PpSerpin3 protein, an inhibitor of serine protease from butterfly pupa golden wasp, and its application

Abstract

This invention belongs to the fields of molecular biology, genetic engineering, and protein engineering, and specifically relates to a serine protease inhibitor PpSerpin3 expressed in the saliva of the butterfly pupa *Triplophysa pulcherrima*, along with its encoded nucleic acid sequence and functional study. This invention provides a serine protease inhibitor PpSerpin3 from *Triplophysa pulcherrima*, which has the amino acid sequence shown in SEQ ID NO:2. The inhibitor of this invention can be used to inhibit the activation of PPO in the hemolymph of the cabbage white caterpillar, thereby inhibiting the hemolymph melanization reaction.

Description

Technical Field

[0001] This invention belongs to the fields of molecular biology, genetic engineering, and protein engineering. Specifically, it relates to a serine protease inhibitor, PpSerpin3, expressed in the saliva of the butterfly pupa *Triplophysa pulmonata*, and the study of its encoded nucleic acid sequence and function. Background Technology

[0002] Pests pose a serious threat to the safe production of crops worldwide, with up to 40% of global crop yields lost annually due to pests.

[0003] Biological pest control is a theoretical and technological system that utilizes the natural enemies of pests and their products to control pests. It overcomes the shortcomings of chemical control and is the core technology for green pest control. Global experience has shown that vigorously researching and developing biological pest control is an effective way to ensure the safety of agricultural products.

[0004] Parasitic wasps are an important natural enemy resource for pests, widely used in agricultural and sanitary pest control, playing a significant role in biological control. Parasitic wasps carry various parasitic factors, including venom, saliva, polynucleotide viruses, and abnormal cells, which can disrupt the host's immune response, regulate host growth and development, control the nutritional composition of the host's hemolymph, and disrupt the host's reproductive and endocrine systems. These factors ensure the normal development of their offspring in the host's hemocoel or body surface, and are key factors for successful parasitism. In-depth research into the parasitic factors of parasitic wasps is beneficial for developing environmentally friendly biocontrol agents, thus opening up new avenues for pest control.

[0005] Insect immune responses include cellular immunity and humoral immunity. Melanization is a unique humoral immune defense mechanism in insect hemolymph, with melanin being the final product. Melanin plays a crucial role in insects' defense against pathogenic bacteria, fungi, viruses, and parasitic wasps. During parasitism, once the wasp egg is encapsulated by hemolymph cells, the precursor of prophenoloxidase (PPO) is converted into active phenoloxidase (PO) through a serine proteinase cascade. PO catalyzes the production of large amounts of quinone intermediates from phenolic substances and mediates melanin formation, thus sealing and killing the wasp egg. Serine proteinase inhibitors can negatively regulate the cleavage of prophenoloxidase mediated by the serine proteinase cascade, inhibiting humoral melanization in the host and thus facilitating successful parasitism by the wasp. Research on this process can help reveal how parasitic wasps control the host's physiological and immune systems, providing a scientific basis for understanding parasitic and symbiotic relationships. It also facilitates the development of new pest control strategies and provides new ideas for green pest control. Summary of the Invention

[0006] The technical problem to be solved by this invention is to explore the function of a salivary serine protease inhibitor of the butterfly pupa *Triplophysa pulmonata* that has an immunosuppressive effect (inhibits hemolymph melanization) on common vegetable pests.

[0007] To address the aforementioned technical problems, this invention provides a serine protease inhibitor PpSerpin3 from the golden pupa of the butterfly wasp, which has the amino acid sequence shown in SEQ ID NO:2.

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

[0009] MRLIIFLICGIIAIASPAIG TGIDERQNFFNVELLQALNEAKPGNVVVGTSSVKAALMILAEAAAGRTRQQIVSTLRLPTDVAQIRDVVSHSISSFKDPKSDTQLQTAIKVWLSKNVALHKDYTDILQRYYKGELQATNFADVAGTVKIINDWAKKCTNGHISSILEPNSVAADTKMVLTTAVYFKGTWLNSFDKTATRSRCFNVPKLGC QQVPLMEVVGNYKYGYVPALDAQVIQIPYTGKRVSMVVLLPQRLGEQALGDLSRDLAFTPMSVLLSSLQETEVLLQLPRFSIGNKVDLRAALEKLGIKDLFDKNANLTTAFPLANVQVGAIMHNAQIEVNEEGTIAAAVSGVSVIPLMGSTSTTFRADRPFLFFLVDHQTNSILFAGRYFQPEGPTTKTV*

[0010] An improvement to the PpSerpin3 serine protease inhibitor from the golden wasp of the present invention is: a protein, a conserved variant thereof, an active fragment thereof, or an active derivative thereof.

