CN114891076B - Mutein and application thereof in preventing and controlling spodoptera frugiperda - Google Patents

Abstract

The invention relates to a mutant protein and application thereof in preventing and controlling spodoptera frugiperda. The protein is a mutant protein of a protein with an amino acid sequence shown as SEQ ID No.4, and the mutant protein is a mutant protein mutated at least one position of six positions of H475A, S482A, N528A, V568A, T604A, P A.

Description

A Mutant Protein and Its Application in Controlling Spodoptera frugiperda

technical field

The invention relates to the field of biological control, in particular to the application of a mutant protein in preventing and controlling Spodoptera frugiperda.

Background technique

Spodoptera frugiperda is an omnivorous pest native to tropical and subtropical regions of America. It has the characteristics of strong reproduction, adaptability and migration ability. Its larvae can feed on 353 species of plants in 76 families, mainly Gramineae, Compositae, Leguminosae, and Amaranthaceae. Since it invaded my country in January 2019, fall armyworm has spread rapidly across the country, posing a major threat to food production safety and ecological security. At present, the control of Spodoptera frugiperda mainly adopts two strategies: chemical control and biological control. Biological control mainly relies on insect pathogenic bacteria, such as Bacillus thuringiensis (Bt). However, with the continuous planting of Bt Cry genetically modified crops, fall armyworm has gradually developed resistance to this type of gene.

Therefore, it is necessary to search for resistance genes against F. frugiperda at a low level in the field.

Contents of the invention

One of the present invention provides a protein, which is a mutein of the protein whose amino acid sequence is shown in SEQ ID No.4. A mutant protein in which at least one site is mutated.

The second invention provides a composition containing the protein according to the first invention.

The third invention provides nucleic acid encoding the protein described in the first invention.

In a specific embodiment, the nucleic acid is a mutated nucleic acid having a nucleotide sequence as shown in SEQ ID No.1, wherein the codon at position 475 A in the H475A mutein is GCT; The codon of A at position 482 is GCT; the codon of A at position 528 in the N528A mutant protein is GCC; the codon at A at position 568 in the mutant protein of V568A is GCT; the codon of A at position 604 in the mutant protein of T604A is GCG; the codon encoding the A at position 616 in the P616A mutein is GCA.

The fourth invention provides a microorganism carrying the nucleic acid according to the third invention and capable of expressing the protein according to the first invention.

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

The fifth aspect of the present invention provides the effects of the protein described in the first aspect of the present invention, the composition described in the second aspect of the present invention, the nucleic acid described in the third aspect of the present invention, and the microorganisms described in the fourth aspect of the present invention in the control of Spodoptera frugiperda application

Beneficial effects of the present invention:

The present invention finds for the first time that the protein whose amino acid sequence is shown as SEQ ID No.4 has been mutated, and it is found that the insecticidal activity of the mutant protein is improved to varying degrees compared with Cry2Ab29, especially the mutant protein Cry2Ab29-N528A, Cry2Ab29-V568A , Cry2Ab29-T604A and Cry2Ab29-P616A have insecticidal activity against Spodoptera frugiperda, and the insecticidal activity of these four mutant proteins is 2.77, 6.87, 3.22 and 2.62 times that of Cry2Ab29.

Both Cry2Ab29 and mutant proteins can bind to BBMV, and this binding can be saturable. Affinity constants (Kd) were calculated by Sigma-Piot: Kd _Cry2Ab29 = 489.93 ± 59.64 nmol/L, Kd _{Cry2Ab29-V568A} = 19.74 ± 8.42 nmol/L. Compared with wild-type Cry2Ab29, the ability of mutant V568A to bind to BBMV was increased by about 24.8 times.

Description of drawings

Figure 1 shows the SDS-PAGE detection results of BBMV slurries.

Detailed ways

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

Unless otherwise specified, the reagents in the examples of the present invention can be purchased through commercial channels.

Spodoptera frugiperda was provided by Cangzhou Yukang Agricultural Technology Service Co., Ltd.

Example 1

Expression of Cry2Ab29 protein

1. Construction of recombinant Bt strains

Design primers F1/R1 (SEQ ID No.2/SEQ ID No.3) based on the nucleotide sequence shown in SEQ ID No.1, such as the nucleotide sequence and primers of SEQ ID No.1 are provided by Sangong Synthesized by Bioengineering (Shanghai) Co., Ltd. Wherein, the nucleotide encoding as shown in SEQ ID No.1 encodes the protein (i.e. Cry2Ab29 protein) shown in SEQ ID No.4.

