A Dark-browed Golden Beetle HparGR16 Gene and Its Application

Abstract

This invention relates to the field of insect genetic engineering technology and provides a dark-browed scarab beetle. HparGR16 Genes and their applications. Genes from the dark-breasted scarab beetle involved in CO2 recognition. HparGR16 The gene, whose DNA sequence is shown in SEQ ID No:1, and the amino acid sequence of the protein encoded by the gene is shown in SEQ ID No:2. This invention is the first to demonstrate... HparGR16 The gene is the CO2 receptor gene of the dark-browed scarab beetle. By interfering with the expression of this gene, or by designing, synthesizing, and screening compounds that can inhibit its protein activity, the CO2 recognition process of the dark-browed scarab beetle can be effectively blocked. This characteristic makes this gene a target for designing and screening control agents, thereby developing novel pest behavior regulators and insecticides, achieving efficient and green control of the dark-browed scarab beetle. This has important application value in the field of genetic engineering for the control of the underground pest, the dark-browed scarab beetle.

Description

Technical Field

[0001] This invention belongs to the field of insect genetic engineering technology, and specifically relates to a dark-gilled scarab beetle. HparGR16 Genes and their applications. Background Technology

[0002] Dark-gilled golden beetle ( Holotrichia parallela The dark-skinned scarab beetle (Motschulsky) belongs to the order Coleoptera, family Scarabaeidae. As an agricultural underground pest, it poses a serious threat to agricultural production and economic development. Adults feed on the leaves of various economic crops and trees, while its larvae (also known as grubs) feed on the roots and stems of crops such as peanuts, corn, and potatoes, causing 10-20% yield losses annually. Currently, prevention and control of the dark-skinned scarab beetle still primarily rely on pesticide application, such as seed treatment with 50% phoxim granules or 48% chlorpyrifos emulsifiable concentrate. However, with increasing pesticide resistance and environmental pollution risks, finding efficient and environmentally friendly biological control methods is urgently needed.

[0003] CO2, a significant component of the air, influences insect life activities, directly affecting feeding and oviposition, and indirectly influencing wing flapping and pheromone sensitivity. Through long-term co-evolution with plants, insects have developed a complete chemoreceptor system, with an extremely sensitive taste system capable of detecting CO2. Taste receptors, typically hair-like, spiny, conical, or plug-like, are distributed on the insect's mouthparts, legs, antennae, and head. Upon receiving external stimuli, gustatory receptors (GRs) on these receptors specifically recognize the stimulating compound, converting the chemical signal into an electrical signal. This signal is then transmitted as impulses through nerve axons to the central nervous system, which in turn sends signals to regulate behavior. If CO2 receptors malfunction, insects lose some of their ability to locate hosts, affecting their feeding and oviposition behaviors. Therefore, CO2 receptors are crucial for insect survival. Although research on GR is increasing, the main focus is on Lepidoptera insects, with a lack of research on taste receptors in Coleoptera insects. In particular, the molecular mechanism of CO2 detection in the larvae of the dark-browed scarab beetle is not yet fully understood, which restricts in-depth exploration of its behavioral regulation.

[0004] In view of this, HparGR16 This gene, highly expressed in the labial palps of the larvae of the dark-browed scarab beetle, is analyzed to reveal its role in CO2 recognition. This analysis will help identify new pest control targets, guide the development of highly effective behavioral regulators for underground pests, and open up new avenues for the green control of the dark-browed scarab beetle. Therefore, this invention proposes a method for the green control of the dark-browed scarab beetle. HparGR16 Genes and their applications. Summary of the Invention

[0005] The purpose of this invention is to provide a dark-gilled golden beetle. HparGR16 Genes and their applications aim to address the problems raised in the background section above.

[0006] The objective of this invention is achieved through the following technical solution:

[0007] A type of dark gill beetle ( Holotrichia parallela Motschulsky) HparGR16 The gene or the protein it encodes, said HparGR16 The DNA sequence of the gene is shown in SEQ ID No:1; HparGR16 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID No:2. HparGR16 The gene-encoded protein can specifically respond to CO2 stimulation and mediate the CO2 tropism behavior of the dark-breasted beetle.

