Otes fuscipes serine protease inhibitor ScSPI5 gene and application thereof

Abstract

According to the invention, a cDNA sequence of the hunting orius venom serine protease inhibitor ScSPI5 is cloned, the cDNA sequence is as shown in SEQ ID No.1, and the coded amino acid is as shown in SEQ ID No.2. The recombinant protein obtained through insect baculovirus in-vitro expression can effectively inhibit the hemolymph phenol oxidase activity of agriculture and forestry pests such as tenebrio molitor, alunanensis and spodoptera frugiperda, and has the advantages that the activity of the hemolymph phenol oxidase can be effectively inhibited, the activity of the hemolymph phenol oxidase can be effectively inhibited, the activity of the hemolymph phenol oxidase can be effectively inhibited, the activity of the hemolymph phenol oxidase can be effectively inhibited, and the activity of the hemolymph phenol oxidase can be effectively inhibited. Therefore, the immunoreaction is inhibited, and the immunity is reduced. Meanwhile, the weight of larvae and pupae of spodoptera frugiperda can be reduced by orally taking the recombinant protein; tenebrio molitor pupae can be killed by injecting the recombinant protein, so that an important reference value is provided for developing a novel biological control insecticide.

Description

Technical Field

[0001] The invention belongs to the fields of molecular biology, genetic engineering and protein engineering, and particularly relates to a functional study of a serine protease inhibitor ScSPI5 expressed in the venom of a yellow-banded rhinoceros assassin bug. Background Art

[0003] The yellow-banded rhinoceros assassin bug (Sycanus croceovittatus) can efficiently prey on a variety of major agricultural and forestry pests. When preying on prey, it pierces its mouthparts into the body of the prey and secretes venom from the venom glands to transport venom into the body of the prey. These venoms are a complex mixture of proteases, protease inhibitors, small molecule peptides, salts, etc., with rich and diverse physiological functions such as paralysis, lethality, tissue lysis, neurotoxicity, antibacterial and inhibition of enzyme activity. Among them, serine protease inhibitors are one of the main components of the venom of the yellow-banded rhinoceros assassin bug, and they are also widely distributed in almost all species. In the venom of parasitic wasps, it can participate in regulating the growth and development, physiological metabolism and immune response of the host. Therefore, studying the serine protease inhibitors, the main components of the venom of the yellow-banded rhinoceros assassin bug, is conducive to the development of biological control agents, which is of great significance for promoting sustainable agricultural development and ecological protection. Summary of the invention

[0004] The purpose of the present invention is to provide a ScSPI5 gene of a serine protease inhibitor of a yellow-banded rhinoceros assassin bug and its application.

[0005] To achieve the above-mentioned purpose, the technical solution adopted by the present invention is: based on high-throughput genome and transcriptome sequencing methods, using yellow-banded rhinoceros assassin bug as experimental material, a serine protease inhibitor (SPI) gene encoding a complete serpin domain and a reaction center ring was screened through gene annotation. Since this gene is the fifth gene distributed on the chromosome, we named it ScSPI5, and its nucleotide sequence is shown in SEQ ID No.1.

[0006] The second object of the present invention is to provide a protein encoded by the ScSPI5 gene, characterized in that the amino acid sequence of the protein is shown in SEQ ID No.2.

[0007] The third object of the present invention is a primer for amplifying the ScSPI5 gene, and the primer sequence is shown in SEQ ID No.3-4.

[0008] The fourth object of the present invention is a kit for amplifying the ScSPI5 gene, the kit containing the primers according to claim 3.

[0009] The fifth object of the present invention is a recombinant expression vector, which contains the ScSPI5 gene according to claim 1.

[0010] The sixth object of the present invention is a recombinant expression cell, characterized in that the recombinant expression cell contains the recombinant expression vector according to claim 5.

[0011] Furthermore, it is characterized in that the host cell is a Spodoptera frugiperda Sf9 cell.

