Insecticidal protein and application thereof in prevention and treatment of lepidoptera insects

Abstract

The invention relates to an insecticidal protein and application thereof in prevention and treatment of lepidoptera insects. The insecticidal protein is a protein I and / or a protein II, the protein I is a first mutant protein of which the structural domain II of Cry2A protein is the structural domain II of Anc0 protein, and the amino acid sequence of the Anc0 protein is shown as SEQ ID No.8; and the protein II is a second mutant protein obtained by mutating the Cry2A protein at at least one of Q318I, A337S, S360 N, G384E and L459A.

Description

[0001] This patent application is a divisional application of the invention entitled "An insecticidal protein and its application in controlling lepidopteran insects" filed on October 27, 2023, with application number 2023114092474. Technical Field

[0002] The present invention relates to the field of biological control, and in particular to application of a protein in controlling lepidopteran insects. Background Art

[0003] The diamondback moth (Plutella xylostella) is distributed throughout China and is one of the main pests of cruciferous vegetables. It specializes in feeding on cruciferous vegetables such as cabbage, broccoli and rapeseed. It poses the most serious harm and threat to the production and development of cruciferous vegetables and is also one of the most difficult pests to control.

[0004] The Asian corn borer (Ostrinia furnacalis) is a polyphagous pest that primarily attacks corn, sorghum, and millet, but can also harm cotton, hemp, sugarcane, sunflower, rice, sugar beets, sweet potatoes, and beans. Its larvae primarily bore into the stems, destroying stalk tissue, impairing nutrient transport, and damaging the plant. In severe cases, the stalks can break in wind. Spring corn cultivation areas in northern China are among the hardest hit areas.

[0005] The Oriental Armyworm (Mythimna separata) is a major pest of Chinese crops. Its larvae are polyphagous and lack diapause, primarily feeding on wheat and corn. Every April, first-generation larvae are common in wheat fields and spring corn seedlings in the Han River and middle and lower Yangtze River basins.

[0006] The tea geometre moth (Ectropis oblique) is a major pest of Chinese tea trees. It is found in Zhejiang, Anhui, Jiangsu, and Fujian provinces. Its larvae primarily feed on young and mature leaves. In severe infestations, they can consume entire leaves and buds, impacting tea yields that year and causing the trees to decline, leading to reduced yields in the following year.

[0007] Currently, the main strategies for controlling diamondback moth, corn borer, oriental armyworm, and tea geometrid are chemical and biological control. Biological control primarily relies on insect pathogens such as Bacillus thuringiensis (Bt). However, with the continued cultivation of transgenic Bt Cry crops, the lepidopteran insects Plutella xylostella, corn borer, oriental armyworm, and tea geometrid have gradually developed resistance.

[0008] Therefore, it is necessary to find new insecticidal proteins against diamondback moth, corn borer, oriental armyworm and tea looper. Summary of the Invention

[0009] One aspect of the present invention provides an insecticidal protein, which 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, wherein the amino acid sequence of Anc0 protein is shown in SEQ ID No. 8; and protein II is a second mutant protein in which at least one of Q318I, A337S, S360N, G384E and L459A of Cry2A protein is mutated.

[0010] In a specific embodiment, the protein I is a first mutant protein in which amino acids 267 to 472 of the Cry2A protein are the same as amino acids 267 to 472 of the Anc0 protein.

[0011] 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.

[0012] In a specific embodiment, the protein II is a second mutant protein in which at least one of Q318I, S360N and G384E of the Cry2A protein is mutated.

[0013] In a specific embodiment, the protein II is a third mutant protein of the protein having an amino acid sequence as shown in SEQ ID No. 4, wherein the third mutant protein has a mutation in at least one of Q318I, A337S, S360N, G384E and L459A.

[0014] In one embodiment, the protein II is a fourth mutant protein of the protein having an amino acid sequence as shown in SEQ ID No. 6, wherein the fourth mutant protein has a mutation in at least one of Q318I, A337S, S360N, G384E and L459A.

