GluCl2 subunit mutation site-based detection reagent for drug resistance of spider mites to abamectin and PCR-RFLP (polymerase chain reaction-restriction fragment length polymorphism) detection method

Abstract

The invention belongs to the technical field of molecular detection, and particularly relates to a GluCl2 subunit mutation site-based detection reagent for drug resistance of spider mites to abamectin and a PCR-RFLP (polymerase chain reaction-restriction fragment length polymorphism) detection method. The GluCl gene sequence in a tetranychus urticae population collected from a field is analyzed to find that a new mutation site T76S exists on a GluCl2 subunit, the mutation exists in a field population with multiple abamectin resistance, and the mutation frequency of the site is increased after abamectin treatment. In the process of identifying the mutation site, the mutation site is identified; according to the present invention, the specific recognition site (T 'CAG) of the DdeI restriction enzyme in the sequence is identified, such that the amplification primer is designed, the PCR-RFLP method for T76S mutation site detection is established, and the method has characteristics of rapid enzyme digestion reaction, simple operation, pollution resistance, short time, accurate experiment result, and easy determination.

Description

Technical Field

[0001] This invention belongs to the field of molecular detection technology, specifically relating to a detection reagent and PCR-RFLP method for detecting the resistance of spider mites to avermectin based on the GluCl2 subunit mutation site. Background Technology

[0002] Two-spotted spider mite ( Tetranychus urticae Two-spotted spider mites (Tetranychidae) belong to the class Arachnida, order Acariformes, and family Tetranychidae. They are a major agricultural pest with a global distribution. As an omnivorous mite, the two-spotted spider mite has a very wide host range, damaging important economic crops and agricultural products including vegetables, fruit trees, flowers, and grains. Not only does it feed on a wide range of host plants, but it can also survive and reproduce under diverse environmental conditions, demonstrating strong adaptability and rapid population growth. Early-stage damage from the two-spotted spider mite is difficult to detect, and once an outbreak occurs, the population grows rapidly, making control extremely challenging.

[0003] Currently, chemical control remains the primary method for managing the two-spotted spider mite population. However, research has revealed that the two-spotted spider mite has developed varying degrees of resistance to traditional acaricides and some novel acaricides, posing challenges to field control. Target resistance is one of the important mechanisms by which pests and mites develop resistance to pesticides, and numerous studies on the mechanisms of pest target resistance have been conducted both domestically and internationally in recent years. Abamectin is a macrocyclic lactone disaccharide insecticide that is highly effective against pests and mites and has a broad insecticidal spectrum, and has been widely used in agriculture and horticulture.

[0004] Existing research has clearly established that glutamate-gated chloride channels (GluCl) are the main target of avermectin. Two-spotted spider mites possess five different GluCl sites, and mutations at the G314D site on GluCl1 and the G326E site on GluCl3 are closely related to avermectin resistance in these mites. However, in some highly avermectin-resistant populations of two-spotted spider mites in China, the G326E mutation at GluCl3 may not be present, indicating that the target mutation resistance mechanism is not the same in different populations. Therefore, existing methods for detecting avermectin resistance in spider mites may still have issues with false negatives or missed detections. Thus, developing new avermectin targets and establishing new rapid detection methods for avermectin resistance in spider mites remains essential and urgent for spider mite control. Summary of the Invention

[0005] The purpose of this invention is to provide a detection reagent and PCR-RFLP detection method for spider mite resistance to avermectin based on the GluCl2 subunit mutation site. This invention establishes a PCR-RFLP detection method for spider mite resistance to avermectin based on the GluCl2 subunit mutation site, which can realize rapid detection and real-time monitoring of spider mite resistance to avermectin.

[0006] This invention provides a reagent for detecting the 76th amino acid of the GluCl2 subunit of spider mites in the detection and / or monitoring of spider mite resistance to avermectin.

[0007] Preferably, when the 76th amino acid of the GluCl2 subunit is mutated from threonine (T) to serine (S), the spider mite's sensitivity to avermectin decreases.

