Pacifastin serine protease inhibitor protein of the parasitoid of the gypsy moth, pcpPI, and uses thereof

Abstract

This invention belongs to the fields of molecular biology, genetic engineering, and protein engineering; specifically, it relates to a Pacifastin serine protease inhibitor PqPI expressed in the venom of the grassland caterpillar wasp, its encoded nucleic acid sequence, and functional studies. This invention provides a Pacifastin serine protease inhibitor PqPI from the grassland caterpillar wasp, which has the amino acid sequence shown in SEQ ID NO:2. This invention also provides the use of the above-mentioned Pacifastin serine protease inhibitor PqPI from the grassland caterpillar wasp: for the preparation of Pacifastin serine protease inhibitors from the grassland caterpillar wasp, which can be used to inhibit the activation of PPO in the hemolymph of grassland caterpillars or cabbage caterpillars.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of molecular biology, genetic engineering and protein engineering. In particular, it relates to a Pacifastin serine protease inhibitor PqPI expressed in the venom of Gynaephora roboris and its encoding nucleic acid sequence and functional research. BACKGROUND

[0002] Grassland is an important ecological system in China, which is a natural ecological security barrier and has extremely important ecological and productive value. In recent years, due to unreasonable utilization of grassland and abnormal changes in climate, grassland pests have become more serious year by year, which seriously restricts the production of China's animal husbandry and the construction of ecological civilization. Gynaephora qinghaiensis is an important pest distributed in the alpine meadow of Qinghai-Tibet Plateau in China, which causes very serious damage to the alpine meadow vegetation. Therefore, it is necessary to study the important natural enemy of Gynaephora qinghaiensis, i.e. Gynaephora roboris parasitoid.

[0003] Parasitoid is an important natural enemy of insects, which has important biocontrol value. Parasitoid carries multiple parasitic factors, including venom, polydnavirus (PDV), teratocyte, etc., which can regulate the physiological activities of host immune response, growth and development. The study of parasitic factors of parasitoid can serve production and open up a new way for biological control of pests. Pacifastin is a family of serine protease inhibitors, most of which are multi-domain precursor proteins, which can regulate the innate immune response of invertebrates and arthropods, and play a crucial role in regulating the phenol oxidase system (such as phagocytosis of invertebrates). The structure and biochemical characteristics of insect pacifastin peptides have been intensively studied, including the regulation of melanization in arthropods. The venom of Gynaephora roboris contains serine protease inhibitors, which are the main regulators of serine protease activity and play a key role in digestion, development, innate immunity and other physiological regulation processes.

[0004] Insect hemocytes are not only responsible for cellular immunity, but also participate in humoral defense through typical factors such as antimicrobial peptides (AMPs), agglutinins, lysozymes, etc. Melanization is a unique and important natural immune mechanism in insects, which is mediated by a cascade of serine proteases to cleave prophenoloxidase, and this process is negatively regulated by serine protease inhibitors. Hemolymph melanization is a conserved immune response in insects and other arthropods, which is involved in many molecular or biochemical reactions, such as serine protease cascade and prophenoloxidase cascade.

[0005] It has been proven that the key enzyme of melanin, the precursor of prophenoloxidase (proPO) produced by ovarian cells, helps to immunize melanin. The melanization reaction mediated by proPO is an important immune response in invertebrates. The insect melanization reaction is part of the insect's natural immune system, and by studying this process, we can gain a deeper understanding of the defense mechanisms of insects against external pathogen invasion. This helps to reveal the basic mechanisms of the immune response, facilitating our overall understanding of the immune system of living organisms. The insect melanization reaction has an impact on the health of individual insects and populations, and studying this immune response can help us understand the impact of environmental stress on insect populations and how to maintain ecological balance. The study of insect melanization reaction also helps to explore the role of the immune system in the process of biological evolution. This helps to understand the evolution of immune adaptation and its role in biodiversity and species survival. SUMMARY

[0006] The technical problem to be solved by the present application is to provide a functional exploration of a grassland moth Cotesia plutellae venom serine protease inhibitor that has an immunosuppressive effect (inhibits hemolymph melanization) on common grassland pests.

