Vip3 protein with improved activity and application thereof

Abstract

The invention discloses Vip3 protein with improved activity and application thereof. Vip3Aa mutant protein is obtained by single-point mutation of a residue Y19 in a structural domain I or a residue V383 in a structural domain III; the Vip3Ca mutant protein is obtained by carrying out single-point mutation on a residue K383 in the structural domain III. The insecticidal activity of the Vip3 mutant protein disclosed by the invention on various lepidoptera pests is obviously improved compared with that of a wild protein.

Description

Technical Field

[0001] The invention belongs to the field of biological control and biotechnology, and specifically relates to a Vip3 protein with improved activity and application thereof. Background Art

[0002] National food security is related to national security, so it is necessary to pay attention to the production of grain and important agricultural products. However, agricultural pests have seriously led to a reduction in grain production and a decline in crop quality. For example, fall armyworms, armyworms, cotton bollworms, etc. seriously threaten the production security of my country's corn and other grain crops. In order to achieve "fighting insects to win grain" and ensure a bumper harvest, the "Notice on the Technical Program for the Prevention and Control of Major Diseases and Pests of Grain Crops in 2024 Issued by the National Agricultural Technology Center" proposed green control coverage requirements for wheat (58%), corn (52%), rice (55%) and other crops. All of them exceed 50%. Biological control is an effective method for green pest control. At present, the use of insect pathogenic microorganisms and biotechnology can achieve green control of major agricultural pests such as fall armyworms.

[0003] Bacillus thuringiensis ( Bacillus thuringiencesis Bt (Bt) is an important insect pathogen. Bt toxins are toxic to an increasing number of insects such as Hemiptera, Diptera, Coleoptera, Hymenoptera, Lepidoptera, and other organisms such as mites and nematodes. Bt insecticidal proteins have been widely used worldwide because of their high specificity for target pests, harmlessness to vertebrates, and easy biodegradation. Bt insecticidal proteins are the most successful alternative to synthetic pesticides, and the global planting area of ​​Bt crops has exceeded 190 million hectares. However, the sustainable use of Bt crops is threatened by the development of pest resistance, and many insects resistant to Bt Cry proteins have been reported. Expressing at least two Bt proteins with different binding sites in the insect gut in crops can achieve more durable control of target pests. Therefore, it is of great theoretical significance and application prospects to study Vip3 proteins from Bt but with different receptors from Cry proteins, which can also effectively control Lepidoptera pests such as fall armyworm.

[0004] Vip3 protein is a soluble protein secreted by Bt during the vegetative growth period. It is divided into three categories: Vip3A, Vip3B and Vip3C, and has insecticidal activity against a variety of lepidopteran pests. Vip3A protein can be hydrolyzed by trypsin or insect midgut fluid into two fragments of approximately 22 kDa and approximately 66 kDa, but the two are still combined together. Vip3 protein exists in the form of a tetramer before and after hydrolysis, and only has membrane perforation function after hydrolysis activation. Therefore, Vip3 protein is called protoxin and toxin before and after hydrolysis and activation, respectively. Studies have shown that trypsin causes conformational changes in domain I of the Vip3Aa protein, providing conditions for its membrane perforation function. After trypsin digestion, during the activation of the Vip3Aa protein, the N-terminus of domain I (P 14 -G 22 ) disengages from the groove formed by domain III and the tetramer core, and then forms a parallel four-helix bundle whose size is sufficient to reach and pierce the lipid bilayer. Therefore, reducing the interaction between domain III and the N terminus of domain I in Vip3Aa protein may accelerate the activation of Vip3Aa protein, thereby improving its insecticidal activity and helping to expand the agricultural application of Vip3 proteins. Summary of the invention

[0005] In order to achieve the purpose of the background technology, reduce the interaction between domain III and the N-terminus of domain I in the Vip3Aa protein, accelerate the activation of the Vip3Aa protein, and improve its insecticidal activity, an insecticidal Vip3 protein and its application are proposed.

