Vip3aa mutant protein and use thereof in controlling pests

Abstract

A Vip3Aa mutant protein and the use thereof in controlling pests. The mutant insecticidal protein Vip3Aa contains an amino acid sequence having the following mutations compared with the amino acid sequence as shown in SEQ ID NO: 1: in the amino acid sequence corresponding to that as shown in SEQ ID NO: 1, the amino acid at position 24 is mutated from tyrosine into any other amino acid; the amino acid at position 27 is mutated from alanine into any other amino acid; the amino acid at position 36 is mutated from methionine into any other amino acid; and / or the amino acid at position 28 is mutated from phenylalanine into any other amino acid. The Vip3Aa mutant protein has a significantly improved insecticidal activity compared with a wild-type Vip3Aa, and has a good insecticidal effect on Lepidoptera insects such as Spodoptera frugiperda.

Description

Vip3Aa mutant protein and its application in controlling pests Technical Field

[0001] The present invention relates to the field of agricultural biotechnology, and more particularly to a Vip3Aa mutant protein and its application in controlling pests. Background Art

[0002] Agricultural pests are the primary factor impacting crop production. With the rapid development of transgenic technology, the ability to create insect-resistant plants through the transfer of 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 been used in transgenic plants to confer insect resistance, including Cry1Ab, Cry1Ac, Cry1F, Cry2Ab, Cry3Bb, and Vip3A. However, with the widespread application of transgenic crops, there remains an urgent need to obtain transgenic plants that express stable levels of Bt proteins and demonstrate effective insect resistance.

[0003] Summary of the Invention

[0004] To solve the above-mentioned 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 24 in the amino acid sequence shown in SEQ ID NO: 1 is mutated from tyrosine to any other amino acid; the amino acid at position 27 is mutated from alanine to any other amino acid; the amino acid at position 36 is mutated from methionine to any other amino acid; and / or the amino acid at position 38 is mutated from phenylalanine to any other amino acid.

[0005] In one embodiment, the amino acid at position 24 in the amino acid sequence corresponding to SEQ ID NO: 1 is mutated from tyrosine to serine; the amino acid at position 27 is mutated from alanine to isoleucine; the amino acid at position 36 is mutated from methionine to leucine; and / or the amino acid at position 38 is mutated from phenylalanine to alanine.

[0006] In another specific embodiment, the mutation further includes: the 12th amino acid in the amino acid sequence corresponding to SEQ ID NO: 1 is mutated from alanine to any other amino acid; the 14th amino acid is mutated from proline to any other amino acid; and / or the 129th amino acid is changed from isoleucine to any other amino acid.

[0007] In another specific embodiment, the amino acid at position 12 in the amino acid sequence corresponding to SEQ ID NO: 1 is mutated from alanine to glycine, aspartic acid, histidine, leucine, methionine, threonine, valine or tyrosine; the amino acid at position 14 is mutated from proline to glutamine, glycine, isoleucine, serine, histidine or methionine; and / or the amino acid at position 129 is changed from isoleucine to methionine.

[0008] In another embodiment, the amino acid at position 12 in the amino acid sequence corresponding to SEQ ID NO: 1 is mutated from alanine to glycine and the amino acid at position 14 is mutated from proline to glutamine; optionally, the amino acid at position 129 is changed from isoleucine to methionine;

[0009] In the amino acid sequence corresponding to SEQ ID NO: 1, the amino acid at position 12 is mutated from alanine to aspartic acid and the amino acid at position 14 is mutated from proline to glycine; optionally, the amino acid at position 129 is mutated from isoleucine to methionine;

[0010] The amino acid at position 12 in the amino acid sequence corresponding to SEQ ID NO: 1 is mutated from alanine to histidine; optionally, the amino acid at position 129 is mutated from isoleucine to methionine;

[0011] In the amino acid sequence corresponding to SEQ ID NO: 1, the amino acid at position 12 is mutated from alanine to leucine and the amino acid at position 14 is mutated from proline to glycine; optionally, the amino acid at position 129 is mutated from isoleucine to methionine;

[0012] In the amino acid sequence corresponding to SEQ ID NO: 1, the amino acid at position 12 is mutated from alanine to methionine and the amino acid at position 14 is mutated from proline to isoleucine; optionally, the amino acid at position 129 is mutated from isoleucine to methionine;