[0007] To solve the above technical problems, the present application provides a grassland moth Cotesia plutellae Pacifastin serine protease inhibitor PqPI, which has the amino acid sequence shown in SEQ ID NO: 2.

[0008] Note: SEQ ID NO: 2 includes a signal peptide, and the underlined amino acid sequence is the signal peptide segment.

[0009] MIAYVFVMYFSTFFISDIYSSSA QIVPDRDDWYKTGLKYDDLPRVFSLKDGCPAKEFYL DCNTCSCVGDSSDPACTYKACPQPPQIVNWKTGKPCPAGQHFYWKCNDCNCAENGREAS CTRNFCPDFGDSSQ*

[0010] Improvements of the grassland moth Cotesia plutellae Pacifastin serine protease inhibitor PqPI of the present application: proteins, their conservative variant proteins, their active fragments, or their active derivatives.

[0011] The application also simultaneously provides a gene encoding the PqPI of the pacifastin serine protease inhibitor of the Cotesia rubecula, which has the nucleotide sequence of 70-399 in SEQ ID NO: 1, or has at least 70 % homology with the nucleotide sequence of 70-399 in SEQ ID NO: 1, or the nucleotide sequence thereof can hybridize with the nucleotide sequence of 70-399 in SEQ ID NO: 1 under the condition of 40-65 DEG C.

[0012] Specifically, the nucleotide sequence of the gene encoding the PqPI of the pacifastin serine protease inhibitor of the Cotesia rubecula is shown in SEQ NO: 1. The PqPI has a signal peptide (1-69 bp in SEQ NO: 1) and contains two pacifastin domains.

[0013] The application also simultaneously provides the use of the PqPI of the pacifastin serine protease inhibitor of the Cotesia rubecula: for preparing the pacifastin serine protease inhibitor of the Cotesia rubecula, which can be used for inhibiting the activation of PPO in the hemolymph of the Cotesia rubecula or Pieris rapae.

[0014] The PqPI of the pacifastin serine protease inhibitor of the Cotesia rubecula and the nucleic acid sequence encoding the same provided by the application can be applied to the amino acid sequence, the coding sequence and the development thereof into the application valuable anti-insect pasture and biological pesticide and applied to the prevention of grassland pests and other fields.

[0015] The application obtains the full-length sequence of the venom pacifastin serine protease inhibitor PqPI of the Cotesia rubecula by genome sequencing of the Cotesia rubecula, and obtains the target gene from the cDNA clone by PCR. After obtaining the amino acid sequence of the pacifastin serine protease inhibitor PqPI of the Cotesia rubecula, the prokaryotic expression is carried out, and the expressed PqPI can inhibit the activation of the prophenoloxidase (PPO) of the agricultural pest Pieris rapae pupa, prevent the formation of the active phenoloxidase (PO), and has the function of inhibiting the immunity of the host body fluid.

[0016] The application is particularly achieved by the following technical scheme: the DNA molecule separated in the application comprises: a nucleotide sequence encoding a pacifastin serine protease inhibitor PqPI of Cotesia rubecula, and the nucleotide sequence has at least 70% homology with the nucleotide sequence from the 70th to 399th nucleotide in SEQ ID NO:1; or the nucleotide sequence can hybridize with the nucleotide sequence from the 70th to 399th nucleotide in SEQ ID NO:1 under the condition of 40-65 DEG C. Preferably, the sequence encodes a protein having the amino acid sequence shown in SEQ ID NO:2. More preferably, the sequence has the nucleotide sequence from the 70th to 399th nucleotide in SEQ ID NO:1.

