CN118108835A - Application of cowpea cysteine type protease inhibitor 649 in agricultural pest control - Google Patents
Application of cowpea cysteine type protease inhibitor 649 in agricultural pest control Download PDFInfo
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- CN118108835A CN118108835A CN202410242911.9A CN202410242911A CN118108835A CN 118108835 A CN118108835 A CN 118108835A CN 202410242911 A CN202410242911 A CN 202410242911A CN 118108835 A CN118108835 A CN 118108835A
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- cowpea
- protease inhibitor
- type protease
- cysteine
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- 239000004471 Glycine Substances 0.000 description 1
- 240000000220 Panda oleosa Species 0.000 description 1
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/81—Protease inhibitors
- C07K14/8107—Endopeptidase (E.C. 3.4.21-99) inhibitors
- C07K14/8139—Cysteine protease (E.C. 3.4.22) inhibitors, e.g. cystatin
-
- 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
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01P—BIOCIDAL, PEST REPELLANT, PEST ATTRACTANT OR PLANT GROWTH REGULATORY ACTIVITY OF CHEMICAL COMPOUNDS OR PREPARATIONS
- A01P7/00—Arthropodicides
- A01P7/02—Acaricides
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/185—Escherichia
- C12R2001/19—Escherichia coli
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- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- Wood Science & Technology (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- General Health & Medical Sciences (AREA)
- Plant Pathology (AREA)
- Biochemistry (AREA)
- Biomedical Technology (AREA)
- Pest Control & Pesticides (AREA)
- Biophysics (AREA)
- Biotechnology (AREA)
- Environmental Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Medicinal Chemistry (AREA)
- Agronomy & Crop Science (AREA)
- Peptides Or Proteins (AREA)
- Physics & Mathematics (AREA)
- Microbiology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Insects & Arthropods (AREA)
- Gastroenterology & Hepatology (AREA)
- General Chemical & Material Sciences (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Dentistry (AREA)
Abstract
The invention discloses a cowpea cysteine type protease inhibitor 649 and application thereof, belonging to the field of polypeptide pesticides, wherein the amino acid sequence of the cowpea cysteine type protease inhibitor 649 recombinant protein is shown as SEQ ID No. 1. The plant source protease is used as a brand new acaricidal gene resource, has a mode of action different from that of the existing acaricide, and has important scientific and practical significance for expanding the novel acaricidal gene resource with biological activity, reducing various safety risks caused by the wide use of the existing chemical acaricide and reducing the dosage of chemical pesticides in the field.
Description
Technical Field
The invention belongs to the fields of genetic engineering and biological control, and particularly relates to application of a cowpea cysteine type protease inhibitor 649.
Background
The means widely used for preventing and controlling agricultural mites at present are chemical pesticides, and protein active substances with mite killing effect are not available. However, because of the characteristics of short generation period, strong fertility, parthenogenesis and the like of the agricultural mites, and the long-term use of the chemical acaricide in the field, the problem of the resistance of the agricultural mites represented by the tetranychus urticae is outstanding, and the tetranychus urticae is even one of the most serious arthropods of the resistance problem, and is inferior to the plutella xylostella (Van Leeuwen T et al.Population bulk segregant mapping uncovers resistance mutations and the mode of action of a chitin synthesis inhibitor in arthropods.Proc Natl Acad Sci USA.2012;109:4407-4412.)., the development of the novel efficient and environment-friendly mite-killing active substance can enrich the means for preventing and controlling the agricultural mites, and reduce the dosage of chemical pesticides in the field.
The plant-derived protease inhibitor (protease inhibitors PIs) is a small molecule polypeptide or protein, which is widely present in plant tissues. The plant protease inhibitor can regulate the activity of endogenous protease of insects, and participate in physiological processes such as programmed cell death and the like; can enter the insect digestive tract, inhibit the proteolytic activity of protease, and interfere the normal growth and development of insects, thereby helping related plants to resist insect damage.
However, current research on plant-derived protease inhibitors has focused mainly on the activity of insects, and has not focused on the action on agricultural pest mites.
Disclosure of Invention
The technical problems to be solved by the invention are as follows: provides the application of the cowpea cysteine type protease inhibitor 649 in the prevention and treatment of agricultural mites, thereby achieving the anti-mite effect.