[0013] In the amino acid sequence corresponding to SEQ ID NO: 1, the amino acid at position 12 is mutated from alanine to threonine and the amino acid at position 14 is mutated from proline to serine; optionally, the amino acid at position 129 is mutated from isoleucine to methionine;

[0014] In the amino acid sequence corresponding to SEQ ID NO: 1, the amino acid at position 12 is mutated from alanine to valine and the amino acid at position 14 is mutated from proline to histidine; optionally, the amino acid at position 129 is mutated from isoleucine to methionine; or

[0015] In the amino acid sequence corresponding to SEQ ID NO: 1, the 12th amino acid is mutated from alanine to tyrosine and the 14th amino acid is mutated from proline to methionine; optionally, the 129th amino acid is changed from isoleucine to methionine.

[0016] In another specific embodiment, the mutation further comprises: introducing enzyme cleavage sites at positions 193-194 and 196-197 corresponding to the amino acid sequence shown in SEQ ID NO: 1.

[0017] In another specific embodiment, the restriction enzyme cleavage site is RA.

[0018] In another specific embodiment, the introduced restriction enzyme cleavage site is to mutate the amino acids at positions 193-194 in the amino acid sequence shown in SEQ ID NO: 1 to RA and insert RA at positions 196-197.

[0019] In a specific embodiment, the amino acid sequence of the mutant insecticidal protein Vip3Aa is shown in SEQ ID NO: 3-6 and SEQ ID NO: 10-79.

[0020] The present invention also provides an isolated polynucleotide comprising a nucleic acid sequence encoding the mutant insecticidal protein Vip3Aa or a complementary sequence thereof.

[0021] In one embodiment, the polynucleotide is DNA, RNA, or a hybrid thereof.

[0022] In one embodiment, the polynucleotide is single-stranded or double-stranded.

[0023] In one embodiment, the polynucleotide has a nucleic acid sequence selected from the group consisting of:

[0024] (1) a nucleic acid sequence encoding an amino acid sequence as shown in any one of SEQ ID NOs: 3-6 and SEQ ID NOs: 10-79, or a complementary sequence thereof;

[0025] (2) a nucleic acid sequence as shown in any one of SEQ ID NOs: 7-9 or a complementary sequence thereof;

[0026] (3) a nucleic acid sequence that hybridizes to the sequence shown in (1) or (2) under stringent conditions; and / or

[0027] (4) A nucleic acid sequence encoding 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.

[0028] In another embodiment, the nucleic acid sequence is optimized for expression in plant cells.

[0029] The present invention also provides an expression vector, which comprises the polynucleotide and an expression control element operably linked thereto.

[0030] The present invention also provides a host cell, which contains the polynucleotide or the expression vector.

[0031] In one embodiment, the host cell is a plant cell.

[0032] The present invention also provides a method for cultivating transgenic plants with or enhanced insect resistance and plants produced by the method, which comprises regenerating the plant cells into plants.

[0033] The present invention also provides the use of the expression vector or the host cell in improving the insect-resistant properties of plants, preparing agents with insect-resistant effects, or cultivating transgenic plants with or enhanced insect-resistant abilities.

[0034] The present invention also provides a method for managing insect resistance or controlling insects, characterized in that it includes contacting an insect with at least the plant, wherein the insect comes into contact with at least the mutant insecticidal protein Vip3Aa by feeding on the tissue of the plant, and after contact, the growth of the insect is inhibited and / or the insect dies, thereby achieving management of the insect resistance or control of the insect damage to the plant.

[0035] In one embodiment, the plant is corn, cotton or soybean.

[0036] In one embodiment, the insect resistance is against Lepidoptera such as Spodoptera frugiperda or the insect is Lepidoptera such as Spodoptera frugiperda.

[0037] The mutant insecticidal protein Vip3Aa obtained by the present invention has significantly improved insecticidal activity compared with the wild-type Vip3Aa. Its transgenic plants have good insecticidal effects on Lepidoptera insects such as the fall armyworm, and show good commercial value in terms of increasing yield.

[0038] Detailed Description of the Invention

[0039] Some terms used in this specification are defined below.