[0015] The second aspect of the present invention provides a composition containing the protein according to one aspect of the present invention.

[0016] The third aspect of the present invention provides a nucleic acid encoding the protein as described in one of the present inventions.

[0017] In one embodiment, for domain II, the sequence of the nucleic acid encoding said domain II is shown in SEQ ID No. 7 from position 799 to position 1416; For the Q318I mutation, the coding base mutation is ATT; For the A337S mutation, the coding base mutation is AGT; For the S360N mutation, the coding base mutation is AAT; For the G384E mutation, the coding base mutated to GAG; For the L459A mutation, the coding base mutation is GCA.

[0018] In one embodiment, the sequence of the nucleic acid encoding the protein whose amino acid sequence is shown in SEQ ID No. 8 is shown in SEQ ID No. 7.

[0019] In one embodiment, the sequence of the nucleic acid encoding the protein whose amino acid sequence is shown in SEQ ID No. 14 is shown in SEQ ID No. 13.

[0020] In one embodiment, the sequence of the nucleic acid encoding the protein whose amino acid sequence is shown in SEQ ID No. 16 is shown in SEQ ID No. 15.

[0021] The fourth aspect of the present invention provides a microorganism that carries the nucleic acid described in the third aspect of the present invention and can express the protein described in the first aspect of the present invention.

[0022] In one embodiment, the microorganism is Escherichia coli and / or Bacillus thuringiensis.

[0023] The fifth aspect of the present invention provides the use of one of the protein according to the first aspect of the present invention, the composition according to the second aspect of the present invention, the nucleic acid according to the third aspect of the present invention, and the microorganism according to the fourth aspect of the present invention in controlling lepidopteran insects.

[0024] In one embodiment, the lepidopteran insect is at least one of Plutella xylostella, Ostrinia furnacalis, Mythimna separata and Ectropisoblique.

[0025] In one embodiment, the protein I is used to control at least one of the diamondback moth (Plutella xylostella), the oriental armyworm (Mythimna separata) and the tea looper (Ectropis oblique).

[0026] In a specific embodiment, at least one of the protein with an amino acid sequence as shown in SEQ ID No. 8, the protein with an amino acid sequence as shown in SEQ ID No. 14, and the protein with an amino acid sequence as shown in SEQ ID No. 16 is used to control at least one of Plutella xylostella, Mythimna separata, and Ectropisoblique.

[0027] In a specific embodiment, the second mutant protein in which the Cry2A protein is mutated at Q318I is used to control Plutella xylostella and / or Ectropis oblique.

[0028] In one embodiment, the third mutant protein having an amino acid sequence as shown in SEQ ID No. 4 with a mutation at Q318I is used to control Plutella xylostella and / or Ectropisoblique.

[0029] In one embodiment, the third mutant protein having an amino acid sequence as shown in SEQ ID No. 6 with a mutation at position Q318I is used to control Plutella xylostella and / or Ectropisoblique.

[0030] In a specific embodiment, the second mutant protein in which the Cry2A protein is mutated at S360N and / or G384E is used to control the tea looper (Ectropis oblique).

[0031] In a specific embodiment, the third mutant protein having an amino acid sequence as shown in SEQ ID No. 4 with mutations at S360N and / or G384E is used to control tea looper (Ectropis oblique).

[0032] In a specific embodiment, the third mutant protein having an amino acid sequence as shown in SEQ ID No. 6 with mutations at S360N and / or G384E is used to control tea looper (Ectropis oblique).

[0033] In one 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 to control Ostrinia furnacalis.

[0034] In one embodiment, the third mutant protein having an amino acid sequence as shown in SEQ ID No. 4 with a mutation at position Q318I is used to control Ostrinia furnacalis and / or Mythimna separata.

[0035] In one embodiment, the third mutant protein having an amino acid sequence as shown in SEQ ID No. 4 with a mutation at A337S is used to control the oriental armyworm (Mythimna separata).