With F1/R1 as a template, with the synthesized nucleotide shown in SEQ ID No.1 as a template, PCR amplification is carried out, and then the amplified product is connected to the pHT315-ORF2 carrier (University of Sussex, UK Donated by Neil), the pHT315-cry2Ab29 recombinant plasmid was obtained, and then the positive recombinant plasmid was transferred into the Bt crystal-free mutant 4Q7, and the positive transformant 4Q7/pHT315-cry2Ab29 was screened out.

2. Expression and extraction of Cry2Ab29 protein

(1) Pick a single colony: Pick a single colony of Bt 4Q7/pHT315-cry2Ab29 in 5 mL of LB liquid medium containing erythromycin, and culture it with shaking at 30°C and 220rpm for 12 hours;

(2) Inoculate 1% of the volume in a 1L Erlenmeyer flask equipped with 300mL1/2LB liquid medium (containing erythromycin), 30°C, 220rpm shaking culture until about 80% of the cells are lysed under the microscope, and the culture is stopped;

(3) Centrifuge the fermentation broth at 6,500×g at low temperature for 15 minutes, and collect the precipitate;

(4) Wash the precipitate once with pre-cooled 1mol/L NaCl, centrifuge at 8,000×g for 15 minutes at low temperature, and remove the supernatant;

(5) Wash with pre-cooled sterilized water for 1-2 times, and mix well during the washing process;

(6) Add the lysate to the precipitation of the cell crystal mixture, stir evenly, and lyse on ice at 100 rpm for more than 8 hours;

(7) After centrifugation at 4°C and 9,500×g for 10 minutes, the supernatant and precipitate were collected respectively;

(8) Precipitation Repeat steps (7), (8) twice;

(9) Pour the supernatant into a clean 50mL centrifuge tube, slowly add about 1/7 volume of 4mol/L NaAc-HAc, stir with a sterile glass rod while adding, and let stand on ice for 4 hours;

(10) After centrifugation at 9500×g for 15 minutes at 4°C, discard the supernatant and collect the precipitate;

(11) The precipitate was washed 2 to 3 times with pre-cooled sterilized water, fully stirred for each washing, and the NaAc-HAc was washed clean;

(12) Add an appropriate amount of 50mmol/L Na ₂ CO ₃ (pH 10.0) to resuspend the precipitate, and repeatedly pipette with a 5mL pipette until fully dissolved.

(13) SDS-PAGE detection of protein extraction results, and quantification with BSA.

Example 2

Expression of mutein

Design mutation primer P-H475A (SEQ ID No.5), thereby carrying out H475A mutation to SEQ ID No.4; Design mutation primer P-S482A (SEQ ID No.6), thereby carrying out S482A mutation to SEQ ID No.4; Design mutation primer P-N528A (SEQ ID No.7), thereby carry out N528A mutation to SEQ ID No.4; Design mutation primer P-V568A (SEQ IDNo.8), thereby carry out V568A mutation to SEQ ID No.4; Design Mutation primer P-T604A (SEQ ID No.9), thereby performing T604A mutation on SEQ ID No.4; designing mutation primer P-P616A (SEQ ID No.10), thereby performing P616A mutation on SEQ ID No.4.

Using the pHT315-cry2Ab29 plasmid as a template, PCR amplification was performed using the above mutation primers to obtain PCR products. Use DPN I endonuclease to remove the plasmid as a template from the obtained PCR product to eliminate the impact on the transformation in the later stage, and obtain the enzyme-cut PCR product, and then use the PCR Cleanup kit to purify the enzyme-cut PCR product according to the product instructions. Use a pipette gun to draw the single-stranded DNA of the purified enzyme-cut PCR product and gently mix it with 100 μL of E. coli Top10 competent cells, then transform by heat shock, then add 600 μL of LB liquid medium, and incubate at 37°C for 1 hour at 150 rpm. All were spread on LB solid medium containing ampicillin and cultured overnight at 37°C. Single colonies were picked and cultured and sequenced to obtain positive recombinant strains Top10/pHT315-cry2Ab29-H475A, Top10/pHT315-cry2Ab29-S482A, Top10/pHT315-cry2Ab29-N528A, Top10/pHT315-cry2Ab29-V568A, Top10/ pHT315-cry2Ab29-T604A, Top10/pHT315-cry2Ab29-P616A.