[0040] In the present invention, "plant" is understood to be any differentiated multicellular organism capable of photosynthesis, in particular monocotyledonous or dicotyledonous plants.

[0041] In the present invention, the term "plant tissue" or "plant part" includes plant cells, protoplasts, plant tissue culture, plant callus, plant pieces, as well as plant embryos, pollen, ovules, seeds, leaves, stems, flowers, branches, seedlings, fruits, kernels, ears, roots, root tips, anthers, etc.

[0042] In the present invention, "plant cell" is understood to be any cell from or found in a plant, which is capable of forming, for example, undifferentiated tissue such as callus, differentiated tissue such as embryos, plant components, plants or seeds.

[0043] In the present invention, "host organism" should be understood as any unicellular or multicellular 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, etc.

[0044] The terms "protein," "polypeptide," and "peptide" are used interchangeably herein to refer to a polymer of amino acid residues, including polymers in which one or more amino acid residues is a chemical analog of a naturally occurring amino acid residue. The proteins and polypeptides of the present invention can be produced recombinantly or by chemical synthesis.

[0045] Specific amino acid positions (numbers) within the proteins of the present invention are determined by aligning the amino acid sequence of the target protein with SEQ ID NO: 1 using standard sequence alignment tools, such as the Smith-Waterman algorithm or the CLUSTALW2 algorithm, wherein 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, WJ and Lipman, DJ (1983) Rapid similarity searches of nucleic acid and protein data banks. Proc. Natl. Acad. Sci. USA, 80: 726-730. Preferably, the default parameters are used in the ClustalW2 (1.82) algorithm: protein gap open penalty = 10.0; protein gap extension penalty = 0.2; protein matrix = Gonnet; protein / DNA end gap = -1; protein / DNA GAPDIST = 4.

[0046] 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) to determine the position of specific amino acids within the protein of the present invention by aligning the amino acid sequence of the protein with Vip3Aa.

[0047] Amino acid sequence identity can be determined conventionally 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.

[0048] It is also clear to those skilled in the art that the structure of a protein can be changed 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 a protein without adversely affecting the activity and / or three-dimensional configuration of the protein molecule. Examples and embodiments of conservative amino acid substitutions are clear to those skilled in the art. 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 non-polar amino acid residue can be substituted for another non-polar amino acid residue, a polar uncharged amino acid residue can be substituted for another polar uncharged amino acid residue, a basic amino acid residue can be substituted for another basic amino acid residue, and an acidic amino acid residue can be substituted for another acidic amino acid residue. Conservative substitutions in which one amino acid is replaced by another amino acid belonging to the same group fall within the scope of the present invention as long as the substitution does not impair the biological activity of the protein.

[0049] Therefore, in addition to the aforementioned mutations, the mutant proteins of the present invention may also include one or more other mutations, such as conservative substitutions, in the amino acid sequence. Furthermore, the present invention also encompasses mutant proteins that include one or more other non-conservative substitutions, as long as the non-conservative substitutions do not significantly affect the desired function and biological activity of the protein of the present invention.

[0050] 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. Therefore, on the other hand, the present invention also relates to fragments in which one or more amino acid residues are deleted from the N and / or C terminus of a mutant protein while retaining its desired functional activity, which are also within the scope of the present invention and are referred to as biologically active fragments. In the present invention, a "biologically active fragment" refers to a portion of a mutant protein of the present invention that retains the biological activity of the mutant protein of the present invention. For example, a biologically active 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, such as 1, 2, 3, 4 or 5) amino acid residues are deleted from the N and / or C terminus of the protein, but which still retains the biological activity of the full-length protein.

[0051] In the embodiment of the present invention, the Vip3Aa shown in SEQ ID NO: 1 is used to add or introduce an enzyme cleavage site (RA or DE). It is known in the art that in different Vip3Aa, the region (T 192 S 193 S 194 K 195 V 196 K 197 K 198 D 199 G 200 ) is very conserved; even, in all Vip3A proteins, the region (T 192 S 193 S 194 K 195 V 196 K 197 K 198 D 199 G 200 ) Especially (K 195 V 196 K 197 K 198 D 199 ) is also very conservative. Therefore, under the guidance of the present invention, those skilled in the art can also find the above region (T 192 S 193 S 194 K 195 V 196 K 197 K 198 D 199 G 200 ) by adding or introducing restriction sites (RA or DE) to obtain different mutants.