The technical scheme of the invention is as follows: the application of the cowpea cysteine type protease inhibitor 649 recombinant protein in the prevention and treatment of agricultural mites is that the amino acid sequence of the cowpea cysteine type protease inhibitor 649 recombinant protein is shown as SEQ ID No. 1.
Further, the agricultural pest mites are spider mites.
Compared with the prior art, the invention has the following beneficial effects:
the invention utilizes a bioinformatics analysis method to obtain candidate gene of the cowpea acaricidal peptide by screening genes in a transcriptome database after the cowpea is eaten by spider mites and comparing the genes with the related genes of the existing spider toxins, and then utilizes a Polymerase Chain Reaction (PCR) and Sanger method to sequence so as to obtain the complete sequence of the gene.
The mature peptide region of the gene is constructed into a prokaryotic expression vector pET-32a (+) and is expressed by a prokaryotic system, so that the insecticidal peptide cowpea cysteine type protease inhibitor coded by the gene can be obtained. The preparation cycle is short, the amino acid sequence is small, and the acaricidal peptide cowpea cysteine type protease inhibitor 649 is suitable for large-scale production in vitro, is used as a totally new acaricidal gene resource, has a mode of action different from that of the existing acaricide, has important scientific and practical significance for expanding the novel acaricidal gene resource with biological activity, reducing various safety risks caused by the wide use of the existing chemical acaricide and reducing the dosage of chemical pesticides in fields.
Drawings
FIG. 1 is an SDS-PAGE electrophoresis of cowpea cysteine protease inhibitor 649 recombinant protein NBX-4; in the figure, M is marker,1 is no load, 2 is not induced, 3 is total protein, 4 is sediment, 5 is supernatant, and 6 is purification.
FIG. 2 shows mortality of Tetranychus urticae eggs after dropping CK and cowpea cysteine type protease inhibitor 649 recombinant protein.
FIG. 3 shows the death phenotype of Tetranychus urticae eggs after dropping CK and cowpea cystein type protease inhibitor 649 recombinant protein.
Detailed Description
The experimental methods in the following examples are conventional methods unless otherwise specified. The test materials used in the examples described below, unless otherwise specified, were purchased from commercial sources.
The reagents and media formulations designed in the examples:
(1) LB liquid medium:
2g of tryptone, 1g of yeast extract and 2g of NaCl are added into 200ml of deionized water, stirred and mixed uniformly, placed in an autoclave, sterilized for 20min at 121 ℃, cooled, added with antibiotics and stored at 4 ℃.
(2) LB solid medium:
2g of tryptone, 1g of yeast extract, 2g of NaCl and 3g of agar are added into 200ml of double distilled water, stirred and mixed uniformly, placed in an autoclave, sterilized for 20min at 121 ℃, cooled to 50 ℃, added with 100mg/L final concentration of ampicillin, poured into a plate, cooled and placed at 4 ℃ for storage.
(3) Electric conversion liquid:
when in use, 100ml of 10 Xelectrotransport liquid (containing Tris0,23mol/L and glycine 1.92 mol/L) is taken, 200ml of methanol is added, and deionized water is used for constant volume to 1L. If necessary, a small amount of SDS (working concentration 0.037%) may be added.
(4) Sealing liquid:
1 XTBST was added with 5% skim milk powder.
(5) Binding Buffer (Washing Buffer):
0.04mM PBS,0.5M sodium chloride, 20mM imidazole, double distilled water to a volume of 1L, pH7.4.
(6) Elution Buffer (Elution Buffer):
0.04mM PBS,0.5M sodium chloride, 500mM imidazole, double distilled water to a volume of 1L, pH7.4.
(7) Ampicillin (Amp, 100 mg/mL): 1g of ampicillin is weighed into a 10mL volumetric flask, fully mixed and dissolved by using sterile water, the volume is fixed to 10mL, and the ampicillin is sterilized by passing through a disposable filter membrane of 0.22 mu m, split charging and light-shielding storage in a refrigerator of minus 20 ℃ for standby.
(8) Kanamycin (Kana, 50 mg/mL): weighing 0.5g of ampicillin in a 10mL volumetric flask, fully and uniformly mixing with sterilized water, then fixing the volume to 10mL, sterilizing by passing through a disposable filter membrane of 0.22 mu m, packaging and storing in a refrigerator at-20 ℃ for later use in a dark place.
