An insecticidal protein and its application in controlling Lepidoptera insects

Abstract

The present invention relates to an insecticidal protein and its application in controlling Lepidoptera insects. The insecticidal protein is protein I and / or protein II, wherein protein I is a first mutant protein in which domain II of Cry2A protein is domain II of Anc0 protein, and the amino acid sequence of Anc0 protein is as shown in SEQ ID No.8; protein II is a second mutant protein in which Cry2A protein has at least one mutation at Q318I, A337S, S360N, G384E and L459A.

Description

Technical Field

[0001] The present invention relates to the field of biological control, and particularly to the application of a protein in controlling Lepidoptera insects. Background Art

[0002] The diamondback moth (Plutella xylostella) is distributed throughout China and is one of the main pests of cruciferous vegetables. It specifically feeds on cruciferous plants such as cabbage, broccoli, and rape, causing the most serious harm and threat to the production and development of cruciferous vegetables and being one of the most difficult pests to control.

[0003] The Asian corn borer (Ostrinia furnacalis) is a polyphagous pest that mainly damages corn, sorghum, and millet, and can also damage crops such as cotton, hemp, sugarcane, sunflower, rice, beet, sweet potato, and beans. The corn borer mainly damages by boring into the stems of larvae, destroying the stem tissue, affecting nutrient transport, damaging the plants, and in severe cases, the stems break when encountering wind. The northern spring corn cultivation area in China is one of the areas with the most serious occurrence.

[0004] The oriental armyworm (Mythimna separata) is an important pest of local crops in China. The larvae have a miscellaneous diet and no diapause, and the main hosts are wheat and corn. Every year in April, in the Hanjiang River and the middle and lower reaches of the Yangtze River, the first-generation larvae of the oriental armyworm generally occur in wheat fields and also generally occur in the seedling stage of spring corn.

[0005] The tea geometrid (Ectropis oblique) is one of the main pests of tea trees in China. The tea geometrid is distributed in Zhejiang, Anhui, Jiangsu, Fujian and other places in China. The larvae mainly feed on young leaves and mature leaves. When it occurs severely, it can eat up all the leaves and tender buds, affecting the tea yield of the current year and causing the decline of the tree vigor, resulting in a reduction in tea production in the coming year.

[0006] At present, the control of diamondback moths, corn borers, oriental armyworms, and tea geometrids mainly adopts two strategies: chemical control and biological control. Biological control mainly relies on insect pathogens such as Bacillus thuringiensis (Bt). However, with the continuous planting of Bt Cry transgenic crops, Lepidoptera insects such as diamondback moths, corn borers, oriental armyworms, and tea geometrids have gradually developed resistance.

[0007] Therefore, it is necessary to find new insecticidal proteins against diamondback moths, corn borers, oriental armyworms, and tea geometrids. Summary of the Invention

[0008] One aspect of the present invention provides an insecticidal protein, which is protein I and / or protein II, wherein,

[0009] The protein I is the first mutant protein of domain II of Cry2A protein, where the domain II of Anc0 protein, and the amino acid sequence of the Anc0 protein is as shown in SEQ ID No.8;

[0010] The protein II is the second mutant protein of Cry2A protein with at least one mutation at Q318I, A337S, S360N, G384E, and L459A.

[0011] In a specific embodiment, the protein I is the first mutant protein in which the amino acids at positions 267 to 472 of Cry2A protein are the amino acids at positions 267 to 472 of Anc0 protein.

[0012] In a specific embodiment, the amino acid sequence of the protein I is at least one of SEQ ID No.8, SEQ ID No.14, and SEQ ID No.16.

[0013] In a specific embodiment, the protein II is the second mutant protein of Cry2A protein with at least one mutation at Q318I, S360N, and G384E.

[0014] In a specific embodiment, the protein II is the third mutant protein of the protein with the amino acid sequence as shown in SEQ ID No.4, and the third mutant protein has at least one mutation at Q318I, A337S, S360N, G384E, and L459A.

[0015] In a specific embodiment, the protein II is the fourth mutant protein of the protein with the amino acid sequence as shown in SEQ ID No.6, and the fourth mutant protein has at least one mutation at Q318I, A337S, S360N, G384E, and L459A.

[0016] The second aspect of the present invention provides a composition containing the protein as described in the first aspect of the present invention.

[0017] The third aspect of the present invention provides a nucleic acid encoding the protein as described in the first aspect of the present invention.

