Molecular markers of chilo suppressalis and chilo irridulans to abamectin and application and detection method thereof

Abstract

The present application relates to the technical field of plant molecular breeding, and provides two kinds of molecular markers of chilo suppressalis resistant to abamectin, application and detection method thereof, the molecular markers of chilo suppressalis resistant to abamectin include resistance gene CsABCB5 and transcription factor ETS1, the nucleotide sequences of resistance gene CsABCB5 and transcription factor ETS1 are shown as SEQ ID NO.6 and SEQ ID NO.7 respectively, and the above technical scheme solves the problems of low detection efficiency of chilo suppressalis resistance in the related art, which is difficult to meet the rapid development in the field.

Description

Technical Field

[0001] This invention relates to the field of plant molecular breeding technology, specifically to two molecular markers for resistance to abamectin in rice stem borers, their applications, and detection methods. Background Technology

[0002] Borer borer Chilo suppressalis The rice stem borer is one of the major pests of rice crops. It damages rice by boring into the stem, causing symptoms such as sheath blight, dead heart, and whiteheads. It also exhibits tolerance to waterlogging and can infest rotating plants. Control of the rice stem borer mainly relies on chemical insecticides, such as triazophos, chlorpyrifos, abamectin, and chlorantraniliprole. Due to the irrational use of insecticides, the rice stem borer has developed varying levels of resistance to multiple insecticides in different regions of my country. Clarifying the resistance level of the rice stem borer to commonly used rice insecticides is crucial for implementing appropriate integrated management measures. Currently, the main method for detecting rice stem borer resistance is in vivo larval bioassay. This method requires collecting a sufficient number of live rice stem borers in the field during outbreaks and raising them to the appropriate age before conducting bioassay experiments. This method consumes significant manpower and resources annually, resulting in low efficiency and difficulty in rapid implementation.

[0003] In the long run, starting from the genetic basis of drug resistance research, screening for drug resistance genes, conducting drug resistance gene detection at the molecular level, and genetically detecting the degree of resistance of rice stem borers to pesticides are important ways to fundamentally solve the problem of resistance detection.

[0004] Based on the above problems, this invention proposes two molecular markers for rice stem borer resistance to avermectin, their applications, and detection methods, thereby further improving the efficiency of rice stem borer resistance detection. Summary of the Invention

[0005] This invention proposes two molecular markers for rice stem borer resistance to abamectin, along with their applications and detection methods, solving the problem of low efficiency in detecting rice stem borer resistance in related technologies, which makes it difficult to conduct rapid field trials.

[0006] The technical solution of the present invention is as follows: This invention proposes a molecular marker for rice stem borer resistance to avermectin, the molecular marker including the resistance gene CsABCB5, the nucleotide sequence of which is shown in SEQ ID NO.6.

[0007] The present invention also proposes a molecular marker for rice stem borer resistance to avermectin, the molecular marker including transcription factor ETS1, the nucleotide sequence of which is shown in SEQ ID NO.7, and the transcription factor ETS1 positively regulates the resistance gene CsABCB5, the nucleotide sequence of which is shown in SEQ ID NO.6.

[0008] The present invention also proposes a gene fragment for reducing the resistance of rice stem borer to avermectin, the gene fragment comprising the dsRNA sequence of the molecular marker of the rice stem borer's resistance to avermectin, the nucleotide sequence of which is shown in SEQ ID NO.12.

[0009] The present invention also proposes a gene fragment for reducing the resistance of rice stem borer to avermectin, the gene fragment comprising the dsRNA sequence of the molecular marker of the rice stem borer's resistance to avermectin, the nucleotide sequence of which is shown in SEQ ID NO.13.

[0010] As a further technical solution, the method for obtaining the gene fragment that reduces the resistance of rice stem borer to abamectin includes the following steps: S1. Using total RNA from rice stem borer as a template, cDNA was obtained through reverse transcription; S2. Based on the CDS sequence of the target gene, design primer pairs with the T7 promoter; S3. Using cDNA as a template, PCR amplification was performed using primers with the T7 promoter to obtain the gene fragment; The primer pair with the T7 promoter includes an upstream primer as shown in SEQ ID NO. 10 and a downstream primer as shown in SEQ ID NO. 11.

[0011] As a further technical solution, the method for obtaining the gene fragment that reduces the resistance of rice stem borer to abamectin includes the following steps: S1. Using total RNA from rice stem borer as a template, cDNA was obtained through reverse transcription; S2. Based on the CDS sequence of the target gene, design primer pairs with the T7 promoter; S3. Using cDNA as a template, PCR amplification was performed using primers with the T7 promoter to obtain the gene fragment; The primer pair with the T7 promoter includes an upstream primer as shown in SEQ ID NO.8 and a downstream primer as shown in SEQ ID NO.9.