(9) 0.02% Agar gel configuration: weighing 0.9g of agar powder, dissolving in 45mL of deionized water, heating to boil by a microwave oven, pouring into a disposable culture dish, placing a glass capillary after semi-solidification, and placing in a refrigerator at 4 ℃ for standby after complete solidification.
(10) IPTG (24 mg/mL): 1.2g of IPTG is weighed and placed in a 50mL centrifuge tube, 40mL of sterilized water is added, and the mixture is fully mixed and dissolved, and the volume is fixed to 50mL. Sterilizing with 0.22 μm disposable filter membrane, packaging, and storing in-20deg.C refrigerator.
Example 1
The gene in the transcriptome database is screened by utilizing a bioinformatics analysis method after the cowpea is eaten by spider mites, and the gene is subjected to sequence comparison with the existing spider toxin related genes to obtain a cowpea acaricidal peptide candidate gene cowpea cysteine type protease inhibitor 649, wherein the CDS sequence of the gene is shown as SEQ ID No.2, the amino acid sequence of the coded protein is shown as SEQ ID No.1, the codon optimization is carried out according to the codon preference of escherichia coli, and the gene sequence of the cowpea cysteine type protease inhibitor 649 mature peptide expressed in the escherichia coli is designed. The gene is cloned into an escherichia coli expression vector pET-32a (+) to construct a pET-32a (+) -649 cowpea cysteine type protease inhibitor recombinant plasmid containing a target gene, and the toxin gene is introduced into an NcoI enzyme cutting site.
Inducible expression of recombinant plasmid pET-32a (+) -649 cowpea cysteine type protease inhibitor
The Origami B (DE 3) BL21 (DE 3) strain, which contains mutated thioredoxin reductase (thioredoxin reductase) (trxB) and glutathione reductase (glutathione reductase) (gor) genes, was selected for competence, which favors the formation of correctly folded disulfide-containing proteins, enhancing protein solubility. Individual positive plaques were picked up in LB plates and added to LB liquid medium containing 100mg/ml ampicillin and 50mg/ml kanamycin, respectively, at 200rpm,37℃in a constant temperature shaker for 6h. The bacterial liquid with correct sequence was subjected to 15ml of expansion culture. The cultured bacterial liquid was inoculated into 200ml of LB liquid medium containing 100mg/ml of ampicillin and 50mg/ml of kanamycin at a ratio of 1:100. Culturing at 220rpm and 37deg.C until the OD600 value of the bacterial liquid is 0.6-0.8. Adding IPTG with the final concentration of 0.1mM into the pET-32a (+) -NbX4 recombinant, and carrying out induction expression for 12h at 25 ℃ and 150 rpm; the expressed bacterial liquid was taken out, centrifuged at 4000rpm at 4℃for 20min, the supernatant was discarded, and 0.01mM PBS was added to the bacterial pellet to resuspend the bacterial pellet to a volume of 20ml. Then carrying out ultrasonic crushing for 30min, running for 8s, and suspending for 8s; after the completion of the crushing, the mixture was centrifuged at 9000rpm at 4℃for 30min, and the supernatant was collected and the precipitate was discarded.
Purification of recombinant cowpea cysteine protease inhibitor pET-32a (+) -649
The supernatant after disruption of the expression-induced expression in large amounts was purified using a Ni-NTA6FF pre-packed gravity column. The preservation solution in the equal gravity column flows out freely, and is added into a Washing Buffer balance nickel column with 1 volume of column, and the maximum flow rate is 150cm/h. After the whole target protein was loaded, the impurity proteins were washed with a Washing Buffer of 3 volume column, and finally the target protein was eluted with 5ml of the Washing Buffer.
SDS-PAGE protein electrophoresis
1. Preparation of separation gel and concentrated gel
Different volumes of 30% acr-Bis (29:1), sephadex buffer and double distilled water were mixed in a test tube. 10% APS and TEMED were added and mixed with gentle stirring to avoid air bubbles. Pouring proper amount of separating gel solution into gel mould
Different volumes of 30% acr-Bis (29:1), concentrated gum buffer and double distilled water were mixed in test tubes. 10% APS and TEMED were added and mixed with gentle stirring to avoid air bubbles. The concentrated gum solution was added to the top of the separation gum until the gel solution reached the top of the front glass plate. The comb is inserted into the gel to avoid generating bubbles. Standing for 10-20 min, and waiting for polymerization of the concentrated glue. After the gel polymerized, the comb was carefully pulled out to avoid damaging the loading well.
