Detection reagent and PCR-RFLP detection method for resistance of spider mites to abamectin based on mutation sites of GluCl2 subunit

Abstract

The application belongs to the technical field of molecular detection, and particularly relates to a detection reagent for detecting the resistance of a spider mite to abamectin based on a mutation site of a GluCl2 subunit and a PCR-RFLP detection method. GluCl Gene sequence analysis finds that there is a new mutation site T76S on the GluCl2 subunit, the mutation exists in multiple abamectin-resistant field populations, and the mutation frequency of the site is increased after abamectin treatment. In the process of identifying the above mutation site, it is found that there is a specific recognition site (T'CAG) of DdeI restriction endonuclease in the sequence, and thus an amplification primer is designed, and a PCR-RFLP method for detecting the T76S mutation site is established. The method is fast in enzyme digestion reaction, simple in operation, not easy to be contaminated, short in time cost, accurate in experimental results, and easier in judgment method.

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 application aims to provide a detection reagent for abamectin resistance of a spider mite based on a mutation site of a GluCl2 subunit and a PCR-RFLP detection method.

[0006] The application provides an application of a reagent for detecting an amino acid at position 76 of a GluCl2 subunit of a spider mite in detection and / or monitoring of abamectin resistance of the spider mite.

[0007] Preferably, when the amino acid at position 76 of the GluCl2 subunit is mutated from threonine (T) to serine (S), the sensitivity of the spider mite to abamectin decreases.

[0008] The application also provides a primer pair for detecting abamectin resistance of a spider mite, wherein the primer pair comprises an upstream primer and a downstream primer, and the nucleotide sequences of the upstream primer and the downstream primer are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively.

[0009] The application also provides a kit for detecting abamectin resistance of a spider mite, wherein the kit comprises a DdeI restriction enzyme and the primer pair described in the above technical solution.

[0010] Preferably, the kit further comprises a PCR amplification reagent.

[0011] Preferably, the kit further comprises a DdeI restriction enzyme reaction buffer.

[0012] The application also provides an application of the primer pair described in the above technical solution or the kit described in the above technical solution in detection and / or monitoring of abamectin resistance of a spider mite.

[0013] The application also provides a PCR-RFLP method for detecting and / or monitoring abamectin resistance of a spider mite, comprising the following steps:

[0014] Taking genomic DNA of the spider mite to be detected as a template, performing a PCR amplification reaction on a primer pair to obtain a PCR amplification product; the primer pair is the primer pair described in the above technical solution or the primer pair in the kit described in the above technical solution;

[0015] Performing an enzyme cutting reaction on the PCR amplification product by using a DdeI restriction enzyme to obtain an enzyme cutting product;

[0016] Performing electrophoresis detection on the enzyme cutting product to obtain an electrophoresis detection band;

[0017] When the electrophoretic detection bands are three bands, and the sizes are 480 bp, 340 bp and 140 bp respectively, the to-be-tested spider mite is a resistant hybrid genotype, and is resistant to abamectin;

[0018] When the electrophoretic detection bands are two bands, and the sizes are 140 bp and 340 bp respectively, the to-be-tested spider mite is a resistant homozygous genotype, and is resistant to abamectin;

[0019] When the electrophoretic detection bands are one band, and the size is 480 bp, the to-be-tested spider mite is a sensitive homozygous genotype, and is sensitive to abamectin.

[0020] Preferably, the reaction system of the PCR amplification reaction comprises: 15 μL 2× premixed Mix, 1 μL upstream primer, 1 μL downstream primer, 2 μL genomic DNA template and 11 μL deionized water;

[0021] The program of the PCR amplification reaction is: 98℃ pre-denaturation for 30 s; 98℃ denaturation for 10 s, 60℃ annealing for 15 s, 72℃ extension for 20 s, a total of 32 cycles; 72℃ extension for 5 min;

[0022] The reaction system of the enzyme digestion reaction is 50 μL, including 5 μL 10× NEBuffer, 1 μL Ddel restriction endonuclease, 1 μg PCR amplification product and the balance of RNase-free water;

[0023] The program of the enzyme digestion reaction is: 37℃ incubation for 3 h.

[0024] Preferably, the spider mite comprises Tetranychus urticae.

[0025] Beneficial effects:

[0026] The application provides application of a reagent for detecting the 76th amino acid of a spider mite GluCl2 subunit in detection and / or monitoring of abamectin resistance of the spider mite; on the basis, the application further provides a primer pair for detecting abamectin resistance of the spider mite, a kit and provides a PCR-RFLP method for detecting and / or monitoring abamectin resistance of the spider mite. 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 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.

