Bdorobp99c-6 and use thereof

Abstract

The present application relates to the technical field of molecular biology, and particularly relates to a molecular marker of malathion resistance of Bactrocera dorsalis, and application and detection method thereof.A molecular marker of malathion resistance of Bactrocera dorsalis, the molecular marker is BdorOBP99c-6 gene and / or BdorOBP99c-6 protein.The present application finds that the odor binding protein gene OBP99c-6 of Bactrocera dorsalis is significantly related to malathion resistance: OBP99c-6 is continuously up-regulated in the malathion resistance population, the encoded protein can bind with malathion and malaoxyphosphon, indicating that OBP99c-6 can be used as a candidate molecular marker related to malathion resistance of Bactrocera dorsalis.The present application further finds that transcription factor Nub can promote the transcription activity of the key region of OBP99c-6 promoter; after silencing Nub, the expression amount of OBP99c-6 decreases, and the sensitivity of Bactrocera dorsalis to malathion increases, indicating that Nub-OBP99c-6 regulatory module is involved in the molecular basis of malathion resistance of Bactrocera dorsalis.

Description

Technical Field

[0001] This invention relates to the field of molecular biology, and in particular to the molecular marker BdorOBP99c-6, which is associated with malathion resistance in the oriental fruit fly, and its applications. Background Technology

[0002] The citrus fruit fly (Bactrocera dorsalis) (Hendel) is a significant agricultural pest characterized by its wide host range, high reproductive capacity, strong environmental adaptability, and rapid spread. It primarily damages fruits and vegetables by having females lay eggs and larvae bore into the fruit, severely impacting yield and quality. Chemical control remains the main method for controlling citrus fruit fly damage, with organophosphate insecticides such as malathion widely used in the field. However, long-term and irrational use of pesticides has led to increasing resistance in citrus fruit flies to malathion, affecting control effectiveness.

[0003] Currently, monitoring of pesticide resistance in the oriental fruit fly (Bactrocera dorsalis) relies heavily on bioassays, which suffer from long detection cycles, cumbersome procedures, and difficulty in providing timely warnings. Existing research primarily focuses on classical resistance mechanisms such as detoxification metabolic enzymes and target mutations, while the development of non-classical resistance-related factors remains insufficient. Insect odorant-binding proteins (OBPs) are a class of soluble carrier proteins capable of binding and transporting hydrophobic chemical molecules, and in recent years have been considered to potentially participate in the recognition, transport, or isolation of exogenous pesticides. Therefore, screening OBP molecular markers associated with malathion resistance in the oriental fruit fly and establishing corresponding detection and application methods are of great significance for resistance monitoring and control.

[0004] Therefore, we propose the molecular markers for resistance to malathion in the oriental fruit fly, their applications, and detection methods. Summary of the Invention

[0005] The purpose of this invention is to provide molecular markers for malathion resistance in the oriental fruit fly, their applications, and detection methods. This invention addresses the industry pain points of difficulty in monitoring malathion resistance in oriental fruit flies and the lack of precise targets for control, enabling early and rapid monitoring of malathion resistance in oriental fruit flies and providing a basis for field pesticide application decisions.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] A molecular marker for resistance to malathion in the oriental fruit fly, wherein the molecular marker is the BdorOBP99c-6 gene and / or the BdorOBP99c-6 protein;

[0008] The nucleotide sequence of the BdorOBP99c-6 gene is shown in SEQ ID NO:1;

[0009] The amino acid sequence of the BdorOBP99c-6 protein is shown in SEQ ID NO:2.

[0010] The molecular markers described herein are used in the detection of malathion content, or in the preparation of reagents or devices for the detection of malathion content.

[0011] The application method includes the following steps:

[0012] Using multiple known concentrations of malathion standards as detection targets, the affinity between OBP99c-6 protein and the standards was determined, and the correspondence between malathion concentration and affinity detection signal values ​​was constructed.

[0013] The affinity of the test sample containing malathion to the OBP99c-6 protein was determined, and the concentration of malathion in the test sample was obtained according to the correspondence between the malathion concentration and the affinity detection signal value.

[0014] The process of constructing the correspondence between malathion concentration and affinity detection signal value includes: fitting an affinity curve with malathion concentration as the abscissa and affinity detection fluorescence value as the ordinate.

[0015] The determination of the affinity between the test sample containing malathion and the OBP99c-6 protein includes: using micro-thermal surge technology to detect the affinity between the OBP99c-6 protein and the malathion sample.

[0016] The concentration range of malathion in the sample to be tested is 100 nM to 1 M.

[0017] The application of the molecular markers in the classification of resistance to malathion or early monitoring of resistance levels in oriental fruit fly populations.

[0018] A primer pair for detecting the molecular marker, characterized in that the primer sequence comprising the primer pair is:

[0019] OBP99c-6 qPCR F: 5'- CGATGAAGAGCCCATTCGCA-3';

[0020] OBP99c-6 qPCR R: 5'-TGTTCACCGATTTTGCTGGC-3'.

[0021] A method for detecting the resistance of the oriental fruit fly to malathion, characterized by comprising the following steps:

[0022] Total RNA was extracted from the fruit fly citrus samples to be tested and reverse transcribed into cDNA.

[0023] Using the cDNA as a template, real-time quantitative PCR amplification was performed using the primer pair described in claim 6 to detect the expression level of the BdorOBP99c-6 gene;

[0024] A malathion-sensitive population of the oriental fruit fly was set up as a control, and the gene expression levels of the test sample and the control population were compared.

[0025] If the expression level of the BdorOBP99c-6 gene in the test sample is significantly higher than that in the sensitive population, the tested oriental fruit fly is determined to be a malathion-resistant population; if the expression level is not significantly higher than that in the sensitive population, the tested oriental fruit fly is determined to be a non-resistant population.

[0026] The statistical standard for "significantly higher" is: the relative expression level of the BdorOBP99c-6 gene in the test sample is more than 1.5 times that of the sensitive population, and the statistical analysis shows p < 0.05.