[0018] In a specific embodiment, for domain II, the sequence of the nucleic acid encoding the domain II is as shown from the 799th to the 1416th position of SEQ ID No.7;

[0019] For the Q318I mutation, the encoding base is mutated to ATT;

[0020] For the A337S mutation, the encoding base is mutated to AGT;

[0021] For the S360N mutation, the coding base mutates to AAT;

[0022] For the G384E mutation, the coding base mutates to GAG;

[0023] For the L459A mutation, the coding base mutates to GCA.

[0024] In a specific embodiment, the sequence of the nucleic acid encoding the protein with the amino acid sequence shown in SEQ ID No. 8 is as shown in SEQ ID No. 7.

[0025] In a specific embodiment, the sequence of the nucleic acid encoding the protein with the amino acid sequence shown in SEQ ID No. 14 is as shown in SEQ ID No. 13.

[0026] In a specific embodiment, the sequence of the nucleic acid encoding the protein with the amino acid sequence shown in SEQ ID No. 16 is as shown in SEQ ID No. 15

[0027] The fourth aspect of the present invention provides a microorganism carrying the nucleic acid as described in the third aspect of the present invention and capable of expressing the protein as described in the first aspect of the present invention.

[0028] In a specific embodiment, the microorganism is Escherichia coli and / or Bacillus thuringiensis.

[0029] The fifth aspect of the present invention provides the use of one of the protein as described in the first aspect of the present invention, the composition as described in the second aspect of the present invention, the nucleic acid as described in the third aspect of the present invention, and the microorganism as described in the fourth aspect of the present invention in controlling Lepidoptera insects.

[0030] In a specific embodiment, the Lepidoptera insects are at least one of Plutella xylostella, Ostrinia furnacalis subspecies, Mythimna separata, and Ectropis oblique.

[0031] In a specific embodiment, the protein I is used to control at least one of Plutella xylostella, Mythimna separata, and Ectropis oblique.

[0032] In a specific embodiment, at least one of the proteins with the amino acid sequences shown in SEQ ID No. 8, the proteins with the amino acid sequences shown in SEQ ID No. 14, and the proteins with the amino acid sequences shown in SEQ ID No. 16 is used for controlling at least one of Plutella xylostella, Mythimna separata, and Ectropis oblique.

[0033] In a specific embodiment, the second mutant protein in which the Cry2A protein has a mutation at Q318I is used for controlling Plutella xylostella and / or Ectropis oblique.

[0034] In a specific embodiment, the third mutant protein in which the protein with the amino acid sequence shown in SEQ ID No. 4 has a mutation at Q318I is used for controlling Plutella xylostella and / or Ectropis oblique.

[0035] In a specific embodiment, the third mutant protein in which the protein with the amino acid sequence shown in SEQ ID No. 6 has a mutation at Q318I is used for controlling Plutella xylostella and / or Ectropis oblique.

[0036] In a specific embodiment, the second mutant protein in which the Cry2A protein has a mutation at S360N and / or G384E is used for controlling Ectropis oblique.

[0037] In a specific embodiment, the third mutant protein in which the protein with the amino acid sequence shown in SEQ ID No. 4 has a mutation at S360N and / or G384E is used for controlling Ectropis oblique.

[0038] In a specific embodiment, the third mutant protein in which the protein with the amino acid sequence shown in SEQ ID No. 6 has a mutation at S360N and / or G384E is used for controlling Ectropis oblique.

[0039] In a specific embodiment, the protein with the amino acid sequence shown in SEQ ID No. 8 and / or the protein with the amino acid sequence shown in SEQ ID No. 14 is used for controlling Ostrinia furnacalis.

[0040] In a specific embodiment, the third mutant protein in which the protein with the amino acid sequence shown in SEQ ID No. 4 has a mutation at Q318I is used for controlling the Asian corn borer (Ostrinia furnacalis) and / or the oriental armyworm (Mythimna separata).

[0041] In a specific embodiment, the third mutant protein in which the protein with the amino acid sequence shown in SEQ ID No. 4 has a mutation at A337S is used for controlling the oriental armyworm (Mythimna separata).

[0042] In a specific embodiment, the third mutant protein in which the protein with the amino acid sequence shown in SEQ ID No. 4 has a mutation at S360N is used for controlling the oriental armyworm (Mythimna separata).

