Mutation Sites of Field Mite Resistance to Abamectin and Their Applications
By detecting single-base site mutations in the promoter region of the CYP392A11 gene, the problem of detection of resistance detection of field mites to avermectin is solved, and the rapid and accurate detection results are achieved, and the reliability of the detection is improved.
Patent Information
- Application Number
- CN202410961432.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2024-07-17
- Publication Date
- 2025-06-20
- Estimated Expiration
- 2044-07-17
AI Technical Summary
The prior art is difficult to quickly and accurately detect the resistance of field mites to avermectin. The traditional method has a long monitoring cycle, a large number of test insects and insufficient sensitivity.
By detecting single-base site mutations in the promoter region of the CYP392A11 gene, the resistance of field mites to avermectin was quickly identified using specific primer sets and PCR amplification techniques.
It realizes rapid and accurate detection of the resistance of field mites to avermectin, avoids the defects of traditional methods, improves the reliability of the test results, and provides technical support for the rational use of insecticides.
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Figure CN118755844B_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the technical field of field resistance detection, and particularly relates to a gene mutation site of field mite pests resistant to abamectin and its application. Background Art
[0002] Tetranychus urticae is a worldwide agricultural mite pest that is extremely prone to developing pesticide resistance. Due to its wide host range, tiny individuals, aggregation on the back of leaves, and the initial damage symptoms being inconspicuous and easily overlooked, it often causes the phenomenon of "small pests causing great disasters", bringing huge economic losses to agricultural production. Currently, chemical insecticides are still the most effective prevention and control means. However, due to the long-term unscientific use of insecticides, combined with the unique physiological characteristics of Tetranychus urticae such as overlapping generations and parthenogenesis, the problem of pesticide resistance in Tetranychus urticae is more prominent than that of other agricultural pests. According to the resistance report cases of the Arthropod Pesticide Resistance Database (APRD) (https: / / www.pesticideresistance.org / ), Tetranychus urticae has developed varying degrees of resistance to at least 96 insecticidal active ingredients such as organophosphates, pyrethroids, antibiotics, and some new acaricides.
[0003] Avermectin is a class of 16-membered macrolide compounds produced by the fermentation of Streptomyces avermitilis. Due to its broad-spectrum, high-efficiency, and low-toxicity characteristics, it is widely used in the control of agricultural pest mites in the field. However, the long-term and unscientific use of avermectin has led to the rapid development of resistance in various pests and mites (Zhang Y, Xu DD, Zhang YJ, Wu QJ, Xie W, Guo ZJ, Wang SL. Frequencies and mechanisms of pesticide resistance in Tetranychus urticae field populations in China. Insect Science, 2022, 29(3): 827-839). The research on the resistance mechanism of Tetranychus urticae to avermectin mainly involves target resistance and metabolic resistance. Currently, there are already literature reports that even the combined action of the two mutations G314D on the resistance target gene glutamate chloride channel gene GluCl1 and G326E on GluCl3 has a phenotypic intensity and relative contribution to avermectin resistance that are both less than 20%. Even in some individual high-level avermectin-resistant populations, the mutation frequency of the G326E mutation site is zero (Xu DD, Zhang Y, Zhang YJ, Wu QJ, Guo ZJ, Xie W, Zhou XM, Wang SL. Transcriptome profiling and functional analysis suggest that the constitutive overexpression of four cytochrome P450s confers resistance to abamectin in Tetranychus urticae from China. Pest Management Science, 2021, 77: 1204-1213; De Rouck Sander, Emre, Dermauw Wannes, Van Leeuwen Thomas. A review of the molecular mechanisms of acaricide resistance in mites and ticks. Insect Biochemistry and Molecular Biology, 2023, 159: 103984.). These results indicate that the mutations at the above two sites play a minor role in the field-resistant populations, and there are other resistance mechanisms in the field-resistant populations, but the research on this resistance mechanism is extremely rare. Summary of the Invention
[0004] The present invention provides a gene mutation site of field mite resistance to abamectin and its application. By detecting the single-base site mutation in the promoter region of the CYP392A11 gene, the resistance of field mites to abamectin can be quickly and accurately detected.
[0005] The present invention provides the application of a reagent for detecting the base sequence of the 802bp site in the promoter region of the CYP392A11 gene of field mites in the preparation of a tool for detecting the resistance of field mites to abamectin.
[0006] Preferably, it includes detecting the base sequence of the 802bp site in the promoter region of the CYP392A11 gene of a field mite population. When the 802bp site in the promoter region of the CYP392A11 gene of the field mite population is all T, the field mites are sensitive to abamectin; when the 802bp site in the promoter region of the CYP392A11 gene of the field mite population appears C, the field mites are resistant to abamectin.