[0006] In order to achieve the above object, the technical solution adopted by the present invention is as follows: The first object of the present invention is a Vip3 protein with improved activity. The Vip3 protein includes Vip3Aa and Vip3Ca mutant proteins. The Vip3Aa mutant protein has the following mutated amino acid sequence: in the corresponding amino acid sequence shown in SEQ ID No.1, the residue Y19 in domain I or the residue V383 in domain III is single-point mutated to any other amino acid, and the Vip3Ca mutant protein has the following mutated amino acid sequence: in the corresponding amino acid sequence shown in SEQ ID No.3, the residue K383 in domain III is single-point mutated to any other amino acid.

[0007] Amino acids are also represented by the following standard abbreviations: alanine (Ala; A), arginine (Arg; R), asparagine (Asn; N), aspartic acid (Asp; D), cysteine ​​(Cys; C), glutamine (Gln; Q), glutamic acid (Glu; E), glycine (Gly; G), histidine (His; H), isoleucine (Ile; 1), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y), and valine (Val; V).

[0008] Furthermore, the tyrosine residue at position 19 in domain I of the Vip3Aa protein was mutated to alanine, and the valine residue at position 383 in domain III was mutated to alanine or glycine.

[0009] Furthermore, the lysine residue at position 383 in domain III of the Vip3Ca protein was mutated to alanine.

[0010] The second object of the present invention is to provide a gene encoding a Vip3 mutant protein, as shown in SEQ ID No. 2 and SEQ ID No. 4.

[0011] The third object of the present invention is to provide an expression cassette encoding a gene.

[0012] The fourth object of the present invention is to propose the application of Vip3 mutant protein in preventing and controlling lepidopteran pests.

[0013] Further, a biological insecticide for controlling lepidopteran pests is prepared, wherein the multiple lepidopteran pests are fall armyworm Spodoptera frugiperda , Spodoptera exigua Spodoptera exigua , Spodoptera litura Spodoptera litura and cotton bollworm Helicoverpa armigera .

[0014] The following beneficial effects can be obtained through the above technical solution: The mutant protein of the present invention has significantly improved insecticidal activity against fall armyworm, beet armyworm, fall armyworm and cotton bollworm, significantly reducing the amount of protein used in actual control operations, which is conducive to biological control of fall armyworm, beet armyworm, fall armyworm and cotton bollworm at a lower cost, thereby enhancing the application of Vip3 proteins in the control of lepidopteran pests. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1Structural diagram of the amino acid residues in domain III of Vip3Aa protein that interact with the N-terminus of domain I.

[0016] Figure 2 Figure 1. Purification results of Vip3Aa protein domain III mutant protein.

[0017] Figure 3 The results of membrane perforation activity of Vip3Aa protein domain III mutant protein.

[0018] Figure 4 Figure 2. Protein purification results of mutants Vip3Aa-V383G and Vip3Aa-Y19A.

[0019] Figure 5 Structural diagram of amino acid residues K383 and N390 in domain III of Vip3Ca protein.

[0020] Figure 6 Figure 2. Protein purification results of mutants Vip3Ca-K383A and Vip3Ca-N390A. DETAILED DESCRIPTION

[0021] The following is combined with Figure 1-6 The present invention is further described with examples:

