Recombinant protein induced by aphids and preparation and application thereof

Abstract

This invention discloses a technical solution for aphid-induced recombinant protein and its preparation and application, using ladybugs... Hvar CSP8 and Hvar Cloning, expression, and functional analysis of CSP15 successfully yielded two soluble recombinant olfactory proteins, revealing their specific ability to recognize various typical plant volatiles induced by aphids, particularly exhibiting high affinity for key pest signaling molecules. Further structural modeling and molecular docking elucidated the molecular basis of protein-ligand interactions, clarifying key binding sites and modes of action. These two aphid-induced recombinant proteins can serve as novel targets for green control of cotton aphids, enabling the development of behavioral regulators to enhance the targeted hunting ability of natural enemy insects against aphids, reduce dependence on chemical pesticides, and provide crucial support for sustainable green control of cotton aphids.

Description

Technical Field

[0001] This invention belongs to the field of crop pest control technology, specifically the field of recombinant protein preparation technology, and more specifically the field of aphid-induced recombinant protein and its preparation and application technology. Background Technology

[0002] The multi-horned ladybug (Coleoptera: Coccinellidae) is a highly valuable predatory natural enemy, highly adaptable to temperature and humidity, with a long feeding cycle in the field and a wide predatory range. Its main prey consists of various aphids such as peach aphids, wheat aphids, and cotton aphids, but it also preys on lepidopteran larvae and eggs, thrips, leafhoppers, and other agricultural pests, playing an important role in biological control. Originating in the Palearctic realm, it is now widely distributed in Asia, Africa, Europe, and many regions of my country, including Beijing, Hebei, and Henan provinces. It commonly inhabits various agricultural ecosystems such as wheat fields, corn fields, alfalfa fields, and cotton fields. The number of generations per year for the multi-horned ladybug is influenced by regional climate, generally ranging from 3 to 5 generations. It belongs to the holometabolous group, and its individual development involves four consecutive stages: egg, larva, pupa, and adult. Ladybugs overwinter in diapause. In my country, as temperatures gradually decrease in mid-October each year, adults gather in groups of 3-5 in cracks on sunny slopes or at the roots of weeds to hibernate. During diapause, both male and female adults cease feeding, mating, and oviposition to adapt to the low temperatures and survive the winter. Ladybugs exhibit cannibalism and feigning death. When threatened in their habitat, adults retract their six legs and curl up, serving as an effective safety mechanism. Cannibalism occurs throughout the larval and adult stages; hungry adults will prey on larvae and eggs of their own species, and older larvae will also prey on younger larvae and eggs.

[0003] As an important predatory natural enemy, the ladybug has a wide range of prey, including cotton aphids and spirea aphids. Aphis spiraecola and wheat aphid Sitobion avenae Common aphids, and also wolfberry psyllids Paratrioza Chinese Grape leafhopper Arboridia apicalis Even the fall armyworm Spodoptera frugiperdaYoung larvae also possess strong predatory potential. In terms of predatory function, the predatory behavior of *Heterodon spp.* against most prey conforms to the Holling-II model, but its predatory capacity varies significantly depending on the prey species: the maximum daily predation of cotton aphids ranges from 146.0 to 333.3 individuals, the highest daily predation of *Lycium barbarum* psyllid nymphs reaches 410.0 individuals, and the daily predation of grape leafhopper adults is only 69.0 individuals. Of particular note is its excellent control ability against young larvae of the fall armyworm, with an a / Th value as high as 269.09. Predatory efficiency is influenced by multiple factors: temperature is a key environmental factor, with 29℃ being its optimal reproductive temperature; simultaneously, the ladybug's own stage and instar also play an important role, with larvae gradually increasing their predation rate as they grow, while the predation rate of adults is constrained by sex and age. In ecosystems such as cotton fields, the ladybug occurs naturally as a key natural enemy, and its effect on controlling cotton aphids is particularly significant, demonstrating broad application prospects in biological control.

[0004] The ladybug (Heteromorpha heteromorpha) is an important predatory natural enemy in cotton fields in Xinjiang Uygur Autonomous Region, playing a crucial role in the integrated management of cotton aphids. However, the molecular mechanism by which the ladybug uses its olfactory system to recognize aphid-induced plant volatiles remains unclear. Deciphering this mechanism and further screening key active volatile components to develop biological control products such as natural enemy attractants that can attract or regulate the behavior of the ladybug is of great significance for improving the level of green control of cotton aphids, reducing dependence on chemical pesticides, and protecting the farmland ecological environment. Therefore, elucidating this olfactory recognition mechanism and promoting its product application is a key direction that urgently needs breakthrough in green control of cotton fields. Summary of the Invention

[0005] Given the current lack of clarity regarding the mechanism by which ladybugs (Hemiberlesia lataniae) use their olfactory system to identify aphid-induced plant volatilization, and the absence of biological control products such as attractants for cotton aphids that can attract or regulate the behavior of ladybugs, the technical solution provided in this application addresses the issue of ladybugs... Hvar CSP8 and Hvar Cloning, expression, and functional analysis of CSP15 successfully yielded two soluble recombinant olfactory proteins, revealing their specific ability to recognize various typical plant volatiles induced by aphids, particularly exhibiting high affinity for key pest signaling molecules. Further structural modeling and molecular docking elucidated the molecular basis of protein-ligand interactions, clarifying key binding sites and modes of action. These two aphid-induced recombinant proteins can serve as novel targets for green control of cotton aphids, enabling the development of behavioral regulators to enhance the targeted hunting ability of natural enemy insects against aphids, reduce dependence on chemical pesticides, and provide crucial support for sustainable green control of cotton aphids.

[0006] To achieve the above objectives, the present invention employs the following technical solution: This invention provides an aphid-induced recombinant protein, wherein the recombinant protein is a chemosensory protein of the ladybug.

[0007] The recombinant protein is a chemoreceptor protein from *Heliotropium indicum*. Hvar CSP8 or Hvar CSP15, whose amino acid sequence is shown in SEQ ID NO. 1 or SEQ ID NO. 2. Encoding the chemoreceptor protein of the ladybug Hvar The nucleotide sequence of CSP8 is shown in SEQ ID NO. 3.

[0008] Encoding the chemoreceptor protein of the ladybug Hvar The nucleotide sequence of CSP15 is shown in SEQ ID NO. 4.