[0012] In the present invention, the ScSPI5 gene can be used for the preparation of serine protease inhibitor drugs, including the preparation of biological control drugs for the yellow powder beetle, Yunnan moth or fall armyworm.

[0013] The beneficial technical effect of the present invention is that the present invention clones a cDNA sequence of the serine protease inhibitor ScSPI5 of the venom of the yellow-banded rhinoceros assassin bug, and the recombinant protein obtained by in vitro expression of insect baculovirus can effectively inhibit the lymph phenol oxidase activity of agricultural and forestry pests such as Tenebrio molitor, Achelura yunnanensis, and Spodoptera frugiperda, thereby inhibiting their immune response and reducing their immunity. At the same time, oral administration of the recombinant protein can reduce the weight of the larvae and pupae of the Spodoptera frugiperda; injection of the recombinant protein can kill the pupae of the mealworm, which provides important reference value for the development of new biological control insecticides. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0015] Figure 1 This is the result of nucleotide and protein sequence analysis of the serine protease inhibitor ScSPI5 of the yellow-banded rhinoceros assassin bug. The reaction center loop of ScSPI5 is marked on the gray background; the amino acid at the P1 site is arginine (Arg); and * indicates a stop codon.

[0016] Figure 2Figure 1 shows the results of ScSPI5 recombinant Bacmid PCR and P1 virus Western bolt detection. Figure A, recombinant Bacmid plasmid PCR identification results. Lane M: DL5000 marker; Lane +: positive control; Lane -: negative control. Figure B, virus Western bolt results (His tag antibody). Lane +: positive control; Lane -: negative control; Lane M: protein marker; Lane 1-2: culture medium; Lane 3-4: cells: culture medium.

[0017] Figure 3 The SDS-PAGE and Western bolt detection images of ScSPI5 purified protein. Lane M: protein marker; Lane 1-2: extracellular protein purification.

[0018] Figure 4 This is a graph showing the inhibitory activity of the recombinant protein ScSPI5, a serine protease inhibitor of the yellow-banded rhinoceros assassin bug, on trypsin. “+” is the positive control protease inhibitor, and “-” is the negative control phosphate buffer solution. Different lowercase letters represent significant differences.

[0019] Figure 5 The results show that the recombinant protein ScSPI5, a serine protease inhibitor of the yellow-banded rhinoceros assassin bug, inhibits phenoloxidase in the hemolymph of three insects. The positive control "+" is the addition of 1-phenyl-2-thiourea solution, and the positive control "-" is the addition of phosphate buffer solution. Different lowercase letters represent significant differences.

[0020] Figure 6 Figure 1 is a statistical chart of the weight of fall armyworms fed with artificial diet containing ScSPI5 recombinant protein. Figure A is a statistical chart of the weight of larvae fed with artificial diet containing ScSPI5 recombinant protein. Figure B is a statistical chart of the weight of pupae fed with artificial diet containing ScSPI5 recombinant protein. Con is the artificial diet without treatment for fall armyworms, PBS is the artificial diet with phosphate buffer solution for fall armyworms, ScSPI5 is the artificial diet with ScSPI5 recombinant protein for fall armyworms, and different lowercase letters represent significant differences.

[0021] Figure 7 Figure 1 is a statistical chart of the mortality of mealworm pupae injected with ScSPI5 recombinant protein. Figure A is the survival rate of mealworm pupae injected with different doses of ScSPI5. Figure B is the lethal medium dose of ScSPI5 to mealworm. Con is untreated mealworm pupae, PBS is mealworm pupae injected with phosphate buffer, and different lowercase letters represent significant differences. DETAILED DESCRIPTION

[0022] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0023] The present invention discloses a gene of the serine protease inhibitor ScSPI5 of the yellow-banded rhinoceros assassin bug, the open reading frame of which is 1263 bp, the sequence of which is shown in SEQ ID No. 1, encoding a protein composed of 420 amino acids, the amino acid sequence of which is the amino acid sequence shown in SEQ ID No. 2. By performing multiple sequence alignment with serine protease inhibitors of known species, the reaction center loop and P1 active site information of ScSPI5 are predicted, and the results are as follows: Figure 1 As shown, the amino acid sequence of the reaction center loop region is: EEGTEAAAATEVGVESRSLLPREKQFIADHPF, and the amino acid residue at the P1 position is arginine (Arg).