[0012] The present invention also proposes the application of the molecular marker for the resistance of rice stem borer to abamectin, or the gene fragment for reducing the resistance of rice stem borer to abamectin, or the gene fragment obtained by the method for obtaining the gene fragment, in reducing the drug resistance of rice stem borer.

[0013] As a further technical solution, the gene fragment is applied to rice stem borer to perform dsRNA interference, thereby reducing the rice stem borer's resistance to avermectin.

[0014] As a further technical solution, the application method includes injection or nanocarrier feeding.

[0015] The present invention also proposes a pharmaceutical composition for reducing the resistance of rice stem borer to avermectin, comprising the gene fragment for reducing the resistance of rice stem borer to avermectin or the gene fragment obtained by the method described above.

[0016] The present invention also proposes the application of the molecular marker for the resistance of rice stem borer to abamectin, or the gene fragment for reducing the resistance of rice stem borer to abamectin, or the gene fragment obtained by the method described above, in the detection of resistance of rice stem borer to abamectin.

[0017] This invention also proposes a dual-luciferase vector, which is constructed by homologous recombination of the promoter fragment of the resistance gene CsABCB5 and a linearized pmirGLO vector, and the nucleotide sequence of the promoter fragment is shown in SEQ ID NO.20.

[0018] The present invention also proposes a detection kit for detecting the resistance of rice stem borer to avermectin, comprising the molecular markers of the rice stem borer's resistance to avermectin.

[0019] The working principle and beneficial effects of this invention are as follows: In this invention, the complete CsABCB5 gene was cloned based on the published partial sequence of the Csup009700.1 gene. Sequence analysis revealed that it belongs to the transporter protein family. For the first time, the transcription factor ETS1 and the transporter protein CsABCB5 were found to be highly expressed in an abamectin-resistant rice stem borer population. ETS1 can positively regulate CsABCB5 expression, participating in and mediating rice stem borer resistance to abamectin. ETS1 and CsABCB5 can serve as resistance detection targets to detect the level of abamectin resistance in rice stem borers in the field, providing clear target genes for monitoring and controlling abamectin resistance in rice stem borers and improving detection efficiency. Attached Figure Description

[0020] The present invention will now be described in further detail with reference to the accompanying drawings and specific embodiments.

[0021] Figure 1 This is a diagram showing the predicted TMHMM transmembrane structure of CsABCB5 in Embodiment 1 of the present invention. Figure 2 This is a phylogenetic tree diagram of the complete CsABCB5 gene in Example 1 of the present invention; Figure 3 This is a comparison chart of the FPKM values ​​of transcription factor ETS1 in rice stem borers in Sichuan and Jiangxi provinces in Example 2 of this invention; Figure 4 This is a comparison chart of the FPKM values ​​of CsABCB5 in the rice stem borer in Sichuan and Jiangxi provinces in Example 2 of the present invention; Figure 5 This is a graph showing the relative expression levels of transcription factor ETS1 in Example 3 of the present invention; In the figure: dsEGFP is the blank control group that interferes with EGFP, and dsETS1 is the treatment group that interferes with the transcription factor ETS1. Figure 6 This is a graph showing the relative expression levels of CsABCB5 in Example 3 of the present invention; In the figure: dsEGFP is the blank control group that interferes with EGFP, and dsETS1 is the treatment group that interferes with the transcription factor ETS1. Figure 7 This is a graph showing the relative expression level of CsABCB5 in the CsABCB5 dsRNA interference experiment in Example 3 of this invention. In the figure: dsEGFP is the blank control group with EGFP interference, and dsCsABCB5 is the treatment group with CsABCB5 interference. Figure 8 The graph shows the expression of CsABCB5 and the sensitivity of rice stem borer to triazophos in Example 3 of this invention. In the figure: dsEGFP represents the mortality rate of the negative control, and dsCsABCB5 represents the mortality rate of the interfering CsABCB5. Figure 9 The graph shows the expression of CsABCB5 and the sensitivity of the rice stem borer to abamectin in Example 3 of this invention. In the figure: dsEGFP represents the mortality rate of the negative control, and dsCsABCB5 represents the mortality rate of the interfering CsABCB5. Figure 10 The graph shows the expression of CsABCB5 and the sensitivity of rice stem borer to chlorantraniliprole in Example 3 of this invention. In the figure: dsEGFP represents the mortality rate of the negative control, and dsCsABCB5 represents the mortality rate of the interfering CsABCB5. Figure 11 This is a graph showing the dual-luciferase detection results in Example 4 of the present invention; In the figure: CsABCB5 promoter is the group transfected with pmirGLO-CsABCB5-promoter vector, CsABCB5 promoter + ETS1 is the group co-transfected with pmirGLO-CsABCB5-promoter + pAC5.1-ETS1 vector, and NC is the negative control group. Detailed Implementation

