQ ID NO: 16, and the terminator T-Ara5 for terminating the expression of this gene with a nucleotide sequence shown as SEQ ID NO: 17.

[0027] The present invention also provides a host cell, which contains the said polynucleotide or the said expression vector.

[0028] In a specific embodiment, the said host cell is a plant cell.

[0029] The present invention also provides a method for cultivating a transgenic plant having or enhanced insect resistance and the plant produced by the said method, which includes regenerating the said plant cell into a plant.

[0030] The present invention also provides the application of the said expression vector or the said host cell in improving the insect resistance of plants, preparing an agent having an insecticidal effect, or cultivating a transgenic plant having or enhanced insect resistance.

[0031] The present invention also provides a method for managing insect resistance or controlling insects, which includes bringing the insects into contact with at least the above-mentioned plants, and the insects come into contact with at least the said mutant insecticidal protein Vip3Aa by feeding on the tissues of the plants. After contact, the growth of the insects is inhibited and / or they are caused to die, thereby achieving the management of the insect resistance or the control of the damage of the insects to the plants.

[0032] In a specific embodiment, the plant is corn, cotton or soybean.

[0033] In a specific embodiment, insect resistance is resistance to Lepidoptera such as Spodoptera frugiperda or the insect is Lepidoptera such as Spodoptera frugiperda.

[0034] The present invention obtained a mutant Vip3Aa insecticidal protein with reduced cytotoxicity to plant cells and high expression in plant cells by performing point mutations on the original Vip3Aa protein sequence, which has excellent insecticidal effects against various pests. Detailed description of the invention

[0036] Some terms used in this specification are defined as follows.

[0037] In the present invention, "plant" should be understood as any differentiated multicellular organism capable of photosynthesis, especially monocotyledonous or dicotyledonous plants.

[0038] In the present invention, the term "plant tissue" or "plant part" includes plant cells, protoplasts, plant tissue cultures, plant calli, plant blocks, and plant embryos, pollen, ovules, seeds, leaves, stems, flowers, branches, seedlings, fruits, kernels, ears, roots, root tips, anthers, etc.

[0039] In the present invention, "plant cell" should be understood as any cell derived from or found in a plant, which is capable of forming, for example: undifferentiated tissues such as callus, differentiated tissues such as embryos, components of plants, plants or seeds.

[0040] In the present invention, "host organism" should be understood as any unicellular or multicellular organism into which a mutant protein-encoding nucleic acid 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, etc.

[0041] The terms "protein", "polypeptide" and "peptide" are used interchangeably in the present invention and 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 the present invention can be produced recombinantly or by chemical synthesis.

[0042] The specific amino acid positions (numbers) within the protein of the present invention are determined by aligning the amino acid sequence of the target protein with Vip3Aa using standard sequence alignment tools, such as aligning the two sequences with the Smith-Waterman algorithm or the CLUSTALW2 algorithm, where the sequences are considered aligned when the alignment score is the highest. The alignment score can be calculated according to the method described in Wilbur, W.J. and Lipman, D.J. (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 end gap = -1; protein / DNA GAPDIST = 4.

[0043] Preferably, the AlignX program (part of the vectorNTI suite) is used with default parameters suitable for multiple alignment (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 with Vip3Aa.

[0044] The identity of the amino acid sequence can be determined by conventional methods using the BLAST algorithm (Altschul et al., 1990, Mol. Biol. 215:403-10) available from the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov / ) using default parameters.

[0045] Those skilled in the art are also aware that the structure of a protein can be altered without adversely affecting its activity and functionality. For example, one or more conservative amino acid substitutions can be introduced into the amino acid sequence of the protein without adversely affecting the activity and / or three-dimensional configuration of the protein molecule. Those skilled in the art are aware of examples and embodiments of conservative amino acid substitutions. Specifically, an amino acid residue can be replaced with another amino acid residue belonging to the same group as the site to be replaced, i.e., a nonpolar amino acid residue is replaced with another nonpolar amino acid residue, a polar uncharged amino acid residue is replaced with another polar uncharged amino acid residue, a basic amino acid residue is replaced with another basic amino acid residue, and an acidic amino acid residue is replaced with another acidic amino acid residue. As long as the substitution does not impair the biological activity of the protein, conservative substitutions in which one amino acid is replaced with another amino acid belonging to the same group fall within the scope of the present invention.

