Pseudomonas sourced insecticidal protein IPD072Aa mutant as well as coding gene and application thereof

Abstract

The invention belongs to the technical field of gene engineering, and discloses a pseudomonas-sourced insecticidal protein IPD072Aa mutant as well as a coding gene and application thereof, the mutant is mutants S22A, A74S and L81A, the amino acid sequence is shown as SEQ ID NO: 1-3, and the nucleotide coding sequence of the gene is shown as SEQ ID NO: 4-6. The invention discovers that the IPD072Aa prototoxin shows obvious poisoning activity on hemiptera pest aphids for the first time, and the aphid killing activities of three mutants of the protein are respectively enhanced by 2.8 times, 2.3 times and 3.6 times compared with the prototoxin. Therefore, the IPD072Aa prototoxin and the mutant thereof can be used as a key gene element for cultivating transgenic insect-resistant crops to prevent and control major agricultural insect aphids.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology. Specifically, it relates to a mutant of the insecticidal protein IPD072Aa derived from Pseudomonas aeruginosa and its encoding gene, as well as a new use of the IPD072Aa protein and its mutant in the control of aphids. Background Technology

[0002] Aphids, belonging to the order Hemiptera, possess piercing-sucking mouthparts that extract sap from the phloem of plants, causing plant death. They are also carriers of various plant viruses, and their honeydew secretions accelerate mold growth, severely harming crop yield and quality. In my country, at least 1009 species of aphids or their subspecies have been recorded, mostly pests of crops, vegetables, fruit trees, and flowers. Current control methods still primarily rely on pesticide spraying. According to the "National Agricultural Pest Resistance Monitoring Report," many aphids have developed severe resistance to pesticides. For example, the cotton aphid has become the number one pest of cotton, exhibiting high levels of resistance to pyrethroids, neonicotinoids, and para-carbamates, making them virtually untreatable. Furthermore, the extensive spraying of chemical pesticides is detrimental to the environment, ecology, and human health. Developing new biological control technologies is an effective strategy for achieving green and sustainable agricultural development.

[0003] Bacillus thuringiensis (Bt), widely distributed in nature, is one of the most successful microorganisms used in pest control. It is a Gram-positive bacterium belonging to the Bacillus cereus group. The toxic proteins extracted from Bt have specific toxic activity against multiple insects and nematodes. In particular, the three-domain Cry crystal protein produced during the spore stage and the Vip3-like protein secreted during the vegetative stage are the most widely used. They have been developed into Bt preparations and transgenic insect-resistant crops and have been successfully used to control various Lepidoptera and Coleoptera pests (Xu C, Wang BC, Yu Z*, Sun M*. (2014) Structural insights into Bacillus thuringiensis Cry, Cyt and parasporin toxins. Toxins (Basel), 6(9):2732-7270.). In recent years, various non-Bt-derived bacterial insecticidal proteins have been discovered. Pseudomonas is a widely distributed genus of bacteria in soil, and many strains within it are plant growth-promoting bacteria with biocontrol effects. In 2016, Ute Schellenberger et al. identified an insecticidal protein IPD072Aa (GenBank: AMN88672.1) from *Pseudomonas chlororaphis*, which exhibited specific and highly effective insecticidal activity against the western maize root borer (*Diabrotica virgifera virgifera*), with a median lethal concentration (LC50). 50) is 26 ng / mg (CJ Boeckman, E Huang, K Sturtz, C Walker, RWoods, J Zhang. (2019) Characterization of the Spectrum of InsecticidalActivity for IPD072Aa: A Protein Derived from Psuedomonas chlororaphis withActivity Against Diabrotica virgifera virgifera (Coleoptera: Chrysomelidae).Journal of Economic Entomology). Further testing of the insecticidal spectrum of IPD072Aa showed that the protein was not sensitive to lepidopteran pests such as the cutworm (Agrotis ipsilon), bollworm (Helicoverpa zea), European corn borer (Ostrinia nubilalis), fall armyworm (Spodoptera frugiperda), soybean cutworm (Pseudoplusia includens), small red nymph (Vanessa cardui), and codling moth (Cydia pomonella); it was ineffective against hemipteran pests such as the bean pod mirid bug (Lygus hesperus); and it was ineffective against other coleopteran pests including the mealworm (Tenebriomolitor), Mexican ladybug (Epilachna varivestis), long-legged ladybug (Hippodamia convergens), spotted ladybug (Coleomegilla maculata), southern corn root borer (Diabrotica undecimpunctata), and Cryptolaemus. While *Montrouzieria* and *Anthonomus grandis* exhibit varying degrees of lethality, the western maize rootworm remains the most susceptible. IPD072Aa, containing 86 amino acid residues, shows very low sequence and structural similarity to known proteins, has a different mechanism of action than Bt-derived insecticidal proteins, and poses no risk of cross-resistance. It has been developed into transgenic maize (DP23211) for the control of coleopteran pests.

