Pepserine protease inhibitor (PpSerpin3) protein of Pepserine protease inhibitor (PpSerpin3) and application of PpSerpin3 protein

Abstract

The invention belongs to the fields of molecular biology, gene engineering and protein engineering, and particularly relates to a serine protease inhibitor PpSerpin3 expressed in spittle of Petonia pupae, a nucleotide sequence coded by the serine protease inhibitor PpSerpin3 and functional research of the serine protease inhibitor PpSerpin3. The invention provides a serine protease inhibitor PpSerpin3 of Petonia pupae. The serine protease inhibitor PpSerpin3 of Petonia pupae has an amino acid sequence as shown in SEQ ID NO: 2. The inhibitor provided by the invention can be used for inhibiting the activation of the haemolymph PPO of the cabbage caterpillar, so as to inhibit the blackening reaction of the haemolymph.

Description

Technical Field

[0001] The invention belongs to the fields of molecular biology, genetic engineering and protein engineering, and particularly relates to a serine protease inhibitor PpSerpin3 expressed in the saliva of the butterfly chrysalis Nephila and the nucleic acid sequence and function research of the encoding thereof. Background Art

[0002] Pests pose a serious threat to the safe production of crops around the world, with up to 40% of global crop production lost to pests each year.

[0003] At present, my country's pest control mainly relies on chemical control, and the amount of pesticides used is large and increasing. Excessive use of pesticides can lead to pesticide residues, rampant pests, environmental pollution, killing of natural enemies, poisoning of humans and animals, and many other hazards. my country's agricultural products have also suffered huge losses due to excessive pesticide residues. Therefore, reducing the use of chemical pesticides is an important way to achieve safe production of agricultural products. Biological pest control is a theoretical and technical system that uses natural enemies of pests and their products to control pests. It can overcome the shortcomings of chemical control and is the core technology of green pest control. Practices around the world have 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 of pests. They are widely used in agricultural and sanitary pest control and play a significant role in the field of biological control. Parasitic wasps carry a variety of parasitic factors, including venom, saliva, polytomous DNA viruses, abnormal cells, etc., which can destroy the host's immune response, regulate the host's growth and development, regulate the host's hemolymph nutrients, and disrupt the host's reproductive and endocrine systems to ensure the normal development of their offspring in the host's blood cavity or body surface. This is a key factor in the successful parasitism of parasitic wasps. In-depth research on parasitic factors of parasitic wasps is conducive to the development of environmentally friendly biological control agents, thereby opening up new ways to control pests.

[0005] Insect immune responses include cellular immunity and humoral immunity. Melanization is a unique humoral immune defense mechanism in insect hemolymph. Melanin is the final product of this reaction and plays a vital role in insects' resistance to pathogenic bacteria, fungi, viruses and parasitic wasps. During the parasitic process, once the eggs of parasitic wasps are encapsulated by blood cells, the prophenoloxidase (PPO) is converted into active phenoloxidase (PO) through a cascade reaction of serine proteinase. PO catalyzes phenolic substances to produce a large number of quinone intermediates and mediates the formation of melanin, sealing and killing the bee eggs. Serine proteinase inhibitors can negatively regulate the shearing process of prophenoloxidase mediated by the serine proteinase cascade reaction, inhibit the melanization of the host's body fluids, and thus help the parasitic wasps to parasitize smoothly. Research on this process can help reveal how parasitic wasps control the physiological and immune systems of the host and provide a scientific basis for understanding the relationship between parasitism and symbiosis. It is also conducive to 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 the present invention is to provide a functional study on a salivary serine protease inhibitor of the butterfly chrysalis vitripennis that has an immunosuppressive effect (inhibiting hemolymph melanization) on common vegetable pests.

[0007] In order to solve the above technical problems, the present invention provides a Chrysomelidae serine protease inhibitor PpSerpin3, which has an amino acid sequence shown in SEQ ID NO:2.

