A pesticidal protein and its use in the control of lepidopteran insects

Abstract

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

Description

[0001] This patent application is a divisional application of the invention entitled "An Insecticidal Protein and Its Application in the Control of Lepidoptera Insects", filed on October 27, 2023, with application number 2023114092474. Technical Field

[0002] This invention relates to the field of biological control, and in particular to the application of a protein in the control of lepidopteran insects. Background Technology

[0003] The diamondback moth (Plutella xylostella) is distributed throughout China and is one of the major pests of cruciferous vegetables. It feeds specifically on cruciferous plants such as cabbage, broccoli, and rapeseed, causing the most serious damage and threat to the production and development of cruciferous vegetables, and is also one of the most difficult major pests to control.

[0004] The Asian corn borer (Ostrinia furnacalis) is a polyphagous pest that primarily damages corn, sorghum, and millet, but can also infest cotton, sugarcane, sunflower, rice, sugar beets, sweet potatoes, and legumes. The larvae mainly bore into the stems, damaging the stalk tissue, hindering nutrient transport, and causing plant damage; in severe cases, the stems break in the wind. Northern China, particularly the spring corn-growing regions, is one of the areas most severely affected by this pest.

[0005] The Eastern Armyworm (Mythimna separata) is a major pest of native crops in China. Its larvae are omnivorous, lack diapause, and primarily feed on wheat and corn. Every April, the first generation of Eastern Armyworm larvae commonly occurs in wheat fields in the Han River and the middle and lower reaches of the Yangtze River, and also frequently occurs during the seedling stage of spring corn.

[0006] The tea geometrid moth (Ectropis oblique) is one of the major pests of tea trees in China. It is distributed in Zhejiang, Anhui, Jiangsu, Fujian, and other provinces in China. The larvae mainly feed on tender and mature leaves. In severe infestations, they can completely deplete the leaves and buds, affecting the tea yield of the current year and causing the tree to weaken, resulting in reduced tea production the following year.

[0007] Currently, the control of diamondback moth, corn borer, armyworm, and tea geometrid moth mainly employs two strategies: chemical control and biological control. Biological control primarily relies on entomopathogenic bacteria, such as Bacillus thuringiensis (Bt). However, with the continued cultivation of Bt Cry-transgenic crops, lepidopteran insects such as the diamondback moth, corn borer, armyworm, and tea geometrid moth have gradually developed resistance.

[0008] Therefore, it is necessary to find novel insecticidal proteins that are resistant to diamondback moth, corn borer, armyworm, and tea geometrid moth. 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 the Cry2A protein is domain II of the Anc0 protein, and the amino acid sequence of the Anc0 protein is shown in SEQ ID No. 8; and protein II is a second mutant protein in which at least one of the Cry2A protein is mutated in Q318I, A337S, S360N, G384E and L459A.

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

[0011] In one specific embodiment, the amino acid sequence of protein I is at least one of SEQ ID No. 8, SEQ ID No. 14 and SEQ ID No. 16.

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

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

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

[0015] The second invention provides a composition containing the protein as described in the first invention.

[0016] The third invention provides a nucleic acid encoding a protein as described in the first invention.

[0017] In one specific embodiment, for domain II, the sequence of the nucleic acid encoding domain II is shown as positions 799 to 1416 of SEQ ID No. 7;

[0018] For the Q318I mutation, the coding base is changed to ATT;

[0019] For the A337S mutation, the coding base is changed to AGT;

[0020] For the S360N mutation, the coding base is changed to AAT;

[0021] For the G384E mutation, the coding base is changed to GAG;

[0022] For the L459A mutation, the coding base is changed to GCA.

[0023] In one specific embodiment, the nucleic acid encoding the protein with the amino acid sequence shown in SEQ ID No. 8 has the sequence shown in SEQ ID No. 7.

[0024] In one specific embodiment, the nucleic acid encoding the protein with the amino acid sequence shown in SEQ ID No. 14 has the sequence shown in SEQ ID No. 13.

