Methods for engineering cry2aa truncated insecticidal proteins and mutants

Abstract

The application discloses a Cry2Aa truncated insecticidal protein modification method and mutants, relates to the field of bioengineering and agricultural pest control; the method takes a Cry2Aa (145-633) truncated body as a skeleton, carries out molecular docking with potential receptors ABCB1, ALP and CAD-TBR of plutella xylostella, determines key interaction sites at positions 366, 367 and 600, fixes domain I and the above-mentioned sites, adopts ProteinMPNN to carry out sequence design, and combines Rosetta InterfaceAnalyzer and PRODIGY evaluation results to screen mutants; the application further provides mutants Cry2Aa-M13, Cry2Aa-M14 and Cry2Aa-M18 obtained by the above-mentioned method, and biological determination results show that the mortality rates of the three mutants on plutella xylostella larvae at 20 mu g / mL are all higher than that of activated Cry2Aa, and the three mutants can be used for preparing biological insecticidal preparations for preventing and treating plutella xylostella.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering and biological control of agricultural pests, specifically relating to a method for preparing Cry2Aa mutant toxin protein based on receptor interaction sites, the mutant, and its application in controlling diamondback moth. Background Technology

[0002] Bacillus thuringiensis (Bt) Bacillus thuringiensis Cry toxins produced by Bt are important active ingredients in the green control of agricultural pests. Cry toxins usually exist in the form of protoxins, which are activated by insect midgut proteases to form an insecticidal core fragment. This core fragment binds to receptor molecules on the midgut epithelial cell membrane, triggering subsequent membrane pore formation or cell damage, leading to insect death. Existing research further shows that the protease activation, N-terminal structural changes, and receptor-mediated oligomerization of Cry toxins are closely related to their insecticidal activity. The modified Cry1A toxin, which removes the N-terminal α-helix, exhibits insecticidal activity against some Bt-resistant insects (Engineering modified Bt toxinsto counter insect resistance. Science, Soberón, 2007, 318(5856): 1640–1642). Therefore, truncating the activated core fragment and N-terminal region of Cry toxins is an important technical approach for the molecular modification of Bt toxins.

[0003] Diamondback moth ( Plutella xylostella Diamondback moth (Bt moth) is a major pest in cruciferous vegetable production. Long-term use of chemical pesticides and single Bt toxin preparations can easily lead to resistance risks. Cry2Aa toxin is active against a variety of lepidopteran pests, but the interfacial binding ability of natural Cry2Aa with different receptors and its key recognition sites vary greatly. Current reports indicate that potential receptors related to Bt toxin interaction in diamondback moth include CAD, ALP, APN, and ABC transporters, and the toxin recognition and action processes differ among different receptors.

[0004] Therefore, molecular modification of the Cry toxin receptor recognition region, analysis of molecular docking of different receptors and screening of common interaction sites through interfacial residue analysis, and targeted evolutionary design of toxin proteins to enhance their interaction with diamondback moth-related receptors and improve insecticidal effects have become urgent technical challenges to be solved in this field. Summary of the Invention

[0005] To address the aforementioned issues, this application provides a method for modifying the Cry2Aa truncated insecticidal protein based on receptor interaction sites and a mutant toxin protein. This method uses the Cry2Aa truncated form activated by chymotrypsin as a backbone, and then screens and analyzes its docking interfaces with three receptors, ABCB1, ALP, and CAD-TBR, and determines common action sites. While keeping Domain I and key sites unchanged, sequence optimization is performed on other regions to obtain candidate mutants with good receptor binding tendency and insecticidal activity.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: First, this application provides a method for modifying the truncated Cry2Aa insecticidal protein, the specific steps of which are as follows: S1: Based on the amino acid sequence of Cry2Aa protoxin (as shown in SEQ ID NO: 5), Cry2Aa protoxin was activated by chymotrypsin treatment to obtain a truncated Cry2Aa (145-633) amino acid sequence from amino acid 145 to amino acid 633 of SEQ ID NO: 5, the amino acid sequence of which is shown in SEQ ID NO: 4; this truncated Cry2Aa (145-633) amino acid sequence was used as the basic framework for subsequent receptor interaction analysis and mutation design.