[0043] In a specific embodiment, the third mutant protein in which the protein with the amino acid sequence shown in SEQ ID No. 4 has a mutation at G384E is used for controlling the oriental armyworm (Mythimna separata).

[0044] In a specific embodiment, the third mutant protein in which the protein with the amino acid sequence shown in SEQ ID No. 4 has a mutation at L459A is used for controlling at least one of the diamondback moth (Plutella xylostella), the Asian corn borer (Ostrinia furnacalis), and the oriental armyworm (Mythimna separata).

[0045] In a specific embodiment, the third mutant protein in which the protein with the amino acid sequence shown in SEQ ID No. 6 has a mutation at A337S is used for controlling the tea geometrid (Ectropis oblique).

[0046] In a specific embodiment, the third mutant protein in which the protein with the amino acid sequence shown in SEQ ID No. 6 has a mutation at L459A is used for controlling the tea geometrid (Ectropis oblique).

[0047] Advantages of the present invention:

[0048] 1) The Cry2A protein containing the Anc0 domain II significantly improves the insecticidal activity of Cry2A-like proteins against Lepidoptera pests. For example, Anc0 (amino acid sequence shown in SEQ ID No. 8) and Cry2Ad1-II (amino acid sequence shown in SEQ ID No. 14) have very good insecticidal activity against Plutella xylostella, Ostrinia furnacalis ssp., Mythimna separata, and Ectropis obliqua, and are significantly superior to known Cry2A-like proteins; the protein Cry2Ah2-II (amino acid sequence shown in SEQ ID No. 16) has very good insecticidal activity against Plutella xylostella, Mythimna separata, and Ectropis obliqua.

[0049] 2) After mutating the Cry2A-like protein at Q318I, for example, after Cry2Ae1 mutates at Q318I, or after Cry2Ah2 mutates at Q318I, the insecticidal activity against Plutella xylostella or Ectropis obliqua can be significantly improved; after mutating the Cry2A-like protein at S360N and / or G384E, for example, after Cry2Ae1 mutates at S360N and / or G384E, or after Cry2Ah2 mutates at S360N and / or G384E, the insecticidal activity against Ectropis obliqua can be significantly improved.

[0050] 3) After Cry2Ae1 mutates at Q318I, A337S, S360N, G384E, and L459A, it acquires insecticidal activity against Mythimna separata.

[0051] 4) After Cry2Ae1 mutates at Q318I, the insecticidal activity against Ostrinia furnacalis ssp. can be significantly improved.

[0052] 5) After Cry2Ae1 mutates at L459A, the insecticidal activity against Plutella xylostella and / or Ostrinia furnacalis ssp. can be significantly improved.

[0053] 6) After Cry2Ah2 mutates at A337S and L459A, the insecticidal activity against Ectropis obliqua can be significantly improved. Description of the Drawings

[0054] Figure 1 Shows the SDS-PAGE detection results of Cry2Ad1, Cry2Ae1, Cry2Ah2, and Anc0.

[0055] Figure 2 Shows the SDS-PAGE detection results of Cry2Ad1, Cry2Ad1-II, Cry2Ah2, and Cry2Ah2-II.

[0056] Figure 3The SDS-PAGE detection results of Cry2Ae1, Cry2Ae1-Q318I, Cry2Ae1-A337S, Cry2Ae1-S360N, Cry2Ae1-G384E, and Cry2Ae1-L459A are shown.

[0057] Figure 4 The SDS-PAGE detection results of Cry2Ah2, Cry2Ah2-Q318I, Cry2Ah2-A337S, Cry2Ah2-S360N, Cry2Ah2-G384E, and Cry2Ah2-L459A are shown. Detailed implementation mode

[0058] The above content of the present invention will be further described in detail below in the form of preferred implementation cases, but it does not constitute a limitation to the present invention.

[0059] Unless otherwise specified, the reagents in the embodiments of the present invention can be purchased through commercial channels.

