CN114891076A - Mutant protein and application thereof in control of Spodoptera frugiperda - Google Patents
Mutant protein and application thereof in control of Spodoptera frugiperda Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/32—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Bacillus (G)
- C07K14/325—Bacillus thuringiensis crystal protein (delta-endotoxin)
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N37/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids
- A01N37/44—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids containing at least one carboxylic group or a thio analogue, or a derivative thereof, and a nitrogen atom attached to the same carbon skeleton by a single or double bond, this nitrogen atom not being a member of a derivative or of a thio analogue of a carboxylic group, e.g. amino-carboxylic acids
- A01N37/46—N-acyl derivatives
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N47/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom not being member of a ring and having no bond to a carbon or hydrogen atom, e.g. derivatives of carbonic acid
- A01N47/40—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom not being member of a ring and having no bond to a carbon or hydrogen atom, e.g. derivatives of carbonic acid the carbon atom having a double or triple bond to nitrogen, e.g. cyanates, cyanamides
- A01N47/42—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom not being member of a ring and having no bond to a carbon or hydrogen atom, e.g. derivatives of carbonic acid the carbon atom having a double or triple bond to nitrogen, e.g. cyanates, cyanamides containing —N=CX2 groups, e.g. isothiourea
- A01N47/44—Guanidine; Derivatives thereof
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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 mutated at least one of six positions of H475A, S482A, N528A, V568A, T604A and P616A.
Description
Technical Field
The invention relates to the field of biological control, and in particular relates to application of mutant protein in control of Spodoptera frugiperda.
Background
Spodoptera frugiperda (Spodoptera frugiperda) is a omnivorous pest native to the tropical and subtropical regions of the America, and has the characteristics of strong reproduction, adaptability, migration capability and the like. The larva can be selected from 353 plants of 76 families, such as Gramineae, Compositae, Leguminosae, and Amaranthaceae. After 1 month in 2019 and invades China, Spodoptera frugiperda rapidly spreads nationwide, and has great threat to grain production safety and ecological safety. At present, two strategies of chemical control and biological control are mainly adopted for controlling Spodoptera frugiperda. Biological control relies primarily on entomopathogenic bacteria, such as Bacillus thuringiensis (Bt). However, with the continued planting of transgenic Bt Cry-type gene crops, Spodoptera frugiperda gradually developed resistance to this type of gene.
Therefore, there is a need to find spodoptera frugiperda-resistant genes that are maintained at low levels in the field resistance gene frequency.
Disclosure of Invention
The invention provides a protein which is a mutant protein of a protein with an amino acid sequence shown as SEQ ID No.4, wherein the mutant protein is mutated at least one of six positions of H475A, S482A, N528A, V568A, T604A and P616A.
The second aspect of the invention provides a composition comprising a protein according to the first aspect of the invention.
The third aspect of the invention provides a nucleic acid encoding a protein according to one of the invention.
In one embodiment, the nucleic acid is a mutant nucleic acid of the nucleic acid having the nucleotide sequence shown in SEQ ID No.1, wherein the codon encoding 475a in the H475A mutant protein is GCT; the codon encoding the 482A in the S482A mutein is GCT; the codon for 528A in the mutant protein of N528A is GCC; the codon encoding 568A of the V568A mutein is GCT; the codon for A at position 604 in the mutant protein of T604A is GCG; the codon encoding A at position 616 in the P616A mutein was GCA.
The fourth aspect of the invention provides a microorganism carrying a nucleic acid according to the third aspect of the invention and capable of expressing a protein according to the first aspect of the invention.
In a specific embodiment, the microorganism is bacillus thuringiensis and/or escherichia coli.
The fifth invention provides the application of the protein according to the first invention, the composition according to the second invention, the nucleic acid according to the third invention and the microorganism according to the fourth invention in preventing and controlling Spodoptera frugiperda
The invention has the beneficial effects that:
the invention firstly discovers that the protein with the amino acid sequence shown as SEQ ID No.4 is mutated, and as a result, the insecticidal activity of the mutant protein is improved to a different degree compared with that of Cry2Ab29, particularly the insecticidal activity of the mutant proteins Cry2Ab29-N528A, Cry2Ab29-V568A, Cry2Ab29-T604A and Cry2Ab29-P616A to Spodoptera frugiperda, and the insecticidal activity of the four mutant proteins is 2.77, 6.87, 3.22 and 2.62 times of that of Cry2Ab 29.