[0025] In one specific embodiment, the nucleic acid encoding the protein with the amino acid sequence shown in SEQ ID No. 16 has the sequence shown in SEQ ID No. 15.

[0026] The fourth invention provides a microorganism that carries nucleic acids as described in the third invention and can express proteins as described in the first invention.

[0027] In one specific embodiment, the microorganism is Escherichia coli and / or Bacillus thuringiensis.

[0028] The fifth invention provides the application of one of the following in the prevention and control of lepidopteran insects: the protein described in the first invention, the composition described in the second invention, the nucleic acid described in the third invention, and the microorganism described in the fourth invention.

[0029] In one specific embodiment, the lepidopteran insect is at least one of the following: diamondback moth (Plutella xylostella), subspecies of corn borer (Ostrinia furnacalis), oriental armyworm (Mythimna separata), and tea geometrid moth (Ectropisoblique).

[0030] In one specific embodiment, protein I is used to control at least one of the diamondback moth (Plutella xylostella), the oriental armyworm (Mythimna separata), and the tea geometrid moth (Ectropis oblique).

[0031] In one specific embodiment, at least one of the proteins with the amino acid sequence shown in SEQ ID No. 8, the amino acid sequence shown in SEQ ID No. 14, and the amino acid sequence shown in SEQ ID No. 16 is used to control at least one of the diamondback moth (Plutella xylostella), the oriental armyworm (Mythimna separata), and the tea geometrid moth (Ectropisoblique).

[0032] In one specific embodiment, the second mutant protein, in which the Cry2A protein is mutated at Q318I, is used to control the diamondback moth (Plutella xylostella) and / or the tea geometrid moth (Ectropis oblique).

[0033] In one specific embodiment, the third mutant protein, with the amino acid sequence shown in SEQ ID No. 4 mutated at Q318I, is used to control the diamondback moth (Plutella xylostella) and / or the tea geometrid moth (Ectropisoblique).

[0034] In one specific embodiment, the third mutant protein, with the amino acid sequence shown in SEQ ID No. 6 mutated at Q318I, is used to control the diamondback moth (Plutella xylostella) and / or the tea geometrid moth (Ectropisoblique).

[0035] In one specific embodiment, the second mutant protein, in which the Cry2A protein is mutated at S360N and / or G384E, is used to control the tea geometrid moth (Ectropis oblique).

[0036] In one specific embodiment, the third mutant protein, with the amino acid sequence shown in SEQ ID No. 4 mutated at S360N and / or G384E, is used to control the tea geometrid moth (Ectropis oblique).

[0037] In one specific embodiment, the third mutant protein, with the amino acid sequence shown in SEQ ID No. 6 mutated at S360N and / or G384E, is used to control the tea geometrid moth (Ectropis oblique).

[0038] In one 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 to control the subspecies of corn borer (Ostrinia furnacalis).

[0039] In one specific embodiment, the third mutant protein, with the amino acid sequence shown in SEQ ID No. 4 mutated at Q318I, is used to control the subspecies of corn borer (Ostrinia furnacalis) and / or oriental armyworm (Mythimna separata).

[0040] In one specific embodiment, the third mutant protein, with a mutation at A337S, as shown in SEQ ID No. 4, is used to control the Eastern armyworm (Mythimna separata).

[0041] In one specific embodiment, the third mutant protein, with a mutation at S360N, as shown in SEQ ID No. 4, is used to control the Eastern armyworm (Mythimna separata).

[0042] In one specific embodiment, the third mutant protein, with a mutation at G384E, as shown in SEQ ID No. 4, is used to control the Eastern armyworm (Mythimna separata).

[0043] In one specific embodiment, the third mutant protein, with a mutation at L459A, as shown in SEQ ID No. 4, is used to control at least one of the diamondback moth (Plutella xylostella), the corn borer subspecies (Ostriniafurnacalis), and the oriental armyworm (Mythimna separata).

[0044] In one specific embodiment, the third mutant protein, with a mutation at A337S, as shown in SEQ ID No. 6, is used to control the tea geometrid moth (Ectropis oblique).

