HparGR22 gene and its application in establishing an in vitro model of phlorizin recognition

Abstract

This invention is applicable to the field of insect genetic engineering technology and provides... HparGR22 Genes and their application in establishing an in vitro model for phlorizin recognition. HparGR22 The DNA sequence of the gene is shown in SEQ ID No:1, and the encoded amino acid sequence is shown in SEQ ID No:2. This gene is expressed at the highest level in the labial palps of the larvae of the dark-browed scarab beetle. Oocytes injected with the cRNA of this gene showed a significant response to stimulation with different concentrations of phlorizin, clearly demonstrating... HparGR22 The product of gene expression can recognize phlorizin, thus establishing its function as a phlorizin receptor gene for the black-breasted beetle. Given that the phlorizin recognition function of this gene is clearly defined for the first time, this invention provides a basis for its application in establishing an in vitro model of phlorizin recognition, lays the foundation for exploring its potential value in the field of genetic engineering for the control of the black-breasted beetle, and opens up new directions for further research and application of this gene.

Description

Technical Field

[0001] This invention belongs to the field of insect genetic engineering technology, and particularly relates to... HparGR22 Genes and their application in establishing an in vitro model for phlorizin recognition. Background Technology

[0002] Dark-gilled golden beetle ( Holotrichia parallela The dark-skinned grub (Motschulsky) belongs to the order Coleoptera, superfamily Scarabaeoidea, and family Melolonthidae. Its larvae, commonly known as white grubs, are a serious global agricultural pest, causing 10-20% yield losses annually. Adults feed on the leaves of various crops, including elm and willow, while larvae feed on the roots and stems of crops such as peanuts, corn, and potatoes. Currently, pesticide application remains the primary means of preventing and controlling the dark-skinned grub, with commonly used pesticides including chlorpyrifos microcapsule suspensions and phoxim microcapsule suspensions. However, pesticide use also brings a series of problems, such as environmental pollution, pesticide residues, and the development of pesticide resistance in pests. Therefore, finding efficient and environmentally friendly biological control methods is urgently needed.

[0003] Phlorizin is a glucoside of phlorizin, with the molecular formula C0. 21 H 24 O 10 With a relative molecular mass of 436.41, phlorizin belongs to the dihydrochalcone analogs of flavonoids and is found in various plant tissues. In the long-term co-evolution of insects and plants, insects have evolved a complete chemosensory system, among which the taste system is extremely sensitive and capable of detecting a variety of non-volatile compounds. Taste receptors are generally distributed on the insect's mouthparts, legs, antennae, and head, and are hair-like, spiny, conical, or plug-like. When an insect's taste receptors receive external stimuli, the taste receptors on them specifically recognize the stimulating compound, converting the chemical signal of the stimulus into an electrical signal, which is transmitted to the central nervous system in the form of pulses via nerve axons. Subsequently, the central nervous system sends out information to regulate behavior. If phlorizin receptors cannot function properly, insects will lose some of their ability to distinguish hosts, thus affecting their feeding, oviposition, and other behaviors. Therefore, phlorizin receptors play a vital role in the survival of insects.

[0004] Although research on insect taste receptors is increasing, it mainly focuses on lepidopteran insects, with less research on taste receptors in coleopteran insects. The phlorizin receptor gene and its function in the dark-browed scarab beetle remain unclear, and there is a lack of in vitro models for studying phlorizin recognition mechanisms. Therefore, this invention proposes… HparGR22Genes and their application in establishing an in vitro model for phlorizin recognition. Summary of the Invention

[0005] The purpose of this invention is to provide HparGR22 The purpose of this study is to address the problems raised in the background section regarding the application of genes and their role in establishing an in vitro model for phlorizin recognition.

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

[0007] HparGR22 Genes, the ones mentioned HparGR22 The DNA sequence of the gene is shown in SEQ ID No:1. HparGR22 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID No:2.

[0008] Furthermore, the aforementioned HparGR22 The gene was expressed at the highest level in the labial palps of the larvae of the dark-browed scarab beetle.

[0009] Expression carrier, including the above-described HparGR22 Gene.

[0010] The in vitro transcribed cRNA was obtained by transcription from the expression vector described above; the nucleotide sequence of the cRNA is shown in SEQ ID No:7.

[0011] The method for constructing an in vitro model for phlorizin recognition includes the following steps:

[0012] The aforementioned cRNA was injected into Xenopus laevis oocytes, and the oocytes were cultured to express it. HparGR22 Proteins encoded by genes.