[0009] Furthermore, this application also provides a method for preparing the aphid-induced recombinant protein, which is prepared by the following steps: (1) Select adult female and male ladybugs that are 3-5 days old after the first generation emerges. Place the collected antennae of the female and male adults into 1.5 mL RNase-free centrifuge tubes and use the HiPure Universal RNA Minikit kit to extract total RNA from the antennae of the female and male ladybugs. (2) After the total RNA obtained in step (1) is qualified, cDNA is synthesized by reverse transcription using 1 μg of total RNA as a template and Fast King one-step method. (3) Using the cDNA obtained in step (2) as a template, the reaction system was amplified for 35 cycles under the following conditions: 1.5 µL dNTP, 2 µL ExTaq buffer, 0.5 µL upper primer, 0.5 µL lower primer, 0.2 µL ExTaq polymerase, 1 µL cDNA, and 14.3 µL H2O. (4) After electrophoresis separation of the target fragment obtained in step (3), cut out a piece of gel with the same size as the target band into a centrifuge tube and recover the PCR product using the Omega recovery kit; (5) The recovered product obtained in step (4) was ligated at 25°C for 45 min with 4 µL of PCR product and 1 µL of pEASY-T. (6) Gently mix the ligation product obtained in step (5) with competent cells and place on ice for 30-40 min; heat shock at 42℃ for 90 s, place on ice for 3 min, then add LB liquid medium without antibiotics, shake at 37℃ and 200 rpm for 60 min; melt the LB solid medium with 50 µg / mL kanamycin and pour it into the culture blood; centrifuge the bacterial solution after shaking for 60 min at 4000 rpm for 2 min, discard 600 µL of supernatant, and suspend the precipitated bacterial cells with the remaining liquid; take 100 µL of the suspension and spread it evenly, and incubate at 37℃ overnight inverted. (7) Place the single colony picked from the culture dish obtained in step (6) into a shaker tube containing 5 mL of LB medium with 50 µg / mL kanamycin, and incubate at 37°C and 200 rpm for 12-16 h; use 10 µL of 2× TransStart® GreenqPCR SuperMix, 0.5 µL of forward primer (10 µM), 0.5 µL of reverse primer (10 µM), 1 µL of cDNA template, and 8 µL of ddH2O; send the positive bacterial culture for sequencing identification to isolate HvarCSP8 and the target fragment of HvarCSP8; (8) Based on the target fragment obtained in step (7), the cloning plasmid pEASY-T1- is digested with restriction endonucleases BamHI and XhoI. Hvar CSP8, pEASY-T1- Hvar The CSP8 and pET28a empty vectors were used to recover the linear target fragment and vector fragment after enzyme digestion, respectively. The target fragment and vector fragment were ligated using a seamless cloning enzyme and transformed into *E. coli* Trans1-T1 competent cells. Positive recombinant plasmids were screened by colony PCR. The positive recombinant plasmid pET28a- Hvar CSP8 and pET28a- Hvar CSP15 identification; the recombinant plasmid was sequenced and identified, and pET28a- Hvar CSP8 and pET28a- Hvar CSP15 was transformed into Escherichia coli BL21(DE3) competent cells, and positive single clones were screened by PCR. (9) Inoculate the positive clones obtained in step (8) into LB liquid medium and culture at 37℃ and 200 rpm. When the absorbance at OD600 is 0.4~0.6, take out 3~5 mL and linearize the recombinant expression vector pET-28a using the double enzyme digestion method according to the instructions of the OMEGA Column Plasmid Extraction Kit (Plasmid Mini Kit I). Hvar CSP 8、pET28a- Hvar The CSP15 digestion system consisted of 5000 ng of the target fragment, 2.5 µL of BamHI, 2.5 µL of XhoI, 5 µL of 10X QuickCut Green Buffer, and 24.62 µL of ddH2O, and was reacted at 37℃ for 8–12 h. (10) The linearized plasmid and the non-linearized plasmid in step (9) showed obvious band differences on the agarose gel. The target band was cut and recovered. The recovered linearized vector and homologous recombination PCR product were mixed in a 10µL system with 2×Ulandy Seamless Cloning Mix 5.0µL, linearized vector (pET-28a) 100 ng, insert fragment (PCR product) 50 ng, and ddH2O. The mixture was reacted in a metal bath at 37℃ for 30 min to carry out homologous recombination. (11) The pET28a- sample identified as positive in step (10) Hvar CSP8 and pET28a- Hvar CSP15 recombinant plasmid was transformed into *Escherichia coli* Transetta (DE3) strain; positive single colonies were cultured overnight at 37°C and 220 rpm in LB medium containing Kana, and cultured until OD600 reached 0.4-0.6. IPTG was added to a final concentration of 0.02 mmol / L, and induction was performed at 16°C for 16-18 h. The induced bacterial culture was centrifuged at 10,000 rpm for 10 min, and the collected cells were resuspended in 20 mM Tris-HCl (pH = 7.4), sonicated, and placed on ice for 30 min at 120 W for 5 s followed by a 5 s pause. The treated sample was analyzed by 15% SDS-PAGE and stained with Coomassie brilliant blue. After sonication, the supernatant was centrifuged at 10,000 rpm for 10 min. Total protein from the supernatant was transferred to the column 3 h later. Before adding the total protein to the column, the total protein concentrations were 20, 50, 100, 150, 200, 250, and 500 mg / L. mmol / L imidazole washing column; His- Hvar CSP8 was washed with imidazole at concentrations of 20, 50, 100, 150, 200, 250, and 500 mmol / L to remove contaminating proteins. His- Hvar CSP15 was washed with imidazole at concentrations of 20, 50, 100, 200, 250, and 500 mmol / L to remove impurities and obtain purified His- Hvar CSP8, His- Hvar CSP15 protein.

[0010] In step (5), the primers used are the upper primer SEQ ID NO. 5 and the lower primer SEQ ID NO. 6 for amplification. Hvar CSP8; the primers used were the upper primer SEQ ID NO. 7 and the lower primer SEQ ID NO. 8 for amplification. Hvar CSP15; In step (7), forward primer SEQ ID NO. 9 and reverse primer SEQ ID NO. 10 are used for amplification. Hvar CSP8; the primers used were the forward primer SEQ ID NO. 11 and the reverse primer SEQ ID NO. 12 for amplification. Hvar CSP15.

[0011] Furthermore, this application also provides the application of the aforementioned aphid-inducing recombinant protein in attracting aphid natural enemies.

[0012] The aphid attractant can be combined with any one or more of the following to attract aphid natural enemies: cis-3-hexenol, linalool, 2-ethyl-1-hexanol, nonanal, α-terpinene and β-caryophyllene, cedrol, α-farnesene and β-pinene, 2-phenylethanol, eucalyptol, geranylacetone, α-caryophyllene, γ-terpinene, DMNT, TMTT and 1,4-diethylbenzene.

[0013] Furthermore, this application also provides an attractant for aphid natural enemies, wherein the attractant contains one of the aforementioned aphid-inducing recombinant proteins or a gene fragment encoding an aphid-inducing recombinant protein.

[0014] The aphid-induced recombinant protein is a chemoreceptor protein from ladybugs. Hvar CSP8 or Hvar CSP8, with an amino acid sequence as shown in SEQ ID NO. 1 or SEQ ID NO. 2; the aphid-induced recombinant protein encoding gene fragment encodes the chemoreceptor protein of *Heteromorpha heteropsis*. Hvar CSP8 or Hvar The CSP8 coding fragment has a nucleotide sequence as shown in SEQ ID NO. 1 or SEQ ID NO. 2.

[0015] The attractant can attract aphid natural enemies by combining it with any one or more of β-caryophyllene, α-farnesene, β-pinene, linalool, terpinene, nonanal, cis-3-hexenol, 2-ethyl-1-hexanol, DMNT, 1,4-diethylbenzene, TMTT, 2-phenylethanol, geranylacetone, eucalyptol, γ-terpinene, and terpinene.

[0016] Compared with the prior art, the present invention has the following features: (1) The technical solution provided in this application discloses the technical solution of aphid-induced recombinant protein and its preparation and application, through the multi-ladybug Hvar CSP8 and Hvar The cloning, expression, and functional analysis of CSP15 clarified the molecular characteristics and expression properties of the two proteins, and the multi-heteromorphic ladybug was successfully cloned. Hvar CSP8 and Hvar The CSP15 gene was synthesized, and a prokaryotic expression system was constructed to obtain a highly pure and soluble recombinant protein (molecular weight approximately 15 kDa). Both are hydrophilic proteins (average hydrophilicities of -0.511 and -0.409, respectively), lacking transmembrane regions, with α-helices dominating their secondary structure (>60%), and the signal peptide located at amino acids 1-19, laying the foundation for functional studies.

[0017] (2) The technical solution provided in this application uses ladybugs Hvar CSP8 and Hvar CSP15 revealed their high affinity binding to aphid-induced plant volatiles. Hvar CSP8 exhibits the strongest binding affinity to β-caryophyllene, and the weakest binding affinity to cedrol, α-farnesene, α-terpinene, β-pinene, linalool, 1-nonanal, and 2-ethyl-1-hexanol. It also has binding affinity to eight other volatile compounds. Hvar CSP15 exhibits good binding affinity for 2-phenylethanol, cis-3-hexen-1-ol, linalool, eucalyptol, benzaldehyde, 1-nonanal, γ-terpinene, α-terpinene, β-caryophyllene, DMNT, geranylacetone, (3E,7E)-4,8,12-trimethyldecadec-1,3,7,11-tetraene, β-pinene, and 1,4-diethylbenzene, with the strongest binding affinity for DMNT and TMTT. DMNT and TMTT are key signaling volatiles released by plants during aphid infestations, indicating... Hvar CSP15 plays an important role in identifying pest-induced volatiles.