[0024] The present invention provides a method for preparing the recombinant protein, comprising the steps of cloning the ScSPI5 gene into a pFastBac vector, transforming the constructed plasmid into DH10Bac competent cells to prepare a recombinant Bacmid plasmid, transfecting the recombinant plasmid into Spodoptera frugiperda Sf 9 cells to produce P1 virus, selecting P1 virus with better expression to amplify P2 virus, and purifying using Ni NTABeads 6FF to obtain the ScSPI5 recombinant protein.

[0025] The present invention further provides the use of the obtained serine protease inhibitor ScSPI5 gene: the protease inhibitor can effectively inhibit the activity of trypsin; inhibit the activity of hemolymph phenoloxidase of Yunnan moth, yellow mealworm, and fall armyworm; reduce the weight of fall armyworm larvae and pupae; and cause the death of yellow mealworm pupae. The protease inhibitor has potential application value in controlling harmful organisms.

[0026] 1. cDNA cloning of the serine protease inhibitor ScSPI5 gene of the yellow-banded rhinoceros assassin bug

[0027] Example 1

[0028] The yellow-banded rhinoceros assassin bug used in the present invention is a population raised for multiple generations in the insect breeding room of the Yunnan Forest Disaster Warning and Key Laboratory, and the yellow-banded rhinoceros assassin bug is raised using mealworm larvae. Rearing conditions: temperature 27 ± 1 ° C, relative humidity 65 ± 5%, photoperiod 14 L: 10 D. According to the sequence of the gene sequence SEQ ID No.1 predicted in the genome of the yellow-banded rhinoceros assassin bug, gene-specific primers containing restriction sites BamH I and Xho I were designed. Forward primer F: 5'-CGGGATCCATGAAGCTGTGCATCCTGCTG-3' (SEQ ID No.3), reverse primer R: 5'-CCGCTCGAGTTACTTGTCGTCGTCGTCCTTGT-3' (SEQ ID No.4). The total RNA of the sample was extracted according to Trizol reagent (Invitrogen, USA), and the specific operation was as follows:

[0029] (1) Disinfect the adult yellow-banded rhinoceros assassin bug with 75% alcohol, wash the insect body with phosphate buffer solution, use filter paper to absorb the residual solution, dissect the posterior lobe of the main gland of the yellow-banded rhinoceros assassin bug under a dissecting microscope, collect it in a 1.5 mL centrifuge tube containing 100 μL Trizol (Invitrogen, USA) solution, freeze it in liquid nitrogen, and quickly grind it with a grinding rod, then add Trizol solution to make up the volume to 1 mL. After mixing, lyse it at room temperature for 5 min.

[0030] (2) Centrifuge the completely lysed sample at 4°C, 12,000 rpm for 15 min, and transfer the supernatant to a new 1.5 mL centrifuge tube.

[0031] (3) Add 200 μL of chloroform to the supernatant, shake vigorously for 15 s, leave at room temperature for 5 min, and centrifuge at 12,000 rpm for 15 min at 4 °C.

[0032] (4) Transfer the supernatant to a new centrifuge tube, add 500 μL of isopropanol, leave at room temperature for 10 min, and centrifuge at 4 °C, 12,000 rpm for 10 min.

[0033] (5) Discard the supernatant, add 1000 μL of freshly prepared 75% ethanol to the centrifuge tube and mix well. Centrifuge at 7600 rpm at 4 °C for 5 min, discard the supernatant and retain the precipitate.