[0022] During the activation of Vip3Aa protein, the N-terminus of domain I (P 14 -G 22 ) detaches from the groove formed by domain III and the tetramer core, and then forms a parallel four-helix bundle. Therefore, domain III may be involved in the activation process of Vip3Aa protein, thereby affecting its membrane perforation function and toxicity. In this embodiment, the wild-type Vip3Aa amino acid sequence used is shown in SEQ ID No. 1: MNKNNTKLSTRALPSFIDYFNGIYGFATGIKDIMNMIFKTDTGGDLTLDEILKNQQLLNDISGKLDGVNGSLNDLIAQGNLNTELSKEILKIANEQNQVLNDVNNKLDAINTMLRVYLPKITSMLSDVMKQNYALSLQIEYLSKQLQEISDKLDIINVNVLINSTLTEITPAYQRIKYVNEKFEELTFATETSSKVKKDGSPADILDELTELTELAKSVTKNDVDGFEFYLNTFHDVMVGNNLFGRSALKTASELITKENVKTSGSEVGNVYNFLIVLTALQAKAFLTLTTCRKLLGLADIDYTSIMNEHLNKEKEEFRVNILPTLSNTFSNPNYAKVKGSDEDAKMIVEAKPGHALIGFEISNDSITVLKVYEAKLKQNYQVDKDSLSEVIYGDMDKLLCPDQSEQIYYTNNIVFPNEYVITKIDFTKKMKTLRYEVTANFYDSSTGEIDLNKKKVESSEAEYRTLSANDDGVYMPLGVISETFLTPINGFGLQADENSRLITLTCKSYLRELLLATDLSNKETKLIVPPSGFISNIVENGSIEEDNLEPWKANNKNAYVDHTGGVNGTKALYVHKDGGISQFIGDKLKPKTEYVIQYTVKGKPSIHLKDENTGYIHYEDTNNNLEDYQTINKRFTTGTDLKGVYLILKSQNGDEAWGDNFIILEISPSEKLLSPELINTNNWTSTGSTNISGNTLTLYQGGRGILKQNLQLDSFSTYRVYFSVSGDANVRIRNSREVLFEKRYMSGAKDVSEMFTTKFEKDNFYIELSQGNNLYGGPIVHFYDVSIK The present invention uses Chimera X software to analyze the amino acid residues in domain III that interact with the N-terminus of domain I (P 14 -G 22 ), and speculates that V 383 DK 385 and T 525 KLIV 529 in domain III, and P 531 interacts with the N-terminus of domain I (P 14 -G22 ) may interact ( Figure 1 ). Reducing the interaction between these amino acids in domain III and the N-terminus of domain I may accelerate the activation process of Vip3Aa protein, thereby enhancing the insecticidal activity of Vip3Aa protein. Therefore, the inventors mutated the above 9 amino acid residues into alanine respectively, and obtained 9 alanine mutants.

[0023] Table 1: Primers for constructing alanine mutants in domain III of Vip3Aa protein The primers required for the construction of alanine mutants are shown in Table 1. PCR was performed using the Vip3Aa protein encoding gene as a template. The sequence of the Vip3Aa protein encoding gene is shown in SEQ ID No.2.

[0024] The obtained PCR product was digested with DMT enzyme for 1 hour, 5 μL of the enzymatic product was added to 50 μL of DMT competent cells, ice-bathed for 30 minutes, heat-shocked in a 45°C water bath for 45 seconds, and immediately ice-bathed for 2 minutes after heat shock. 800 μL of antibiotic-free LB medium was added, and cultured in a shaking incubator at 37°C and 200 rpm for 1 hour. After centrifugation, 650 μL of the supernatant was discarded, and 150 μL of the bacterial solution was taken out and heated to 40 °C. Kan Spread the plate on the LB agar plate with resistance, invert and culture at 37℃ overnight, pick a single colony and put it in 5mL LB test tube ( Kan The cells were incubated at 37°C for 8-10 hours, and the bacterial suspension was aspirated for preservation and sequencing. The plasmid was extracted from the bacterial suspension with correct sequencing, and transformed into Escherichia coli BL21 (DE3), and glycerol bacteria were prepared and stored in a -80°C refrigerator.

[0025] (1) Place the stored glycerol tube strain in LB (containing Kan Streak on plates with resistance to the virus and incubate them upside down at 37°C overnight.

[0026] (2) Pick a single colony and inoculate it into 5 mL LB (containing Kan The cells were cultured in liquid medium with resistance at 37°C and 180 rpm overnight.

[0027] (3) Transfer 10 mL of overnight culture solution to 1 L LB (containing Kan The cells were cultured in liquid medium at 37°C and 180 r / min until OD600 reached about 1.0. IPTG with a final concentration of 0.5 mmol / L was added to induce Vip3Aa protein expression, and the cells were cultured overnight at 16°C and 180 r / min.