[0004] With the widespread cultivation of Bt genetically modified crops, lepidopteran and coleopteran pests have been effectively controlled. However, against this backdrop, various secondary pests have risen to become major pests. Currently, aphids have become significant pests of many crops, vegetables, and fruit trees, including cotton, wheat, corn, soybeans, and sorghum, and are difficult to control. Several Bt insecticidal proteins were tested earlier for their activity against different aphids. Manuel Porcar et al. tested the toxic effects of Cry1Ab, Cry3A, Cry4Aa, Cry11Aa, and Cyt1Aa on the pea aphid (Acyrthosiphon pisum), with only Cry4Aa showing activity. The LC50 test lasted 7 days. 50 Between 70-100 μg / ml (M Porcar, AM Grenier, B Federici, Y Rahbe. (2009) Effects of Bacillus thuringiensis δ-Endotoxins on the Pea Aphid (Acyrthosiphon pisum).); Huarong Li et al. tested pea aphids with Cry1Ac and Cry3Aa at a concentration of 500 μg / ml, and the aphid mortality rate was 71% after 7 days, but the mortality rate of the negative control group also reached 34% (Interaction of the Bacillus thuringiensis deltaendotoxins Cry1Ac and Cry3Aa with the gut of the pea aphid, Acyrthosiphonpisum (Harris)); Sattar Sampurna reported the LC50 of the binary toxins Vip1Ae / Vip2Ae against cotton aphids. 50 The concentration was 0.576 μg / ml (Molecular Characterization of a Novel Vegetative Insecticidal Protein from Bacillus thuringiensis Effective Against Sap-Sucking Insect Pest.); Chougule NP et al. inserted an aphid gut-binding polypeptide into the Cyt2Aa sequence, and the genetically engineered mutant showed an LC50 concentration against pea aphids and peach aphids (Myzus persicae). 50The concentrations were 9.55 and 42.68 μg / ml, respectively (Retargeting of the Bacillus thuringiensis toxin Cyt2Aa against hemipteran insect pests.); Rausch MA et al. introduced a cathepsin cleavage site into Cry4Aa, and the resulting mutant protein increased the lethality from 17.8% to 51.1% at a concentration of 125 μg / ml (Modification of Cry4Aa toward Improved Toxin Processing in the Gut of the Pea Aphid, Acyrthosiphon pisum.); Leopoldo Palma et al. reported the LC50 of a Cry41-related protein against the peach aphid. 50 The concentration was 32.7 μg / ml (Molecular and Insecticidal Characterization of a Novel Cry-Related Protein from Bacillus Thuringiensis Toxic against Myzus persicae). This report is over 10 years old, but no follow-up research or development of related insect-resistant products has been observed. Therefore, screening for effective insecticidal gene resources for aphid control is urgently needed. Summary of the Invention

[0005] In view of the shortcomings of the prior art, the first objective of the present invention is to provide a mutant of the insecticidal protein IPD072Aa derived from Pseudomonas aeruginosa and its encoding gene. The IPD072Aa mutant exhibits significant toxic activity against hemiptera pests such as aphids and can be used as a key gene element for the cultivation of transgenic insect-resistant crops to control aphids, a major agricultural pest.

[0006] Specifically, the objective of this invention is achieved according to the following technical solution: a mutant of the insecticidal protein IPD072Aa derived from *Pseudomonas aeruginosa* is obtained through alanine scanning mutagenesis technology, namely mutants S22A, A74S, and L81A, whose amino acid sequences are as follows:

[0007] MGITVTNNSSNPIEVAINHWGADGDTSFFSVGNGKQETWDRSDSRGFVLSLKKNGAQHPYYVQASSKIEVDNNAVKDQGRLIEPLS (S22A, as shown in SEQ ID NO: 1);

[0008] MGITVTNNSSNPIEVAINHWGSDGDTSFFSVGNGKQETWDRSDSRGFVLSLKKNGAQHPYYVQASSKIEVDNNSVKDQGRLIEPLS (A74S, as shown in SEQ ID NO:2);

[0009] MGITVTNNSSNPIEVAINHWGSDGDTSFFSVGNGKQETWDRSDSRGFVLSLKKNGAQHPYYVQASSKIEVDNNAVKDQGRAIEPLS (L81A, as shown in SEQ ID NO:3).