[0008] The present invention also provides a primer pair for detecting the resistance of spider mites to avermectin, the primer pair comprising an upstream primer and a downstream primer, the nucleotide sequences of which are shown in SEQ ID NO:1 and SEQ ID NO:2, respectively.

[0009] The present invention also provides a kit for detecting the resistance of spider mites to avermectin, the kit comprising the DdeI restriction endonuclease and the primer pair described in the above technical solution.

[0010] Preferably, the kit also includes PCR amplification reagents.

[0011] Preferably, the kit further includes DdeI restriction endonuclease reaction buffer.

[0012] The present invention also provides the application of the primer pairs or kits described in the above technical solutions in detecting and / or monitoring spider mites' resistance to avermectin.

[0013] This invention also provides a PCR-RFLP method for detecting and / or monitoring spider mite resistance to avermectin, comprising the following steps: Using the genomic DNA of the spider mite to be tested as a template, a PCR amplification reaction was performed with primer pairs to obtain PCR amplification products; the primer pairs are the primer pairs described in the above technical solution or the primer pairs in the kit described in the above technical solution; The PCR amplification product was digested with DdeI restriction endonuclease to obtain the digested product. The enzyme digestion products were subjected to electrophoretic detection to obtain electrophoretic detection bands; When the electrophoretic detection band consists of three bands with sizes of 480 bp, 340 bp, and 140 bp, the tested spider mite is a resistant heterozygous genotype and is resistant to abamectin. When the electrophoretic detection band consists of two bands with sizes of 140 bp and 340 bp, the tested spider mite is a homozygous genotype of resistance and is resistant to abamectin. When the electrophoretic detection band is a single band with a size of 480 bp, the spider mite to be tested is a sensitive homozygous genotype and is sensitive to abamectin.

[0014] Preferably, the PCR amplification reaction system comprises: 15 μL 2× premixed Mix, 1 μL upstream primer, 1 μL downstream primer, 2 μL genomic DNA template, and 11 μL deionized water; The PCR amplification reaction program was as follows: 98℃ pre-denaturation for 30 s; 98℃ denaturation for 10 s, 60℃ annealing for 15 s, 72℃ extension for 20 s, for a total of 32 cycles; 72℃ extension for 5 min. The reaction system for the enzyme digestion reaction is 50 μL, including 5 μL 10×NEBuffer, 1 μL DdeI restriction endonuclease, 1 μg PCR amplification product, and the remainder RNase-free water. The procedure for the enzyme digestion reaction is as follows: incubation at 37°C for 3 hours.

[0015] Preferably, the spider mite includes the two-spotted spider mite.

[0016] Beneficial effects: This invention provides a reagent for detecting the 76th amino acid of the GluCl2 subunit of spider mites in the detection and / or monitoring of avermectin resistance in spider mites. Based on this, the invention also provides primer pairs, a kit for detecting avermectin resistance in spider mites, and a PCR-RFLP method for detecting and / or monitoring avermectin resistance in spider mites. This invention is applied to two-spotted spider mite populations collected from the field. GluCl Gene sequence analysis revealed a novel mutation site, T76S, on the GluCl2 subunit. This mutation was present in multiple avermectin-resistant field populations, and its frequency increased after avermectin treatment. Subsequently, transgenic fruit fly lines containing this mutation showed decreased sensitivity to avermectin, indicating a close correlation between this mutation site and avermectin tolerance / resistance. This invention identifies avermectin resistance in test spider mite populations by detecting the presence of the T76S mutation. The presence of the T76S mutation confirms that the population exhibits some degree of avermectin resistance.