[0027] Preferably, the qPCR amplification reaction system is as follows: NovoStart SYBR qPCR SuperMix Plµs 5.0 μL, 10 μM upstream / downstream primers 0.3 μL each, cDNA template 0.5 μL, and nuclease-free water to a final volume of 10 μL; the qPCR amplification conditions are: 95℃ pre-denaturation for 2 min; 95℃ denaturation for 30 s, 60℃ annealing and extension for 30 s, for 40 cycles; and 60℃ for 30 s, followed by 95℃ for 15 s to form a melting curve.

[0028] A method for regulating the sensitivity of the oriental fruit fly to malathion involves inhibiting the expression of the transcription factor Nub in the oriental fruit fly, thereby downregulating the expression of the BdorOBP99c-6 gene and thus increasing the sensitivity of the oriental fruit fly to malathion.

[0029] The present invention has at least the following beneficial effects:

[0030] This invention discovered that the odor-binding protein gene OBP99c-6 in *Bactrocera dorsalis* is associated with malathion resistance: OBP99c-6 is continuously upregulated in malathion-resistant populations, and its encoded protein can bind to malathion and malathion, indicating that OBP99c-6 can serve as a candidate molecular marker for malathion resistance in *Bactrocera dorsalis*. This invention further found that the transcription factor Nub can enhance the transcriptional activity of a key upstream cis-regulatory segment of BdorOBP99c-6; silencing Nub leads to a decrease in the expression level of BdorOBP99c-6, accompanied by an increase in *Bactrocera dorsalis* sensitivity to malathion, suggesting that the Nub–BdorOBP99c-6 regulatory module may be involved in the formation of malathion resistance in *Bactrocera dorsalis*.

[0031] This invention uses the expression level of OBP99c-6 as a molecular indicator for assessing the risk of malathion resistance in the oriental fruit fly (Bactrocera dorsalis). Changes in OBP99c-6 expression in the test population can be detected using methods such as real-time quantitative PCR, providing technical support for the auxiliary identification of malathion-resistant populations in the oriental fruit fly, early monitoring of resistance, and field application decisions. This detection method is relatively simple to operate, has a short detection cycle, and is suitable for batch sample analysis.

[0032] Furthermore, this invention provides an RNA interference sensitization strategy targeting Nub, an upstream regulator of BdorOBP99c-6. Inhibiting Nub expression through RNA interference reduces the expression level of BdorOBP99c-6 and increases the sensitivity of *Bactrocera dorsalis* to malathion, providing a new potential molecular target and application basis for the development of RNA interference-based resistance management technologies and precision control strategies. Attached Figure Description

[0033] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the following description of the embodiments will be briefly introduced. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0034] Figure 1 This is a diagram showing the results of MR and MS validation of BdorOBP99c-6 expression in the oriental fruit fly;

[0035] Figure 2 This is a graph showing the results of a Western blot using OBP99c-6.

[0036] Figure 3 This is a graph showing the affinity test results between OBP99c-6 protein and malathion.

[0037] Figure 4 This is a schematic diagram of sequence differences in the upstream regulatory region of BdorOBP99c-6;

[0038] Figure 5 This is a schematic diagram of two rounds of dual-luciferase screening in the upstream regulatory region of BdorOBP99c-6;

[0039] Figure 6 This diagram illustrates the regulation of transcriptional activity of the key promoter fragment of BdorOBP99c-6 by the transcription factor Nub, as well as Nub's RNA interference and its sensitivity to malathion in the oriental fruit fly. Detailed Implementation

[0040] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0041] Unless otherwise specified, the experimental methods in the following examples are conventional methods; the chemical and biological reagents used are conventional chemical and biological reagents in the art and are commercially available.

[0042] Among them, malathion: solid standard, purchased from LGC Technology (Shanghai) Co., Ltd., 99.4% purity, chemical formula C 10 H 19 O6PS2, CAS Registry Number 121-75-5.

[0043] Example 1. Establishment of MR and MS populations of the oriental fruit fly and confirmation of its resistance phenotype.

[0044] The malathion-sensitive population MS was continuously reared in the laboratory under pesticide-free conditions. The malathion-resistant population MR was obtained from the MS population through continuous malathion selection, and pesticide screening pressure was maintained in subsequent generations.

[0045] The susceptibility of MR and MS populations to malathion was determined using a spot drip method. Specifically, malathion solution was applied dropwise to the dorsal plate of adult oriental fruit flies, and mortality rates were recorded and the median lethal dose (LD50) was calculated after treatment with different doses. ).

[0046] After 61 generations of continuous screening with malathion, the MR population exhibited resistance to malathion. Reaching 9043.60 ng / fly; MS population's resistance to malathion The resistance rate was 64.37 ng / fly, and the resistance fold of the MR population relative to the MS population was 140.49 times. All test insects were reared at a temperature of 27 ± 0.5 ℃, a relative humidity of 70%, and a photoperiod of 14 h:10 h (light:dark) and fed with artificial feed.

[0047] Example 2. Expression patterns of OBPs at different developmental stages

[0048] Adult oriental fruit flies of the malathion-resistant population (MR) and the malathion-sensitive population (MS) were selected. Samples were collected on days 1, 3, 5, 7, and 9 after emergence, and were designated as A1, A3, A5, A7, and A9, respectively. Each treatment group had at least four biological replicates, with each replicate consisting of two oriental fruit flies.

[0049] Total RNA was extracted from two whole insects, and the expression profiles of OBP genes in resistant and susceptible populations were analyzed. The susceptible population refers to those collected from Guangdong Province in 2009 and reared and propagated without any pesticide exposure. The resistant population was screened by treating each generation with malathion; the malathion concentration increased with each generation, and the resistance fold of the currently used resistant population is 140 times that of the susceptible population. It reached 9043.60 ng / fly.

[0050] Total RNA was extracted from *Bactrocera dorsalis* using TRIzol reagent. Two whole insects were placed in a 1.5 mL nuclease-free centrifuge tube, treated with liquid nitrogen, and 400 µL of TRIzol was added. The mixture was homogenized and incubated at room temperature for 10 min. Then, 200 µL of chloroform was added, the mixture was vortexed for 15 s, and incubated at room temperature for 10 min. The mixture was then centrifuged at 12000 g for 15 min at 4 °C. The supernatant of the separated liquid was aspirated, and 500 µL of isopropanol was added. The mixture was gently vortexed and incubated at room temperature for 10 min. The mixture was then centrifuged at 12000 g for 10 min at 4 °C, the supernatant was discarded, and the white precipitate was retained. The white precipitate was washed with 1 mL of 75% ethanol. The mixture was then centrifuged at 7600 g for 5 min at 4 °C, and the supernatant was discarded. The precipitate was dried in a clean bench for 5-10 min, and 30-100 µL of nuclease-free water was added to dissolve the precipitate and mix thoroughly. Take 1.5 µL of RNA and use NanodropONE (ThermoFisher, USA) to determine its concentration and purity. Take 5 µL for agarose gel electrophoresis to check RNA integrity. Store qualified RNA at -80℃ for later use.