[0036] In one embodiment, the third mutant protein having an amino acid sequence as shown in SEQ ID No. 4 with a mutation at S360N is used to control the oriental armyworm (Mythimna separata).

[0037] In one embodiment, the third mutant protein having an amino acid sequence as shown in SEQ ID No. 4 with a mutation at G384E is used to control the oriental armyworm (Mythimna separata).

[0038] In one embodiment, the third mutant protein having an amino acid sequence as shown in SEQ ID No. 4 with a mutation at L459A is used to control at least one of the diamondback moth (Plutella xylostella), the corn borer (Ostriniafurnacalis) and the oriental armyworm (Mythimna separata).

[0039] In a specific embodiment, the third mutant protein having an amino acid sequence as shown in SEQ ID No. 6 with a mutation at A337S is used to control tea looper (Ectropis oblique).

[0040] In a specific embodiment, the third mutant protein having an amino acid sequence as shown in SEQ ID No. 6 with a mutation at L459A is used to control tea looper (Ectropis oblique). Beneficial effects of the present invention:

[0041] 1) Cry2A proteins containing the Anc0 domain II significantly improve the insecticidal activity of Cry2A-like proteins against lepidopteran 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) both exhibit excellent insecticidal activity against Plutella xylostella, Ostrinia nubilalis (subspecies of the human corn borer), Oriental armyworm, and Tea geometrid, and are significantly superior to known Cry2A-like proteins. Cry2Ah2-II (amino acid sequence shown in SEQ ID No. 16) also exhibits excellent insecticidal activity against Plutella xylostella, Oriental armyworm, and Tea geometrid.

[0042] 2) After the Cry2A protein is mutated with Q318I, for example, after Cry2Ae1 is mutated with Q318I, or after Cry2Ah2 is mutated with Q318I, the insecticidal activity against Plutella xylostella or tea geometrid can be significantly improved; after the Cry2A protein is mutated with S360N and / or G384E, for example, after Cry2Ae1 is mutated with S360N and / or G384E, or after Cry2Ah2 is mutated with S360N and / or G384E, the insecticidal activity against tea geometrid can be significantly improved.

[0043] 3) After Cry2Ae1 underwent mutations at Q318I, A337S, S360N, G384E, and L459A, it acquired insecticidal activity against the oriental armyworm.

[0044] 4) After the Q318I mutation occurs in Cry2Ae1, its insecticidal activity against the corn borer subspecies is significantly improved.

[0045] 5) The L459A mutation in Cry2Ae1 significantly enhances its insecticidal activity against Plutella xylostella and / or Ostrinia nubilalis.

[0046] 6) After Cry2Ah2 undergoes mutations at A337S and L459A, its insecticidal activity against tea looper is significantly improved. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 The results of SDS-PAGE detection of Cry2Ad1, Cry2Ae1, Cry2Ah2, and Anc0 are shown.

[0048] Figure 2 The results of SDS-PAGE detection of Cry2Ad1, Cry2Ad1-II, Cry2Ah2, and Cry2Ah2-II are shown.

[0049] Figure 3The SDS-PAGE detection results of Cry2Ae1, Cry2Ae1-Q318I, Cry2Ae1-A337S, Cry2Ae1-S360N, Cry2Ae1-G384E, and Cry2Ae1-L459A are shown.

[0050] Figure 4 The SDS-PAGE detection results of Cry2Ah2, Cry2Ah2-Q318I, Cry2Ah2-A337S, Cry2Ah2-S360N, Cry2Ah2-G384E and Cry2Ah2-L459A are shown. DETAILED DESCRIPTION

[0051] The above contents of the present invention are further described in detail below in the form of preferred implementation cases, but they do not constitute a limitation of the present invention.

[0052] Unless otherwise specified, the reagents in the embodiments of the present invention can be purchased through commercial channels. Example 1: Expression of Cry2Ad1, Cry2Ae1, and Cry2Ah2 proteins

[0053] 1. Construction of recombinant Bt strains.

