Application of Venus flytrap ADP-glucose pyrophosphorylase in the control of citrus fruit fly

Abstract

The present invention discloses an application of Venus flytrap ADP-glucose pyrophosphorylase in the prevention and control of citrus fruit fly. As a new biological insecticide resource, the protein has a mode of action different from that of existing insecticides, and has important scientific and practical significance for expanding new insecticide gene resources with biological activity, reducing various safety risks caused by the use of existing chemical insecticides, and reducing the use of chemical pesticides in the field.

Description

Technical Field

[0001] The invention belongs to the field of genetic engineering and biological control, and specifically relates to application of Venus flytrap ADP-glucose pyrophosphorylase in the prevention and control of citrus fruit flies. Background Art

[0002] At present, chemical pesticides are widely used in the control of citrus fruit flies in agriculture. However, due to its strong reproductive capacity, the citrus fruit flies have a prominent problem of resistance to chemical pesticides. The insect has developed resistance to organophosphates, pyrethroids, carbamate insecticides and avermectins (Han HAA, et al. Trichlorfon and SpinosadResistance Survey and Preliminary Determination of the Resistance Mechanismin Pakistani Field Strains of Bactrocera dorsalis. Scientific Reports. 2018,8(1): 11223.). Therefore, the development of new, efficient and environmentally friendly insecticide active substances can enrich the control methods of citrus fruit flies and reduce the use of chemical pesticides in the field.

[0003] As an insectivorous plant, the Venus flytrap ( Dionaea muscipula) can attract and capture pests, and use the glands in the insect trap to secrete digestive juices to digest the pests. The digestive juices in the insect trap contain a variety of hydrolases, secondary metabolites and other substances, which can hydrolyze structures such as insect exoskeleton and chitin, and finally digest and absorb the nutrients of the insects (Hedrich R, et al. Onthe Origin of Carnivory: Molecular Physiology and Evolution of Plants on anAnimal Diet. Annual Review of Plant Biology. 2021, 72(1): 133-153.). These enzymes have the potential to be developed into new plant-derived insecticidal active substances. ADP-glucose pyrophosphorylase is a key enzyme in starch biosynthesis. It catalyzes the conversion of photosynthetically derived glucose-1-phosphate (Glc-1-P) into the active form of the glucosyl donor ADP-glucose (ADP-Glc), and plays an important role in plant growth and stress response (Dhaka A, et al. A comprehensive study on core enzymes involved in starch metabolism in the model nutricereal, foxtail millet ( Setaria italica L. However, current research on ADP-glucose pyrophosphorylase mainly focuses on its role in plant development, stress resistance and other aspects of the plant itself, and rarely extends it to pest control. Summary of the invention

[0004] The technical problem to be solved by the present invention is to provide application of Venus flytrap ADP-glucose pyrophosphorylase in the prevention and control of citrus fruit fly.

[0005] The technical solution of the present invention is: application of Venus flytrap ADP-glucose pyrophosphorylase DMAGP1 in the prevention and control of citrus fruit fly, and the amino acid sequence of the DMAGP1 is shown in SEQ ID No.4.

[0006] Compared with the prior art, the present invention has the following beneficial effects:

[0007] The present invention uses a bioinformatics analysis method to screen genes in a transcriptome database of Venus flytrap digestion fluid to obtain candidate genes for controlling citrus fruit flies, and then uses polymerase chain reaction (PCR) and Sanger sequencing to obtain the complete sequence of the gene. The mature peptide region of the gene is constructed into a prokaryotic expression vector pET-28a-SUMO, and the insecticidal protein DMAGP1 encoded by the gene can be obtained through prokaryotic system expression. The preparation cycle is short, the amino acid sequence is small, and it is suitable for large-scale in vitro production. At the same time, as a new insecticidal gene resource, the insecticidal protein has a mode of action different from that of existing insecticides, and has important scientific and practical significance for expanding new insecticidal gene resources with biological activity, reducing various safety risks caused by the widespread use of existing chemical insecticides, and reducing the amount of chemical pesticides used in the field. BRIEF DESCRIPTION OF THE DRAWINGS

[0008] Figure 1 SDS-PAGE electrophoresis of the Venus flytrap recombinant protein DMAGP1; 1 is the purified protein, and 2 is the marker;

[0009] Figure 2 A shows the mortality of Bactrocera dorsalis at different time points after injection of CK and recombinant protein DMAGP1. Asterisks indicate significant differences (P<0.05). B shows the death phenotype of Bactrocera dorsalis after injection of recombinant protein DMAGP1. DETAILED DESCRIPTION

[0010] The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are purchased from commercial channels unless otherwise specified.

[0011] The reagents and culture medium formulas involved in the examples are:

[0012] (1) LB liquid medium:

[0013] Add 2 g of tryptone, 1 g of yeast extract and 2 g of NaCl to 200 ml of deionized water, stir to mix, place in an autoclave, sterilize at 121°C for 20 min, add antibiotics after cooling, and store at 4°C.

[0014] (2) LB solid medium:

[0015] Add 2 g of tryptone, 1 g of yeast extract, 2 g of NaCl and 3 g of agar to 200 ml of deionized water, stir to mix, place in an autoclave, sterilize at 121°C for 20 min, cool to 50°C, add kanamycin with a final concentration of 50 mg / L, pour into plates, cool and store at 4°C.