[0008] A method according to the above description HparGR16 Application of the gene or its encoded protein in the preparation of a formulation of the dark-gilled beetle that recognizes and responds to CO2 processes.

[0009] A sort of HparGR16 The dsRNA of the gene, the nucleotide sequence of which is shown in SEQ ID No:9, can specifically interfere with... HparGR16 Gene expression; the aforementioned HparGR16 The DNA sequence of the gene is shown in SEQ ID No:1.

[0010] An application of the dsRNA described above in the preparation of a formulation for the dark-browed scarab beetle that recognizes and responds to CO2 processes.

[0011] Compared with the prior art, the beneficial effects of the present invention are:

[0012] This invention is the first to demonstrate HparGR16 The gene is the CO2 receptor gene of the dark-browed golden terrapin, and its CO2 recognition function has not been previously reported, making this the first public disclosure. Based on this, by interfering with the expression of this gene, or by designing, synthesizing, and screening compounds that can inhibit its protein activity, the CO2 recognition process of the dark-browed golden terrapin can be effectively blocked. This characteristic makes... HparGR16 Genes can be used as targets for the design and screening of control agents, leading to the development of novel pest behavior regulators and insecticides, achieving efficient and environmentally friendly control of the dark-browed scarab beetle. Therefore, HparGR16 Genes and their encoded protein products can serve as important candidates for the above-mentioned applications and have significant application value in the field of genetic engineering for the control of the underground pest, the dark-browed scarab beetle. Attached Figure Description

[0013] Figure 1 for HparGR16 Relative expression levels of genes in various tissues of the dark-browed scarab beetle larvae (data are presented as mean ± standard error, and statistical analysis was performed by one-way ANOVA and Tukey multiple comparison test; different letters indicate significant differences). p < 0.05).

[0014] Figure 2 To express HparGR16 The gene in the oocyte at 10 -2 Reactions of different compounds under M.

[0015] Figure 3 To express HparGR16 Dose-response curves of oocytes of the gene to different concentrations of sodium bicarbonate (data are presented as mean ± standard error, and statistical analysis was performed by one-way ANOVA and Tukey multiple comparison test; different letters indicate significant differences). p < 0.05).

[0016] Figure 4 The tropism of dark-browed scarab beetle larvae to different concentrations of CO2 (data obtained through multiple methods). t Statistical analysis, *: p < 0.05, **: p < 0.01); where a is the tropism experiment of wild-type larvae to CO2; b is the feeding ds HparGR16 Experiment on the larval attraction to CO2; c represents feeding ds GFP Experiments on the larval attraction to CO2.

[0017] Figure 5 Electrophysiological experiments of the labial palps of the larvae of the dark-browed scarab beetle in response to CO2; where 'a' represents the labial palpebrae potential of wild-type larvae (data are presented as mean ± standard error, and statistical analysis was performed using one-way ANOVA and Tukey's multiple comparison test; different letters indicate significant differences). p < 0.05); b is feeding ds GFP With feeding ds HparGR16 Comparison of larval responses (data obtained through multiple methods) t Statistical analysis, *: p < 0.05, **: p < 0.01). Detailed Implementation

[0018] In order to provide a clearer understanding of the technical features, objectives and beneficial effects of the present invention, the technical solution of the present invention will now be described in detail below, but it should not be construed as limiting the scope of implementation of the present invention.

[0019] The specific implementation of the present invention will be described in detail below with reference to specific embodiments. Unless otherwise specified, the methods in the embodiments are conventional methods.

[0020] The dark-gilled beetle used in this invention was collected by Wang Shang from the fields of Rizhao City, Shandong Province, China in June 2024. Wang Shang's contact information is: No. 5333, Xi'an Road, Lvyuan District, Changchun City, Jilin Province, 130062, China.