[0052] 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 canonical or wild-type sequence or a reference sequence.

[0053] The terms "wild-type" and "mutant" are relative, referring to the most frequent phenotype in a particular population, or to the system, organism, or gene that possesses such a phenotype. In some examples, a wild-type allele refers to the standard allele at a locus, or the most frequent allele in a particular population, and can be represented by a specific amino acid or nucleic acid sequence. For example, the wild-type Vip3Aa20 protein can be represented by SEQ ID NO: 1.

[0054] 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 represent the sense or antisense strand. Nucleic acids include DNA, RNA, or hybrids thereof, and may be of natural or synthetic origin. For example, a nucleic acid may include mRNA or cDNA. A nucleic acid may include a nucleic acid that has been amplified (e.g., using the polymerase chain reaction). The nucleotide designations "R" refer to a purine such as guanine or adenine; "Y" refers to a pyrimidine such as cytosine or thymine (uracil in the case of RNA); "M" refers to adenine or cytosine; "K" means guanine or thymine; and "W" means adenine or thymine.

[0055] The term "isolated", when referring to a nucleic acid, refers to a nucleic acid that is separated from a substantial portion of the genome in which it is naturally present and / or substantially separated from other cellular components that naturally accompany the nucleic acid. For example, any nucleic acid that has been produced by synthesis (e.g., by continuous base condensation) is considered to be 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 a genome are also considered to be isolated.

[0056] It is well known to those skilled in the art 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 alternative nucleic acid sequences encoding the same protein is within the capabilities of those skilled in the art, and thus, the present invention encompasses 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 achieve better expression.

[0057] The term "transgenic" plant refers to a plant comprising a heterologous polynucleotide. Preferably, the heterologous polynucleotide is stably integrated in the genome so that the polynucleotide is passed to successive generations. The heterologous polynucleotide can be integrated into the genome separately or integrated as a part of a recombinant expression cassette." transgenic" is used herein to refer to any cell, cell line, callus, tissue, plant part or plant, whose genotype is changed due to the presence of heterologous nucleic acids, including those initially changed transgenic organisms or cells, and those produced from initial transgenic organisms or cell hybridization or asexual reproduction. As used herein, the term "transgenic" is not intended to include changing genomes (chromosomes or chromosomes) by conventional plant breeding methods (for example, hybridization) or by naturally occurring events (such as, self-fertilization, random cross fertilization, non-recombinant virus infection, non-recombinant bacterial transformation, non-recombinant transposition or spontaneous mutation).

[0058] The Vip3Aa gene herein can be introduced into plants according to common methods in the industry, and can be subjected to transgenic manipulation via appropriate plant transformation expression vectors.

[0059] The selection of any appropriate promoter, including vectors, is a common practice in the industry for plant transgenics. For example, commonly used promoters in plant transgenics include, but are not limited to, the SP6 promoter, the T7 promoter, the T3 promoter, the PM promoter, the maize ubiquitin promoter, the cauliflower mosaic virus (CaMV) 35S promoter, the nopaline synthase (nos) promoter, the figwort mosaic virus 35S promoter, the sugarcane stalk-shaped virus promoter, the bamboo yellow mottle virus promoter, the light-inducible promoter ribulose-1,5-ketose carboxylase (ssRUBISCO small subunit), the rice cytoplasmic triosephosphate isomerase (TPI) promoter, the Arabidopsis adenine phosphoribosyltransferase (APRT) promoter, the octopine synthase promoter, and the BCB (blue copper binding protein) promoter.

[0060] Plant transgenic vectors include a polyadenylation signal sequence that can cause 3'-end polyadenylation, including, but not limited to, the NOS 3'-terminal derivative of the nopaline synthase gene of Agrobacterium tumefaciens, the octopine synthase 3'-terminal derivative of the octopine synthase gene of Agrobacterium tumefaciens, the 3'-terminal end of the tomato or potato protease inhibitor I or II gene, the CaMVPolyA signal sequence, the 3'-terminal end of the rice α-amylase gene, and the 3'-terminal end of the phaseolin gene.