[0027] In identifying the aforementioned mutation sites, this invention uses Snap Gene software. GluCl2 Enzyme 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

[0028] 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.

[0029] Figure 1 This is a graph showing the comparison of GluCl2 site mutations between resistant and susceptible populations in Example 1;

[0030] 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;

[0031] 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

[0032] The application provides application of a reagent for detecting amino acid at position 76 of a Tetranychus glomeratus GluCl2 subunit in detection and / or monitoring of resistance of the Tetranychus glomeratus to abamectin.

[0033] As an implementation form, when the amino acid at position 76 of the GluCl2 subunit is mutated from threonine (T) to serine (S), the sensitivity of the Tetranychus glomeratus to abamectin decreases. GluCl2 The gene encoding the GluCl2 subunit has a GenBank accession number of XM_015929942.2. As an implementation form, the Tetranychus glomeratus is Tetranychus urticae.

[0034] The application finds a new mutation site T76S on the GluCl2 subunit through gene sequence analysis of a Tetranychus glomeratus population collected from a field. GluCl The mutation exists in multiple abamectin-resistant field populations, and the mutation frequency at the site increases after abamectin treatment. A transgenic Drosophila strain containing the mutation is subsequently constructed, and the sensitivity of the transgenic Drosophila strain to abamectin decreases, indicating that the mutation site is closely related to the resistance / sensitivity to abamectin.

[0035] The application further provides a primer pair for detecting the resistance of the Tetranychus glomeratus to abamectin, wherein the primer pair comprises an upstream primer and a downstream primer, and the nucleotide sequences of the upstream primer and the downstream primer are respectively shown in SEQ ID NO: 1 and SEQ ID NO: 2.

[0036] The application further provides a kit for detecting the resistance of the Tetranychus glomeratus to abamectin, wherein the kit comprises DdeI restriction endonuclease and the primer pair in the above technical solution. As an implementation form, the kit further comprises PCR amplification reagents, and the type and source of the PCR amplification reagents are not particularly limited in the application, and the products Mix can be prepared by self or purchased through a conventional marketing channel. As an implementation form, the kit further comprises a DdeI restriction endonuclease reaction buffer, and the type and source of the DdeI restriction endonuclease reaction buffer are not particularly limited in the application, and a conventional DdeI restriction endonuclease reaction buffer can be used and obtained through a conventional marketing channel.

[0037] The application further provides application of the primer pair in the above technical solution or the kit in the above technical solution in detection and / or monitoring of the resistance of the Tetranychus glomeratus to abamectin. As an implementation form, the Tetranychus glomeratus is Tetranychus urticae. As an implementation form, the Tetranychus glomeratus is one or more of adult mites, juvenile mites or nymphs.

[0038] The application further provides a PCR-RFLP method for detecting and / or monitoring the resistance of a spider mite to abamectin, comprising the following steps:

[0039] The genomic DNA of the spider mite to be detected is used as a template, and a primer pair is used for a PCR amplification reaction to obtain a PCR amplification product; the primer pair is the primer pair in the above technical solution or the primer pair in the kit in the above technical solution.

[0040] The PCR amplification product is subjected to an enzyme cutting reaction by using a DdeI restriction endonuclease to obtain a cutting product.

[0041] The cutting product is subjected to electrophoresis detection to obtain an electrophoresis detection band.

[0042] When the electrophoresis detection band is three bands with sizes of 480 bp, 340 bp and 140 bp, the spider mite to be detected is a resistant heterozygous genotype and has resistance to abamectin.

[0043] When the electrophoresis detection band is two bands with sizes of 140 bp and 340 bp, the spider mite to be detected is a resistant homozygous genotype and has resistance to abamectin.

[0044] When the electrophoresis detection band is one band with a size of 480 bp, the spider mite to be detected is a sensitive homozygous genotype and is sensitive to abamectin.

[0045] The genomic DNA of the spider mite to be detected is used as a template, and a primer pair is used for a PCR amplification reaction to obtain a PCR amplification product; the primer pair is the primer pair in the above technical solution or the primer pair in the kit in the above technical solution. The application does not have special limitations on the extraction method of the genomic DNA of the spider mite, and a conventional extraction method in the art can be used, such as using a commercially available genomic extraction kit for extraction. As an implementation manner, the reaction system of the PCR amplification reaction in the application is 15 μL of 2x premixed Mix, 1 μL of an upstream primer, 1 μL of a downstream primer, 2 μL of a genomic DNA template and 11 μL of deionized water. As an implementation manner, the program of the PCR amplification reaction in the application is pre-denaturation at 98℃ for 30 s; denaturation at 98℃ for 10 s, annealing at 60℃ for 15 s, extension at 72℃ for 20 s, a total of 32 cycles; and extension at 72℃ for 5 min.