[0018] (3) The technical solution provided in this application uses ladybugs Hvar CSP8 and Hvar CSP15 elucidated the molecular mechanism of protein-ligand binding, and obtained a highly reliable three-dimensional structural model (the proportion of the optimal reasonable region on the Laplace diagram is >70%) through homology modeling and molecular docking. Hvar CSP8 binds to α-farnesene at a rate of -6.647 kcal / mol, with key binding residues including amino acid sites such as TRP98, LEU41, TYR44, VAL29, LEU65, and ILE64 playing a key role. Hvar The binding energy of CSP15 to nonanal is -6.3 kcal / mol, and key amino acids include IL31, TYR28, TYR24, and PRO58. These results provide structural templates for targeted modification or simulation of active ligands.

[0019] (4) The technical solution provided in this application provides a new target and product development basis for the green control of cotton aphids. Hvar CSP8 and Hvar CSP15 specifically recognizes various plant volatiles induced by aphids and is an important functional protein in the olfactory system of ladybugs for locating prey. The constructed behavioral regulators can enhance the search efficiency of natural enemy insects for aphids, reduce the use of chemical pesticides, and achieve green and sustainable aphid control in cotton fields. This application lays the theoretical and technical foundation for the development of push-pull strategy biocontrol products based on olfactory proteins. Hvar CSP8 and Hvar CSP15 has a clear and significant beneficial effect in elucidating the molecular mechanism by which ladybugs recognize aphid-induced volatiles, and in the subsequent development of highly effective biocontrol products. Attached Figure Description

[0020] Figure 1 The image shows a ladybug. Hvar CSP8 signal peptide prediction diagram.

[0021] Figure 2 The image shows a ladybug. Hvar CSP15 signal peptide prediction diagram.

[0022] Figure 3 The image shows the analysis using TMHMM software. Hvar CSP8 and Hvar CSP15 transmembrane region results.

[0023] Figure A is... Hvar Figure B shows the results of the CSP8 transmembrane region; Figure B is... Hvar CSP15 transmembrane region results.

[0024] Figure 4 As shown Hvar CSP8 and Hvar Predicted phosphorylation sites of CSP15 protein.

[0025] Figure A is... Hvar Prediction of phosphorylation sites in CSP8 protein; Figure B shows... Hvar Prediction of phosphorylation sites in CSP15 protein.

[0026] Figure 5 As shown Hvar CSP8 and Hvar Figure showing the hydrophobicity analysis results of CSP15.

[0027] Figure A is... Hvar The hydrophobicity analysis results of CSP8 protein; Figure B shows... Hvar CSP15 hydrophobicity analysis results.

[0028] Figure 6 The image shows a ladybug. Hvar CSP8 and Hvar CSP15 secondary structure prediction diagram.

[0029] Figure A shows a ladybug. Hvar CSP8 secondary structure prediction diagram; Figure B shows *Heteromorpha multifida*. Hvar CSP15 secondary structure prediction diagram.

[0030] Figure 7 The image shows a ladybug. Hvar SDS-PAGE analysis of CSP8 protein expression and purification products.

[0031] In Figure A, M represents the protein marker; 1-2: Hvar CSP8 uninduced and induced bacterial cultures; 3-4: supernatant and precipitate after bacterial culture disruption; 5-6: concentrated protein and protein with his-tag removed. In Figure B: M: protein marker; 1: flow-through buffer; 2-8: imidazole elution buffers at concentrations of 20, 50, 100, 150, 200, 250, and 500 mM.

[0032] Figure 8 The image shows a ladybug. Hvar SDS-PAGE analysis of CSP15 protein expression and purification products.

[0033] In Figure A: M: Protein Marker; 1-2: Hvar CSP15 uninduced and induced bacterial cultures; 3-4: supernatant and precipitate after bacterial culture disruption; 5-6: concentrated protein and protein with his-tag removed. In Figure B: M: protein marker; 1: flow-through buffer; 2-8: imidazole elution buffers at concentrations of 20, 50, 100, 200, 250, and 500 mM.

[0034] Figure 9 As shown Hvar CSP8 and Hvar Binding curve of CSP15 with fluorescent probe l-NPN.

[0035] Figure A is... Hvar Binding curves of CSP8 and fluorescent probe l-NPN; Figure B shows... Hvar Binding curve of CSP15 with fluorescent probe l-NPN.

[0036] Figure 10 The image shows a ladybug. Hvar Binding curves of CSCSP8 with the compound.

[0037] Figure 11The image shows a ladybug. Hvar Binding curves of CSP15 with the compound.

[0038] Figure 12 As shown Hvar CSP8 and Hvar La-type conformation diagram of CSP15 ligand.

[0039] Figure 13 As shown Hvar Molecular docking diagram of CSP8 and its ligands.

[0040] Figure 14 As shown Hvar Molecular docking diagram of CSP15 and its ligands. Detailed Implementation

[0041] The following examples are provided to further illustrate the content of this invention, but should not be construed as limiting the invention. Any modifications or substitutions made to the methods, steps, or conditions of this invention without departing from the spirit and essence of the invention are within the scope of this invention.

[0042] The instruments and equipment used in this application are: ultrasonic cell disruptor (Ningbo Xinzhi Biotechnology Co., Ltd.); Heraeus Multifuge X3R benchtop high-speed refrigerated centrifuge (Thermo Fisher Scientific); SpectraMaxi3x multi-functional microplate reader (Meigu Molecular Instruments Co., Ltd.); Forma-902-80℃ cryogenic storage box (Thermo Fisher Scientific); and IS-RDS6 stacked thermostatic shaker (Jingqi Co., Ltd., USA).

[0043] The reagents used in this application are: protein marker and seamless cloning mixed enzyme, Suzhou Youyilandi Biotechnology Co., Ltd.; BamHI, XhoI restriction endonuclease, and pEASY®-T1 Simple Cloning Kit cloning vector kit, Takara Bio Engineering (Dalian) Co., Ltd.; BCA protein concentration assay kit, Lanjieke Technology Co., Ltd.; Fast Blue protein staining solution, Shanghai Yuanye Biotechnology Co., Ltd.; Amicon Ultra-15 ultrafiltration tube (3kDa), Merck Millipore Laboratory Equipment (Shanghai) Co., Ltd.; and imidazole, Beijing Solarbio Technology Co., Ltd.

[0044] The LB medium used in this application was obtained by: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, pH 1.

[0045] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0046] The pupae of the test insects in this application were collected from cotton, corn, and lambsquarters plants around the Korla Experimental Base of the Institute of Plant Protection, Xinjiang Academy of Agricultural Sciences (Korla City, Bayingolin Mongol Autonomous Prefecture, Xinjiang Uygur Autonomous Region, 41.75ºN, 85.81ºE), and then kept in a stable indoor environment.

[0047] Data in this application are expressed as mean ± standard error (Mean ± SEM). The Shapiro-Wilk test was used to assess the normality of the data, and the Levene test was used for homogeneity of variance analysis. For comparisons among multiple groups, if normality and homogeneity of variance were satisfied, one-way ANOVA was used for overall testing, and the Tukey HSD test was used for post-hoc multiple comparisons. If the variances were unequal, Welch's ANOVA was used for correction, and the Games-Howell test was used for multiple comparisons. A p-value < 0.05 was considered statistically significant. Statistical analysis and graphing were performed using GraphPad Prism 10.1.2 and R language 4.5.1.

[0048] Example 1: An aphid-induced recombinant protein A recombinant protein induced by aphids, wherein the recombinant protein is a chemosensory protein of ladybug.

[0049] A recombinant protein induced by aphids, wherein the recombinant protein is a chemoreceptor protein of the ladybug. Hvar CSP8 or Hvar CSP15, whose amino acid sequence is shown in SEQ ID NO. 1 or SEQ ID NO. 2. Encoding the chemoreceptor protein of the ladybug Hvar The nucleotide sequence of CSP8 is shown in SEQ ID NO. 3.

[0050] Encoding the chemoreceptor protein of the ladybug Hvar The nucleotide sequence of CSP15 is shown in SEQ ID NO. 4.