[0017] The pacifastin serine protease inhibitor PqPI of Cotesia rubecula separated in the application comprises: a protein having the amino acid sequence of SEQ ID NO:2, or a conservative variant protein thereof, or an active fragment thereof, or an active derivative thereof. Preferably, the protein is a protein having the sequence of SEQ ID NO:2.

[0018] The host cell transformed by the DNA molecule in the application is a prokaryotic cell.

[0019] In the application, the "isolated" and "purified" DNA means that the DNA or fragment has been separated from the sequences on both sides in the natural state, and also means that the DNA fragment has been separated from the components accompanied by nucleotides in the natural state, and has been separated from the proteins accompanied by it in the cell.

[0020] In the application, the nucleic acid sequence encoded by the pacifastin serine protease inhibitor PqPI of Cotesia rubecula refers to: a nucleotide sequence encoding a protein having the activity of the pacifastin serine protease inhibitor PqPI of Cotesia rubecula, such as the nucleotide sequence from the 70th to 399th nucleotide in SEQ ID NO:1 and its degenerate sequence. The degenerate sequence refers to a sequence in which one or more codons in the coding frame of the sequence of SEQ ID NO:1 from the 70th to 399th nucleotide are replaced by degenerate codons encoding the same amino acid. Due to the degeneracy of the code, the degenerate sequence with a homology as low as about 70% to the nucleotide sequence from the 70th to 399th nucleotide in SEQ ID NO:1 can also encode the sequence described in SEQ ID NO:1.

[0021] It also includes nucleotide sequences capable of hybridizing with the nucleotide sequence from positions 70–399 of SEQ ID NO:1 under moderately stringent conditions, more preferably under highly stringent conditions. It also includes nucleotide sequences with at least 70% homology to the nucleotide sequence from positions 70–399 of SEQ ID NO:1, preferably at least 80%, more preferably at least 90.1%, and most preferably at least 95%. It also includes variants of the open reading frame sequence in SEQ ID NO:1 that encode a protein having the same function as the natural Pacifastin serine protease inhibitor PqPI from the grassland caterpillar wasp. These variations include (but are not limited to): deletions, insertions and / or substitutions of several nucleotides (typically 1–90, preferably 1–60, more preferably 1–20, best preferably 1–10), and additions of several nucleotides (typically up to 60, preferably up to 30, more preferably up to 10, best preferably up to 5) to the 5' and / or 3' ends.

[0022] In this invention, the term "Pacifastin serine protease inhibitor PqPI" or "protein" refers to a protein having the SEQ ID NO:2 sequence with Pacifastin serine protease inhibitor PqPI activity. This term also includes variations of the SEQ ID NO:2 sequence having the same function as the native Pacifastin serine protease inhibitor PqPI. These variations include (but are not limited to): deletions, insertions, and / or substitutions of several amino acids (typically 1–50, preferably 1–30, more preferably 1–20, most preferably 1–10), and the addition of one or more amino acids (typically up to 20, preferably up to 10, more preferably up to 5) at the C-terminus and / or N-terminus. For example, in the art, substitution with amino acids of similar or comparable properties generally does not alter the function of the protein. Similarly, the addition of one or more amino acids at the C-terminus and / or N-terminus generally does not alter the function of the protein. The term also includes the active fragments and active derivatives of PqPI, a serine protease inhibitor of the grassland caterpillar wasp Pacifastin.

[0023] In this invention, the PqPI conserved variant protein of the Pacifastin serine protease inhibitor from the grassland caterpillar wasp refers to a protein formed by replacing up to 10, preferably up to 8, and more preferably up to 5 amino acids with similar or identical properties with the amino acid sequence of SEQ ID NO:2.

[0024] In the present application, various vectors known in the art can be used, such as commercially available vectors, including plasmids, cosmids, etc. In the production of the PqPI protein of the P. xylosta Cotesia vesta Pacifastin serine protease inhibitor, the PqPI coding sequence can be operably linked to an expression control sequence to form a PqPI expression vector.