[0061] The vector also includes a gene encoding a selectable marker as a reporter molecule. Examples of selectable markers include, but are not limited to, antibiotics (e.g., neomycin, carbenicillin, kanamycin, spectinomycin, hygromycin, bleomycin, chloramphenicol, etc.) or herbicide resistance (glyphosate, glufosinate, glufosinate, etc.) genes.

[0062] Vector transformation methods include Agrobacterium-mediated transformation, electroporation, microparticle bombardment, polyethylene glycol-medium absorption, and the like to introduce recombinant plasmids into plants.

[0063] The plant transformation recipients in the present invention include plant cells (including suspension culture cells), protoplasts, callus tissues, hypocotyls, seeds, cotyledons, buds and mature plants.

[0064] The scope of transgenic plants includes not only the plants obtained at the time of gene introduction, but also its clones and offspring (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 show the characteristics of the first generation transgenic plants after hybridization and fusion. The scope of the present invention also includes plant parts, such as seeds, flowers, stems, fruits, leaves, roots, tubers, and tuberous stems, which are derived from plants that have been genetically modified in advance by the methods mentioned in the present invention, or their offspring, and are composed of at least a portion of genetically modified cells.

[0065] As used herein, "insecticide" or "insect-resistant" refers to being 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 corn borer and / or fall armyworm.

[0066] As used herein, "inhibition of insect growth" refers to sublethal effects, meaning effects that are not lethal but induce certain effects on growth, development, behavior, physiology, biochemistry, and tissue, such as slowed and / or stopped growth. Furthermore, the plants should be morphologically normal and can be cultivated under conventional methods for product consumption and / or production.

[0067] The present invention can be implemented in a variety of different forms, and the implementation methods are not limited to the methods described herein. The examples herein are provided for thorough and complete effectiveness, so that those skilled in the art can fully understand the scope of the present invention. The same reference numbers refer to the same elements throughout the present invention.

[0068] The terms used herein are intended to describe specific embodiments and are not intended to set limitations. Unless otherwise expressly stated herein, the terms "a," "an," and "the" used in the above English version also include their plural forms. The terms "comprises" and / or "comprising," or "includes" and / or "including" used herein specifically refer to the presence of the features, factors, and / or components described herein, and do not exclude the presence or addition of one or more other features, factors, and components. The term "and / or" used in the above includes all items in the one or more combination list.

[0069] The present invention has been described in detail through a series of embodiments, but the present invention is not limited to the disclosed embodiments. Any quantitative changes, substitutions, replacements, etc. within the scope of the present invention are not described herein, or may be modified according to public needs.

[0070] Sequence Description DETAILED DESCRIPTION

[0071] The following examples are presented so as to provide those skilled in the art with a complete disclosure and description of how to make and use the present invention, and 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 below are all or the only experiments performed. It should be understood by those skilled in the art that many variations and / or modifications may be made to the invention shown in the specific aspects without departing from the spirit or scope of the invention as broadly described. Therefore, the aspects herein are to be considered in all respects as illustrative and not restrictive.

[0072] Example 1: Acquisition and synthesis of Vip3Aa20 single-point mutant protein

[0073] 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 Vip3Aa20 protein sequence (GenBank: ABG20429.1, amino acid sequence shown in SEQ ID NO: 1) that performs well in transgenic maize MIR162 was selected. Position 24 of Vip3Aa20 was mutated from Y to S (amino acid sequence shown in SEQ ID NO: 3); position 27 of Vip3Aa20 was mutated from A to I (amino acid sequence shown in SEQ ID NO: 4); position 36 of Vip3Aa20 was mutated from M to L (amino acid sequence shown in SEQ ID NO: 5); and position 38 of Vip3Aa20 was mutated from F to A (amino acid sequence shown in SEQ ID NO: 6). These amino acid sequences were codon-optimized for maize, and the nucleotide sequences were synthesized by Nanjing GenScript Biotechnology Co., Ltd.