[0051] Example 2: A method for preparing an aphid-induced recombinant protein This application also provides a method for preparing aphid-induced recombinant protein: (1) Select adult female and male ladybugs that are 3-5 days old after the first generation emerges. Place the collected antennae of the female and male adults into 1.5 mL RNase-free centrifuge tubes and use the HiPure Universal RNA Minikit kit to extract total RNA from the antennae of the female and male ladybugs. (2) After the total RNA obtained in step (1) is qualified, cDNA is synthesized by reverse transcription using 1 μg of total RNA as a template and Fast King one-step method. (3) Using the cDNA obtained in step (2) as a template, the reaction system was amplified for 35 cycles under the following conditions: 1.5 µL dNTP, 2 µL ExTaq buffer, 0.5 µL upper primer, 0.5 µL lower primer, 0.2 µL ExTaq polymerase, 1 µL cDNA, and 14.3 µL H2O. (4) After electrophoresis separation of the target fragment obtained in step (3), cut out a piece of gel with the same size as the target band into a centrifuge tube and recover the PCR product using the Omega recovery kit; (5) The recovered product obtained in step (4) was ligated at 25°C for 45 min with 4 µL of PCR product and 1 µL of pEASY-T. (6) Gently mix the ligation product obtained in step (5) with competent cells and place on ice for 30-40 min; heat shock at 42℃ for 90 s, place on ice for 3 min, then add LB liquid medium without antibiotics, shake at 37℃ and 200 rpm for 60 min; melt the LB solid medium with 50 µg / mL kanamycin and pour it into the culture blood; centrifuge the bacterial solution after shaking for 60 min at 4000 rpm for 2 min, discard 600 µL of supernatant, and suspend the precipitated bacterial cells with the remaining liquid; take 100 µL of the suspension and spread it evenly, and incubate at 37℃ overnight inverted. (7) Place the single colony picked from the culture dish obtained in step (6) into a shaker tube containing 5 mL of LB medium with 50 µg / mL kanamycin, and incubate at 37°C and 200 rpm for 12-16 h; use 10 µL of 2× TransStart® GreenqPCR SuperMix, 0.5 µL of forward primer (10 µM), 0.5 µL of reverse primer (10 µM), 1 µL of cDNA template, and 8 µL of ddH2O; send the positive bacterial culture for sequencing identification to isolate HvarCSP8 and the target fragment of HvarCSP8; (8) Based on the target fragment obtained in step (7), the cloning plasmid pEASY-T1- is digested with restriction endonucleases BamHI and XhoI. Hvar CSP8, pEASY-T1- HvarThe CSP8 and pET28a empty vectors were used to recover the linear target fragment and vector fragment after enzyme digestion, respectively. The target fragment and vector fragment were ligated using a seamless cloning enzyme and transformed into *E. coli* Trans1-T1 competent cells. Positive recombinant plasmids were screened by colony PCR. The positive recombinant plasmid pET28a- Hvar CSP8 and pET28a- Hvar CSP15 identification; the recombinant plasmid was sequenced and identified, and pET28a- Hvar CSP8 and pET28a- Hvar CSP15 was transformed into Escherichia coli BL21(DE3) competent cells, and positive single clones were screened by PCR. (9) Inoculate the positive clones obtained in step (8) into LB liquid medium and culture at 37℃ and 200 rpm. When the absorbance at OD600 is 0.4~0.6, take out 3~5 mL and linearize the recombinant expression vector pET-28a using the double enzyme digestion method according to the instructions of the OMEGA Column Plasmid Extraction Kit (Plasmid Mini Kit I). Hvar CSP 8、 pET28a- Hvar The CSP15 digestion system consisted of 5000 ng of the target fragment, 2.5 µL of BamHI, 2.5 µL of XhoI, 5 µL of 10X QuickCut Green Buffer, and 24.62 µL of ddH2O, and was reacted at 37℃ for 8–12 h. (10) The linearized plasmid and the non-linearized plasmid in step (9) showed obvious band differences on the agarose gel. The target band was cut and recovered. The recovered linearized vector and homologous recombination PCR product were mixed in a 10µL system with 2×Ulandy Seamless Cloning Mix 5.0µL, linearized vector (pET-28a) 100 ng, insert fragment (PCR product) 50 ng, and ddH2O. The mixture was reacted in a metal bath at 37℃ for 30 min to carry out homologous recombination. (11) The pET28a- sample identified as positive in step (10) Hvar CSP8 and pET28a- HvarCSP15 recombinant plasmid was transformed into *Escherichia coli* Transetta (DE3) strain; positive single colonies were cultured overnight at 37°C and 220 rpm in LB medium containing Kana, and cultured until OD600 reached 0.4-0.6. IPTG was added to a final concentration of 0.02 mmol / L, and induction was performed at 16°C for 16-18 h. The induced bacterial culture was centrifuged at 10,000 rpm for 10 min, and the collected cells were resuspended in 20 mM Tris-HCl (pH = 7.4), sonicated, and placed on ice for 30 min at 120 W for 5 s followed by a 5 s pause. The treated sample was analyzed by 15% SDS-PAGE and stained with Coomassie brilliant blue. After sonication, the supernatant was centrifuged at 10,000 rpm for 10 min. Total protein from the supernatant was transferred to the column 3 h later. Before adding the total protein to the column, the total protein concentrations were 20, 50, 100, 150, 200, 250, and 500 mg / L. mmol / L imidazole washing column; His- Hvar CSP8 was washed with imidazole at concentrations of 20, 50, 100, 150, 200, 250, and 500 mmol / L to remove contaminating proteins. His- Hvar CSP15 was washed with imidazole at concentrations of 20, 50, 100, 200, 250, and 500 mmol / L to remove impurities and obtain purified His- Hvar CSP8, His- Hvar CSP15 protein.

[0052] In step (5), the primers used are the upper primer SEQ ID NO. 5 and the lower primer SEQ ID NO. 6 for amplification. Hvar CSP8; the primers used were the upper primer SEQ ID NO. 7 and the lower primer SEQ ID NO. 8 for amplification. Hvar CSP15; In step (7), forward primer SEQ ID NO. 9 and reverse primer SEQ ID NO. 10 are used for amplification. Hvar CSP8; the primers used were the forward primer SEQ ID NO. 11 and the reverse primer SEQ ID NO. 12 for amplification. Hvar CSP15.

[0053] Example 3: Application of an aphid-induced recombinant protein This application also provides the application of the above-mentioned aphid-inducing recombinant protein or the aphid-inducing recombinant protein preparation method in the attraction of aphid natural enemies or the preparation of attractants.

[0054] The application described is that it can be combined with any one or more of cis-3-hexenol, linalool, 2-ethylhexanol, nonanal, α-terpinene and β-caryophyllene, cedrol, α-farnesene and β-pinene, 2-phenylethanol, eucalyptol, geranylacetone, α-caryophyllene, γ-terpinene, DMNT, TMTT and 1,4-diethylbenzene to attract aphid natural enemies.

[0055] This application also provides an attractant for aphid natural enemies, wherein the attractant contains an aphid-inducing recombinant protein or a gene fragment encoding an aphid-inducing recombinant protein.

[0056] The aphid-induced recombinant protein is a chemoreceptor protein from ladybugs. Hvar CSP8 or Hvar CSP8, the amino acid sequence of which is shown in SEQ ID NO. 1 or SEQ ID NO. 2.

[0057] The aphid-induced recombinant protein encoding gene fragment encodes the chemoreceptor protein of the ladybug. Hvar CSP8 or Hvar The CSP8 coding fragment has a nucleotide sequence as shown in SEQ ID NO. 1 or SEQ ID NO. 2.

[0058] The attractant can attract aphid natural enemies by combining it with any one or more of cis-3-hexenol, linalool, 2-ethylhexanol, nonanal, α-terpinene and β-caryophyllene, cedrol, α-farnesene and β-pinene, 2-phenylethanol, eucalyptol, geranylacetone, α-caryophyllene, γ-terpinene, DMNT, TMTT and 1,4-diethylbenzene.

[0059] Example 4: Hvar CSP8 and Hvar Cloning and Sequence Characterization of CSP15 1. Amplification of *Heterocystis jirovecii* Hvar CSP8 and Hvar CSP15 gene Specific primers for the ORF region of this gene were designed using Premier 6.0 software. HvarCSP8 and HvarCSP15 See Table 1. The PCR reaction system is shown in Table 2. The PCR reaction program was: 35 cycles of amplification under the following conditions: 94℃ for 3 min, 94℃ for 30 s, 56℃ for 30 s, and 72℃ for 20 s. The amplification products were detected by 1% agarose gel electrophoresis. All primers were synthesized by Xinjiang Youkang Biotechnology Co., Ltd.