[0051] The qualified RNA samples were treated with the RQ1 DNase kit (Promega, USA) to remove genomic DNA from the samples. The resulting purified RNA samples were then reverse transcribed to synthesize first-strand cDNA according to the instructions of the PrimeScript® RT reagent Kit (Takara, Japan).

[0052] All primers for OBPs were designed using the online website Primer3 (http: / / primer3.ut.ee / ). RT-qPCR experiments and result evaluation were performed using RPS3 (GenBank: XM_011212815) and α-tubulin (GenBank: GU269902) as internal reference genes. The names of the detected OBP99c-6 and the names of the internal reference genes and primer sequences are shown in the table below:

[0053] sequence list

[0054] OBP99c-6 qPCR F CGATGAAGAGCCCATTCGCA SEQ ID NO.4 OBP99c-6 qPCR R TGTTCACCGATTTTGCTGGC SEQ ID NO.5 α-tubulin qPCR F CGCATTCATGGTTGATAACG SEQ ID NO.6 α-tubulin qPCR R GGGCACCAAGTTAGTCTGGA SEQ ID NO.7 RPS3 qPCR F TGGATCACCAGAGTGGATCA SEQ ID NO.8 RPS3 qPCR R TAAGTTGACCGGAGGTTTGG SEQ ID NO.9

[0055] Using the synthesized cDNA as a template, and RPS3 and α-tubulin as internal control genes, the reaction system is as follows:

[0056] NovoStart SYBR qPCR SuperMix Plµs (Brand: Nearshore, China) 5.0 μL, upstream and downstream primers (10 μM) 0.3 μL each, cDNA template 0.5 μL, nuclease-free water to 10 μL.

[0057] PCR program: 95℃ pre-denaturation for 2 min; 95℃ denaturation for 30 s, 60℃ annealing and extension for 30 s, 40 cycles; 60℃ for 30 s, 95℃ for 15 s (to form a melting curve).

[0058] The final relative expression level was adopted. The expression patterns of the OBP99c-6 gene in different populations were investigated: the expression patterns of OBP99c-6 in long-term malathion-resistant and susceptible populations were analyzed, and the quantitative results are as follows: Figure 1 As shown in the figure, statistical analysis revealed that OBP99c-6 expression was significantly upregulated in the resistant population, twice that of the susceptible population. Based on these results, BdorOBP99c-6 exhibited a stable upregulated expression pattern across multiple age stages in the MR population. Therefore, BdorOBP99c-6 can serve as a candidate molecular marker associated with malathion resistance in *Bactrocera citrinum*, and can be used for auxiliary assessment of malathion resistance risk, screening of resistant populations, and subsequent research on resistance mechanisms in *Bactrocera citrinum*.

[0059] Example 3. Heterologous expression of OBP99c-6 protein

[0060] The open reading frame sequence information of OBP99c-6 was obtained from the internal database of the inventor's laboratory (OBPs gene bank of the Oriental fruit fly). Full-length primers were designed to amplify the full-length sequence. The full-length amplification primers are as follows:

[0061] OBP99c-6 F1: 5'-ATGAAATTCTTCATTGTGATCCT-3',

[0062] OBP99c-6 R1: 5'-TCATTTTTGGAAGTAAGCTTTCC-3'.

[0063] The target gene was amplified using whole cDNA from the oriental fruit fly (Bactrocera dorsalis) as a template. The reaction system was as follows: 12.5 μL 2× HieffCanace® AdvanceFast PCR Master Mix (With Dye) (brand: YEASEN, China), 9.5 μL nuclease-free water, 1 μL each of forward and reverse primers, and 2 μL cDNA, for a total of 25 μL. Reaction conditions: 98℃ pre-denaturation for 30 s; 98℃ denaturation for 10 s, 60℃ annealing for 5 s, 72℃ extension for 10 s, 35 cycles; final extension at 72℃ for 5 min, and storage at 12℃.

[0064] Sequencing yielded the ORF sequence of the BdorOBP99c-6 gene (denoted as SEQ ID NO.1):

[0065] 5'-ATGAAATTCTTCATTGTGATCCTTGTAGTCATTGCATTGACATACGCTGAAGATGAATGGATACCCAAAAATGATACAGAACTTGAAGTTATCGCCCAGGAATGCCTCAGAGATTTTCCACTTAGCAAGGAACAACTGCAAAAATTTTCGAGCTTCGAATATCCCGATGAAGAGCCCATTCGCAAGTACATGCTTTGTACCGCTAAGAGAG TGGGTTTCTTCACCGAACAGGAGGGCTATCATGCCGATCGTGTTGCCAAACAATTAAAAATGGATTTGGATGAGGCTGAGATCGTTGCCATCGCTGAGGGTTGCGCGGACAAGAACGTCGAGGGTAGCAGCGCTGATGTATGGGCCTATCGTGTTCACAAGTGTGTGATGGCCAGCAAAATCGGTGAACATGGGAAAGCTTACTTCCAAAAATGA -3'.

[0066] Then, based on the signal peptide prediction, homologous arm primers with their own signal peptide removed and containing both NdeⅠ and EcoRI restriction sites were designed:

[0067] NdeⅠ OBP99c-6 F: 5'- gtgccgcgcggcagccatatgGAAGATGAATGGATACCCCAAAAATG -3',

[0068] EcoRI OBP99c-6 R: 5'-ttgtcgacggagctcgaattcTCATTTTTGGAAGTAAGCTTTCCC-3'.

[0069] The target gene was amplified using 2× Hieff Canace® AdvanceFast PCR Master Mix (With Dye) (brand: YEASEN, China) as a template, with the reaction system as described above. After electrophoresis detection, the product was sent to Kexin Technology Co., Ltd. for sequencing.