[0060] Example 1

[0061] Expression of Cry2Ad1, Cry2Ae1, and Cry2Ah2 proteins

[0062] 1. Construction of recombinant Bt strains

[0063] The nucleic acids composed of the nucleotide sequences shown in SEQ ID No.1, SEQ ID No.3, SEQ ID No.5 and SEQ ID No.7 were synthesized by Sangon Biotech (Shanghai) Co., Ltd. Among them, the nucleic acid with the base sequence shown in SEQ ID No.1 encodes a protein with the amino acid sequence shown in SEQ ID No.2 (i.e., Cry2Ad1 protein); the nucleic acid with the base sequence shown in SEQ ID No.3 encodes a protein with the amino acid sequence shown in SEQ ID No.4 (i.e., Cry2Ae1 protein); the nucleic acid with the base sequence shown in SEQ ID No.5 encodes a protein with the amino acid sequence shown in SEQ ID No.6 (i.e., Cry2Ah2 protein); the nucleic acid with the base sequence shown in SEQ ID No.7 encodes a protein with the amino acid sequence shown in SEQ ID No.8 (i.e., Anc0 protein). The synthesized insecticidal protein genes were respectively ligated to the PUC18-p1Ac-GFP vector to obtain p1Ac-GFP-Cry2Ad1, p1Ac-GFP-Cry2Ae1, p1Ac-GFP-Cry2Ah2 and p1Ac-GFP-Anc0 recombinant plasmids, which were then transformed into Escherichia coli TOP10. The transformants were named TOP10 / p1Ac-GFP-Cry2Ad1, TOP10 / p1Ac-GFP-Cry2Ae1, TOP10 / p1Ac-GFP-Cry2Ah2 and TOP10 / p1Ac-GFP-Anc0 in sequence.

[0064] The transformant TOP10 / PUC18-p1Ac-GFP obtained by transforming Escherichia coli TOP10 with the PUC18-p1Ac-GFP empty vector was used as a negative control for protein expression analysis.

[0065] 2. Protein expression and extraction

[0066] (1) Pick a single colony: Pick a single colony of TOP10 / p1Ac-GFP-Cry2Ad1 into 5 mL of LB liquid medium containing ampicillin, and culture it at 37 °C with shaking at 220 rpm for 12 hours to obtain an activated bacterial solution;

[0067] (2) Inoculate the activated bacterial solution into a 1 L Erlenmeyer flask containing 800 mL of LB liquid medium (containing ampicillin) at a volume ratio of 1%, and culture it at 37 °C with shaking at 220 rpm for 72 h to obtain a fermentation broth;

[0068] (3) Centrifuge the fermentation broth at 8,000 × g at 4 °C for 10 minutes, discard the supernatant, and resuspend the precipitate with pre-cooled 20 mmol / L Tris-HCl (pH = 8.0) buffer to obtain a first bacterial suspension;

[0069] (4) Transfer the first suspension into a 50 mL centrifuge tube, centrifuge at 8,000×g at 4°C for 10 minutes, discard the supernatant, and resuspend the precipitate with pre-cooled 20 mmol / L Tris-HCl (pH = 8.0) to obtain the second bacterial suspension;

[0070] (5) Ultrasonically disrupt the cell wall of the second bacterial suspension (power 70%, 5 min, ultrasonicate for 3 s, pause for 5 s) to obtain the disrupted bacterial solution;

[0071] (6) Centrifuge the disrupted bacterial solution at 8,000×g at 4°C for 10 minutes and collect the precipitate;

[0072] (7) Wash the precipitate with pre-cooled sterile water 2 to 3 times;

[0073] (8) Add 5 ml of 20 mmol / L Tris-HCl (pH = 8.0) buffer to resuspend the precipitate to obtain the Cry2Ad1 protein suspension to be tested;

[0074] (9) Extract the proteins of TOP10 / p1Ac-GFP-Cry2Ae1, TOP10 / p1Ac-GFP-Cry2Ah2, TOP10 / p1Ac-GFP-Anc0, and TOP10 / PUC18-p1Ac-GFP respectively with the same operations as in (1) to (8) above, and obtain the Cry2Ae1 protein suspension to be tested, the Cry2Ah2 protein suspension to be tested, the Anc0 protein suspension to be tested, and the negative control protein suspension respectively;

[0075] (10) Perform SDS-PAGE detection on each protein suspension to be tested and the negative control protein suspension, and the results are shown in Figure 1 .

[0076] According to Figure 1 the results, Cry2Ad1, Cry2Ae1, Cry2Ah2, and Anc0 were successfully expressed in Escherichia coli, and the expression products were in the precipitate after cell disruption. Therefore, the protein suspension to be tested is the protein suspension containing Cry2Ad1, Cry2Ae1, Cry2Ah2, and Anc0.

[0077] Before activity determination, quantitatively analyze the protein suspension containing Cry2Ad1, Cry2Ae1, Cry2Ah2, and Anc0 with BSA.