[0017] In identifying the aforementioned mutation sites, this invention uses Snap Gene software. GluCl2Enzyme restriction site scanning of the gene sequence identified a specific recognition site for the DdeI restriction endonuclease (T'CAG). Based on this, amplification primers were designed, and a PCR-RFLP method for detecting the T76S mutation site was established. This method requires only one enzyme digestion step after amplification of the specific fragment, followed by a single agarose gel electrophoresis to observe the bands and determine the presence of mutations. In contrast, traditional PCR amplification followed by sequencing requires 1-2 days for results, and the sequencing results need to be compared with the original sequence using SnapGene software, which is cumbersome. Therefore, compared to traditional sequencing methods, the method described in this invention offers rapid enzyme digestion, simple operation, low contamination risk, shorter processing time, accurate results, and easier interpretation. The PCR-RFLP method described in this invention can provide rapid guidance for the scientific selection of pesticides for spider mite populations in the field, providing a scientific basis for reducing the use of chemical pesticides. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0019] Figure 1 This is a graph showing the comparison of GluCl2 site mutations between resistant and susceptible populations in Example 1; Figure 2 This is a graph showing the mutation frequency results of different two-spotted spider mite populations under abamectin-induced treatment in Example 3; Figure 3 This is an electrophoresis result of the T76S site mutation detection of the GluCl2 subunit in the two-spotted spider mite in Example 5. Detailed Implementation

[0020] This invention provides a reagent for detecting the 76th amino acid of the GluCl2 subunit of spider mites in the detection and / or monitoring of spider mite resistance to avermectin.

[0021] As one implementation, when the 76th amino acid of the GluCl2 subunit is mutated from threonine (T) to serine (S), the spider mite's sensitivity to avermectin decreases. As one implementation, the amino acid encoding the GluCl2 subunit... GluCl2 The gene's accession number in NCBI is XM_015929942.2. In one embodiment, the spider mite is *Tetranychus two-spotted*. In another embodiment, the spider mite is one or more of an adult, larva, or nymph.

[0022] This invention targets two-spotted spider mite populations collected from the field. GluClGene sequence analysis revealed a novel mutation site, T76S, on the GluCl2 subunit. This mutation was present in multiple avermectin-resistant field populations, and its mutation frequency increased after avermectin treatment. Subsequently, transgenic fruit fly strains containing this mutation showed decreased sensitivity to avermectin, indicating that this mutation site is closely related to avermectin tolerance / resistance.

[0023] The present invention also provides a primer pair for detecting the resistance of spider mites to avermectin, the primer pair comprising an upstream primer and a downstream primer, the nucleotide sequences of which are shown in SEQ ID NO:1 and SEQ ID NO:2, respectively.

[0024] This invention also provides a kit for detecting resistance of spider mites to avermectin, the kit comprising a DdeI restriction endonuclease and the primer pair described in the above-described technical solution. As one embodiment, the kit further comprises PCR amplification reagents; this invention does not specifically limit the type and source of the PCR amplification reagents, and either self-prepared reagents or commercially available ready-made mixes are acceptable. As one embodiment, the kit further comprises a DdeI restriction endonuclease reaction buffer; this invention does not specifically limit the type and source of the DdeI restriction endonuclease reaction buffer, and conventional DdeI restriction endonuclease reaction buffers available in the art or obtained through conventional commercial channels are acceptable.

[0025] This invention also provides the application of the primer pairs or kits described in the above-described technical solutions in detecting and / or monitoring avermectin resistance in spider mites. In one embodiment, the spider mite is *Tetranychus two-spotted*. In another embodiment, the spider mite is one or more of adult mites, larvae, or nymphs.

[0026] This invention also provides a PCR-RFLP method for detecting and / or monitoring spider mite resistance to avermectin, comprising the following steps: Using the genomic DNA of the spider mite to be tested as a template, a PCR amplification reaction was performed with primer pairs to obtain PCR amplification products; the primer pairs are the primer pairs described in the above technical solution or the primer pairs in the kit described in the above technical solution; The PCR amplification product was digested with DdeI restriction endonuclease to obtain the digested product. The enzyme digestion products were subjected to electrophoretic detection to obtain electrophoretic detection bands; When the electrophoretic detection band consists of three bands with sizes of 480 bp, 340 bp, and 140 bp, the tested spider mite is a resistant heterozygous genotype and is resistant to abamectin. When the electrophoretic detection band consists of two bands with sizes of 140 bp and 340 bp, the tested spider mite is a homozygous genotype of resistance and is resistant to abamectin. When the electrophoretic detection band is a single band with a size of 480 bp, the spider mite to be tested is a sensitive homozygous genotype and is sensitive to abamectin.

