Insect-derived antibacterial protein and application thereof in prevention and control of Candidatus Liberibacter asiaticum

Abstract

The invention discloses an insect-derived antibacterial protein. The amino acid sequence of the insect-derived antibacterial protein is shown as SED NO.1. Relates to the technical field of prevention and control of plant diseases and insect pests, and further discloses application of the insect-source antibacterial protein in prevention and control of Candidatus Liberobacter asiaticum. Research finds that in-vivo screening of diaphorina citri which does not carry germs after continuous feeding of plants infected with Candidatus Liberobacter asiaticum finds that a Waprin gene is remarkably up-regulated; protein coded and translated by the gene has a remarkable splitting effect on Candidatus Liberibacter asiaticum. The invention also finds that the insect-source antibacterial protein directly acts on the pathogenic bacteria or the disease-transmitting mediator of the Candidatus Liberobacter asiaticum on the living body level, avoids the ecological risk of chemical agents and the physical control blind area of the pathogenic bacteria, and also makes up the defect that the mediator insect-pathogenic bacteria interaction mechanism cannot be deeply explored depending on natural-source active substances due to the limitation of research materials in the prior art. Further, the blank of efficient targeted prevention and control means is developed, and a new path is provided for green prevention and control and fundamental research breakthrough of the citrus huanglongbing.

Description

Technical Field

[0001] This invention relates to the field of pest and disease control technology, specifically to an insect-derived antibacterial protein and its application in controlling Huanglongbing (HLB) of citrus. Background Technology

[0002] Citrus Huanglongbing (HLB) is the most devastating disease affecting the citrus industry worldwide, causing billions of dollars in economic losses annually. Its pathogen is the Gram-negative bacterium *Bacillus phloem*. Candidatus Liberibacter spp. C Las), most citrus varieties currently on the market are susceptible to C The disease is influenced by Las and causes varying degrees of symptoms. HLB has a latent period; early-stage infected citrus trees do not show symptoms. Young trees infected typically die within 2-3 years, while mature trees die within 5-8 years. Because no effective treatment has yet been found, HLB is often referred to as the "cancer" of citrus and was classified as a Class A disease by my country's Ministry of Agriculture and Rural Affairs in 2021.

[0003] Currently, to combat HLB, the industry has developed a series of control measures, including effective control of the vector insect citrus psyllid, using plant defense inducers to enhance the resistance of citrus trees, employing heat therapy to inhibit pathogen activity, using biological control technologies to regulate ecological balance, strictly implementing quarantine plans to prevent the spread of the disease, and timely removal of diseased trees. However, existing methods for controlling citrus Huanglongbing encounter multiple technical challenges: on the one hand, spraying pesticides to control disease vectors (such as the Asian citrus psyllid) can have adverse effects on the ecological environment and other organisms; on the other hand, the pathogen of citrus Huanglongbing (… C Las (a type of plant) is located deep within the sieve tubes of fruit trees, making it difficult to apply pesticides, and it cannot be cultivated in the laboratory to date, making both eradication and basic research extremely difficult.

[0004] Therefore, this application is hereby submitted. Summary of the Invention

[0005] The technical problem this invention aims to solve is that existing methods for controlling citrus Huanglongbing (HLB) have adverse effects on the ecological environment and other organisms. The HLB pathogen resides deep within the sieve tubes of fruit trees, making it difficult to apply pesticides, and it cannot yet be cultured in a laboratory, resulting in exceptionally difficult eradication and basic research. The objective is to provide an insect-derived antibacterial protein and its application method in controlling HLB.

[0006] This invention is achieved through the following technical solution: In a first aspect, the present invention provides an insect-derived antimicrobial protein, the amino acid sequence of which is shown in SED NO.1.

[0007] As one of the preferred technical solutions, the insect-derived antibacterial protein is encoded and translated through the Waprin gene of the citrus psyllid, the nucleotide sequence of which is shown in SED NO.2.