[0028] (4) Collect the cells by centrifugation at 8000 rpm for 5 min at 4°C, resuspend them in Lysis buffer (containing PMSF at a final concentration of 1 mmol / L), and disrupt them by ultrasonication.

[0029] (5) Centrifuge at 18,000 r / min for 40 min at 4°C to collect the supernatant for purification of Vip3Aa protein.

[0030] (6) Add the supernatant to the nickel affinity chromatography column at a flow rate of 0.8 mL / min and hang the column twice. The column was washed with 15 column volumes of washing buffer at a flow rate of 1.0 mL / min.

[0031] (7) Add 5-10 mL of elution buffer to the nickel column, let it stand for 10 min, and control the flow rate to 0.5 mL / min, and collect the eluate.

[0032] (8) The eluate is aspirated into a dialysis bag and dialyzed in dialysis buffer for 3 to 4 times, with the dialysis buffer replaced every 3 hours. At the end of dialysis, the Vip3Aa protein used in the experiment is obtained.

[0033] (9) The purified protein was packaged, quick-frozen in liquid nitrogen, and stored at -80°C.

[0034] (10) The purified Vip3Aa protein and mutants were subjected to SDS-PAGE detection. The results are shown in Figure 2 .

[0035] Vip3Aa protein is activated by protease hydrolysis in the insect midgut, and then binds to specific receptors in the midgut to form channels in the midgut epidermal cells. The formation of these channels destroys the integrity of the cell membrane, causing rapid imbalance of ions and small molecules inside and outside the cell, ultimately leading to cell rupture and insect death. The present invention analyzes the membrane perforation activity of Vip3Aa protein and its mutants by the leakage of calcein in calcein-encapsulated liposomes.

[0036] Preparation of Calcein Liposomes (1) A lipid mixture of DOPE and DOPC (1:1 (w / w)) was dissolved in 5 mL of chloroform and evaporated under reduced pressure at 40°C to form a film.

[0037] (2) Add 3 mL of 60 mM calcein solution and hydrate by ultrasonication to ensure that all dried liposomes enter the solution to obtain a liposome suspension. The obtained liposome suspension was frozen and thawed for 5 cycles (liquid nitrogen quick freezing, 40°C water bath).

[0038] (3) The liposome particle size was further reduced by ultrasonication for 4 min (400 W, on for 2 s, off for 2 s) in an ice-water bath. The liposome solution was extruded 21 times using a 0.1 mm polycarbonate membrane and a 1 mL hand-push extruder.

[0039] (4) Add the uniformly extruded liposome solution to a G25 desalting column that had been previously cleaned with 20 mM Tris-HCl for three desalting treatments. Rinse three times with 20 mM Tris-HCl buffer, then take a sample and add the sample when the liquid level of the 20 mM Tris-HCl buffer solution on the column just goes down. When the sample liquid level just goes down, add a large amount of buffer solution. Then take 1 mL of the sample and add the sample when the liquid level of the buffer solution on the G25 column just goes down. When the sample liquid level just goes down, add a large amount of buffer solution. Collect 40 mL of the effluent that has color at the beginning. The effluent that has color at the beginning is the purified calcein liposome and place it in a 4°C refrigerator for later use.

[0040] (5) Take 200 μL of liposome (concentration 30 μM) solution and place it on the ELISA plate. Measure the fluorescence intensity of the reaction, which is recorded as F0. The excitation wavelength of the fluorescence measurement is 485 nm and the emission wavelength is 510 nm.

[0041] (6) Add 20 μL of Vip3Aa protein and protein complex to the ELISA plate and measure the fluorescence intensity every 40 s, recorded as Ft, for a total of 20 min.

[0042] (7) Add 5 μL of 0.01% Triton-100 and react for 5 min. Measure the final fluorescence intensity F100.

[0043] (8) Leakage rate of calcein % = (Ft-F0) / (F100-F0)*100.