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

[0009] MRLIIFLICGIIAIASPAIGTGIDERQNFFNVELLQALNEAKPGNVVVGTSSVKAALMILAEAAAGRTRQQIVSTLRLPTDVAQIRDVVSHSISSFKDPKSDTQLQTAIKVWLSKNVALHKDYTDILQRYYKGELQATNFADVAGTVKIINDWAKKCTNGHISSILEPNSVAADTKMVLTTAVYFKGTWLNSFDKTATRSRCFNVPKLGC QQVPLMEVVGNYKYGYVPALDAQVIQIPYTGKRVSMVVLLPQRLGEQALGDLSRDLAFTPMSVLLSSLQETEVLLQLPRFSIGNKVDLRAALEKLGIKDLFDKNANLTTAFPLANVQVGAIMHNAQIEVNEEGTIAAAVSGVSVIPLMGSTSTTFRADRPFLFFLVDHQTNSILFAGRYFQPEGPTTKTV*

[0010] As an improvement of the Chrysalis vitripennis serine protease inhibitor PpSerpin3 of the present invention: a protein, a conservative variant protein thereof, an active fragment thereof or an active derivative thereof.

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

[0012] Specifically, the nucleotide sequence of the gene encoding the serine protease inhibitor PpSerpin3 of the pupal cercis chrysalis is shown in SEQ NO: 1. PpSerpin3 has a signal peptide (the 1st to 60th bp in SEQ NO: 1) and contains a serpin domain.

[0013] The present invention also provides the use of the above-mentioned butterfly pupa viridis serine protease inhibitor PpSerpin3: for preparing the butterfly pupa viridis serine protease inhibitor, the inhibitory protein can be used to inhibit the activation of PPO in the hemolymph of Pieris rapae, and then inhibit the hemolymph melanization reaction.

[0014] The present invention provides a Chrysalis chrysogenum serine protease inhibitor PpSerpin3 and a nucleic acid sequence encoding the same, which can be used to develop valuable insect-resistant plants and biological pesticides using its amino acid sequence and coding sequence and applied to multiple fields such as vegetable pest control.

[0015] The present invention uses genome sequencing of the butterfly chrysalis pygmy wasp to obtain the full-length sequence of the salivary serine protease inhibitor PpSerpin3, and obtains the target gene from cDNA cloning by PCR. After obtaining the amino acid sequence of the butterfly chrysalis pygmy wasp serine protease inhibitor PpSerpin3, it is purified under non-denaturing conditions after prokaryotic expression, and the expressed PpSerpin3 can inhibit the activation of PPO of the pupae of the agricultural pest Pieris rapae, prevent it from forming active PO, and has the function of inhibiting the host humoral immunity.

[0016] The present invention is specifically implemented by the following technical scheme: the DNA molecule isolated in the present invention comprises: a nucleotide sequence encoding a serine protease inhibitor PpSerpin3 of the butterfly chrysalis, and the nucleotide sequence has at least 70% homology with the nucleotide sequence from nucleotides 62 to 1203 in SEQ ID NO:1; or the nucleotide sequence can hybridize with the nucleotide sequence from nucleotides 62 to 1203 in SEQ ID NO:1 under the condition of 40-65°C. Preferably, the sequence encodes a protein having an amino acid sequence shown in SEQ ID NO:2. More preferably, the sequence has a nucleotide sequence from nucleotides 62 to 1203 in SEQ ID NO:1.

[0017] The serine protease inhibitor PpSerpin3 of Chrysalis chrysogenum isolated by the present invention comprises: a protein having an 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 a sequence of SEQ ID NO: 2.

[0018] The host cells transformed by the DNA molecules of the present invention are prokaryotic cells.

[0019] In the present invention, "isolated" or "purified" DNA means that the DNA or fragment has been separated from the sequences on both sides of it in the natural state, and also means that the DNA fragment has been separated from the accompanying nucleotide components in the natural state and has been separated from the proteins accompanying it in the cell.

[0020] In the present invention, the nucleic acid sequence encoding the serine protease inhibitor PpSerpin3 of the butterfly pupae wasp refers to: a nucleotide sequence encoding a protein having the activity of the serine protease inhibitor PpSerpin3 of the butterfly pupae wasp, such as the nucleotide sequence at positions 62-1203 in SEQ ID NO:1 and its degenerate sequence. The degenerate sequence refers to a sequence generated after one or more codons in the nucleotides at positions 62-1203 of the coding frame of the sequence of SEQ ID NO:1 are replaced by degenerate codons encoding the same amino acid. Due to the degeneracy of the codons, a degenerate sequence with a homology of as low as about 70% with the nucleotide sequence at positions 62-1203 in SEQ ID NO:1 can also encode the sequence described in SEQ ID NO:1.