[0008] Secondly, the present invention provides the application of the above-mentioned insect-derived antibacterial protein in regulating the Huanglongbing pathogen of citrus.

[0009] As one of the preferred technical solutions, the application method of insect-derived antibacterial protein is as follows: the insect-derived antibacterial protein is applied to citrus plants, thereby causing the citrus psyllids that feed on citrus leaves to... C Las cells lyse, resulting in a decrease in bacterial load.

[0010] As one of the preferred technical solutions, the bacterial lysis morphology in the citrus psyllid is revealed by observing the state of the intestinal tissue cells of the adult larvae under a microscope.

[0011] As one of the preferred technical solutions, the application concentration of the insect-derived antibacterial protein is 1 mM.

[0012] Thirdly, the present invention also provides a method for preparing the above-mentioned insect-derived antimicrobial protein, comprising the following steps: The gene with the nucleotide sequence SEQ ID NO.2 was obtained by PCR amplification from the citrus psyllid. The gene with the nucleotide sequence SEQ ID NO.2 was inserted into a prokaryotic expression plasmid to construct a recombinant plasmid; The recombinant plasmid was transferred into competent cells, and prokaryotic expression was performed in the competent cells. After induction with IPTG, the bacterial cells containing insect-derived antimicrobial proteins were collected.

[0013] As one of the preferred technical solutions, the amplification primer pair required for prokaryotic expression is: pColdII- waprin -F: CATCATCATCATCATCATCATGATGAAATTATTCCAGTTC pColdII- waprin -R: AGACTGCAGGTCGACAAGCTTTTATGGATATGCACATTTC.

[0014] As one of the preferred technical solutions, the bacterial cells are cultured for 12 hours after induction and the precipitate is collected by centrifugation. Then, the precipitate is vortexed and resuspended, ultrasonically disrupted, centrifuged, and the supernatant is collected. The supernatant is passed through a Ni-NTA pre-packed gravity column, eluted with imidazole, and further desalted to obtain a purified insect-derived antibacterial protein solution.

[0015] As one of the preferred technical solutions, the insect-derived antibacterial protein solution can be directly fed to adult citrus psyllids carrying the bacteria or sprayed on the leaves of citrus plants for the citrus psyllids to feed on.

[0016] Compared with the prior art, the present invention has the following advantages and beneficial effects: 1. This invention is the first to propose the use of insect-derived antibacterial proteins in the control of citrus Huanglongbing (HLB). The insect-derived antibacterial proteins provided by this invention act directly on the disease-transmitting vector at the living level, avoiding the ecological risks of chemical agents and the blind spots of physical control of pathogens. Furthermore, it fills the gap in existing technologies, which, due to limitations in research materials, have failed to deeply explore the interaction mechanism between vector insects and pathogens based on naturally derived active substances. This fills the gap in developing efficient targeted control methods and provides a new path for green control and basic research breakthroughs in citrus HLB. Attached Figure Description

[0017] To more clearly illustrate the technical solutions of the exemplary embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly described below. It should be understood that the following drawings only show some embodiments of the present invention and should not be considered as a limitation of the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort. In the drawings: Figure 1 This is a Western Blot validation image of the insect-derived antibacterial protein of the present invention; Figure 2 This invention involves feeding the bacteria-carrying test worms with Waprin protein into their intestines. C Electron micrograph of Las morphology; Figure 3 The present invention relates to the intestinal tract of bacteria-carrying test worms fed with sucrose water. C Electron micrograph of Las morphology; Figure 4 This is a diagram showing the state of intestinal cells of the uninfected test worms of the present invention; Figure 5 This is a comparison chart showing the amount of bacteria acquired by citrus psyllids 120 hours after feeding on infected plants following RNA interference according to the present invention. Detailed Implementation

[0018] To make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be further described in detail below with reference to the embodiments and accompanying drawings. The illustrative embodiments and descriptions of the present invention are only used to explain the present invention and are not intended to limit the present invention.