[0010] Furthermore, the present invention also provides a coding gene for the above-mentioned insecticidal protein IPD072Aa mutant. More preferably, the nucleotide sequence of the coding gene for the above-mentioned insecticidal protein IPD072Aa mutant is as follows:

[0011] atgggtattaccgttaccaacaacagcagcaacccgattgaagtggccattaaccattggggtgccgatggtgatacgagcttcttcagcgtgggtaatggcaaacaggaaacgtggggatcgtagcgatagtcgcg gctttgttctgagcctgaagaagaatggtgcccagcatccgtattatgttcaggcgagcagcaaaattgaagttgataataatgcggtaaaagatcagggtcgtctgattgaacctctgagctaa (S22A gene, such as SEQ ID NO:4);

[0012] atgggtattaccgttaccaacaacagcagcaacccgattgaagtggccattaaccattggggtagcgatggtgatacgagcttcttcagcgtgggtaatggcaaacaggaaacgtggggatcgtagcgatagtcgcg gctttgttctgagcctgaagaagaatggtgcccagcatccgtattatgttcaggcgagcagcaaaattgaagttgataataattcggtaaaagatcagggtcgtctgattgaacctctgagctaa (A74S gene, such as SEQ ID NO:5);

[0013] atgggtattaccgttaccaacaacagcagcaacccgattgaagtggccattaaccattggggtagcgatggtgatacgagcttcttcagcgtgggtaatggcaaacaggaaacgtggggatcgtagcgatagtcgcg gctttgttctgagcctgaagaagaatggtgcccagcatccgtattatgttcaggcgagcagcaaaattgaagttgataataatgcggtaaaagatcagggtcgtgccattgaacctctgagctaa (L81A gene, such as SEQ ID NO:6).

[0014] A second objective of this invention is to provide a recombinant vector containing the coding gene for an insecticidal protein IPD072Aa mutant; more preferably, the vector is the Escherichia coli expression vector pColdII; simultaneously, this invention also provides a genetically engineered bacterium containing the above-mentioned recombinant vector.

[0015] The third objective of this invention is to provide a new use for the product, namely the above-mentioned insecticidal protein IPD072Aa mutant, the above-mentioned insecticidal protein IPD072Aa mutant encoding gene, the above-mentioned recombinant vector, or the above-mentioned application, wherein the application is any one of the following: (1) application in the preparation of insecticides for the control of aphids; (2) application in the preparation of transgenic aphid-resistant crops or transgenic aphid-resistant microorganisms.

[0016] Since the inventors have discovered for the first time that the wild-type protein IPD072Aa has significant toxic activity against aphids, this invention also provides new uses for the known protein IPD072Aa and its encoding gene, namely: the application of the insecticidal protein IPD072Aa in the preparation of insecticides for aphid control, transgenic insect-resistant crops, or transgenic insecticidal microorganisms; and the application of the encoding gene of the insecticidal protein IPD072Aa in the preparation of insecticides for aphid control, transgenic insect-resistant crops, or transgenic insecticidal microorganisms. More preferably, the coding sequence of the insecticidal protein IPD072Aa gene is shown in SEQ ID NO:8 or SEQ ID NO:9.

[0017] It should be noted that the amino acid sequence of the insecticidal protein IPD072Aa is as follows:

[0018] MGITVTNNSSNPIEVAINHWGSDGDTSFFSVGNGKQETWDRSDSRGFVLSLKKNGAQHPYYVQASSKIEVDNNAVKDQGRLIEPLS (IPD072Aa, as shown in SEQ ID NO:7).

[0019] Further preferably, in order to enable the insecticidal protein IPD072Aa to be expressed in high soluble form in Escherichia coli, the inventors optimized the gene encoding the insecticidal protein IPD072Aa using codons. The optimized nucleotide coding sequence is as follows:

[0020] atgggtattaccgttaccaacaacagcagcaacccgattgaagtggccattaaccattggggtagcgatggtgatacgagcttcttcagcgtgggtaatggcaaacaggaaacgtggggatcgtagcgatagtcgcggc tttgttctgagcctgaagaagaatggtgcccagcatccgtattatgttcaggcgagcagcaaaattgaagttgataataatgcggtaaaagatcagggtcgtctgattgaacctctgagctaa (IPD072Aa gene, such as SEQ ID NO:8).

[0021] Further preferably, to verify the insecticidal effect of the insecticidal protein IPD072Aa transferred into tobacco, the inventors optimized the codons of the gene encoding the insecticidal protein IPD072Aa. The optimized nucleotide coding sequence is as follows:

[0022] atgggaattactgttactaataattcttctaatcctattgaagttgctattaatcactggggtagtgatggtgatacttctttcttctctgttggtaatggtaagcaggaaacttgggataggtctgattctaggggt tttgttctttctcttaagaagaatggtgctcaacacccttattatgttcaagctagttctaagattgaggttgataataatgcagttaaggatcagggtagacttattgaaccactttcttaa (IPD072Aa gene, such as SEQ ID NO:9).