[0074] Example 2: Transformation of Escherichia coli with the recombinant expression vector to obtain Vip3Aa20 single-point mutant protein

[0075] Taking the mVip3Aa20(A27I) point mutant protein as an example, the synthesized mVip3Aa20(A27I) nucleotide sequence (SEQ ID NO: 7) was ligated into the protein expression vector pET15b (Novagen, USA, CAT: 69864-3) according to the instructions for the pET15b vector from Novagen, resulting in the recombinant expression vector pQYDB0206. Following the above-described method for constructing the recombinant expression vector pQYDB0206, recombinant expression vectors pQYDB0203, pQYDB0207, and pQYDB0205 for the mVip3Aa20(Y24S), mVip3Aa20(M36L), and mVip3Aa20(F38A) point mutant proteins were obtained.

[0076] The recombinant expression vectors pQYDB0203, pQYDB0205, pQYDB0206, and pQYDB0207 were transformed into competent Escherichia coli BL21(DE3) cells (Transgen, China, Cat: CD501) using the heat shock method. After induction of protein expression, the cells were harvested and affinity purified using a Ni column. The purified proteins were analyzed by SDS-PAGE, and the protein concentration was estimated. The purified proteins were then stored at -20°C until use.

[0077] Example 3: Identification of the anti-insect effect of feeding Vip3Aa20 single-point mutant protein on Spodoptera frugiperda

[0078] The point mutant proteins obtained above were tested for their insecticidal effects on fall armyworm. The insecticidal activity of the wild-type protein and each mutant protein against 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. 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 is gradiently diluted with Na2CO3 / NaHCO3 buffer (pH=10) to at least 4 concentrations. Use a manual continuous dispenser to dispense the above concentration dilutions, dispense 50μl into each well and shake well to ensure that the protein covers the entire surface of the feed. After the addition is completed, the 24-well cell culture plate is placed in a clean bench to dry. After the protein has completely penetrated into the surface of the feed, the second-instar larvae of the fall armyworm are inoculated. One head is inoculated into each well, the lid is covered, tied tightly, and placed at a temperature of 25-27°C, a relative humidity of 65-70%, and a light intensity of L / D=16h / 8h. 50μl of buffer solution is added as a control, and the test is repeated twice. After 7 days, the death of each group of test insects is observed, the insect mortality rate is calculated, and the corresponding LC50 and LC95 are obtained. Some representative data are shown in Table 1. The results show that the LC50 and LC95 values ​​of the mutant protein described in the present application are significantly lower than those of the wild-type Vip3Aa20 protein. Judging from the protein test results, the insecticidal activity of the mutant against Spodoptera frugiperda is about 2-3 times higher than that of the wild-type.

[0079] Table 1 LC50 and LC95 of mVip3Aa20(A27I), mVip3Aa20(M36L) and mVip3Aa20(F38A) proteins against Spodoptera frugiperda

[0080] Example 4: Identification of the Anti-insect Effect of Vip3Aa20 Multi-point Mutant Protein on Spodoptera frugiperda

[0081] Based on the single-point mutants mVip3Aa20(Y24S), mVip3Aa20(A27I), mVip3Aa20(M36L), and mVip3Aa20(F38A), combined mutations at other sites were performed, resulting in a total of 70 Vip3Aa20 multi-point mutants (specific amino acid sequences are shown in SEQ ID NOs: 10-79). Among them, SSRARA (S193R+S194A+introduction of RA cleavage site between 196-197) refers to the mutation of serine at positions 193 / 194 to RA and the insertion of RA between 196-197.

[0082] Following the experimental methods described for the Vip3Aa20 single-point mutant protein in the previous examples, the anti-insecticide activity of the Vip3Aa20 multi-point mutant proteins against Spodoptera frugiperda was evaluated. Table 2 shows representative data. The results demonstrate that the LC50 and LC95 values ​​of the mutant proteins described herein are significantly lower than those of the wild-type Vip3Aa20 protein, indicating that the mutant proteins exhibit significantly enhanced insecticidal activity against Spodoptera frugiperda compared to the wild-type Vip3Aa20 protein.

[0083] Table 2 LC50 and LC95 of Vip3Aa20 multi-point mutant proteins against Spodoptera frugiperda

[0084] In addition, the remaining combined mutations tested also showed varying degrees of improvement in insecticidal activity against Spodoptera frugiperda.