[0019] The "scope" disclosed in this invention is defined in the form of a lower limit and an upper limit. A given scope is defined by selecting a lower limit and an upper limit, which define the boundaries of the specific scope. The scope defined in this way can include or exclude end values, and can be arbitrarily combined; that is, any lower limit can be combined with any upper limit to form a scope.

[0020] Unless otherwise specified, all embodiments and optional embodiments of the present invention can be combined with each other to form new technical solutions.

[0021] Unless otherwise specified, all technical features and optional technical features of this invention can be combined to form new technical solutions.

[0022] Unless otherwise specified, the terms "comprising" and "including" as used in this invention can be open-ended or closed-ended. For example, "comprising" and "including" can mean that other substances not listed may also be included, or that only the listed substances may be included.

[0023] Unless otherwise specified, all steps of the present invention may be performed sequentially or randomly, preferably sequentially. For example, the method includes steps (a) and (b), indicating that the method may include steps (a) and (b) performed sequentially, or it may include steps (b) and (a) performed sequentially. For example, the mention that the method may also include step (c) indicates that step (c) may be added to the method in any order. For example, the method may include steps (a), (b), and (c), or it may include steps (a), (c), and (b), or it may include steps (c), (a), and (b), etc.

[0024] The technical solution of the present invention will be further described in detail below with reference to the embodiments.

[0025] It should be noted that, unless otherwise specified, the experimental methods used in the embodiments are conventional methods. Unless otherwise specified, the materials, reagents, methods, and instruments used are all conventional materials, reagents, methods, and instruments in the art, and can be obtained commercially by those skilled in the art. Example 1

[0026] Effective control of the vector insect, the citrus psyllid, often involves using plant defense inducers to enhance the resistance of citrus trees, employing heat therapy to inhibit pathogen activity, or utilizing biological control techniques to regulate ecological balance, strictly enforcing quarantine plans to prevent disease spread, and promptly removing diseased trees. However, existing methods for controlling citrus Huanglongbing (HLB) encounter multiple technical challenges: on the one hand, spraying pesticides to control disease vectors (such as the Asian citrus psyllid) can have adverse effects on the ecological environment and other organisms; on the other hand, the pathogen of citrus HLB (HLB)... C Las (a type of plant) is located deep within the sieve tubes of fruit trees, making it difficult to apply pesticides, and it cannot be cultivated in the laboratory to date, making both eradication and basic research extremely difficult.

[0027] Based on this, the present invention provides an insect-derived antimicrobial protein, the amino acid sequence of which is shown in SED NO.1. The insect-derived antimicrobial protein is encoded by the Waprin gene of the citrus psyllid, the nucleotide sequence of which is shown in SED NO.2.

[0028] The insect-derived bactericidal protein provided by this invention can act directly on the disease vector at the living level, which avoids the ecological risks of chemical agents and the blind spots of physical control of pathogens. It also fills the gap in the existing technology due to the limitation of research materials, which has failed to rely on natural active substances to explore the interaction mechanism between vector insects and pathogens and thus develop efficient targeted control methods. This provides a new path for the green control and basic research breakthroughs of citrus Huanglongbing.

[0029] Specifically, during their research on treatment methods for citrus Huanglongbing (HLB), the inventors discovered that screening citrus psyllids that did not carry the pathogen after continuously feeding on HLB-infected plants revealed a significant upregulation of the Waprin gene. The protein encoded by this gene has a significant lytic effect on HLB pathogens.

[0030] Primers were designed based on the gene sequence provided by the citrus psyllid genome. The specific primer design is shown in Table 1.

[0031] Table 1 Primer information for target gene sequences

[0032] The specific experimental procedure is as follows: PCR amplification was performed using PrimeSTAR Max DNA Polymerase reagent. Dc_Waprin Sequences were obtained using DcWaprin-F and DcWaprin-R primer pairs.

[0033] The specific amplification reaction system is as follows:

[0034] The PCR reaction program was as follows: 98℃ pre-denaturation for 10 min; 98℃ denaturation for 15 s, 58℃ annealing for 30 s, 72℃ extension for 30 s, 35 cycles; 72℃ extension for 5 min; 4℃ pre-store.