[0023] Compared with existing technologies, the beneficial effects of this invention are as follows: This invention is the first to discover that the IPD072Aa protoxin exhibits significant toxic activity against aphids, a hemiptera pest. Furthermore, using alanine scanning mutagenesis technology, three mutants with even higher insecticidal activity—S22A, A74S, and L81A—were obtained. These three mutants showed aphid-killing activity enhanced by 2.88 times, 2.16 times, and 3.41 times, respectively, compared to the protoxin, achieving unexpected technical effects. Therefore, the IPD072Aa protoxin and its three mutants can serve as key gene elements for the cultivation of transgenic insect-resistant crops to control aphids, a major agricultural pest. Attached Figure Description

[0024] Figure 1 This is a map of the IPD072Aa / pColdII recombinant vector of the present invention.

[0025] Figure 2 This is an SDS-PAGE image of the IPD072Aa protein of this invention.

[0026] Figure 3 The image shows the results of the insecticidal activity assay of the insecticidal protein IPD072Aa of this invention against cotton aphids.

[0027] Figure 4 The graph shows the results of the insecticidal activity assay of the insecticidal protein IPD072Aa of the present invention against brown planthopper, two-spotted spider mite, and corn aphid.

[0028] Figure 5 This is a map of the recombinant vector IPD072Aa / PBI121 of the present invention.

[0029] Figure 6 This is a diagram showing the verification results of positive clones after the IPD072Aa gene of this invention was transformed into Agrobacterium.

[0030] Figure 7 This is a diagram illustrating the in vivo insecticidal effect of transient expression of IPD072Aa protein in tobacco according to the present invention.

[0031] Figure 8 This is an SDS-PAGE image of the single-point mutant of IPD072Aa of the present invention.

[0032] Figure 9 This is a graph showing the bioactivity of 86 monomeric mutants of this invention against cotton aphids. Detailed Implementation

[0033] To better understand the technical content of this invention, specific embodiments are provided below for further detailed explanation. Unless otherwise specified, the methods involved in the embodiments are conventional methods in the art and can be operated with reference to the general techniques described in the literature. Reagents or instruments whose manufacturers are not specified can all be obtained commercially.

[0034] Example 1: Synthesis and vector construction of the IPD072Aa protein-coding gene

[0035] The insecticidal protein IPD072Aa sequence contains 86 amino acid residues, with a corresponding full-length DNA sequence of 261 nucleotides. Gene synthesis was performed by Wuhan Qingke Biotechnology Co., Ltd. Codon optimization of the target gene was conducted according to the *E. coli* expression host (as shown in SEQ ID NO:8), and the target gene was subcloned into the cold shock expression vector pColdII using BamHI and XhoI restriction endonuclease sites. Figure 1 As shown. The successfully constructed recombinant plasmid calcium was transformed into Escherichia coli Top10 strains and stored.

[0036] Example 2: Expression of IPD072Aa in Escherichia coli

[0037] (1) The recombinant plasmid calcium successfully constructed in Example 1 was transformed into BL21(DE3) competent cells, added to 10 ml of LB liquid medium and cultured overnight (with ampicillin added to a final concentration of 100 μg / ml), at 37°C and 220 rpm, until OD. 600 The concentration reached approximately 0.6. The next day, it was transferred to 500 ml of LB liquid medium at a ratio of 1:100 (v / v) for further culture (with ampicillin added to a final concentration of 100 μg / ml) until OD reached... 600 It takes approximately 4-6 hours to reach a pH of 0.6-0.8.

[0038] (2) The culture conditions were then changed to 16℃ and 150 rpm. After the temperature of the culture medium in the shake flask dropped, isopropyl-β-D-thiogalactoside (IPTG) was added to a final concentration of 0.1 mmol to induce protein expression. The induction time was about 12 h.

[0039] (3) After induction, the bacterial cells were collected using a low-temperature centrifuge at 12,000 rpm for 3 min, and resuspended in lysis buffer (50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, pH 8.0). The cells were then lysed using a high-pressure cell disruptor (JNBIO, Inc., China) and centrifuged at 12,000 rpm for 45 min to obtain the supernatant.

[0040] (4) Purify the protein using nickel column affinity chromatography. Pass the supernatant through the nickel column three times, wash away non-specifically bound impurities with lysis buffer, and elute the target protein from the nickel column with 10 ml of Elution Buffer (50 mM NaH2PO4, 300 mM NaCl, 500 mM imidazole, pH 8.0). Finally, remove the imidazole from the eluted protein using dialysis (50 mM NaH2PO4, 300 mM NaCl, pH 8.0), concentrate the protein using an ultrafiltration tube, add 40 μl of concentrated protein to 10 μl of 5× loading buffer and mix well for later use. Aliquot the remaining protein into 1.5 ml EP tubes and freeze at -80°C.

