CN114957428B - Amblyseius barkeri miticidal peptide NbX-4 and application thereof - Google Patents

Abstract

The invention discloses a neoseiulus barkeri acaricidal peptide NbX-4 and application thereof, the acaricidal peptide is used as a brand-new acaricidal gene resource, has an action mode 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 acaricide and reducing the dosage of chemical pesticides in fields.

Description

Amblyseius barkeri miticidal peptide NbX-4 and application thereof

Technical Field

The invention belongs to the field of genetic engineering and biological control, and particularly relates to neoseiulus barkeri acaricidal peptide NbX-4 and application thereof.

Background

At present, the means widely used for preventing and controlling agricultural mites is chemical pesticide, and protein active substances with mite killing effect do not exist. However, because of the characteristics of short generation cycle, strong fecundity and parthenogenesis of agricultural pest mites, the long-term use of chemical acaricides in the field leads to outstanding resistance problems of agricultural pest mites represented by Tetranychus urticae, which is even one of the most serious arthropods, and is inferior to that of diamondback moth (Van Leuwen T et al. Therefore, the development of the novel efficient and environment-friendly acaricidal active substance can enrich the control means of agricultural pest mites and reduce the dosage of field chemical pesticides.

As natural enemies of arthropods, arachnids are mostly equipped with naturally occurring insecticidal (acaricidal) active peptides (Spider toxins), and due to their high activity, they are safe for non-target organisms, and have received much attention in the development of novel insecticidally active substances (King GF, et al., spider-Venom peptides: structures, pharmacology, and porosity for control of insect pests, annu. Rev. Entomol.2013, 58. The spider toxins found to act on many of the various channels and receptors of the cell membrane of the arthropod nervous system, such as ion channels, nerve ligand-gated channels, and G protein-associated receptors. However, current research on spider toxins has focused primarily on the active substances of insect predators, such as predatory spiders, and has not focused on substances that are biologically active against agricultural pest mites in predatory mites, the minor natural enemies in the arachnids.

Disclosure of Invention

The technical problem to be solved by the invention is as follows: a novel acaricidal protein with acaricidal effect can be expressed in a large amount by a molecular genetic engineering method or in crops by a transgenic technology, so that the acaricidal effect is achieved.

The technical scheme of the invention is as follows: the neoseiulus barkeri miticide peptide NbX-4 has the amino acid sequence shown in SEQ ID No. 2.

The nucleotide sequence of the gene of the mite-killing peptide NbX-4 of the neoseiulus barkeri is shown in SEQ ID No. 1.

An acaricidal peptide is mature peptide of Amblyseius barkeri NbX-4, and its amino acid sequence is shown in SEQ ID No. 3.

A gene encoding the above miticidal peptide. Due to the degeneracy of codons, the encoding genes can be in various combinations, and preferably, the nucleotide sequence of the genes is shown as SEQ ID No. 4.

An expression vector contains a gene for coding a protein shown by SEQ ID No.2 or SEQ ID No. 3.

The polypeptide shown in SEQ ID No.2 or SEQ ID No.3 is applied to prevention and treatment of tetranychus cinnabarinus.

Compared with the prior art, the invention has the following beneficial effects:

the invention utilizes a bioinformatics analysis method to screen genes in a full-length transcriptome database of the neoseiulus pasteurianus, compares the genes with related genes of the existing spider toxin to obtain a candidate gene of the acaricidal peptide of the neoseiulus pasteurianus, and then utilizes Polymerase Chain Reaction (PCR) and Sanger method to sequence to obtain a complete sequence of the gene. The mature peptide region of the gene is constructed into a prokaryotic expression vector pET-32a (+), and the insecticidal peptide NbX-4 coded by the gene can be obtained through prokaryotic system expression. The acaricide peptide has short preparation period and small amino acid sequence, is suitable for in-vitro large-scale production, has an action mode different from that of the existing acaricide as a brand-new acaricide gene resource, and has important scientific and practical significance for expanding novel acaricide gene resources with biological activity, reducing various safety risks caused by the wide use of the existing acaricide and reducing the dosage of field chemical pesticides.

Drawings

FIG. 1 is a graph of the mortality of two-spotted spider mites (red) at different time points after CK and recombinant toxin NbX-4 injection, with asterisks indicating significant differences (P < 0.05);

FIG. 2 shows the death phenotype of Tetranychus urticae (Red) after CK and recombinant toxin NbX-4 injection

FIG. 3 plasmid sequencing results;

FIG. 4 is an electrophoresis diagram of NBX-4SDS-PAGE of recombinant protein of Amblyseius barkeri; in the figure, M is marker,1 is no load, 2 is no induction, 3 is total protein, 4 is precipitation, 5 is supernatant, and 6 is purification.

Detailed Description

The experimental procedures in the following examples are all conventional ones unless otherwise specified. The test materials used in the following examples were all commercially available unless otherwise specified.