[0013] The in vitro model for phlorizin recognition, constructed using the methods described above, is capable of specifically recognizing phlorizin.

[0014] Compared with the prior art, the beneficial effects of the present invention are:

[0015] In this invention HparGR22 The DNA sequence of the gene is shown in SEQ ID No:1, and the amino acid sequence of the encoded protein is shown in SEQ ID No:2. Quantitative real-time PCR analysis revealed that the gene was most highly expressed in the labial palps of the larvae of the black-faced scarab beetle. Furthermore, experiments involving gene cloning, construction of expression vectors, in vitro transcription of cRNA, and injection into Xenopus laevis oocytes, combined with detection using a two-electrode voltage-clamp system, confirmed that oocytes injected with the cRNA of this gene showed a significant response to stimulation with different concentrations of phlorizin, thus clarifying the gene's expression level. HparGR22The product of gene expression can recognize phlorizin, thus establishing its function as a phlorizin receptor gene for the black-breasted beetle. Given that the phlorizin recognition function of this gene is clearly defined for the first time, this invention provides a basis for its application in establishing an in vitro model of phlorizin recognition, lays the foundation for exploring its potential value in the field of genetic engineering for the control of the black-breasted beetle, and opens up new directions for further research and application of this gene. Attached Figure Description

[0016] Figure 1 for HparGR22 Gel electrophoresis images of the relative expression levels of the gene in various tissues of the larvae of the dark-browed scarab beetle (channel 1 is the full-size gold 2K marker; channels 2-7 are the electrophoretic bands in the antennae, labial palps, mandibles, head, thorax, and abdomen, respectively; channel 8 is the negative control).

[0017] Figure 2 for HparGR22 The relative expression levels of the gene in various tissues of the dark-browed scarab beetle larvae (multiple comparison tests were performed after one-way ANOVA). p <0.05, different letters indicate significant differences.

[0018] Figure 3 for HparGR22 Electrophoresis images of genes in bacterial PCR (well 1 is BioMed 2K Marker; wells 2-5 are...) HparGR22 Genes, of which channels 3-5 are positive clones).

[0019] Figure 4 To express HparGR22 Dose-response curves of phlorizin in oocytes of the gene at different concentrations (multiple comparison tests were performed after one-way ANOVA). p <0.05, different letters indicate significant differences. Detailed Implementation

[0020] 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.

[0021] 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.

[0022] 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.

[0023] Example 1: HparGR22Gene correlation analysis;

[0024] HparGR22 The genes were obtained through transcriptome analysis of the lower labial barbels of the Dark-browed Golden Beetle. HparGR22 The DNA sequence of the gene is shown in SEQ ID No:1. This DNA sequence is... HparGR22 An open reading frame of a gene consists of 1245 nucleotides:

[0025]

[0026] The amino acid sequence of the protein encoded by this gene is shown in SEQ ID No:2:

[0027] MDNGTTIKEITCLFNETAPAPKPEWPDFVLLFVKTIIMAGIIIASIFGNLLVIISVMRHRKLRVITNYFVVSLAFADMLVAMFAMTFNASVQIFGKWLFGYFM CDVWNSLDVYFSTASILHLCCISVDRYYAIVRPLKYPIIMTKVTVALMLCCIWISPAVLSFLPIFIGWYTTDINQIYRAKCKYDCQFIVNKPYSVISSSISFWI PCTIMIFTYFAIFREANKQEKELYSRHGAAMLLHNQNNTNGDMLSNSGGSSKTLMHEINQDFHHTPTKERNIMKMKREHKAAKTLGIIMGIFILCWLPFFSWYV ITSLCTHCFNPDILVSIVFWIGYFNSTLNPVIYAYFNREFREAFKNTLEFLFCSLCRRPPSDLDHYDMRRPSLRYDDRTRSTYSETYLKHVDRRRSSEFGSSL.

[0028] Example 2: HparGR22 Analysis of the relative expression levels of genes in different tissues of the larvae of the dark-browed scarab beetle;

[0029] (1) RNA extraction and cDNA synthesis;

[0030] 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 All-In-One 5X RT MasterMix (Applied Biological Materials, Richmond, Canada) instructions.