[0046] After obtaining the PCR amplification product, as an embodiment, the PCR amplification product is purified to obtain a purified PCR amplification product. As an embodiment, the purification step comprises: detecting the PCR amplification product on an agarose gel electrophoresis, and cutting and recovering the obtained target band to obtain a target band gel strip; and purifying the target band gel strip with a purification reagent to obtain the purified PCR amplification product. As an embodiment, the agarose gel electrophoresis is 1.2% agarose gel electrophoresis. As an embodiment, the purification reagent is a purification kit; in a specific embodiment of the present application, the purification kit is a Monarch PCR & DNA Purification Kit (NEB, MA, England).

[0047] After obtaining the purified PCR amplification product, the purified PCR amplification product is subjected to a restriction enzyme reaction to obtain a restriction enzyme reaction product. As an embodiment, the reaction system of the restriction enzyme reaction is 50 μL, including 5 μL 10×NEBuffer, 1 μL Ddel restriction enzyme, 1 μg PCR amplification product, and the balance of RNase-free water. As an embodiment, the procedure of the restriction enzyme reaction is: incubation at 37°C for 3 h.

[0048] After obtaining the restriction enzyme reaction product, the restriction enzyme reaction product is subjected to electrophoretic detection to obtain an electrophoretic detection band; when the electrophoretic detection band is three bands with sizes of 480 bp, 340 bp and 140 bp, the to-be-tested spider mite is a resistant heterozygous genotype and has resistance to abamectin; when the electrophoretic detection band is two bands with sizes of 140 bp and 340 bp, the to-be-tested spider mite is a resistant homozygous genotype and has resistance to abamectin; when the electrophoretic detection band is one band with a size of 480 bp, the to-be-tested spider mite is a sensitive homozygous genotype and is sensitive to abamectin.

[0049] As an embodiment, the electrophoretic detection is agarose gel electrophoresis detection; as another embodiment, the agarose gel electrophoresis detection is 2.0% agarose gel electrophoresis detection.

[0050] As an embodiment, the spider mite is Tetranychus urticae. As an embodiment, the spider mite is one or more of adult spider mites, juvenile spider mites or nymph spider mites.

[0051] Compared with the traditional resistance / antibiotic evaluation method, i.e. the indoor bioassay test method, the PCR-RFLP method for detecting and / or monitoring the resistance of the spider mite to abamectin has the following advantages: (1) The traditional bioassay needs to prepare abamectin, dip leaf discs, select test spider mites, culture and determine mortality, etc., which takes 2 days, while the method only needs 6-8 hours to obtain the results, and the time consumption is short; (2) The traditional bioassay needs 400-500 spider mites for detecting the resistance of each pesticide, while the detection technology only needs 30-40 spider mites; (3) The traditional bioassay needs to test female adult spider mites, while the PCR-RFLP method is not limited to adult spider mites, and can also be completely applicable to juvenile spider mites and nymphs. Based on this, the PCR-RFLP method can save a lot of time, does not need a large number of test insects, and reduces the instability or test error caused by the factors such as the damage to spider mites in the process of pesticide preparation and mite selection, and can quickly obtain stable results. In practical application, the PCR-RFLP method can quickly detect whether different populations have resistance / antibiotic to abamectin, quickly judge whether the test population can be prevented and controlled by abamectin or the same mechanism of action of the pesticide, and provide a scientific basis for the scientific management of the field population of spider mites.

[0052] In order to further illustrate the present application, the technical solutions provided by the present application will be described in detail below in conjunction with the drawings and examples, but they should not be understood as limiting the scope of protection of the present application.

[0053] Example 1

[0054] In order to determine whether there are relatively stable site mutations of different subunits of GluCl between the abamectin-resistant and sensitive populations of T. urticae, the genomic sequences and cDNA sequences of the sensitive population (IPP-SS) and the field population of T. urticae were compared. The sensitive population of T. urticae was 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.).