[0034] (6) The residual ethanol was evaporated at room temperature. After the precipitate was dried, 30 μL of DEPC water was added to dissolve the precipitate and mixed. Its integrity was detected by 1.2% agarose gel electrophoresis, and the total RNA concentration and quality were determined using a NanoDrop 2000 ultra-micro spectrophotometer (ThermoFisher Scientific, USA). 1 μg of total RNA was taken and cDNA was synthesized using the Prim-Script RTReagent Kit with gDNA Eraser Reverse Transcription Kit (TaKaRa, China). The synthesized cDNA template was stored at -20 °C for future use.

[0035] (7) The ScSPI5 gene was cloned by PCR using the Dream Taq Green PCR Master Mix Kit (TaKaRa, China). A 25 µL reaction system was used, and the volumes of each reagent were as follows: 12.5 µL of 2 × Dream Taq Green PCR premix, 0.5 µL of each forward and reverse primer, 1 µL of cDNA template, and 10.5 µL of Nuclease-Free Water. PCR reaction conditions: 95 °C pre-denaturation for 3 min, 95 °C denaturation for 30 s, 58 °C annealing for 30 s, 72 °C extension for 1 min, 35 cycles, 72 °C extension for 15 min, and finally cooling to 4 °C.

[0036] 2. Baculovirus expression and purification of ScSPI5, a serine protease inhibitor of the yellow-banded rhinoceros bug

[0037] Example 2

[0038] The cloned ScSPI5 gene was ligated into the pFastBac vector, and the constructed plasmid was transformed into DH10Bac competent cells (Invitrogen, USA). Positive colonies were screened by Sanger sequencing and the accuracy of the gene sequence was verified ( Figure 2 A) Screen the recombinant plasmid containing the correct gene sequence. Take 2 mL of Spodoptera frugiperda Sf9 cells of appropriate density and inoculate them into a six-well plate. Let the cells adhere to the wall at 27 °C. After sufficient amount of plasmid is fully reacted with Cellfectin II Reagent (Thermo Fisher Scientific, USA) transfection reagent, add Sf-900 II SFM (ThermoFisher Scientific, USA) medium to make the volume 1 mL and mix well, then gently add it to the Spodoptera frugiperda Sf 9 cells.

[0039] Cultivate at 27°C until cells show signs of virus infection (4-7 days). Centrifuge the remaining culture and transfer it to a 2 mL sterile centrifuge tube. Store the P1 virus at 4°C in the dark and detect its expression using Western Blot. Figure 2 B). P1 virus with high expression quality was screened and expressed at 2.0 × 10 6 cells / mL was inoculated into a 30 mL Erlenmeyer flask of Spodoptera frugiperda Sf 9 cells. The cells were cultured at 27 °C and 120 rpm for 3 to 5 days to promote the amplification of P2 virus. Afterwards, 5 mL of P2 virus was purified using Ni NTA Beads 6FF. The samples were then eluted with phosphate buffer containing 20 mM imidazole (pH 7.4), 40 mM imidazole (pH 7.4), and 500 mM imidazole (pH 7.4) in sequence. Protein samples were analyzed by SDS-PAGE electrophoresis and then detected by Western bolt using His-tag monoclonal antibody (Proteintech, China).

[0040] Take 200 mL of a solution with a density of 2.0 × 10 6 Cells / mL of High five were inoculated into a triangular flask, and then an appropriate amount of P2 virus was added to the triangular flask for amplification and cultured at 27°C and 120 rpm for 3 to 5 days until signs of infection appeared. 6 Spodoptera frugiperda Sf 9 cells with a concentration of cells / mL were transferred to a triangular flask, and an appropriate amount of P2 virus was added for expression amplification. The culture was then collected and centrifuged to separate the cells and the culture medium. The entire culture medium was purified with Ni NTABeads 6FF (Sigma-Aldrich, USA), and the samples were eluted with phosphate buffer solutions containing 20 mM imidazole (pH 7.4), 40 mM imidazole (pH 7.4), and 500 mM imidazole (pH 7.4) in sequence. The obtained protein samples were again detected by SDS-PAGE (12%) electrophoresis and Western bolt (Proteintech, China), and finally the ScSPI5 extracellular purified recombinant protein ( Figure 3 The concentration of the purified recombinant protein was quantified using a BCA protein assay kit (TaKaRa, China).