[0021] It also includes nucleotide sequences that can hybridize with the nucleotide sequence from nucleotides 62 to 1203 in SEQ ID NO:1 under moderate stringency conditions, preferably under highly stringent conditions. It also includes nucleotide sequences that have at least 70% homology, preferably at least 80%, more preferably at least 90.1%, and most preferably at least 95% homology with the nucleotide sequence from nucleotides 62 to 1203 in SEQ ID NO:1. It also includes variant forms of the open reading frame sequence in SEQ ID NO:1 that can encode a protein having the same function as the natural Pleurotus eryngii serine protease inhibitor PpSerpin3. These variations include (but are not limited to): deletion, insertion and / or substitution of several (usually 1-90, preferably 1-60, more preferably 1-20, and most preferably 1-10) nucleotides, and addition of several (usually within 60, preferably within 30, more preferably within 10, and most preferably within 5) nucleotides at the 5' and / or 3' end.

[0022] In the present invention, the PpSerpin3 or protein of the serine protease inhibitor of the pupae golden bee refers to: a protein of the SEQ ID NO:2 sequence having the activity of the PpSerpin3 serine protease inhibitor of the pupae golden bee. The term also includes variant forms of the SEQ ID NO:2 sequence having the same function as the natural PpSerpin3 serine protease inhibitor of the pupae golden bee. These variant forms include (but are not limited to): deletion, insertion and / or substitution of several (usually 1-50, preferably 1-30, more preferably 1-20, and most preferably 1-10) amino acids, and addition of one or several (usually within 20, preferably within 10, and more preferably within 5) amino acids at the C-terminus and / or N-terminus. For example, in the art, when amino acids with similar or similar properties are substituted, the function of the protein is usually not changed. For another example, adding one or several amino acids at the C-terminus and / or N-terminus usually does not change the function of the protein. The term also includes active fragments and active derivatives of the Nymphalidae serine protease inhibitor PpSerpin3.

[0023] In the present invention, the conservative variant protein of the serine protease inhibitor PpSerpin3 of the Chrysalis chrysogenum refers to a protein formed by replacing at most 10, preferably at most 8, and more preferably at most 5 amino acids with similar or close properties with the amino acid sequence of SEQ ID NO: 2.

[0024] In the present invention, various vectors known in the art can be selected, such as commercially available vectors, including plasmids, cosmids, etc. When producing the PpSerpin3 protein of the present invention, the PpSerpin3 coding sequence of the PpSerpin3 serine protease inhibitor can be operably linked to the expression regulatory sequence to form the PpSerpin3 expression vector of the PpSerpin3 serine protease inhibitor.

[0025] As used herein, "operably linked to" refers to a situation where certain parts of a linear DNA sequence can affect the activity of other parts of the same linear DNA sequence. For example, if the signal peptide DNA is expressed as a precursor and participates in the secretion of the protein, then the signal peptide (secretion leader sequence) DNA is operably linked to the protein DNA; if the promoter controls the transcription of the sequence, then it is operably linked to the coding sequence; if the ribosome binding site is placed in a position that allows translation, then it is operably linked to the coding sequence. Generally, "operably linked to" means adjacent, and for the secretory leader sequence, it means adjacent in the reading frame.

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

[0027] Western blotting or fluorescence quantitative PCR can also be used to analyze the expression of the gene product of the P. viridis serine protease inhibitor PpSerpin3, that is, to analyze the presence and quantity of RNA transcripts of the P. viridis serine protease inhibitor PpSerpin3 in cells.

[0028] In addition, the nucleic acid molecule used as a probe in the present invention can be used to detect whether a nucleic acid molecule encoding the serine protease inhibitor PpSerpin3 of the pupae vitripennis is present in a sample.

[0029] The present invention relates to a method for detecting whether a nucleotide sequence of a serine protease inhibitor PpSerpin3 of the pupae wasp is present in a sample, which comprises hybridizing the sample with the above-mentioned probe and then detecting whether the probe is bound. Preferably, the sample is a product of PCR amplification, wherein the PCR amplification primers correspond to the nucleotide coding sequence of the serine protease inhibitor PpSerpin3 of the pupae wasp and can be located on both sides or in the middle of the coding sequence. The primer length is generally 15-50 nucleotides.

[0030] In addition, according to the nucleotide sequence and amino acid sequence of the Chrysalis viridis serine protease inhibitor PpSerpin3 of the present invention, homologous genes or homologous proteins of the Chrysalis viridis serine protease inhibitor PpSerpin3 can be screened based on nucleic acid homology or expressed protein homology.