[0045] In one specific embodiment, the third mutant protein, with a mutation at L459A, as shown in SEQ ID No. 6, is used to control the tea geometrid moth (Ectropis oblique).

[0046] The beneficial effects of this invention are:

[0047] 1) Cry2A proteins containing the Anc0 domain II significantly improve the insecticidal activity of Cry2A-type proteins against lepidopteran pests. For example, Anc0 (amino acid sequence as shown in SEQ ID No. 8) and Cry2Ad1-II (amino acid sequence as shown in SEQ ID No. 14) have very good insecticidal activity against diamondback moth, corn borer, armyworm, and tea geometrid moth, and are significantly better than known Cry2A-type proteins. Cry2Ah2-II (amino acid sequence as shown in SEQ ID No. 16) protein has very good insecticidal activity against diamondback moth, armyworm, and tea geometrid moth.

[0048] 2) Mutating Cry2A proteins with the Q318I mutation, for example, Cry2Ae1 with the Q318I mutation or Cry2Ah2 with the Q318I mutation, can significantly improve the insecticidal activity against diamondback moth or tea geometrid moth; mutating Cry2A proteins with the S360N and / or G384E mutation, for example, Cry2Ae1 with the S360N and / or G384E mutation or Cry2Ah2 with the S360N and / or G384E mutation, can significantly improve the insecticidal activity against tea geometrid moth.

[0049] 3) Cry2Ae1 acquired insecticidal activity against Eastern armyworm after mutations in Q318I, A337S, S360N, G384E and L459A.

[0050] 4) After the Q318I mutation, Cry2Ae1 can significantly improve its insecticidal activity against the subspecies of corn borer.

[0051] 5) After mutation in L459A, Cry2Ae1 can significantly improve the insecticidal activity against diamondback moth and / or subspecies corn borer.

[0052] 6) Cry2Ah2 can significantly improve its insecticidal activity against tea geometrid moth after mutations occur in A337S and L459A. Attached Figure Description

[0053] Figure 1 The SDS-PAGE detection results for Cry2Ad1, Cry2Ae1, Cry2Ah2, and Anc0 are shown.

[0054] Figure 2 The SDS-PAGE detection results for Cry2Ad1, Cry2Ad1-II, Cry2Ah2, and Cry2Ah2-II are shown.

[0055] Figure 3The SDS-PAGE results for Cry2Ae1, Cry2Ae1-Q318I, Cry2Ae1-A337S, Cry2Ae1-S360N, Cry2Ae1-G384E, and Cry2Ae1-L459A are shown.

[0056] Figure 4 The SDS-PAGE results for Cry2Ah2, Cry2Ah2-Q318I, Cry2Ah2-A337S, Cry2Ah2-S360N, Cry2Ah2-G384E, and Cry2Ah2-L459A are shown. Detailed Implementation

[0057] The present invention will be further described in detail below through preferred embodiments, but these embodiments do not constitute a limitation thereof.

[0058] Unless otherwise specified, all reagents used in the embodiments of this invention are commercially available.

[0059] Example 1: Expression of Cry2Ad1, Cry2Ae1 and Cry2Ah2 proteins

[0060] 1. Construction of recombinant Bt strains.

[0061] The 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. Specifically, 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); and 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 gene was ligated into the PUC18-p1Ac-GFP vector to obtain recombinant plasmids p1Ac-GFP-Cry2Ad1, p1Ac-GFP-Cry2Ae1, p1Ac-GFP-Cry2Ah2, and p1Ac-GFP-Anc0, which were then transformed into Escherichia coli TOP10. The transformed strains were named TOP10 / p1Ac-GFP-Cry2Ad1, TOP10 / p1Ac-GFP-Cry2Ae1, TOP10 / p1Ac-GFP-Cry2Ah2, and TOP10 / p1Ac-GFP-Anc0, respectively.