The reagents and media formulations designed in the examples:

(1) LB liquid medium:

adding 2g tryptone, 1g yeast extract and 2g NaCl into 200ml deionized water, stirring and mixing uniformly, placing in a high-pressure sterilization pot, sterilizing at 121 ℃ for 20min, cooling, adding antibiotics, and storing at 4 ℃.

(2) LB solid Medium:

adding 2g tryptone, 1g yeast extract, 2g NaCl and 3g agar into 200ml double distilled water, stirring and mixing uniformly, placing in an autoclave, sterilizing at 121 ℃ for 20min, cooling to 50 ℃, adding ampicillin with the final concentration of 100mg/L, inverting the plate, cooling, and storing at 4 ℃.

(3) Electric liquid transfer:

when in use, 100ml of 10 Xelectrotransfer solution (containing Tris0,23mol/L and glycine 1.92 mol/L) is taken, 200ml of methanol is added, and the volume is fixed to 1L by deionized water. If necessary, a small amount of SDS (working concentration 0.037%) may be added

(4) Sealing liquid:

5% skimmed milk powder was added to 1 XTSST.

(5) Binding Buffer (Washing Buffer):

0.04mM PBS,0.5M sodium chloride, 20mM imidazole, double distilled water to volume of 1L, pH7.4.

(6) Elution Buffer (elusion Buffer):

0.04mMPBS,0.5M sodium chloride, 500mM imidazole, double distilled water to volume of 1L, pH7.4.

(7) Ampicillin (Amp, 100 mg/mL): weighing 1g of ampicillin in a 10mL volumetric flask, fully and uniformly mixing and dissolving the ampicillin in sterile water, then fixing the volume to 10mL, filtering the solution through a disposable filter membrane of 0.22 mu m for sterilization, subpackaging and storing the solution in a refrigerator at the temperature of-20 ℃ in a dark place for later use;

(8) Kanamycin (Kana, 50 mg/mL): weighing 0.5g of ampicillin in a 10mL volumetric flask, fully and uniformly mixing and dissolving the ampicillin in sterile water, then fixing the volume to 10mL, filtering the solution through a disposable filter membrane with the diameter of 0.22 mu m for sterilization, subpackaging the solution and storing the solution in a refrigerator at the temperature of minus 20 ℃ in a dark place for later use;

(9) 0.02% agar gel preparation: weighing 0.9g of agar powder, dissolving in 45mL of deionized water, heating to boil in a microwave oven, pouring into a disposable culture dish, placing a glass capillary after the agar powder is semi-solidified, and placing in a refrigerator at 4 ℃ for later use after the agar powder is completely solidified.

(10) IPTG (24 mg/mL): 1.2g of IPTG was weighed into a 50mL centrifuge tube, 40mL of sterile water was added, mixed well and dissolved, and the volume was adjusted to 50mL. Filtering with 0.22 μm disposable filter membrane for sterilization, subpackaging and storing at-20 deg.C in refrigerator for use.

Example 1

Screening genes in a total-length transcriptome database of the neoseiulus pasteurii by utilizing a bioinformatics analysis method, comparing the genes with related genes of the existing spider toxins to obtain a candidate gene NbX-4 of the neoseiulus pasteurii miticide peptide, wherein the CDS sequence of the gene is shown as SEQ ID No.1, the amino acid sequence of the encoded protein is shown as SEQ ID No.2, the mature peptide sequence (shown as SEQ ID No. 3) is subjected to codon optimization according to the codon preference of escherichia coli, and the gene sequence (shown as SEQ ID No. 4) of the NbX-4 mature peptide expressed in escherichia coli is designed. The gene is cloned into an escherichia coli expression vector pET-32a (+), and pET-32a (+) -NbX4 recombinant plasmids containing target genes are constructed, and the toxin genes are introduced into an NcoI enzyme cutting site.

Inducible expression of recombinant plasmid pET-32a (+) -NbX4

Origami B (DE 3) strains were selected for competence, which contained mutated thioredoxin reductase (trxB) and glutathione reductase (gor) genes, to facilitate the formation of correctly folded disulfide-containing proteins and to enhance protein solubility. Individual positive plaques were picked on LB plates and added to LB liquid medium containing 100mg/ml ampicillin and 50mg/ml kanamycin, respectively, in a constant temperature shaker at 200rpm,37 ℃ for 6h. The correctly sequenced bacterial solution (FIG. 3) was subjected to 15ml scale-up culture. And (3) mixing the cultured bacterial liquid according to the ratio of 1:100 to 200ml of LB liquid medium containing 100mg/ml of ampicillin and 50mg/ml of kanamycin. Culturing at 220rpm and 37 deg.C until the OD600 value of the bacterial liquid is 0.6-0.8. Adding IPTG with the final concentration of 0.1mM into pET-32a (+) -NbX4 recombinants, and inducing and expressing for 12h at the temperature of 25 ℃ and the rpm of 150; taking out the expressed bacterial liquid, centrifuging at 4 ℃ and 4000rpm for 20min, discarding the supernatant, adding 0.01mM PBS into the bacterial precipitation for heavy suspension, and fixing the volume to 20ml. Then carrying out ultrasonic crushing for 30min, running for 8s, and pausing for 8s; after the disruption was completed, the mixture was centrifuged at 9000rpm for 30min at 4 ℃ to obtain a supernatant, and the precipitate was discarded (FIG. 4).

