Mutant insecticidal protein vip3aa and use thereof

Abstract

A mutant insecticidal protein Vip3Aa and a use thereof. The mutant insecticidal protein Vip3Aa comprises an amino acid sequence having the following mutations compared with an amino acid sequence shown in SEQ ID NO:1: the 12th amino acid in the amino acid sequence shown in SEQ ID NO: 1 is mutated from alanine to leucine, methionine, threonine, valine, or tyrosine, and the 14th amino acid thereof is mutated from proline to glycine, isoleucine, serine, histidine, or methionine. Point mutation is carried out on a Vip3Aa protein sequence 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 a variety of pests.

Description

A mutant insecticidal protein Vip3Aa and its application Technical Field

[0001] The present invention relates to the field of biotechnology, and more particularly to a mutant insecticidal protein Vip3Aa and applications thereof. Background Art

[0002] Insect pests are a significant factor affecting crop production. A key goal of transgenic crop research is insect resistance. The most widely cultivated crops are those harbouring insect-resistant protein genes from Bacillus thuringiensis (Bt). The insecticidal protein (Vegetative Insecticidal Protein 3, or Vip3) produced by Bacillus thuringiensis during its vegetative stage is a tetramer composed of five domains. The N-terminus primarily controls structural stability, while the C-terminus houses a potential specific receptor-binding domain. There are 14 model samples and over 110 protein types. Vip3 proteins have distinct insecticidal mechanisms from Cry proteins and exhibit strong insecticidal activity against the fall armyworm (Spodoptera frugiperda), making them highly complementary to the development of transgenic insect-resistant crops. Currently, the primary protein used in the market is Vip3Aa. However, Vip3Aa is toxic to plant cells to a certain extent, making it difficult to obtain transformants with high expression levels during the genetic transformation process, and negatively affecting the viability of transgenic corn pollen. There is an urgent need to obtain insect-resistant proteins that have high expression levels in plants, good insect-resistant effects, but no negative effects.

[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 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.

[0005] In one embodiment, the mutant insecticidal protein Vip3Aa,

[0006] In the amino acid sequence corresponding to SEQ ID NO: 1, the 12th amino acid is mutated from alanine to leucine and the 14th amino acid is mutated from proline to glycine;

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

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

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

[0010] 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.

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

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

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

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

[0015] In one embodiment, the polynucleotide has a nucleic acid sequence selected from the group consisting of:

[0016] (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;

[0017] (2) a nucleic acid sequence as shown in any one of SEQ ID NOs: 7-11 or a complementary sequence thereof;

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

[0019] (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.

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

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

[0022] The present invention also provides an expression vector, which comprises a tandem gene expression frame for expressing the mutant insecticidal proteins Vip3Aa and Pat.

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

[0024] In another specific embodiment, the gene tandem expression cassette further comprises:

[0025] a promoter CaMV35S promoter for initiating Pat expression as shown in SEQ ID NO: 13, and a terminator sequence CaMV poly(A) signal for terminating gene expression as shown in SEQ ID NO: 14;

[0026] The nucleotide sequence is shown in SEQ ID NO: 15, which is the promoter OsUbi2promoter for promoting the expression of the mutant insecticidal protein Vip3Aa; the nucleotide sequence is shown in SEQ ID NO: 16, which is the chloroplast localization peptide CTP-TS-SSU; and the nucleotide sequence is shown in SEQ ID NO: 17, which is the terminator T-Ara5 for terminating the expression of the gene.

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

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

[0029] 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.

[0030] The present invention also provides the use of the expression vector or the host cell in improving the insect-resistant properties of plants, preparing medicaments with insect-resistant effects, or cultivating transgenic plants with or enhanced insect-resistant capabilities.

[0031] The present invention also provides a method for managing insect resistance or controlling insects, which comprises contacting an insect with at least the above-mentioned plant, wherein the insect contacts at least the mutant insecticidal protein Vip3Aa by feeding on the tissues 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.

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

[0033] In one embodiment, the insect resistance is against Lepidoptera such as Spodoptera frugiperda or the insect is Lepidoptera such as Spodoptera frugiperda.