[0060] Table 1: Primer Information 2. Gel extraction and purification of PCR products After electrophoretic separation, a gel piece of the same size as the target band was cut into a centrifuge tube, and the PCR products were recovered using an Omega recovery kit. The specific steps are as follows: (1) Add Binding Buffer XP2, which is approximately three times the volume of the glue block; (2) Melt the glue in a water bath at 55~65℃ until the glue block is completely melted in Binding Buffer XP2, and shake to mix it once every 2~3 minutes during the process; (3) After the above reaction solution has cooled, transfer the melted mixture to a HiBind® DNA centrifuge column and centrifuge at 10,000 rpm for 1 min at room temperature. Discard the filtrate. If the sample volume exceeds 700 μL, it can be loaded onto the column in multiple batches until all the sample has passed through the column. Put the column back into the collection tube, add 300 μL of Binding Buffer XP2, centrifuge at 10,000 rpm for 1 min at room temperature, and discard the filtrate. (4) Add 700 μL of SPW Wash Buffer (which has been diluted with anhydrous ethanol as required) to the column, centrifuge at 10,000 rpm for 1 min at room temperature, discard the filtrate, and repeat the washing once. After discarding the filtrate, put the empty column back into the collection tube and centrifuge at 13,000 rpm for 2 min at room temperature to completely remove residual ethanol; (5) Transfer the centrifuge column to a new 1.5 mL sterile centrifuge tube, add 15~30 μL of Elution Buffer or sterile ultrapure water (adjust according to the expected final concentration) to the center of the column membrane, let stand at room temperature for 1 min, and centrifuge at 13,000 rpm for 1 min to elute the DNA.

[0061] Table 2: PCR reaction system 3. Ligation of PCR products with cloning vectors (1) Perform ligation in a 0.2 mL PCR tube according to the ligation system in Table 3; (2) Centrifuge in a small centrifuge for 45 min at 25°C.

[0062] Table 3: Systems for ligating PCR products into cloning vectors 4. Transformation of the ligation product into E. coli (1) Gently mix the ligation product and competent cells and place on ice for 30-40 min.

[0063] (2) Heat shock at 42℃ for 90 s, place on ice for 3 min, then add LB liquid medium without antibiotics, shake at 37℃ and 200 rpm for 60 min.

[0064] (3) Melt the LB solid medium containing kanamycin (Kana, final concentration 50 µg / mL) and pour it into the culture blood.

[0065] (4) Centrifuge the bacterial culture after shaking for 60 min at 4000 rpm for 2 min, discard 600 µL of supernatant, and suspend the precipitated bacterial cells in the remaining liquid.

[0066] (5) Take 100 µL of the suspension and spread it evenly. Incubate overnight at 37°C with the container upside down.

[0067] 5. PCR identification of positive monoclonal antibodies Single colonies were picked and placed in shake tubes containing 5 mL of LB medium (Kana, 50 µg / mL) and incubated at 37°C and 200 rpm for 12–16 h. The PCR reaction system for the bacterial culture is shown in Table 4–5. Positive bacterial cultures were sent for sequencing.

[0068] Table 4: Colony PCR Reaction System 6. Hvar CSP8 and Hvar Bioinformatics analysis of the CSP15 gene Compare clones in the NCBI database. Hvar CSP8 and Hvar The CSP15 gene sequence was used to construct a molecular phylogenetic tree using MEGA 11.0 software. The amino acid sequence, protein physicochemical properties, and domain characteristics were analyzed using online websites (see Table 5).

[0069] Table 5: Bioinformatics analysis websites and software used in this paper Protein hydrophobicity analysis yielded HvarCSP15 and HvarCSP8 The results of protein hydrophobicity analysis showed that HvarCSP8 It exhibits strong hydrophilicity at positions 8, 9, and 12, and strong hydrophobicity at positions 21, 97, and 109. HvarCSP15 It exhibits strong hydrophilicity at positions 8, 10, and 13, and strong hydrophobicity at positions 23, 99, and 119, with average hydrophilicities of -0.511 and -0.409 respectively, indicating... HvarCSP8 and HvarCSP15 It is a hydrophilic protein; see appendix. Figure 5 As shown. SignalP6.1 predictions revealed the presence of *Heteromorpha multifida*. HvarCSP8 and HvarCSP15 The signal peptide is located at amino acid positions 1-19, see appendix. Figure 1 Appendix Figure 2 As shown. THMM prediction results show HvarCSP8 and HvarCSP15 The protein does not have a transmembrane region and is not a transmembrane protein; see appendix. Figure 3 As shown. Hvar CSP8 (Figure 4A) and Hvar The phosphorylation site prediction results for CSP15 (Figure 4B) are shown. The horizontal axis represents the amino acid sequence position, and the vertical axis represents the phosphorylation potential (0-1). The purple line represents the prediction threshold of 0.5, and the red, green, and blue lines represent serine, threonine, and tyrosine sites, respectively. A comparison shows that... Hvar CSP8 has a limited number of potential phosphorylation sites, with only a few high-confidence sites exceeding the threshold, such as serine at position 22 and threonine at position 70, and no tyrosine phosphorylation sites reaching the threshold; while Hvar CSP15 exhibits a wider and greater distribution of potential phosphorylation sites. Not only do it show multiple high-confidence peaks at threonine and tyrosine residues at positions 20-25 and serine residues at positions 85-90, but it also contains more serine residues exceeding the threshold. Overall, this demonstrates a significant difference in phosphorylation modification potential between the two CSP proteins. Hvar The abundance of potential phosphorylation sites in CSP15 is significantly higher than that in CSP15. Hvar See attached CSP8. Figure 4 As shown. Using the SOPMA online tool... HvarCSP8 and HvarCSP15 Secondary structure prediction of the proteins showed that their structural composition consisted only of α-helices, random coils, and extended chains, with no β-turns detected. Among these, α-helices accounted for the largest proportion. HvarCSP8 and HvarCSP15 The proportions of the two groups were 62.20% and 61.83% respectively, with random coils accounting for 37.01% and 37.40%, and extended chains accounting for 0.79% and 0.76%. (See appendix) Figure 6 As shown.

[0070] Example 5: Prokaryotic Expression and Protein Purification 1. pET28a- Hvar CSP8 and pET28a- Hvar Construction of CSP15 prokaryotic expression vector Using restriction endonucleases BamH I and Xho I enzyme digestion clone plasmid pEASY-T1- HvarThe CSP8 and pET28a empty vectors were used to recover the linear target fragment and vector fragment after enzyme digestion, respectively. The target fragment and vector fragment were ligated using a seamless cloning enzyme and transformed into *E. coli* Trans1-T1 competent cells. Positive recombinant plasmids were screened by colony PCR, using the same reaction system and procedure as in Table 4-3. For positive recombinant plasmids pET28a-... Hvar CSP8 and pET28a- Hvar CSP15 identification was performed, and the recombinant plasmid was entrusted to Xinjiang Youkang Biotechnology Co., Ltd. for sequencing identification. pET28a- Hvar CSP 8 With pET28a- Hvar CSP15 was transformed into *E. coli* BL21(DE3) competent cells, and positive single clones were screened by PCR. The reaction system is shown in Table 6. Positive clones were inoculated into LB liquid medium and cultured at 37°C and 200 rpm. When the absorbance at OD600 was 0.4–0.6, 3–5 mL of the recombinant expression vector pET-28a was linearized using a double enzyme digestion method according to the instructions of the OMEGA Plasmid Mini Kit I. The enzyme digestion system is shown in Table 7, and the reaction was carried out overnight at 37°C. The linearized plasmid and the non-linearized plasmid showed a clear difference in bands on an agarose gel. The target band was excised and recovered according to the method described in Example 4 above. The recovered linearized vector was mixed with the homologous recombination PCR product according to the system in Table 8, and homologous recombination was performed in a metal bath at 37°C for 30 min.