[0007] The present invention provides a primer set for amplifying the promoter region of the CYP392A11 gene of field mites, including a forward primer with a nucleotide sequence as shown in SEQ ID No.1 and a reverse primer as shown in SEQ ID No.2.
[0008] The present invention also provides a kit for detecting the resistance of field mites to abamectin, including the above primer set.
[0009] Preferably, it also includes a reaction buffer, a DNA polymerase, and dNTPs.
[0010] The present invention provides a method for detecting the resistance of field mites to abamectin, including mixing the above primer set or the primer set in the above kit with the genomic DNA of field mites to prepare a PCR amplification system, performing a PCR amplification reaction, and sequencing the amplification product;
[0011] When the sequencing result shows that the 802bp site in the promoter region of the CYP392A11 gene of the test population is all T, the test population is a sensitive population;
[0012] When the sequencing result shows that the 802bp site in the promoter region of the CYP392A11 gene of the test population appears C, the test population is a resistant population.
[0013] Preferably, the species of the field mites includes Tetranychus urticae.
[0014] Preferably, the number of the test population is not less than 30.
[0015] Preferably, the PCR amplification system is 25 μL in volume and includes: 5.00 μL of reaction buffer, 1.25 μL of 10 μM forward primer, 1.25 μL of 10 μM reverse primer, 0.50 μL of 10 mM dNTPs, 0.25 μL of DNA polymerase, 1.00 μL of cDNA, and the balance of ddH2O.
[0016] Preferably, the procedure of the PCR amplification reaction includes: pre-denaturation at 98 °C for 30 s; denaturation at 98 °C for 10 s, annealing at 57 °C for 30 s, extension at 72 °C for 10 s, for 35 cycles; and finally extension at 72 °C for 2 min.
[0017] Beneficial effects: In the present invention, the cytochrome P450 detoxifying enzyme CYP392A11 gene is used as a resistance detection marker for field mites against abamectin, and a primer pair for amplifying the CYP392A11 gene to verify the presence of point mutations is provided, thereby completing the rapid identification of the resistance of field mites to abamectin. In the present invention, a single nucleotide mutation (T mutated to C) at the 802 bp position in the promoter region of the CYP392A11 gene in the resistant population leads to an increase in promoter activity, an increase in the expression level of the CYP392A11 gene, and thus the resistance of Tetranychus urticae to abamectin. Therefore, the present invention uses the site mutation as the target site for abamectin resistance, that is, detecting the single-base site mutation in the promoter region of the CYP392A11 gene by PCR amplification technology is an effective and rapid method for detecting the resistance of field mites to abamectin.
[0018] The present invention detects the resistance level of field mites to abamectin through the single-base site mutation in the promoter region of the detoxifying metabolism P450 gene CYP392A11 of field mites, avoiding the deficiencies in traditional resistance monitoring such as a long monitoring period (a series of processes from testing leaf dipping, picking test insects, microscopic examination of results, and judging dead test insects, which takes more than 30 hours), a large number of test insects required (500 - 600 healthy test insects with consistent physiological states are required to complete a bioassay for determining a standard curve), and insufficient test sensitivity (sometimes it is difficult to quickly determine the early resistance or low-frequency resistance of the test population), greatly supplementing the reliability of the resistance detection results, and providing a theoretical basis for the rapid detection of the resistance of field mites to abamectin. Using the detection method of the present invention, the resistance level of Tetranychus urticae in the field to abamectin can be quickly detected, thereby providing technical support for the rational use of pesticides. Description of the Drawings
[0019] Figure 1 It is a specific primer for amplifying the promoter region fragment of the CYP392A11 gene of Tetranychus urticae in the present invention;
[0020] Figure 2Alignment of the fragment sequence of the CYP392A11 promoter region between the resistant population and the susceptible population of Tetranychus urticae
[0021] Figure 3 Schematic diagram of the mutation in the promoter region of the CYP392A11 gene in the abamectin-resistant and susceptible populations Detailed implementation manners
[0022] The present invention provides the application of a reagent for detecting the base sequence at the 802bp site of the promoter region of the CYP392A11 gene in field pest mites in the preparation of a tool for detecting the resistance of field pest mites to abamectin.
[0023] Through statistics in the examples of the present invention, it is found that the base at the 802bp site of the promoter region of the CYP392A11 gene is associated with the resistance level, and it appears in the form of T base in the susceptible population, while in the resistant population, C base appears. In the present invention, as long as C base appears at the said site of the gene in the detected population, the detected population is identified as the resistant population.