[0062] The transformant TOP10 / PUC18-p1Ac-GFP obtained by transforming Escherichia coli TOP10 with the empty vector PUC18-p1Ac-GFP was used as a negative control for protein expression analysis.

[0063] 2. Protein expression and extraction.

[0064] (1) Picking a single colony: Pick a single colony of TOP10 / p1Ac-GFP-Cry2Ad1 into 5 mL of LB liquid medium containing ampicillin, and culture at 37°C and 220 rpm for 12 hours to obtain an activated bacterial solution; (2) Inoculate the activated bacterial solution at 1% volume into a 1 L Erlenmeyer flask containing 800 mL of LB liquid medium (containing ampicillin), and culture at 37°C and 220 rpm for 72 h to obtain a fermentation broth; (3) Centrifuge the fermentation broth at 4°C and 8,000 × g for 10 minutes, discard the supernatant, and resuspend the precipitate in 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 at 4°C and 8,000 × g for 10 minutes, discard the supernatant, and resuspend the precipitate in pre-cooled 20 mmol / L Tris-HCl (pH=8.0) buffer to obtain the first bacterial suspension; (5) The second bacterial suspension was obtained by suspending the second bacterial suspension in Tris-HCl (pH=8.0); (6) The cell walls of the second bacterial suspension were broken by sonication (70% power, 5 min, 3 s for over 3 s, 5 s for rest) to obtain the broken bacterial solution; (7) The broken bacterial solution was centrifuged at 8,000 × g at 4 degrees Celsius for 10 minutes and the precipitate was collected; (8) The precipitate was washed 2 to 3 times with pre-cooled sterile water; (9) 5 ml of 20 mmol / L solution was added. (9) The precipitate was resuspended in Tris-HCl (pH=8.0) buffer to obtain the Cry2Ad1 test protein suspension; (10) The proteins of TOP10 / p1Ac-GFP-Cry2Ae1, TOP10 / p1Ac-GFP-Cry2Ah2, TOP10 / p1Ac-GFP-Anc0 and TOP10 / PUC18-p1Ac-GFP were extracted using the same operation as (1) to (8) above to obtain the Cry2Ae1 test protein suspension, Cry2Ah2 test protein suspension, Anc0 test protein suspension and negative control protein suspension, respectively; (11) The test protein suspensions and negative control protein suspensions were subjected to SDS-PAGE detection, and the results are shown in the figure. Figure 1 .

[0065] according to Figure 1 The results showed that Cry2Ad1, Cry2Ae1, Cry2Ah2 and Anc0 were successfully expressed in Escherichia coli, and the expression products were found in the precipitate after cell lysis. Therefore, the protein suspension to be tested was a protein suspension containing Cry2Ad1, Cry2Ae1, Cry2Ah2 and Anc0.

[0066] Prior to activity assay, protein suspensions containing Cry2Ad1, Cry2Ae1, Cry2Ah2 and Anc0 were quantified using BSA.

[0067] Example 2: Domain substitution, site-directed mutagenesis, and expression of mutant proteins

[0068] Design primer pairs F-domain II-1 (as shown in SEQ ID No. 9) / R-domain II-1 (as shown in SEQ ID No. 10) and primer pairs F-domain II-2 (as shown in SEQ ID No. 11) / R-domain II-2 (as shown in SEQ ID No. 12).

[0069] Domain substitution was performed on the protein with the amino acid sequence shown in SEQ ID No. 2. The procedure was as follows: Using recombinant plasmid p1Ac-GFP-Anc0 as a template and primers F-domain II-1 and R-domain II-1, a DNA fragment (encoding amino acids 267 to 472 of the protein shown in SEQ ID No. 8, constituting domain II of the protein) was amplified using high-fidelity polymerase (Phusion) to obtain PCR product 1; using recombinant plasmid p1Ac-GFP-Cry2Ad1 as a template and primers F-domain II-2 and R-domain II-2, a DNA fragment (which is the entire p1Ac-GFP-Cry2Ad1 DNA fragment excluding amino acids 267 to 472 as shown in SEQ ID No. 2) was amplified using high-fidelity polymerase (Phusion) to obtain PCR product 2. PCR product 1 and PCR product 2 were recovered by agarose gel electrophoresis to obtain recovered product 1 and recovered product 2, respectively. Recovered product 1 and recovered product 2 were then subjected to homologous recombination using Exnase II to obtain the p1Ac-GFP-Cry2Ad1-II plasmid. The homologous recombination product p1Ac-GFP-Cry2Ad1-II was then transformed into *E. coli* competent cells TOP10. Single-spot culture was picked and 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.