[0071] Table 6: Enzyme digestion system Table 7: Homologous Recombination System 2. His- Hvar CSP8 and His- Hvar CSP15 induced expression and purification pET28a-, which was identified as positive in Example 4 HvarCSP8 and pET28a- HvarCSP15 The recombinant plasmid was transformed into *Escherichia coli* Transetta (DE3) strain. Positive single colonies were cultured overnight at 37°C and 220 rpm in LB medium containing Kana, and then cultured until OD500. 600The concentration of isopropyl β-D-thiogalactoside (IPTG) was increased to 0.4-0.6, and the mixture was induced at 16°C for 16-18 h. After induction, the bacterial culture was centrifuged at 12000 rpm for 10 min, and the collected cells were resuspended in 20 mM Tris-HCl (pH = 7.4), sonicated, and placed on ice for 30 min (120 W, 5 s sonication, 5 s pause). The treated sample was analyzed by 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and stained with Coomassie brilliant blue. After sonication, the mixture was centrifuged at 10000 rpm for 10 min. The total protein in the supernatant was transferred to the column for 3 h (total protein was equilibrated with 20 mmol / L imidazole for 40 min before being added to the column). Then His- Hvar CSP8 was washed with imidazole at concentrations of 20, 50, 100, 150, 200, 250, and 500 mmol / L to remove contaminating proteins. His- Hvar CSP15. Do not use 20, 50, 100, 200, 250, or 500 mmol / L imidazole washes to remove contaminating proteins. For protein ultrafiltration concentration, replace the imidazole with 20 mM Tris-HCl buffer, quantify, and store at -80°C.

[0072] 3. Protein concentration determination Protein concentration was determined using the Biosharp BCA Protein Assay Kit, following the manufacturer's instructions. To maintain background consistency with the sample solvent, BSA standards were diluted with sample lysis buffer (20 mM Tris-HCl, pH = 7.4) to prepare a series of concentration standards. 20 μL of the sample or different concentration standards were added to each well of a 96-well plate, along with 200 μL of BCA working solution (Reagent A: Reagent B = 50:1). After incubation at 37°C for 30 min, the absorbance was measured at 562 nm. The protein concentration was calculated based on the standard curve.

[0073] When the positive bacteria carrying the recombinant vector reached an OD600 value of 0.4-0.6, 1 mL of bacterial culture was used as a pre-induction control. IPTG (isopropyl galactothioglycoside) was added to the remaining bacterial culture to a final concentration of 0.02 mmol / L, and the culture was incubated at 16℃ with shaking for 18-20 h to induce protein expression. After incubation, the bacterial pellet was collected by centrifugation at 10,000 r / min for 10 min, and the expression of the target protein was analyzed by 15% SDS-PAGE (15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis). The results showed that the recombinant protein had a molecular weight of approximately 15 kD and mainly existed in the form of soluble supernatant. (See Appendix) Figure 7 A; see appendix Figure 8 As shown in Figure A. Recombinant proteins were purified using nickel-column affinity chromatography. The total bacterial protein after induction was first loaded onto a nickel column equilibrated with buffer (containing 20 mmol / L imidazole). The His-tagged fusion protein specifically bound to the nickel column via affinity. Fractions of different imidazole concentrations were collected in fractions, and 20 µL of each fraction was analyzed by SDS-PAGE electrophoresis. The results showed that His- Hvar CSP8 requires an effective elution concentration of 100 mmol / L imidazole, His- Hvar CSP15 requires an imidazole concentration of 250 mmol / L to be eluted; and in the second elution with 250 mmol / L imidazole eluent, the fusion protein His- with a high purity of approximately 15 kDa can be specifically eluted. Hvar CSP8 and His- Hvar CSP15 has less interference from other proteins; see appendix. Figure 7 B; see appendix Figure 8 As shown in B. The purified fusion protein was concentrated by ultrafiltration, and the His-tag was removed by intestinal mycokinase and then stored at -80°C for later use.

[0074] The purified protein was quantified using the BCA method, and the results showed... HvarCSP8 and What The concentrations of rCSP15 protein were 1.00 mg / mL and 0.9 mg / mL, respectively. Using Scatchard linear regression, the binding constants Kd values ​​between the two recombinant proteins and the fluorescent probe were calculated to be 6.99 μM and 12.09 μM, respectively. The regression equations were Y = -0.2162X + 0.5283(R² + π / 2)². 2 =0.9385), Y=-0.1347X+0.2406 (R 2 =0.8945), see appendix Figure 9 As shown, the target proteins can bind well to 1-NPN.

[0075] Example 6: Ladybug Hvar CSP8 and Hvar Analysis of the binding characteristics of CSP15 protein to compounds Based on the description in the above embodiments, a fluorescence competitive binding experiment was performed using a multifunctional microplate reader to determine... Hvar CSP8 and Hvar The binding affinity of CSP15 protein to aphid-induced plant volatiles is shown in Table 8. Compound 1-NPN was used as a probe, with an excitation wavelength of 337 nm and an emission wavelength of 420 nm. Both 1-NPN and the plant volatiles were dissolved in HPLC-grade methanol to obtain a 1 mM stock solution. The protein was then diluted to a final concentration of 2 µM using HCl-this buffer (20 μM, pH 7.4).

[0076] The specific operating steps are as follows: (1) First, measure the protein content. Hvar CSP8 Hvar The fluorescence binding ability of CSP15 protein to the fluorescent probe 1-NPN was assessed. 200 µL of protein (final concentration 2 µM) was added to a 96-well microplate, and the plate was scanned and the emission spectrum recorded after the microplate reader stabilized. (2) Add 1-NPN to the protein in an incremental manner, and scan once after each addition, and record the maximum fluorescence value of the scan results; (3) Analyze the recorded maximum fluorescence intensity data using the Scatchard equation. If the Scatchard equation analysis shows a linear relationship between the horizontal axis (concentration of bound 1-NPN) and the vertical axis (bound / free 1-NPN), it indicates that... Hvar CSPs exhibit a saturation effect when binding to fluorescent probes, meeting the requirements of fluorescence competitive binding assays; (4) Re-measurement Hvar The binding affinity between CSPs and plant volatiles. 200 µL of protein (final concentration 2 µM) and 0.4 µL of 1-NPN (final concentration 2 µM) were added to a 96-well microplate. (5) Add odor molecules to the microplate in increasing order of concentration. Scan the plate after each addition and record the maximum fluorescence value of the scan results. (6) According to the formula: calculate Hvar The binding constants of CSPs to odor molecules, where [IC50] is […]. What The concentration of the ligand odorant molecule when the fluorescence intensity of the rCSPs / 1-NPN] complex decreases by half, where [1-NPN] is the concentration of free 1-NPN, and [K1-NPN] is... Hvar Dissociation constant of CSPs / 1-NPN complex.

[0077] Table 8: Ligands used in fluorescence competition binding assay Hvar The binding capacity of CSP8 protein to plant volatiles was measured, and its Ki value was 40.09 ± 1.05 μmol·L⁻¹ for 2-ethylhexanol. - ¹, Linalool 14.40±0.29 μmol·L - ¹, Cedrol 13.22±1.65 μmol·L - ¹, Nononal 10.72±0.33 μmol·L - ¹, α-terpinene 8.74±0.42 μmol·L - ¹, β-caryophyllene 10.72±0.33 μmol·L - ¹, α-farnesene 9.08±0.20 μmol·L - ¹, β-pinene 9.49±0.72 μmol·L - ¹, among which α-terpinene exhibits the strongest binding activity (Ki = 8.74 ± 0.42 μmol·L⁻¹). - ¹). Regarding Hvar The CSP15 protein assay showed that its Ki value was 13.45 ± 1.30 μmol·L⁻¹ for 2-phenylethanol. - ¹, cis-3-hexenol 10.38±1.56 μmol·L - ¹, Linalool 31.36±0.91 μmol·L - ¹, Eucalyptol 29.74±1.56 μmol·L - ¹, Nononal 28.55±1.11 μmol·L - ¹, Geraniol acetone 19.29±2.17 μmol·L - ¹, α-Caryophyllene 23.56±0.49 μmol·L - ¹, γ-terpinene 17.52±1.85 μmol·L - ¹, α-terpinene 10.77±0.91 μmol·L - ¹, β-caryophyllene 23.56±0.49 μmol·L - ¹, DMNT 40.85±0.45 μmol·L - ¹, TMTT 18.59±0.9 μmol·L -¹, 1,4-Diethylbenzene 30.74 ± 3.27 μmol·L - ¹, among which cis-3-hexenol exhibits the strongest binding activity (Ki = 10.38 ± 1.56 μmol·L⁻¹). - ¹), see Table 9 and appendix for details. Figure 11 As shown.