[0022] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0023] Example 1: Cloning and Analysis of the CsABCB5 Gene (1) Design of 5' RACE gene-specific primers (GSP): Based on the published partial sequence of the Csup009700.1 gene (SEQ ID NO.1) (Ma W, Zhao X, Yin C, et al. A chromosome-level genome assembly reveals the genetic basis of cold tolerance in anotorious rice insect pest, Chilo suppressalis. Mol Ecol Resour. 2020; 20:268–282.), RACE gene-specific primers (GSPs) for Csup009700.1 were designed according to the requirements of RACE experiments: Csup009700.1-GSP1:CTTGCCGCATCCACTGGGCCCCACGA (SEQ ID NO.2); Csup009700.1-GSP2:GTGCGCGCCAGCTGCCCCATGAT (SEQ ID NO. 3).

[0024] The primers were sent to Sangon Biotech (Shanghai) Co., Ltd. for synthesis.

[0025] (2) Synthesis of 5' RACE first-strand cDNA: Total RNA was extracted from *Taxobacterium tumefaciens* in Sichuan Province using Trizol reagent (Invitrogen, catalog number 15596026). First-strand cDNA synthesis for 5' RACE was performed according to the instructions of the HiScript-TS 5' / 3' RACE Kit (Novizan, catalog number RA101-01): The components required for first-strand cDNA synthesis were removed and dissolved on ice. After thorough dissolution, the mixture was gently tapped to mix, briefly centrifuged, and then placed on ice. The 5' RACE amplification reaction system was prepared according to the table below (Table 1): Table 1

[0026] After mixing the above reagents, briefly centrifuge, and then perform PCR amplification in a PCR instrument according to the following procedure (see Table 2): Table 2

[0027] The obtained product is the PCR amplification product. The reverse transcription reaction system is prepared according to the table below (Table 3): Table 3

[0028] After mixing the above reagents, briefly centrifuge, and then perform reverse transcription in a PCR instrument according to the following procedure (see Table 4): Table 4

[0029] The resulting product is 5' RACE-Ready cDNA, which can be stored at -20°C for 3 months.

[0030] (3) 5' RACE first round amplification: Using the 5' RACE-Ready cDNA obtained in the previous step as a template, configure the amplification system according to the table below (Table 5): Table 5

[0031] Gently mix with a pipette, briefly centrifuge to collect the mixture, place on ice, and then perform the reaction in a PCR instrument according to the following procedure (see Table 6): Table 6

[0032] After the PCR reaction was completed, the first round of 5' RACE amplification product of Csup009700.1 was obtained. The bands were not very specific after electrophoresis analysis, so a second round of nested PCR amplification was performed.

[0033] (4) Second round of nested PCR amplification using 5' RACE: Take 5 μL of the first-round 5' RACE amplification product of Csup009700.1, add it to 245 μL of ddH2O, vortex to mix, and obtain the diluted product. Prepare the second-round nested PCR amplification system of 5' RACE according to the table below (Table 7): Table 7

[0034] Gently mix with a pipette, briefly centrifuge to collect the mixture, place on ice, and then perform the PCR reaction in a PCR instrument according to the following procedure (see Table 8): Table 8

[0035] After the PCR reaction, 5' RACE amplification products were obtained. Electrophoresis analysis showed that the bands were specific. The PCR products were recovered and sent for sequencing according to the Wizard SV Gel and PCR Clean-Up System kit (catalog number A9281, Promega).

[0036] 2. Design of primers for the CsABCB5 gene: Total RNA was extracted from *Stachys chinensis* (rice stem borer) in Sichuan Province using Trizol reagent. cDNA was synthesized using the SuperScript™ III First-Stand Synthesis System (catalog number 18080-051, Invitrogen). Primers for the CsABCB5 gene were designed based on the 5' RACE amplification product from the previous step. CsABCB5-cF: ATGAAACGGAATGAATCTCTAAAGAG (SEQ ID NO.4), CsABCB5-cR: TTATTCCGTGGTTTGTTGGCG (SEQ ID NO. 5).

[0037] The aforementioned primers were sent to Sangon Biotech (Shanghai) Co., Ltd. for primer synthesis.