Both Cry2Ab29 and the mutant protein were able to bind BBMV and this binding was saturable. The affinity constant (Kd) was calculated by Sigma-Pitt: kd Cry2Ab29 ＝489.93±59.64nmol/L、Kd Cry2Ab29-V568A 19.74 ± 8.42 nmol/L. Compared with the wild type Cry2Ab29, mutant V568A had an approximately 24.8-fold increase in BBMV binding capacity.
Drawings
FIG. 1 shows the results of SDS-PAGE detection of BBMV slurry.
Detailed Description
The above-described aspects of the invention are explained in more detail below by means of preferred embodiments, but they are not intended to limit the invention.
The reagents in the examples of the present invention were all commercially available unless otherwise specified.
Spodoptera frugiperda is provided by Cangzhou Yunkang agricultural science and technology services, Inc.
Example 1
Expression of Cry2Ab29 protein
1. Construction of recombinant Bt strains
A primer F1/R1(SEQ ID No.2/SEQ ID No. 3) was designed based on the nucleotide sequence shown in SEQ ID No.1, and both the nucleotide sequence shown in SEQ ID No.1 and the primer were synthesized by Biotechnology engineering (Shanghai) GmbH. Wherein, the nucleotide shown as SEQ ID No.1 encodes the protein shown as SEQ ID No.4 (namely Cry2Ab29 protein).
Using F1/R1 as a template and synthetic nucleotide shown as SEQ ID No.1 as a template, carrying out PCR amplification, then connecting the amplified product to a pHT315-ORF2 vector (donated by Neil of Susaix university, England) to obtain a pHT315-cry2Ab29 recombinant plasmid, then transferring the positive recombinant plasmid into a Bt crystal-free mutant strain 4Q7, and screening out positive transformants 4Q7/pHT315-cry2Ab 29.
Expression and extraction of Cry2Ab29 protein
(1) Selecting a single bacterial colony: selecting a Bt 4Q7/pHT315-cry2Ab29 single colony to be cultured in 5mL LB liquid medium containing erythromycin at 30 ℃ and 220rpm for 12 hours with shaking;
(2) Inoculating into a 1L triangular flask containing 300mL1/2LB liquid medium (containing erythromycin) at 1% volume, and culturing at 30 deg.C under shaking at 220rpm until about 80% of thallus is lysed;
(3) centrifuging the fermentation liquor at 6,500 Xg for 15 minutes at low temperature, and collecting the precipitate;
(4) washing the precipitate with pre-cooled 1mol/L NaCl for 1 time, centrifuging at 8,000 Xg for 15 min, and removing the supernatant;
(5) washing with pre-cooled sterilized water for 1-2 times, and mixing thoroughly;
(6) adding lysis solution into the cell crystal mixture precipitate, stirring uniformly, and cracking for more than 8 hours on ice at 100 rpm;
(7) centrifuging at 4 deg.C for 10 min at 9,500 Xg, and collecting supernatant and precipitate;
(8) the precipitation is repeated twice in the steps (7) and (8);
(9) pouring the supernatant into a clean 50mL centrifuge tube, slowly adding about 1/7 volume of 4mol/L NaAc-HAc, stirring with a sterile glass rod while adding, and standing on ice for 4 hours;
(10) centrifuging at 4 deg.C and 9500 Xg for 15 min, removing supernatant, and collecting precipitate;
(11) washing the precipitate with pre-cooled sterilized water for 2-3 times, wherein each washing needs to be fully stirred, and washing NaAc-HAc cleanly;
(12) adding proper amount of 50mmol/L Na 2 CO 3 (pH 10.0) resuspend the pellet and blow repeatedly with a 5mL pipette until well solubilized.
(13) Protein extraction was checked by SDS-PAGE and quantified with BSA.
Example 2
Expression of the mutant protein
Designing a mutation primer P-H475A (SEQ ID No.5) so as to perform H475A mutation on SEQ ID No. 4; designing a mutation primer P-S482A (SEQ ID No.6) to make the S482A mutation to SEQ ID No. 4; design mutation primer P-N528A (SEQ ID No.7), thereby carrying out N528A mutation on SEQ ID No. 4; design mutation primer P-V568A (SEQ ID No.8) to make the V568A mutation to SEQ ID No. 4; designing a mutation primer P-T604A (SEQ ID No.9) to carry out T604A mutation on SEQ ID No. 4; the mutation primer P-P616A (SEQ ID No.10) was designed to carry out the P616A mutation to SEQ ID No. 4.