[0007] S2, construct molecular docking models with the diamondback moth receptors ABCB1, ALP, and CAD-TBR obtained in step S1, respectively; count the interfacial contact residues of Cry2Aa in each receptor complex, and determine the key interaction sites of the receptors through intersection analysis; the key interaction sites finally obtained include amino acid residues at positions 366, 367, and 600 of Cry2Aa.

[0008] S3, using the Cry2Aa(145-633) obtained in step S1 as the basic framework, the key structural and functional regions of Cry2Aa and the key interaction sites for multi-receptor co-recognition determined in step S2 are used as fixed regions, and the remaining non-fixed regions are used as optimizable regions. AI-assisted directed sequence design is performed using protein design software to obtain multiple candidate mutants; the aforementioned key structural and functional regions of Cry2Aa are preferably Domain I, a truncated form of Cry2Aa(145-633); In one embodiment of this application, the protein design software used is ProteinMPNN software, and the input Cry2Aa structural and functional information includes: the three-dimensional structural coordinate file of Cry2Aa (145-633), fixed residue site information, and the range of designable residues.

[0009] To use the truncated Cry2Aa(145-633) as the design backbone for mutations, the Domain I region of Cry2Aa(145-633) was fixed in protein design software, corresponding to amino acid residues 1 to 128 of the truncated Cry2Aa(145-633) sequence (SEQ ID NO: 4); at the same time, amino acid residues 366, 367, and 600 of the Cry2Aa sequence (SEQ ID NO: 5) were set as fixed residues; in addition to the above fixed regions and fixed residues, other amino acid residues of Cry2Aa(145-633) were set as designable regions, and sequence optimization design was performed by protein design software (such as the ProteinMPNN software in the examples).

[0010] S4. The receptor binding capacity of the candidate mutants obtained in step S3 is evaluated, and mutants are screened based on the structural evaluation results. Subsequently, the selected mutants are expressed and verified by bioassays of diamondback moth to obtain the modified Cry2Aa toxin protein mutant.

[0011] In one embodiment of this application, the aforementioned "receptor binding capacity evaluation" refers to the following steps: after obtaining candidate mutant sequences, a preliminary screening is performed on the candidate sequences to select sequences that simultaneously meet the following requirements: 1) the candidate sequence length is consistent with the Cry2Aa(145-633) truncated form; 2) the Domain I and the fixed residues at positions 366, 367, and 600 of Cry2Aa retained in the Cry2Aa(145-633) truncated form remain unchanged; 3) the candidate sequences do not contain any missing, terminated, or unidentified amino acid residues; 4) there are no completely repetitive sequences between different candidate sequences. Then, the candidate mutants obtained from the preliminary screening are comprehensively evaluated based on interfacial energy, number of hydrogen bonds, binding free energy, and dissociation constant, selecting candidate mutants with low interfacial energy, low binding free energy, low dissociation constant, and a large number of hydrogen bonds.

[0012] In the embodiments of this application, candidate mutants were used to establish complex models with the diamondback moth receptors ABCB1, ALP, and CAD-TBR, respectively. Rosetta Interface Analyzer and PRODIGY software were used for screening to obtain preferred mutants. In the Rosetta Interface Analyzer evaluation results, a lower interface-dG (protein binding free energy) value indicates a more favorable interfacial binding energy between the candidate mutant and the receptor complex; a higher number of interface-hbonds (number of interfacial hydrogen bonds) indicates richer interfacial hydrogen bond interactions. In the PRODIGY prediction results, a lower predicted binding free energy ΔG and a smaller predicted dissociation constant Kd value indicate a stronger predicted binding affinity between the candidate mutant and the corresponding receptor.

[0013] Secondly, this application provides the application of the Cry2Aa toxin mutant protein obtained by the above method in killing diamondback moth.

[0014] Third, this application also provides Cry2Aa toxin mutant proteins Cry2Aa-M13, Cry2Aa-M14, and Cry2Aa-M18, whose amino acid sequences are shown in SEQ ID NO:1 to SEQ ID NO:3. These three mutant proteins were designed based on the Cry2Aa activation core fragment Cry2Aa(145-633) as the backbone and the receptor interaction sites of diamondback moth receptors ABCB1, ALP, and CAD-TBR.

[0015] Fourth, this application provides the application of the Cry2Aa toxin mutant protein with the above-mentioned amino acid sequences as shown in SEQ ID NO:1 to SEQ ID NO:3 in the killing of diamondback moth.