[0038] 3. PCR amplification of the CsABCB5 gene: The CsABCB5 gene was amplified using *Taxobacterium tumefaciens* cDNA as a template and FastPfu enzyme. The reaction system is shown in Table 9 below.

[0039] Table 9

[0040] After mixing the above reagents, briefly centrifuge, and then perform PCR amplification in a PCR instrument according to the following procedure (see Table 10): Table 10

[0041] After PCR amplification, the PCR products were recovered and sent for sequencing according to the Wizard SV Gel and PCR Clean-Up System kit instructions. Open reading frame analysis was performed on the sequencing results to extract the coding sequence and translate it to obtain the theoretical amino acid sequence. Software was used to predict the transmembrane structure of this amino acid sequence, and the prediction results are shown below. Figure 1 As shown, this protein contains the typical 12 transmembrane regions of the ABC transporter family; phylogenetic analysis (such as...) reveals that... Figure 2 As shown in the figure, the gene is named CsABCB5 (nucleotide sequence as shown in SEQ ID NO. 6).

[0042] Example 2: Expression Difference Analysis Resistance of the rice stem borer in Sichuan and Jiangxi provinces to insecticides (avermectin, triazophos, and chlorantraniliprole) was monitored. The monitoring data showed that the rice stem borer in Sichuan was sensitive to avermectin, triazophos, and chlorantraniliprole, while the rice stem borer in Jiangxi showed high resistance to these insecticides. Therefore, the rice stem borer in Sichuan was used as the sensitive control group, and the rice stem borer in Jiangxi was used as the resistant group.

[0043] The fat body is a major detoxification and metabolic organ in insects. Transcriptome sequencing analysis was performed on fat bodies collected from the Sichuan and Jiangxi regions of the rice stem borer. Each group contained three replicates, and each sample contained fat body tissue from five fifth-instar larvae of the rice stem borer. Total RNA was extracted from the samples from the Sichuan and Jiangxi regions using the TRIzol method, and eukaryotic transcriptome libraries were constructed. After the libraries passed quality control, high-throughput sequencing was performed using the Illumina sequencing platform, with reads of 150 bp paired ends. The Illumina high-throughput sequencing results were initially stored as raw image data files, and were converted into raw sequenced reads after sequence base identification using bcl-convert software. The results were stored in FASTQ (fq) file format.

[0044] The original sequencing files were filtered using FASTP, aligned using BWA, and subjected to differential expression analysis to obtain a differential gene expression matrix. Analysis of the obtained differential gene expression matrix of *Staphylococcus aureus* revealed that ETS1 (nucleotide sequence shown in SEQ ID NO.7) and CsABCB5 were upregulated in *Staphylococcus aureus* from Jiangxi Province. A comparison of the expression levels of ETS1 and CsABCB5 in *Staphylococcus aureus* from Sichuan and Jiangxi Provinces is shown in the figure below. Figure 3 , Figure 4 As shown, Figure 3 middle This indicates that p < 0.01, and there is a significant difference in FPKM between the rice stem borer in Sichuan and the rice stem borer in Jiangxi. Figure 4 middle This indicates that p < 0.001, and there is a significant difference in FPKM between the rice stem borer in Sichuan and the rice stem borer in Jiangxi.

[0045] Example 3: Validation of the regulatory relationship between transcription factors ETS1 and CsABCB5 genes 1. Preparation of dsRNA from ETS1 and CsABCB5 genes (1) Design of primers for target genes: Total RNA was extracted from *Staphylococcus aureus* (rice borer) in Sichuan Province using Trizol reagent. cDNA was synthesized using the SuperScript™ III First-Stand Synthesis System reverse transcription kit. Primers with the T7 promoter were designed using Primer Premier 5 software based on the CDS sequences of the *Staphylococcus aureus* ETS1 and CsABCB5 genes. ETS1-dsF: TAATACGACTCACTATAGGG CAACAAGAAGATGGCCGAGG (SEQ ID NO.8), ETS1-dsR: TAATACGACTCACTATAGGG AACAACGACCGCTCCTTTTC (SEQ ID NO.9), CsABCB5-dsF: TAATACGACTCACTATAGGG ACTTTCTTCTCGTGGGCCTG (SEQ ID NO.10), CsABCB5-dsR: TAATACGACTCACTATAGGG GCAATGGTGTTCGCTCGATC (SEQ ID NO.11), underlined as the T7 promoter sequence, was sent to Sangon Biotech (Shanghai) Co., Ltd. for primer synthesis.