[0041] III. Functional study of the serine protease inhibitor ScSPI5 gene in the venom of the yellow-banded rhinoceros assassin bug of the present invention

[0042] Example 3

[0043] 20 μL of different doses (2 μg, 4 μg, 6 μg, 8 μg) of ScSPI5 recombinant protein were added to 5 μL of 1 mg / mL trypsin (Solerbo, China) solution and mixed well. Then 130 μL of Gly-HCl buffer (Solerbo, China) was added and incubated in a metal bath at 37 °C for 5 min. After the incubation, 50 μL of 1 mM chromogenic substrate Nα-benzoyl-L-arginine-4-nitroaniline hydrochloride (Aladdin, China) solution was added and mixed well, and the mixture was placed in a metal bath at 37 °C for 5 min. 150 μL of the mixed solution was placed in a microplate reader (TECAN, Switzerland) and the absorbance was measured at a wavelength of 405 nm. In the positive control group, ScSPI5 was replaced with an equal volume of protease inhibitor diluted 10 times, and in the negative control group, an equal volume of Gly-HCl buffer was replaced. Three biological replicates were set for each group, and a bar graph of the residual enzyme activity of the ScSPI5 recombinant protein dose-dependent was drawn. The experimental results are shown in Figure 2. Figure 4 As shown in the figure: With the increase of the dose of ScSPI5 recombinant protein, the residual enzyme activity of trypsin continued to decrease. When 8 μg of recombinant protein was added, the inhibitory effect of trypsin activity was equivalent to that of the positive control, and the inhibition rate of trypsin activity reached 59% ( Figure 4 ). The results showed that the recombinant serine protease inhibitor had a strong inhibitory effect on trypsin, which is both the most important digestive enzyme in insects and a key serine protease that regulates human metabolism. Therefore, the recombinant serine protease inhibitor has potential application value as a pest control agent and a drug for regulating human metabolism.

[0044] Example 4

[0045] The larvae of the yellow powder beetle, fall armyworm and Yunnan moth were placed on ice for freezing anesthesia. The pupae and larvae were cut open with sterile scissors to allow the hemolymph to flow out. 50 μL was collected in a centrifuge tube and centrifuged at 4500 rpm for 10 min at 4 ℃ to remove blood cells. The supernatant was collected and the total protein concentration in the supernatant of each group was determined by BCA method. 2 μL of supernatant was added to the centrifuge tube, and then 5 μg, 10 μg, and 15 μg of ScSPI5 recombinant protein were added respectively. The reaction was carried out at room temperature for 10 min. After the reaction was completed, 10 μL of phosphate buffer solution was added, and then 20 mM L-DOPA (Aladdin, China) solution was added to make the final volume 194 μL. The mixed solution was added to the enzyme-labeled well strip and the absorbance was measured at a wavelength of 490 nm using an enzyme reader (TECAN, Switzerland). The recombinant protein was replaced with an equal dose of bovine serum albumin solution (BioFroxx, China) as a negative control, and 0.02 M 1-phenyl-2-thiourea solution (Yuanye, China) as a positive control, with three biological replicates for each group. One unit of phenol oxidase activity was defined as 0.001∆A490 / min. The results were obtained by Figure 5 As shown in Figure 2, when 5 μg of ScSPI5 recombinant protein was added, the activity of phenoloxidase in the hemolymph of three insects could be effectively inhibited ( Figure 5 ). Phenol oxidase is an important factor in the innate immune melanization reaction of insects. The serine protease inhibitor ScSPI5 can effectively inhibit its immune melanization reaction and affect the defense of insects against the invasion of foreign pathogens. The serine protease inhibitor ScSPI5 in the present invention can provide sequence reference and experimental basis for the development of new insecticides.