[0016] Fifth, this application provides an insecticide for controlling diamondback moth, the insecticide comprising at least one of the Cry2Aa toxin mutant proteins with amino acid sequences as shown in SEQ ID NO:1 to SEQ ID NO:3.

[0017] This application selects ABCB1, ALP, and CAD-TBR as receptor models from a large number of reported potential receptors associated with Bt toxin action in diamondback moth, representing the toxin-binding regions of transport proteins, membrane-bound enzymes, and cadherin, respectively. Based on molecular docking and binding interface analysis between Cry2Aa and these receptors, multiple common or high-frequency interaction sites are screened for sequence design, resulting in several candidate Cry2Aa mutants. Further selection of the optimal mutant toxin protein is achieved based on receptor binding interface evaluation results. This mutant preparation method is highly targeted, produces well-defined candidate mutants, exhibits good insecticidal effect, has a clear preparation route, and shows promise for agricultural applications. It can provide a new mutant preparation method and candidate mutant resources for green control of diamondback moth and Cry toxin protein modification.

[0018] Compared with the prior art, the present invention has the following beneficial effects: (1) This application uses the Cry2Aa core fragment after protease activation as the target of modification, so that the mutation design is closer to the actual form in which the toxin exerts its activity in the midgut of insects.

[0019] (2) This application first compares the unactivated toxin with the activated toxin through SDS-PAGE analysis and bioassay, providing experimental basis for subsequent design of activated truncated toxins as the parent organism.

[0020] (3) This application combines the ABCB1, ALP and CAD-TBR multi-receptor models to perform interface screening simultaneously, which can evaluate mutants from the perspective of multi-receptor recognition.

[0021] (4) This application fixes the Domain I region and the key interaction sites at positions 366, 367 and 600 of Cry2Aa in the truncated Cry2Aa(145-633) and performs ProteinMPNN sequence optimization on other non-fixed regions, taking into account both the structural stability and sequence diversity of the mutant.

[0022] (5) This application verifies the insecticidal effect of the preferred mutant by expression preparation and diamondback moth bioassay. The Cry2Aa mutant obtained by this method has a good lethal effect on diamondback moth larvae and can be used to prepare biological insecticides for the control of diamondback moth. Attached Figure Description

[0023] Figure 1 SDS-PAGE validation image of Cry2Aa(145-633) truncated somatic protein expression preparation.

[0024] Figure 2 A schematic diagram of the truncated body structure drawn based on the Cry2Aa(145-633) three-dimensional structural model.

[0025] Figure 3 Statistical results of bioassays of diamondback moth for unactivated Cry2Aa and truncated Cry2Aa(145-633).

[0026] Figure 4 A molecular docking model diagram of the truncated Cry2Aa(145-633) and CAD-TBR.

[0027] Figure 5 A molecular docking model of the truncated Cry2Aa(145-633) and ALP.

[0028] Figure 6 This is a molecular docking model diagram of the truncated Cry2Aa(145-633) and ABCB1.

[0029] Figure 7 The diagram shows the Wayne intersection analysis of the interface residues of Cry2Aa(145-633) with three receptor docking models.

[0030] Figure 8 The figure shows the bioassay results of the candidate Cry2Aa mutant at a comparative concentration of 20 μg / mL in Diamondback moth. Detailed Implementation

[0031] The raw materials and reagents involved in the example were all obtained through commercial channels.

[0032] The Cry2Aa protoxin sample was purchased from Meiyan Company, and its amino acid sequence is shown in SEQ ID NO: 5.

[0033] The chymotrypsin was purchased from Sigma-Aldrich under the product name α-Chymotrypsin from bovine pancreas.

[0034] The artificial feed used in the examples was prepared in the applicant's laboratory and its components include wheat germ powder, streptomycin, sucrose, Vickers salt, agar, multivitamins, and sterile water.

[0035] Example 1: Obtaining and Verifying the Cry2Aa Activated Core Toxin Fragment Cry2Aa protoxin was mixed with chymotrypsin at a mass ratio of 20:1 and incubated at 37°C for 2 h. Activation results showed that Cry2Aa could be cleaved between amino acids 144 and 145, forming an activated core toxin fragment with amino acid 145 as the starting site (experimental steps were the same as in Example 1 of patent CN 118290543 A). Based on this activation site, a truncated Cry2Aa (145-633) variant starting from amino acid 145 of the Cry2Aa protoxin was constructed, and the amino acid sequence of this truncated variant is shown in SEQ ID NO: 4.