[0046] (2) PCR amplification of the target fragment and preparation of dsRNA: Using *Taxobacterium tumefaciens* cDNA as a template, the gene was amplified using FastPfu enzyme. The reaction system is shown in Table 9 above. After mixing the above reaction solutions, the mixture was briefly centrifuged, and then PCR amplification was performed in a PCR instrument according to the above procedure (see Table 10). After PCR amplification, the PCR product was recovered according to the instructions of the Wizard SV Gel and PCR Clean-Up System kit. Subsequently, dsRNA was synthesized according to the instructions of the T7 High Yield RNA Transcription Kit from Nanjing Novizan Pharmaceutical Co., Ltd., and the reaction system is shown in Table 11 below: Table 11

[0047] Gently mix all components with a pipette, briefly centrifuge, and incubate at 37°C for 4 hours. Add 1 μL of DNase I to the reaction system and incubate at 37°C for 15 minutes to digest the transcribed DNA template. After electrophoresis analysis and purification, the synthesized dsRNA yields the dsRNA sequence of the resistance gene CsABCB5 (nucleotide sequence as shown in SEQ ID NO. 12) and the dsRNA sequence of the transcription factor ETS1 (nucleotide sequence as shown in SEQ ID NO. 13), which are stored for later use.

[0048] 2. dsRNA interference experiment This invention employs two methods for dsRNA interference experiments: direct injection of dsRNA and a nanocarrier feeding method. In the experiment detecting CsABCB5 expression after ETS1 interference, direct injection of ETS1 dsRNA was used. In the experiment performing bioassays on rice stem borers after CsABCB5 interference, a nanocarrier feeding method was used.

[0049] (1) Injection of dsRNA to interfere with ETS1 of the rice stem borer Fourth instar mid-stage larvae of uniform size were selected and starved for 4 hours before being placed on ice. 2.5 μg of ETS1 dsRNA was injected into the space between the penultimate and penultimate leg segments of the larvae's abdomen using a capillary tube pulled out by a needle extractor. Fifteen larvae were injected per treatment, with three replicates. dsEGFP was injected as a control. Larvae were subsequently collected 1, 2, and 3 days after ETS1 dsRNA injection for subsequent interference efficiency and target gene detection experiments.

[0050] (2) Interference of rice stem borer CsABCB5 by nanocarrier feeding method Prepare a solution of 20 μg dsRNA / g artificial feed. Mix CsABCB5 dsRNA with an equal mass of the nanocarrier Spc in DEPC-treated water and let stand for 10 min. Then, spread the mixture containing the nanocarrier and dsRNA evenly on the surface of the artificial feed using a pipette and allow it to evaporate before use. Select mid-second instar larvae of uniform size, starve them for 24 h, and then place them on the prepared nanocarrier-dsRNA mixture for culture. Supplement with fresh nanocarrier-dsRNA mixture daily for 5 consecutive days. The control group was interfered with using dsEGFP. Subsequently, the test larvae were used for subsequent interference efficiency and bioassay experiments.

[0051] (3) Interference efficiency and target gene detection Samples were prepared 24, 48, and 72 hours after ETS1 dsRNA injection, and qPCR was used to detect the interference efficiency of ETS1 and the expression of CsABCB5. Five days after CsABCB5 interference using the nanocarrier feeding method, samples were prepared, and qPCR was used to detect the interference efficiency of CsABCB5.

[0052] The primers for ETS1's quantitative PCR are designed as follows: ETS1-qF: CGACGACTACTACCACCACG (SEQ ID NO.14), ETS1-qR: CGTAACTCTCACCGTAGCCC (SEQ ID NO.15), The quantitative PCR primers for CsABCB5 are as follows: CsABCB5-qF: GCGTGCTGGACAGATACAAG (SEQ ID NO.16), CsABCB5-qR: CTCCCTGTCAGCCACTAACA (SEQ ID NO.17), Internal reference gene selection EF: EF-qF: TGAACCCCCATACAGCGAATCC (SEQ ID NO.18), EF-qR: TCTCCGTGCCAACCAGAAATAGG (SEQ ID NO. 19).

[0053] All tests included three biological replicates, each containing five samples, and the differences were analyzed using the independent samples t-test in SPSS software.

[0054] The results showed that the interference efficiency of ETS1 dsRNA reached its peak of over 50% two days after injection. Figure 5On day 2, when ETS1 was significantly interfered with, the expression level of CsABCB5 was also significantly suppressed. Figure 6 Nanocarrier feeding showed that after 5 days of continuous feeding with CsABCB5 dsRNA, CsABCB5 expression was significantly inhibited, with an interference efficiency of approximately 50%. Figure 7 ).