2. Sample application
Mu.l of each of 10. Mu.l of marker, empty, uninduced, total protein, pellet, supernatant, purified, etc. samples were sequentially added to the wells.
3. Setting electrophoresis conditions, and starting electrophoresis
And adding an electrophoresis buffer solution into the electrophoresis tank, connecting a power supply, wherein black is a negative electrode, and red is a positive electrode. Setting the voltage to 80V, starting electrophoresis, changing the voltage to 120V after electrophoresis reaches the upper end of the separating gel, and stopping electrophoresis until the electrophoresis reaches the lower end of the electrophoresis tank, wherein the period is about one half hour.
4. Dyeing and decolorizing
And taking the glue out of the glass plate, putting the glass plate into coomassie brilliant blue staining solution for staining for 30min, taking the glue out of the staining solution, putting the glue into decolorizing solution, and decolorizing for a plurality of times until protein bands are clear.
5. Viewing and photographing
The protein bands were observed to determine that the target protein was between 35kd and 25kd, which was satisfactory, after which the pictures were taken.
As a result, as shown in FIG. 1, the uninduced protein first did not appear as an additional distinct band. Secondly, recombinant proteins carry a histidine tag (His-tag) which will bind specifically only to the Ni column and will elute other proteins during purification. Recombinant proteins can be eluted when high concentrations of competing reactants are added. Finally, the protein molecular weight of cysteine protease inhibitor 649 was 31.5kd, while the significant increase in protein band of interest was between 35kd and 25kd, and was near 35kd; in summary, the protein of interest is therefore considered to be the expressed cysteine protease inhibitor 649.
Determination of the concentration of the recombinant cowpea cysteine protease inhibitor pET-32a (+) -649
The concentration of the target protein was determined by coomassie brilliant blue method. 5 XG 250 was diluted to 1X. 1 XG 250 was mixed with the target protein and PBS in a ratio of 4:1. The set-up technique was repeated and absorbance was measured at 595 nm. The protein content in the sample was calculated by substituting the absorbance into a bovine serum standard curve, and the concentration of cowpea cysteine type protease inhibitor 649 in the experiment was 3040. Mu.g/ml.
Example 2
Process and method for biological testing
The ovicidal activity of the recombinant cowpea cysteine type protease inhibitor 649 on the tetranychus urticae was determined by adopting a microscopic drip method, eggs hatched by the same batch of tetranychus urticae adult mites were selected, each treatment was repeated for 2 times, and 10 eggs of tetranychus urticae were selected for each repetition. 10 eggs are selected from the leaf butterfly of the eggs left by removing the adult spider mites of the two-spotted spider mites, 0.2 mu l of clear water and cowpea cysteine type protease inhibitor 649 are respectively dripped on each egg, the dripping is repeated twice a day, and the conditions of different treated eggs are observed. The experiments are divided into a control group and an experimental group, wherein the control group is used for dripping clear water, and the experimental group is used for dripping the cowpea cysteine type protease inhibitor 649. FIG. 2 shows the mortality results of spot 649, showing that the protease inhibitor has better ovicidal activity. FIG. 3 shows the death phenotype of Tetranychus urticae after dropping CK and cowpea cysteine protease inhibitor 649.
Claims (2)
1. The application of the cowpea cysteine type protease inhibitor 649 recombinant protein in the prevention and treatment of agricultural mites is that the amino acid sequence of the cowpea cysteine type protease inhibitor 649 recombinant protein is shown as SEQ ID No. 1.
2. The use according to claim 1, wherein the agricultural pest mites are spider mites.
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| CN202410242911.9A CN118108835A (en) | 2024-03-04 | 2024-03-04 | Application of cowpea cysteine type protease inhibitor 649 in agricultural pest control |
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| CN202410242911.9A CN118108835A (en) | 2024-03-04 | 2024-03-04 | Application of cowpea cysteine type protease inhibitor 649 in agricultural pest control |
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