[0041] (5) The target protein band was detected by SDS-PAGE gel electrophoresis. BSA protein standard solutions with concentration gradients of 5, 2.5, 1.25, 0.625, and 0.3125 mg / ml were simultaneously spotted onto a single gel. The gel image was obtained by scanning with an optical density scanner. The grayscale of the corresponding protein bands was analyzed using Quantity One software to construct a BSA standard curve and calculate the concentration of the target protein. For example... Figure 2 As shown: M is the 26614 protein marker (Thermo Fisher Scientific). Lanes 1-6 correspond to BSA concentrations of 6.0, 3.0, 1.5, 0.75, 0.375, and 0.1875 mg / ml, respectively, with a loading volume of 8 μl. Lanes 7-9 are the IPD072Aa target protein, with loading volumes of 3 μl, 5 μl, and 8 μl, respectively.

[0042] Example 3: Determination of the bioactivity of IPD072Aa protein against cotton aphids

[0043] The cotton aphids used in the experiment were reared on potted cotton plants indoors at a temperature of 26℃ and a photoperiod of 16L:8D. Second-instar cotton aphids were used in the experiment, and a double-layer film feeding method was employed. Specifically, a layer of paraffin film was gently spread and placed over one end of a glass double-ended tube (20mm inner diameter, 30mm height). 180 μl of the test sample (a 1:3 mixture of protein and artificial feed) was dropped into the center of the film, and then another layer of paraffin film was placed over it to form a feed naan. Approximately 20 second-instar aphid nymphs were added to each tube, and the other end of the double-ended tube was sealed with a black cloth. Each sample was treated in triplicate. The tubes were placed in an artificial climate incubator at 25℃, a photoperiod of 16L:8D, and 70% humidity. The feeding and survival of the aphids were observed daily. After 3 days, the number of dead aphids was counted, and the mortality rate was calculated. The artificial feed formulation for the aphids was based on the optimal artificial pure feed A3 group proposed by Ye Chao et al. (Ye Chao et al., 2016).

[0044] like Figure 3As shown, the IPD072Aa protein concentration used in the experiment was 200 μg / ml, with protein buffer as a negative control and 2 μg / ml imidacloprid as a positive control to ensure the reliability of the experiment. Compared with the buffer-treated group, IPD072Aa showed significant toxicity to cotton aphids, with an average mortality rate of 78.05% after 72 hours of treatment. Its LC50 against cotton aphids was further determined to be 109.1 μg / ml, as shown in Table 1.

[0045] Table 1. LC50 of the IPD072Aa protein of this invention against cotton aphids 50

[0046] Protein LC50 (μg / ml) 95% confidence internal (μg / ml) Regression coefficient IPD072Aa 109.1 93.19-127.1 0.9960

[0047] Example 4: Bioactivity assay of IPD072Aa protein against brown planthopper, two-spotted spider mite and corn aphid

[0048] Brown planthoppers: The brown planthoppers used in the experiment were reared indoors at a temperature of 27℃, humidity of 60%, and a photoperiod of 16L:8D. Third-instar brown planthoppers were used and fed using a double-layer film feeding method (for details, please refer to: Liu Yonglei, 2015). The specific procedure was as follows: A hole was punched in a transparent plastic bottle (69 mm high, 42 mm in diameter) using a hole puncher (d = 20 cm). The hole was then sealed from the inside with 3M medical breathable tape to allow the test insects to breathe. 1 ml of 1% water agar was added to the bottom of the bottle to maintain humidity. A layer of paraffin film was gently spread and placed over the bottle opening. 180 μl of the test sample (a 1:2 mixture of protein and artificial feed) was dropped into the center of the film, and another layer of paraffin film was added to create a feed container. Twenty third-instar brown planthopper nymphs were added to each bottle, with three replicates per sample. The bottles were placed in an incubator at 28°C, a photoperiod of 16L:8D, and 70% humidity. Feeding and survival of the test insects were observed daily, and the feed was changed every two days. After five days, the number of dead brown planthoppers was counted, and the mortality rate was calculated.

[0049] like Figure 4 As shown in Figure A, the IPD072Aa protein concentration used in the experiment was 200 μg / ml, with the same concentration of the brown planthopper-killing protein orf1715 (developed by the inventors, see Chinese Patent CN119823241A) as a positive control. Compared with the buffer control group, IPD072Aa showed significant insecticidal activity against brown planthoppers, with an average mortality rate of 66.8% after 120 hours of treatment.