[0011] The present invention also provides a gene encoding the above-mentioned serine protease inhibitor PpSerpin3 of the golden pheasant pupa, which has the nucleotide sequence of positions 62–1203 in SEQ ID NO:1; or has at least 70% homology with the nucleotide sequence of positions 62–1203 in SEQ ID NO:1; or its nucleotide sequence can hybridize with the nucleotide sequence of positions 62–1203 in SEQ ID NO:1 under conditions of 40–65°C.

[0012] Specifically, the nucleotide sequence of the gene encoding PpSerpin3, an inhibitor of serine protease from the golden pheasant pupa, is shown in SEQ NO:1. PpSerpin3 contains a signal peptide (bps 1-60 of SEQ NO:1) and one serpin domain.

[0013] The present invention also provides the use of the above-mentioned PpSerpin3 serine protease inhibitor from the golden worm pupae: for preparing a serine protease inhibitor from the golden worm pupae, which can be used to inhibit the activation of PPO in the hemolymph of the cabbage caterpillar, thereby inhibiting the hemolymph melanization reaction.

[0014] The PpSerpin3 serine protease inhibitor for the butterfly pupa golden wasp and its encoded nucleic acid sequence provided by this invention can be used to develop insect-resistant plants and biological pesticides with application value and applied in multiple fields such as vegetable pest control.

[0015] This invention utilizes genome sequencing of the pupa *Pieris rapae* to obtain the full-length sequence of the salivary serine protease inhibitor PpSerpin3, and clones the target gene from cDNA via PCR. After obtaining the amino acid sequence of PpSerpin3, it was purified under non-denaturing conditions following prokaryotic expression. The expressed PpSerpin3 can inhibit the activation of PPO in the pupae of the agricultural pest *Pieris rapae*, preventing the formation of active PO and thus inhibiting the host's humoral immunity.

[0016] This invention is specifically achieved through the following technical solution: the DNA molecule isolated in this invention comprises a nucleotide sequence encoding a serine protease inhibitor PpSerpin3 from the golden wasp pupa, and the nucleotide sequence has at least 70% homology with the nucleotide sequence from positions 62 to 1203 in SEQ ID NO:1; or the nucleotide sequence can hybridize with the nucleotide sequence from positions 62 to 1203 in SEQ ID NO:1 under conditions of 40–65°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 positions 62 to 1203 in SEQ ID NO:1.

[0017] The serine protease inhibitor PpSerpin3 isolated by the golden pheasant pupa of this invention comprises: a protein having the amino acid sequence SEQ ID NO:2, or a conserved variant thereof, or an active fragment thereof, or an active derivative thereof. Preferably, the protein is a protein having the sequence SEQ ID NO:2.

[0018] The host cell for DNA molecule transformation in this invention is a prokaryotic cell.

[0019] In this invention, "isolated" or "purified" DNA means that the DNA or fragment has been separated from the sequences flanking it in its natural state, and also means that the DNA fragment has been separated from the components that accompany the nucleotides in their natural state, and has been separated from the proteins that accompany them in the cell.

[0020] In this invention, the nucleic acid sequence encoded by the PpSerpin3 serine protease inhibitor from the golden wasp (Pteris vittata) refers to the nucleotide sequence encoding a protein with the activity of the PpSerpin3 serine protease inhibitor from the golden wasp, such as the nucleotide sequence at positions 62–1203 of SEQ ID NO:1 and its degenerate sequence. This degenerate sequence refers to a sequence in which one or more codons in the coding frame of SEQ ID NO:1 are replaced by degenerate codons encoding the same amino acid. Due to the degeneracy of codons, a degenerate sequence with homology as low as approximately 70% to the nucleotide sequence at positions 62–1203 of 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 62–1203 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 62–1203 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 PpSerpin3 serine protease inhibitor from the golden 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 "PpSerpin3" or "protein" refers to a protein having the sequence of SEQ ID NO:2, which contains the activity of the PpSerpin3 serine protease inhibitor from the golden wasp. This term also includes variations of the SEQ ID NO:2 sequence having the same function as the native PpSerpin3 serine protease inhibitor from the golden wasp. 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 PpSerpin3, a serine protease inhibitor from the golden pupa wasp.