[0085] Example 5: Detection of protein content and insecticidal activity in transgenic corn leaves

[0086] Maize transformation vectors were conventionally constructed and transformed into maize callus using Agrobacterium tumefaciens. Corresponding maize transformants were obtained through infection, co-cultivation, recovery, selection, and regeneration. The transformation efficiency of selected mutation sites was calculated (Table 5). Protein content in leaves of maize transformants expressing the various mutant proteins was determined using ELISA (enzyme-linked immunosorbent assay) to compare the expression of the Vip3Aa20 mutant protein in each maize transformant.

[0087] Subsequently, the resistance of each mutant protein transformant to the fall armyworm was tested using the insect resistance detection method of transgenic detached leaves. Each mutant protein corn transformation material with a leaf age of V3-V4 was selected. The second leaf with the heart leaf facing outward was used as the test material. The leaf tip was removed, and a leaf with a length of 2-3 cm was cut and placed in a sampling device. Two replicates were made for each material. The early second-instar larvae of the fall armyworm were selected and picked into the sampling device with a small brush. Ten second-instar larvae of the fall armyworm were placed in each device. After the inoculation was completed, the experimental device was placed in an environment with a temperature of 27±1℃ and a humidity of 70±5%. Four days after inoculation, the insect resistance level of each mutant protein corn transformation material was determined according to the grading standard for the degree of damage to corn leaves by the fall armyworm (Table 3) and the evaluation standard for resistance to the fall armyworm (Table 4). Representative results are shown in Table 5.

[0088] Table 3 Grading standards for the degree of damage to corn leaves by fall armyworm

[0089] Table 4 Evaluation criteria for corn leaf resistance to Spodoptera frugiperda

[0090] Table 5 Transformation efficiency and resistance rate of Vip3Aa20 mutants to Spodoptera frugiperda

[0091] Compared with wild-type Vip3Aa20 transgenic corn, the toxicity of each mutant protein to corn cells was significantly reduced, the transformation efficiency was greatly improved, and the insect resistance effect showed varying degrees of improvement.

[0092] At the same time, after many tests, it was found that introducing the gene of the present invention into plants such as corn, soybeans and cotton produced excellent insect-resistant properties without causing changes in agronomic traits, and has good industrial value.

[0093] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not limiting. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents 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: In the amino acid sequence corresponding to SEQ ID NO: 1, the 24th amino acid is mutated from tyrosine to any other amino acid; the 27th amino acid is mutated from alanine to any other amino acid; the 36th amino acid is mutated from methionine to any other amino acid; and / or the 38th amino acid is mutated from phenylalanine to any other amino acid.

2. The mutant insecticidal protein Vip3Aa according to claim 1, characterized in that In the amino acid sequence corresponding to SEQ ID NO: 1, the 24th amino acid is mutated from tyrosine to serine; the 27th amino acid is mutated from alanine to isoleucine; the 36th amino acid is mutated from methionine to leucine; and / or the 38th amino acid is mutated from phenylalanine to alanine.

3. The mutant insecticidal protein Vip3Aa according to claim 1, characterized in that The mutations also include: In the amino acid sequence corresponding to SEQ ID NO: 1, the 12th amino acid is mutated from alanine to any other amino acid; the 14th amino acid is mutated from proline to any other amino acid; and / or the 129th amino acid is mutated from isoleucine to any other amino acid.

4. The mutant insecticidal protein Vip3Aa according to claim 3, characterized in that The amino acid at position 12 in the amino acid sequence corresponding to SEQ ID NO: 1 is mutated from alanine to glycine, aspartic acid, histidine, leucine, methionine, threonine, valine or tyrosine; the amino acid at position 14 is mutated from proline to glutamine, glycine, isoleucine, serine, histidine or methionine; and / or the amino acid at position 129 is changed from isoleucine to methionine.