[0054] Nucleic acids consisting 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. The nucleic acid with the base sequence shown in SEQ ID No. 1 encodes the 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 the 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 the protein with the amino acid sequence shown in SEQ ID No. 6 (i.e., Cry2Ah2 protein); and the nucleic acid with the base sequence shown in SEQ ID No. 7 encodes the protein with the amino acid sequence shown in SEQ ID No. 8 (i.e., Anc0 protein). The synthesized insecticidal protein genes were ligated into the PUC18-p1Ac-GFP vector to obtain the p1Ac-GFP-Cry2Ad1, p1Ac-GFP-Cry2Ae1, p1Ac-GFP-Cry2Ah2 and p1Ac-GFP-Anc0 recombinant plasmids, which were 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, respectively.

[0055] The transformant TOP10 / PUC18-p1Ac-GFP obtained by empty-transforming Escherichia coli TOP10 was used as a negative control for protein expression analysis.

[0056] 2. Protein expression and extraction.

[0057] (1) Picking a single colony: Pick a single colony of TOP10 / p1Ac-GFP-Cry2Ad1 and place it in 5 mL of LB liquid culture medium containing ampicillin, shake and culture it at 37 degrees Celsius and 220 rpm for 12 hours to obtain an activated bacterial solution; (2) Inoculate 1% of the activated bacterial solution into a 1 L Erlenmeyer flask containing 800 mL of LB liquid culture medium (containing ampicillin), shake and culture it at 37 degrees Celsius and 220 rpm for 72 hours to obtain a fermentation liquid; (3) Centrifuge the fermentation liquid at 4 degrees Celsius and 8,000 × g for 10 minutes, discard the supernatant, and resuspend the precipitate with pre-cooled 20 mmol / L Tris-HCl (PH=8.0) buffer to obtain the first bacterial suspension; (4) Transfer the first suspension to a 50 mL centrifuge tube, centrifuge it at 4 degrees Celsius and 8,000 × g for 10 minutes, discard the supernatant, and resuspend the precipitate with pre-cooled 20 mmol / L (5) The cell walls of the second bacterial suspension were broken by ultrasonic wave (power 70%, 5 min, supersonic wave 3 s, stop 5 s) to obtain a broken bacterial solution; (6) The broken bacterial solution was centrifuged at 8,000 × g for 10 minutes at 4 degrees Celsius to collect the precipitate; (7) The precipitate was washed 2 to 3 times with pre-cooled sterile water; (8) 5 ml of 20 mmol / L The precipitate was resuspended in Tris-HCl (pH=8.0) buffer to obtain a Cry2Ad1 test protein suspension; (9) The proteins of TOP10 / p1Ac-GFP-Cry2Ae1, TOP10 / p1Ac-GFP-Cry2Ah2, TOP10 / p1Ac-GFP-Anc0 and TOP10 / PUC18-p1Ac-GFP were extracted respectively by the same operation as above (1) to (8) to obtain a Cry2Ae1 test protein suspension, a Cry2Ah2 test protein suspension, an Anc0 test protein suspension and a negative control protein suspension respectively; (10) The test protein suspensions and the negative control protein suspension were subjected to SDS-PAGE detection. The results are shown in FIG. Figure 1 .

[0058] according to Figure 1 The results showed that Cry2Ad1, Cry2Ae1, Cry2Ah2 and Anc0 were successfully expressed in E. coli, and the expression products were in the precipitate after cell disruption. Therefore, the protein suspension to be tested was the protein suspension containing Cry2Ad1, Cry2Ae1, Cry2Ah2 and Anc0.

[0059] Before the activity assay, the protein suspension containing Cry2Ad1, Cry2Ae1, Cry2Ah2 and Anc0 was quantified with BSA. Example 2: Domain replacement, site-directed mutagenesis, and expression of mutant proteins

[0060] The primer pair F-domain II-1 (shown in SEQ ID No. 9) / R-domain II-1 (shown in SEQ ID No. 10) and the primer pair F-domain II-2 (shown in SEQ ID No. 11) / R-domain II-2 (shown in SEQ ID No. 12) were designed.