[0023] In this invention, the PpSerpin3 conserved variant protein, a serine protease inhibitor of the butterfly pupa, 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 this invention, various vectors known in the art can be used, such as commercially available vectors, including plasmids, granules, etc. In producing the PpSerpin3 protein, the serine protease inhibitor from the golden wasp of this invention, the coding sequence of the PpSerpin3 protein can be operatively linked to an expression regulatory sequence to form an expression vector for the PpSerpin3 protein.

[0025] As used in this invention, "operably linked to" refers to a situation where certain portions of a linear DNA sequence can influence 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 protein secretion, then the signal peptide (secretion leader sequence) DNA is operably linked to the protein DNA; if a promoter-controlled sequence is transcribed, then it is operably linked to the coding sequence; if the ribosome binding site is positioned where translation is possible, then it is operably linked to the coding sequence. Generally, "operably linked to" means adjacent, and for a secretion leader sequence, it means adjacent within the reading frame.

[0026] In this invention, the host cell is a prokaryotic cell. Commonly used prokaryotic host cells refer to *Escherichia coli* cells.

[0027] The expression of the PpSerpin3 gene product, an inhibitor of serine protease in butterfly pupa, can also be analyzed using Western blotting or quantitative real-time PCR. This involves analyzing the presence and quantity of the RNA transcript of PpSerpin3 in cells.

[0028] Furthermore, the nucleic acid molecule that can be used as a probe in this invention can be used to detect the presence of a nucleic acid molecule encoding PpSerpin3, an inhibitor of serine protease from the golden wasp, in a sample.

[0029] This invention relates to a method for detecting the presence of the PpSerpin3 nucleotide sequence, a serine protease inhibitor from the golden wasp *Pteris vittata*, in a sample. The method involves hybridizing the sample with the aforementioned probe and then detecting whether the probe has bound. Preferably, the sample is a product of PCR amplification, wherein the PCR amplification primers correspond to the nucleotide coding sequence of the PpSerpin3 serine protease inhibitor from the golden wasp and may be located on either side or in the middle of this coding sequence. The primer length is generally 15–50 nucleotides.

[0030] Furthermore, the nucleotide and amino acid sequences of the PpSerpin3 serine protease inhibitor from the pupa of the golden wasp according to the present invention can be used to screen for homologous genes or homologous proteins of the PpSerpin3 serine protease inhibitor based on nucleic acid homology or homology of expressed proteins.

[0031] The full-length nucleotide sequence or fragment thereof of the PpSerpin3 serine protease inhibitor from the golden pupa of the present invention can generally be obtained by PCR amplification, recombination, or artificial synthesis. For PCR amplification, primers can be designed based on the relevant nucleotide sequences disclosed in this invention, especially the open reading frame sequences, and the relevant sequences can be obtained by amplification using commercially available cDNA libraries or cDNA libraries prepared according to conventional methods known to those skilled in the art.

[0032] Once the relevant sequence is obtained, it can be obtained in large quantities using recombination methods. This typically involves cloning it into a vector, transferring it into cells, and then isolating the sequence from the proliferated host cells using conventional methods.

[0033] Furthermore, mutations can be introduced into the protein sequence of this invention through chemical synthesis. Using the *P. seraphim* serine protease inhibitor PpSerpin3 of this invention, substances that interact with PpSerpin3, or receptors, can be screened using various conventional screening methods.

[0034] Other aspects of the invention will be apparent to those skilled in the art from the disclosure herein. The invention exhibits significant inhibitory activity in the hemolymphocyte oxidase activation assay of the cabbage white caterpillar, demonstrating a significant inhibitory effect on the humoral immunity of the cabbage white caterpillar. The PpSerpin3 serine protease inhibitor from the butterfly pupa of the present invention is a protein with immunosuppressive effects against lepidopteran pest larvae, and therefore has significant application value.