[0034] The present invention obtains a mutant Vip3Aa insecticidal protein with reduced toxicity to plant cells by performing point mutation on the original Vip3Aa protein sequence, which can be highly expressed in plant cells and has excellent insecticidal effects on various pests.

[0035] Detailed Description of the Invention

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

[0037] In the present invention, "plant" is understood to be any differentiated multicellular organism capable of photosynthesis, in particular monocotyledonous or dicotyledonous plants.

[0038] 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.

[0039] 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.

[0040] 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.

[0041] 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.

[0042] 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 Vip3Aa 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. The default parameters for the ClustalW2 (1.82) algorithm are preferably used: protein gap open penalty = 10.0; protein gap extension penalty = 0.2; protein matrix = Gonnet; protein / DNA end gap = -1; protein / DNA GAPDI ST = 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) 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.

[0044] 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.

[0045] 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.

[0046] 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.

[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. 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.

[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 the canonical or wild-type sequence or a reference sequence.

[0049] 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.

[0050] 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.

[0051] 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.

[0052] 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).

[0053] The herbicide-resistant Pat gene and Vip3Aa gene herein can be introduced into plants according to methods commonly used in the industry, and can be subjected to transgenic manipulation via appropriate plant transformation expression vectors.

[0054] 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.

[0055] 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 CaMVPoly A signal sequence, the 3'-terminal end of the rice α-amylase gene, and the 3'-terminal end of the phaseolin gene.

[0056] 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.

[0057] Vector transformation methods include Agrobacterium-mediated transformation, electroporation, microparticle bombardment, polyethylene glycol-medium absorption, and the like to introduce recombinant plasmids into plants.

[0058] The plant transformation recipients of the present invention include plant cells (including suspension culture cells), protoplasts, callus tissues, hypocotyls, seeds, cotyledons, buds and mature plants.

[0059] 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.

[0060] 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.

[0061] 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.

[0062] 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.

[0063] 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.

[0064] 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. BRIEF DESCRIPTION OF THE DRAWINGS

[0065] Figure 1 shows the comparison of plant cytotoxicity of transgenic maize QYI186 (Vip3Aa) and QYI8133, QYI8155, QYI8282, QYI8293, and QYI8337 callus differentiation after 4 weeks.

[0066] Sequence Description DETAILED DESCRIPTION

[0067] 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, this disclosure is to be considered in all respects as illustrative and not restrictive.

[0068] Example 1: Construction of corn transgenic vector

[0069] 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 original Vip3Aa protein sequence (GenBank: ABG20429.1, amino acid sequence shown in SEQ ID NO: 1) was selected. After protein structure prediction, double mutations were performed at positions 12 and 14 of the amino acid sequence, changing from 12A+14P to 12L+14G, 12M+14I, 12T+14S, 12V+14H, and 12Y+14M, respectively. The resulting amino acid sequences are shown in SEQ ID NOs: 2-6. These amino acid sequences were codon-optimized for maize, and the corresponding coding nucleotide sequences are shown in SEQ ID NOs: 7-11. The nucleotide sequences were synthesized by GenScript Biotech.

[0070] When the mutant Vip3Aa gene sequences (SEQ ID NOs: 7-11) were artificially synthesized, the chloroplast localization peptide CTP-TS-SSU (SEQ ID NO: 16) was simultaneously synthesized upstream of the 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 the OsUbi2 promoter-driven Vip3Aa mutant gene expression cassette. The Vip3Aa mutant gene expression cassette was then inserted into the region containing the Pat gene (SEQ ID NO: 17) by homologous recombination seamless cloning. NO: 12) was used to obtain an expression cassette containing the Vip3Aa mutant and the glufosinate-resistant gene Pat, and then the two expression cassettes were connected between LB and RB of the pCAMBIA1300 backbone by homologous recombination to construct the vectors pQY008133 (12L+14G), pQY008155 (12M+14I), pQY008282 (12T+14S), pQY008293 (12V+14H), and pQY008337 (12Y+14M).

[0071] The vector pQYI0186 was constructed according to the above method. The difference between pQYI0186 and the above vector is that the mutant Vip3Aa is replaced by the original Vip3Aa.