[0036] After the truncated Cry2Aa(145-633) protein was prepared by expression, the protein bands were verified by SDS-PAGE. The results are as follows: Figure 1 As shown. Figure 1 The presence of a Cry2Aa (145-633) truncated protein band at a molecular weight position of approximately 55 kDa indicates that the truncated protein can be effectively expressed and prepared.

[0037] Figure 2 To obtain the structural diagram of Cry2Aa (145-633) using PyMOL software for visualization and site annotation, Domain I, Domain II, and Domain III were labeled according to the typical three-domain configuration of Cry toxins based on the CDD domain analysis module in the NCBI database. The amino acid sequence of the Cry2Aa protoxin, as shown in SEQ ID NO:5, was used to calculate... Figure 2 The Domain I region of Cry2Aa shown is located in the amino acid residue region from position 1 to position 272. Since the experimental design uses the Cry2Aa (145-633) activation core fragment as the mutant design backbone, the actual immobilized region in the subsequent ProteinMPNN design process is the Domain I region retained in Cry2Aa (145-633), that is, amino acid residues from position 145 to position 272 as shown in SEQ ID NO:5, which corresponds to amino acid residues from position 1 to position 128 in the truncated sequence shown in SEQ ID NO:4.

[0038] To verify the effect of activation treatment on the insecticidal activity of Cry2Aa, a PBS blank control group, an unactivated Cry2Aa group, and a chymotrypsin-activated Cry2Aa group were set up. Unactivated Cry2Aa (Cry2Aa protoxin) and Cry2Aa (145-633) truncated form were treated on the surface of artificial feed at the same final concentration.

[0039] Bioassays were performed using a surface coating method on artificial feed: an equal volume of the protein solution to be tested was evenly coated onto the surface of artificial feed, and after natural drying, healthy, uniform second-instar diamondback moth larvae were inoculated. Each treatment was set up in triplicate, with 20 larvae per replicate. After treatment, the larvae were cultured for 72 h, and larval mortality was observed and recorded. The mortality rate was calculated as the number of dead larvae / the total number of tested larvae × 100%.

[0040] Bioassay statistical results as follows Figure 3 As shown, the mortality rate of the PBS blank control group was low, and the unactivated Cry2Aa group only showed a weak lethal effect. After activation treatment with chymotrypsin, the lethality of the Cry2Aa (145-633) truncated form against diamondback moth larvae was significantly increased, indicating that the activated core fragment of Cry2Aa (145-633) with amino acid 145 as the starting site can serve as the basic framework for subsequent receptor interaction analysis and mutant design.

[0041] Example 2: Molecular docking of the truncated Cry2Aa(145-633) with three receptors and analysis of key receptor interaction sites.

[0042] Complex models of Cry2Aa(145-633) with diamondback moth-related receptors CAD-TBR, ALP, and ABCB1 were constructed using the ZDOCK molecular docking program. During docking, the 3D structure model of Cry2Aa(145-633) was used as the ligand structure file, and the 3D structure models of CAD-TBR, ALP, and ABCB1 were used as the receptor structure files, respectively. These were input into ZDOCK for global rigid-body protein-protein docking. The docking process was performed using default ZDOCK parameters. Based on the docking score output by ZDOCK, the top-ranked complex conformations were selected. Considering the rationality of the interface contact region between Cry2Aa and the receptors, representative complex models were selected for subsequent interface residue analysis. The complex models of CAD-TBR, ALP, and ABCB1 obtained in the examples are shown below. Figure 4-6 As shown.

[0043] The docking results were then visualized and analyzed using PyMOL software. The interfacial contact residues between the Cry2Aa chain and the receptor chain within the range of 4 Å were counted, and the interfacial contact residues of Cry2Aa in different receptor models were compared and analyzed.