[0046] Accordingly, in addition to containing the above mutations, the mutant proteins of the present invention may also contain one or more other mutations in the amino acid sequence, such as conservative substitutions. In addition, the present invention also encompasses mutant proteins containing one or more other non-conservative substitutions, provided that such non-conservative substitutions do not significantly affect the desired functions and biological activities of the proteins of the present invention.

[0047] 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 still retaining its functional activity. Accordingly, in another aspect, the present invention also relates to fragments in which one or more amino acid residues have been deleted from the N- and / or C-terminus of the mutant protein while retaining its desired functional activity, which 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 can be a portion 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 have been deleted from the N- and / or C-terminus of the protein, but which still retains the biological activity of the full-length protein.

[0048] 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 a normal sequence or a wild-type sequence or a reference sequence.

[0049] The terms "polynucleotide", "nucleic acid", "nucleic acid molecule", or "nucleic acid sequence" are used interchangeably and refer to oligonucleotides, nucleotides, or polynucleotides and fragments or portions thereof, which may be single-stranded or double-stranded, and represent sense or antisense strands. Nucleic acids include DNA, RNA, or hybrids thereof, and may have a natural or synthetic origin. 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 designation "R" means purine such as guanine or adenine; "Y" means pyrimidine such as cytosine or thymine (uracil if RNA); "M" means adenine or cytosine; "K" means guanine or thymine; and "W" means adenine or thymine.

[0050] The term "isolated", when referring to a nucleic acid, means a nucleic acid that is separated from a substantial portion of the genome in which it naturally occurs and / or is substantially separated from other cellular components that naturally accompany the nucleic acid. For example, any nucleic acid that has been produced synthetically (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 nucleic acids that have otherwise been excised from the genome are also considered isolated.

[0051] Those skilled in the art are well aware that due to the degeneracy of the genetic code, there are a variety of different nucleic acid sequences that can encode the amino acid sequences disclosed herein. Generating other nucleic acid sequences that encode the same protein is within the capabilities of those of ordinary skill in the art, and thus the present invention encompasses nucleic acid sequences that encode the same amino acid sequence due to the degeneracy of the genetic codons. For example, in order to achieve high expression of a heterologous gene in a target host organism such as a plant, the gene can be optimized using the codons preferred by the host organism to enable better expression.

[0052] The term "transgenic" plant refers to a plant that contains a heterologous polynucleotide. Preferably, the heterologous polynucleotide is stably integrated into the genome such that the polynucleotide is passed on to successive generations. The heterologous polynucleotide can 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 heterologous nucleic acid, including those initially altered transgenic organisms or cells, as well as those produced by hybridization or asexual propagation from the initial transgenic organism or cell. As used herein, the term "transgenic" is not intended to include genomic (chromosomal or extrachromosomal) alterations by conventional plant breeding methods (e.g., hybridization) or by natural events such as self-fertilization, random cross-fertilization, non-recombinant viral infection, non-recombinant bacterial transformation, non-recombinant transposition, or spontaneous mutation.

[0053] In this invention, the herbicide resistance Pat gene and Vip3Aa gene can be introduced into plants according to the methods commonly used in this industry, and transgenic operations can be carried out through appropriate plant transformation expression vectors.

[0054] Selecting any appropriate promoter, including the vector, is a commonly used method in the industry for plant transgenesis. For example: Promoters commonly used in plant transgenesis include but are not limited to SP6 promoter, T7 promoter, T3 promoter, PM promoter, maize ubiquitin promoter, cauliflower mosaic virus (CaMV) 35S promoter, nopaline synthase (nos) promoter, figwort mosaic virus 35S promoter, sugarcane bacilliform virus promoter, bamboo mosaic virus promoter, light-inducible promoter ribulose-1,5-bisphosphate carboxylase (ssRUBISCO small subunit), rice cytoplasmic triose phosphate isomerase (TPI) promoter, Arabidopsis adenine phosphoribosyltransferase (APRT) promoter, octopine synthase promoter, and BCB (blue copper-binding protein) promoter.