[0035] PCR products that passed 1% agarose gel electrophoresis were recovered using the TaKaRa MiniBEST Agarose Gel DNA Extraction Kit according to the manufacturer's instructions. After adding sticky ends, the target gene fragment was ligated using the pGEM-T Easy vector. After overnight ligation at 4°C, the ligation product was transferred into thawed competent cells (Trans 5α), incubated on ice for 30-40 min, then heat-shocked in a 42°C water bath for 50 s, followed by immediate cold shock on ice for 5 min. 200 μL of LB liquid medium was then added, and the cells were incubated at 37°C for 1 h using a shaker. The bacterial culture was then evenly spread onto LB solid medium and incubated overnight at 37°C. Rounded white spots around the blue spots were picked and placed in 700 μL of LB liquid medium containing ampicillin, and shaken for 5 h. After passing PCR bacterial testing and sequencing, the bacterial culture was expanded to 30 ml.

[0036] Sequencing revealed that the nucleotide sequence encoding Waprin gene (SED NO.2) is: ATGAAATTATTCCAGTTCTGTGCTGCATTCCTTGTTTTGATCGTTGTTGTGCTGTGATGCATTTCCAAGCAAGTTCGGTAACTGTCCTATGGCAAGTACAGTGAAAACTTGCACTCCCAAATGTTACAGCGACTATGAGTGTTCGGGTAACAAGCGCTGT TGTCCTAACTACTGTGGCTGGAAATCCTGTAGTGATACGAGTCCTGTAGCCCATGATAAGGGCCCATCAGAAAAAGTTGTCTACTGCGAAAATGTCAAGTGCCAACCGAAGCAGATTTGCAAGTTTGATCCGAAGACCAAAAGATCGAAATGTGCATTCCATAA.

[0037] The amino acid sequence encoded by this gene (SED NO.1) is: MKLFQFCAAFLVLIVLCCDAFPSKFGNCPMASTVKTCTPKCYSDYECSGNKRCCPNYCGWKSCSDTSPVAHDKGPSEKVVYCENVKCQPKQICKFDPKTKRSKCAYP. Example 2: Effects of feeding citrus psyllids with insect-derived antibacterial protein

[0038] Based on Example 1, plasmids were extracted using the QIAGEN Plasmid Plus Midi Kit, following its instructions. The specific procedure was as follows: Place a 50 mL centrifuge tube containing bacterial culture in a refrigerated centrifuge and centrifuge at 4°C and 5000 g for 20 min. Discard the supernatant and retain the bacterial precipitate. Then, add 2 mL of Buffer P1 to the centrifuge tube to resuspend the precipitate, followed by 2 mL of Buffer P2 and gently mix. Incubate at room temperature for 5 min. Add 2 mL of Buffer S3 to a 50 mL centrifuge tube and mix by inverting the tube 5-6 times. The solution will show white flocculent turbidity. Transfer the liquid and precipitate into the filter cartridge, incubate at room temperature for 10 min, and then use a plunger to separate the liquid and precipitate into a new 50 mL tube; Add 2 mL of Buffer BB to a new 50 mL tube, invert 4 to 6 times to mix, transfer the mixed liquid to a QIAGEN Plasmid Plus Midi rotating column, and connect the QIAGEN Plasmid Plus Midi rotating column to a vacuum pump to extract the solution. After extraction, add 700 μL of Buffer ETR and extract simultaneously, then add 700 μL of Buffer PE and extract simultaneously. Place the rotating column into an empty collection tube, centrifuge at 9000 g for 1 min, then add 60-80 μL of Buffer EB, let stand at room temperature for 1 min, and centrifuge at 12,000 g for 1 min to elute plasmid DNA. After measuring the concentration of the extracted plasmid solution using a nucleic acid concentration analyzer, it was stored at -20℃ for later use.