[0061] Domain replacement was performed on the protein with the amino acid sequence shown in SEQ ID No. 2. The procedure was as follows: using the recombinant plasmid p1Ac-GFP-Anc0 as a template and primers F-domain II-1 and R-domain II-1, a DNA fragment encoding the amino acid sequence of the protein shown in SEQ ID No. 8, constituting domain II of the protein, was amplified using ultra-high-fidelity polymerase (Phusion) 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, a DNA fragment excluding the fragment encoding amino acids 267 to 472 shown in SEQ ID No. 2, was amplified using ultra-high-fidelity polymerase (Phusion) to obtain PCR product 2. PCR products 1 and 2 were recovered from agarose gels to obtain recovered products 1 and 2, respectively. These products were then homologously recombined using a homologous recombinase (Exnase II) to generate the p1Ac-GFP-Cry2Ad1-II plasmid. The homologous recombinant product, p1Ac-GFP-Cry2Ad1-II, was then transformed into competent E. coli TOP10 cells. A single bacterial strain was isolated and the bacterial suspension was sequenced to obtain the positive recombinant strain TOP10 / p1Ac-GFP-Cry2Ad1-II. The base sequence of the target gene, cry2Ad1-II, in the p1Ac-GFP-Cry2Ad1-II plasmid 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.

[0062] Domain replacement was performed on the protein with the amino acid sequence shown in SEQ ID No. 6. The procedure was as follows: using the recombinant plasmid p1Ac-GFP-Anc0 as a template, F-domain II-1 and R-domain II-1 as primers, and ultra-high-fidelity polymerase (Phusion) was used to amplify a DNA fragment (which encodes the amino acid sequence of amino acids 267 to 472 of the protein shown in SEQ ID No. 8), resulting in PCR product 3. Using the recombinant plasmid p1Ac-GFP-Cry2Ah2 as a template, F-domain II-2 and R-Fdomain II-2 as primers, and ultra-high-fidelity polymerase (Phusion) was used to amplify a DNA fragment (which is the entire p1Ac-GFP-Cry2Ad1 DNA fragment excluding the fragment encoding the other amino acids from positions 267 to 471 shown in SEQ ID No. 6), resulting in PCR product 4. PCR products 3 and 4 were recovered from agarose gels to obtain recovered products 3 and 4, respectively. These recovered products 3 and 4 were then homologously recombined using a homologous recombinase (Exnase II) to generate the p1Ac-GFP-Cry2Ah2-II plasmid. The homologous recombinant product p1Ac-GFP-Cry2Ah2-II was then transformed into competent E. coli TOP10 cells. A single bacterial strain was isolated and the bacterial suspension was sequenced to obtain the positive recombinant strain TOP10 / p1Ac-GFP-Cry2Ah2-II. The base sequence of the target gene cry2Ah2-II in the p1Ac-GFP-Cry2Ah2-II plasmid 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.

[0063] Mutation primers F-Q318I (as shown in SEQ ID No. 17) / R-Q318I (as shown in SEQ ID No. 18) were designed to mutate CAG at positions 952 to 954 in the sequence shown in SEQ ID No. 3 to ATT, thereby subjecting the protein encoded by SEQ ID No. 3 to a Q318I mutation as shown in SEQ ID No. 4. The procedure was as follows: PCR amplification was performed using the p1Ac-GFP-Cry2Ae1 recombinant plasmid as a template, F-Q318I and R-Q318I as primers, and an ultra-high-fidelity polymerase (Phusion) to obtain PCR product 5. The methylated plasmid template was removed from the resulting PCR product 5 using DPN I enzyme to obtain an enzyme-digested product. The enzyme digestion products were homologously recombined using a homologous recombinase (Exnase II) to obtain the p1Ac-GFP-Cry2Ae1-Q318I plasmid. The homologous recombinant product p1Ac-GFP-Cry2Ae1-Q318I was then transformed into Escherichia coli competent cells TOP10. After a single bacterial strain was picked, the bacterial liquid was sequenced 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, the CAG at positions 952 to 954 in the sequence shown in SEQ ID No. 5 was mutated to ATT, thereby changing the amino acid sequence encoded by SEQ ID No. 5 to SEQ ID No. The protein shown in 6 was mutated to Q318I to obtain the positive plasmid p1Ac-GFP-Cry2Ah2-Q318I and the positive recombinant strain TOP10 / p1Ac-GFP-Cry2Ah2-Q318I. Other operations were the same as TOP10 / p1Ac-GFP-Cry2Ae1-Q318I.