[0044] In the present invention, the mixture of each mutant protein and trypsin was incubated with artificially prepared liposomes encapsulating calcein, and the membrane perforation activity of each protein complex was analyzed by the liposome leakage efficiency. The results are as follows: Figure 3 As shown. Figure 3 It can be seen that the calcein release rate caused by the mutant Vip3Aa-V383A is significantly higher than that of the Vip3Aa protein and other mutants, that is, the mutation of residue 383 of the Vip3Aa protein to alanine helps to enhance the membrane perforation activity of the Vip3Aa protein.

[0045] In order to analyze the reasons for the increased membrane perforation activity of Vip3Aa-V383A protein from a structural perspective, the present invention uses two servers, Phyre2 and alphafold3, to predict the protein structure of the Vip3Aa protein mutant. The difference between the two is that Phyre2 uses the reported Vip3Aa protein structure as a template to predict the structure of the Vip3Aa protein mutant, while alphafold3 does not rely on the template. Then, the present invention uses Chimera X to analyze that the contact between the mutant domain III and the N-terminus of domain I is reduced, and the hydrogen bond between residue Y19 and residue A383 disappears in the mutant Vip3Aa-V383A. These results show that reducing the interaction between residue Y19 and residue 383 helps to improve the membrane perforation activity of Vip3Aa protein.

[0046] Indoor bioactivity assay of fall armyworm (24-well plate feed method), insect feeds containing different concentrations of insecticidal proteins were prepared. Blank feed and feed supplemented with 20 mmol / L Tris-HCL buffer were used as blank control and negative control, respectively, and newly hatched larvae of fall armyworm were selected as test insects.

[0047] (1) Weigh 20 g of artificial feed and place it in a sterile culture dish. Press the feed until there are no lumps and spread it evenly in a 9 cm sterile culture dish. (2) Use a pipette to draw 3 mL of the prepared test sample mixed solution and mix the protein sample with The feed is fully mixed and evenly divided into 24 wells. It is then placed at room temperature for a period of time to evaporate the feed. Excess water; (3) Use a medicine spoon to evenly distribute the feed into the 24-well culture plate, and make sure the feed is placed on one side; (4) First, shake the larvae onto an A4 paper, and use a brush to pick out the one-day-old larvae that are fast crawling, active, and can be placed in a 24-well culture plate, one end per well; (5) After the larvae are collected, cover them with the top cover of the 24-well plate (covered with blow-molded board) and then secure them with two rubber bands to prevent the larvae from escaping; (6) Place the 24-well culture plate in an insect incubator at 28°C and 65% RH; (7) Regularly observe the dryness and wetness of the feed, whether the light cycle of the incubator is normal, and whether the conditions are suitable. After 7 days of incubation, count the survival and death of the test insects. The sorted data is used to calculate the mortality rate and LC50 using SPSS software.

[0048] Table 2 Insecticidal activity of Vip3Aa protein and its domain III mutant against Spodoptera frugiperda As shown in Table 2 , among the nine alanine mutant proteins, the insecticidal activity of mutant Vip3Aa-V383A was significantly enhanced by 1.5 times, which was related to the enhancement of its membrane perforation activity.

[0049] From the membrane perforation activity and insecticidal activity results of the mutant Vip3Aa-V383A, it can be seen that eliminating the hydrogen bond between residue 383 and residue Y19 is beneficial to the insecticidal activity of Vip3Aa protein. The present invention mutates Y19 to alanine and V383 to glycine. The predicted protein structure analysis shows that in both mutants, the hydrogen bond between residue 19 and residue 383 disappears. PCR amplification was performed using the Vip3Aa protein expression plasmid as a template, and the corresponding mutants were obtained after DMT enzyme treatment, colon transformation and DNA sequencing verification. The primers required for mutant construction are shown in Table 3. The protein expression and purification of the mutant was the same as in Example 1, and the purified mutant protein was subjected to SDS-PAGE detection. The results are shown in Table 3. Figure 4 The insecticidal activity was determined in the same manner as in Example 3. Newly hatched larvae of Spodoptera frugiperda were selected as test insects. After 7 days of culture, the survival and death of the test insects were counted. The data were collated to calculate the mortality rate and LC50. The results are shown in Table 4.