PCR amplification was carried out using pHT315-cry2Ab29 plasmid as a template and the above mutant primers to obtain PCR products. And removing the plasmid serving as a template from the obtained PCR product by using DPN I endonuclease to eliminate the influence on the transformation in the later period to obtain an enzyme digestion PCR product, and then purifying the enzyme digestion PCR product by using a PCR Cleanup kit according to a commercial instruction. The single-stranded DNA of the purified digested PCR product was aspirated by a pipette gun and gently mixed with 100. mu.L of competent cells of Escherichia coli Top10, then heat-shocked for transformation, followed by addition of 600. mu.L of LB liquid medium, culture at 37 ℃ and 150rpm for 1 hour, plating all on LB solid medium containing ampicillin, and culture at 37 ℃ overnight. Single colony culture is picked and bacterial liquid sequencing is carried out, and positive recombinant strains Top10/pHT315-cry2Ab29-H475A, Top10/pHT315-cry2Ab 29-S482A, Top10/pHT315-cry2Ab29-N528A, Top10/pHT315-cry2Ab29-V568A, Top10/pHT315-cry2Ab 29-T604A and Top10/pHT315-cry2Ab29-P616A are respectively obtained.
pHT315-cry2Ab 10-H10, pHT315-cry2Ab 10-S10, pHT315-cry2Ab 10-P10, pHT315-cry2Ab 10-V10, pHT315-cry2Ab 10-S482 4, pHT315-cry2Ab 10-N10, pHT315-cry2Ab 10-V10, pHT315-cry2Ab 10-T10-S10, pHT315-cry2Ab 10-N10, pHT315-cry2Ab 10-V10, pHT315-cry2Ab 10-T10-V568, pHT315-cry2Ab 10-T604-T10 and P616-10 Ab 616 from Top10/pHT315-cry2Ab 3823-S29-S A, respectively, then, 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-cry2Ab29-P616A are screened out after respectively transforming E.coli ET competent cells; then extracting pHT315-cry2Ab29-H475A, pHT315-cry2Ab29-S482A, pHT315-cry2Ab 29-N528-Ab 38723, pHT315-cry2Ab29-V568A, ET/pHT315-cry2Ab29-T604A and ET/pHT315-cry2Ab29-P A from ET/pHT315-cry2Ab29-H475 Ab29, pHT315-cry2Ab29-S482A, pHT315-cry2Ab29-N528A, pHT315-cry2Ab 387 6-V568A, pHT315-cry2Ab 29-T49742 Ab 468 and pHT315-cry2Ab29 Ab 5475 plasmids, respectively, transforming competent cells into competent cells of ET 4Q7 Bt 685 5475, positive transformants were screened for 4Q7/pHT315-cry2Ab29-H475A, 4Q7/pHT315-cry2Ab29-S482A, 4Q7/pHT315-cry2Ab29-N528A, 4Q7/pHT315-cry2Ab29-V568A, 4Q7/pHT315-cry2Ab 29-T604A and 4Q7/pHT315-cry2Ab 29-P616A.
The mutant proteins expressed by 4Q7/pHT315-Cry2Ab29-H475A, 4Q7/pHT315-Cry2Ab29-S482A, 4Q7/pHT315-Cry2Ab 29-N528A, 4Q7/pHT315-Cry2Ab29-V568A, 4Q7/pHT315-Cry2Ab29-T604A and 4Q7/pHT315-Cry2Ab 29-P616A are Cry2Ab29-H475A, Cry2Ab29-S482A, Cry2Ab29-N528A, Cry2Ab29-V568A, Cry2Ab29-T604A and Cry2Ab29-P616A respectively.
The mutant protein was expressed and extracted in the same manner as in example 1, and also, the concentration of the mutant protein was quantified by BSA before the activity measurement.
Example 3
Determination of Spodoptera frugiperda insecticidal Activity
At 50mmol/L Na 2 CO 3 (pH 10.0) as a blank; the concentration of the Cry2Ab29 protein and the mutant protein thereof is set to be the sameEach of the protein samples to be tested was obtained at 3.70. mu.g/mL, 11.11. mu.g/mL, 33.33. mu.g/mL, 100.00. mu.g/mL, 300.00. mu.g/mL and 600. mu.g/mL.
Weighing 15g of artificial feed (formula shown in table 1) in a sterilized culture dish, adding more than 3mL of protein samples to be detected or blank control at each concentration, fully and uniformly mixing, uniformly subpackaging in a sterilized 24-hole cell culture plate, and standing at room temperature until all excessive water in the feed is evaporated; inoculating healthy, active and undried Spodoptera frugiperda first-hatched larvae (within 12h after hatching) into the holes filled with the feed by using a writing brush, paving moist toilet paper on 1 insect in each hole, covering a plastic cover, binding tightly by using a rubber band, vertically putting into a biochemical incubator, culturing at 28 ℃, with a photoperiod of 16L:8D and relative humidity of 65%, observing every day, and checking whether the light, humidity, temperature and feed are mildewed or not and whether water vapor is condensed or not; the number of dead insects was investigated after 7 days, and the results are shown in Table 2. Mortality was then calculated. Each treatment was performed 3 times for 24 beetles. Protein lethal middle concentration (LC) was analyzed using SPSS 24 software 50 ) The results are shown in Table 2.