[0055] Plant transgenic vectors include polyadenylation signal sequences that can cause 3'-end polyadenylation. For example, it includes but is not limited to the NOS 3'-terminal derivative of the nopaline synthase gene of Agrobacterium, the octopine synthase 3'-terminal derivative of the octopine synthase gene of Agrobacterium, the 3'-end of the tomato or potato protease inhibitor I or II gene, CaMV PolyA signal sequence, the 3'-end of the rice α-amylase gene, and the 3'-end of the phaseolin gene.

[0056] The vector also includes coding genes that can be selectively marked as reporter molecules. Examples of selectable markers include but are not limited to antibiotics (such as: neomycin, carbenicillin, kanamycin, spectinomycin, hygromycin, bleomycin, chloramphenicol, etc.) or herbicide resistance (glyphosate, glufosinate, bialaphos, etc.) genes.

[0057] Methods for vector transformation include using Agrobacterium-mediated transformation, electroporation, particle bombardment, polyethylene glycol-mediated absorption, and other methods to introduce the recombinant plasmid into plants.

[0058] Plant transformation receptors in this invention include plant cells (including suspension-cultured cells), protoplasts, callus, hypocotyls, seeds, cotyledons, buds, and mature plants.

[0059] The scope of transgenic plants includes not only the plants obtained in the current generation into which genes have been introduced, but also their clones and progeny (T1 generation, T2 generation, or subsequent generations). The scope of the present invention also includes all mutants and variants of the above-mentioned transgenic plants that exhibit the characteristics of the primary transgenic plants after hybridization and fusion. The scope of the present invention also includes parts of plants, such as seeds, flowers, stems, fruits, leaves, roots, tubers, and corms, which are derived from plants that have been previously genetically modified by the methods mentioned in the present invention, or their progeny, and which consist of at least a part of the genetically modified cells.

[0060] As used in the present invention, "insecticidal" or "insect-resistant" means being toxic to crop pests, thereby achieving "control" and / or "prevention" of crop pests. Preferably, "insecticidal" or "insect-resistant" means killing crop pests. The above pests include Lepidoptera, such as Ostrinia furnacalis and / or Spodoptera frugiperda.

[0061] As used in the present invention, "inhibition of insect growth" means sub-lethal, that is, not yet lethal but capable of causing certain effects in aspects such as growth and development, behavior, physiology, biochemistry, and tissue, such as slow growth and / or cessation of growth. At the same time, the plant should be normal in morphology and can be cultivated by conventional methods for the consumption and / or production of products.

[0062] The present invention can be implemented in various different forms, and the implementation methods are not limited by the methods described herein. The implementation examples herein are provided to achieve thorough and complete effects, and those skilled in the industry can fully understand the scope of the present invention. The same reference numerals in the present invention refer to the same elements.

[0063] The terms used herein are for describing specific embodiments and are not intended to set limitations. Unless specifically stated otherwise in the text, the use of "a", "an", and "the" in the above content in both Chinese and English versions also includes their plural forms. The terms "comprises" and / or "comprising," or "includes" and / or "including" used herein specifically refer to the presence of the characteristics, elements, and / or components described herein, and do not exclude the presence and addition of one or more other characteristics, elements, and components. The term "and / or" used in the above content includes all items in one or more combination lists.

[0064] The present invention has been elaborated in detail through a series of embodiments, but the present invention is not limited to the disclosed embodiments. Any number of variations, substitutions, permutations, etc. within the scope of the present invention that are not described herein or can be modified according to the needs of the public. BRIEF DESCRIPTION OF THE DRAWINGS

[0065] Figure 1Comparison of the cytotoxicity of plant cells after 4 weeks of callus differentiation of genetically modified maize QYI186 (transformed with Vip3Aa), QYI8133, QYI8155, QYI8282, QYI8293, and QYI8337.