[0031] The full-length nucleotide sequence of the serine protease inhibitor PpSerpin3 of the present invention or its fragment can usually be obtained by PCR amplification, recombination or artificial synthesis. For PCR amplification, primers can be designed based on the relevant nucleotide sequence disclosed in the present invention, especially the open reading frame sequence, and a commercially available cDNA library or a cDNA library prepared by conventional methods known to those skilled in the art is used as a template to amplify and obtain the relevant sequence.

[0032] Once the relevant sequence is obtained, it can be obtained in large quantities by recombinant methods. This is usually done by cloning it into a vector, then transferring it into cells, and then isolating the relevant sequence from the propagated host cells by conventional methods.

[0033] In addition, mutations can be introduced into the protein sequence of the present invention by chemical synthesis. Using the present invention's PpSerpin3 serine protease inhibitor, substances or receptors interacting with PpSerpin3 serine protease inhibitor can be screened out by various conventional screening methods.

[0034] Other aspects of the present invention are obvious to those skilled in the art due to the disclosure of this article. The present invention has a significant inhibitory effect in the phenoloxidase activation test of the hemolymph of Pieris rapae, and has a significant inhibitory effect on the humoral immunity of Pieris rapae. Vegetable pests in my country are very serious, and the negative impact of using chemical pesticides is very large. The serine protease inhibitor PpSerpin3 of the present invention is a protein that has an immunosuppressive effect on the larvae of lepidopteran pests, and therefore has great application value.

[0035] In summary, the present invention discloses a PpSerpin3, a serine protease inhibitor of the butterfly pupa golden wasp, which has an amino acid sequence shown in SEQ ID NO:2. The present invention also discloses a gene encoding the above-mentioned PpSerpin3, a serine protease inhibitor of the butterfly pupa golden wasp, which has a nucleotide sequence of positions 62-1203 in SEQ ID NO:1. The inhibitor of the present invention can be used to inhibit the activation of PPO in the hemolymph of Pieris rapae, thereby inhibiting the melanization reaction of the hemolymph. The present invention also provides a method for improving the control effect of plants on Pieris rapae: a recombinant plasmid with a nucleotide sequence as shown in SEQ ID NO:1 is transferred into a target plant, thereby improving the control effect of plants on Pieris rapae. That is, the PpSerpin3 protein of the butterfly pupa golden wasp is transferred into a target plant, etc., and has a control effect on Pieris rapae. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] The specific implementation modes of the present invention are further described in detail below with reference to the accompanying drawings.

[0037] Figure 1 This is a diagram showing the cloning result of the PpSerpin3 gene with the signal peptide removed according to the present invention. The two lanes in the upper figure are both PpSerpin3 genes, and the two lanes in the lower figure are both CAT genes.

[0038] Figure 2 The SDS-PAGE and Western blot images of the PpSerpin3 recombinant protein of the present invention;

[0039] The M marked in the figure is the standard protein, CAT is the CAT negative control fused with a 6*his tag, and PpSerpin3 is the PpSerpin3 recombinant protein fused with a 6*his tag.

[0040] Figure 3 This is a diagram showing the inhibitory effect of the prokaryotic expression of the PpSerpin3 gene expression product of the present invention on the activation of phenoloxidase in the hemolymph of Pieris rapae pupae; that is, it reflects the inhibitory reaction of PpSerpin3 on the melanization of the hemolymph of Pieris rapae;

[0041] Figure 3 middle:

[0042] A. Activation of prophenoloxidase PPO and activity of phenoloxidase PO;

[0043] B. The dosage effect of PpSerpin3 in inhibiting hemolymph melanization in Pieris rapae;

[0044] Note: The negative control was the addition of PBS buffer and CAT; the addition of 1 μg of the tagged protein his-PpSerpin3 of M.luteus; the positive control was PTU buffer. DETAILED DESCRIPTION

[0045] The present invention is further described below in conjunction with the specific experimental data of the laboratory and in conjunction with specific examples. These examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples without specifying specific conditions are usually based on conventional conditions, such as the conditions described in Sambrook et al. Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer.

[0046] Embodiment 1,

[0047] 1. Cloning of the Serine Protease Inhibitor PpSerpin3 Gene of the Chrysalis chrysalis:

[0048] To study the function of the PpSerpin3 gene, based on the gene sequence predicted in the genome of the butterfly pupa golden wasp, the sequence described in SEQ ID NO: 1, the signal peptide (the 1st to 60th bp in SEQ NO: 1) was removed from the candidate gene sequence, primers were designed using PerlPrime, and PCR was used to clone its coding sequence. The RNA of the butterfly pupa golden wasp was extracted and then reverse transcribed into cDNA. The coding sequence was obtained by PCR using the designed primers. The vector PGEM-Teasy (Promega) was connected, and the positive clones were picked and sent to Shangya Company for sequencing. The results are shown in the figure. Figure 1 As shown, the length of the PpSerpin3 gene was consistent with the predicted size of 1143 bp, and the negative control was the catalase CAT gene of about 660 bp.