TABLE 1
| Feed components | 1 part of the dosage | 2 portions of dosage |
| Agar-agar | 55 g | 110g |
| Soybean flour | 110g | 220g |
| Wheat bran/wheat germ powder | 210g | 420g |
| Yeast powder | 40 g | 80g |
| Sorbic acid | 4g | 8g |
| Casein as a food additive | 55g | 110g |
| Ascorbic acid | 4g | 8g |
| Compound vitamin | 3 mL | 6mL |
| Formaldehyde (I) | 3 mL | 6mL |
| Acetic acid | 6mL | 12mL |
| Tekeduo (a Chinese character) | 1 mL | 2mL |
| Distilled water | 1800(1600+200)mL | 3600(3400+200)mL |
TABLE 2
| Protein | LC 50 (μg/g) | 95% confidence Limit (μ g/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 |
As can be seen from the results in Table 2, the insecticidal activities of the mutant proteins were improved to different degrees compared with those of Cry2Ab29, and particularly, the insecticidal activities of the mutant proteins Cry2Ab29-N528A, Cry2Ab29-V568A, Cry2Ab29-T604A and Cry2Ab29-P616A against Spodoptera frugiperda, which are sequentially 2.77, 6.87, 3.22 and 2.62 times the insecticidal activity of Cry2Ab 29.
Example 4
Formulation of Buffer I: 300mM mannitol, 17mM Tris-HCl, 5mM EGTA, 2mM Dithiothreitol (DTT), ultra pure water, pH 7.5.
1. Extraction of midgut Brush Border Membrane Vesicle (BBMV) of Spodoptera frugiperda
(1) Placing the third instar larvae of Spodoptera frugiperda in a culture dish on ice, and quickly cutting out midgut tissues;
(2) placing the midgut in a physiological saline solution containing benzyl xanthyl fluoride (PMSF) to remove surrounding fat and remove food residues in the intestine, and then rinsing the midgut tissue in a Phosphate Buffered Saline (PBS) to be clean;
(3) according to the middle intestine: (ii) adding a precooler I (w/v) ═ 1:10, homogenizing on ice with a sterile and precooled homogenizer, specifically 10 times at 2200rpm, to obtain a midgut homogenate, wherein 100 μ L is taken out therefrom for enzyme activity determination;
(4) To the remaining homogenate of the midgut was added 1 volume of 24mmol/L MgCl 2 And standing on ice for 15 min.
(5)2,500 Xg, centrifuging at low temperature for 15 minutes, and retaining the supernatant to obtain a supernatant;
(6) transferring the supernatant into a new precooled 50mL centrifuge tube, centrifuging the supernatant at the low temperature for 30 minutes at 30,000 Xg, and collecting the precipitate;
(7) the precipitate was washed with 1/2 volumes of Buffer I and 1/2 volumes of MgCl 2 Re-suspending;
(8) repeating the step (7) and the step (8);
(9) the precipitate was collected and concentrated as 1mL Buffer I: 3g midgut, the pellet was resuspended in an appropriate amount of precooler I to obtain BBMV serum.
(10) The BBMV slurry was examined by SDS-PAGE and the results are shown in FIG. 1, and the total protein concentration of the BBMV slurry and the midgut homogenate in step (3) were determined by the Bradford method. The total protein concentration of BBMV was 2.58mg/mL and the total protein concentration of the midgut homogenate in step (3) was 2.06 mg/mL.