[0070] The amino acid sequence of the BdorOBP99c-6 protein (SEQ ID NO.2):

[0071] 5'-EDEWIPKNDTELEVIAQECLRDFPLSKEQLQKFSSFEYPDEEPIRKYMLCTAKRVGFFTEQEGYHADRVAKQLKMDLDEAEIVAIAEGCADKNVEGSSADVWAYRVHKCVMASKIGEHGKAYFQK*-3'.

[0072] The size of the target band was checked using 1% agarose gel electrophoresis. For PCR samples containing a single target band, liquid recovery was performed according to the instructions of the TIANGEN Universal DNA Purification Kit. The concentration and quality of the recovered product were measured using a nucleic acid concentration analyzer.

[0073] Enzyme digestion: The vector plasmid pET28a (Thermo Fisher Scientific) was double-digested using the following system: 10×QuickcutsmartBuffer 2 μL, NdeⅠ 1 μL, EcoRI 1 μL, pET28a vector 1 μg, and nuclease-free water to a final volume of 20 μL. The incubation program was set to 37℃ for 1.5 h. After the reaction, all products were subjected to 1% agarose gel electrophoresis and recovered. The digested products were then ligated into the pET28a vector using the following reaction system: 2×HiFi Seamless Cloning Mix 5 µL (brand: GenStar, China), target gene 3 µL, pET28a 1 µL, and nuclease-free water 1 µL.

[0074] The thoroughly mixed reaction solution was placed in a PCR instrument and reacted at 50 °C for 10 min. Subsequently, transformation was performed using DH5α ChemicallyCompetent Cell (Qingke Biotechnology, China) according to the manufacturer's instructions. After transformation, the bacterial culture was plated on LB agar (containing 50 μg / mL kanamycin) and cultured at 37 °C with shaking at 200 rpm. The specific method for bacterial detection PCR was the same as above. Positive PCR products were sent for testing, and the sequencing-qualified bacterial cultures were amplified, plasmid extracted, and the cultures were preserved. The recombinant plasmid pET28a-BdorOBP99c-6 was obtained. The recombinant plasmid sequence (partial vector sequence (underlined) + OBP99c-6 sequence (signal peptide removed)) is as follows: (SEQ ID NO.3)

[0075] 5'- TTAACTTTAAGAAGGAGATATACCATGGGCAGCAGCCATCATCATCATCATCACAGCAGCGGCCT GGTGCCGCGCGGCAGCCATATG GAAGATGAATGGATACCCCAAAAATGATACAGAACTTGAAGTTATCGCCCAGGAATGCCTCAGAGATTTTCCACTTAGCAAGGAACAACTGCAAAAATTTTCGAGCTTCGAATATCCCGATGAAGAGCCCATTCGCAAGTACATGCTTTGTACCGCTAAGAGAGTGGGTTTCTTCACCGAACAGGAGGGC TATCATGCCGATCGTGTTGCCAAACAATTAAAAATGGATTTGGATGAGGCTGAGATCGTTGCCATCGCTGAGGGTTGCGCGGACAAGAACGTCGAGGGTAGCAGCGCTGATGTATGGGCCTATCGTGTTCACAAGTGTGTGATGGCCAGCAAAATCGGTGAACATGGGAAAGCTTACTTCCAAAAATGA GAATTCGAGCTCCGTCGACAAGCTTGCGGCCGCACTCGAGCACCACCACCACCACC ACTGAGATCCG -3'.

[0076] Example 4. In vitro affinity test of OBP99c-6 protein with malathion.

[0077] I. Recombinant Protein Purification

[0078] The recombinant plasmid pET28a-BdorOBP99c-6 obtained in Example 3 was transformed into Escherichia coli BL21(DE3), and the transformation and bacterial detection methods were the same as in Example 4. Positive clones were added to 400 mL LB broth (containing 50 μg / mL kanamycin) and cultured at 37°C with shaking at 200 rpm until the bacterial OD600 reached 0.6–1.0. Isopropyl-β-D-thioga-lactopyranoside (IPTG) was added to a final concentration of 0.1 mM, and the culture was continued at 37°C with shaking at 200 rpm for approximately 16 h to induce protein expression. The induced bacterial culture was centrifuged at 4°C, 7500 × g for 15 min, the supernatant was discarded, and the bacterial pellet was resuspended in 25 mL of 1×PBS buffer. The resuspended E. coli cells were disrupted using an ultrasonic cell disruptor (5 s pulses, 3 s intervals, total time 15 min). The cells were centrifuged at 4°C, 12000 × g for 15 min, and the supernatant and pellet were collected for SDS-PAGE electrophoresis to detect protein expression.

[0079] After draining the buffer from the pre-packed HyPur T Ni-TED 6FF (His-Tag) gravity column (Sangon Biotech), 10 mL of ultrapure water and 1×PBS were added to the column successively. Then, 5 mL of supernatant containing recombinant protein was added to the Ni-NTA column each time until all supernatant was drained. The target protein was eluted with 1×PBS buffer containing different concentrations of imidazole (40 mM, 80 mM, 160 mM, 250 mM). The solution containing the target protein was collected for SDS-PAGE electrophoresis to determine the protein molecular weight. Specific procedures were followed according to the SDS-PAGE pre-cast gel instructions (Qingke Biotechnology), using electrophoresis conditions of 150 V for 30 min. The Ni-NTA column was then equilibrated with 1×PBS and 20% ethanol and stored at 4℃.

[0080] Transfer the SDS-PAGE gel to a polyvinylidene fluoride (PVDF) membrane immersed in transfer buffer (10 mL transfer buffer, 10 mL anhydrous ethanol, and 30 mL pure water mixed) for 2 min. Transfer the membrane at 25 V and 200 mA for 3 min. Wash the PVDF membrane with 1×TBST (a mixture of Tris-HCl and Tween-20) and block it with blocking buffer (50 mL 1×TBST + 2.5 g skim milk powder) at 37 °C for 1 h. Immerse the PVDF membrane with His-tag antibody and incubate overnight at 4 °C. Recover the primary antibody, wash the PVDF membrane with 1×TBST, and incubate the PVDF membrane in goat anti-mouse IgG serum (Beyotime) for 2 h. Recover the secondary antibody, wash the PVDF membrane with 1×TBST, and use a 1:1 mixture of solutions A and B from the High Sensitive ECL Chemiluminescence Substrate Kit. The mixture was added dropwise onto the PVDF membrane, and after a 10-second reaction, the image was captured using an imaging system. Figure 2 The results shown are obtained after WB ( Figure 2 The protein was labeled with a protein-specific antibody, and the colorimetric reaction showed a specific band only at 15-20 kDa, indicating successful acquisition of the OBP99c-6 recombinant protein. The purified protein was then analyzed using a BCA protein assay kit (Beyotime), following the instructions.