[0025] As used herein, "operably linked" means that the portions of a linear DNA sequence are capable of affecting the activity of other portions of the same linear DNA sequence. For example, if a signal peptide DNA is expressed as a precursor and participates in the secretion of the protein, then the signal peptide (secretion leader) DNA is operably linked to the protein DNA; if a promoter control sequence controls transcription of a coding sequence, then it is operably linked to the coding sequence; if a ribosome binding site is placed in a position to be translated, then it is operably linked to the coding sequence. In general, "operably linked" means contiguous, and for secretion leaders, in reading frame.

[0026] In the present application, the host cell is a prokaryotic cell. The commonly used prokaryotic host cell refers to an E. coli cell.

[0027] Western blotting techniques or fluorescent quantitative PCR can also be used to analyze the expression of the PqPI gene product, i.e., to analyze the presence and quantity of the PqPI RNA transcript in the cell.

[0028] In addition, a nucleic acid molecule used as a probe in the present application can be used to detect whether a nucleic acid molecule encoding PqPI is present in a sample.

[0029] The present application relates to a method for detecting whether a PqPI nucleotide sequence is present in a sample, which comprises hybridizing the probe described above with the sample, and then detecting whether the probe has bound. Preferably, the sample is a PCR amplification product, wherein the PCR amplification primers correspond to the PqPI nucleotide coding sequence and can be located on both sides or in the middle of the coding sequence. The length of the primers is generally 15-50 nucleotides.

[0030] In addition, based on the nucleotide sequence and the amino acid sequence of the pacifastin serine protease inhibitor PqPI of the Cotesia rubecula according to the present application, homologous genes or homologous proteins of the pacifastin serine protease inhibitor PqPI can be screened based on nucleic acid homology or expression protein homology.

[0031] The full-length nucleotide sequence of the pacifastin serine protease inhibitor PqPI of the Cotesia rubecula according to the present application or a fragment thereof can be obtained by PCR amplification, recombination or artificial synthesis. For the PCR amplification, primers can be designed according to the nucleotide sequence disclosed in the present application, especially the open reading frame sequence, and a commercially available cDNA library or a cDNA library prepared according to the conventional method known to those skilled in the art is used as a template to amplify the relevant sequence.

[0032] Once the relevant sequence is obtained, the recombination method can be used to obtain the relevant sequence in large quantities. Generally, the relevant sequence is cloned into a vector, and then introduced into cells, and then separated from the proliferated host cells by the conventional method.

[0033] In addition, mutations can be introduced into the protein sequence of the present application by chemical synthesis. By using the pacifastin serine protease inhibitor PqPI of the Cotesia rubecula according to the present application, substances interacting with the pacifastin serine protease inhibitor PqPI of the Cotesia rubecula or receptors and the like can be screened by various conventional screening methods.

[0034] Other aspects of the present application will be apparent to those skilled in the art from the disclosure herein. The present application has a significant inhibitory effect in the activation test of the PPO of the Pieris rapae hemolymph, and has a significant inhibitory effect on the humoral immunity of the Pieris rapae. The Cotesia rubecula is very harmful in China, and the use of chemical pesticides has a great negative impact. The pacifastin serine protease inhibitor PqPI of the Cotesia rubecula according to the present application is a protein that has an immunosuppressive effect on the larvae of Lepidoptera pests, and therefore has great application value.

[0035] In summary, the present application discloses a pacifastin serine protease inhibitor PqPI of the Cotesia rubecula, which has the amino acid sequence shown in SEQ ID NO: 2. The present application also discloses a gene encoding the pacifastin serine protease inhibitor PqPI of the Cotesia rubecula, which has the nucleotide sequence of 70-399 in SEQ ID NO: 1. The inhibitor of the present application can be used to inhibit the activation of the PPO of the Pieris rapae hemolymph, and further inhibit the hemolymph melanization reaction. BRIEF DESCRIPTION OF DRAWINGS

[0036] The specific embodiments of the present application will be further described in conjunction with the accompanying drawings.