[0050] Two-spotted spider mite: Two-spotted spider mites used in the experiment were bred on white kidney beans. Two-spotted spider mites were purchased from Beijing Kuoye Tianyuan Biotechnology Co., Ltd., and white kidney beans were purchased from Shouguang Xinxinran Horticulture Co., Ltd. The breeding conditions were: temperature 26±1℃, humidity 65±5%, and photoperiod 16L:8D. The leaf immersion method was selected for bioassay, and the following is a detailed description: (1) Prepare 3% agar, heat it in a microwave oven to dissolve it, and after cooling it to 50-60℃, take 10ml and add it to a plastic petri dish with a diameter of 6cm; (2) Pick fresh, uncontaminated broad bean leaves, and prepare small leaf dishes of 2cm using a hole punch, taking care to avoid the main vein area; (3) Immerse the leaves in the protein sample to be tested for 5-10s, take them out and dry them until there is no free water on the surface of the leaves, and then spread them flat in a petri dish containing agar; (4) Use a No. 0 brush to pick 20-30 healthy nymphs onto the leaves, and set up 3 replicates for each sample. Set 1 μg / ml avermectin as a positive control, and ddH2O and protein buffer as negative controls; (5) To avoid the water vapor generated by the volatilization of agar affecting the survival of mites, use a red-hot fine needle to poke several small holes in the lid of the dish, and half-cover the lid. (6) Record the total number and the number of deaths under a stereomicroscope every 24 hours.

[0051] like Figure 4 As shown in Figure B, after 72 hours of treatment, the average lethality rate of the buffer control group was 23.8%, while the average lethality rate of IPD072Aa at a concentration of 500 μg / ml was 50.79%, exhibiting weaker insecticidal activity.

[0052] Corn aphids: The corn aphids used in the experiment were raised indoors in potted corn plants at a temperature of 28 ℃, slightly higher than the temperature required for cotton aphid rearing. The photoperiod was 16L:8D, and the bioassay method was the same as that used for cotton aphids in Example 3. Figure 4 As shown in C, after 72 hours of treatment, the average lethality rate of the buffer control group was 26.44%, while the average lethality rate of the experimental group with a concentration of 100 μg / ml of IPD072Aa was 100%.

[0053] The experimental results of Examples 3 and 4 show that the IPD072Aa protein and its encoding gene provided by the present invention have great application potential in aphid control.

[0054] Example 5: Analysis of the insect resistance effect of transient expression of the IPD072Aa gene in tobacco

[0055] Compared to stable expression, transient expression of exogenous genes in tobacco cells allows for a brief intracellular residence without integration into the genome, offering advantages such as shorter cycle time and higher success rate. Gene synthesis was performed by Aoke (Wuhan) Biotechnology Co., Ltd., with codon optimization of the target gene according to the tobacco expression host (as shown in SEQ ID NO:9). The target gene was then subcloned into the plant expression vector PBI121 using BamHI and XbaI restriction endonuclease sites. Figure 5 As shown.

[0056] The recombinant plasmid was transformed into Agrobacterium GV3101 competent cells using the heat shock method. The specific steps are as follows: (1) Take 7 μl of recombinant plasmid into GV3101 cells and place it on ice for 30 min; (2) Quickly freeze in liquid nitrogen for 5 min and then place it in a 37℃ water bath for 5 min, and then quickly place it back on ice for 5 min. Add 600 μl of liquid LB and place it in a shaker at 28℃ / 200 rpm for 3-6 h; (3) Centrifuge at 4000 rpm for 1 min, remove excess supernatant, and leave 100 μl of bacterial culture to be spread on LB plates. Add Kan antibiotic with a final concentration of 50 μg / ml and Rif antibiotic with a final concentration of 25 μg / ml, and place it in a 28℃ incubator for 2 days to wait for transformants to be generated.

[0057] The positive transformants were verified using PCR. The procedure was as follows: (1) The transformants to be verified were picked into 25 ml of LB liquid medium containing Kan and Rif, and cultured at 28℃ / 200 rpm for 48 h on a shaker; (2) The cells were collected by centrifugation at 9,000 rpm for 5 min; (3) 250 μl of solution I (25 mM Tris-HCl pH 8.0, 10 mM EDTA, 50 mM Glucose) was added and mixed; (4) 250 μl of solution II (1% SDS, 0.2 M NaOH) was added and mixed, allowed to stand for 5 min, and centrifuged at 12,000 rpm for 10 min; (5) 250 μl of solution III (3 M potassium acetate, 11.5% glacial acetic acid) was added and mixed, allowed to stand for 5 min, and centrifuged at 12,000 rpm for 10 min; (6) 500 μl of the supernatant was collected and recovered using a DNA recovery kit; (7) 1 The recovered DNA sample (μl) was subjected to PCR reaction using Easytaq polymerase (purchased from Beijing TransGen Biotech Co., Ltd.). The reaction system and procedure are shown in Tables 2-4. The identification results are as follows: Figure 6As shown: M represents Trans 2K Plus II DNA Marker, "+" and "-" represent positive (1 μl recombinant plasmid) and negative control (1 μl ddH2O), and lanes 1-6 represent 6 transformants respectively. (8) Store the correct transformants in glycerol tubes and place them in a -80℃ freezer.