[0078] Example 2

[0079] Domain replacement, site-directed mutagenesis, and expression of mutant proteins

[0080] Design primer pairs F-domain II-1 (shown in SEQ ID No. 9) / R-domain II-1 (shown in SEQ ID No. 10) and primer pairs F-domain II-2 (shown in SEQ ID No. 11) / R-domain II-2 (shown in SEQ ID No. 12).

[0081] Perform domain replacement on the protein with the amino acid sequence shown in SEQ ID No. 2. The operation is as follows: Using the recombinant plasmid p1Ac-GFP-Anc0 as a template, and F-domain II-1 and R-domain II-1 as primers, use a high-fidelity polymerase (Phusion) to amplify a DNA fragment (the amino acid sequence encoded by it is the amino acids at positions 267 to 472 of the protein shown in SEQ ID No. 8, which constitutes domain II of the protein), to obtain PCR product 1; Using the recombinant plasmid p1Ac-GFP-Cry2Ad1 as a template, and primers F-domain II-2 and R-domain II-2 as primers, use a high-fidelity polymerase (Phusion) to amplify a DNA fragment (it is the DNA fragment of p1Ac-GFP-Cry2Ad1 minus the fragment encoding the amino acids at positions 267 to 472 shown in SEQ ID No. 2), to obtain PCR product 2. Perform agarose gel recovery on PCR product 1 and PCR product 2 respectively to obtain recovery product 1 and recovery product 2. Use a homologous recombination enzyme (Exnase II) to perform homologous recombination on recovery product 1 and recovery product 2 to obtain the p1Ac-GFP-Cry2Ad1-II plasmid. Then transform the homologous recombination product p1Ac-GFP-Cry2Ad1-II into Escherichia coli competent cell TOP10. After picking a single colony and culturing it in a shaker, sequence the bacterial solution to obtain the positive recombinant strain TOP10 / p1Ac-GFP-Cry2Ad1-II. Among them, in the p1Ac-GFP-Cry2Ad1-II plasmid, the base sequence of the target gene cry2Ad1-II is shown in SEQ ID No. 13, and the amino acid sequence of the target protein Cry2Ad1-II is shown in SEQ ID No. 14.

[0082] Domain replacement was performed on the protein with the amino acid sequence shown in SEQ ID No. 6. The operation was as follows: Using the recombinant plasmid p1Ac-GFP-Anc0 as a template, and F-domain II-1 and R-domain II-1 as primers, a DNA fragment (encoding the amino acids at positions 267 to 472 of the protein shown in SEQ ID No. 8) was amplified using a high-fidelity polymerase (Phusion) to obtain PCR product 3; Using the recombinant plasmid p1Ac-GFP-Cry2Ah2 as a template, and F-domain II-2 and R-Fdomain II-2 as primers, a DNA fragment (which is the fragment of the entire DNA of p1Ac-GFP-Cry2Ad1 excluding the other amino acids encoding positions 267 to 471 shown in SEQ ID No. 6) was amplified using a high-fidelity polymerase (Phusion) to obtain PCR product 4. PCR product 3 and PCR product 4 were respectively subjected to agarose gel recovery to obtain recovery product 3 and recovery product 4. Recovery product 3 and recovery product 4 were subjected to homologous recombination using a homologous recombinase (Exnase II) to obtain the p1Ac-GFP-Cry2Ah2-II plasmid. Then, the homologous recombination product p1Ac-GFP-Cry2Ah2-II was transformed into Escherichia coli competent cells TOP10. After picking single colonies and culturing them in liquid, the bacterial solution was sequenced to obtain the positive recombinant strain TOP10 / p1Ac-GFP-Cry2Ah2-II. Among them, in the p1Ac-GFP-Cry2Ah2-II plasmid, the base sequence of the target gene cry2Ah2-II is shown in SEQ ID No. 15, and the amino acid sequence of the target protein Cry2Ah2-II is shown in SEQ ID No. 16.