[0024] The present invention provides a primer set for amplifying the promoter region of the CYP392A11 gene in field pest mites, including a forward primer with the nucleotide sequence shown in SEQ ID No.1 and a reverse primer shown in SEQ ID No.2.
[0025] The present invention detects the resistance level of field pest mites to abamectin through the single-base site mutation in the promoter region of the CYP392A11 gene of Tetranychus urticae. The species of the pest mites preferably includes Tetranychus urticae. The present invention preferably uses the fragment of the promoter region of the CYP392A11 gene of Tetranychus urticae as the target sequence, and designs specific PCR primers according to the PCR primer design principle. The sequences are as follows:
[0026] Forward primer: 5′-ATTTCTAACTTGCTATCTTACAACTGTTACA3′,
[0027] Reverse primer: 5′-AACTTTTTGCATGAAAATGTGATTG 3′.
[0028] The present invention also provides a kit for detecting the resistance of field pest mites to abamectin, including the above primer set.
[0029] Preferably, the kit of the present invention further includes other reagents for preparing the PCR system, such as reaction buffer, DNA polymerase and dNTPs. In the examples, the polymerase in the PCR reaction preferably uses Q5 high-fidelity DNA polymerase (New England Biolabs, NEB), so the reaction buffer uses the matching 5×Q5 reaction buffer.
[0030] The present invention provides a method for detecting the resistance of field mite pests to abamectin, including mixing the above primer set or the primer set in the above kit with the genomic DNA of field mite pests to prepare a PCR amplification system, performing a PCR amplification reaction, and sequencing the amplification product;
[0031] When the sequencing result shows that the 802bp site in the promoter region of the CYP392A11 gene of the test population is T, the test population is a sensitive population;
[0032] When the sequencing result shows that the 802bp site in the promoter region of the CYP392A11 gene of the test population appears as C, the test population is a resistant population.
[0033] The method of the present invention is preferably a population detection method, and it is required that the number of field mite pests in each test population is not less than 30, and the larger the population number, the higher the detection sensitivity.
[0034] The present invention preferably uses the genomic DNA of the population to be tested as a template, mixes it with the above primer pair to configure a PCR amplification system. The PCR amplification system is 25 μL in volume and preferably includes: reaction buffer 5.00 μL, 10 μM forward primer 1.25 μL, 10 μM reverse primer 1.25 μL, 10 mM dNTPs 0.50 μL, DNA polymerase 0.25 μL, cDNA 1.00 μL, and the balance of ddH2O. The prepared PCR amplification system of the present invention is placed in a PCR instrument for amplification reaction. The program of the PCR amplification reaction preferably includes: pre-denaturation at 98°C for 30 s; denaturation at 98°C for 10 s, annealing at 57°C for 30 s, extension at 72°C for 10 s, 35 cycles, and finally extension at 72°C for 2 min.
[0035] The present invention preferably recovers and purifies the PCR amplification product, then ligates the amplification product into a vector, transforms competent cells, and sequences the positive clones.
[0036] To further illustrate the present invention, the following examples are used to describe in detail the gene mutation sites of the resistance of field mite pests to abamectin provided by the present invention and their applications, but they should not be construed as limiting the protection scope of the present invention.
[0037] In the embodiments of the present invention, the Tetranychus urticae populations used include a Tetranychus urticae susceptible population and a Tetranychus urticae resistant population. Among them, the Tetranychus urticae susceptible population (IPP-SS) was continuously reared on kidney bean leaves in the insect rearing room of the Insect Group, Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, and had not been exposed to any toxic pesticides during the rearing period, showing a sensitive level to abamectin; the Tetranychus urticae field resistant population JZ-HB was collected from Jingzhou area, Hubei Province. The field collection method was random sampling, sampling once every 1000 m, and at least three samples were taken from each area. Approximately 1000 adult mites collected from each point were mixed together to represent the Tetranychus urticae population in that area. This susceptible population and resistant population have been publicly reported in the published article Zhang Y, Xu DD, Zhang YJ, Wu QJ, Xie W, Guo ZJ, Wang SL. Frequencies and mechanisms of pesticide resistance in Tetranychus urticae field populations in China. Insect Science, 2022, 29(3): 827-839.
[0038] After the above Tetranychus urticae populations were collected from the field and brought back to the laboratory, they were first isolated and reared in an artificial climate chamber (the rearing temperature was 26±1°C, the relative humidity was 60%-70%, and the photoperiod was light:dark = 16 h:8 h). After 1-2 generations of relatively stable populations, the experiments were carried out.