[0049] 2. Prokaryotic expression and purification of PpSerpin3

[0050] After obtaining the full-length PpSerpin3, the primers for constructing the prokaryotic expression vector were designed with the full-length ORF (without the signal peptide):

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

[0052] 5'–taccctcgagggatcgACGGGCATAGACGAGCG–3',

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

[0054] 5'–gcaggtcgacaagcttcaatgatgatgatgatgatgCACCGTCTTGGTCGTAGG–3'.

[0055] Using the gene fragment plasmid connected to the vector PGEM-T easy (Promega) as a template, KOD One TM PCR Master Mix (TOYOBO, Shanghai, China) was used for PCR. The PCR amplification system was 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 50μl

[0061] After mixing the above reagents, gently pipette and centrifuge at low speed, put into PCR amplification instrument, and the reaction conditions are: 98℃ denaturation for 10s, 55℃ annealing for 5s, 68℃ extension for 10s, and repeat 35 cycles. After the fragment size of PCR amplification product was checked by 1% agarose gel electrophoresis, the gel was cut and recovered using Gel Extraction & Clean-up Kit (EASY-DO, Zhejiang, China); the PCR fragment amplification product was obtained and set aside.

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

[0063] 10×Fast Digest Buffer: 2μl

[0064] FastDigest BamHI:1μl

[0065] FastDigest HindIII:1μl

[0066] pCold I empty plasmid: 1 μg

[0067] Nuclease-free water: to 20μl

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

[0069] After the size of the digested product was checked by 1% agarose gel electrophoresis, the gel was cut and recovered using Gel Extraction & Clean-up Kit (EASY-DO, Zhejiang, China). After that, the target fragment obtained by PCR amplification was homologously recombined with the linearized pCold I plasmid (enzyme cutting sites: BamHI and HindIII) using the ClonExpress Ultra One Step Cloning Kit (Vazyme, Nanjing, China). The recombination system was as follows:

[0070] 2×ClonExpress Mix: 5μl

[0071] Linearized pCold I plasmid: 100 ng

[0072] PCR fragment amplification product: 50ng

[0073] Nuclease-free water: to 10μl

[0074] Use a pipette to gently mix, centrifuge briefly and collect the reaction solution to the bottom of the tube. Reaction conditions: 50℃, 5min. After the reaction is completed, immediately place it on ice to cool for 15min and directly transform it. The ligation product is first transformed into the competent cell Trans1-T1 Phage Resistant Chemically Competent Cell (Transgen, Beijing, China), and then sent to Zhejiang Shangya Biotechnology Co., Ltd. for sequencing. The recombinant plasmid whose sequencing results are consistent with the sequence described in SEQ ID NO:1 in the sequence list is transformed into BL21 (DE3) Chemically Competent Cell (Transgen, Beijing, China), as follows: (1) Take 50μl of competent cells melted in an ice bath, add the target DNA, mix gently, and place it in an ice bath for 30min. (2) Place it in a 42℃ water bath for 45s, 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 culture medium (without antibiotics) to the centrifuge tube, mix well, and incubate at 37°C, 200 rpm for 1 h to allow the bacteria to recover.

[0076] (4) The revived bacteria were centrifuged at 1000 g for 10 min, the supernatant was discarded, the pellet was resuspended in 200 μl of sterile LB liquid culture medium (without antibiotics), and 50 μl was applied to an LB plate with ampicillin resistance.

[0077] (5) Place the plate in a 37°C constant temperature incubator and culture overnight for approximately 16 to 24 hours.

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

[0079] (7) Shake at 37°C and 200 rpm until turbidity occurs. At this point, the recombinant plasmid E. coli has been obtained.