[0044] like Figure 4As shown, in the Cry2Aa-CAD-TBR docking model, the blue structure represents the truncated Cry2Aa (145-633) structure, and the orange structure represents the CAD-TBR. The interfacial contact residues involve regions such as positions 344, 357, 359, 366-369, 475-484, and 600. Figure 5 As shown, in the Cry2Aa-ALP docking model, the blue structure represents the truncated Cry2Aa (145-633) structure, and the green structure represents ALP. The interfacial contact residues mainly include residues near positions 489-491 and 599-600. Figure 6 As shown, in the Cry2Aa-ABCB1 docking model, the blue structure represents the truncated Cry2Aa (145-633), and the yellow structure represents ABCB1. The interfacial contact residues are mainly concentrated at positions 366-367 and 556-630 of Cry2Aa; combined with Figure 7 Intersection analysis of different receptor models showed that sites such as LEU366, PRO367, and ASN600 had high interfacial association in the recognition of the three receptors.

[0045] Based on the statistical results of the interface residues of the three receptor complexes, positions 366, 367, and 600 of Cry2Aa appeared in the interface contact regions of multiple receptors, exhibiting high co-occurrence frequency and receptor coverage, reflecting key characteristics of Cry2Aa's co-recognition with different receptors. Therefore, amino acid residues 366, 367, and 600 of Cry2Aa were identified as key interaction sites associated with multiple receptors and will be used as fixed residues in subsequent ProteinMPNN directed sequence design.

[0046] Example 3: Design and structural evaluation of candidate Cry2Aa mutants based on ProteinMPNN This embodiment uses the ProteinMPNN online server (implemented by ColabDesign, model version v-48-020) provided by the NeuroSnap platform for candidate mutant-assisted design, and uses the Cry2Aa(145-633) activated truncated structure obtained in Example 1 ( Figure 2 As the input backbone, the input information includes the three-dimensional structural coordinate file of Cry2Aa(145-633), the chain information to be designed, the fixed residue site information, and the range of designable residues.

[0047] The design process is as follows: Domain I (amino acid residues 1 to 128 in the truncated sequence shown in SEQ ID NO:4) retained in the Cry2Aa (145-633) truncated version is used as the fixed structural-functional region. The 366th, 367th, and 600th amino acid residues of Cry2Aa (SEQ ID NO:5) determined in Example 2 are set as fixed residues to maintain the basic structural-functional region and key interaction sites related to multiple receptors of the Cry2Aa truncated version unchanged. In addition to the above fixed regions and fixed residues, the remaining non-fixed regions are set as designable regions, allowing ProteinMPNN to optimize the amino acid sequence design based on the input protein backbone and generate multiple candidate mutant sequences.

[0048] After obtaining the candidate mutant sequences, the multiple candidate sequences output by ProteinMPNN were first sorted and preliminarily screened. The preliminary screening criteria included: the candidate sequence length was consistent with the Cry2Aa(145-633) truncated version; the Domain I retained in the Cry2Aa(145-633) truncated version and the fixed residues at positions 366, 367, and 600 of Cry2Aa remained unchanged; amino acid residue substitutions were allowed in other designable regions; the candidate sequences did not contain any missing, terminated, or unidentified amino acid residues; and there were no completely repetitive sequences between different candidate sequences.

[0049] Based on the above conditions, and further considering the distribution of mutation sites, sequence differences, and feasibility of subsequent receptor complex modeling and structural evaluation, this embodiment selects six representative candidate mutants from multiple candidate sequences. These mutants are then used to construct protein-protein complex models with the diamondback moth receptors ABCB1, ALP, and CAD-TBR, respectively, for subsequent molecular docking, receptor complex modeling, and structural evaluation. In practice, other mutants can also be selected for modeling and evaluation.

[0050] This embodiment utilizes Rosetta InterfaceAnalyzer and PRODIGY software to evaluate the six candidate mutants mentioned above. The results are shown in Tables 1 and 2 below. The input files for both software programs are the three-dimensional structural coordinate files of the protein-protein complexes constructed from the candidate mutants and receptors. Cry2Aa mutant and receptor information were set for interface feature evaluation and binding capacity prediction. The data in Table 1 were obtained from the interface evaluation of the candidate mutants with the ABCB1, ALP, and CAD-TBR receptor complexes using Rosetta InterfaceAnalyzer. The evaluation parameters included the interface energy (interface-dG) and the number of interface hydrogen bonds (interface-hbonds). The data in Table 2 were obtained from the binding affinity prediction of the candidate mutants with the receptor complexes using the PRODIGY online server. The evaluation parameters included the predicted binding free energy (ΔG) and the predicted dissociation constant (Kd). The structural evaluation results obtained from the two software programs were used for comprehensive screening of candidate mutants.