[0050] Table 3 Primers for Y19A and V383G mutants of Vip3Aa protein Table 4 Insecticidal activity of Vip3Aa protein Y19A and V383G mutants against Spodoptera frugiperda The results are shown in Table 4. Compared with the Vip3Aa protein, the insecticidal activities of Vip3Aa-Y19A and Vip3Aa-V383G were significantly enhanced, increasing by 1.3 and 1.4 times, respectively.

[0051] In order to explore whether reducing or eliminating the hydrogen bonding between domain III and the N-terminus of domain I is also beneficial to improving the insecticidal activity of other Vip3 proteins, the present invention selected Vip3Ca protein as the research object. The inventors analyzed the Vip3Ca protein domain III using Chimera X software and found that K383 and N390 ( Figure 5 ) and the N-terminus of domain I (P 14 -G 22 ) has hydrogen bonding. In the amino acid sequence SEQ ID No. 3, the present invention mutates K383 and N390 to alanine to eliminate the hydrogen bonding.

[0052] SEQ ID No.3 is as follows: MNMNNTKLNARALPSFIDYFNGIYGFATGIKDIMNMIFKTDTGGDLTLDEILKNQQLLNEISGKLDGVNGSLNDLIAQGNLNTELSKEILKIANEQNQVL NDVNNKLDAINTMLNIYLPKITSMLSDVMKQNYALSLQIEYLSRQLQEISDKLDVINLNVLINSTLTEITPSYQRIKYVNEKFDKLTFATESTLRAKQGI FNEDSFDNNTLENLTDLAELAKSITKNDVDSFEFYLHTFHDVLIGNNLFGRSALKTASELITKDEIKTSGSEIGKVYSFLIVLTSLQAKAFLTLTTCRKLLGLSDIDYTSIMNEHLNNEKNEFRDNILPALSNKFSNPSYAKTIGSDNYAKVILESEPGYALVGFEIINDPIPVLKAYKAKLKQNYQVDNQSLSEIVYLDI DKLFCPENSEQKYYTKNLTFPDGYVITKITFEKKLNNLIYEATANFYDPSTGDIDLNKKQVESTFPQTDYITMDIGDDDGIYMPLGVISETFLTPINSFG LEVDAKSKTLTLKCKSYLREYLLESDLKNKETGLIAPPNVFISNVVKNWDIEEDSLEPWVANNKNAYVDNTGGIERSKALFTQGDGEFSQFIGDKLKPNTD YIIQYTVKGKPAIYLKNKSTGYITYEDTNGNSEEFQTIAVKFTSETDLSQTHLVFKSQNGYEAWGDNFIILEAKLFETPESPELIKFNDWERFGTTYITG NELRIDHSRGGYFRQSLNIDSYSTYDLSFSFSGLWAKVIVKNSRGVVLFEKVKNNGSSYEDISESFTTASNKDGFFIELTAERTSSTFHSFRDISIKEKIE PCR amplification was performed using the Vip3Ca protein expression plasmid (the Vip3Ca gene sequence is shown in SEQ ID No. 4) as a template, and the primers are shown in Table 5. The PCR products were treated with DMT enzyme, transformed into E. coli, and verified by DNA sequencing to obtain the corresponding mutants. The protein expression of the mutants was the same as in Example 1, and the Vip3Ca protein and its mutants were subjected to SDS-PAGE detection. The results are shown in Figure 6 .

[0053] SEQ ID No. 4 is as follows: Table 5 Primers required for constructing K383A and N390A mutants in domain III of Vip3Ca protein Primer name Sequence (5'-3') K383A-F TAAAGCAAAACTAGCTCAAAATTATCAAGTT K383A-R AGCTAGTTTGCTTTATACGCTTTTAATACCGG N390A-F CAAAATTATCAAGTTGATGCTCAGTCGTTATCAG N390A-R GCATCAACTTGATAATTTTGTTTTAGTTTTGCTTTAT The insecticidal activity was determined in the same manner as in Example 3. Newly hatched larvae of Spodoptera frugiperda were selected as test insects. After 7 days of culture, the survival and death of the test insects were counted. The data were collated to calculate the mortality rate and LC50. The results are shown in Table 6.