APN enzyme Activity
(1) Weighing L-leucine p-nitroaniline (L-leucina-p-nitroanilide) and dissolving the L-leucine p-nitroanilide in dimethyl sulfoxide (DMSO) to ensure that the final concentration of the L-leucine p-nitroaniline is 10mmol/L, thus obtaining an L-leucine p-nitroaniline solution;
(2) pipetting 400. mu.L of ultrapure water, 250. mu.L of 1mol/L NaCl, 200. mu.L of 1mol/L Tris-HCl (pH 8.0) into a sterilized 1.5mL centrifuge tube, mixing the mixture uniformly, adding 100. mu.L of leucine p-nitroaniline solution, and adding 5. mu.L of BBMV slurry or 5. mu.L of midgut homogenate;
(3) After mixing well, incubation at room temperature for 10 minutes, OD was measured at 0, 2, 4 and 6 minutes 405 A value;
(4) calculating the OD of two adjacent time points of the same sample 405 The difference of the values (Δ A), and two adjacent time points OD 405 Calculating delta A/delta t according to the time difference (delta t) of the values, and calculating the APN enzyme activity of BBMV serous fluid to be 158.9U/mg BBMV based on the average value of the delta A/delta t (namely the delta A/delta t of 0 to 2min, 2 to 4min and 4 to 6 min) of three adjacent time points, wherein the APN enzyme activity of the midgut homogenate is 65.05U/mg midgut.
(5) Calculating the ratio of the APN enzyme activity in the BBMV to the APN enzyme activity of the homogenate of the midgut to indicate the quality of the extracted BBMV, wherein the larger the ratio of the APN enzyme activity to the APN enzyme activity of the homogenate of the midgut, the better the quality of the extracted BBMV is. Based on step (4), the ratio of the two is 2.44 times, which indicates that the quality of BBMV extraction can be used for the next experiment.
ALP enzymatic Activity
(1) Weighing P-nitrophenyl phosphate (P-nitrophenyl phosphate) and dissolving the P-nitrophenyl phosphate in sterile water to enable the final concentration of the P-nitrophenyl phosphate to be 1 mg/mL, so as to obtain a P-nitrophenyl phosphate solution;
(2) sucking 2.5 mu L of BBMV serous fluid or 2.5 mu L of midgut homogenate by a liquid transfer machine, uniformly mixing with 250 mu L of p-nitrophenyl phosphate solution in a sterilized 1.5mL centrifuge tube, and incubating for 15 minutes at room temperature;
(3) The reaction was stopped by adding 250. mu.L of 250mmol/L EDTA (pH 8.0);
(4) measuring OD 405 The value is obtained. Repeating for 3 times, and calculating by averaging the ALP enzyme activity of BBMV serous fluid to be 251.7U/mg BBMV and the ALP enzyme activity of midgut homogenate to be 134.6U/mg midgut.
(5) Calculating the ratio of the ALP enzyme activity in the BBMV to the ALP enzyme activity of the midgut homogenate liquid to be used for explaining the quality of the extracted BBMV, wherein the larger the ratio of the two is, the better the quality of the extracted BBMV is. Based on step (4), the ratio of the two is 1.87 times, which further indicates that the quality of BBMV extraction can be used for the next experiment.
Cry2Ab29 and ELLSA binding assays between mutant proteins and BBMV
(1) Diluting the BBMV slurry with TBS buffer solution to obtain 10 mu g/mL BBMV diluent;
(2) coating: BBMV was coupled to a 96-well microplate by adding 0.1mL of BBMV dilution to each reaction well of the 6-well microplate and allowing the solution to stand overnight at 4 ℃. The next day, the well diluent was discarded and washed 5 times with TBST buffer;
(3) and (3) sealing: adding 0.1mL of TBST containing 2% BSA, blocking at 37 ℃ for 2 hours, and then washing 5 times with TBST buffer;
(4) sample adding: adding 0.1mL of Cry2Ab29 or mutant protein thereof diluted in a gradient manner to different concentrations into reaction wells coated with BBMV, incubating for 2 hours at 37 ℃, and then washing for 5 times with TBST buffer;
(5) Adding an antibody: 0.1mL of a freshly diluted Cry2Ab antibody (1:2000) was added to each reaction well, incubated at 37 ℃ for 1 hour, and then washed 5 times with TBST buffer;
(6) adding a secondary antibody: to each reaction well, 0.1mL of horseradish peroxidase (HRP) -labeled IgG diluted 1:5000 was added, incubated at 37 ℃ for 1 hour, and then washed 5 times with TBST buffer;
(7) color development: adding 0.1mL of TMB substrate solution into each reaction hole, and incubating for 20 minutes at 37 ℃;
(8) and (3) terminating the reaction: adding 0.1mL of 2mol/L HCl into each reaction hole;
(9) measuring an OD value: reading the OD value of each hole at 450nm on an enzyme-labeling instrument;
(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 its affinity constant (Kd) was calculated.