Respectively from Top10/pHT315-cry2Ab29-H475A, Top10/pHT315-cry2Ab29-S482A, Top10/pHT315-cry2Ab29-N528A, Top10/pHT315-cry2Ab29-V568A, Top10/pHT315-cry2Ab29-T604A and Top10/pHT315-cry2Ab29-P616A extraction The plasmids of pHT315-cry2Ab29-H475A, pHT315-cry2Ab29-S482A, pHT315-cry2Ab29-N528A, pHT315-cry2Ab29-V568A, pHT315-cry2Ab29-T604A and pHT315-cry2Ab29-P616A were transformed into large Enterobacter ET competent cells, screening Positive transformants ET/pHT315-cry2Ab29-H475A, ET/pHT315-cry2Ab29-S482A, ET/pHT315-cry2Ab29-N528A, ET/pHT315-cry2Ab29-V568A, ET/pHT315-cry2Ab29-T604A and ET/pHT315-cry2 Ab29- P616A; then from ET/pHT315-cry2Ab29-H475A, ET/pHT315-cry2Ab29-S482A, ET/pHT315-cry2Ab29-N528A, ET/pHT315-cry2Ab29-V568A, ET/pHT315-cry2Ab29-T604A and ET/pHT31 5- cry2Ab29-P616A extract pHT315-cry2Ab29-H475A, pHT315-cry2Ab29-S482A, pHT315-cry2Ab29-N528A, pHT315-cry2Ab29-V568A, pHT315-cry2Ab29-T604A and pHT315-cry2Ab29- P616A plasmids were transformed into Bt 4Q7 competent cells , Screened positive transformants 4Q7/pHT315-cry2Ab29-H475A, 4Q7/pHT315-cry2Ab29-S482A, 4Q7/pHT315-cry2Ab29-N528A, 4Q7/pHT315-cry2Ab29-V568A, 4Q7/pHT315-cry2Ab29-T604A and 4Q7/pHT315- cry2Ab29-P616A.

4Q7/pHT315-cry2Ab29-H475A, 4Q7/pHT315-cry2Ab29-S482A, 4Q7/pHT315-cry2Ab29-N528A, 4Q7/pHT315-cry2Ab29-V568A, 4Q7/pHT315-cry2Ab29-T604A and 4Q7/pH T315-cry2Ab29-P616A respectively expressed The mutant proteins were Cry2Ab29-H475A, Cry2Ab29-S482A, Cry2Ab29-N528A, Cry2Ab29-V568A, Cry2Ab29-T604A and Cry2Ab29-P616A in sequence.

The mutant protein was expressed and extracted in the same manner as in Example 1. Likewise, the concentration of the mutant protein was quantified by BSA before the activity measurement.

Example 3

Determination of insecticidal activity against Spodoptera frugiperda

50mmol/L Na ₂ CO ₃ (pH 10.0) was used as the blank control; the concentration of Cry2Ab29 protein and its mutant protein was set to 3.70μg/mL, 11.11μg/mL, 33.33μg/mL, 100.00μg/mL, 300.00μg /mL and 600 μg/mL to obtain each protein sample to be tested.

Weigh 15g of artificial feed (recipe in Table 1) into a sterilized petri dish, add 3mL of protein samples to be tested or blank control at various concentrations above, mix well, and evenly distribute in sterilized 24-well cell culture plates placed at room temperature until the excess water in the feed evaporated; use a brush to insert healthy, active individuals, and newly hatched Spodoptera frugiperda larvae (within 12 hours after hatching) into the wells containing the above-mentioned feed 1 worm per hole was covered with wet toilet paper, covered with a plastic cover, tied tightly with rubber bands, placed upright in a biochemical incubator, cultivated at 28°C, photoperiod 16L:8D, relative humidity 65%, observed and checked every day Illumination, humidity, temperature and whether the feed is moldy or not, and whether there is water vapor condensation; after 7 days, the number of dead insects is investigated, and the results are shown in Table 2. Mortality rates were then calculated. 24 worms per treatment, repeated 3 times. The lethal concentration (LC ₅₀ ) of the protein was analyzed by SPSS 24 software, and the results are shown in Table 2.