[0027] This invention uses the genomic DNA of the spider mite as a template and performs PCR amplification using primer pairs to obtain PCR amplification products. The primer pairs are those described in the above-mentioned technical solutions or those in the kits described in the above-mentioned technical solutions. This invention does not specifically limit the extraction method of the spider mite genomic DNA; conventional extraction methods in the art can be used, such as using a commercially available genomic DNA extraction kit. As one embodiment, the reaction system for the PCR amplification reaction of this invention is: 15 μL 2× premixed Mix, 1 μL upstream primer, 1 μL downstream primer, 2 μL genomic DNA template, and 11 μL deionized water. As one embodiment, the PCR amplification reaction program of this invention is: 98℃ pre-denaturation for 30 s; 98℃ denaturation for 10 s, 60℃ annealing for 15 s, 72℃ extension for 20 s, for a total of 32 cycles; 72℃ extension for 5 min.

[0028] After obtaining the PCR amplification product, as one embodiment, the present invention purifies the PCR amplification product to obtain a purified PCR amplification product. As one embodiment, the purification step of the present invention includes: detecting the PCR amplification product on agarose gel electrophoresis, and recovering the obtained target band by gel cutting to obtain a target band strip; purifying the target band strip with a purification reagent to obtain the purified PCR amplification product. As one embodiment, the agarose gel electrophoresis is 1.2% agarose gel electrophoresis. As one embodiment, the purification reagent is a purification kit; in a specific embodiment of the present invention, the purification kit is the Monarch PCR & DNA Purification Kit (NEB, MA, England).

[0029] After obtaining the purified PCR amplification product, the present invention performs an enzyme digestion reaction on the purified PCR amplification product to obtain the enzyme digestion product. As one embodiment, the reaction system of the enzyme digestion reaction of the present invention, in 50 μL volumes, consists of 5 μL of 10×NE Buffer, 1 μL of DdeI restriction endonuclease, 1 μg of PCR amplification product, and the remainder being RNase-free water. As one embodiment, the enzyme digestion reaction procedure of the present invention is: incubation at 37°C for 3 h.

[0030] After obtaining the enzyme digestion product, the present invention performs electrophoretic detection on the enzyme digestion product to obtain electrophoretic detection bands; when the electrophoretic detection bands are three bands with sizes of 480 bp, 340 bp and 140 bp respectively, the tested spider mite is a resistant heterozygous genotype and is resistant to avermectin; when the electrophoretic detection bands are two bands with sizes of 140 bp and 340 bp respectively, the tested spider mite is a resistant homozygous genotype and is resistant to avermectin; when the electrophoretic detection bands are one band with a size of 480 bp, the tested spider mite is a sensitive homozygous genotype and is sensitive to avermectin.

[0031] In one embodiment, the electrophoretic detection is agarose gel electrophoresis; in another embodiment, the agarose gel electrophoresis detection is 2.0% agarose gel electrophoresis.

[0032] In one embodiment, the spider mite is a two-spotted spider mite. In another embodiment, the spider mite is one or more of an adult mite, a larva, or a nymph.

[0033] The PCR-RFLP method for detecting and / or monitoring spider mite resistance to avermectin described in this invention has the following advantages compared to the traditional resistance evaluation method—the indoor bioassay test: (1) Traditional bioassay requires the preparation of avermectin reagent, soaking of leaf discs, selection of test spider mites, cultivation, and mortality determination, which takes 2 days, while the method described in this invention only takes 6-8 hours to obtain results, thus saving time; (2) Traditional bioassay requires 400-500 spider mites to detect resistance to each agent, while this detection technology does not require a large number of test insects, only 30-40 need to be tested; (3) Traditional bioassay requires testing of adult female mites, while the PCR-RFLP method described in this invention is not limited to adult mites, but is also fully feasible for larvae and nymphs. Based on this, the PCR-RFLP method described in this invention can save a lot of time, does not require a large number of test insects, and reduces instability or experimental errors caused by factors such as accidental injury to spider mites during reagent preparation and mite selection, and can also quickly obtain stable results. In practical applications, the PCR-RFLP method described in this invention can rapidly detect whether different populations are resistant to abamectin, and quickly determine whether the test population can be controlled with abamectin or agents with the same mechanism of action, providing a scientific basis for the scientific management of spider mite populations in the field.