[0055] It was found that the amino acid mutation of T76S site on GluCl2 existed in multiple field resistant populations compared with the sensitive population, and the results are shown in Figure 1 , wherein the mutation site is shown by the red frame position; the sequence marked in blue is the upstream primer and the downstream primer; GluCl2-SS and GluCl2-RR represent the sensitive population and the resistant population of T. urticae, respectively. Figure 1

[0056] Example 2

[0057] The steps of amplification of T. urticae GluCl2 subunit and detection of T76S mutation are as follows:

[0058] 1. Test populations

[0059] The T. urticae populations used in this example are the sensitive population (IPP-SS), the resistant breeding population NIL-Aba and the field population. The T. urticae sensitive population was long-term reared on bean leaves in the insect rearing room of the Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, and was not exposed to any toxic substances during the rearing period. It showed a sensitive level (LC 50 value of 0.05 mg / L) to abamectin. The resistant breeding population NIL-Aba was obtained by multiple generations of crossing between the abamectin-sensitive population and the field-resistant population, and showed high resistance to abamectin. The T. urticae sensitive population and the resistant breeding population NIL-Aba have been published 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.).

[0060] The four field populations of T. urticae were collected in the field in 2022-2023, and the bioassay using leaf dipping method was used to determine their resistance to abamectin. Compared with the T. urticae sensitive population reared in the laboratory for a long time, these populations showed more than 1000-fold high resistance to abamectin (Table 1). The indoor rearing conditions of the above spider mite populations were as follows: 26 ± 1℃, humidity of 60 ± 5 %, and light cycle of L:D = 16 h:8 h.

[0061] ​Table 1. Dates, locations, and host information for field populations of the two-spotted spider mite.

[0062]

[0063] 2. Test Methods

[0064] 2.1 DNA extraction from individual spider mites

[0065] 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.

[0066] 2.2 PCR amplification and sequencing

[0067] According to GluCl2 containing mutation sites ( GluCl2 The 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.

[0068] 3. Experimental Results

[0069] The sequence alignment and genotype analysis were performed using Snap Gene software, and the mutation frequency of gene locus was calculated. The results showed that the T76S locus mutation existed in the four T. urticae populations collected from the field and resistant to abamectin, and the mutation frequencies were different in different populations, with the lowest of 16.22% and the highest of 62.85% (Table 2).

[0070] Table 2 Resistance of different T. urticae populations to abamectin and mutation frequency

[0071]

[0072] Example 3

[0073] Abamectin-induced changes of T76S mutation in different T. urticae populations

[0074] 1 Test method

[0075] The leaf dipping method was used for pesticide induction test, and the method was as follows: after the T76S mutation frequency of T. urticae population was detected by PCR amplification and sequencing, the LC 50 and LC 70 concentrations (shown in Table 1) of abamectin were used to dip the leaves of each population for 10 s, and then 20-25 female adult T. urticae of the same population to be tested were introduced, and the test was repeated 3 times; after being placed at room temperature for 24 h, the surviving T. urticae were picked under a microscope, and the single DNA extraction, fragment cloning and sequencing were performed, and the mutation frequency was calculated, and the mutation frequency changes of each population before and after abamectin induction were compared. The LC 70 concentrations of each population were 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.

[0076] 2 Test results

[0077] The tested populations had different degrees of T76S locus mutation frequency, and the mutation frequency of each population increased to different degrees after the treatment of abamectin at the LC 50 and LC 70 concentrations, in which the mutation frequencies of HD-BJ population increased from 16.22% to 51.92% and 52.72% after the treatment of LC 50 and LC 70 , with the largest increase ( Figure 2 ). It was indicated that the T76S locus mutation might be related to the resistance or tolerance of T. urticae to abamectin.

[0078] Example 4

[0079] The transgenic fruit fly verification of T76S mutation of B. bicolorana GluCl2 subunit is as follows:

[0080] 1 Test method

[0081] Amplification of B. bicolorana GluCl2 subunit and mutant GluCl2 T76S Sequence, after correct sequence is obtained by cloning sequencing, is connected with pJFRC28-10XUAS-IVS-GFP-p10 vector after double enzyme digestion of I and I, and the plasmid is extracted by cloning recovery. The correct introduction of mutation is completed by Qidong Fangjing Biotechnology Co., Ltd. Not I and Xba I double enzyme digestion, and the plasmid is extracted by cloning recovery. The correct introduction of mutation is completed by Qidong Fangjing Biotechnology Co., Ltd.