[0081] II. Analysis of the binding of recombinant proteins to insecticides

[0082] Recombinant proteins were labeled according to the instructions of the RED-tris-NTA protein labeling kit. Affinity analysis of the recombinant proteins with the insecticides was performed. 2 μL of a 5 μM fluorescent dye solution was dissolved in 98 μL of 1×PBST buffer to make the fluorescent dye concentration 100 nM; the recombinant protein was diluted to 200 nM with 1×PBST buffer to a volume of 100 μL; the fluorescent dye and recombinant protein solution were mixed in equal volumes and incubated at room temperature in the dark for 30 min. The insecticides were dissolved in acetone to prepare a 10 mM stock solution. The insecticides used were malathion and malathion.

[0083] The 10 mM insecticide was diluted in PBST at a 1:1 ratio to create 16 gradients (10 mM, 5 mM, 2.5 mM, 1.25 mM, 625 μM, 312.5 μM, 156.25 μM, 78.13 μM, 39.06 μM, 19.53 μM, 9765 nM, 4883 nM, 2441 nM, 1221 nM, 610 nM, 305 nM). The labeled recombinant protein solution was mixed with an equal volume (10 μL) of insecticide dilution solution and incubated for 5–10 min. Samples were siphoned using Monolith™ NT.115 Series Capillaries (Nano Temper Technology), and the binding of fluorescently labeled OBP99c-6 protein to the insecticide was detected using a Monolith NT.115 (Nano Temper Technology) micro-thermal surge instrument. The parameters were set as follows: Excitation power 60, MST power 40, constant temperature 25°C, and MST test time 20 s. The Kd value of OBP99c-6 to the insecticide was calculated using MO.Affinity Analysis (version 2.2.4) software.

[0084] After obtaining the recombinant OBP99c-6 protein through in vitro expression, its binding affinity to malathion and malathion was detected using micro-thermal surge technology. The results are as follows: Figure 3 As shown, OBP99c-6 formed well-fitting binding curves with malathion and malathion, with Kd values ​​of 37.22 μM and 202.91 μM, respectively. These results indicate that OBP99c-6 can bind to both malathion and malathion, and its affinity for malathion is higher than that for malathion, providing experimental evidence at the protein-ligand binding level for the association between OBP99c-6 and malathion resistance in the oriental fruit fly.

[0085] Example 5: Screening of key upstream regulatory regions of BdorOBP99c-6, verification of Nub-mediated transcriptional activation and RNA interference function.

[0086] I. Cloning of the upstream regulatory region of BdorOBP99c-6 and sequence difference analysis among MR and MS populations

[0087] To analyze the differences in the upstream regulatory region of BdorOBP99c-6 between the malathion-resistant population MR and the malathion-sensitive population MS, genomic DNA from MR and MS orientalis flies was used as a template to perform PCR amplification, cloning, and sequencing analysis on a region approximately 2000 bp upstream of the ATG start codon in BdorOBP99c-6. The A site of the ATG start codon in BdorOBP99c-6 was used as 0 bp.

[0088] The primer sequences used to amplify the upstream candidate regulatory region of BdorOBP99c-6 are as follows:

[0089] OBP99c6-UP-F: 5′-AAGTCACTCATCCTCCCCGA-3′;

[0090] OBP99c6-UP-R: 5′-TGATGTGATGTACGTACCAATGC-3′.

[0091] The upstream regulatory region of BdorOBP99c-6 in the MR and MS populations was amplified using the primers described above. The PCR reaction mixture consisted of 12.5 μL of 2× Hieff Canace® AdvanceFast PCR Master Mix (With Dye) (brand: YEASEN, China), 1 μL each of upstream and downstream primers, 2 μL of genomic DNA template, and nuclease-free water to a final volume of 25 μL. Reaction conditions: 98℃ pre-denaturation for 30 s; 98℃ denaturation for 10 s, 60℃ annealing for 5 s, 72℃ extension for 30 s, 35 cycles; final extension at 72℃ for 5 min, and storage at 12℃.

[0092] After agarose gel electrophoresis, the PCR products were recovered, the target band was ligated into a cloning vector, and positive clones were selected for sequencing after transformation into competent cells. The upstream region sequence of BdorOBP99c-6 obtained from MR and MS populations was compared and analyzed.

[0093] The results showed differences in the amplification band patterns of the upstream candidate regulatory region of BdorOBP99c-6 between the MR and MS populations. Sequence alignment further revealed deletions and / or base variations in this region. These results suggest that the structural differences in the upstream candidate regulatory region of BdorOBP99c-6 may be related to its higher expression in the malathion-resistant MR population. Figure 4 As shown.

[0094] II. Two rounds of dual-luciferase screening of the upstream regulatory region of BdorOBP99c-6

[0095] To screen key functional fragments in the upstream candidate regulatory region of BdorOBP99c-6, two rounds of segmented amplification were performed on the upstream region of the start codon of BdorOBP99c-6 using genomic DNA from MR and MS populations as templates, and luciferase reporter vectors were constructed for each segment.

[0096] In the first round of screening, candidate regulatory fragments were amplified from -2122 bp to -1753 bp, -1773 bp to -1343 bp, -1353 bp to -963 bp, -993 bp to -443 bp, and -473 bp to 0 bp upstream of BdorOBP99c-6. These fragments were then double-digested with KpnI and HindIII and ligated between the corresponding restriction sites of the pGL3.0-Basic luciferase reporter vector to obtain MR-derived and MS-derived reporter vectors. Sequencing confirmed the correct insertion sequence and orientation of each recombinant plasmid.