[0083] Design mutant primers F-Q318I (shown in SEQ ID No. 17) / R-Q318I (shown in SEQ ID No. 18), mutate CAG at positions 952 to 954 in the sequence shown in SEQ ID No. 3 to ATT, thereby performing a Q318I mutation on the protein with the amino acid sequence shown in SEQ ID No. 4 encoded by SEQ ID No. 3. The operation is as follows: Using the p1Ac-GFP-Cry2Ae1 recombinant plasmid as a template, and F-Q318I and R-Q318I as primers, perform PCR amplification with a high-fidelity polymerase (Phusion) to obtain PCR product 5. Digest the methylated plasmid template of the obtained PCR product 5 with DPN I enzyme to obtain a digested product. Use a homologous recombination enzyme (Exnase II) to perform homologous recombination on the digested product to obtain the p1Ac-GFP-Cry2Ae1-Q318I plasmid. Then transform the homologous recombination product p1Ac-GFP-Cry2Ae1-Q318I into Escherichia coli competent cell TOP10. After picking a single colony and culturing it in a shaker, sequence the bacterial solution to obtain the positive plasmid p1Ac-GFP-Cry2Ae1-Q318I and the positive recombinant strain TOP10 / p1Ac-GFP-Cry2Ae1-Q318I; Using p1Ac-GFP-Cry2Ah2 as a template, and F-Q318I / R-Q318I as primers, mutate CAG at positions 952 to 954 in the sequence shown in SEQ ID No. 5 to ATT, thereby performing a Q318I mutation on the protein with the amino acid sequence shown in SEQ ID No. 6 encoded by SEQ ID No. 5 to obtain the positive plasmid p1Ac-GFP-Cry2Ah2-Q318I and the positive recombinant strain TOP10 / p1Ac-GFP-Cry2Ah2-Q318I. Other operations are the same as TOP10 / p1Ac-GFP-Cry2Ae1-Q318I.

[0084] Design mutant primers F-A337S (as shown in SEQ ID No. 19) / R-A337S (as shown in SEQ ID No. 20). Using the p1Ac-GFP-Cry2Ae1 recombinant plasmid as a template, mutate the GCG at positions 1009 to 1011 in the sequence as shown in SEQ ID No. 3 to AGT, thereby performing an A337S mutation on the protein with the amino acid sequence as shown in SEQ ID No. 4, to obtain the positive plasmid p1Ac-GFP-Cry2Ae1-A337S and the positive recombinant strain TOP10 / p1Ac-GFP-Cry2Ae1-A337S; Using F-A337S / R-A337S as primers, and p1Ac-GFP-Cry2Ah2 as a template, mutate the GCT at positions 1009 to 1011 in the sequence as shown in SEQ ID No. 5 to AGT, perform an A337S mutation on the protein with the amino acid sequence as shown in SEQ ID No. 6, to obtain the positive plasmid p1Ac-GFP-Cry2Ah2-A337S and the positive recombinant strain TOP10 / p1Ac-GFP-Cry2Ah2-A337S. Other operations are the same as TOP10 / p1Ac-GFP-Cry2Ae1-Q318I.

[0085] Design mutant primers F-S360N (as shown in SEQ ID No. 21) / R-S360N (as shown in SEQ ID No. 22). Using the p1Ac-GFP-Cry2Ae1 recombinant plasmid as a template, mutate the AGT at positions 1078 to 1080 in the sequence as shown in SEQ ID No. 3 to AAT, thereby performing an S360N mutation on the protein with the amino acid sequence as shown in SEQ ID No. 4, to obtain the positive plasmid TOP10 / p1Ac-GFP-Cry2Ae1-S360N and the positive recombinant strain TOP10 / TOP10 / p1Ac-GFP-Cry2Ae1-S360N; Using F-S360N / R-S360N as primers, and p1Ac-GFP-Cry2Ah2 as a template, mutate the AGT at positions 1078 to 1080 in the sequence as shown in SEQ ID No. 5 to AAT, perform an S360N mutation on the protein with the amino acid sequence as shown in SEQ ID No. 6, to obtain the positive plasmid p1Ac-GFP-Cry2Ah2-S360N and the positive recombinant strain TOP10 / p1Ac-GFP-Cry2Ah2-S360N. Other operations are the same as TOP10 / p1Ac-GFP-Cry2Ae1-Q318I.