[0055] Figure 5 middle P < 0.05 indicates a significant difference, while ns indicates no significant difference. Figure 6 middle This indicates a significant difference (P < 0.05). Figure 7 middle This indicates that P < 0.01, which is a significant difference.

[0056] (4) Bioassay of rice stem borer after interference with CsABCB5 Bioassays were performed 5 days after CsABCB5 interference using a nanocarrier feeding method. Bioassays for triazophos and abamectin were performed using the capillary drop method, while bioassays for chlorantraniliprole were performed using the feed-film method. The bioassay concentrations for triazophos were 0.139 μg / insect, for abamectin 10 ng / insect, and for chlorantraniliprole 1.592 mg / mL. Insect mortality was checked 2 days after triazophos treatment, and 3 days after abamectin and chlorantraniliprole treatments, and the total number of insects and the number of dead insects were recorded. Insects were considered dead when they could not move coordinatedly when lightly touched with a brush. Each insecticide was used to treat 30 insects.

[0057] Bioassay results showed that after interfering with CsABCB5: Figure 8 Bioassays showed that inhibiting CsABCB5 expression did not affect the sensitivity of rice stem borer to triazophos; Figure 9 Bioassays showed that inhibiting the expression of CsABCB5 increased the sensitivity of rice stem borer to abamectin; Figure 10 Bioassays showed that inhibiting CsABCB5 expression did not affect the sensitivity of rice stem borer to chlorantraniliprole.

[0058] Figure 8 , Figure 10 In ns, no significant difference is indicated; Figure 9 middle This indicates a significant difference (P < 0.05). The above results indicate that transcription factor ETS1 and transporter CsABCB5 play important roles in the resistance of rice stem borer to avermectin.

[0059] Example 4 Functional Verification 1. Construction of dual-luciferase vector (1) Design of primers for target genes: DNA was extracted from *Stachys edulis* (a type of rice stem borer) in Sichuan Province using a TransGold DNA Extraction Kit (catalog number EE101-01, Beijing TransGold Biotechnology Co., Ltd.). Primers with homologous arm sequences were designed using Primer Premier 5 software based on the *Stachys edulis* CsABCB5 promoter region sequence (nucleotide sequence as shown in SEQ ID NO. 20). CsABCB5-5UpmF: cggcgtagaggatcgagatctCGAACCTTGTGTAACCTGTTTCTC (SEQ ID NO.21), where lowercase letters in the sequence represent homologous arm sequences; CsABCB5-5UpmR: cagtaccggattgccaagcttTCTTGGATCAACCTCTCTGAAATAAA (SEQ ID NO.22), where lowercase letters in the sequence represent homologous arm sequences.

[0060] Total RNA was extracted from *Staphylococcus aureus* (Chinese rice stem borer) in Sichuan Province using Trizol reagent. First-strand cDNA synthesis was performed using the SuperScript™ III First-Stand Synthesis System reverse transcription kit. Primers with homologous arm sequences were designed based on the *Staphylococcus aureus* ETS1 cDNA sequence using Primer Premier 5 software. ETSfcF: tagtccagtgtggtggaattcATGGGAATCAAAGTCATGATGAAA (SEQ ID NO.23), where lowercase letters in the sequence represent homologous arm sequences; ETSfcR: gccctctagactcgagcggccgc TCAGTCATCGTCCTTCTTTTCCA (SEQ ID NO.24), where lowercase letters in the sequence represent homologous arm sequences.

[0061] Using the pmirGLO vector linearized by BsmBl digestion as a template, pmirGLO vector primers were designed: pmirLF: AAGCTTGGCAATCCGGTACTG (SEQ ID NO.25), pmirLR: AGATCTCGATCCTCTACGCCG (SEQ ID NO. 26).

[0062] The aforementioned primers were sent to Sangon Biotech (Shanghai) Co., Ltd. for primer synthesis.

[0063] (2) PCR amplification of the target gene and pmirGLO dual-luciferase vector: The promoter region sequence of CsABCB5 was amplified using *Taxobacterium tumefaciens* DNA as a template, the ETS1 and CsABCB5 sequences were amplified using *Taxobacterium tumefaciens* cDNA as a template, and the pmirGLO vector sequence was amplified using a BsmBl-digested linearized vector as a template. Genes were amplified using FastPfu enzyme. The reaction system is shown in Table 9 above. After mixing the above reaction solutions, the mixture was briefly centrifuged, and then the reaction was performed in a PCR instrument according to the above program (see Table 10). The annealing time was 2 min for the CsABCB5 promoter amplification, 1 min 30 s for the ETS1 sequence amplification, and 6 min for the pmirGLO vector amplification. After PCR amplification, the PCR products were recovered according to the Wizard SV Gel and PCR Clean-Up System kit instructions.