[0070] Domain substitution was performed on the protein with the amino acid sequence shown in SEQ ID No. 6. The procedure was as follows: Using recombinant plasmid p1Ac-GFP-Anc0 as a template and F-domain II-1 and R-domain II-1 as primers, a DNA fragment (encoding amino acids from position 267 to position 472 of the protein shown in SEQ ID No. 8) was amplified using high-fidelity polymerase (Phusion) to obtain PCR product 3; using 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 entire DNA fragment of p1Ac-GFP-Cry2Ad1 excluding the amino acids encoding positions 267 to 471 as shown in SEQ ID No. 6) was amplified using high-fidelity polymerase (Phusion) to obtain PCR product 4. PCR products 3 and 4 were recovered by agarose gel electrophoresis, yielding recovered product 3 and recovered product 4, respectively. Homologous recombination of recovered product 3 and recovered product 4 was performed using Exnase II to obtain the p1Ac-GFP-Cry2Ah2-II plasmid. The p1Ac-GFP-Cry2Ah2-II plasmid was then transformed into *E. coli* competent cells TOP10. Single-spot culture was picked and sequenced, yielding 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.

[0071] 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 of the sequence shown in SEQ ID No. 3 to ATT, thereby mutating the protein encoded by the amino acid sequence shown in SEQ ID No. 4 as Q318I. The operation is as follows: using the p1Ac-GFP-Cry2Ae1 recombinant plasmid as a template, and using F-Q318I and R-Q318I as primers, PCR amplification was performed using high-fidelity polymerase (Phusion) to obtain PCR product 5. The methylated plasmid template of the obtained PCR product 5 was excised with DPN I enzyme to obtain the enzyme digestion product. Homologous recombination of the enzyme digestion product was performed using Exnase II to obtain the plasmid p1Ac-GFP-Cry2Ae1-Q318I. The homologous recombination product p1Ac-GFP-Cry2Ae1-Q318I was then transformed into E. coli competent cells TOP10. Single-spot culture was picked and 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 position from position 952 to 954 in the sequence shown in SEQ ID No. 5 was mutated to ATT, thus obtaining the amino acid sequence encoded by SEQ ID No. 5 as shown in SEQ ID No. 5. The protein shown in 6 was mutated with 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 those for TOP10 / p1Ac-GFP-Cry2Ae1-Q318I.

[0072] 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, GCG at positions 1009 to 1011 of the sequence shown in SEQ ID No. 3 was mutated to AGT, thereby causing an A337S mutation in the protein encoded by the amino acid sequence shown in SEQ ID No. 4, resulting in the positive plasmid p1Ac-GFP-Cry2Ae1-A337S and the positive recombinant strain TOP10 / p1Ac-GFP-Cry2Ae1-A337S. Using F-A337S / R-A337S primers and p1Ac-GFP-Cry2Ah2 as a template, GCT at positions 1009 to 1011 of the sequence shown in SEQ ID No. 5 was mutated to AGT, thereby causing an A337S mutation in the protein encoded by the amino acid sequence shown in SEQ ID No. 4, resulting in the positive plasmid p1Ac-GFP-Cry2Ae1-A337S and the positive recombinant strain TOP10 / p1Ac-GFP-Cry2Ae1-A337S. The protein shown in Figure 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 those for TOP10 / p1Ac-GFP-Cry2Ae1-Q318I.