[0086] Design mutant primers F-G384E (as shown in SEQ ID No. 23) / R-G384E (as shown in SEQ ID No. 24). Using the p1Ac-GFP-Cry2Ae1 recombinant plasmid as a template, mutate GGG at positions 1150 to 1152 in the sequence as shown in SEQ ID No. 3 to GAG, thereby performing a G384E mutation on the protein with the amino acid sequence as shown in SEQ ID No. 4, to obtain the positive plasmid p1Ac-GFP-Cry2Ae1-G384E and the positive recombinant strain TOP10 / p1Ac-GFP-Cry2Ae1-G384E; using F-G384E / R-G384E as primers, and p1Ac-GFP-Cry2Ah2 as a template, mutate GGG at positions 1150 to 1152 in the sequence as shown in SEQ ID No. 5 to GAG, perform a G384E mutation on the protein with the amino acid sequence as shown in SEQ ID No. 6, to obtain the positive plasmid p1Ac-GFP-Cry2Ah2-G384E and the positive recombinant strain TOP10 / p1Ac-GFP-Cry2Ah2-G384E. Other operations are the same as TOP10 / p1Ac-GFP-Cry2Ae1-Q318I.

[0087] Design mutant primers F-L459A (as shown in SEQ ID No. 25) / R-L459A (as shown in SEQ ID No. 26). Using the p1Ac-GFP-Cry2Ae1 recombinant plasmid as a template, mutate TTA at positions 1375 to 1377 in the sequence as shown in SEQ ID No. 3 to GCA, thereby performing an L459A mutation on the protein with the amino acid sequence as shown in SEQ ID No. 4, to obtain the positive plasmid p1Ac-GFP-Cry2Ae1-L459A and the positive recombinant strain TOP10 / p1Ac-GFP-Cry2Ae1-L459A; using F-L459A / R-L459A as primers, and p1Ac-GFP-Cry2Ah2 as a template, mutate TTA at positions 1375 to 1377 in the sequence as shown in SEQ ID No. 5 to GCA, perform an L459A mutation on the protein with the amino acid sequence as shown in SEQ ID No. 6, to obtain the positive plasmid p1Ac-GFP-Cry2Ah2-L459A and the positive recombinant strain TOP10 / p1Ac-GFP-Cry2Ah2-L459A. Other operations are the same as TOP10 / p1Ac-GFP-Cry2Ae1-Q318I.

[0088] Express and extract the mutant proteins in the same way as in Example 1. The results show that each mutant protein is successfully expressed in Escherichia coli, and the expression products are in the precipitate after cell disruption. The results are shown inFigures 2 to 4 Therefore, each protein suspension to be measured is a protein suspension containing mutant proteins.

[0089] Similarly, before activity measurement, the concentration of each mutant protein was quantified by BSA.

[0090] Example 3

[0091] Insecticidal activity measurement

[0092] Using 20 mmol / L Tris-HCl (pH 8.0) as the blank control; each protein suspension was diluted to 100 μg / ml with 20 mmol / L Tris-HCl (pH 8.0) and mixed thoroughly to obtain each protein sample to be measured.

[0093] Steps for measuring the insecticidal activity against Plutella xylostella: Soak clean cabbage leaves with a diameter of 6 cm in the protein sample to be measured and the negative control. After 10 min, take them out and let them air-dry naturally until the water has completely evaporated. Then put them into a sterilized petri dish. Use a brush to transfer 30 healthy and active second-instar Plutella xylostella larvae into the sterilized petri dish containing the above-mentioned cabbage leaves. Each treatment was repeated 3 times. Then place them in an artificial climate incubator and culture at 26 °C, with a photoperiod of 14L:10D and a relative humidity of 60%. Observe every day and check the light, humidity, and temperature; after 72 h, investigate the number of dead insects.

[0094] The difference in the steps for measuring the insecticidal activity against Ectropis obliqua from those for measuring the insecticidal activity against Plutella xylostella is that the cabbage leaves are replaced with fresh tea leaves.

[0095] Steps for measuring the insecticidal activity against Ostrinia furnacalis: Weigh 7 g of the special feed for Ostrinia furnacalis (the feed formula is shown in Table 1) into a sterilized petri dish, add 0.7 mL of the protein sample to be measured, and add 0.7 mL of 20 mmol / L Tris-HCl (pH 8.0) to the negative control. Mix thoroughly and wait for the excess water to completely evaporate. Use a brush to transfer 30 healthy and active newly hatched Ostrinia furnacalis larvae into the petri dish containing the above-mentioned feed. Each treatment was repeated 3 times. Then place them in an artificial climate incubator and culture at 26 °C, with a photoperiod of 14L:10D and a relative humidity of 60%. Observe every day and check the light, humidity, and temperature; after 7 d, investigate the number of dead insects.