[0064] (3) Construction of the dual-luciferase vectors pmirGLO-CsABCB5-promoter and pAC5.1-ETS1 expression vector: The dual-luciferase vectors pmirGLO-CsABCB5-promoter and pAC5.1-ETS1 expression vector were constructed using the ClonExpress II homologous recombination kit from Novizan. The target fragment and the linearized pmirGLO vector or pAC5.1 vector were mixed according to the system shown in Table 12 below: Table 12

[0065] Gently mix the components with a pipette, briefly centrifuge, and incubate at 37°C for 40 min to obtain the recombinant product; cool to 4°C or immediately place on ice to cool.

[0066] (4) Transformation of recombinant products: Thaw the competent cells on ice. Add 10 μL of the recombinant product prepared in step (3) above to 100 μL of competent cells. Gently tap the tube wall to mix (do not shake). Incubate on ice for 30 min. Heat shock in a 42°C water bath for 45 sec, then immediately cool on ice for 3 min. Add 900 μL of SOC or LB medium (without antibiotics) and incubate at 37°C for 1 h (200 rpm). Centrifuge at 5,000 rpm (2,400 × g) for 5 min and discard 900 μL of supernatant. Resuspend the cells in the remaining medium and spread them gently on a plate containing the correct antibiotic using a sterile spreader. Incubate upside down at 37°C for 16 h. Pick several clones from the recombinant reaction transformation plate for colony PCR identification. For colonies that are positive by colony PCR, inoculate the remaining bacterial culture into liquid LB medium containing appropriate antibiotics and incubate overnight for first-generation sequencing.

[0067] 2. Transfect the vector into S2 cells for expression. Cell passage: Observe the S2 cell density and culture medium color under a microscope. When the medium turns yellow and becomes clear, indicating a cell density of over 90%, it is ready for passage. Use a 1 mL pipette to aspirate and discard the original culture medium. Slowly add 2 mL of PBS or S2 cell culture medium along the side wall of a T25 cell flask, gently rotating the flask to wash away any remaining medium. Gently and repeatedly pipette the cells to disperse them; observe under a microscope until they are independent cells. Then, passage at a volume ratio of 1:3 or 1:4. Depending on experimental needs, inoculate an appropriate amount of cells into a culture dish, add fresh cell growth medium to a final volume of 6 mL, shake well, and incubate at 28°C for 5 days.

[0068] Cell transfection: 24 hours before transfection, gently and repeatedly pipette the logarithmic growth phase cells to disperse them, and adjust the cell density to 1×10⁶ cells / year using medium containing 10% FBS. 5 Cells / mL, reseeded into 96-well cell culture plates, to achieve a cell count of 1×10⁶ cells per well. 4 Incubate the cells at 28°C for 24 hours until the cell density reaches 30%-50%, then they are ready for transfection. Replace the cell culture medium with serum-free medium 2 hours before transfection.

[0069] Add an appropriate amount of endotoxin-free plasmid to a sterile 1.5 mL EP tube, followed by an appropriate amount of FuGENE® HD Transfection Reagent and S2 cell culture medium. Mix gently and incubate at room temperature for 10 min. Finally, add 100 ng pmirGLO plasmid, 100 ng pAC5.1 plasmid, and 0.6 μL FuGENE® HD Transfection Reagent to each well of a 96-well cell culture plate. Transfer the plasmid and FuGENE® HD Transfection Reagent mixture to cell culture medium, mix well, and incubate at 28°C for 48 h. The resulting mixture is then used for dual-luciferase detection, representing the co-transfection of the pmirGLO-CsABCB5-promoter + pAC5.1-ETS1 vector group.

[0070] The only difference between the group transfected with pmirGLO-CsABCB5-promoter and the group co-transfected with pmirGLO-CsABCB5-promoter + pAC5.1-ETS1 vector was that 100 ng of pAC5.1-ETS1 plasmid was replaced with an empty pAC5.1 plasmid, and empty pmirGLO plasmid and empty pAC5.1 plasmid were set as negative control groups.

[0071] 3. Dual-luciferase assay The dual-luciferase assay was performed using the Novizan Duo-Lite Luciferase Assay System (catalog number DD1205), and the specific procedure is as follows: (1) Take out the cell culture plate to be tested from the incubator and place it at room temperature for 30 minutes to allow the temperature of the culture plate to equalize to room temperature.

[0072] (2) Detection of firefly luciferase activity: Add 100 μL of Duo-Lite Luciferase detection reagent to each well of a 96-well plate and mix well. Incubate at room temperature for 10 min, and then detect the luminescence of firefly luciferase in a microplate reader.