Table 1

feed ingredients 1 serving 2 servings agar 55g 110g soy flour 110g 220g Wheat Bran/Wheat Germ Flour 210g 420g yeast 40g 80g Sorbic acid 4g 8g Casein 55g 110g ascorbic acid 4g 8g multi-vitamins 3 mL 6mL formaldehyde 3 mL 6mL Acetic acid 6mL 12mL Turkdo 1 mL 2mL distilled water 1800(1600+200)mL 3600(3400+200)mL

Table 2

protein <![CDATA[LC₅₀(μg/g)]]> 95% confidence limit (μg/g) Cry2Ab29 58.23 41.13-86.64 Cry2Ab29-H475A 35.39 25.16-49.01 Cry2Ab29-S482A 31.74 21.75-45.80 Cry2Ab29-N528A 21.01 14.62-29.72 Cry2Ab29-V568A 8.47 5.76-11.62 Cry2Ab29-T604A 18.11 6.59-34.69 Cry2Ab29-P616A 22.16 16.15-30.22

According to the results in Table 2, it can be seen that the insecticidal activity of the mutant proteins is higher than that of Cry2Ab29, especially the insecticidal activity of the mutant proteins Cry2Ab29-N528A, Cry2Ab29-V568A, Cry2Ab29-T604A and Cry2Ab29-P616A to Spodoptera frugiperda. The insecticidal activities of the four mutant proteins were 2.77, 6.87, 3.22 and 2.62 times that of Cry2Ab29 in turn.

Example 4

The formula of Buffer I: 300mM mannitol, 17mM Tris-HCl, 5mM EGTA, 2mM dithiothreitol (DTT), ultrapure water, pH 7.5.

1. Extraction of brush border membrane vesicles (BBMV) from the midgut of Spodoptera frugiperda

(1) Place the third instar larvae of Spodoptera frugiperda in a petri dish on ice, and quickly cut out the midgut tissue;

(2) Place the midgut in physiological saline containing phenylmethylsulfanyl fluoride (PMSF) to remove the surrounding fat and remove food residues in the intestine, and then rinse the midgut tissue in phosphate buffered saline (PBS) ;

(3) According to the midgut: Buffer I (w/v) = 1:10, add pre-cooled Buffer I, and use a sterile and pre-cooled homogenizer on ice to homogenize, specifically 2200rpm for 10 times, so that Obtain midgut homogenate, wherein 100 μL is taken out for enzyme activity determination;

(4) Add 1 volume of 24 mmol/L MgCl ₂ to the remaining midgut homogenate, and let stand on ice for 15 minutes.

(5) Centrifuge at 2,500×g for 15 minutes at low temperature, retain the supernatant, and obtain the supernatant;

(6) Transfer the supernatant to a new pre-cooled 50mL centrifuge tube, centrifuge at 30,000×g for 30 minutes at low temperature, and collect the precipitate;

(7) The Buffer I and 1/2 volume MgCl _of 1/2 volume are used for precipitation Resuspension;

(8) repeat step (7) and step (8);

(9) Collect the precipitate, according to the ratio of 1mL Buffer I: 3g midgut, take an appropriate amount of pre-cooled Buffer I and re-suspend the precipitate to obtain BBMV slurry.

(10) SDS-PAGE detects the BBMV serous fluid, and the results are shown in Figure 1, and the total protein concentration of the midgut homogenate in the BBMV serous fluid and step (3) is measured by the Bradford method. The BBMV total protein concentration obtained was 2.58 mg/mL, and the total protein concentration of the midgut homogenate in step (3) was 2.06 mg/mL.