[0066] Sequence Listing

[0067] Sequence number Gene information Related vector information SEQ ID NO: 1 Vip3Aa amino acid sequence pQYI0186 SEQ ID NO: 2 mVip3Aa(A12L+P14G) amino acid sequence pQY008133 SEQ ID NO: 3 mVip3Aa(A12M+P14I) amino acid sequence pQY008155 SEQ ID NO: 4 mVip3Aa(A12T+P14S) amino acid sequence pQY008282 SEQ ID NO: 5 mVip3Aa(A12V+P14H) amino acid sequence pQY008293 SEQ ID NO: 6 mVip3Aa(A12Y+P14M) amino acid sequence pQY008337 SEQ ID NO: 7 mVip3Aa(A12L+P14G) nucleotide sequence SEQ ID NO: 8 mVip3Aa(A12M+P14I) nucleotide sequence SEQ ID NO: 9 mVip3Aa(A12T+P14S) nucleotide sequence SEQ ID NO: 10 mVip3Aa(A12V+P14H) nucleotide sequence SEQ ID NO: 11 mVip3Aa(A12Y+P14M) nucleotide sequence SEQ ID NO: 12 Pat SEQ ID NO: 13 CaMV35S promoter nucleotide sequence SEQ ID NO: 14 CaMV poly(A) signal SEQ ID NO: 15 OsUbi2 promoter SEQ ID NO: 16 Chloroplast localization peptide CTP-TS-SSU SEQ ID NO: 17 T-Ara5 Detailed Description of the Invention

[0068] The following examples are provided to give those skilled in the art a complete disclosure and description of how to make and use the present invention, and the following examples are not intended to limit the scope of what the inventors regard as their invention, nor are they intended to represent or imply that the experiments described below are all or the only experiments conducted. Those skilled in the art should understand that many changes and / or modifications can be made to the invention shown in the specific aspects without departing from the spirit or scope broadly described in the present invention. Therefore, the present invention is considered illustrative rather than restrictive in all aspects.

[0069] Example 1: Construction of Maize Transgenic Vector

[0070] According to the Vip3Aa sequence information listed on the Bt gene naming website (http: / / www.lifesci.sussex.ac.uk / home / Neil_Crickmore / Bt / vip.html), the original Vip3Aa protein sequence (GenBank: ABG20429.1, the amino acid sequence is shown in SEQ ID NO: 1) was selected. After protein structure prediction, the 12th and 14th amino acids of its amino acid sequence were double-mutated from the original 12A + 14P to 12L + 14G, 12M + 14I, 12T + 14S, 12V + 14H, and 12Y + 14M, respectively. The mutated amino acid sequences are shown in SEQ ID NOs: 2-6. The amino acid sequence was optimized for maize codons, and the corresponding coding nucleotide sequences are shown in SEQ ID NOs: 7-11. The nucleotide sequences were synthesized by GenScript Biotech Corporation.

[0071] When artificially synthesizing the above mutant Vip3Aa gene (SEQ ID NO: 7-11) sequences respectively, a chloroplast localization peptide CTP-TS-SSU (SEQ ID NO: 16) was simultaneously synthesized upstream of ATG. The artificially synthesized CTP-TS-SSU-Vip3Aa mutant gene fragments were constructed downstream of the rice Ubiquitin2 promoter (SEQ ID NO: 15) and upstream of the T-Ara5 terminator (SEQ ID NO: 17) to obtain a Vip3Aa mutant gene expression cassette driven by the OsUbi2 promoter. Then, the Vip3Aa mutant gene expression cassette was inserted into a vector containing the Pat gene (SEQ ID NO: 12) by the method of seamless cloning with homologous recombination to obtain an expression cassette containing the Vip3Aa mutant and the glufosinate-resistant gene Pat. Then, the two expression cassettes were ligated between the LB and RB of the pCAMBIA1300 backbone by the method of homologous recombination to construct vectors pQY008133 (12L+14G), pQY008155 (12M+14I), pQY008282 (12T+14S), pQY008293 (12V+14H), pQY008337 (12Y+14M).