[0034] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0035] Example 1 To clarify whether there are relatively stable site mutations in different GluCl subunits between avermectin-resistant and susceptible populations of Tetranychus spp., the genome and cDNA sequences of susceptible and field populations of Tetranychus spp. (IPP-SS) were compared. This susceptible population of Tetranychus spp. has been previously disclosed in a published article (Zhang Y, Tian T, Zhang K, Xie W, Wu QJ, Zhang YJ, Wang SL. Lack of fitness cost and inheritance of resistance to abamectin based on the establishment of a near-isogenic strain of Tetranychusurticae. Journal of Integrative Agriculture, 2023, 22(6): 1809-1819.).

[0036] The results showed that, compared with the susceptible population, multiple field-resistant populations had an amino acid mutation at the T76S site in GluCl2, as shown in the following results. Figure 1 As shown, in Figure 1 In the diagram, the mutation sites are shown in the red boxes; the sequences marked in blue are the upstream and downstream primers; GluCl2-SS and GluCl2-RR represent the susceptible and resistant populations of the two-spotted spider mite, respectively.

[0037] Example 2 The steps for amplification of the GluCl2 subunit and detection of the T76S mutation in the two-spotted spider mite are as follows: 1. Experimental population The two-spotted spider mite populations used in this embodiment included the susceptible population (IPP-SS), the resistant breeding population NIL-Aba, and the field population. The susceptible population was reared long-term on bean leaves in the insect rearing laboratory of the Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences. During the rearing period, it was not exposed to any toxic substances and showed sensitivity to abamectin at the LC50 level. 50The value was 0.05 mg / L. The resistant breeding population NIL-Aba was obtained through multiple generations of hybridization between an abamectin-sensitive population and a field-resistant population, and it exhibited high resistance to abamectin. This two-spotted spider mite sensitive population and the resistant breeding population NIL-Aba have been disclosed in the published article (Zhang Y, Tian T, Zhang K, Xie W, Wu QJ, Zhang YJ, Wang SL. Lack of fitness cost and inheritance of resistance to abamectin based on the establishment of a near-isogenic strain of Tetranychus urticae. Journal of Integrative Agriculture, 2023, 22(6): 1809-1819.).

[0038] Four field populations of the two-spotted spider mite were collected from the field in 2022-2023. Bioassays using the leaf-dipping method were employed to determine their resistance to abamectin. Compared to susceptible populations of the two-spotted spider mite kept indoors for extended periods, these populations exhibited more than 1000 times higher resistance to abamectin (Table 1). The indoor rearing conditions for the aforementioned spider mite populations were: 26 ± 1℃, humidity 60 ± 5%, and photoperiod L:D = 16 h : 8 h.

[0039] Table 1. Dates, locations, and host information for field populations of the two-spotted spider mite.

[0040] 2. Test Methods 2.1 DNA extraction from individual spider mites Genomic DNA was extracted from single adult mites of susceptible and resistant populations of Tetranychus tataricus according to the Kapa genomic DNA kit instructions. Thirty-two female adult mites were randomly selected from each population and placed in 0.2 mL PCR eight-tube strips. After thorough grinding, 30 μL of extraction buffer was added, and the mixture was centrifuged. The samples were then placed in a Bio-Rad PCR instrument (S1000) for incubation at 75℃ for 10 min followed by 95℃ for 5 min. After the reaction, the DNA samples were stored at -20℃ for later use.