[0037] Figure 1 Figure for the PqPI gene cloning result of the signal peptide-removed PqPI of the present application;

[0038] Figure 2 SDS-PAGE and Western blot figures of the PqPI recombinant protein of the present application;

[0039] M is a standard protein, lane 1 is a CAT negative control of the fusion GST tag, and lane 2 is a PqPI recombinant protein of the fusion GST tag;

[0040] Figure 3 Figure for the inhibitory effect of the prokaryotic expression PqPI gene expression product of the PqPI of the Pteromalus puparum Pacifastin serine protease inhibitor on the phenoloxidase activation of the Pieris rapae pupa hemolymph;

[0041] A. Activation of the phenoloxidase proenzyme PPO and the phenoloxidase PO activity;

[0042] B. Dose effect of the PqPI.1 on the melanization of the Pieris rapae hemolymph;

[0043] Note: the negative control is PBS and CAT buffer; 1 μg of the tag protein GST-PqPI is added to M. luteus; and the positive control is PTU buffer. DETAILED DESCRIPTION

[0044] The present application will be further described in conjunction with the specific experimental data of the laboratory and the specific embodiments. These embodiments are only used for illustrating the present application and are not used for limiting the scope of the present application. The experimental methods not specified in the following embodiments are usually according to the conventional conditions, for example, the conditions described in Sambrook et al. Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or the conditions suggested by the manufacturers.

[0045] Example 1,

[0046] 1. PqPI gene cloning of the Pteromalus puparum Pacifastin serine protease inhibitor:

[0047] To study the function of PqPI gene, the candidate gene sequence was removed of signal peptide according to the sequence of SEQ ID NO: 1, and the primers were designed by Perl Prime. The coding sequence was cloned by PCR. The RNA of P. jaecanica was reversely transcribed into cDNA, and the coding sequence was obtained by PCR according to the designed primers. The vector PGEM-T easy (Promega) was connected, and the positive clones were picked and sent to Shengya Company for sequencing test. The results are shown in Figure 1 FIG. 1, the length of PqPI gene is consistent with the predicted size of 327 bp, and the negative control is about 750 bp of catalase CAT gene.

[0048] 2. Prokaryotic expression and purification of PqPI

[0049] After obtaining the full-length PqPI, the primers for constructing the prokaryotic expression vector were designed based on the full-length ORF (without signal peptide):

[0050] Forward primer PGEX-4T-2-BamHI-F:

[0051] 5'-gatctggttccgcgtggatccATGCAAATCGTACCAGATCGAGA-3',

[0052] Reverse primer PGEX-4T-2-NotI-R:

[0053] 5'-tcagtcagtcacgatgcggccgcTCACTGTGAAGAGTCTCCAAAGTCTG-3'.

[0054] The KOD enzyme was used for PCR amplification with the plasmid of the gene fragment connected to the vector PGEM-T easy (Promega) as a template. The PqPI PCR amplification system was as follows: KOD One TM PCR Master Mix (TOYOBO, Shanghai, China) was used for PCR. The PCR amplification system was as follows:

[0055] KOD One TM PCR Master Mix: 25 μl

[0056] PCR Forward primer (10 μM): 1.5 μl

[0057] PCR Reverse primer (10 μM): 1.5 μl

[0058] cDNA: 750 ng

[0059] Nuclease-free water: to 50μl

[0060] After mixing the above reagents, gently pipette and centrifuge at low speed, and then put into the PCR amplifier. The reaction conditions are as follows: denaturation at 98°C for 10s, annealing at 55°C for 5s, extension at 68°C for 10s, and repeating for 35 cycles. After the PCR amplification product is tested for fragment size by 1% agarose gel electrophoresis, it is recovered by gel extraction and cleanup kit (EASY-DO, Zhejiang, China).

[0061] Extract the pGEX-4T-2 plasmid, and perform double enzyme digestion on the pGEX-4T-2 plasmid with BamH I and Not I-R.