[0064] Mutation primers F-A337S (as shown in SEQ ID No. 19) / R-A337S (as shown in SEQ ID No. 20) were designed. Using the p1Ac-GFP-Cry2Ae1 recombinant plasmid as a template, the GCG at positions 1009 to 1011 in the sequence shown in SEQ ID No. 3 were mutated to AGT, thereby causing the A337S mutation in the protein encoded by the amino acid sequence shown in SEQ ID No. 4. This resulted in the positive plasmid p1Ac-GFP-Cry2Ae1-A337S and the positive recombinant strain TOP10 / p1Ac-GFP-Cry2Ae1-A337S. Using primers F-A337S / R-A337S and p1Ac-GFP-Cry2Ah2 as a template, the GCT at positions 1009 to 1011 in the sequence shown in SEQ ID No. 5 were mutated to AGT, thereby causing the A337S mutation in the protein encoded by the amino acid sequence shown in SEQ ID No. The protein shown in 6 was mutated to A337S to obtain the positive plasmid p1Ac-GFP-Cry2Ah2-A337S and the positive recombinant strain TOP10 / p1Ac-GFP-Cry2Ah2-A337S. Other operations were the same as TOP10 / p1Ac-GFP-Cry2Ae1-Q318I.

[0065] Mutation primers F-S360N (as shown in SEQ ID No. 21) / R-S360N (as shown in SEQ ID No. 22) were designed. Using the p1Ac-GFP-Cry2Ae1 recombinant plasmid as a template, AGT at positions 1078 to 1080 in the sequence shown in SEQ ID No. 3 was mutated to AAT, thereby subjecting the encoded protein with the amino acid sequence shown in SEQ ID No. 4 to an S360N mutation, thereby obtaining the positive plasmid TOP10 / p1Ac-GFP-Cry2Ae1-S360N and the positive recombinant strain TOP10 / TOP10 / p1Ac-GFP-Cry2Ae1-S360N. Using primers F-S360N / R-S360N and p1Ac-GFP-Cry2Ah2 as a template, AGT at positions 1078 to 1080 in the sequence shown in SEQ ID No. 5 was mutated to AAT, thereby subjecting the encoded protein with the amino acid sequence shown in SEQ ID No. The protein shown in No. 6 was mutated to S360N, resulting in the positive plasmid p1Ac-GFP-Cry2Ah2-S360N and the positive recombinant strain TOP10 / p1Ac-GFP-Cry2Ah2-S360N. Other operations were performed the same as for TOP10 / p1Ac-GFP-Cry2Ae1-Q318I.