[0041] 2.2 PCR amplification and sequencing According to GluCl2 containing mutation sites ( GluCl2The gene accession number in NCBI is XM_015929942.2. A specific primer pair was designed based on the subunit sequence: upstream primer: 5'-ATCCCACATTGATTTACCTAACTCC-3' (SEQ ID NO:1), downstream primer: 5'-ATATTTCATCCTTTTAATCACTTTTGC-3' (SEQ ID NO:2). PCR amplification was performed using the following reaction system and conditions: 15 μL of 2× premixed Mix (composed of Super Pfx DNA Polymerase, Mg...) was added to a 30 μL reaction system. 2+ The reaction mixture consisted of dNTPs and reaction buffer, 1 μL of upstream primer, 1 μL of downstream primer, and 2 μL of DNA template. The final volume was brought to 30 μL with deionized water. After mixing, the mixture was aliquoted into 0.2 mL eight-tube strips and placed in a PCR instrument. The reaction program was: 98℃ pre-denaturation for 30 s; 98℃ denaturation for 10 s, 60℃ annealing for 15 s, and 72℃ extension for 20 s, for a total of 32 cycles; finally, a final extension at 72℃ for 5 min. After the reaction, the amplified products were detected by 1.2% agarose gel electrophoresis. Samples meeting the expected fragment size were sent to Beijing Qingke Biotechnology Co., Ltd. for paired-end sequencing. The sequencing results were analyzed, and the mutation frequency was calculated.

[0042] 3. Experimental Results Sequence alignment and genotyping were performed using Snap Gene software to calculate gene locus mutation frequencies. The results showed that the T76S site mutation was present in all four two-spotted spider mite populations resistant to avermectin collected from the field. The mutation frequency varied among different populations, ranging from a minimum of 16.22% to a maximum of 62.85% (Table 2).

[0043] Table 2. Resistance and mutation frequency of different two-spotted spider mite populations to avermectin.

[0044] Example 3 Avermectin-induced changes in the T76S mutation among different populations of the two-spotted spider mite. 1. Experimental Methods The leaf-dipping method was used for induction experiments, as follows: After detecting the T76S mutation frequency of the two-spotted spider mite population using PCR amplification and sequencing, the LC-weighted average of abamectin in the bioassay was used to analyze the levels of abamectin in each population. 50 (As shown in Table 1) and LC 70The leaves were immersed in the solution for 10 seconds, followed by the introduction of 20-25 adult female two-spotted spider mites from the same population. This process was repeated three times. After 24 hours at room temperature, surviving two-spotted spider mites were selected under a microscope for single-mite DNA extraction, fragment cloning, and sequencing. Mutation frequencies were calculated, and the changes in mutation frequencies before and after abamectin induction were compared among different populations. The LC ratios for each population were also analyzed. 70 The concentrations are as follows: NIL-Aba: 400 mg / L, SY-HN: 810 mg / L, MY-BJ: 500 mg / L, CP-BJ: 1720 mg / L and HD-BJ: 3640 mg / L.

[0045] 2. Experimental Results The tested populations exhibited varying degrees of T76S site mutation frequencies, LC 50 and LC 70 Treatment with avermectin at various concentrations resulted in varying degrees of increase in mutation frequencies across all population groups, with the HD-BJ population showing the highest mutation frequency in the LC range. 50 and LC 70 The mutation frequencies after treatment increased from 16.22% to 51.92% and 52.72%, respectively, showing the largest increase. Figure 2 This suggests that the T76S mutation may be associated with resistance or tolerance to avermectin.

[0046] Example 4 The following steps were taken to validate the transgenic fruit fly containing the T76S mutation in the GluCl2 subunit of the two-spotted spider mite: 1. Experimental Methods Amplification of the GluCl2 subunit of Tetranychus bicolor and mutant GluCl2 T76S After obtaining the correct sequence through cloning and sequencing, it is compared with... Not I and Xba The pJFRC28-10XUAS-IVS-GFP-p10 vector, after double digestion with enzyme I, was ligated, cloned, and the plasmid was extracted. Subsequent correct introduction of mutations was carried out, and Qidong Fangjing Biotechnology Co., Ltd. was responsible for constructing two transgenic fruit fly strains.