[0035] In summary, this invention discloses a serine protease inhibitor PpSerpin3 from the golden pheasant larva, which has the amino acid sequence shown in SEQ ID NO:2. This invention also discloses the gene encoding the aforementioned golden pheasant larva serine protease inhibitor PpSerpin3, which has the nucleotide sequence from positions 62 to 1203 in SEQ ID NO:1. The inhibitor of this invention can be used to inhibit the activation of PPO in the hemolymph of the cabbage white caterpillar, thereby inhibiting the hemolymph melanization reaction. This invention also provides a method to improve the control effect of plants against the cabbage white caterpillar: by transferring a recombinant plasmid with the nucleotide sequence shown in SEQ ID NO:1 into the target plant, thereby improving the plant's control effect against the cabbage white caterpillar. That is, the golden pheasant larva protein PpSerpin3 transferred into the target plant has a control effect against the cabbage white caterpillar. Attached Figure Description

[0036] The specific embodiments of the present invention will be further described in detail below with reference to the accompanying drawings.

[0037] Figure 1 The image shows the cloning results of the PpSerpin3 gene with signal peptide removed according to the present invention. The two lanes in the upper image are for the PpSerpin3 gene, and the two lanes in the lower image are for the CAT gene.

[0038] Figure 2 The images show the SDS-PAGE and Western blot diagrams of the PpSerpin3 recombinant protein of this invention.

[0039] In the figure, M represents the standard protein, CAT is the CAT negative control fused with the 6*his tag, and PpSerpin3 is the PpSerpin3 recombinant protein fused with the 6*his tag.

[0040] Figure 3 The diagram shows the inhibitory effect of the prokaryotically expressed PpSerpin3 gene, a serine protease inhibitor from the golden wasp of the present invention, on the activation of hemolymph phenol oxidase in cabbage caterpillar pupae; that is, it demonstrates the inhibitory effect of PpSerpin3 on hemolymph melanization in cabbage caterpillars.

[0041] Figure 3 middle:

[0042] A. Activation of prophenol oxidase (PPO) and activity of phenol oxidase (PO);

[0043] B.PpSerpin3 inhibits the dose-effect of hemolymph melanization in cabbage caterpillars;

[0044] Note: The negative control was prepared with PBS buffer and CAT; 1 μg of the tag protein his–PpSerpin3 of M. luteus was added; the positive control was prepared with PTU buffer. Detailed Implementation

[0045] The present invention will be further illustrated below with specific laboratory experimental data and concrete embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments that do not specify specific conditions are generally performed under conventional conditions, such as those described in Sambrook et al. Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer.

[0046] Example 1

[0047] 1. Cloning of the PpSerpin3 gene, a serine protease inhibitor from the butterfly pupa golden wasp:

[0048] To investigate the function of the PpSerpin3 gene, based on the predicted gene sequence from the *P. sarcoptes* pupa genome—the sequence described in SEQ ID NO:1—the signal peptide (bp 1-60 of SEQ NO:1) was removed from the candidate gene sequence. Primers were designed using PerlPrime, and the coding sequence was cloned by PCR. RNA was extracted from the *P. sarcoptes* pupa and then reverse transcribed into cDNA. The coding sequence was obtained by PCR using the designed primers. The cDNA was ligated into the vector PGEM–Teasy (Promega), and positive clones were selected and sent to Shangya Biotechnology Co., Ltd. for sequencing verification. The results are as follows: Figure 1 As shown, the length of the PpSerpin3 gene is consistent with the predicted size of 1143 bp, while the negative control is the catalase CAT gene, which is about 660 bp.

[0049] 2. Prokaryotic expression and purification of PpSerpin3

[0050] After obtaining the full-length PpSerpin3, primers for constructing a prokaryotic expression vector were designed using the full-length ORF (with the signal peptide removed).

[0051] Forward primer pCold I–BamHI–F:

[0052] 5'–taccctcgagggatcgACGGGCATAGACGAGCG–3',

[0053] Reverse primer pCold I–HindIII–R:

[0054] 5'–gcaggtcgacaagcttcaatgatgatgatgatgatgCACCGTCTTGGTCGTAGG–3'.