[0097] Reporter vectors of different origins and lengths were co-transfected into Sf9 cells with the Renilla luciferase internal control plasmid pRL-CK, with the pGL3.0-Basic empty vector serving as a negative control. Dual-luciferase reporter assays were performed using Sf9 cells. When the Sf9 cell density reached 2 × 10^6 cells / mL, cells were seeded at a density of 0.4 × 10^6 cells / well in 24-well cell culture plates, with 500 μL of Sf-900 II cell culture medium added to each well. The plates were then incubated statically at 27 ℃ for 24 h.

[0098] Add 1 μg pGL3.0-Basic reporter plasmid and 0.02 μg pRL-CK internal control plasmid to 300 μL Sf-900 II cell culture medium, mix gently, then add 4 μL FuGENE® HD transfection reagent, mix gently by pipetting, and incubate at room temperature for 15 min. Remove the cell culture medium from the 24-well plate after 24 h of culture, add the corresponding transfection mixture to each well, and incubate at 27 °C for 4 h. After incubation, replace with 500 μL fresh Sf-900 II cell culture medium and incubate at 27 °C for 48 h.

[0099] 48 h after transfection, remove the cell culture medium, add 200 μL of dual-luciferase reporter gene cell lysis buffer to each well, and incubate on ice for 10 min to allow the cells to fully lyse. Collect the cell lysis buffer into a 1.5 mL centrifuge tube, centrifuge at 12000 g for 2 min, and use the supernatant for luciferase activity assay.

[0100] Prepare the assay solution according to the Dual-Glo® Luciferase Assay System kit instructions. In an opaque 96-well white plate, add 100 μL of Luciferase Reagent to each well, followed by 20 μL of cell lysis supernatant. Mix gently and then use a multi-plate reader to measure the firefly luciferase signal. Subsequently, add 100 μL of StopReagent to the same well (Stop Substrate and Stop Buffer were prepared at a 1:100 ratio), mix gently, and then measure the Renida luciferase signal. Relative luciferase activity was calculated using the following formula:

[0101] Relative luciferase activity = firefly luciferase signal value / kidney luciferase signal value.

[0102] The results showed that in the first round of screening, the -473 bp to 0 bp fragment from MR-derived proteins exhibited significantly higher relative luciferase activity, significantly higher than the corresponding fragment from MS-derived proteins, and also significantly higher than other upstream candidate fragments and the pGL3.0-Basic empty vector control. Other more distal fragments did not show transcriptional activation activity comparable to the -473 bp to 0 bp fragment from MR-derived proteins. These results indicate that the -473 bp to 0 bp region upstream of the BdorOBP99c-6 start codon is a proximal candidate regulatory region associated with higher transcriptional activity in the MR population. Figure 5 As shown in A in the diagram.

[0103] After the first round of screening identified the -473 bp to 0 bp region, a second round of fine segmentation was performed on this proximal region to construct reporter vectors for -473 bp to -308 bp, -323 bp to -139 bp, and -168 bp to 0 bp, respectively. These fragments were also derived from MR and MS populations, and were ligated into the pGL3.0-Basic vector after double digestion with KpnI and HindIII.

[0104] The results showed that the -473 bp to -308 bp fragment exhibited transcriptional activation activity in both MR and MS-derived sequences. The MR-derived -473 bp to -308 bp fragment showed significantly higher relative luciferase activity than the corresponding MS-derived fragment, and was also significantly higher than the -323 bp to -139 bp and -168 bp to 0 bp fragments. The -323 bp to -139 bp and -168 bp to 0 bp fragments did not show significant enhancing activity in either MR or MS-derived sequences. These results indicate that the -473 bp to -308 bp region upstream of the BdorOBP99c-6 start codon is a key cis-regulatory region associated with higher transcriptional activity in the MR population. Figure 5As shown in B in the diagram.

[0105] III. Transcriptional activation of the key cis-regulatory fragment upstream of BdorOBP99c-6 from -473 bp to -308 bp by transcription factor Nub

[0106] The key cis-regulatory fragment from -473 bp to -308 bp upstream of BdorOBP99c-6 was analyzed using a transcription factor binding site prediction method. Combined with the expression of candidate transcription factors in MR and MS populations, the candidate transcription factor Nub was screened.

[0107] Using the -473 bp to -308 bp upstream fragment of BdorOBP99c-6 from the MR population as a template, a luciferase reporter vector P(-473 / -308) was constructed. Simultaneously, the candidate Nub response sequence in the MR-type key fragment was replaced with the corresponding sequence from the MS population to construct the modified reporter vector Cri-P(-473 / -308). Both P(-473 / -308) and Cri-P(-473 / -308) fragments were double-digested with KpnI and HindIII and ligated between the corresponding restriction sites of the pGL3.0-Basic luciferase reporter vector.

[0108] The Nub coding sequence was amplified using *Bactrocera dorsalis* cDNA as a template, with KpnI and XbaI restriction endonuclease sites introduced at both ends of the amplification primers. The pIB / V5-His eukaryotic expression vector was linearized by double digestion with KpnI and XbaI, and the linearized vector was recovered. The Nub fragment was ligated into the linearized pIB / V5-His vector via homologous recombination to obtain the Nub eukaryotic expression vector pIB-Nub. The recombinant expression vector was verified by colony PCR, double digestion, and sequencing, confirming the correct insertion of the Nub coding sequence. The empty pIB / V5-His vector was used as a control.

[0109] Dual-luciferase reporter assay was performed using Sf9 cells. When the Sf9 cell density reached 2×10^6 cells / mL, 0.4×10^6 cells were seeded into 24-well cell culture plates, and 500 μL of Sf-900 II cell culture medium was added to each well. The plates were then incubated at 27 ℃ for 24 h to allow the cells to adhere.

[0110] Add 1 μg pGL3.0-Basic reporter plasmid, 1 μg pIB-Nub expression plasmid or an equal volume of pIB / V5-His empty vector, and 0.02 μg pRL-CK internal control plasmid to 300 μL Sf-900 II cell culture medium. Gently mix, then add 4 μL FuGENE® HD transfection reagent, gently pipette to mix, and incubate at room temperature for 15 min. Specific settings are as follows:

[0111] P(-473 / -308)+pIB-Nub group: transfected with P(-473 / -308) reporter vector, pIB-Nub expression vector and pRL-CK internal reference plasmid;

[0112] Cri-P(-473 / -308)+pIB-Nub group: transfected with Cri-P(-473 / -308) reporter vector, pIB-Nub expression vector and pRL-CK internal reference plasmid;

[0113] P(-473 / -308)+pIB / V5-His empty vector group: transfected with P(-473 / -308) reporter vector, pIB / V5-His empty vector and pRL-CK internal reference plasmid.