[0021] Example 1: HparGR16 Gene correlation analysis;

[0022] HparGR16 The genes were obtained through transcriptome analysis of the lower labial barbels of the Dark-browed Golden Beetle. HparGR16 The DNA sequence of the gene is shown in SEQ ID No:1. This DNA sequence is... HparGR16 An open reading frame of a gene consists of 1062 nucleotides:

[0023]

[0024] The amino acid sequence of the protein encoded by this gene is shown in SEQ ID No:2:

[0025] MIVFFKHNNTPVVKSSTRELSYIILIGMMFAYISVFPILAKPSVMTCALCRFIPGLGFAMMYAALLTKTNRIARILAGSKKKIPTRKRIFMSTASQLLITIFLIGVEVVISVLMLYNQAPNFKYTYEKGRTILECDITARGLVVPLTYNFFLILLCTLYALKTRNVPENFNEAKFIG FAMYTTCVIWIAFMSIYFGSNTKVVTFSACILSAIVAWVFLFLPKLYIILLRPERNNRAFFTTDKTIRCHIGSRVSSALTEKSSTLTGKSIANSVYGENISNYKEAGKTQNIPEKRTLSCQTGVELLSVLLNPKSLIELHAETRTYVPRITEQNCCSGNGCELRNITIRLPDARF.

[0026] Example 2: HparGR16 Analysis of the relative expression levels of genes in different tissues of the larvae of the dark-browed scarab beetle;

[0027] 1) RNA extraction and cDNA synthesis;

[0028] Several active larvae of the dark-browed scarab beetle were selected, and their antennae, labial palps, mandibles, head, thorax, and abdomen were dissected and preserved. RNA was extracted using the Trizol method, and cDNA (SEQ ID No:1 is the cDNA sequence) was synthesized according to the instructions of the StarScript II Genomic DNA Reverse Transcription Premix Kit (GenStar, Beijing, China).

[0029] 2) Quantitative real-time PCR;

[0030] Using the synthesized cDNA as a template, the upstream primer sequence was 5'-GCAGCAACACCAAAGTA-3' (as shown in SEQ ID No:3), and the downstream primer sequence was 5'-GAAGACACCCTAGAACCTAT-3' (as shown in SEQ ID No:4). The cDNA was analyzed in different tissues using 2×RealStar Fast dye qPCR premix (Low ROX) (GenStar, Beijing, China) and a StepOne Plus Real-time PCR instrument (Applied Biosystems, Waltham, USA). HparGR16The relative expression levels of genes. Analysis revealed that... HparGR16 The gene was most highly expressed in the mustache (see Figure 1 ).

[0031] Example 3: Expression HparGR16 The response of oocytes to CO2;

[0032] 1) HparGR16 Gene cloning;

[0033] Using cDNA derived from reverse transcription of the lower labial barbel RNA of the dark-browed golden beetle as a template, amplification was performed using upstream primer 5'-AATTCCCCGGGGATCCATGATCGTGTTCTTTAAACACAACA-3' (as shown in SEQ ID No:5) and downstream primer 5'-TTGCTCTAGAGAATTCTTAGAAACGAGCATCAGGTAATCTT-3' (as shown in SEQ ID No:6). HparGR16 Gene. The reaction system was as follows: 1 μL cDNA template; 1 μL each of upstream and downstream primers; 12.5 μL 2×PCR buffer; 0.5 μL dNTPs; 0.5 μL high-fidelity enzyme; 8.5 μL ddH2O. The amplification program was as follows: (1) 95℃ pre-denaturation for 3 min; (2) 95℃ denaturation for 15 s, 56℃ annealing for 15 s, 72℃ extension for 2 min, 40 cycles; (3) 72℃ extension for 5 min. The PCR product of the target gene and the enzyme-digested pGEMHE vector were subjected to agarose gel electrophoresis, and the correct nucleic acid bands were cut from the gel for gel recovery. The gel recovery was performed using a common agarose gel DNA recovery kit (DP209, Tiangen, Beijing), and the operation procedure was in accordance with the kit instructions.