The results show that the affinity constant Kd of Cry2Ab29 is 489.93 ± 59.64nmol/L, the affinity constant Kd of mutein Cry2Ab29-V568A is 19.74 ± 8.42nmol/L, and the affinity constant Kd of mutein Cry2Ab29-T604A is 100.06 ± 35.46 nmol/L. Compared with Cry2Ab29, the binding capacity of the mutant protein Cry2Ab29-V568A to BBMV is improved by about 24.8 times, and the binding capacity of the mutant protein Cry2Ab29-T604A to BBMV is improved by about 4.90 times. The results are consistent with the results of the insecticidal activity analysis, and thus indicate that the higher the insecticidal activity of the mutein against spodoptera frugiperda, the lower the Kd value, the affinity constant for binding to BBMV.
While the invention has been described with reference to specific embodiments, those skilled in the art will appreciate that various changes can 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 matter, and method to the essential scope and spirit of the present invention. All such modifications are intended to be included within the scope of the present invention as defined in the appended claims.
Sequence listing
<110> institute of plant protection of Chinese academy of agricultural sciences
<120> mutant protein and application thereof in control of Spodoptera frugiperda
<130> LHA2160510
<160> 10
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1902
<212> DNA
<213> Bacillus thuringiensis (Bacillus thuringiensis)
<400> 1
atgaatagtg tattgaatag cggaagaact actatttgtg atgcgtataa tgtagcggct 60
catgatccat ttagttttca acacaaatca ttagataccg tacaaaagga atggacggag 120
tggaaaaaaa ataatcatag tttataccta gatcctattg ttggaactgt ggctagtttt 180
ctgttaaaga aagtggggag tcttgttgga aaaaggatac taagtgagtt acggaattta 240
atatttccta gtggtagtac aaatctaatg caagatattt taagagagac agaaaaattc 300
ctgaatcaaa gacttaatac agacactctt gcccgtgtaa atgcggaatt gacagggctg 360
caagcaaatg tagaagagtt taatcgacaa gtagataatt ttttgaaccc taaccgaaac 420
gctgttcctt tatcaataac ttcttcagtt aatacaatgc aacaattatt tctaaataga 480
ttaccccagt tccagatgca aggataccaa ctgttattat tacctttatt tgcacaggca 540
gccaatttac atctttcttt tattagagat gttattctaa atgcagatga atggggaatt 600
tcagcagcaa cattacgtac gtatcgagat tacttgaaaa attatacaag agattactct 660
aactattgta taaatacgta tcaaagtgcg tttaaaggtt taaacactcg tttacacgat 720
atgttagaat ttagaacata tatgttttta aatgtatttg agtatgtatc tatctggtcg 780
ttgtttaaat atcaaagtct tctagtatct tccggtgcta atttatatgc aagtggtagt 840
ggaccacagc agacccaatc atttacttca caagactggc catttttata ttctcttttc 900
caagttaatt caaattatgt gttaaatgga tttagtggtg ctaggctttc taataccttc 960
cctaatatag ttggtttacc tggttctact acaactcacg cattgcttgc tgcaagggtt 1020
aattacagtg gaggaatttc gtctggtgat ataggtgcat ctccgtttaa tcaaaatttt 1080
aattgtagca catttctccc cccattgtta acgccatttg ttaggagttg gctagattca 1140
ggttcagatc gggagggcgt tgccaccgtt acaaattggc aaacagaatc ctttgagaca 1200
actttagggt taaggagtgg tgcttttaca gctcgcggta attcaaacta tttcccagat 1260
tattttattc gtaatatttc tggagttcct ttagttgtta gaaatgaaga tttaagaaga 1320
ccgttacact ataatgaaat aagaaatata gcaagtcctt caggaacacc tggtggagca 1380
cgagcttata tggtatctgt gcataacaga aaaaataata tccatgctgt tcatgaaaat 1440
ggttctatga ttcatttagc gccaaatgac tatacaggat ttactatttc gccgatacat 1500
gcaactcaag tgaataatca aacacgaaca tttatttctg aaaaatttgg aaatcaaggt 1560