[0073] 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 of the sequence shown in SEQ ID No. 3 was mutated to AAT, thereby causing an S360N mutation in the encoded amino acid sequence shown in SEQ ID No. 4, resulting in 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, AGT at positions 1078 to 1080 of the sequence shown in SEQ ID No. 5 was mutated to AAT, thereby causing an S360N mutation in the encoded amino acid sequence shown in SEQ ID No. 4, resulting in the positive plasmid TOP10 / p1Ac-GFP-Cry2Ae1-S360N and the positive recombinant strain TOP10 / TOP10 / p1Ac-GFP-Cry2Ae1-S360N. The protein shown in No. 6 was mutated to S360N to obtain the positive plasmid p1Ac-GFP-Cry2Ah2-S360N and the positive recombinant strain TOP10 / p1Ac-GFP-Cry2Ah2-S360N. Other operations were the same as for TOP10 / p1Ac-GFP-Cry2Ae1-Q318I.

[0074] 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 sequence at positions 1150 to 1152 in the sequence shown in SEQ ID No. 3 was mutated to GAG, thereby causing a G384E mutation in the protein encoded by the amino acid sequence shown in SEQ ID No. 4, resulting in 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, the GGG sequence at positions 1150 to 1152 in the sequence shown in SEQ ID No. 5 was mutated to GAG, thereby causing a G384E mutation in the protein encoded by the amino acid sequence shown in SEQ ID No. 4, resulting in the positive plasmid p1Ac-GFP-Cry2Ae1-G384E and the positive recombinant strain TOP10 / p1Ac-GFP-Cry2Ae1-G384E. The protein shown in Figure 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 for TOP10 / p1Ac-GFP-Cry2Ae1-Q318I.

[0075] 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 was mutated to GCA, thereby causing an L459A mutation in the protein encoded by the amino acid sequence shown in SEQ ID No. 4, resulting in 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, the TTA at positions 1375 to 1377 in the sequence shown in SEQ ID No. 5 was mutated to GCA, thereby causing an L459A mutation in the protein encoded by the amino acid sequence shown in SEQ ID No. 4, resulting in the positive plasmid p1Ac-GFP-Cry2Ae1-L459A and the positive recombinant strain TOP10 / p1Ac-GFP-Cry2Ae1-L459A. The protein shown in Figure 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 for TOP10 / p1Ac-GFP-Cry2Ae1-Q318I.

[0076] The mutant proteins were expressed and extracted in the same manner as in Example 1. The results showed that each mutant protein was successfully expressed in *E. coli*, and the expression products were found in the precipitate after cell lysis. (See attached image). Figures 2 to 4 Therefore, each protein suspension to be tested is a protein suspension containing the mutant protein.

[0077] Similarly, the concentration of each mutant protein was quantified by BSA prior to activity assay.

[0078] Example 3: Insecticidal activity assay

[0079] 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 the protein samples to be tested.

[0080] Procedure for determining the insecticidal activity of diamondback moth: Clean cabbage leaves with a diameter of 6 cm were soaked in the protein sample to be tested and the negative control. After 10 minutes, they were removed and air-dried until the moisture was completely evaporated. They were then placed in sterile petri dishes. Thirty healthy, active second-instar diamondback moth larvae were inoculated into the sterile petri dishes containing the cabbage leaves using a brush. Each treatment was repeated 3 times. The dishes were 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 light, humidity, and temperature were checked daily. The number of dead insects was investigated after 72 hours.

[0081] The difference between the assay for the insecticidal activity of the tea geometrid moth and the assay for the insecticidal activity of the diamondback moth lies in the substitution of cabbage leaves with fresh tea leaves.

[0082] Procedure for determining the insecticidal activity against Asian corn borer: Weigh 7 g of Asian corn borer-specific feed (feed formula shown in Table 1) into a sterile petri dish, add 0.7 mL of the protein sample to be tested, and add 0.7 mL of 20 mmol / L Tris-HCl (pH 8.0) for the negative control. Mix thoroughly and allow excess water to evaporate completely. Using a brush, inoculate 30 healthy, active newly hatched corn borer larvae into the petri dish containing the above feed. Repeat each treatment 3 times. Then place the dish in an artificial climate incubator at 26 degrees Celsius, photoperiod 14L:10D, and relative humidity 60%. Observe daily and check light, humidity, and temperature. After 7 days, investigate the number of dead insects.