[0114] Remove the cell culture medium from the 24-well plate after 24 h of culture, add the corresponding transfection mixture to each well, and incubate at 27 ℃ for 4 h. After incubation, replace with 500 μL of fresh Sf-900 II cell culture medium and incubate at 27 ℃ for 48 h.

[0115] 48 h after transfection, remove the cell culture medium, add 200 μL of dual-luciferase reporter gene cell lysis buffer to each well, and incubate on ice for 10 min to allow the cells to fully lyse. Collect the cell lysis buffer into a 1.5 mL centrifuge tube, centrifuge at 12000 g for 2 min, and use the supernatant for luciferase activity assay.

[0116] Prepare the assay solution according to the Dual-Glo® Luciferase Assay System kit instructions. In an opaque 96-well white plate, add 100 μL of Luciferase Reagent to each well, followed by 20 μL of cell lysis supernatant. Mix gently and then use a multi-plate reader to measure the firefly luciferase signal. Subsequently, add 100 μL of StopReagent to the same well (Stop Substrate and Stop Buffer were prepared at a 1:100 ratio), mix gently, and then measure the Renida luciferase signal. Relative luciferase activity was calculated using the following formula:

[0117] Relative luciferase activity = firefly luciferase signal value / kidney luciferase signal value.

[0118] The results showed that the relative luciferase activity of the P(-473 / -308)+pIB / V5-His co-transfection group was significantly increased compared with the P(-473 / -308)+pIB / V5-His empty vector group. The relative luciferase activity of the Cri-P(-473 / -308)+pIB-Nub group was significantly decreased compared with the P(-473 / -308)+pIB / V5-His empty vector group, but there was no significant difference. These results indicate that Nub can activate the transcriptional activity of the key cis-regulatory fragment upstream of BdorOBP99c-6 from -473 bp to -308 bp; candidate regulatory sequences with differences between the MR and MS populations in this fragment may be involved in the Nub-mediated transcriptional activation process, as shown in the results below. Figure 5 As shown in C.

[0119] IV. Effects of RNA interference silencing of Nub on BdorOBP99c-6 expression and malathion sensitivity in Bacteroides citrus.

[0120] To further verify the role of Nub in the regulation of BdorOBP99c-6 expression and the sensitivity of Bacteroides citrinum to malathion, RNA interference technology was used to silence the expression of Nub in Bacteroides citrinum.

[0121] Primers for dsNub synthesis were designed based on the Nub gene coding sequence, with dsEGFP used as a negative control. The T7 promoter sequence TAATACGACTCACTATAGGG was added to the 5′ end of each primer. The primer sequences used are as follows:

[0122] dsGFP-F: 5′-TAATACGACTCACTATAGGGCAGTTCTTGTTGAATTAGATG-3′;

[0123] dsGFP-R: 5′-TAATACGACTCACTATAGGGTTTGGTTTGTCTCCCATGATG-3′;

[0124] dsNub-x2-OBP99c6-F: 5′-TAATACGACTCACTATAGGGTCTACACCTACAACGACTGC-3′;

[0125] dsNub-x2-OBP99c6-R: 5′-TAATACGACTCACTATAGGGCAGTTGCTGGGTTGCTTGAG-3′.

[0126] Using PCR products containing the Nub target fragment as templates, the dsNub in vitro transcription template was amplified using the aforementioned dsNub primers; using PCR products containing the EGFP sequence as templates, the dsEGFP in vitro transcription template was amplified using the aforementioned dsGFP primers. The PCR products were purified by agarose gel electrophoresis after detection.

[0127] dsRNA in vitro transcription was performed using the TranscriptAid T7 Transcription Kit (Thermo Scientific, USA). The transcription reaction system consisted of: 4.0 μL of 5× TranscriptAid Reaction Buffer, 2.0 μL each of ATP, GTP, CTP, and UTP, 2.0 μL of TranscriptAid Enzyme, 1.0 μg of dsDNA template, and RNase-Free Water to a final volume of 20 μL. The reaction system was incubated overnight at 37 °C.

[0128] After transcription, 2 μL of DNase I was added to the reaction system and incubated at 37 °C for 15 min to remove residual DNA template; then 2 μL of EDTA was added and the reaction was terminated at 65 °C for 10 min. The obtained dsRNA was purified by phenol / chloroform extraction and ethanol precipitation. Specifically, the reaction solution was transferred to a 1.5 mL centrifuge tube, 115 μL of DEPC-treated water and 15 μL of 3 mol / L sodium acetate solution were added, and the mixture was stirred well. 150 μL of a 1:1 water-saturated phenol and chloroform mixture was added, followed by 300 μL of chloroform. The mixture was shaken and centrifuged at 4 °C and 12000 g for 10 min. The supernatant was transferred to a new centrifuge tube, 350 μL of anhydrous ethanol was added, and the mixture was stirred well. The tube was then incubated at −20 °C for at least 2 h. Subsequently, the tube was centrifuged at 4 °C and 12000 g for 10 min, the supernatant was discarded, and the white precipitate at the bottom of the tube was retained. The precipitate was washed with 500 μL of pre-cooled 70% ethanol and centrifuged at 4 °C and 7500 g for 5 min. After discarding the supernatant, the precipitate was dried at room temperature for 10–15 min and then dissolved in 25–35 μL of RNase-free water to dissolve the dsRNA.

[0129] The concentration and purity of dsRNA were determined using a micro-volume nucleic acid concentration analyzer, and the integrity and band size of dsRNA were detected by agarose gel electrophoresis. Qualified dsNub and dsEGFP were aliquoted and stored at -80 °C for later use.

[0130] Healthy adult oriental fruit flies of the malathion-resistant MR population were selected and randomly divided into a dsNub treatment group and a dsEGFP control group. dsNub or dsEGFP was injected into the insects via microinjection, with 2 μg of dsRNA injected into each insect.