5. The mutant insecticidal protein Vip3Aa according to any one of claims 3 to 4, characterized in that: In the amino acid sequence corresponding to SEQ ID NO: 1, the amino acid at position 12 is mutated from alanine to glycine and the amino acid at position 14 is mutated from proline to glutamine; optionally, the amino acid at position 129 is mutated from isoleucine to methionine; In the amino acid sequence corresponding to SEQ ID NO: 1, the amino acid at position 12 is mutated from alanine to aspartic acid and the amino acid at position 14 is mutated from proline to glycine; optionally, the amino acid at position 129 is mutated from isoleucine to methionine; The amino acid at position 12 in the amino acid sequence corresponding to SEQ ID NO: 1 is mutated from alanine to histidine; optionally, the amino acid at position 129 is mutated from isoleucine to methionine; In the amino acid sequence corresponding to SEQ ID NO: 1, the amino acid at position 12 is mutated from alanine to leucine and the amino acid at position 14 is mutated from proline to glycine; optionally, the amino acid at position 129 is mutated from isoleucine to methionine; In the amino acid sequence corresponding to SEQ ID NO: 1, the amino acid at position 12 is mutated from alanine to methionine and the amino acid at position 14 is mutated from proline to isoleucine; optionally, the amino acid at position 129 is mutated from isoleucine to methionine; In the amino acid sequence corresponding to SEQ ID NO: 1, the amino acid at position 12 is mutated from alanine to threonine and the amino acid at position 14 is mutated from proline to serine; optionally, the amino acid at position 129 is mutated from isoleucine to methionine; In the amino acid sequence corresponding to SEQ ID NO: 1, the amino acid at position 12 is mutated from alanine to valine and the amino acid at position 14 is mutated from proline to histidine; optionally, the amino acid at position 129 is mutated from isoleucine to methionine; or In the amino acid sequence corresponding to SEQ ID NO: 1, the 12th amino acid is mutated from alanine to tyrosine and the 14th amino acid is mutated from proline to methionine; optionally, the 129th amino acid is changed from isoleucine to methionine.

6. The mutant insecticidal protein Vip3Aa according to any one of claims 1 to 5, characterized in that The mutations also include: Enzyme cleavage sites are introduced at positions 193-194 and 196-197 corresponding to the amino acid sequence shown in SEQ ID NO: 1; the enzyme cleavage sites are preferably RA.

7. The mutant insecticidal protein Vip3Aa according to claim 6, characterized in that The introduced enzyme cleavage site is to mutate the amino acids at positions 193-194 in the amino acid sequence shown in SEQ ID NO: 1 into RA and insert RA at positions 196-197.

8. The mutant insecticidal protein Vip3Aa according to any one of claims 1-2, wherein the amino acid sequence thereof is shown in SEQ ID NOs: 3-6 and SEQ ID NOs: 10-79.

9. An isolated polynucleotide comprising a nucleic acid sequence encoding the mutant insecticidal protein Vip3Aa according to any one of claims 1 to 8 or a complementary sequence thereof.

10. The polynucleotide according to claim 9, characterized in that The polynucleotide is DNA, RNA or a hybrid thereof.

11. The polynucleotide according to claim 9 or 10, characterized in that The polynucleotide is single-stranded or double-stranded.

12. The polynucleotide according to any one of claims 9 to 11, 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: 3-6 and SEQ ID NOs: 10-79, or a complementary sequence thereof; (2) a nucleic acid sequence as shown in any one of SEQ ID NOs: 7-9 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 encoding 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.

13. The polynucleotide of claim 12, wherein the nucleic acid sequence is optimized for expression in plant cells.

14. An expression vector comprising the polynucleotide according to any one of claims 9 to 13 and an expression control element operably linked thereto.

15. A host cell comprising the polynucleotide according to any one of claims 9 to 13 or the expression vector according to claim 14; preferably, the host cell is a plant cell.

16. A method for cultivating a transgenic plant having or improving insect resistance and a plant produced by said method, comprising regenerating the plant cell according to claim 15 into a plant.

17. Use of the expression vector according to claim 14 or the host cell according to claim 15 for improving the insect resistance of plants, preparing an insect-resistant agent, or cultivating transgenic plants with or enhanced insect resistance; wherein the plant is preferably corn, cotton, or soybean, and the insect resistance is preferably resistance to Lepidoptera such as Spodoptera frugiperda.

18. A method of managing insect resistance or controlling insects, characterized in that The method comprises contacting an insect with at least the plant according to claim 16, 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 contact, thereby achieving management of the resistance of the insect or control of the damage to the plant by the insect; wherein the plant is preferably corn, cotton or soybean, and the insect is preferably Lepidoptera such as Spodoptera frugiperda.

Images