[0031] (2) Quantitative real-time PCR;

[0032] Using the cDNA synthesized in the previous step as a template, the upstream primer sequence was 5'-GAAGGGCAGCCAGCATA-3' (as shown in SEQ ID No:3), and the downstream primer sequence was 5'-TCAACCAAGATTTCCACCAT-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). HparGR22 The relative expression levels of genes. Analysis revealed that... HparGR22 The gene was expressed at the highest level in the mustache, showing a significant difference (see [link]). Figure 1 and Figure 2 ).

[0033] Example 3: Expression HparGR22 The response of oocytes to phlorizin;

[0034] (1) HparGR22 Gene cloning;

[0035] Using cDNA derived from reverse transcription of the lower labial barbel RNA of the dark-browed scarab beetle as a template, amplification was performed using upstream primer 5'-AATTCCCCGGGGATCCATGGATAACGGAACTACGATAAAAG-3' (as shown in SEQ ID No:5) and downstream primer 5'-TTGCTCTAGAGAATTCTCAAAGACTCGAACCGAACTCC-3' (as shown in SEQ ID No:6). HparGR22 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.

[0036] (2) HparGR22 Construction of gene expression vectors;

[0037] a. HparGR22 Homologous recombination reaction between gene fragments and linearized vectors;

[0038] 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 transformation.

[0039] b. Transformation and extraction of recombinant plasmids;

[0040] use Trans 1-T1 (CD501-02, TransGen, Beijing) competent cells were transformed with 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 ILB 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 that amplified the correct bands were amplified and cultured (see...). Figure 3 The samples were then sent to Sangon Biotech (Shanghai) for sequencing. Sequence alignment was used to confirm the accuracy of the sequenced genes. Recombinant plasmids were extracted from the correctly sequenced strains, following the instructions of the plasmid miniprep kit (DP103, Tiangen, Beijing).

[0041] (3) Linearization of recombinant plasmids and product recovery;

[0042] The recombinant plasmid was digested with TaKaRa restriction enzyme. The reaction system consisted of: 1 μg recombinant plasmid; 1 μL 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. Complete digestion was assessed using 1% agarose gel electrophoresis. The DNA product was then recovered via 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 overnight at -20°C. 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.

[0043] (4) In vitro transcription of RNA and injection;

[0044] DNA products were transcribed in vitro using the mMESSAGE mMACHINE T7 kit (AM1344, Thermo Fisher Scientific, Waltham). The reaction mixture 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 15000 rpm at 4°C for 15 min, and the waste liquid was discarded. 1 mL of anhydrous ethanol was added, and the mixture was centrifuged at 15000 rpm at 4°C for 15 min, 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 at 18°C ​​for 3 days.

[0045] The nucleotide sequence of the cRNA is shown in SEQ ID No:7:

[0046]

[0047] (5) Two-electrode voltage clamp experiment;

[0048] Post-injection oocytes were tested using a dual-electrode voltage clamp system. 10 -7 M, 10 -6 M, 10 -5 M, 10 -4 M, 10 -3 M and 10 -2 Oocytes stimulated with M concentration of phlorizin showed a significant response to phlorizin stimulation (see...). Figure 4 Specifically, as the concentration of phlorizin increases, the response value of oocytes also increases, reaching 10. -7 The value at M is 19.733 nA ± 0.974 nA, and at 10 -6 The value at M is 27.700 nA ± 1.131 nA, and at 10 -5 The value at M is 38.667 nA ± 1.782 nA, and at 10 -4 The value at M is 48.433 nA ± 1.915 nA, and at 10 -3 The value at M is 72.967 nA ± 8.399 nA, and at 10 -2 The reaction value is the largest at M, which is 148.400 nA ± 4.003 nA.

[0049] 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. HparGR22 Genes, characterized by, The HparGR22 The DNA sequence of the gene is shown in SEQ ID No:

1. HparGR22 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID No:

2.

2. As described in claim 1 HparGR22 Genes, characterized by, The HparGR22 The gene was expressed at the highest level in the labial palps of the larvae of the dark-browed scarab beetle.

3. An expression carrier, characterized in that, Includes the claims 1 or 2 HparGR22 Gene.

4. In vitro transcribed cRNA, characterized in that, It is obtained by transcription from the expression vector of claim 3; the nucleotide sequence of the cRNA is shown in SEQ ID No:

7.

5. A method for constructing an in vitro model for phlorizin recognition, characterized in that, Includes the following steps: The cRNA described in claim 4 was injected into Xenopus laevis oocytes, and the oocytes were cultured to express it. HparGR22 Proteins encoded by genes.

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