[0039] Using the pColdII-waprin-F / pColdII-waprin-R primer pair, the extracted plasmid was used as a template to amplify the fragment. The Waprin sequence with homologous arms was amplified by PCR using PrimeSTAR Max DNA Polymerase reagent. The PCR reaction program was as follows: 98℃ pre-denaturation for 3 min; 98℃ denaturation for 30 s, 60℃ annealing for 30 s, 72℃ extension for 30 s, 35 cycles; 72℃ extension for 5 min; and storage at 4℃.

[0040] After the reaction was complete, the product was recovered. The pColdII plasmid was linearized using NdeI and HindIII restriction enzymes. Using the ClonExpress® II One Step Cloning Kit, following the instructions, the recovered product was homologously recombined with the linearized plasmid to construct a recombinant plasmid. The recombinant plasmid was transformed into competent cells (Trans 5α), and after plasmid extraction, it was transformed into TransBL21 (DE3) competent cells for prokaryotic expression. The specific steps are as follows: Correctly sequenced colonies were cultured at 37°C and 200 rpm / min until the OD600 value reached approximately 0.8. Isopropyl-β-D-thiogalactoside (IPTG) was added to the bacterial culture to a final concentration of 1 mM, followed by induction culture at 16°C and 180 rpm / min for 12 h. The bacterial pellet was collected by centrifugation at 4000 g for 30 min at 4°C. 50 mL of phosphate buffer (pH = 7.5) was added to the pellet, and the pellet was vortexed to resuspend it. The bacterial cells were disrupted using an ultrasonic disruptor at 30% power for 40 min (3 s disruption followed by a 5 s interval). The disrupted solution was then centrifuged at 12000 g for 30 min at 4°C, and the supernatant was collected. The supernatant was passed through a Ni-NTA pre-packed gravity column and eluted with imidazole to obtain the purified recombinant protein.

[0041] like Figure 1 As shown, Western blotting of the protein solution using Waprin antibody confirmed successful expression of Waprin protein. Subsequently, a desalting column was used to replace imidazole with phosphate buffer.

[0042] Adult citrus psyllids infected with the above-described protein solution or 16% sucrose solution were fed for 24 hours. Adults feeding on the 16% sucrose solution served as the control group, while non-infected adults served as the control group. Subsequently, their intestines were dissected, and the morphology of the Huanglongbing bacteria in the intestines was observed using transmission electron microscopy. The specific steps are as follows: Sample preparation and prefixation: The intestinal tissue of adult worms fed with Waprin protein, 16% sucrose water, and 24 h after being sterile was dissected. During dissection, the worms were immersed in phosphate buffered saline (PBS) at pH 7.4. Immediately after dissection, the intestines were placed in centrifuge tubes containing 2.5% glutaraldehyde and incubated overnight at 4°C. Sample cleaning: Remove glutaraldehyde from the centrifuge tubes, then wash the intestines 6 times with phosphate buffer (pH 7.4). Change the phosphate buffer every 15 minutes. Post-fixation: Transfer the centrifuge tube to a fume hood, remove the phosphate buffer, slowly add 500 μL of 1% osmium tetroxide to the centrifuge tube, seal the centrifuge tube with sealing film, and incubate at room temperature in a fume hood for 3 h; Sample cleaning: Aspirate 1% osmium tetroxide and wash the sample with phosphate buffer, once every 5 minutes, for a total of 3 times; Dehydration: After removing excess phosphate buffer, dehydrate with a pre-prepared gradient of 50%, 70%, 80%, 90%, 95% and 100% anhydrous ethanol, treating each concentration gradient for 15 min. Replacement: Remove excess anhydrous ethanol, immediately add 1 mL of acetone and treat for 30-60 min, replacing the acetone every 10 min. Encapsulation: A magnetic rotor was placed in the resin encapsulation medium and placed on a magnetic rotary stirrer. The mixture was incubated at 600 rpm and 37°C for 1 h. Subsequently, the intestines and encapsulation medium were placed together in a preheated encapsulation mold. The mold was then placed in an oven and polymerized at gradients of 40°C, 50°C, and 60°C, incubating for 12 h at each temperature gradient, with a final polymerization at 70°C for 24 h.