2. APN enzyme activity

(1) Weigh L-leucine p-nitroanilide (L-leucina-p-nitroanilide) and dissolve it in dimethyl sulfoxide (DMSO) to make the final concentration 10mmol/L to obtain L-leucine p-nitroaniline solution;

(2) Pipette 400 μL ultrapure water, 250 μL 1mol/L NaCl, 200 μL 1mol/L Tris-HCl (pH=8.0) into a sterilized 1.5mL centrifuge tube and mix evenly, add 100 μL-leucine to Nitroaniline solution, then add 5 μL BBMV slurry or 5 μL midgut homogenate respectively;

(3) Mix well, incubate at room temperature for 10 minutes, and measure the OD ₄₀₅ values at 0, 2, 4 and 6 minutes;

(4) Calculate the difference (ΔA) between the OD ₄₀₅ values at two adjacent time points of the same sample, and the time difference (Δt) between the OD ₄₀₅ values at two adjacent time points, and then calculate ΔA/Δt , and then based on the average value of ΔA/Δt of three adjacent two time points (that is, ΔA/Δt of 0 and 2min, 2 and 4min, and 4 and 6min), the APN enzyme activity of BBMV serum was calculated to be 158.9U/mg BBMV. The APN enzyme activity of intestinal homogenate was 65.05U/mg midgut.

(5) Calculate the ratio of APN enzyme activity in BBMV to APN enzyme activity in midgut homogenate to illustrate the quality of extracted BBMV. The larger the ratio, the better the quality of extracted BBMV. Based on step (4), the ratio of the two is 2.44 times, indicating that the quality of the extracted BBMV can be used in the next experiment.

3. ALP enzyme activity

(1) Take p-nitrophenyl phosphate (P-nitrophenyl phsphate) and dissolve it in sterile water so that the final concentration is 1 mg/mL to obtain p-nitrophenyl phosphate solution;

(2) Use a pipette to draw 2.5 μL BBMV slurry or 2.5 μL midgut homogenate, mix with 250 μL p-nitrophenyl phosphate solution in a sterilized 1.5 mL centrifuge tube, and incubate at room temperature for 15 minutes;

(3) Add 250 μL 250 mmol/L EDTA (pH=8.0) to terminate the reaction;

(4) Measure OD ₄₀₅ value. The ALP enzyme activity of the BBMV serum was calculated 3 times, and the ALP enzyme activity of the midgut homogenate was 134.6 U/mg midgut.

(5) Calculate the ratio of ALPase activity in BBMV to ALPase activity in midgut homogenate to illustrate the quality of the extracted BBMV. The larger the ratio, the better the quality of the extracted BBMV. Based on step (4), the ratio of the two is 1.87 times, further indicating that the quality of the extracted BBMV can be used in the next experiment.

4. ELLSA binding analysis between Cry2Ab29 and mutant proteins and BBMV

(1) Dilute the BBMV slurry with TBS buffer to obtain a 10 μg/mL BBMV dilution;

(2) Coating: Add 0.1mL BBMV diluent to each reaction well of the 6-well microplate, and overnight at 4°C, so as to couple the BBMV to the 96-well microplate. The next day, discard the dilution in the well, and wash 5 times with TBST buffer;

(3) Blocking: add 0.1 mL of TBST containing 2% BSA, block for 2 hours at 37°C, and then wash 5 times with TBST buffer;

(4) Adding samples: Add 0.1 mL of Cry2Ab29 or its mutant protein in gradient dilutions to different concentrations to the reaction wells coated with BBMV, incubate at 37°C for 2 hours, and then wash 5 times with TBST buffer;

(5) Antibody addition: Add 0.1 mL of freshly diluted Cry2Ab antibody (1:2000) to each reaction well, incubate at 37°C for 1 hour, and then wash 5 times with TBST buffer;

(6) Add secondary antibody: Add 0.1 mL of horseradish peroxidase (HRP)-labeled IgG diluted 1:5000 to each reaction well, incubate at 37°C for 1 hour, and then wash 5 times with TBST buffer;

(7) Color development: add 0.1mL TMB substrate solution to each reaction well, and incubate at 37°C for 20 minutes;

(8) Stop the reaction: add 0.1mL 2mol/L HCl to each reaction well;

(9) Measure the OD value: read the OD value of each well at 450nm on a microplate reader;

(10) Standard deviation analysis was performed on each set of data, and the affinity between Cry2Ab29 or its mutant protein and BBMV was calculated by Sigma-Plot software, and the affinity constant (Kd) was calculated.