[0083] The difference between the insecticidal activity assay for the Eastern armyworm and the Asian corn borer is that the feed used was a special feed for the Eastern armyworm (see Table 1 for the feed formula).

[0084] The mortality rate and the corrected mortality rate were calculated. The results of the biopsy are shown in Tables 2 to 4.

[0085] The formula for calculating the adjusted mortality rate is as follows:

[0086]

[0087] Table 1

[0088]

[0089] Table 2

[0090]

[0091] Significance analysis was performed using the independent samples t-test (p<0.05) to analyze significant differences between proteins.

[0092] As shown in Table 2, the Anc0 protein exhibits very high insecticidal activity against diamondback moth, Asian corn borer, armyworm, and tea geometrid moth. The mutant protein Cry2Ad1-II of Cry2Ad1 shows insecticidal activity against these same pests. The mutant protein Cry2Ah2-II of Cry2Ah2 enhances insecticidal activity against diamondback moth and tea geometrid moth, and also gains insecticidal activity against armyworm. Therefore, replacing domain II of the Cry2A class protein with domain II of the Anc0 protein, or more precisely, replacing amino acids 267 to 472 of the Cry2A class protein with amino acids 267 to 472 of the Anc0 protein, can significantly improve the insecticidal activity of the Cry2A class protein against lepidopteran pests.

[0093] Table 3

[0094]

[0095] " / " indicates unknown activity. The significance analysis used the independent samples t-test (p<0.05) to analyze the significant differences between proteins.

[0096] As shown 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 exhibited insecticidal activity against the Eastern Armyworm. Cry2Ae1-Q318I and Cry2Ae1-L459A enhanced the insecticidal activity against the Diamondback Moth and the Asian Corn Borer, while Cry2Ae1-Q318I, Cry2Ae1-S360N and Cry2Ae1-G384E enhanced the insecticidal activity against the Tea Geometrid Moth.

[0097] Table 4

[0098]

[0099] " / " indicates unknown activity. The significance analysis used the independent samples t-test (p<0.05) to analyze the significant differences between proteins.

[0100] According to the results in Table 4, compared with the Cry2Ah2 protein, the five point mutant proteins of Cry2Ah2, namely Cry2Ah2-Q318I, Cry2Ah2-A337S, Cry2Ah2-S360N, Cry2Ah2-G384E and Cry2Ah2-L459A, all enhanced the insecticidal activity against the tea geometrid moth, while Cry2Ah2-Q318I enhanced the insecticidal activity against the diamondback moth.

[0101] Based on the activities of Cry2Ae1-Q318I in Table 3 and Cry2Ah2-Q318I in Table 4, it can be seen that the Q318I mutation of Cry2A proteins can enhance the insecticidal activity against diamondback moth or tea geometrid moth; based on the activities of Cry2Ae1-S360N and Cry2Ae1-G384E in Table 3 and Cry2Ah2-S360N and Cry2Ah2-G384E in Table 4, it can be seen that the S360N or G384E mutation of Cry2A proteins can enhance the insecticidal activity against tea geometrid moth.

Claims

1. An insecticidal protein having the amino acid sequence shown in SEQ ID No.

14.

2. A composition containing the insecticidal protein as described in claim 1.

3. The nucleic acid encoding the insecticidal protein as described in 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 as described in claim 3 or 4 and capable of expressing the protein as described in claim 1.

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

7. The application 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, or the microorganism according to claim 5 or 6 in the control of lepidopteran insects; The Lepidoptera insects mentioned are at least one of the following: diamondback moth (Plutella xylostella), subspecies corn borer (Ostriniafurnacalis), oriental armyworm (Mythimna separata), and tea geometrid moth (Ectropis oblique).

Images