[0072] The vector pQYI0186 was constructed according to the above method. The difference between pQYI0186 and the above vectors is that the mutant Vip3Aa was replaced with the original Vip3Aa.

[0073] Example 2. Comparison of cytotoxicity of transgenic plants

[0074] The transgenic vectors pQY008133 (12L+14G), pQY008155 (12M+14I), pQY008282 (12T+14S), pQY008293 (12V+14H), pQY008337 (12Y+14M) and pQYI0186 were used to transform maize callus by the Agrobacterium-mediated transformation method. The transformants QYI8133, QYI8155, QYI8282, QYI8293, QYI8337 and QYI186 were obtained through screening and culturing. During the genetic transformation process, the emergence of the intermediate materials of the maize transformants QYI8133, QYI8155, QYI8282, QYI8293, QYI8337 and QYI186 was compared.

[0075] Table 1 Positive transformation seedlings of Vip3Aa mutants and QYI186

[0076] Insect-resistant gene transformant Transformed immature embryos (pcs) Positive transformed seedlings (plants) QYI186 1000 60 QYI8133 1000 609 QYI8155 1000 651 QYI8282 1000 613 QYI8293 1000 579 QYI8337 1000 624

[0077] The results showed that the callus growth of QYI186 was severely inhibited and harmed, and only 60 positive seedlings were obtained among 1000 transformed embryos. However, the callus growth of QYI8133, QYI8155, QYI8282, QYI8293, and QYI8337 was good, and a large number of positive seedlings were obtained (579-651 positive seedlings / 1000 transformed embryos). Figure 1 and Table 1, indicating that the cytotoxicity of the transformed Vip3Aa mutant to the recipient plant was significantly reduced compared with the original Vip3Aa gene.

[0078] Example 3: Protein content of transgenic corn leaves

[0079] After the determination of protein expression level, at the V7-V8 stage of the T2 generation of transgenic corn, the average expression levels of mutant Vip3Aa proteins in the leaves of transgenic corn QYI8133, QYI8155, QYI8282, QYI8293, and QYI8337 are shown in Table 2 (leaf fresh weight). Compared with the original average expression level of Vip3Aa of 11 μg / g (leaf fresh weight) in the leaves of transgenic corn QYI186, the expression level of the Vip3Aa mutant protein in transgenic corn was significantly increased.

[0080] Table 2 Protein expression in Vip3Aa mutant and QYI186 positive transformed seedlings

[0081] Vector number Mutation site Protein expression level (average) μg / g pQYI186 Vip3Aa 11 pQYI8133 mVip3Aa(A12L+P14G) 43.68 pQYI8155 mVip3Aa(A12M+P14I) 45.93 pQYI8282 mVip3Aa(A12T+P14S) 67.55 pQYI8293 mVip3Aa(A12V+P14H) 38.07 pQYI8337 mVip3Aa(A12Y+P14M) 56.29

[0082] Example 4: Insecticidal activity detection

[0083] 1. LC50 determination

[0084] The lethal median concentration (LC50) of the original protein and each mutant protein to fall armyworm was determined by the feed surface method. The freshly prepared artificial feed was poured into a beaker and soaked in hot water. The feed was dispensed into a 24-well cell culture plate using a manual continuous dispenser, with 1 mL of feed dispensed into each well, and the diameter of the well was 1.6 cm. After the feed solidified, the surface area formed was 2 cm 2 The mutant protein to be tested was stained with Na 2 CO 3 / NaHCO 3The buffer solution (pH = 10) was serially diluted to 4 concentrations. The above concentration dilutions were aliquoted using a manual continuous pipettor, with 50 μL aliquoted into each well and shaken well to ensure that the protein completely covered the surface of the feed. The 24-well cell culture plate after sample addition was placed in a laminar flow hood to dry. After the protein had penetrated the feed surface, second-instar larvae of Spodoptera frugiperda were inoculated. One larva was inoculated into each well, the lid was covered and tightened, and it was placed under the conditions of a temperature of 25 - 27 °C, a relative humidity of 65 - 70%, and a light cycle of L / D = 16 h / 8 h. The group with 50 μL of buffer solution added was used as a control, and the experiment was repeated 2 times. After 7 days, the death of the test insects in each group was observed, the insect mortality rate was counted, and the corresponding median lethal concentration LC50 was obtained. The results showed that the LC50 value of the mutant protein of this application did not differ significantly from that of the original Vip3Aa protein, and the insecticidal effect against Spodoptera frugiperda was maintained or even improved, as shown in Table 3.