[0039] Example 1
[0040] Entrust a biological company to synthesize primers SEQ ID No.1 and SEQ ID No.2 designed from the fragments shown in the specific fragment sequences as Figure 1 shown.
[0041] 1. Extraction of Tetranychus urticae genomic DNA
[0042] Use the Blood / Cell / Tissue Genomic DNA Extraction Kit of Tiangen Biochemical Technology Co., Ltd. to extract the genomic DNA of the Tetranychus urticae indoor susceptible population IPP-SS and the field resistant population JZ-HB.
[0043] 2. PCR amplification of the promoter region of the Tetranychus urticae CYP392A11 gene and product recovery
[0044] Using a polymerase, a 25 μL reaction system was prepared with Q5 Ultra-Fidelity DNA Polymerase (New England Biolabs, NEB): 5.00 μL of 5×Q5 reaction buffer, 1.25 μL of 10 μM forward primer, 1.25 μL of 10 μM reverse primer, 0.50 μL of 10 mM dNTPs, 0.25 μL of Q5 Ultra-Fidelity DNA Polymerase, 1.00 μL of cDNA, and 15.75 μL of ddH₂O.
[0045] The PCR reaction program was as follows: pre-denaturation at 98 °C for 30 s; denaturation at 98 °C for 10 s; annealing at 57 °C for 30 s, extension at 72 °C for 10 s, with a total of 35 cycles, and finally extension at 72 °C for 2 min. After the PCR reaction, the size of the target band fragment was detected by 1.2% agarose gel electrophoresis and the PCR products were recovered.
[0046] The product was purified using the Monarch PCR & DNA Purification Kit (NEB, England), and the operation was carried out according to the instructions.
[0047] 3. Ligation, transformation of the target fragment and vector, and screening and detection of positive clones
[0048] (1) Ligation of the target fragment and vector
[0049] The purified target gene fragment was ligated with -Blunt Cloning Vector (TransGen Biotech Co., Ltd., Beijing). The 5 μL ligation system was as follows: 4 μL of the recovered product and -Blunt Cloning Vector 1 μL;
[0050] The above reaction system was gently tapped to mix it evenly, and after ligation at room temperature for 15 min, it was placed on ice.
[0051] (2) Transformation of the recombinant vector
[0052] 1) After the ligation reaction, 50 μL of Trans 1-T1 competent cells (the transformation efficiency is the highest when in an ice-water mixture) (TransGen Biotech Co., Ltd., Beijing) was added to the ligation product, gently mixed, and immediately placed on ice for 20 - 30 min;
[0053] 2) Heat shock in a 42 °C water bath for 30 s and immediately place on ice for 2 min;
[0054] 3) Add 200 μL of LB medium (without antibiotics), place it in a shaker and culture for 1 h, and the shaker was set at 200 rpm and 37 °C;
[0055] 4) Mix 40 μL 20 mg / mL X-gal and 16 μL 500 mM IPTG and add to the prepared solid culture medium (containing antibiotics) plate, spread evenly, and let stand at 37°C for 30 min;
[0056] 5) Pipette 200 μL of the shaken bacterial solution and spread it evenly on the plate, then place it in a 37°C incubator for overnight culture.
[0057] (3) Screening and detection of positive clones
[0058] The positive clones were identified by bacterial liquid PCR method. The specific steps were as follows: 30 positive clone plaques were picked and placed in 200 μL liquid culture medium containing ampicillin antibiotics, and cultured in a shaker at 200 rpm and 37°C for at least 3 h. Then, M13F / M13R universal primers were used for bacterial liquid PCR identification, and then 1.2% agarose gel electrophoresis was used to compare the positive clones. -BluntCloningVector vector to determine the correctness of the target fragment inserted. The bacterial solution that meets the size of the target gene fragment is entrusted to Beijing Qingke Biotechnology Co., Ltd. for sequencing.
[0059] 4. Comparative analysis of the promoter region sequence of the CYP392A11 gene of two-spotted spider mite
[0060] The PCR primer pair used to identify whether the promoter region of the two-spotted spider mite resistance gene CYP392A11 is mutated was used to further compare and analyze the promoter region sequence of the CYP392A11 gene. The results are as follows Figure 2 and Figure 3 As shown, it was found that there was a point mutation at site 802 in the promoter region of the gene in the avermectin-resistant population. This site existed in the form of T base in the avermectin-sensitive population, but mainly appeared in the form of C base in the resistant population. The resistance level of two-spotted spider mite to avermectin was determined by detecting the base form of this site.