[0096] The difference in the steps for measuring the insecticidal activity against Mythimna separata from those for measuring the insecticidal activity against Ostrinia furnacalis is that the feed is the special feed for Mythimna separata (the feed formula is shown in Table 1).

[0097] Calculate the mortality rate and the corrected mortality rate. The results of the bioassay are shown in Tables 2 to 4.

[0098] The formula for calculating the corrected mortality rate is as follows:

[0099]

[0100] Table 1

[0101]

[0102] Table 2

[0103]

[0104] The significance analysis used an independent samples T-test (p < 0.05) to analyze the significant differences between proteins.

[0105] According to the results in Table 2, the Anc0 protein showed very high insecticidal activity against Plutella xylostella, Ostrinia furnacalis, Mythimna separata, and Ectropis obliqua. The mutant protein Cry2Ad1-II of Cry2Ad1 acquired insecticidal activity against Plutella xylostella, Ostrinia furnacalis, Mythimna separata, and Ectropis obliqua. The mutant protein Cry2Ah2-II of Cry2Ah2 enhanced the insecticidal activity against Plutella xylostella and Ectropis obliqua and acquired insecticidal activity against Mythimna separata. It can be seen that replacing domain II of Cry2A proteins with domain II of the Anc0 protein, or more precisely, replacing the amino acids at positions 267 to 472 of Cry2A proteins with those at positions 267 to 472 of the Anc0 protein, can significantly improve the insecticidal activity of Cry2A proteins against Lepidoptera pests.

[0106] Table 3

[0107]

[0108] “ / ” indicates unknown activity. The significance analysis used an independent samples T-test (p < 0.05) to analyze the significant differences between proteins.

[0109] According to the results in Table 3, compared with the Cry2Ae1 protein, the five point mutant proteins of Cry2Ae1, namely Cry2Ae1-Q318I, Cry2Ae1-A337S, Cry2Ae1-S360N, Cry2Ae1-G384E, and Cry2Ae1-L459A, all acquired insecticidal activity against Mythimna separata. Cry2Ae1-Q318I and Cry2Ae1-L459A enhanced the insecticidal activity against Plutella xylostella and Ostrinia furnacalis. Cry2Ae1-Q318I, Cry2Ae1-S360N, and Cry2Ae1-G384E enhanced the insecticidal activity against Ectropis obliqua.

[0110] Table 4

[0111]

[0112] " / " represents unknown activity. Independent-sample T test (p < 0.05) was used for significance analysis to analyze the significant differences between proteins.

[0113] According to the results in Table 4, compared with the Cry2Ah2 protein, the five point-mutated proteins of Cry2Ah2, namely Cry2Ah2-Q318I, Cry2Ah2-A337S, Cry2Ah2-S360N, Cry2Ah2-G384E, and Cry2Ah2-L459A, all enhanced the insecticidal activity against Ectropis obliqua, and Cry2Ah2-Q318I enhanced the insecticidal activity against Plutella xylostella.

[0114] Based on the activity of Cry2Ae1-Q318I in Table 3 and the activity of Cry2Ah2-Q318I in Table 4, it can be known that mutating the Cry2A protein at Q318I can enhance the insecticidal activity against Plutella xylostella or Ectropis obliqua; based on the activity of Cry2Ae1-S360N and Cry2Ae1-G384E in Table 3 and the activity of Cry2Ah2-S360N and Cry2Ah2-G384E in Table 4, it can be known that mutating the Cry2A protein at S360N or G384E can enhance the insecticidal activity against Ectropis obliqua.

Claims

1. An insecticidal protein, the amino acid sequence of which is shown in SEQ ID No.

8.

2. A composition comprising the insecticidal protein according to claim 1.

3. Nucleic acid encoding the insecticidal protein according to claim 1.

4. The nucleic acid according to claim 3, wherein The sequence of the nucleic acid is shown in SEQ ID No.

7.

5. A microorganism into which the nucleic acid according to claim 3 or 4 is introduced and which can express the insecticidal protein according to claim 1.

6. The microorganism according to claim 5, characterized in that, The microorganism is Escherichia coli and / or Bacillus thuringiensis.

7. Use of one of the insecticidal protein according to claim 1, the composition according to claim 2, the nucleic acid according to claim 3 or 4, and the microorganism according to claim 5 or 6 in controlling Lepidoptera insects; The Lepidoptera insects are at least one of Plutella xylostella, subspecies Ostrinia furnacalis, Mythimna separata, and Ectropis oblique.

Images