Western Blot detection

Mu.l of the supernatant was taken, 10. Mu.l of protein loading buffer was added, boiled at 100 ℃ for 5min, 1L of protein buffer was added to SDS-polyacrylamide gel, and 15. Mu.l of protein standard was added. Running out the concentrated gel at 80V, and then using 120V until the protein sample reaches the bottom of the gel; after electrophoresis, the gel is cut to a proper size and transferred into an electrotransfer solution, a PVDF membrane is soaked in methanol for 3min and then transferred into the electrotransfer solution, and membrane transfer is carried out after covering, wherein the gel is 200mA and 1h. After the membrane is transferred, taking out the membrane, washing for 5min for 3 times by TBST, sealing for 2-3 h by sealing liquid at room temperature at 80r/min; taking out the membrane after sealing, washing for 3 times by TBST (TBST), 10min each time, adding primary antibody (1: 2000), incubating for 2h at room temperature, 80r/min or overnight at 4 ℃; after the primary antibody incubation is finished, taking out the membrane, washing for 3 times by TBST (TBST), each time for 10min, transferring to a secondary antibody (1: 10000), incubating for 1h at room temperature for 80r/min; after the secondary antibody incubation is finished, washing for 3 times (10 min each) by TBST, adding a luminescent solution, keeping away from light for 2min, and performing chemiluminescence detection after the surface liquid is absorbed by filter paper (figure 4).

Purification of recombinant toxin pET-32a (+) -NbX4

The supernatant after disruption of the mass expression-induced expression was purified using a Ni-NTA 6FF pre-packed gravity column. The liquid in the gravity column is firstly equalled to flow out freely, and a Washing Buffer balance nickel column with the volume of 1 column is added, and the maximum flow rate is 150cm/h. After the complete loading of the target protein, the hybrid protein was washed with Washing Buffer of 3-volume column, and finally the target protein was eluted with 5ml of Elution Buffer (FIG. 4).

Determination of concentration of recombinant toxin pET-32a (+) -NbX4

The concentration of the target protein was determined by Coomassie Brilliant blue. Dilute 5 xg 250 to 1 ×.1 × G250 was mixed with the protein of interest and PBS according to a 4:1, in a ratio of 1. The set up technique was repeated and absorbance was measured at 595 nm. The measured absorbance was substituted into a bovine serum standard curve to calculate the protein content in the sample, and the NbX-4 concentration in this experiment was 2255. Mu.g/ml.

Example 2

Bioassay process and method

The insecticidal activity of the recombinant toxin NbX-4 to tetranychus cinnabarinus is determined by a microinjection method, adult mites with similar heads and sizes are selected, each adult mite is treated for 3 times, each adult mite is selected for 30-50 times repeatedly, the adult mites are starved for 12 hours before injection, and four limbs of the adult mites are placed on 0.02% agar in a fixed and neat mode. To inject the mites with a point of stress, a glass capillary is typically placed against the needle during the agar manufacturing process. Approximately 10nl of recombinant toxin was injected per adult mite, the injection site was level with the genital orifice, and the side was in the back region. After the injection is finished, picking the mites to the prepared leaf discThe whole process needs to be gentle, and secondary damage to mites is reduced. The experiment is divided into a control group and an experimental group, wherein the control group is injected with PBS, and the experimental group is injected with the recombinant toxin NbX-4. FIG. 1 is a mortality result of NbX-4 injection, showing that the venom peptide has a better insecticidal activity. FIG. 2 shows the death phenotype of Tetranychus urticae (Red) after CK and recombinant toxin NbX-4 injection. At the same time, to determine the lethal middle concentration LD of the venom peptide ₅₀ Corresponding concentration gradients were set for injection. LD ₅₀ Values and chi-square values are shown in table 1 (mortality at each concentration is shown in table 2):

table 1: nbX4 toxicity determination of tetranychus cinnabarinus

Note: chi square value (x) ² ) Numerical value indicates the passage of the chi-square test (P < 0.05)

Table 2: mortality rate of NbX-4 to tetranychus cinnabarinus adults under different concentrations

Sequence listing

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Claims (6)

1. An acaricidal peptide, the amino acid sequence of which is shown in SEQ ID No. 3.

2. A gene encoding the miticidal peptide of claim 1.

3. The gene of claim 2, wherein the nucleotide sequence is as shown in SEQ ID No. 4.

4. An expression vector contains a gene for coding a protein shown as SEQ ID No. 3.

5. The expression vector of claim 4, wherein the nucleotide sequence of the gene is shown in SEQ ID No. 4.

6.application of the polypeptide shown in SEQ ID No.3 in prevention and treatment of tetranychus cinnabarinus.

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