[0046] Example 5

[0047] Twenty fifth-instar fall armyworm larvae of similar weight were starved for 12 h. Freshly prepared artificial diet was cut into small pieces (10 × 10 × 10 mm) and 4 μg of ScSPI5 recombinant protein was added. Afterwards, the larvae were placed in a plastic box and transferred to a constant temperature incubator at 27 °C and 65% relative humidity for breeding. In order to keep the protein activity constant and provide the larvae with sufficient fresh food source, the feed containing ScSPI5 recombinant protein was changed once a day. An equal volume of ultrapure water and untreated feed were fed as a control. The weight of the larvae was recorded on the 5th day of the experiment, and the weight of the pupae was measured after pupation. The results are shown in Figure 6 As shown, the body weight of Spodoptera frugiperda larvae fed with diet containing ScSPI5 was reduced by about 15% compared with the body weight of larvae without treatment or larvae fed with diet containing ultrapure water ( Figure 6 A), the larvae continued to develop and pupate, and the weight of the pupae fed with ScSPI5 was also reduced by about 15% compared with the control group ( Figure 6B). Therefore, ScSPI5 can be used to develop novel pest management strategies by inhibiting the growth and development of pests.

[0048] Example 6

[0049] The newly molted mealworm pupae were disinfected with 75% alcohol, washed with phosphate buffer solution, and then the excess water was absorbed with filter paper. 5 µL of ScSPI5 recombinant protein at different doses (0.0625 µg, 0.125 µg, 0.25 µg, 0.5 µg, 1 µg) was injected into the body through the 7th abdominal segment of the mealworm pupae using a microsyringe, and the pupae were transferred to a glass dish and placed in a constant temperature incubator at 27 °C and a relative humidity of 65%. The survival rate of the pupae was monitored and recorded every 24 h until the pupae emerged as adults. The control group included pupae injected with an equal volume of phosphate buffer solution and pupae without treatment. Three biological replicates were set for each group, and 20 pupae were set for each replicate. The results are shown in the figure. Figure 7 As shown in Figure 2, with the increase of ScSPI5 dosage, the survival rate of P. truncatula decreased ( Figure 7 A), which showed that ScSPI5 had a dose-dependent lethal effect. At 1 μg, ScSPI5 could reduce the survival rate of P. fulgens to 26.6% (Fig. Figure 7 A). Based on the mortality caused by different doses of ScSPI5, the median lethal dose of ScSPI5 was 5.6 ± 1.1 µg / g ( Figure 7 B). The serine protease inhibitor ScSPI5 of the present invention can effectively kill yellow mealworms, which can provide reference value and experimental basis for controlling coleopteran pests and developing new biological control insecticides.

[0050] Finally, it should be noted that the above embodiments are only used to illustrate rather than limit the technical solutions of the present invention. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that the present invention can still be modified or replaced by equivalents. Any modification or partial replacement that does not depart from the spirit and scope of the present invention should be included in the scope of the claims of the present invention.

Claims

1. The ScSPI5 gene of the serine protease inhibitor of the yellow-banded rhinoceros assassin bug is characterized by: The nucleotide sequence of the ScSPI5 gene is shown in SEQ ID No.

1.

2. A protein encoded by the ScSPI5 gene according to claim 1, characterized in that: The amino acid sequence of the protein is shown in SEQ ID No.

2.

3. A primer for amplifying the ScSPI5 gene according to claim 1, wherein the primer sequence is shown in SEQ ID No.3-4.

4. A kit for amplifying the ScSPI5 gene according to claim 1, characterized in that: The kit contains the primers according to claim 3.

5. A recombinant expression vector, characterized in that: The recombinant expression vector contains the ScSPI5 gene according to claim 1.

6. A recombinant expression cell, characterized in that: The recombinant expression cell contains the recombinant expression vector according to claim 5.

7. The recombinant expression cell according to claim 6, characterized in that: The host cells are Spodoptera frugiperda Sf9 cells.

8. Use of the ScSPI5 gene according to claim 1 in the preparation of serine protease inhibitor drugs.

9. The use according to claim 8, characterized in that: The application is application in the preparation of biological control drugs for the yellow powder beetle, Yunnan moth moth or fall armyworm.

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