[0043] Slide preparation and staining: 80 nm sections were cut using an ultramicrotome, and qualified sample sections were placed on a copper grid. The sections were then rinsed three times with pure water for 5 min each time. Staining was performed with 2% uranium acetate for 10 min, followed by rinsing three times with pure water for 5 min each time. Finally, staining was performed with lead citrate for 7 min, followed by rinsing three times with pure water for 5 min each time.

[0044] Imaging observation: After the sample was thoroughly dried, the ultrastructure of Huanglongbing bacteria in intestinal cells was observed using a transmission electron microscope HT7800 at 80 kV.

[0045] Figure 4 A diagram showing the intestinal cell state of an uninfected citrus psyllid, such as... Figure 2 As shown, the Huanglongbing bacteria in the intestines of citrus psyllids fed with Waprin protein solution lysed; Figure 3 As shown, the Huanglongbing bacteria in the intestines of citrus psyllids fed with 16% sucrose solution remained morphologically intact. Example 3: Knockdown of the gene encoding Waprin protein in citrus psyllid using RNA interference technology

[0046] Based on Example 1, the Waprin gene fragment containing the T7 promoter sequence was amplified using the dsDcWaprin-F / dsDcWaprin-R primer pair. Subsequently, dsWaprin was synthesized and purified using the TranscriptAid T7 High Yield Transcription kit according to the manufacturer's instructions. The integrity of the dsRNA was detected by 1% agarose gel electrophoresis, and the qualified dsRNA was stored at -80℃ for later use. The dsWaprin solution was injected into newly emerged citrus psyllid adults (emergence time less than 12 h) for 24 h. The RNAi-injected adults were then transferred to infected plants and fed continuously for 120 h, with dsGFP-injected adults serving as a control. DNA was then extracted from each insect using the Tiangen Genomic DNA Extraction Kit (DP304), with the specific steps as follows: Add 200 μL of buffer GA, grind the sample thoroughly, then add 20 μL of proteinase K and mix thoroughly. Add 200 μL of buffer GB, mix thoroughly, incubate at 65°C for 10 min, and then centrifuge briefly to remove water droplets from the inner wall of the tube cap. Add 200 μL of anhydrous ethanol and mix thoroughly for 15 s; Transfer all the solution and flocculent precipitate obtained in the previous step to an adsorption column CB3 that has been placed in a collection tube, centrifuge at 12000 × g for 1 min, and discard the waste liquid. Add 500 μL of buffer GD to the adsorption column CB3, centrifuge at 12000 × g for 1 min, and discard the waste liquid; Add 600 μL of buffer PW to the adsorption column CB3, centrifuge at 12000 × g for 1 min, and discard the waste liquid. Repeat this operation once; centrifuge at 12000 × g for 2 min, discard the waste liquid, and place the adsorption column CB3 at room temperature to air dry any residual washing liquid in the adsorption material; Add 35 μL of elution buffer TE to the adsorption membrane, let it stand at room temperature for 2 min, centrifuge at 12000 × g for 2 min, and then collect the solution in a centrifuge tube. The concentration and purity of the extracted DNA were determined using a spectrophotometer and stored at -80°C for subsequent detection.

[0047] Using qPCR detection C Las titer. C The primer and probe sequences for Las detection quantification are shown in Table 1. Primer pairs CLas-F and CLas-R were used.

[0048] TaqMan fluorescent probe method for detection C The reaction system for Las titer is as follows: The Dc_Waprin sequence was amplified by PCR, and the specific amplification reaction system is as follows:

[0049] The reaction procedure was as follows: pre-denaturation at 95℃ for 10 min; denaturation at 95℃ for 15 s; reaction at 60℃ for 60 s with fluorescence collection, for a total of 40 cycles; cooling at 37℃ for 30 s.