[0073] (3) Detection of Renaissance luciferase activity: Add 100 μL of Duo-Lite Stop & Lite detection reagent to each well of a 96-well plate and mix well. Incubate at room temperature for 10 min and then detect Renaissance luciferase activity using a microplate reader.

[0074] Using the fluorescence value of *Rhizophora stylosa* as an internal reference, the relative fluorescence value of *Firefly* was calculated, and the results of dual-luciferase assay were as follows: Figure 11As shown, the dual-luciferase results indicated that the luciferase activity co-transfected with the CsABCB5 promoter and transcription factor ETS1 was significantly higher than that with only the CsABCB5 promoter, suggesting that transcription factor ETS1 directly regulates the expression of CsABCB5.

[0075] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A molecular marker for rice stem borer resistance to avermectin, characterized in that, The molecular marker includes the resistance gene CsABCB5, the nucleotide sequence of which is shown in SEQ ID NO.

6.

2. A molecular marker for rice stem borer resistance to avermectin, characterized in that, The molecular marker includes transcription factor ETS1, the nucleotide sequence of which is shown in SEQ ID NO.

7. ETS1 positively regulates the resistance gene CsABCB5, the nucleotide sequence of which is shown in SEQ ID NO.

6.

3. A gene fragment that reduces the resistance of rice stem borer to avermectin, characterized in that, The gene fragment includes the dsRNA sequence of the molecular marker for the resistance of rice stem borer to avermectin as described in claim 1, and its nucleotide sequence is shown in SEQ ID NO.

12.

4. A gene fragment that reduces the resistance of rice stem borer to avermectin, characterized in that, The gene fragment includes the dsRNA sequence of the molecular marker of rice stem borer resistance to avermectin as described in claim 2, and its nucleotide sequence is shown in SEQ ID NO.

13.

5. The method for obtaining the gene fragment that reduces the resistance of rice stem borer to avermectin according to claim 3, characterized in that, Includes the following steps: S1. Using total RNA from rice stem borer as a template, cDNA was obtained through reverse transcription; S2. Based on the CDS sequence of the target gene, design primer pairs with the T7 promoter; S3. Using cDNA as a template, PCR amplification was performed using primers with the T7 promoter to obtain the gene fragment; The primer pair with the T7 promoter includes an upstream primer as shown in SEQ ID NO.10 and a downstream primer as shown in SEQ ID NO.

11.

6. The method for obtaining the gene fragment that reduces the resistance of rice stem borer to avermectin according to claim 4, characterized in that, Includes the following steps: S1. Using total RNA from rice stem borer as a template, cDNA was obtained through reverse transcription; S2. Based on the CDS sequence of the target gene, design primer pairs with the T7 promoter; S3. Using cDNA as a template, PCR amplification was performed using primers with the T7 promoter to obtain the gene fragment; The primer pair with the T7 promoter includes an upstream primer as shown in SEQ ID NO.8 and a downstream primer as shown in SEQ ID NO.

9.

7. The molecular marker for rice stem borer resistance to avermectin according to any one of claims 1 to 2, or the gene fragment for reducing rice stem borer resistance to avermectin according to any one of claims 3 to 4, or the gene fragment obtained by the method according to any one of claims 5 to 6, in reducing rice stem borer resistance.

8. The application according to claim 7, characterized in that, The gene fragment was applied to rice stem borer to induce dsRNA interference, thereby reducing the rice stem borer's resistance to avermectin.

9. A pharmaceutical composition for reducing the resistance of rice stem borer to avermectin, characterized in that, This includes the gene fragment for reducing the resistance of rice stem borer to abamectin as described in any one of claims 3 to 4, or the gene fragment obtained by the method described in any one of claims 5 to 6.

10. The application of the molecular marker for rice stem borer resistance to avermectin according to any one of claims 1 to 2, or the gene fragment for reducing rice stem borer resistance to avermectin according to any one of claims 3 to 4, or the gene fragment obtained by the method for obtaining the gene fragment according to any one of claims 5 to 6, in the detection of rice stem borer resistance to avermectin.

11. A dual-luciferase carrier, characterized in that, The promoter fragment of the molecular marker for the resistance of rice stem borer to avermectin as described in claim 1 was constructed by homologous recombination with the linearized pmirGLO vector, and the nucleotide sequence of the promoter fragment is shown in SEQ ID NO.

20.

12. A test kit for detecting the resistance of rice stem borer to abamectin, characterized in that, Including the molecular markers for avermectin resistance in rice stem borer as described in any one of claims 1 to 2.

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