[0047] The resistance of two transgenic Drosophila strains and a control strain to avermectin was determined using the culture medium immersion method. First, a high-concentration avermectin stock solution was prepared. Then, using a 0.1% Triton X-100 aqueous solution, a series of avermectin solutions were prepared through a two-fold serial dilution method. 200 μL of the solution was poured into rearing tubes containing Drosophila culture medium, submerging the surface of the medium and soaking for 4 h. The solution was then discarded, and the rearing tubes were placed in a fume hood to air dry. After anesthetizing adult Drosophila with carbon dioxide, 10–15 adult Drosophila were introduced into each tube. Four biological replicates were set up for each concentration treatment, and a blank control group without treatment was included. The treated Drosophila were placed in a Drosophila incubator at 27℃ and cultured for 48 h. The number of surviving and dead flies in each tube was recorded, and the mortality rate at each concentration was recorded.

[0048] 2. Experimental Results Compared to the control group (nos.Cas9), both Drosophila strains transformed with the GluCl2 subunit showed decreased sensitivity to avermectin. After avermectin treatment, the LC50 of the control group Drosophila strain was significantly lower. 50 The LC50 value was 38.09 mg / L for a Drosophila strain transformed with the non-mutated GluCl2 subunit T76. 50 The value was 71.98 mg / L, with an resistance fold of 1.89. The introduction of the T76S single mutation induced some resistance in Drosophila to abamectin. The LC50 of adult Drosophila from the T76S single point mutant strain was 71.98 mg / L. 50 The value was 105.20 mg / L, which was 2.76 times that of the control group and 1.46 times that of the T76 Drosophila strain, indicating decreased sensitivity. This clarified that the T76S mutation is related to decreased sensitivity of the population to avermectin (i.e., resistance to avermectin).

[0049] Table 3. Toxicity determination of abamectin against different strains of transgenic fruit fly adults.

[0050] Example 5 The steps for establishing the PCR-RFLP detection technique for the two-spotted spider mite are as follows: 1. Experimental Methods The amplified cells were processed using Snap Gene software. GluCl2 After scanning the restriction sites of the gene fragment sequence, the presence of T76S was found to create a new DdeI restriction endonuclease-specific recognition site (T'CAG) in the sequence, thus establishing a PCR-RFLP method for detecting the T76S mutation site.

[0051] First, genomic DNA was extracted from the susceptible population indoors and the resistant population in the field, followed by PCR amplification. The upstream primer used was 5'-ATCCCACATTGATTTACCTAACTCC-3' (SEQ ID NO:1), and the downstream primer was 5'-ATATTTCATCCTTTTAATCACTTTTGC-3' (SEQ ID NO:2). The PCR amplification reaction system and conditions were the same as described above: 15 μL of 2× premixed mix, 1 μL of upstream primer, 1 μL of downstream primer, and 2 μL of DNA template were added to a 30 μL system, and then deionized water was added to bring the volume to 30 μL. The reaction program was: 98℃ pre-denaturation for 30 s; 98℃ denaturation for 10 s, 60℃ annealing for 15 s, and 72℃ extension for 20 s, for a total of 32 cycles; finally, a final extension at 72℃ for 5 min was performed. After PCR amplification, the PCR amplification products were detected on a 1.2% agarose gel. The target band was excised from the gel and recovered. The purified PCR amplification products were then purified using a Monarch PCR & DNA Purification Kit (NEB, MA, England) according to the manufacturer's instructions. Following purification, the purified PCR amplification products were digested with the following enzymes: 5 μL 10×NEBuffer, 1 μL DdeI restriction endonuclease, and 1 μg of the purified product. The volume was then adjusted to 50 μL with RNase-free water and incubated at 37°C for 3 h. The digested products were then analyzed by 2.0% agarose gel electrophoresis, and the number of bands was used to determine the individual's genotype.