[0050] like Figure 5 As shown, the sample Ct value is substituted into the established standard curve (y = -4.11x + 55.508 R² = 0.9964) for calculation. C The titer of Las was measured. Results showed that silencing the Waprin gene significantly increased the total bacterial load per insect. This indicates that knocking down the gene encoding the Waprin protein using RNAi technology can significantly enhance the resistance of citrus psyllids to Las. C Las's ability to acquire.

[0051] The above experiments further demonstrated the correlation between the Waprin gene and the bacterial load in citrus psyllids, thus providing a new approach for the subsequent control of citrus Huanglongbing pathogens (CLas). Specific methods include directly feeding infected adult citrus psyllids with insect-derived antibacterial protein solutions or spraying them on the leaves of citrus plants for the psyllids to feed on. These methods avoid the ecological risks of chemical agents and the blind spots of physical control of pathogens. They also fill the gap in existing technologies, which, due to limitations in research materials, have failed to deeply explore the interaction mechanism between vector insects and pathogens based on naturally derived active substances. This fills the gap in developing efficient targeted control methods and provides a new path for the green control and basic research breakthroughs of citrus Huanglongbing.

[0052] The specific embodiments described above further illustrate the purpose, technical solution, and beneficial effects of the present invention. It should be understood that the above description is only a specific embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. An insect-derived antibacterial protein, characterized in that, The amino acid sequence of the insect-derived antibacterial protein is shown as SED NO.

1.

2. The insect-derived antibacterial protein according to claim 1, characterized in that, The insect-derived antibacterial protein is translated by the Waprin gene of the citrus psyllid, and the nucleotide sequence of the Waprin gene of the citrus psyllid is shown as SED NO.

2.

3. The insect-derived antibacterial protein of claim 1 or 2 for use in preventing and controlling the Huanglongbing pathogen of citrus.

4. Use according to claim 3, characterized in that, The application method of the insect-derived antibacterial protein is as follows: the insect-derived antibacterial protein is used for spraying on citrus plants, so that the amount of bacteria in the citrus psylla feeding on the citrus leaves is reduced C Las bacterial lysis, and the bacterial load is reduced.

5. Use according to claim 3, characterized in that, The bacterial lysis form in the body of the citrus psyllid is manifested by observing the state of the intestinal tissue cells of the adult under a microscope.

6. Use according to claim 3, characterized in that, The application concentration of the insect-derived antibacterial protein is 1 mM.

7. A method for preparing an insect-derived antibacterial protein as claimed in claim 1 or 2, characterized by, The method comprises the following steps: PCR amplification of the gene with the nucleotide sequence of SEQ ID NO. 2 from the citrus psyllid; inserting the gene with the nucleotide sequence of SEQ ID NO. 2 into a prokaryotic expression plasmid to construct a recombinant plasmid; transferring the recombinant plasmid into a competent cell, performing prokaryotic expression in the competent cell, collecting the bacterial bodies containing the insect-derived antibacterial protein after IPTG induction.

8. The method of claim 7, wherein, The amplification primer pair required for prokaryotic expression is as follows: pColdII- waprin -F: CATCATCATCATCATCATATGATGAAATTATTCCAGTTC pColdII- waprin -R: AGACTGCAGGTCGACAAGCTTTTATGGATATGCACATTTC.

9. The preparation method according to claim 7, characterized in that, After induction, the bacterial bodies are cultured for 12 h and centrifuged to collect the precipitate, which is then resuspended by vortexing, broken by ultrasonic, and centrifuged to collect the supernatant. The supernatant is passed through a Ni-NTA preloaded gravity column, eluted with imidazole, and further desalted to obtain a purified insect-derived antibacterial protein solution.

10. The method of claim 9, wherein, The insect-derived antibacterial protein solution can be directly fed to the adult citrus psyllid carrying the bacteria or sprayed on the leaves of the citrus plant for the citrus psyllid to feed on.

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