[0066] Mutation primers F-G384E (as shown in SEQ ID No. 23) / R-G384E (as shown in SEQ ID No. 24) were designed. Using the p1Ac-GFP-Cry2Ae1 recombinant plasmid as a template, the GGG at positions 1150 to 1152 in the sequence shown in SEQ ID No. 3 were mutated to GAG, thereby causing the G384E mutation in the protein encoded by the amino acid sequence shown in SEQ ID No. 4. This resulted in the positive plasmid p1Ac-GFP-Cry2Ae1-G384E and the positive recombinant strain TOP10 / p1Ac-GFP-Cry2Ae1-G384E. Using primers F-G384E / R-G384E and p1Ac-GFP-Cry2Ah2 as a template, the GGG at positions 1150 to 1152 in the sequence shown in SEQ ID No. 5 were mutated to GAG, thereby causing the G384E mutation in the protein encoded by the amino acid sequence shown in SEQ ID No. The protein shown in 6 was mutated to G384E to obtain the positive plasmid p1Ac-GFP-Cry2Ah2-G384E and the positive recombinant strain TOP10 / p1Ac-GFP-Cry2Ah2-G384E. Other operations were the same as TOP10 / p1Ac-GFP-Cry2Ae1-Q318I.

[0067] Mutation primers F-L459A (as shown in SEQ ID No. 25) / R-L459A (as shown in SEQ ID No. 26) were designed. Using the p1Ac-GFP-Cry2Ae1 recombinant plasmid as a template, the TTA at positions 1375 to 1377 in the sequence shown in SEQ ID No. 3 were mutated to GCA, thereby causing the L459A mutation in the protein encoded by the amino acid sequence shown in SEQ ID No. 4, thereby obtaining the positive plasmid p1Ac-GFP-Cry2Ae1-L459A and the positive recombinant strain TOP10 / p1Ac-GFP-Cry2Ae1-L459A. Using primers F-L459A / R-L459A and p1Ac-GFP-Cry2Ah2 as a template, the TTA at positions 1375 to 1377 in the sequence shown in SEQ ID No. 5 were mutated to GCA, thereby causing the L459A mutation in the protein encoded by the amino acid sequence shown in SEQ ID No. The protein shown in 6 was mutated to L459A to obtain the positive plasmid p1Ac-GFP-Cry2Ah2-L459A and the positive recombinant strain TOP10 / p1Ac-GFP-Cry2Ah2-L459A. Other operations were the same as TOP10 / p1Ac-GFP-Cry2Ae1-Q318I.

[0068] The mutant proteins were expressed and extracted in the same manner as in Example 1. The results showed that the mutant proteins were successfully expressed in E. coli, and the expression products were in the precipitate after cell disruption. Figures 2 to 4 Therefore, each protein suspension to be tested is a protein suspension containing a mutant protein.

[0069] Likewise, the concentration of each mutant protein was quantified by BSA before activity assay. Example 3: Insecticidal activity assay

[0070] 20 mmol / L Tris-HCl (pH 8.0) was used as a 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 tested.

[0071] The steps for determining the insecticidal activity of diamondback moth are as follows: clean cabbage leaves with a diameter of 6 cm are soaked in the protein sample to be tested and the negative control. After 10 minutes, they are taken out and air-dried until the water is completely evaporated. The leaves are placed in a sterile petri dish. Thirty healthy, active 2nd-instar diamondback moth larvae are inoculated into the sterile petri dish containing the above cabbage leaves using a brush. Each treatment is repeated 3 times. The leaves are then placed in an artificial climate incubator and cultured at 26 degrees Celsius, a photoperiod of 14L:10D, and a relative humidity of 60%. The leaves are observed every day to check the light, humidity, and temperature. The number of dead insects is counted after 72 hours.

[0072] The difference between the assay steps for insecticidal activity against tea geometrid and diamondback moth is that cabbage leaves are used instead of fresh tea leaves.

[0073] The following steps were used to determine the insecticidal activity against Asian corn borer: 7 g of Asian corn borer feed (see Table 1 for feed formula) was weighed and placed in a sterile culture dish. 0.7 mL of the protein sample to be tested was added, and 0.7 mL of 20 mmol / L Tris-HCl (pH 8.0) was added as a negative control. The mixture was thoroughly mixed and allowed to evaporate completely. Thirty healthy, active, newly hatched corn borer larvae were then placed in the culture dish containing the feed using a brush. Each treatment was repeated three times. The larvae were then placed in an artificial climate incubator at 26°C, a photoperiod of 14 L:10 D, and a relative humidity of 60%. The larvae were observed daily to check the light intensity, humidity, and temperature. The number of dead larvae was counted after 7 days.