gattctttaa ggtttgaaca aaacaacacg acagctcgtt atacgcttag agggaatgga 1620
aatagttaca atctttattt aagagtatct tcaataggaa attcaactat tcgagttact 1680
ataaacggta gagtttatac tgtttcaaat gttaatacca ctacaaataa cgatggagtt 1740
aatgataatg gagctcgttt ttcagatatt aatatcggta atatagtagc aagtgataat 1800
actaatgtaa cgctagatat aaatgtgaca ttaaactccg gtactccatt tgatctcatg 1860
aatattatgt ttgtgccaac taatctttca ccactttatt aa 1902
<210> 2
<211> 44
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 2
tatttaagga ggaattctag atgaatagtg tattgaatag cgga 44
<210> 3
<211> 36
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 3
catgattacg ccaagctttt aataaagtgg tggaag 36
<210> 4
<211> 633
<212> PRT
<213> Bacillus thuringiensis (Bacillus thuringiensis)
<400> 4
Met Asn Ser Val Leu Asn Ser Gly Arg Thr Thr Ile Cys Asp Ala Tyr
1 5 10 15
Asn Val Ala Ala His Asp Pro Phe Ser Phe Gln His Lys Ser Leu Asp
20 25 30
Thr Val Gln Lys Glu Trp Thr Glu Trp Lys Lys Asn Asn His Ser Leu
35 40 45
Tyr Leu Asp Pro Ile Val Gly Thr Val Ala Ser Phe Leu Leu Lys Lys
50 55 60
Val Gly Ser Leu Val Gly Lys Arg Ile Leu Ser Glu Leu Arg Asn Leu
65 70 75 80
Ile Phe Pro Ser Gly Ser Thr Asn Leu Met Gln Asp Ile Leu Arg Glu
85 90 95
Thr Glu Lys Phe Leu Asn Gln Arg Leu Asn Thr Asp Thr Leu Ala Arg
100 105 110
Val Asn Ala Glu Leu Thr Gly Leu Gln Ala Asn Val Glu Glu Phe Asn
115 120 125
Arg Gln Val Asp Asn Phe Leu Asn Pro Asn Arg Asn Ala Val Pro Leu
130 135 140
Ser Ile Thr Ser Ser Val Asn Thr Met Gln Gln Leu Phe Leu Asn Arg
145 150 155 160
Leu Pro Gln Phe Gln Met Gln Gly Tyr Gln Leu Leu Leu Leu Pro Leu
165 170 175
Phe Ala Gln Ala Ala Asn Leu His Leu Ser Phe Ile Arg Asp Val Ile
180 185 190
Leu Asn Ala Asp Glu Trp Gly Ile Ser Ala Ala Thr Leu Arg Thr Tyr
195 200 205
Arg Asp Tyr Leu Lys Asn Tyr Thr Arg Asp Tyr Ser Asn Tyr Cys Ile
210 215 220
Asn Thr Tyr Gln Ser Ala Phe Lys Gly Leu Asn Thr Arg Leu His Asp
225 230 235 240
Met Leu Glu Phe Arg Thr Tyr Met Phe Leu Asn Val Phe Glu Tyr Val
245 250 255
Ser Ile Trp Ser Leu Phe Lys Tyr Gln Ser Leu Leu Val Ser Ser Gly
260 265 270
Ala Asn Leu Tyr Ala Ser Gly Ser Gly Pro Gln Gln Thr Gln Ser Phe
275 280 285
Thr Ser Gln Asp Trp Pro Phe Leu Tyr Ser Leu Phe Gln Val Asn Ser
290 295 300
Asn Tyr Val Leu Asn Gly Phe Ser Gly Ala Arg Leu Ser Asn Thr Phe
305 310 315 320
Pro Asn Ile Val Gly Leu Pro Gly Ser Thr Thr Thr His Ala Leu Leu
325 330 335
Ala Ala Arg Val Asn Tyr Ser Gly Gly Ile Ser Ser Gly Asp Ile Gly
340 345 350
Ala Ser Pro Phe Asn Gln Asn Phe Asn Cys Ser Thr Phe Leu Pro Pro
355 360 365
Leu Leu Thr Pro Phe Val Arg Ser Trp Leu Asp Ser Gly Ser Asp Arg
370 375 380
Glu Gly Val Ala Thr Val Thr Asn Trp Gln Thr Glu Ser Phe Glu Thr
385 390 395 400
Thr Leu Gly Leu Arg Ser Gly Ala Phe Thr Ala Arg Gly Asn Ser Asn
405 410 415
Tyr Phe Pro Asp Tyr Phe Ile Arg Asn Ile Ser Gly Val Pro Leu Val
420 425 430
Val Arg Asn Glu Asp Leu Arg Arg Pro Leu His Tyr Asn Glu Ile Arg
435 440 445
Asn Ile Ala Ser Pro Ser Gly Thr Pro Gly Gly Ala Arg Ala Tyr Met
450 455 460
Val Ser Val His Asn Arg Lys Asn Asn Ile His Ala Val His Glu Asn
465 470 475 480
Gly Ser Met Ile His Leu Ala Pro Asn Asp Tyr Thr Gly Phe Thr Ile
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 a mutant protein of a protein with an amino acid sequence shown as SEQ ID No.4, wherein the mutant protein is mutated at least one of six positions of H475A, S482A, N528A, V568A, T604A and P616A.