[0055] Using a gene fragment plasmid ligated to the vector PGEM–T easy(Promega) as a template, KOD One was used... TM PCR was performed using PCR Master Mix (TOYOBO, Shanghai, China). The PCR amplification system is as follows:

[0056] KOD One TM PCR Master Mix: 25 μl

[0057] PCR Forward primer(10μM):1.5μl

[0058] PCR Reverse primer(10μM):1.5μl

[0059] cDNA: 750ng

[0060] Nuclease-free water: to 50 μl

[0061] After mixing the above reagents thoroughly, gently pipette and centrifuge at low speed, then place in a PCR amplification instrument. The reaction conditions are: denaturation at 98℃ for 10 s, annealing at 55℃ for 5 s, extension at 68℃ for 10 s, repeated for 35 cycles. After verifying the fragment size by 1% agarose gel electrophoresis, the PCR amplification products are extracted and recovered using a Gel Extraction & Clean-up Kit (EASY-DO, Zhejiang, China); the PCR fragment amplification products are then ready for use.

[0062] The pCold I plasmid was extracted and double-digested with FastDigest BamHI and FastDigest HindIII (ThermoFisher, CA, USA) restriction endonucleases. The digestion system is as follows:

[0063] 10×Fast Digest Buffer: 2μl

[0064] FastDigest BamHⅠ: 1μl

[0065] FastDigest HindIII: 1μl

[0066] pCold I empty vector plasmid: 1 μg

[0067] Nuclease-free water: to 20 μl

[0068] Gently aspirate and mix well, then incubate at 37°C for 15-30 min to obtain the linearized pCold I plasmid.

[0069] After the enzyme digestion products were examined for fragment size by 1% agarose gel electrophoresis, they were recovered by gel extraction and clean-up using a Gel Extraction & Clean-up Kit (EASY-DO, Zhejiang, China). Subsequently, the target fragment obtained by PCR amplification was homologously recombinated with linearized pCold I plasmid (restriction sites: BamHI and HindIII) using the ClonExpress Ultra One Step Cloning Kit (Vazyme, Nanjing, China). The recombination system is as follows:

[0070] 2×ClonExpress Mix: 5μl

[0071] Linearized pCold I plasmid: 100 ng

[0072] PCR fragment amplification product: 50 ng

[0073] Nuclease-free water: to 10 μl

[0074] Use a pipette to gently mix, and briefly centrifuge to collect the reaction solution to the bottom of the tube. Reaction conditions: 50℃, 5min. After the reaction is complete, immediately place on ice to cool for 15min and directly perform transformation. The ligation product is first transformed into competent cells Trans1-T1 Phage Resistant Chemically Competent Cell (Transgen, Beijing, China) and sent to Zhejiang Shangya Biotechnology Co., Ltd. for sequencing. The recombinant plasmid that is consistent with the sequence listed in SEQ ID NO:1 is transformed into BL21(DE3) Chemically Competent Cell (Transgen, Beijing, China) as follows: (1) Take 50μl of competent cells thawed on ice, add the target DNA, gently mix, and place in an ice bath for 30min. (2) Place in a 42℃ water bath for 45s, and then quickly transfer the tube to an ice bath for 2min. Do not shake the centrifuge tube during this process.

[0075] (3) Add 500 μl of sterile LB liquid medium (without antibiotics) to the centrifuge tube, mix well, and incubate at 37°C and 200 rpm for 1 h to allow the bacteria to recover.

[0076] (4) Centrifuge the revived bacteria at 1000g for 10min, discard the supernatant, resuspend the precipitate in 200μl of sterile LB liquid medium (without antibiotics), and take 50μl to spread on LB plates with ampicillin resistance.

[0077] (5) Place the plate in a 37℃ constant temperature incubator and incubate overnight, for about 16-24 hours.

[0078] (6) Add 1 ml of ampicillin-resistant LB liquid medium to a 1.5 ml sterile centrifuge tube, and use a pipette to transfer the monoclonal bacteria selected for ampicillin resistance into the tube.

[0079] (7) Shake at 37℃ and 200 rpm until turbid. At this point, the recombinant plasmid of E. coli has been obtained.

[0080] For positive expression strains whose sequencing results were consistent with the sequence described in SEQ ID NO:1, IPTG was added to induce expression. The expression conditions were: shaking at 37℃ until the OD value was approximately 0.6, then adding IPTG to a final concentration of 0.1 mM, and inducing expression at 6℃ for 20 h. The protein was then purified using Compare His-Tag Purification Resin (Roche, Switzerland), with the recombinant protein pCold I–CAT serving as a negative control. SDS-PAGE results showed that the PpSerpin3 recombinant protein was successfully expressed and purified in *E. coli*, with a size of approximately 44 kDa, consistent with the predicted sequence size in SEQ ID NO:2. Furthermore, the PpSerpin3 recombinant protein was detected using a His-tag antibody, and Western blotting results indicated successful expression and purification. Figure 2 As shown.