The results showed that the affinity constant Kd of Cry2Ab29=489.93±59.64nmol/L, the affinity constant Kd=19.74±8.42nmol/L of the mutant protein Cry2Ab29-V568A, and the affinity constant Kd=100.06±35.46nmol of the mutant protein Cry2Ab29-T604A /L. Compared with Cry2Ab29, the binding ability of the mutant protein Cry2Ab29-V568A to BBMV was increased by about 24.8 times, and the binding ability of the mutant protein Cry2Ab29-T604A was increased by about 4.90 times. This result is consistent with the results of the insecticidal activity analysis, therefore, the higher the insecticidal activity of the mutant protein against Spodoptera frugiperda, the lower the affinity constant Kd value of its binding to BBMV.

While the invention has been described with reference to specific embodiments, it will be understood by those skilled in the art that various changes may be made without departing from the true spirit and scope of the invention. In addition, many modifications may be made to adapt a particular situation, material, composition of materials and method to the spirit, spirit and scope of the invention. All such changes are included within the scope of the claims of the present invention.

sequence listing

<110> Institute of Plant Protection, Chinese Academy of Agricultural Sciences

<120> A Mutant Protein and Its Application in Controlling Spodoptera frugiperda

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485 490 495

Ser Pro Ile His Ala Thr Gln Val Asn Asn Gln Thr Arg Thr Phe Ile

500 505 510

Ser Glu Lys Phe Gly Asn Gln Gly Asp Ser Leu Arg Phe Glu Gln Asn

515 520 525

Asn Thr Thr Ala Arg Tyr Thr Leu Arg Gly Asn Gly Asn Ser Tyr Asn

530 535 540

Leu Tyr Leu Arg Val Ser Ser Ile Gly Asn Ser Thr Ile Arg Val Thr

545 550 555 560

Ile Asn Gly Arg Val Tyr Thr Val Ser Asn Val Asn Thr Thr Thr Asn

565 570 575

Asn Asp Gly Val Asn Asp Asn Gly Ala Arg Phe Ser Asp Ile Asn Ile

580 585 590

Gly Asn Ile Val Ala Ser Asp Asn Thr Asn Val Thr Leu Asp Ile Asn

595 600 605

Val Thr Leu Asn Ser Gly Thr Pro Phe Asp Leu Met Asn Ile Met Phe

610 615 620

Val Pro Thr Asn Leu Ser Pro Leu Tyr

625 630

<210> 5

<211> 43

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 5

ataacagaaa aaataatatc gctgctgttc atgaaaatgg ttc 43

<210> 6

<211> 41

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 6

atgctgttca tgaaaatggt gctatgattc atttagcgcc a 41

<210> 7

<211> 43

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 7

attctttaag gtttgaacaa gccaacacga cagctcgtta tac 43

<210> 8

<211> 44

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 8

ataaacggta gagtttatac tgcttcaaat gttaatacca ctac 44

<210> 9

<211> 44

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 9

gcaagtgata atactaatgt agcgctagat ataaatgtga catt 44

<210> 10

<211> 47

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 10

aatgtgacat taaactccgg tactgcattt gatctcatga atattat 47

Claims (7)

1. A protein, which is an amino acid sequence such as a mutant protein of the protein shown in SEQ ID No. 4, and the mutant protein is a site in six sites of H475A, S482A, N528A, V568A, T604A, and P616A A mutated mutant protein. 2. A composition comprising the protein of claim 1. 3. A nucleic acid encoding the protein of claim 1. 4. nucleic acid according to claim 2, is characterized in that, described nucleic acid is the mutated nucleic acid of nucleotide sequence such as the nucleic acid shown in SEQ ID No.1, wherein, the codon of 475 position A in coding H475A mutein is GCT; the codon encoding A at position 482 in S482A mutant protein is GCT; the codon encoding A at position 528 in N528A mutant protein is GCC; the codon encoding A at position 568 in V568A mutant protein is GCT; The codon of A at 604 is GCG; the codon of A at 616 in the encoded P616A mutant protein is GCA. 5. A microorganism, which carries the nucleic acid as claimed in claim 3 or 4 and can express the protein as claimed in claim 1. 6. The microorganism according to claim 5, characterized in that, the microorganism is Bacillus thuringiensis and/or Escherichia coli. 7. according to the protein described in claim 1, the composition described in claim 2, the nucleic acid described in claim 3 or 4, the application of a kind of microorganism described in claim 5 or 6 in preventing and treating Spodoptera frugiperda .

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