2. A composition comprising the protein of claim 1.
3. A nucleic acid encoding the protein of claim 1.
4. The nucleic acid according to claim 2, wherein the nucleic acid is a mutant nucleic acid of the nucleic acid having the nucleotide sequence shown in SEQ ID No.1, wherein the codon encoding 475a in the H475A mutant protein is GCT; the codon encoding the 482a of the S482A mutein is GCT; the codon for 528A in the encoded N528A mutant protein is GCC; the codon encoding 568A of the V568A mutein is GCT; the codon for A at position 604 in the mutant protein of T604A is GCG; the codon encoding A at position 616 in the P616A mutein was GCA.
5. A microorganism carrying the nucleic acid of claim 3 or 4 and capable of expressing the protein of claim 1.
6. The microorganism according to claim 5, wherein the microorganism is Bacillus thuringiensis and/or Escherichia coli.
7. Use of one of the protein according to claim 1, the composition according to claim 2, the nucleic acid according to claim 3 or 4, the microorganism according to claim 5 or 6 for the control of spodoptera frugiperda.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210290037.7A CN114891076B (en) | 2022-03-23 | 2022-03-23 | Mutein and application thereof in preventing and controlling spodoptera frugiperda |
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| CN202210290037.7A CN114891076B (en) | 2022-03-23 | 2022-03-23 | Mutein and application thereof in preventing and controlling spodoptera frugiperda |
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| CN114891076B CN114891076B (en) | 2023-05-12 |
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Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH0665292A (en) * | 1992-08-11 | 1994-03-08 | Kubota Corp | Insecticidal protein against larva of coleopteran insect and new dna encoding the same |
| US6043415A (en) * | 1996-10-07 | 2000-03-28 | Ramot Univ. Auth. For Applied Research And Industrial Development Ltd. | Synthetic Bacillus thuringiensis cryic gene encoding insect toxin |
| CN1332800A (en) * | 1998-11-04 | 2002-01-23 | 孟山都公司 | Methods for transforming plants to express bacillus thuringiensis delta-endotoxins |
| CN105777880A (en) * | 2016-04-11 | 2016-07-20 | 中国农业科学院植物保护研究所 | Insecticidal crystal protein, nucleic acid, and preparation method and application of insecticidal crystal protein |
| CN110622998A (en) * | 2019-10-14 | 2019-12-31 | 中国农业科学院植物保护研究所 | Application of protein in preventing and treating spodoptera frugiperda and/or prodenia litura |
| CN111171118A (en) * | 2019-12-23 | 2020-05-19 | 隆平生物技术(海南)有限公司 | Plant insect-resistant gene mCry2Ab, and vector and application thereof |
| CN113527448A (en) * | 2021-08-18 | 2021-10-22 | 中国农业科学院植物保护研究所 | Application of protein in preventing and treating Spodoptera frugiperda and/or cotton bollworm |
-
2022
- 2022-03-23 CN CN202210290037.7A patent/CN114891076B/en active Active
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH0665292A (en) * | 1992-08-11 | 1994-03-08 | Kubota Corp | Insecticidal protein against larva of coleopteran insect and new dna encoding the same |
| US6043415A (en) * | 1996-10-07 | 2000-03-28 | Ramot Univ. Auth. For Applied Research And Industrial Development Ltd. | Synthetic Bacillus thuringiensis cryic gene encoding insect toxin |
| CN1332800A (en) * | 1998-11-04 | 2002-01-23 | 孟山都公司 | Methods for transforming plants to express bacillus thuringiensis delta-endotoxins |
| CN105777880A (en) * | 2016-04-11 | 2016-07-20 | 中国农业科学院植物保护研究所 | Insecticidal crystal protein, nucleic acid, and preparation method and application of insecticidal crystal protein |
| CN110622998A (en) * | 2019-10-14 | 2019-12-31 | 中国农业科学院植物保护研究所 | Application of protein in preventing and treating spodoptera frugiperda and/or prodenia litura |
| CN111171118A (en) * | 2019-12-23 | 2020-05-19 | 隆平生物技术(海南)有限公司 | Plant insect-resistant gene mCry2Ab, and vector and application thereof |
| CN113527448A (en) * | 2021-08-18 | 2021-10-22 | 中国农业科学院植物保护研究所 | Application of protein in preventing and treating Spodoptera frugiperda and/or cotton bollworm |
Non-Patent Citations (1)
| Title |
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| "GenBank accession NO:AIS85926.1" * |
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| CN114891076B (en) | 2023-05-12 |
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