[0062] Plasmid enzyme digestion and homologous recombination: the pGEX-4T-2 empty plasmid is double enzyme-digested with FastDigest BamH I and FastDigest Not I-R (Thermo Fisher, CA, USA) restriction enzymes, and the enzyme digestion reaction solution is as follows:

[0063] 10×Fast Digest Buffer: 2μl

[0064] FastDigest BamH I: 1μl

[0065] FastDigest Not I-R: 1μl

[0066] pGEX-4T-2 plasmid: 1μg

[0067] Nuclease-free water: to 20μl

[0068] Gently mix and pipette, incubate at 37°C for 15-30min, and obtain linearized pGEX-4T-2 plasmid.

[0069] After the enzyme digestion product is tested for fragment size by 1% agarose gel electrophoresis, it is recovered by gel extraction and cleanup kit (EASY-DO, Zhejiang, China). Thereafter, the target fragment obtained by PCR amplification is homologously recombined with the linearized pGEX-4T-2 plasmid (enzyme digestion sites: BamH I and Not I-R) using ClonExpress Ultra One Step Cloning Kit (Vazyme, Nanjing, China) kit, and the recombination system is as follows:

[0070] 2×ClonExpress Mix: 5μl

[0071] Linearized pGEX-4T-2 plasmid: 100 ng

[0072] PCR fragment amplification product: 50 ng

[0073] Nuclease-free water: to 10 μl

[0074] Gently pipette, centrifuge briefly to collect the reaction solution to the bottom of the tube, reaction conditions: 50°C, 5 min. After the reaction is completed, immediately placed on ice for 15 min, directly for transformation. The ligation product is first transformed into competent cells Trans1-T1 Phage Resistant Chemically Competent Cell (Transgen, Beijing, China), sent to Zhejiang Shangya Biotechnology Co., Ltd. for sequencing. After verification, the correct recombinant plasmid is transformed into BL21 Chemically Competent Cell (Transgen, Beijing, China), as follows:

[0075] (1) Take 50 μl of the melted competent cells on ice bath, add the target DNA, gently mix, and place in ice bath for 30 min.

[0076] (2) 42°C water bath for 45 s, then quickly transfer the tube to ice bath for 2 min, do not shake the centrifuge tube during this process.

[0077] (3) Add 500 μl of sterile LB liquid medium (without antibiotics) to the centrifuge tube, mix well, and incubate at 37°C, 200 rpm for 1 h to recover the bacteria.

[0078] (4) Centrifuge the recovered bacteria at 1000g for 10 min, discard the supernatant, resuspend the pellet with 200 μl of sterile LB liquid medium (without antibiotics), and take 50 μl to plate on LB plates with ampicillin resistance.

[0079] (5) Plate in a 37°C incubator overnight, about 16-24 h.

[0080] (6) Add 1 ml of LB liquid medium with ampicillin resistance to a 1.5 ml sterile centrifuge tube, and transfer the single colony selected by ampicillin resistance to the tube using a pipette.

[0081] For the positive expression strains with correct sequencing, IPTG was added to induce expression. The expression conditions were as follows: 37°C, shaking at 200 rpm until the OD value was about 0.4-0.5, 20°C, shaking at 200 rpm for 30 min, adding IPTG, and inducing expression for 16-24 h. The recombinant protein was then purified using GST resin (GenScript), with the recombinant protein pGEX-4T-2-CAT as a negative control. SDS-PAGE results showed that the PqPI recombinant protein was successfully expressed and purified in E. coli, and the size of the expressed recombinant protein was about 38 kDa. To increase the throughput of PPO and PO activity detection, the detection method was further modified to be used in a 384-well plate. The substrate (L-dopa), activator (M. luteus), and inhibitor (positive control PTU, negative control CAT, and target protein) were mixed in a 384-well plate. The diluted plasma sample was added to another 384-well plate, and the samples were mixed by centrifugation.