[0034] 2) HparGR16 Construction of gene expression vectors;

[0035] a. HparGR16 Homologous recombination reaction between gene fragments and linearized vectors;

[0036] Homologous recombination ligation of the purified target gene fragment and the linearized vector was performed using the pEASY-Uni Seamless Cloning and Assembly Kit (CU101-03, TransGen, Beijing). The reaction system and procedure followed the manufacturer's instructions. The reaction mixture consisted of: 5 μL of 2×Assembly Mix; 0.01–0.25 pmol each of the linearized vector and the gene fragment (the gene fragment molars were twice that of the linearized vector); and nucleic acid-free water to a final volume of 10 μL. The reaction mixture was gently mixed and incubated in a PCR instrument at 50 °C for 15 min. After the reaction, the centrifuge tubes were immediately placed on ice for a few seconds to cool before subsequent transformation.

[0037] b. Transformation and extraction of recombinant plasmids;

[0038] use Trans 1-T1 (CD501-02, TransGen, Beijing) competent cells were transformed into recombinant plasmids. The specific transformation procedure was as follows: Competent cells were placed on ice until completely thawed. 2 μL of homologous recombination product was added to every 50 μL of thawed competent cells, and the cells were incubated on ice for 30 min; then incubated in a 42℃ water bath for 30 s, followed immediately by an ice bath for 2 min; 450 μL of LB medium was added, and the cells were cultured at 37℃ and 200 rpm for 1 h using a shaker; 100 μL of medium was evenly spread on a plate, and then incubated overnight upside down at 37℃. The next day, single clones were selected for PCR detection. Colonies with correctly amplified bands were amplified, cultured, and sent to Sangon Biotech (Shanghai) for sequencing. Sequence alignment confirmed the correctness of the sequenced genes. Recombinant plasmids were extracted from the correctly sequenced strains using a plasmid miniprep kit (DP103, Tiangen, Beijing) according to the manufacturer's instructions.

[0039] 3) Linearization of recombinant plasmids and product recovery;

[0040] The recombinant plasmid was digested with TaKaRa restriction enzyme. The reaction system was as follows: 1 μg recombinant plasmid; 1 μL EcoR I restriction enzyme; 2 μL 10× Buffer; and nucleic acid-free water to a final volume of 20 μL. The reaction system was incubated in a metal bath at 37°C for 4 h. After complete digestion, 1% agarose gel electrophoresis was used to detect the digestion. The DNA product was then recovered by ethanol-sodium acetate precipitation. The procedure was as follows: 2 μL sodium acetate (3 mol / L, pH=5.2) and 40 μL ice-cold ethanol were added, vortexed, and stored at -20°C overnight. The next day, the reaction solution was centrifuged at 4°C and 15,000 rpm for 15 min, and the supernatant was discarded. 750 μL of anhydrous ethanol was added, and the solution was centrifuged at 4°C and 15,000 rpm for 5 min, and the supernatant was discarded. The ethanol was dried, and 10 μL of DEPC-treated water was added to dissolve the precipitate.

[0041] 4) In vitro transcription of RNA and injection;

[0042] DNA products were transcribed in vitro using the mMESSAGE mMACHINE T7 kit (AM1344, Thermo Fisher Scientific, Waltham). The reaction system consisted of: 2 μL Enzyme Mix; 2 μL 10×Buffer; 10 μL 2×NTP / CAP; 1 μg linearized plasmid; and DEPC-treated water to a final volume of 20 μL. The reaction mixture was incubated at 37°C for 2 h. Then, 1 μL TURBO and 30 μL LiCl were added, and the mixture was incubated at -20°C for 30 min. The mixture was then centrifuged at 15,000 rpm for 15 min at 4°C, and the waste liquid was discarded. 1 mL of anhydrous ethanol was added, and the mixture was centrifuged at 15,000 rpm for 15 min at 4°C, and the waste liquid was discarded. 8 μL of DEPC-treated water was added to dissolve the precipitate. 20 ng of cRNA was injected into Xenopus laevis oocytes, and the cells were cultured for 3 days.