[0081] 3. To test whether the serine protease inhibitor PpSerpin3 has the function of inhibiting hemolymph melanization, recombinant PpSerpin3 protein (1 μg) was incubated with hemolymph from cabbage caterpillar pupae. The specific steps are as follows:

[0082] Plasma was obtained by cutting the hind limbs of cabbage caterpillar larvae with scissors. The plasma was diluted 4-fold and added to a refrigerated TBS buffer solution containing 20 mM Tris, 150 mM NaCl, and pH 7.6. The solution was centrifuged at 3000 g for 10 min at 4°C to remove blood cells, resulting in cell-free hemolymph. For the PPO activation assay, 5 μl of the recombinant PpSerpin3 protein (0.2 μg / μl) obtained in step 2 above was mixed with 10 μl of diluted cabbage caterpillar hemolymph in a 384-well plate. Then, 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 immobilized on an inverted sample plate. The PPO (prophenol oxidase) cascade in each well was simultaneously activated by centrifuging both immobilized 384-well plates. Plate measurements were taken every 5 minutes at A470 and 25°C using a Thermo Scientific Varioskan Flash microplate reader (Thermo Scientific, USA) for 2 hours. For the PO activity assay, 10 μl of diluted cabbage white worm hemolymph and 5 μl of activator (0.1 μg / μl M. luteus) were mixed in a 384-well plate and incubated at 25°C for approximately 10 min. Then, 5 μl of recombinant protein (0.2 μg / μl) 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 immobilized on an inverted sample plate. Both immobilized 384-well plates were centrifuged to allow simultaneous reaction, and the results were detected using a microplate reader. One unit of PO activity was defined as 0.001 ΔA470 / min.

[0083] like Figure 3 The results indicate that PpSerpin3 inhibits melanization of the cabbage caterpillar hemolymph by inhibiting the activation of prophenoloxidase PPO into phenoloxidase PO, rather than by directly inhibiting phenoloxidase PO activity. That is, PpSerpin3 has no inhibitory effect on already generated PO. Furthermore, its inhibitory effect on PPO activation exhibits a dose-dependent effect, such as... Figure 3 As shown in B. Among them, 1 μg of recombinant protein showed the strongest inhibitory effect on hemolymphatic melanosis, followed by 0.125–0.5 μg of recombinant protein, while 0–0.0625 μg of recombinant protein had no significant inhibitory effect on hemolymphatic melanosis.

[0084] In conjunction with the above text Figure 3The results showed that PpSerpin3 could inhibit the activation of prophenol oxidase (PPO), preventing the formation of active PO, thereby suppressing the important humoral immune response of melanosis in cabbage caterpillars. Therefore, it is expected that the transfer of the pupa worm protein PpSerpin3 into target plants will have a control effect on cabbage caterpillars and other lepidopteran species.

[0085] Finally, it should be noted that the above examples are merely some specific embodiments of the present invention. Obviously, the present invention is not limited to the above embodiments and many variations are possible. All variations that can be directly derived or conceived by those skilled in the art from the disclosure of the present invention should be considered within the scope of protection of the present invention.

Claims

1. The use of PpSerpin3, a serine protease inhibitor from the golden pheasant pupa, characterized in that: It is used to inhibit the activation of PPO in the hemolymph of cabbage caterpillars, thereby inhibiting the hemolymph melanization reaction; The amino acid sequence of the serine protease inhibitor PpSerpin3 from the golden pupa wasp is shown in SEQ ID NO:

2.

2. The use as described in claim 1, characterized in that: The nucleotide sequence of the gene encoding the serine protease inhibitor PpSerpin3 of the golden wasp pupa is shown in SEQ ID NO:

1.

3. A method for improving the control effect of plants against cabbage caterpillars, characterized by: Transforming a recombinant plasmid containing the nucleotide sequence shown in SEQ ID NO: 1 in the application described in claim 2 into the target plant can improve the plant's control effect against cabbage caterpillars.

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