[0082] Further, the PqPI recombinant protein was detected using a GST tag antibody, and the Western Blot results showed that the recombinant protein was successfully expressed and purified, as shown in Figure 2

[0083] 3. To detect whether the Pacifastin serine protease inhibitor PqPI has the function of inhibiting hemolymph melanization, the PqPI recombinant protein (1 μg) was incubated with the hemolymph of the pupae of the cabbage worm. The specific steps were as follows:

[0084] ​PqPI was diluted to 4 times in cold TBS buffer (20 mM Tris, 150 mM NaCl, pH = 7.6) and 5 μl of the recombinant protein (0.2 μg / μl) was mixed with 10 μl of the diluted hemolymph in a 384-well plate. The inhibitory activity of PqPI on the melanization of the host was detected. First, 5 μl of activator (0.1 μg / μl M. luteus) and 5 μl of substrate solution (50 mM L-Dopa dissolved in PBS, pH = 7.5) were mixed and added to another 384-well plate which was inverted and fixed on the sample plate. The two fixed 384-well plates were centrifuged to activate the PPO (phenoloxidase proenzyme) cascade in each well. The plate was measured every 5 minutes for 2 hours at A470 and 25°C using a microplate reader Thermo Scientific Varioskan Flash (Thermo Scientific, USA). For the dose-dependent test, 5 μl of the recombinant protein was mixed with 15 μl of the diluted hemolymph and 5 μl of the activator (0.1 μg / μl M. luteus). After incubation at 25°C for about 10 minutes, 800 μl of the substrate solution (20 mM Dopa dissolved in PBS, pH = 6.5) was added. The fluorescence value of the cells adhering to the bottom of each well was measured using a microplate reader Thermo Scientific Varioskan Flash (Thermo Scientific, USA) at A470 for 20 minutes for 200 μl of the sample in a 96-well plate. One unit of PO activity was defined as 0.001 ΔA470 / min.

[0085] As Figure 3 A shows that PqPI inhibits the activation of phenoloxidase proenzyme PPO to inhibit the melanization of the hemolymph of the cabbage moth and does not directly affect the activity of phenoloxidase PO. In addition, the inhibitory effect has a dose effect, as shown in Figure 3 B. Among them, 1 μg of the recombinant protein has the strongest inhibitory effect on the melanization of the hemolymph, 0.125-0.5 μg of the recombinant protein has a secondary inhibitory effect, and 0-0.0625 μg of the recombinant protein has no obvious inhibitory effect on the melanization of the hemolymph (F (5,12) = 66.6417, p < 0.001). It is expected that the grassland caterpillar parasitoid protein PqPI introduced into target plants will have a control effect on the cabbage moth and the grassland caterpillar.

[0086] Finally, it should be noted that the above enumeration is only several specific embodiments of the present application. Obviously, the present application is not limited to the above embodiments, and there are many variations. All variations that can be directly derived or inferred by those of ordinary skill in the art from the disclosure of the present application should be considered within the scope of the present application.

Claims

1. A pacifastin serine protease inhibitor PqPI of the parasitoid Cotesia rubecula, characterized in that The amino acid sequence thereof is as follows: QIVPDRDDWYKTGLKYDDLPRVFSLKDGCPAKEFYLDCNTCSCVGDSSDPACTYKA CPQPPQIVNWKTGKPCPAGQHFYWKCNDCNCAENGREASCTRNFCPDFGDSSQ.

2. A gene encoding the PqPI Pacifastin Serine Protease Inhibitor of the P. coeniae wasp of claim 1, characterized in that The nucleotide sequence of the gene is shown in SEQ NO: 1, 70-399.

3. Use of a Pacifastin Serpin Proteinase Inhibitor PqPI of the grass eggar moth, Cotesia melita, according to claim 1, characterized in that The PqPI protein is used for inhibiting the activation of PPO in the hemolymph of the grassland caterpillar or the cabbage caterpillar, thereby inhibiting the melanization reaction of the hemolymph.

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