[0058] Agrobacterium was transferred into tobacco using the injection method, and its insect-resistant effect was observed (for details, see: Chen Xiangsong, 2018). The following is a summary: (1) Agrobacterium carrying recombinant plasmids was inoculated into 10 ml of LB liquid medium, and the corresponding concentrations of Kan and Rif antibiotics were added. The medium was then cultured at 28°C for 24 h in a shaker. (2) 1% of the culture was transferred to a new 10 ml of LB liquid medium. In addition to Kan and Rif, 2-morpholinoethanesulfonic acid (MES, 20 μl, 0.5 M) and acetylsuccinone (AS, 4 μl, 100 M) were added. The medium was then cultured at 28°C and 250 rpm in a shaker until the OD600 was approximately 1.0. (3) The cells were collected by centrifugation at 4,000 rpm for 10 min. The cells were resuspended in 10 mM MgCl2 until the OD600 was 1.0. 100 mM MgCl2 was added at a ratio of 2 μl per ml of bacterial culture. AS, let stand for more than 3 hours; (4) Take a vigorous 3-week-old tobacco plant, draw in the bacterial solution with a 1ml syringe, remove the needle, press the front of the leaf with your finger, and let the bacterial solution penetrate from the back of the leaf; (5) Remove the leaf after 24 hours. Take a plastic petri dish with a diameter of 9cm, add 1.2% agar to keep it moist, open the lid (a square with a side length of about 4cm) to avoid condensation, and lay the picked leaves flat on the agar with the back of the leaves facing up; (6) Take about 20 tobacco aphids and inoculate them onto the leaves. Set up 3 replicates for each sample. Use Agrobacterium without recombinant plasmids as a negative control. Place the inoculated plates in a light incubator at a temperature of 26℃ and a light cycle of 16L:8D. Count and analyze the mortality rate after 48 hours. The aphids used were raised indoors in potted Tobacco 'Red Flower', and the rearing environment was the same as that of the cotton aphid in Example 3.

[0059] like Figure 7 As shown, the average mortality rate of aphids in the negative control group (CK-) was 7.04% after 48 hours, while the average mortality rate of aphids in the experimental group IPD072Aa was 46.67%. Therefore, this preliminary verification shows that the IPD072Aa protein also exhibits a certain insect-resistant effect in plants.

[0060] Table 2 Amplification System

[0061] Component Volume Final Concentration Template 1 μl Primer-F (10µM) 1 μl 0.2 μM Primer-R (10µM) 1 μl [[ID=3%]]0.2 μM 10×Easytaq® Buffer 2 μl l× 2.5mM dNTPs 1.6 μl 0.2 μM TransStart® FastPfu DNA Polymerase 0.2 μl 2.5 units Nuclease-free Water 13.2 μl Total volume 20 μl

[0062] Table 3 Amplification Primers

[0063] orf1715-F ATGGGAATTACTGTTACT orf1715-R GGATCCAGAAAGTGGTTC

[0064] Table 4 Amplification Procedure

[0065]

[0066] Example 6: Construction and expression of the monoalanine mutant of IPD072Aa

[0067] To further enhance the aphid-killing effect of IPD072Aa, this invention utilizes alanine scanning technology to replace 86 amino acid residues in the sequence with alanine, and replaces the original alanine residues with serine, thereby establishing a mutant library and evaluating insecticidal activity.

[0068] The mutant was constructed via circular PCR. Different primers were designed to introduce the desired mutation site into the sequence, using the pColdII recombinant plasmid carrying the IPD072Aa gene from Example 1 as a template. The PCR product was purified using a DNA fragment recovery kit (model D2500-01, Omega). Unused template in the PCR product was removed by digestion with DpnI enzyme at 37°C for 1 hour. The digested product was then directly transformed into DH5α competent cells, and the linear plasmid was ligated into a circular form using E. coli's own recombination system. Sequencing confirmed successful mutant construction.

[0069] Following the method described in Example 2, the 80 successfully constructed mutant plasmids were transformed into BL21, and all were found to be solublely expressed. Figure 8 The image shows the SDS-PAGE results of some mutants. "-" and "+" represent bacterial samples before and after induction, respectively; "F" represents the column permeate; and "E" represents the eluted target protein. Further culture was conducted to purify and prepare the mutant protein, and the protein concentration was determined using a BCA kit (Beijing Solarbio Science & Technology Co., Ltd.).

[0070] Table 5 Amplification Procedure

[0071]

[0072] Table 6 Amplification System

[0073] Component Volume Final Concentration Template 1 μl 500ng / μl Primer-F (10µM) 1 μl 0.2 μM Primer-R (10µM) 1 μl 0.2 μM 5×TransStart® FastPfu Buffer l× 4 μl 2.5mM dNTPs 1.6 μl 0.2 μM TransStart® FastPfu DNA Polymerase 0.2 μl 2.5 units Nuclease-free Water 11.2 μl Total volume

[0074] Table 7 DpnI digestion system

[0075] 20 μl Component Volume DNA product 5 μl Buffer 2.5 μl DpnI 1 μl Nuclease-free Water