[0051] Table 1. Rosetta Interface Analyzer interface evaluation results of six candidate Cry2Aa mutants with three receptor complexes.

[0052] Table 2. Predicted PRODIGY binding free energy and dissociation constants of six candidate Cry2Aa mutants with three receptor complexes.

[0053] As shown in Table 2, the PRODIGY prediction results show that Cry2Aa-M18 has a lower predicted ΔG value and a smaller Kd value in the CAD-TBR model, indicating a strong predicted binding trend in this receptor model. Cry2Aa-M14 also performs well in the CAD-TBR model. Cry2Aa-M13 shows good predicted binding free energy and dissociation constant in all three receptor models: ABCB1, ALP, and CAD-TBR. Combining the interface energy and number of interface hydrogen bonds from Rosetta Interface Analyzer in Table 1 and the predicted binding affinity results from PRODIGY in Table 2, Cry2Aa-M13, Cry2Aa-M14, and Cry2Aa-M18 can be considered as preferred candidate mutants with better overall performance.

[0054] Based on the combined evaluation results of Rosetta InterfaceAnalyzer and PRODIGY, Cry2Aa-M14 performed exceptionally well in the CAD-TBR model, Cry2Aa-M18 showed good overall performance across the three receptor models, and Cry2Aa-M13 exhibited a relatively balanced tendency for binding to multiple receptor interfaces. Therefore, Cry2Aa-M13, Cry2Aa-M14, and Cry2Aa-M18 were selected as preferred candidate mutants for subsequent expression and bioassay verification in this invention. Their amino acid sequences are shown in SEQ ID NO: 1-SEQ ID NO: 3.

[0055] In other implementations, structure-based protein sequence design tools such as RosettaDesign and EvoDesign can be used to design candidate mutants.

[0056] Example 4: Gene synthesis, expression, and protein preparation of candidate mutants The amino acid sequences of the candidate mutants Cry2Aa-M13, Cry2Aa-M14, and Cry2Aa-M18 obtained from Example 3 were codon-optimized, and gene synthesis was commissioned to a commercial biotechnology company. The synthesized fragments were ligated into the pET-28a prokaryotic expression vector to construct recombinant expression plasmids.

[0057] The pET-28a recombinant expression plasmid, synthesized by a commercial company and containing the nucleotide sequences shown in Cry2Aa-M13, Cry2Aa-M14, and Cry2Aa-M18, was introduced into *E. coli* BL21(DE3) competent cells via chemical transformation. The pET-28a prokaryotic expression system used in this field is the commonly used T7 promoter-induced expression system. The transformation, resistance screening, and IPTG-induced expression steps are standard techniques in this field and can be performed according to publicly available experimental manuals such as the pET System Manual and *Molecular Cloning: A Laboratory Manual*. The plasmid was plated on LB agar plates containing 50 μg / mL kanamycin and 1% glucose and incubated overnight at 37°C. Positive single clones were picked and inoculated into LB liquid medium containing 50 μg / mL kanamycin and 1% glucose, and cultured overnight at 37°C with shaking at 250 rpm as the seed culture. The following day, the seed culture was inoculated into LB liquid medium containing 50 μg / mL kanamycin and 1% glucose at an appropriate inoculation ratio. The culture was shaken at 37°C and 250 rpm. When the OD600 of the bacterial culture reached 0.6–0.9, IPTG was added to a final concentration of 1 mM. The culture was then transferred to 25°C for 20 h of induction to obtain the candidate Cry2Aa mutant protein.

[0058] Example 5 Bioassay of Diamondback Moth with Candidate Cry2Aa Mutant To verify the differences in insecticidal activity of candidate mutants under the same treatment dosage, this example uses the LC50 of chymotrypsin-activated Cry2Aa, which was previously measured. 50 The results served as the basis for dosage setting. Activated Cry2Aa, which is the activated core toxin fragment Cry2Aa(145-633) formed after Cry2Aa protoxin is treated with chymotrypsin, was used as the toxin control in this example.