[0082] The medium immersion method is used to carry out the determination of the resistance of the two transgenic fruit fly strains and the control strain to abamectin. First, a high concentration of abamectin mother liquor is prepared, and then a series of concentrations of abamectin solution is prepared by using 0.1% Triton X-100 aqueous solution through double gradient dilution method. 200 μL of drug solution is poured into the feeding tube containing fruit fly medium, which is immersed in the medium surface and soaked for 4 h, then the drug solution is poured out and the feeding tube is dried under the fume hood. After the fruit fly adults are anesthetized with carbon dioxide, 10-15 fruit fly adults are introduced into each tube, and 4 biological replicates are set for each concentration treatment, and a blank control group without drug treatment is set. The treated fruit flies are placed in a 27℃ fruit fly incubator for culture, and the survival and death numbers of fruit flies in each tube are detected and recorded after 48 h, and the mortality under each concentration is recorded.

[0083] 2 Test results

[0084] Compared with the control group of fruit flies (nos.Cas9), the sensitivity of the two fruit fly strains with transgenic GluCl2 subunit to abamectin decreases. The LC 50 value of the control group of fruit flies is 38.09 mg / L, the LC 50 value of the fruit fly strain with transgenic GluCl2 subunit T76 is 71.98 mg / L, and the resistance ratio is 1.89 times. The introduction of T76S single mutation makes the fruit fly have a certain resistance to abamectin, and the LC 50 value of the fruit fly strain with T76S single point mutation is 105.20 mg / L, which is 2.76 times of the control group of fruit flies and 1.46 times of the T76 fruit fly strain, showing a decrease in sensitivity. It is confirmed that the T76S mutation is related to the decrease in sensitivity of the population to abamectin (i.e. resistance to abamectin).

[0085] Table 3 Toxicity determination of abamectin on different transgenic fruit fly strains

[0086]

[0087] Example 5

[0088] The steps of establishing the PCR-RFLP detection technique of T. urticae are as follows:

[0089] 1 Test method

[0090] After scanning the restriction sites of the amplified GluCl2 gene fragment sequence by Snap Gene software, it was found that the presence of T76S caused a new specific recognition site (T’CAG) of DdeI restriction enzyme in the sequence, thereby establishing a PCR-RFLP method for detecting T76S mutation site.

[0091] First, the genomic DNA of T. urticae sensitive population in the laboratory and field resistant population was extracted, and then PCR amplification was performed, using the upstream primer 5'-ATCCCACATTGATTTACCTAACTCC-3' (SEQ ID NO: 1) and the downstream primer 5'-ATATTTCATCCTTTTAATCACTTTTGC-3' (SEQ ID NO: 2). The PCR amplification reaction system and reaction conditions were the same as described above: in a 30 μL system, 15 μL of 2×pre-mixed Mix, 1 μL of upstream primer and 1 μL of downstream primer, 2 μL of DNA template, and deionized water were added to make up to 30 μL. The reaction program was 98℃ pre-denaturation for 30 s; 98℃ denaturation for 10 s, 60℃ annealing for 15 s, 72℃ extension for 20 s, a total of 32 cycles; finally, 72℃ extension for 5 min. After the PCR amplification was completed, the PCR amplification product was detected on a 1.2% agarose gel, the target band was cut and recovered, and then purified using the Monarch PCR&DNA Purification Kit (NEB, MA, England) according to the steps in the instructions to obtain the purified PCR amplification product; after purification of the amplification product, the purified PCR amplification product was subjected to enzyme digestion reaction: 5 μL of 10×NEBuffer, 1 μL of DdeI restriction enzyme, 1 μg of purified product, and deionized water without RNase were added to make up to 50 μL, and then mixed and incubated at 37℃ for 3 h. The enzyme digestion product was detected by 2.0% agarose gel electrophoresis, and the genotype of the individual was determined by the number of bands.