[0043] The nucleotide sequence of the cRNA is shown in SEQ ID No:9:

[0044]

[0045] 5) Two-electrode voltage clamp experiment;

[0046] The injected oocytes were tested using a dual-electrode voltage clamp system. First, 10... -2 Stimulating oocytes with glucose, fructose, sucrose, sodium chloride, and sodium bicarbonate (simulating CO2 stimulation) at concentrations M, it was found that when glucose, fructose, sucrose, and sodium chloride were applied, the curve showed only weak current fluctuations; however, when sodium bicarbonate was applied, the curve showed a significant and unique current peak, indicating that oocytes only specifically respond to sodium bicarbonate stimulation. HparGR16 The gene-encoded protein can recognize CO2 stimulation (see...) Figure 2 The sodium bicarbonate concentration was then diluted to 10... -7 M, 10 -6 M, 10 -5 M, 10 -4 M, 10 -3 M and 10 -2 After M, the oocytes were stimulated again, and the results were as follows. Figure 3 As shown in the figure, as the sodium bicarbonate concentration increases, the response value of the oocyte increases, 10 -7 The value at M is 4.167 ± 2.831 nA, 10 -6 The value at M is 12.700 ± 4.153 nA, 10 -5 The value at M is 26.533 ± 4.943 nA, 10 -4 The value at M is 39.667 ± 4.318 nA, 10 -3 The value at M is 54.100±5.962 nA, and at 10 -2 The reaction value is the largest at M, which is 77.233 ± 4.087 nA.

[0047] Example 4: HparGR16 Functional analysis of genes in CO2 tropism and electrophysiological experiments of dark-browed scarab beetle larvae;

[0048] 1) Interference from dsRNA;

[0049] a. Synthesis of dsRNA;

[0050] Using cDNA reverse transcribed from the barbel RNA of the dark-browed beetle as a template, amplification was performed using the upstream primer 5'-TAATACGACTCACTATAGGGGCAGCAACACCAAAGTA-3' (as shown in SEQ ID No:7) and the downstream primer 5'-TAATACGACTCACTATAGGGGAAGACACCCTAGAACCTAT-3' (as shown in SEQ ID No:8). The reaction system consisted of: 1 μL cDNA template; 1 μL each of upstream and downstream primers; 12.5 μL 2×PCR buffer; 0.5 μL dNTPs; 0.5 μL high-fidelity enzyme; and 8.5 μL ddH2O. The amplification program was as follows: (1) 95℃ pre-denaturation for 3 min; (2) 95℃ denaturation for 15 s, 56℃ annealing for 15 s, and 72℃ extension for 2 min, for 40 cycles; (3) 72℃ extension for 5 min. After agarose gel electrophoresis, the PCR products were recovered using the Promega Wizard® SV Gel and PCRClean-Up System (A9281, Promega, Madison) kit. dsRNA was synthesized using the Promega T7RiboMAX™ Express RNAi System (P1700, Promega, Madison) kit (forward strand: 5'-GCAGCAACACCAAAGTAGTGACGTTCTCCGCGTGCATTATTTTGAGCGCCATAGTCGCCTGGGTCTTCCTTTTCTTGCCGAAATTGTACATAATTTTATTGAGACCCGAACGCAACAATCGCGCGTTCTTCACAACCGATAAGACGATTCGCTGCCATATAGGTTCTAGGGTGTCTTC-3' (as shown in SEQ ID). (as shown in No:10); reverse chain: 3'-CGUCGUUGUGGUUUCACUACUGCAGAAGGCGCACGUAAUAAAACUCGCGGUAUCAAGCGGACCCAGAAGGAAAAGAACGGGCUUUAACAUGUAUAAAAUCUCGGGCUUGCGUUGUAGCGCAAGAAGUGUUGGCUAUUCUGCUAAGCGACGGUAUAUCCAAGAUCACAGAAG-5' (as shown in SEQ ID No:11)).