[0016] (3) Washing Buffer:

[0017] 0.04 mM PBS, 0.5 M sodium chloride, 20 mM imidazole, dilute to 1 L with double distilled water, pH 7.4.

[0018] (4) Elution Buffer:

[0019] 0.04 mM PBS, 0.5 M sodium chloride, 500 mM imidazole, and double distilled water to 1 L, pH 7.4.

[0020] (5) Kanamycin (Kana, 50 mg / mL): weigh 0.5 g of kanamycin into a 10 mL volumetric flask, mix thoroughly with sterile water to dissolve, and then make up to 10 mL. Sterilize by filtering through a 0.22 μm disposable filter membrane, and store in aliquots in a dark place at -20°C until use.

[0021] (6) IPTG (24 mg / mL): Weigh 1.2 g IPTG into a 50 mL centrifuge tube, add 40 mL sterile water, mix thoroughly to dissolve, and adjust the volume to 50 mL. Sterilize through a 0.22 μm disposable filter membrane, and store in a -20 °C refrigerator for later use.

[0022] Example 1

[0023] Using bioinformatics analysis methods, genes in the transcriptome database of Venus flytrap digestion fluid were screened to obtain the candidate gene DMAGP1 (SEQ ID No. 2) for controlling citrus fruit flies. The amino acid sequence of the protein encoded by this gene is shown in SEQ ID No. 1. The mature peptide sequence was codon-optimized according to the codon preference of Escherichia coli, and the gene sequence of DMAGP1 mature peptide for expression in Escherichia coli was designed (shown in SEQ ID No. 3), and the encoded recombinant protein sequence is shown in SEQ ID No. 4. The gene was cloned into the Escherichia coli expression vector pET-28a-SUMO to construct the pET-28a-SUMO-DMAGP1 recombinant plasmid containing the target gene, and the protein gene was introduced into the BamHI / XhoI restriction site.

[0024] Inducible expression of recombinant plasmid pET-28a-SUMO-DMAGP1

[0025] E.Coli BL21 (DE3) strain was selected for competence. Single positive plaques were picked from the LB plate and added to LB liquid culture medium containing 50 mg / ml kanamycin, and the culture was continued at 200 rpm, 37°C, for 6 h in a constant temperature shaker. 15 ml of the correctly sequenced bacterial solution was expanded and cultured. The cultured bacterial solution was inoculated into 200 ml LB liquid culture medium containing 50 mg / ml kanamycin at a ratio of 1:100. The culture was cultured at 220 rpm and 37°C until the OD600 value of the bacterial solution was 0.5-0.6. IPTG with a final concentration of 0.2 mM was added to the pET-28a-SUMO-DMAGP1 recombinant, and the expression was induced for 15-18 h at 16°C and 150 rpm; the expressed bacterial solution was taken out and centrifuged at 4°C and 4000 rpm for 20 min, the supernatant was discarded, and 0.01 mM PBS was added to the bacterial pellet to resuspend it and the volume was fixed to 20 ml. Then, ultrasonic disruption was performed for 30 minutes, running for 8 seconds and pausing for 8 seconds. After the disruption was completed, centrifuge at 4°C, 9000 rpm for 30 minutes, take the supernatant and discard the precipitate.

[0026] Purification of recombinant protein pET-28a-SUMO-DMAGP1

[0027] The supernatant after the large amount of expression induction and expression was purified using a Ni-NTA 6FF pre-loaded gravity column. Wait for the preservation solution in the gravity column to flow out freely, add 1 volume of Washing Buffer to equilibrate the nickel column, and the maximum flow rate is 150 cm / h. After all the target protein is loaded, use 3 volumes of Washing Buffer to wash the impurities, and finally elute the target protein with 5 ml of Elution Buffer ( Figure 1 ).

[0028] Determination of the concentration of recombinant protein pET-28a-SUMO-DMAGP1

[0029] The concentration of the target protein was determined using the Coomassie Brilliant Blue method. Dilute 5×G250 to 1×. Mix 1×G250 with the target protein and PBS in a ratio of 4:1. Set up technical replicates and measure the absorbance at 595nm. Substitute the measured absorbance into the bovine serum standard curve to calculate the protein content in the sample. In this experiment, the concentration of DMAGP1 was 410μg / ml.

[0030] Example 2

[0031] Bioassay process and methods

[0032] The insecticidal activity of recombinant protein DMAGP1 against Bactrocera dorsalis was determined by microinjection. Adults of similar size were selected, and 3 replicates were used for each treatment. 30 adults of Bactrocera dorsalis were selected for each replicate. Each adult was injected with 400nl of recombinant protein, and the injection site was the abdomen. After the injection, the adults were placed in the insect cage. The whole process should be gentle to reduce secondary damage to Bactrocera dorsalis. The experiment was divided into a control group and an experimental group. The control group was injected with TBS, and the experimental group was injected with recombinant protein DMAGP1. Figure 2 A in the middle is the mortality result after injection of DMAGP1, showing that the protein has good insecticidal activity; B is the death phenotype of citrus fruit fly after injection of recombinant protein DMAGP1.

[0033] It can be seen from the above experiments that the recombinant protein DMAGP1 of the present invention has good insecticidal activity against Bactrocera dorsalis and has very good application prospects.

Claims

1. Application of Venus flytrap ADP-glucose pyrophosphorylase DMAGP1 in the prevention and control of citrus fruit fly, wherein the amino acid sequence of DMAGP1 is shown in SEQ ID No.4.

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