[0092] 2 Test results

[0093] After enzyme digestion of the PCR product with DdeI restriction enzyme, the display results of the enzyme digestion product presented three possible cases: three clear bands were detected, and their sizes were 480 bp, 340 bp and 140 bp (such as Figure 3If only one band of 340 bp appears (as shown in lanes 5 and 6), it indicates that the tested individual is of a resistant heterozygous genotype; if two bands of 140 bp and 340 bp appear (as shown in lanes 3 and 4), it indicates that the tested individual is of a resistant homozygous genotype, i.e., the Ddel enzyme cutting site exists in the individual; if only one band of 480 bp is detected (as shown in lanes 1 and 2), it indicates that the Ddel enzyme cutting site does not exist, indicating that the individual is of a sensitive homozygous genotype. Figure 3 If only one band of 340 bp appears (as shown in lanes 5 and 6), it indicates that the tested individual is of a resistant heterozygous genotype; if two bands of 140 bp and 340 bp appear (as shown in lanes 3 and 4), it indicates that the tested individual is of a resistant homozygous genotype, i.e., the Ddel enzyme cutting site exists in the individual; if only one band of 480 bp is detected (as shown in lanes 1 and 2), it indicates that the Ddel enzyme cutting site does not exist, indicating that the individual is of a sensitive homozygous genotype. Figure 3 If only one band of 340 bp appears (as shown in lanes 5 and 6), it indicates that the tested individual is of a resistant heterozygous genotype; if two bands of 140 bp and 340 bp appear (as shown in lanes 3 and 4), it indicates that the tested individual is of a resistant homozygous genotype, i.e., the Ddel enzyme cutting site exists in the individual; if only one band of 480 bp is detected (as shown in lanes 1 and 2), it indicates that the Ddel enzyme cutting site does not exist, indicating that the individual is of a sensitive homozygous genotype.

[0094] From the above examples, it can be concluded that the PCR-RFLP detection method for the resistance of the spider mite to the avermectin is established based on the GluCl2 subunit mutation site, and the rapid detection and real-time monitoring of the resistance of the spider mite to the avermectin can be realized.

[0095] Although the above examples have described the present application in detail, it is only a part of the embodiments of the present application, but not all the embodiments, and other embodiments can be obtained by people according to the present embodiments without creativity, which all belong to the protection scope of the present application.

Claims

1. A reagent for detecting the 76th amino acid of the GluCl2 subunit of Tetranychus urticae in detecting and / or monitoring the resistance of Tetranychus urticae to abamectin; when the 76th amino acid of the GluCl2 subunit is mutated from threonine to serine, the sensitivity of Tetranychus urticae to abamectin decreases. The reagent comprises a primer pair for detecting the resistance of Tetranychus urticae to abamectin and Ddel restriction endonuclease, wherein the primer pair comprises an upstream primer and a downstream primer, and the nucleotide sequences of the upstream primer and the downstream primer are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively. The gene sequence encoding the GluCl2 subunit has the accession number XM_015929942.2 in NCBI.

2. Use according to claim 1, characterized in that, The reagent further comprises a PCR amplification Mix.

3. Use according to claim 1 or 2, characterized in that, The reagent further comprises a Ddel restriction endonuclease reaction buffer.

4. A PCR-RFLP method for detecting and / or monitoring the resistance of Tetranychus urticae Koch to abamectin, characterized in that, The method comprises the following steps: PCR amplification reaction is performed on the genomic DNA of the Tetranychus urticae to be tested with a primer pair to obtain a PCR amplification product, wherein the primer pair comprises an upstream primer and a downstream primer, and the nucleotide sequences of the upstream primer and the downstream primer are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively; The PCR amplification product is subjected to enzyme digestion reaction with Ddel restriction endonuclease to obtain an enzyme digestion product; The enzyme digestion product is subjected to electrophoresis detection to obtain an electrophoresis detection band; When the electrophoresis detection band is three bands with sizes of 480 bp, 340 bp and 140 bp, respectively, the Tetranychus urticae to be tested is a resistant heterozygous genotype and has resistance to abamectin; When the electrophoresis detection band is two bands with sizes of 140 bp and 340 bp, respectively, the Tetranychus urticae to be tested is a resistant homozygous genotype and has resistance to abamectin; When the electrophoresis detection band is one band with a size of 480 bp, the Tetranychus urticae to be tested is a sensitive homozygous genotype and is sensitive to abamectin.

5. The method of claim 4, wherein, The reaction system of the PCR amplification reaction comprises 15 μL of 2×pre-mixed Mix, 1 μL of an upstream primer, 1 μL of a downstream primer, 2 μL of a genomic DNA template and 11 μL of deionized water.

6. The method of claim 4, wherein, The program of the PCR amplification reaction is as follows: pre-denaturation at 98℃ for 30 s; denaturation at 98℃ for 10 s, annealing at 60℃ for 15 s, extension at 72℃ for 20 s, a total of 32 cycles; and extension at 72℃ for 5 min.

7. The method of claim 4, wherein, The reaction system of the enzyme digestion reaction is 50 μL, comprising 5 μL of 10×NEBuffer, 1 μL of Ddel restriction endonuclease, 1 μg of a PCR amplification product and the balance of RNase-free water; and the program of the enzyme digestion reaction is as follows: incubation at 37℃ for 3 h.

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