[0072] Example 2: Comparison of cytotoxicity of transgenic plants

[0073] Maize calli were transformed with transgenic vectors pQY008133 (12L + 14G), pQY008155 (12M + 14I), pQY008282 (12T + 14S), pQY008293 (12V + 14H), pQY008337 (12Y + 14M), and pQYI0186 using Agrobacterium tumefaciens. Transformants QYI8133, QYI8155, QYI8282, QYI8293, QYI8337, and QYI186 were obtained after screening and culture. During the genetic transformation process, seedling emergence of intermediate materials from maize transformants QYI8133, QYI8155, QYI8282, QYI8293, QYI8337, and QYI186 was compared.

[0074] Table 1 Positive transformation results of Vip3Aa mutant and QYI186

[0075] The results showed that the callus growth of the QYI186 transformant was severely inhibited and harmed, with only 60 positive transformed seedlings out of 1000 transformed embryos. In contrast, the callus growth of the QYI8133, QYI8155, QYI8282, QYI8293, and QYI8337 transformants was good, and a larger number of positive transformed seedlings were obtained (579 to 651 positive transformed seedlings per 1000 transformed embryos), as shown in Figure 1 and Table 1. This indicates that the Vip3Aa mutant had significantly reduced cytotoxicity to the recipient plants compared with the original Vip3Aa gene.

[0076] Example 3: Protein content of transgenic corn leaves

[0077] Protein expression levels were measured in the leaves of transgenic maize varieties QYI8133, QYI8155, QYI8282, QYI8293, and QYI8337 at the V7-V8 stage of the T2 generation (fresh leaf weight), as shown in Table 2. Compared to the original average Vip3Aa expression level of 11 μg / g (fresh leaf weight) in the leaves of transgenic maize QYI186, the expression level of the Vip3Aa mutant protein in the transgenic maize was significantly increased.

[0078] Table 2 Protein expression levels in Vip3Aa mutant and QYI186 positive transformed seedlings

[0079] Example 4: Insecticidal activity detection

[0080] 1. LC50 determination

[0081] The LC50 values ​​of the original protein and each mutant protein against Spodoptera frugiperda were determined using the feed surface assay. Freshly prepared artificial feed was poured into a beaker and soaked in hot water. The feed was then dispensed into a 24-well cell culture plate using a manual continuous dispenser, with 1 mL of feed dispensed into each well. The wells had a diameter of 1.6 cm. After the feed solidified, the resulting surface area was 2 cm. 2. The mutant protein to be tested was gradiently diluted with Na2CO3 / NaHCO3 buffer (pH=10) to 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 air 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 was added as a control, and the test was repeated twice. After 7 days, the death of each group of test insects was observed, the insect mortality rate was calculated, and the corresponding lethal concentration LC50 was obtained. The results showed that the LC50 value of the mutant protein of the present application was not significantly different from that of the original Vip3Aa protein, and the insecticidal effect on the fall armyworm was maintained or even improved, as shown in Table 3.

[0082] Table 3 Data on the median lethal concentration (LC50) of mutant proteins at positions 12 and 14 of Vip3Aa protein against Spodoptera frugiperda

[0083] 2. Detection of insect resistance activity of transgenic corn leaves

[0084] 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 at the V3-V4 leaf stage was selected, and the second leaf with the heart leaf facing outward was used as the test material. The leaf tip was removed, and leaves with a length of 2-3 cm were cut and placed in the sampling device. Two replicates were made for each material. The early 2nd instar larvae of the fall armyworm were selected and picked into the sampling device with a small brush. Ten larvae were placed in each device. After the inoculation was completed, the experimental device was placed in an environment with a temperature of 27±1°C and a humidity of 70±5%. Four days after inoculation, the insect resistance level of each mutant protein corn transformation material was determined (Tables 4 and 5), and the resistance ratio of Vip3Aa and each mutant protein transformation material was counted (Table 6).

[0085] Table 4 Grading standards for the degree of damage to corn leaves by fall armyworm

[0086] Table 5 Evaluation criteria for corn leaf resistance to Spodoptera frugiperda

[0087] Table 6 Resistance ratio of Vip3Aa and its mutant protein transformed materials

[0088] Judging from the results of transgenic insect resistance tests, the high resistance rate of the five mutants to fall armyworm was significantly higher than that of the wild type.

[0089] 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: 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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