[0085] Table 3 Median lethal concentration (LC50) data of mutant proteins at positions 12 and 14 of Vip3Aa protein against Spodoptera frugiperda

[0086] Mutation site <![CDATA[LC50 (95% confidence interval) μg / cm 2 > Vip3Aa(QYI186) 0.34(0.31-0.39) mVip3Aa(A12L+P14G) 0.26(0.21-0.40) mVip3Aa(A12M+P14I) 0.17(0.14-0.28) mVip3Aa(A12T+P14S) 0.21(0.14-0.38) mVip3Aa(A12V+P14H) 0.29(0.21-0.47) mVip3Aa(A12Y+P14M) 0.23(0.14-0.37)

[0087] 2. Detection of insecticidal activity of transgenic maize leaves

[0088] The resistance of each mutant protein transformant to Spodoptera frugiperda was tested using the insecticidal detection method for transgenic detached leaves. The maize transformation materials of each mutant protein at the V3 - V4 leaf stage were selected, and the second leaf with the heart leaf facing outwards was used as the test material. The leaf tip was removed, and a leaf segment with a length of 2 - 3 cm was cut and placed into the sampling device, with 2 replicates for each plant material. Second-instar early larvae of Spodoptera frugiperda were selected and picked into the sampling device with a small brush, and 10 larvae were placed in each device. After insect inoculation, the experimental device was placed in an environment with a temperature of 27 ± 1 °C and a humidity of 70 ± 5%. Four days after insect inoculation, the insect resistance level of each maize transformation material of the mutant protein was determined (Tables 4 and 5), and the resistance ratio of Vip3Aa and each mutant protein transformation material was counted (Table 6).

[0089] Table 4 Grading standard for the damage degree of maize leaves by Spodoptera frugiperda

[0090]

[0091]

[0092] Table 5 Resistance evaluation standard of maize leaves to Spodoptera frugiperda

[0093] Leaf-eating level at the heart leaf stage (first condition) Mortality rate (second condition) Resistance type 1 4 DAA, more than 90% High resistance (HR) 2 4 DAA, more than 80% Resistant (R) 3-4 None Susceptible (S)

[0094] Table 6 Resistance ratio of Vip3Aa and each mutant protein transformation material

[0095] Mutation site High resistance ratio Vip3Aa 40.91％ mVip3Aa(A12L+P14G) 54.17％ mVip3Aa(A12M+P14I) 64.52％ mVip3Aa(A12T+P14S) 83.75％ mVip3Aa(A12V+P14H) 48.65％ mVip3Aa(A12Y+P14M) 56.06％

[0096] From the results of transgenic insect resistance tests, the high resistance ratios of the 5 mutants to Spodoptera frugiperda were all significantly higher than those of the wild type.

[0097] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the spirit and scope of the technical solutions of the present invention.

Claims

1. A mutant insecticidal protein Vip3Aa, comprising an amino acid sequence having the following mutations compared to the amino acid sequence shown in SEQ ID NO: 1: the 12th amino acid in the amino acid sequence corresponding to SEQ ID NO: 1 is mutated from alanine to leucine, methionine, threonine, valine or tyrosine; and the 14th amino acid is mutated from proline to glycine, isoleucine, serine, histidine or methionine.