[0052] 2. Experimental Results After digestion of the PCR product with DdeI restriction endonuclease, the results of the digestion product display show three possible scenarios: three clear bands were detected, with sizes of 480 bp, 340 bp, and 140 bp, respectively (e.g., Figure 3 If lanes 5 and 6 are shown in the middle lane, it indicates that the tested individual has a heterozygous genotype of resistance; if two bands appear, at 140 bp and 340 bp respectively (as shown in the middle lane), it indicates that the tested individual has a heterozygous genotype of resistance. Figure 3 As shown in lanes 3 and 4 of the middle swimming pool), this indicates that the tested individual has a homozygous genotype of resistance, meaning that a DdeI restriction site is present within the individual; if only a single 480bp band is detected (e.g. Figure 3 As shown in lanes 1 and 2 of the middle swim grout line, the absence of the DdeI restriction site indicates that the individual is a sensitive homozygous genotype. In this embodiment, while performing restriction enzyme digestion reactions on the PCR products of sensitive and resistant populations of *Tetranychus two-spotted*, the PCR products were also subjected to paired-end sequencing by Beijing Qingke Biotechnology Co., Ltd. The sequencing results were consistent with the results of the restriction enzyme digestion reaction and were consistent with the resistant or sensitive phenotypes of the test populations. Based on this, the present invention successfully established a PCR-RFLP analysis system for detecting this mutation site in *Tetranychus two-spotted*.

[0053] From the above examples, it can be concluded that the present invention establishes a PCR-RFLP detection method for spider mite resistance to avermectin based on the GluCl2 subunit mutation site, which can realize rapid detection and real-time monitoring of spider mite resistance to avermectin.

[0054] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. Application of reagents for detecting the 76th amino acid of the GluCl2 subunit in spider mites in the detection and / or monitoring of avermectin resistance in spider mites.

2. The application according to claim 1, characterized in that, When the 76th amino acid of the GluCl2 subunit is mutated from threonine to serine, the spider mite's sensitivity to avermectin decreases.

3. A primer pair for detecting resistance of spider mites to avermectin, characterized in that, The primer pair includes an upstream primer and a downstream primer, the nucleotide sequences of which are shown in SEQ ID NO:1 and SEQ ID NO:2, respectively.

4. A kit for detecting resistance of spider mites to avermectin, characterized in that, The kit includes the DdeI restriction endonuclease and the primer pair as described in claim 3.

5. The reagent kit according to claim 4, characterized in that, The kit also includes PCR amplification reagents.

6. The kit according to claim 4 or 5, characterized in that, The kit also includes DdeI restriction endonuclease reaction buffer.

7. The use of the primer pair of claim 3 or the kit of any one of claims 4 to 6 in the detection and / or monitoring of spider mite resistance to avermectin.

8. A PCR-RFLP method for detecting and / or monitoring resistance of spider mites to avermectin, characterized in that, Includes the following steps: Using the genomic DNA of the spider mite to be tested as a template, a PCR amplification reaction was performed with primer pairs to obtain PCR amplification products; the primer pairs are the primer pairs described in claim 3 or the primer pairs in the kit described in any one of claims 4 to 6; The PCR amplification product was digested with DdeI restriction endonuclease to obtain the digested product. The enzyme digestion products were subjected to electrophoretic detection to obtain electrophoretic detection bands; When the electrophoretic detection band consists of three bands with sizes of 480 bp, 340 bp, and 140 bp, the tested spider mite is a resistant heterozygous genotype and is resistant to abamectin. When the electrophoretic detection band consists of two bands with sizes of 140 bp and 340 bp, the tested spider mite is a homozygous genotype of resistance and is resistant to abamectin. When the electrophoretic detection band is a single band with a size of 480 bp, the spider mite to be tested is a sensitive homozygous genotype and is sensitive to abamectin.

9. The method according to claim 8, characterized in that, The PCR amplification reaction system includes: 15 μL 2× premixed Mix, 1 μL upstream primer, 1 μL downstream primer, 2 μL genomic DNA template and 11 μL deionized water; The PCR amplification reaction program was as follows: 98℃ pre-denaturation for 30 s; 98℃ denaturation for 10 s, 60℃ annealing for 15 s, 72℃ extension for 20 s, for a total of 32 cycles; 72℃ extension for 5 min. The reaction system for the enzyme digestion reaction is 50 μL, including 5 μL 10×NEBuffer, 1 μL DdeI restriction endonuclease, 1 μg PCR amplification product, and the remainder RNase-free water. The procedure for the enzyme digestion reaction is as follows: incubation at 37°C for 3 hours.

10. The application according to claim 1 or 2, the application according to claim 7, or the method according to claim 8 or 9, characterized in that, The spider mite includes the two-spotted spider mite.

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