[0078] Table 9: Hvar CSP8 and Hvar The binding ability of CSP15 with 31 aphid-induced plant volatiles Note: IC 50 This refers to the concentration when odor molecules replace half (50%) of 1-NPN; K i Odor molecules and recombination Hvar CSP8 and Hvar The dissociation constant of CSP15; the space represents K. i The value cannot be calculated.

[0079] Example 7: Hvar CSP8 and Hvar CSP15 protein homology modeling and molecular docking 1. Hvar CSP8 and Hvar CSP15 protein homology modeling Based on the descriptions in the above embodiments, and using FPKM value analysis and tissue expression profiling results, we selected the olfactory protein with the highest expression level in *Heteromorpha multiflora* for subsequent protein structure modeling. The amino acid sequence of the *Heteromorpha multiflora* olfactory gene (with signal peptide removed) was BLAST-aligned in the NCBI database (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi). The E-values ​​of the aligned sequences were then compared. <E -5 The results with the lowest E-value (minimum value of 0) were saved to observe the similarity between the ladybug olfactory protein and other insect olfactory proteins. Homology modeling was performed using the Swiss-model online server (https: / / swissmodel.expasy.org): the amino acid sequence of the target protein was submitted to Swiss-model, and the "Template Search" module automatically searched for homology templates in the PDB database. The template with the highest GMQE (Global Model Quality Assessment) and QMEAN scores was selected to construct a three-dimensional structural model, and the model quality was validated using Swiss-model's built-in evaluation tools.

[0080] 2. Ladybug Hvar CSP8 and Hvar Molecular docking of CSP15 protein with aphid-induced plant volatiles Following the method of Qu et al., the Procheck, Verify_3D, and ERRAT programs were used to evaluate the model. Procheck analyzed the dihedral distribution of amino acid residues using Laplace conformation diagrams; if more than 80% of non-glycine and non-proline residues were located in the optimal reasonable region, the model's stereochemical quality was considered good. Verify_3D compared the compatibility of the primary structure and three-dimensional structure of the amino acid sequence; a score greater than 0.2 was considered reasonable. ERRAT analyzed non-bonded interactions between atoms; an ERRAT value greater than 50% indicated high model reasonableness. Homology modeling of the CSP protein was performed using the Swiss-Model online server (https: / / swissmodel.expasy.org / ), using the template with the highest homology to obtain the three-dimensional structural model of the CSP protein. Using this model as the receptor, semi-flexible molecular docking was performed using AutoDock 4.2.6 software to analyze the binding ability of the CSP model to the ligand. First, the protein and ligand PDB formats were converted to PDBQT format. The docking box was set in AutoDock Tools (x=y=z=100, grid spacing 0.0375 Å), and the grid energy was calculated using the AutoGrid program. The docking process employed a Lamarckian genetic algorithm (LGA), combining genetic algorithms with local search to score and rank ligand conformations using a global search and energy optimization approach. In the docking parameters, Maximum_evals was set to middle (2,500,000), with the rest set to default values. After running AutoDock, several optimal conformations were generated, sorted by binding energy, and the conformation with the lowest binding energy was selected for analysis. A lower binding energy indicates a more stable receptor-ligand binding. Image processing and analysis were performed using PyMOL 2.3.0 software.

[0081] 3. Results Analysis The two methods were constructed using Laplace charts. Hvar CSP8 and Hvar The CSP15 protein was compared. Each color region in the image represents an amino acid in each protein. Red indicates the optimal range; bright yellow indicates a more suitable range; light yellow indicates a barely acceptable range; and white indicates an unsuitable range. Hvar CSP8 and Hvar The amino acid proportions in the optimal region of CSP15 were 78.4% and 71.3%, respectively. Both constructed CSP models had amino acid proportions in their optimal regions exceeding 50%, meeting the evaluation criteria. (See appendix.) Figure 12 As shown. In molecular docking, HvarThe binding energies of CSP8 with its ligands are as follows: β-caryophyllene (-7.210 kcal / mol) > cedrol (-6.574 kcal / mol) > α-farnesene (-6.158 kcal / mol) > α-terpinene (-5.698 kcal / mol) > β-pinene (-5.374 kcal / mol) > linalool (-4.952 kcal / mol) > 1-nonanal (-4.502 kcal / mol) > 2-ethyl-1-hexanol (-4.623 kcal / mol), as detailed in Table 10. β-caryophyllene exhibits the highest binding energy among the ligands. Key amino acid sites involved in binding include LEU91, TYR44, LEU34, VAL29, VAL39, and ALA68. These sites, which play a major role in molecular docking, form hydrophobic interactions; see Appendix. Figure 13 As shown. In Hvar In the molecular docking experiments of the CSP15 protein, a total of 12 ligand compounds were tested. According to the binding affinity data, 1-nonanal showed the strongest binding ability (-6.30 kcal / mol), followed by 2-phenylethanol (-5.60 kcal / mol), α-terpinene (-5.20 kcal / mol), and 1,4-diethylbenzene (-5.02 kcal / mol); β-caryophyllene (-4.84 kcal / mol), γ-terpinene (-4.36 kcal / mol), geranylacetone (-4.29 kcal / mol), TMTT (-4.13 kcal / mol), linalool (-4.11 kcal / mol), and eucalyptol (-3.93 kcal / mol) showed moderate binding; DMNT (-3.60 kcal / mol) and cis-3-hexen-1-ol (-3.19 kcal / mol) showed relatively weak binding. See Table 10 for details on molecular docking. Hvar The key amino acids that CSP15 binds to include: TYR28, ILE31, TRY24, and MET85, etc. (See appendix for details.) Figure 14 As shown.

[0082] Table 10: Hvar CSP8 and Hvar Binding energy table of CSP15 ligands Fluorescence competitive binding assays are widely used to detect the binding ability of insect odor-binding proteins and chemoreceptor proteins to external odor molecules due to their advantages of simple operation, low cost, high safety, and stable results. In fluorescence competitive binding assays, N-phenyl-1-naphthylamine (1-NPN) is a commonly used fluorescent probe. When 1-NPN binds to a protein, under specific excitation light (usually 337 nm), the tryptophan residues inside the protein act as intrinsic fluorescence donors, transferring energy to the bound 1-NPN acceptor molecule through the Förster nonradiative energy transfer (FRET) mechanism, resulting in static quenching of the protein's intrinsic fluorescence. Hvar CSP8 and Hvar CSP15 protein binds well to 1-NPN. (In *Dipladenia multifiliis*) Hvar CSP8 and Hvar The fluorescence competitive binding assay of CSP15 protein and its ligand preliminarily screened 16 potential volatiles, which can provide a theoretical basis for the green control of multi-ladybugs in the field.

[0083] The above embodiments are merely examples to clearly illustrate the present invention and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. An aphid-induced recombinant protein, characterized in that, The recombinant protein is a chemosensory protein of *Heterothecia praecox*. Hvar CSP8 or Hvar CSP15, whose amino acid sequence is shown in SEQ ID NO.1 or SEQ ID NO.

2.

2. The aphid-induced recombinant protein as described in claim 1, characterized in that, Encoding the chemoreceptor protein of the ladybug Hvar The nucleotide sequence of CSP8 is shown in SEQ ID NO.

3.

3. The aphid-induced recombinant protein as described in claim 1, characterized in that, Encoding the chemoreceptor protein of the ladybug Hvar The nucleotide sequence of CSP15 is shown in SEQ ID NO.

4.