2. The mutant insecticidal protein Vip3Aa according to claim 1, characterized in that The 12th amino acid in the amino acid sequence corresponding to SEQ ID NO: 1 is mutated from alanine to leucine and the 14th amino acid is mutated from proline to glycine; The 12th amino acid in the amino acid sequence corresponding to SEQ ID NO: 1 is mutated from alanine to methionine and the 14th amino acid is mutated from proline to isoleucine; The 12th amino acid in the amino acid sequence corresponding to SEQ ID NO: 1 is mutated from alanine to threonine and the 14th amino acid is mutated from proline to serine; The 12th amino acid in the amino acid sequence corresponding to SEQ ID NO: 1 is mutated from alanine to valine and the 14th amino acid is mutated from proline to histidine; or, The 12th amino acid in the amino acid sequence corresponding to SEQ ID NO: 1 is mutated from alanine to tyrosine and the 14th amino acid is mutated from proline to methionine.

3. The mutant insecticidal protein Vip3Aa according to claim 1 or 2, wherein the amino acid sequence is shown in SEQ ID NO: 2-6.

4. An isolated polynucleotide comprising a nucleic acid sequence encoding the mutant insecticidal protein Vip3Aa according to any one of claims 1 to 3 or a complementary sequence thereof.

5. The polynucleotide according to claim 4, characterized in that The polynucleotide is DNA, RNA or a hybrid thereof.

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

7. The polynucleotide according to any one of claims 4 to 6, characterized in that It has a nucleic acid sequence selected from the group consisting of: (1) a nucleic acid sequence encoding an amino acid sequence as shown in any one of SEQ ID NOs: 2-6 or a complementary sequence thereof; (2) a nucleic acid sequence as shown in any one of SEQ ID NOs: 7-11 or a complementary sequence thereof; (3) a nucleic acid sequence that hybridizes to the sequence shown in (1) or (2) under stringent conditions; and / or (4) A nucleic acid sequence that encodes the same amino acid sequence as the sequence shown in (1) or (2) due to the degeneracy of the genetic code, or a complementary sequence thereof.

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

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

10. An expression vector comprising a tandem expression frame for expressing the mutant insecticidal protein Vip3Aa and Pat according to claim 1; preferably, the nucleotide sequence of the mutant insecticidal protein Vip3Aa is any one of SEQ ID NOs: 7-11, and the nucleotide sequence of the gene Pat is SEQ ID NO:

12.

11. The expression vector according to claim 10, wherein the gene tandem expression cassette further comprises: The nucleotide sequence of CaMV 35S promoter for initiating Pat expression is shown in SEQ ID NO: 13, and the nucleotide sequence of CaMV poly(A) signal for terminating the gene expression is shown in SEQ ID NO: 14; The nucleotide sequence is shown in SEQ ID NO: 15 as the promoter OsUbi2promoter for promoting the expression of the mutant insecticidal protein Vip3Aa, the nucleotide sequence is shown in SEQ ID NO: 16 as the chloroplast localization peptide CTP-TS-SSU, and the nucleotide sequence is shown in SEQ ID NO: 17 as the terminator T-Ara5 for terminating the expression of the gene.

12. A host cell comprising the polynucleotide according to any one of claims 4 to 8 or the expression vector according to any one of claims 9 to 11; preferably, the host cell is a plant cell.

13. A method for cultivating a transgenic plant having or improving insect resistance and a plant produced by the method, comprising regenerating the plant cell according to claim 12 into a plant.

14. Use of the expression vector according to any one of claims 9 to 11 or the host cell according to claim 12 in improving the insect resistance of plants, preparing an agent with insect resistance effect, or cultivating transgenic plants with or with improved insect resistance; wherein the plant is preferably corn, cotton or soybean, and the insect resistance is preferably resistance to Lepidoptera such as fall armyworm.

15. A method of managing insect resistance or controlling insects, characterized in that The method comprises contacting an insect with at least the plant of claim 13, wherein the insect contacts at least the mutant insecticidal protein Vip3Aa by feeding on the tissue of the plant, and the growth of the insect is inhibited and / or the insect dies after the contact, thereby achieving management of the resistance of the insect or controlling the harm to the plant by the insect; wherein the plant is preferably corn, cotton or soybean, and the insect is preferably Lepidoptera such as fall armyworm.

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