[0131] Twelve hours after RNA interference treatment, insects from the dsNub and dsEGFP treatment groups were collected, and total RNA was extracted and reverse transcribed to obtain cDNA. RNA extraction, reverse transcription, and RT-qPCR detection were performed according to the methods described in Example 2. RPS3 and α-tubulin were used as internal reference genes. The method calculates the relative expression levels of Nub and BdorOBP99c-6.

[0132] The results showed that compared with the dsEGFP treatment group, the expression level of Nub in the dsNub treatment group was significantly decreased, with a silencing efficiency of 56.05%, indicating that dsNub can effectively inhibit Nub expression in Bacteroides orientalis. Further analysis revealed that the expression level of BdorOBP99c-6 was also significantly decreased after dsNub treatment, indicating that the downregulation of Nub expression is associated with a decrease in the transcriptional level of BdorOBP99c-6. Figure 6 As shown in A in the diagram.

[0133] To detect changes in the sensitivity of oriental fruit flies to malathion after Nub silencing, the sensitivity to malathion was determined by a spot method 12 h after RNA interference treatment.

[0134] The number of dead test insects in each group was recorded at 0 h, 12 h, 24 h, 36 h, and 48 h after malathion treatment, and the cumulative mortality rate was calculated. The cumulative mortality rate was calculated using the following formula:

[0135] Cumulative mortality rate (%) = Cumulative number of dead test insects / Total number of treated test insects × 100%.

[0136] The results showed that, compared with the dsEGFP treatment group, the cumulative mortality rate of the dsNub treatment group was significantly increased at 24 h, 36 h, and 48 h after malathion exposure, with significant differences at 24 h and 36 h, and highly significant differences at 48 h. These results indicate that RNA interference silencing of Nub can increase the sensitivity of *Bactrocera dorsalis* to malathion. Figure 6 As shown in B in the diagram.

[0137] The results, including the activation of key cis-regulatory fragments (-473 bp to -308 bp) upstream of BdorOBP99c-6 by Nub co-transfection, the significant downregulation of BdorOBP99c-6 expression after dsNub treatment, and the increased cumulative mortality rate after malathion treatment, suggest that Nub may be involved in the transcriptional regulation of BdorOBP99c-6 and further affect the sensitivity of *Bacteroides citrinum* to malathion. Therefore, Nub can serve as a potential RNA interference target for enhancing the sensitivity of *Bacteroides citrinum* to malathion.

[0138] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the claimed invention. The scope of protection claimed by the appended claims and their equivalents is defined.

Claims

1. A molecular marker for resistance to malathion in the oriental fruit fly, characterized in that, The molecular marker is the BdorOBP99c-6 gene and / or the BdorOBP99c-6 protein; The nucleotide sequence of the BdorOBP99c-6 gene is shown in SEQ ID NO:1; The amino acid sequence of the BdorOBP99c-6 protein is shown in SEQ ID NO:

2.

2. The use of the molecular marker as described in claim 1 in detecting malathion content, or in preparing reagents or devices for detecting malathion content.

3. The application according to claim 2, characterized in that, The application method includes the following steps: Using multiple known concentrations of malathion standards as detection targets, the affinity between OBP99c-6 protein and the standards was determined, and the correspondence between malathion concentration and affinity detection signal values ​​was constructed. The affinity of the test sample containing malathion to the OBP99c-6 protein was determined, and the concentration of malathion in the test sample was obtained according to the correspondence between the malathion concentration and the affinity detection signal value.

4. The application according to claim 3, characterized in that, The process of constructing the correspondence between malathion concentration and affinity detection signal value includes: fitting an affinity curve with malathion concentration as the abscissa and affinity detection fluorescence value as the ordinate. The determination of the affinity between the test sample containing malathion and the OBP99c-6 protein includes: using micro-thermal surge technology to detect the affinity between the OBP99c-6 protein and the malathion sample. The concentration range of malathion in the sample to be tested is 100 nM to 1 M.

5. The application of the molecular markers as described in claim 1 in the classification of resistance to malathion or early monitoring of resistance levels in oriental fruit fly populations.

6. A primer pair for detecting the molecular marker of claim 1, characterized in that, The primer sequences of the primer pair are as follows: OBP99c-6 qPCR F: 5'- CGATGAAGAGCCCATTCGCA-3'; OBP99c-6 qPCR R: 5'-TGTTCACCGATTTTGCTGGC-3'.

7. A reagent kit, characterized in that, Includes the primer pair as described in claim 6.

8. A method for detecting the resistance of the oriental fruit fly to malathion, characterized in that, Includes the following steps: Total RNA was extracted from the fruit fly citrus samples to be tested and reverse transcribed into cDNA. Using the cDNA as a template, real-time quantitative PCR amplification was performed using the primer pair described in claim 6 to detect the expression level of the BdorOBP99c-6 gene; A malathion-sensitive population of the oriental fruit fly was set up as a control, and the gene expression levels of the test sample and the control population were compared. If the expression level of the BdorOBP99c-6 gene in the test sample is significantly higher than that in the sensitive population, the test fruit fly is determined to be a malathion-resistant population. If the expression level is not significantly higher than that of the sensitive population, the tested fruit fly (Bactrocera dorsalis) is determined to be a non-resistant population. The statistical standard for "significantly higher" is: the relative expression level of the BdorOBP99c-6 gene in the test sample is more than 1.5 times that of the sensitive population, and the statistical analysis shows p < 0.

05.

9. The method according to claim 8, characterized in that: The qPCR amplification reaction system was as follows: 5.0 μL of NovoStart SYBRqPCR SuperMix Plµs, 0.3 μL each of 10 μM upstream and downstream primers, 0.5 μL of cDNA template, and nuclease-free water to a final volume of 10 μL. The qPCR amplification conditions were as follows: 95℃ pre-denaturation for 2 min; 95℃ denaturation for 30 s, 60℃ annealing and extension for 30 s, for 40 cycles; and 60℃ for 30 s followed by 95℃ for 15 s to form the melting curve.

10. A method for regulating the sensitivity of the oriental fruit fly to malathion, characterized in that, By inhibiting the expression of the transcription factor Nub in the oriental fruit fly, and subsequently downregulating the expression of the BdorOBP99c-6 gene, the sensitivity of the oriental fruit fly to malathion is increased.

Images