[0059] Preliminary bioassay results showed that Cry2Aa activated by chymotrypsin had a lower LC50 value for diamondback moth larvae. 50 The concentration is approximately 20 μg / mL. Therefore, in this embodiment, 20 μg / mL was selected as the single concentration comparison dose to compare the insecticidal activity changes of the candidate mutant relative to activated Cry2Aa.

[0060] Using PBS as a blank control and 20 μg / mL activated Cry2Aa as the initial sample control, treatment groups were set up with 20 μg / mL Cry2Aa-M13, 20 μg / mL Cry2Aa-M14, and 20 μg / mL Cry2Aa-M18. Bioassays were performed using the artificial feed surface coating method: an equal volume of the test protein solution was evenly coated onto the surface of artificial feed, allowed to air dry, and then inoculated with healthy, uniformly developed second-instar diamondback moth larvae. Each treatment was set up in triplicate, with 20 larvae per replicate, and cultured for 72 h at 25℃, approximately 70% relative humidity, and a 16 h light / 8 h dark photoperiod.

[0061] Mortality rate = number of dead insects / total number of tested insects × 100%; when there are natural deaths in the PBS control group, the corrected mortality rate formula is used for correction: Corrected mortality rate (%) = (mortality rate of treatment group - mortality rate of control group) / (100% - mortality rate of control group) × 100%.

[0062] The bioassay results are shown in Figure 8. Under the treatment condition of 20 μg / mL, the mortality rate of the activated Cry2Aa treatment group was close to its corresponding LC50 level. The mortality rates of the Cry2Aa-M13, Cry2Aa-M14 and Cry2Aa-M18 treatment groups were all higher than those of the activated Cry2Aa control group, indicating that the obtained candidate mutants further improved the lethality of diamondback moth larvae while maintaining the insecticidal activity of Cry2Aa.

Claims

1. A method for modifying a truncated Cry2Aa insecticidal protein, characterized in that, The specific steps are as follows: 1) Using the truncated Cry2Aa(145-633) amino acid sequence as shown in SEQ ID NO:4 as the basic backbone, the key structural and functional regions of Cry2Aa and the key interaction sites for recognition by multiple receptors were used as fixed regions, and the remaining regions were used as optimizable regions. Targeted sequence design was carried out using protein design software to obtain multiple candidate mutants. The key functional region of the Cry2Aa structure is the region composed of amino acid residues located at positions 1-128 of the truncated amino acid sequence of Cry2Aa (145-633); The key interaction sites for the multi-receptor co-recognition are located at positions 366, 367, and 600 of the amino acid sequence shown in SEQ ID NO: 5, respectively. 2) The receptor binding capacity of the candidate mutants obtained in step 1) was evaluated, and the mutants were screened according to the evaluation results for bioassay verification in diamondback moth to obtain the modified Cry2Aa toxin protein mutant.

2. The method for modifying Cry2Aa truncated insecticidal protein according to claim 1, characterized in that, Step 1) The protein design software is ProteinMPNN software.

3. The method for modifying Cry2Aa truncated insecticidal protein according to claim 1, characterized in that, Step 2) The receptor binding capacity evaluation refers to: firstly, preliminary screening of candidate mutants is carried out, selecting amino acid residues with the same sequence length as the truncated Cry2Aa(145-633) and without deletions, terminations or unidentifiable residues; then, a comprehensive evaluation and screening of the interfacial energy, number of hydrogen bonds, binding free energy and dissociation constant of the candidate mutants is carried out.

4. The modified Cry2Aa toxin protein mutant obtained by any of the methods described in claims 1-3.

5. The application of the modified Cry2Aa toxin protein mutant obtained by any of the methods in claims 1-3 in the killing of diamondback moth.

6. A mutant of the modified Cry2Aa toxin protein, characterized in that, The mutant is any one of the proteins whose amino acid sequences are shown in SEQ ID NO: 1 to SEQ ID NO:

3.

7. The application of the modified Cry2Aa toxin protein mutant as described in claim 6 in the killing of diamondback moth.

8. An insecticide, characterized in that... The insecticide contains at least one of the mutant Cry2Aa toxin protein with amino acid sequences as shown in SEQ ID NO: 1 to SEQ ID NO: 3.

Images