4. The method for preparing aphid-induced recombinant protein as described in claim 1, characterized in that, It is prepared by the following steps: (1) Select adult female and male ladybugs that are 3-5 days old after the first generation emerges. Place the collected antennae of the female and male adults into 1.5 mL RNase-free centrifuge tubes and use the HiPure Universal RNA Minikit kit to extract total RNA from the antennae of the female and male ladybugs. (2) After the total RNA obtained in step (1) is qualified, cDNA is synthesized by reverse transcription using 1 μg of total RNA as a template and FastKing one-step method. (3) Using the cDNA obtained in step (2) as a template, the reaction system was amplified for 35 cycles under the following conditions: 1.5 µL dNTP, 2 µL ExTaq buffer, 0.5 µL upper primer, 0.5 µL lower primer, 0.2 µL ExTaq polymerase, 1 µL cDNA, and 14.3 µL H2O. (4) After electrophoresis separation of the target fragment obtained in step (3), cut out a piece of gel with the same size as the target band into a centrifuge tube and recover the PCR product using the Omega recovery kit; (5) The recovered product obtained in step (4) was ligated at 25°C for 45 min with 4 µL of PCR product and 1 µL of pEASY-T. (6) Gently mix the ligation product obtained in step (5) with competent cells and place on ice for 30-40 min; heat shock at 42℃ for 90 s, place on ice for 3 min, then add LB liquid medium without antibiotics, shake at 37℃ and 200 rpm for 60 min; melt the LB solid medium with 50 µg / mL kanamycin and pour it into the culture blood; centrifuge the bacterial solution after shaking for 60 min at 4000 rpm for 2 min, discard 600 µL of supernatant, and suspend the precipitated bacterial cells with the remaining liquid; take 100 µL of the suspension and spread it evenly, and incubate at 37℃ overnight inverted. (7) Place the single colony picked from the culture dish obtained in step (6) into a shaker tube containing 5 mL of LB medium supplemented with 50 µg / mL kanamycin, and incubate at 37°C and 200 rpm for 12-16 h; use 10 µL of 2× TransStart® Green qPCR SuperMix, 0.5 µL of forward primer (10 µM), 0.5 µL of reverse primer (10 µM), 1 µL of cDNA template, and 8 µL of ddH2O; send the positive bacterial culture for sequencing identification to isolate and obtain the colony. Hva rCSP8 and Hvar CSP8 target fragment; (8) The target fragment obtained in step (7) is processed using restriction endonuclease. BamH I and Xho I enzyme digestion clone plasmid pEASY-T1- Hvar CSP8, pEASY-T1- Hvar The CSP8 and pET28a empty vectors were used to recover the linear target fragment and vector fragment after enzyme digestion, respectively. The target fragment and vector fragment were ligated using a seamless cloning enzyme and transformed into E. coli Trans1-T1 competent cells. Positive recombinant plasmids were screened by bacterial PCR. For positive recombinant plasmid pET28a- Hvar CSP8 and pET28a- Hvar CSP15 identification; the recombinant plasmid was sequenced and identified, and pET28a- Hvar CSP 8 With pET28a- Hvar CSP15 was transformed into Escherichia coli BL21(DE3) competent cells, and positive single clones were screened by PCR; (9) Inoculate the positive clones obtained in step (8) into LB liquid medium and culture at 37℃ and 200 rpm. When the absorbance at OD600 is 0.4~0.6, take out 3~5 mL and linearize the recombinant expression vector pET-28a using the double enzyme digestion method according to the instructions of the OMEGA Column Plasmid Extraction Kit (Plasmid Mini Kit I). Hvar CSP 8、 pET28a- Hvar The CSP15 digestion system consisted of 5000 ng of the target fragment, 2.5 µL of BamHI, 2.5 µL of XhoI, 5 µL of 10X QuickCut Green Buffer, and 24.62 µL of ddH2O, and was incubated at 37 °C for 8–12 h. (10) The linearized plasmid and the non-linearized plasmid in step (9) showed obvious band differences on the agarose gel. The target band was cut and recovered. The recovered linearized vector and homologous recombination PCR product were mixed in a 10µL system with 2× UElandySeamless Cloning Mix 5.0µL, linearized vector (pET-28a) 100 ng, insert fragment (PCR product) 50 ng, and ddH2O. The mixture was reacted in a metal bath at 37℃ for 30 min to carry out homologous recombination. (11) The pET28a- sample identified as positive in step (10) HvarCSP8 and pET28a- HvarCSP15 The recombinant plasmid was transformed into *Escherichia coli* Transetta (DE3) strain; positive single colonies were cultured overnight at 37°C and 220 rpm in LB medium containing Kana, and cultured until OD500. 600 The concentration of the culture medium was 0.4-0.6, and IPTG was added to a final concentration of 0.02 mmol / L. The cells were induced at 16℃ for 16-18 h. After induction, the bacterial culture was centrifuged at 10000 rpm for 10 min. The collected bacterial cells were resuspended in 20 mM Tris-HCl (pH = 7.4), sonicated, and placed on ice for 30 min. The sonication was performed at 120 W for 5 s, followed by a 5 s pause. The treated sample was analyzed by 15% SDS-PAGE and stained with Coomassie brilliant blue. After sonication, the cells were centrifuged at 10000 rpm for 10 min. The total protein in the supernatant was transferred to the column by rotation for 3 h. Before adding the total protein to the column, the column was washed with 20, 50, 100, 150, 200, 250, and 500 mmol / L imidazole. His- Hva rCSP8 was washed with imidazole at concentrations of 20, 50, 100, 150, 200, 250, and 500 mmol / L to remove contaminating proteins. His- Hvar CSP15 was washed with imidazole at concentrations of 20, 50, 100, 200, 250, and 500 mmol / L to remove impurities and obtain purified His- Hvar CSP8, His- Hvar CSP15 protein.

5. The method for preparing aphid-induced recombinant protein as described in claim 4, characterized in that, In step (5), the primers used for amplification are the upper primer SEQ ID NO.5 and the lower primer SEQ ID NO.

6. Hvar CSP8; the primers used were the upper primer SEQ ID NO.7 and the lower primer SEQ ID NO.8 for amplification. Hvar CSP15; In step (7), forward primer SEQ ID NO.9 and reverse primer SEQ ID NO.10 are used for amplification. Hva rCSP8; the primers used were forward primer SEQ ID NO.11 and reverse primer SEQ ID NO.12 for amplification. Hva rCSP15.

6. The application of the aphid-inducing recombinant protein as described in any one of claims 1 to 3 in the attraction of aphid natural enemies.

7. The application as described in claim 6, characterized in that, The aphid attractant can be combined with any one or more of β-caryophyllene, α-farnesene, β-pinene, linalool, 2-ethyl-1-hexanol, terpinene, nonanal, cis-3-hexenol, DMNT, 1,4-diethylbenzene, TMTT, 2-phenylethanol, geranylacetone, eucalyptol, γ-terpinene, terpinene, and cis-3-hexenol to attract aphid natural enemies.

8. An attractant for aphid natural enemies, characterized in that, The attractant contains the aphid-inducing recombinant protein as described in any one of claims 1 to 3, or a gene fragment encoding the aphid-inducing recombinant protein.

9. An attractant for aphid natural enemies as described in claim 8, characterized in that, The aphid-induced recombinant protein is a chemoreceptor protein from ladybugs. Hvar CSP8 or Hvar CSP8, with an amino acid sequence as shown in SEQ ID NO. 1 or SEQ ID NO. 2; the aphid-induced recombinant protein encoding gene fragment encodes the chemoresense protein of *Heteromorpha heteropsis*. Hvar CSP8 or Hvar The CSP8 coding fragment has a nucleotide sequence as shown in SEQ ID NO. 1 or SEQ ID NO.

2.

10. An attractant for aphid natural enemies as described in claim 8, characterized in that, The attractant can attract aphid natural enemies by combining it with any one or more of the following: cis-3-hexenol, linalool, 2-ethyl-1-hexanol, nonanal, α-terpinene and β-caryophyllene, cedrol, α-farnesene and β-pinene, 2-phenylethanol, eucalyptol, geranylacetone, α-caryophyllene, γ-terpinene, DMNT, TMTT and 1,4-diethylbenzene.

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