[0080] For the positive expression strain whose sequencing results are consistent with the sequence described in SEQ ID NO:1 in the sequence table, IPTG was added to induce expression. The expression conditions were shaking at 37°C in a shaker until the OD value was about 0.6, adding IPTG to a final concentration of 0.1 mM, inducing expression at 6°C for 20 hours, and then purifying with cOmplete His-Tag Purification Resin resin (Roche, Switzerland), where the recombinant protein pCold I-CAT was used as a negative control. SDS-PAGE results showed that the PpSerpin3 recombinant protein could be successfully expressed and purified in Escherichia coli, and the size of the expressed recombinant protein was about 44 kDa, which was consistent with the sequence size prediction of SEQ ID NO:2. Furthermore, the PpSerpin3 recombinant protein was detected using a his tag antibody, and the Western Blot results showed that the recombinant protein was successfully expressed and purified, as shown in Figure 2. Figure 2 shown.

[0081] 3. In order to detect whether the serine protease inhibitor PpSerpin3 has the function of inhibiting hemolymph melanization, the PpSerpin3 recombinant protein (1 μg) was incubated with the hemolymph of Pieris rapae pupae. The specific steps are as follows:

[0082] The hind limbs of the larvae of Pieris rapae were cut with scissors to obtain plasma, which was diluted to 4 times and added to a refrigerated TBS buffer of 20mM Tris, 150mM NaCl, pH=7.6. The blood cells were removed by centrifugation at 4°C and 3000g for 10min to obtain cell-free hemolymph. For the PPO activation test, 5μl of the PpSerpin3 recombinant protein (0.2μg / μl) obtained in step 2 above was mixed with 10μl of diluted Pieris rapae hemolymph in a 384-well plate, and then 5μl of the activator (0.1μg / μl M.luteus) and 5μl of the substrate solution (50mM L-Dopa dissolved in PBS, pH=7.5) were mixed and added to another 384-well plate inverted and fixed on the sample plate. The PPO (prophenoloxidase) cascade reaction in each well was activated simultaneously by centrifuging the two fixed 384-well plates. The plates were measured every 5 minutes at A470 and 25°C for 2 h using a microplate reader Thermo Scientific Varioskan Flash (Thermo Scientific, USA). For the PO activity test, 10 μl of diluted Pieris rapae 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 about 10 min. 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 inverted and fixed on the sample plate. The two fixed 384-well plates were centrifuged to react simultaneously and detected using a microplate reader. 1 unit of PO activity was defined as 0.001 △ A470 / min.

[0083] like Figure 3 AThe results showed that PpSerpin3 inhibited the melanization of Pieris rapae hemolymph by inhibiting the activation of prophenoloxidase PPO into phenoloxidase PO, rather than directly inhibiting the activity of phenoloxidase PO. In other words, PpSerpin3 had no inhibitory effect on the already generated PO. In addition, its inhibitory effect on PPO activation had a dose effect, such as Figure 3 As shown in B. Among them, 1 μg of recombinant protein had the strongest inhibitory effect on hemolymph melanization reaction, followed by 0.125-0.5 μg of recombinant protein, while 0-0.0625 μg of recombinant protein had no obvious inhibitory effect on hemolymph melanization reaction.

[0084] Combined with the above Figure 3As a result, PpSerpin3 can inhibit the activation of prophenoloxidase PPO and prevent it from forming active PO, thereby inhibiting the melanization of hemolymph in Pieris rapae, an important humoral immune response of insects. Therefore, it can be expected that the transfer of the PpSerpin3 protein from the pupal wasp into the target plant will have a control effect on Lepidoptera such as Pieris rapae.

[0085] Finally, it should be noted that the above examples are only some specific embodiments of the present invention. Obviously, the present invention is not limited to the above embodiments, and there are many variations. All variations that can be directly derived or associated with the content disclosed by a person skilled in the art should be considered as the protection scope of the present invention.

Claims

1. A serine protease inhibitor of Chrysalis chrysanthemi, PpSerpin3, characterized in that: Its amino acid sequence is shown in SEQ ID NO:

2.

2. The gene encoding the serine protease inhibitor PpSerpin3 of Chrysalis chrysogenum as claimed in claim 1, characterized in that: The nucleotide sequence thereof is shown in SEQ ID NO:

1.

3. The use of the Chrysalis chrysogenum serine protease inhibitor PpSerpin3 as claimed in claim 1, characterized in that: Used to inhibit the activation of PPO in the hemolymph of Pieris rapae, thereby inhibiting the hemolymph melanization reaction.

4. A method for improving the control effect of plants on cabbage worms, characterized in that: The recombinant plasmid with the nucleotide sequence shown in SEQ ID NO: 1 is transferred into the target plant, thereby improving the control effect of the plant on cabbage worm.

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