[0051] Feeding and optimal timing screening of a.dsRNA;

[0052] Select several active dark-gill beetle larvae and feed them 3µg of ds every 24 hours. ​Ten larvae were dissected every 24 hours for labial palps. RNA was extracted using the Trizol method, and cDNA was synthesized using the StarScript II Genomic DNA De-Genomicized Reverse Transcription Premix Kit (GenStar, Beijing, China). The control group was fed ds ​ (GACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACG The synthesized cDNA was subjected to quantitative real-time PCR using the upstream primer 5'-GCAGCAACACCAAAGTA-3' (as shown in SEQ ID No:12) and the downstream primer 5'-GAAGACACCCTAGAACCTAT-3' (as shown in SEQ ID No:4) to detect... ​ Changes in relative expression levels. Interference was detected 72 hours after the initial detection. ​ The relative expression level was the lowest, so larvae with 72h of interference were selected for subsequent experiments.

[0053] 2) Analysis of the tropism of dark-browed scarab beetle larvae to different concentrations of CO2;

[0054] A two-armed behavioral apparatus was used for testing: one side was filled with pure air, and the other side was filled with CO2 gas at concentrations of 1%, 5%, and 10%, respectively. A control group was set up: both sides were filled with air. After each test, the apparatus was placed in a ventilated environment to remove CO2, and the gas positions were exchanged. This behavioral experiment was conducted on both wild-type and disturbed larvae. For each treatment and concentration, 30 larvae of similar size were selected for testing. The results are as follows: ​ As shown in the figure (yellow represents the number of larvae that prefer air, and red represents the number of larvae that prefer CO2), CO2 has a significant attraction for wild-type larvae and a greater attraction for larvae fed with ds. ​ The larvae retain part of the attraction, for feeding ds ​ The larvae lose their attraction.

[0055] 3) Electrophysiological experiment of CO2 on the labial palps of the dark-browed scarab beetle larvae;

[0056] Electrophysiological recordings were performed using a CS-55 (Syntech, Kirchzarten, Germany) and amplifier (Syntech, Kirchzarten, Germany) to study the electrophysiological responses of the labial palps of the larvae of the dark-browed scarab beetle to different concentrations (1%, 5%, and 10%) of CO2. Pure air served as a negative control. Both wild-type and disturbed larvae underwent this electrophysiological experiment, and the results are as follows: ​ As shown in the figure, the labial palps of wild-type larvae respond to CO2 stimulation, and the response value increases with increasing CO2 concentration. ​ (a) Feeding ds ​ The larvae of the ds larvae responded significantly weaker to CO2 than those of the ds larva ​ Feeding group ( ​ (b)

[0057] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make several modifications and improvements without departing from the concept of the present invention, and these should also be considered within the scope of protection of the present invention. These modifications and improvements will not affect the effectiveness of the implementation of the present invention or the practicality of the patent.

Claims

1. A gene HparGR16 of the dark-spotted scarab beetle (Holotrichia parallela Motschulsky) or the protein encoded therein, characterized in that, The DNA sequence of the HparGR16 gene is shown in SEQ ID No:1; the amino acid sequence of the protein encoded by the HparGR16 gene is shown in SEQ ID No:

2. The protein encoded by the HparGR16 gene can specifically respond to CO2 stimulation and mediate the CO2 tropism behavior of the dark-breasted beetle.

2. The application of the HparGR16 gene or the protein encoded by it according to claim 1 in the design and screening of drugs for the prevention and control of the dark-browed scarab beetle.

3. A dsRNA of the HparGR16 gene, characterized in that, The forward and reverse strand sequences of the dsRNA are shown in SEQ ID No:10 and SEQ ID No:11, respectively; the dsRNA can specifically interfere with the expression of the HparGR16 gene; the DNA sequence of the HparGR16 gene is shown in SEQ ID No:1.

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