[0054] Table 6 Insecticidal activity of K383A and N390A mutants in domain III of Vip3Ca protein against Spodoptera frugiperda Compared with Vip3Ca protein, the insecticidal activity of Vip3Ca-K383A was significantly enhanced, increasing by 1.8 times, but the insecticidal activity of Vip3Ca-N390A decreased, and the reason for the decrease needs to be studied.

[0055] The present invention detects the insecticidal activity of Vip3 proteins (Vip3Aa-V383A, Vip3Aa-Y19A, Vip3Aa-V383G and Vip3Ca-K383A) with increased insecticidal activity against fall armyworm against beet armyworm, Spodoptera litura and cotton bollworm. The insecticidal activity determination is the same as in Example 3, and beet armyworm, Spodoptera litura and cotton bollworm are selected as newly hatched larvae as test insects. After 7 days of cultivation, the survival and death of the test insects are counted, and the data are sorted for calculating the mortality rate and LC50. The results are shown in Table 7.

[0056] Table 7 Insecticidal activity of Vip3 proteins with enhanced insecticidal activity against Spodoptera exigua, Spodoptera litura and Helicoverpa armigera As can be seen from Table 7, the insecticidal activities of the Vip3 mutant proteins against Spodoptera litura, Spodoptera exigua and cotton bollworm were all improved. Compared with Vip3Aa protein, the insecticidal activities of Vip3Aa-V383A against Spodoptera litura, Spodoptera exigua and cotton bollworm were increased by 2.7 times, 1.8 times and 1.8 times, respectively; the insecticidal activities of Vip3Aa-Y19A against Spodoptera litura, Spodoptera exigua and cotton bollworm were increased by 1.6 times, 1.2 times and 1.1 times, respectively; the insecticidal activities of Vip3Aa-V383G against Spodoptera litura, Spodoptera exigua and cotton bollworm were increased by 1.7 times, 1.2 times and 1.1 times, respectively; compared with Vip3Ca protein, the insecticidal activities of Vip3Ca-K383A against Spodoptera litura, Spodoptera exigua and cotton bollworm were increased by 2.5 times, 1.7 times and 2.2 times, respectively. In summary, the present invention is based on reducing or eliminating the N-terminus (P 14 -G 22 ) helps to obtain mutant proteins with significantly improved insecticidal activity.

[0057] The above are all preferred embodiments of the present invention. For ordinary technicians in this technical field, without departing from the principle of the present invention, various equivalent modifications to the present invention belong to the protection scope of the claims attached to this application.

Claims

1. A Vip3 protein with improved activity, characterized in that: The Vip3 protein includes Vip3Aa and Vip3Ca mutant proteins. The Vip3Aa mutant protein has the following mutated amino acid sequence: in the corresponding amino acid sequence shown in SEQ ID No.1, residue Y19 in domain I or residue V383 in domain III is single-point mutated to any other amino acid, and the Vip3Ca mutant protein has the following mutated amino acid sequence: in the corresponding amino acid sequence shown in SEQ ID No.3, residue K383 in domain III is single-point mutated to any other amino acid.

2. The Vip3 protein with enhanced activity according to claim 1, characterized in that: The tyrosine residue at position 19 in domain I of the Vip3Aa protein was mutated to alanine, and the valine residue at position 383 in domain III was mutated to alanine or glycine.

3. The Vip3 protein with enhanced activity according to claim 1, characterized in that: The lysine residue at position 383 in domain III of the Vip3Ca protein was mutated to alanine.

4. A gene encoding the Vip3 mutant protein according to any one of claims 1 to 3.

5. An expression cassette containing the coding gene according to claim 4.

6. Use of the Vip3 mutant protein according to any one of claims 1 to 3 in controlling lepidopteran pests.

7. The use according to claim 6, characterized in that: The multiple lepidopteran pests are fall armyworm Spodoptera frugiperda , Spodoptera exigua Spodoptera exigua , Spodoptera litura Spodoptera litura and cotton bollworm Helicoverpa armigera .

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