[0074] The difference between the insecticidal activity assay for Oriental armyworm and that for Asian corn borer is that the feed used is a feed specifically for Oriental armyworm (see Table 1 for the feed formula).

[0075] The calculated mortality rate and adjusted mortality rate were calculated, and the bioassay results are shown in Tables 2 to 4.

[0076] The formula for calculating the adjusted mortality rate is as follows:

[0077] Table 1

[0078] Table 2

[0079] The independent sample T test (p < 0.05) was used to analyze the significant differences between proteins.

[0080] The results in Table 2 show that the Anc0 protein exhibited very high insecticidal activity against Plutella xylostella, Asian corn borer, Oriental armyworm, and tea geometrid. The Cry2Ad1 mutant protein, Cry2Ad1-II, also exhibited insecticidal activity against Plutella xylostella, Asian corn borer, Oriental armyworm, and tea geometrid. The Cry2Ah2 mutant protein, Cry2Ah2-II, exhibited enhanced insecticidal activity against Plutella xylostella and tea geometrid, and also exhibited insecticidal activity against Oriental armyworm. This indicates that replacing domain II of Cry2A proteins with domain II of the Anc0 protein, or more specifically, replacing amino acids 267 to 472 of Cry2A proteins with amino acids 267 to 472 of Anc0 proteins, significantly improves the insecticidal activity of Cry2A proteins against lepidopteran pests.

[0081] Table 3

[0082] “ / ” indicates unknown activity, and the significance analysis was performed using the independent sample T test (p < 0.05) to analyze the significant differences between proteins.

[0083] According to the results in Table 3, compared with the Cry2Ae1 protein, the five point mutant proteins of Cry2Ae1, Cry2Ae1-Q318I, Cry2Ae1-A337S, Cry2Ae1-S360N, Cry2Ae1-G384E and Cry2Ae1-L459A, all obtained insecticidal activity against oriental armyworm. Cry2Ae1-Q318I and Cry2Ae1-L459A enhanced the insecticidal activity against Plutella xylostella and Asian corn borer, and Cry2Ae1-Q318I, Cry2Ae1-S360N and Cry2Ae1-G384E enhanced the insecticidal activity against tea looper.

[0084] Table 4

[0085] “ / ” indicates unknown activity, and the significance analysis was performed using the independent sample T test (p < 0.05) to analyze the significant differences between proteins.

[0086] According to the results in Table 4, compared with the Cry2Ah2 protein, the five point mutant proteins of Cry2Ah2, Cry2Ah2-Q318I, Cry2Ah2-A337S, Cry2Ah2-S360N, Cry2Ah2-G384E and Cry2Ah2-L459A, all enhanced the insecticidal activity against tea looper, and Cry2Ah2-Q318I enhanced the insecticidal activity against diamondback moth.

[0087] According to the activities of Cry2Ae1-Q318I in Table 3 and Cry2Ah2-Q318I in Table 4, the Q318I mutation of the Cry2A protein can enhance the insecticidal activity against Plutella xylostella or tea geometrid. According to the activities of Cry2Ae1-S360N and Cry2Ae1-G384E in Table 3 and the activities of Cry2Ah2-S360N and Cry2Ah2-G384E in Table 4, the S360N or G384E mutation of the Cry2A protein can enhance the insecticidal activity against tea geometrid.

Claims

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

14.

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

3. A nucleic acid encoding the insecticidal protein according to claim 1.

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

13.

5. A microorganism carrying the nucleic acid according to claim 3 or 4 and capable of expressing the 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 lepidopteran insect is at least one of the group consisting of the diamondback moth (Plutella xylostella), the corn borer (Ostriniafurnacalis), the oriental armyworm (Mythimna separata) and the tea looper (Ectropis oblique).

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