[0076] Example 7: Evaluation of the insecticidal activity of the monoalanine mutant of IPD072Aa against cotton aphids

[0077] Following the bioassay method described in Example 3, the activity of 80 single-point mutants obtained in Example 5 against cotton aphids was tested. As shown in Table 8, at a final concentration of 100 μg / ml, the 72-hour corrected mortality rate of the wild-type protein IPD072Aa was 47.85%. The activities of five single-point mutants, I3A, P12A, E14A, K53A, and L85A, were similar to or slightly improved compared to the wild type. The insecticidal activity of three single-point mutants, S22A, A74S, and L81A, was significantly improved, with 72-hour corrected mortality rates of 96.84%, 81.35%, and 90.01%, respectively. As shown in Table 9, LC was further tested. 50 The activities of S22A, A74S, and L81A were 2.88 times, 2.16 times, and 3.41 times greater than those of the IPD072Aa protoxin, respectively.

[0078] Table 8. 72-hour corrected mortality rate of single-point mutants against cotton aphids

[0079] 16.5 μl protein corrected mortality protein corrected mortality protein corrected mortality 47.86% WT 20.56% V31A 4.36% H58A 15.64% M1A 33.78% G32A 11.67% P59A 31.35% G2A 40.87% N33A 0.01% Y60A 48.16% I3A 4.2% G34A 14.35% Y61A 43.21% T4A 13.17% K35A 42.86% [[ID=..]]V62A 0 V5A 20.47% Q36A 47.2% A64S 47.7% T6A 8.06% E37A 0 S66A 4.92% N7A 4.6% T38A 22.94% K67A 7.62% N8A 45.48% W39A 34.01% I68A 34.05% S9A 17.42% D40A 18.47% E69A 44.14% S10A 19.78% R41A 1.52% V70A 24.64% N11A 9.68% S42A 4.44% D71A 59.06% P12A 8.86% D43A 20.34% N72A 39.13% I13A 14.76% S44A 25.96% N73A 55.35% E14A 8.88% R45A 82.54% A74S 18.07% N18A 22.41% G46A 32.59% V75A 35.77% H19A F47A 12.86% K76A 5.63% W20A 0 V48A 15.72% D77A 0 G21A 0 L49A 18.89% Q78A 0 S22A 96.85% S50A 0.16% G79A 0 D23A 26.27% L51A 0 R80A 0 G24A 9.69% K52A 7.01% L81A 90.02% T26A 35.74% K53A 55.63% I82A 7.33% S27A 12.92% N54A 0 E83A 8.4% F28A 10.77% G55A 1.6% P84A 0 F29A 4.22% A56S 6.87% L85A 51.11% S30A 21.48% Q57A 33.62% S86A 5.84%

[0080] Table 9. Median lethal concentrations of mutants S22A, A74S, and L81A against cotton aphids.

[0081] Protein LC50 (μg / ml) 95% confidence internal (μg / ml) Regression coefficient S22A 37.82 32.9-42.2 0.9985 A74S 50.42 46.74-54.16 0.9995 L81A 31.99 18.74-48.86 0.9764

Claims

1. A mutant of the insecticidal protein IPD072Aa derived from Pseudomonas, the amino acid sequence of which is shown in SEQ ID NO:1 or SEQ ID NO:2 or SEQ ID NO:

3.

2. The encoding gene of the insecticidal protein IPD072Aa mutant as described in claim 1.

3. The encoding gene of the insecticidal protein IPD072Aa mutant according to claim 2, characterized in that, The coding sequence of the gene is shown in SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:

6.

4. A recombinant vector, characterized in that, The gene encoding the insecticidal protein IPD072Aa mutant as described in claim 2.

5. A genetically engineered bacterium, characterized in that, It includes the recombinant vector as described in claim 4.

6. The use of the insecticidal protein IPD072Aa mutant of claim 1, the encoding gene of claim 2 or 3, the recombinant vector of claim 4, or the genetically engineered bacteria of claim 5 in the preparation of insecticides for controlling aphids.

7. The use of the insecticidal protein IPD072Aa mutant of claim 1, the encoding gene of claim 2 or 3, the recombinant vector of claim 4, or the genetically engineered bacteria of claim 5 in the preparation of transgenic aphid-resistant crops or transgenic aphid-resistant microorganisms.

8. The application of insecticidal protein IPD072Aa in the preparation of insecticides for controlling aphids, transgenic insect-resistant crops or transgenic insecticidal microorganisms, wherein the amino acid sequence of the insecticidal protein IPD072Aa is shown in SEQ ID NO:

7.

9. Application of the gene encoding the insecticidal protein IPD072Aa in the preparation of insecticides for aphid control, transgenic insect-resistant crops, or transgenic insecticidal microorganisms.

10. The application according to claim 9, characterized in that, The coding sequence of the gene is shown in SEQ ID NO:8 or SEQ ID NO:9.

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