[0061] Example 2
[0062] Determination of resistance to avermectin in field populations
[0063] (1) Determination of resistance of field populations to avermectins
[0064] Three field populations from Daxing, Beijing (DX-BJ), Changping, Beijing (CP-BJ) and Yinchuan, Ningxia (YC-NX) were tested for avermectin resistance.
[0065] The method for measuring drug resistance uses the optimized agar leaf-dipping method, and the specific operation steps are as follows: Prepare agar with a concentration of 0.1% in an appropriate volume. Drop the melted agar after being melted by a microwave oven onto the bottom of a plastic dish with a pipette, and place it at room temperature to cool until solidified. Dilute the tested insecticide with distilled water into 5 - 7 concentrations. Use a punch with a diameter of 2.5 cm to punch the kidney bean leaves into small leaf discs. Dip the leaf discs fully into the liquid medicine with forceps for 10 s, take them out and let them dry. Then lay the dried leaf discs with the back of the leaf facing up flat on the agar in the plastic dish. Set 4 replicates for each drug concentration, and place leaf discs impregnated with distilled water solution and dried in the blank control. After the above work is completed, pick healthy female adult mites with consistent physiological states onto each leaf disc with a No. 0 writing brush. There are 25 - 30 spider mites on each leaf disc. Then cover it with the upper cover of a petri dish with extremely small holes pierced, and transfer it to an intelligent artificial climate chamber for breeding and observation. The temperature is set at 26 ± 1 °C, the humidity is 60 ± 5%, and the light cycle is 16 h:8 h (L:D). After breeding for 24 h, check and record the number of surviving and dead spider mites under a stereomicroscope (Olympus SZX-7, Japan). Gently touch the mite body with the tip of a writing brush. If the test insect has only 1 leg moving or does not move at all, it is judged as dead. Note that spider mite samples that escape from the leaf discs and stick to the agar are not included in the total count.
[0066] For the results of the drug resistance measurement test, the median lethal concentration LC 50 , 95% confidence interval, slope ± standard error, chi-square value and degrees of freedom were all analyzed using Polo Plus 2.0 software. Resistance multiple = LC 50 value of the resistant population / LC 50 value of the sensitive population.
[0067] (2) Results and analysis of drug resistance measurement
[0068] The indoor sensitive population of Tetranychus urticae is highly sensitive to abamectin, and its LC 50 is 0.05 mg / L (Zhang et al., 2020). Through the measurement of abamectin drug resistance of three field resistant populations, the results are shown in Table 1. The LC 50 values of the three field populations in Daxing, Beijing (DX-BJ), Changping, Beijing (CP-BJ) and Yinchuan, Ningxia (YC-NX) are 806.31 mg / L, 1367.41 mg / L and 1411.33 mg / L respectively. Compared with the indoor sensitive population, they all show extremely high levels of resistance to abamectin, and the resistance multiples reach 16126.2, 27348.2 and 28226.6 times respectively.
[0069] Table 1 Resistance levels of field populations of Tetranychus urticae to abamectin
[0070]
[0071] (3) Detection of point mutations in resistant populations
[0072] For the above three field abamectin-resistant populations, genomic DNA was extracted and subjected to PCR amplification and sequencing analysis using the method in Example 1 to verify the reliability of the detection method in Example 1. The specific experimental procedure was as shown in Example 1.
[0073] By analyzing the sequencing results, at position 802 in the promoter region of the P450 gene CYP291A11 in the three resistant populations of DX-BJ, CP-BJ, and YC-NX, the base C was mainly present, and the proportions of base C were 70.59%, 64.71%, and 64.29% respectively, indicating that these three populations had developed high-level resistance to abamectin. This was completely consistent with the results obtained from the resistance determination method. At the same time, it also showed that as long as the occurrence of base C was detected at position 802 in this region in a population, it could be determined that the population had developed resistance to abamectin. Thus, it can be seen that the identification method set in Example 1 was reasonable, and by analyzing the type of this single base at position 802 in the promoter region of the CYP291A11 gene, the abamectin-resistant and sensitive populations of Tetranychus urticae could be clearly distinguished.
[0074] Although the above embodiments have described the present invention in detail, they are only a part of the embodiments of the present invention, not all of them. People can also obtain other embodiments based on this embodiment without creative efforts, and these embodiments all fall within the protection scope of the present invention.
Claims
1. A method for detecting the resistance of Tetranychus urticae to avermectin, characterized in that: The method comprises preparing a PCR amplification system by mixing a primer set with genomic DNA of two-spotted spider mite, performing a PCR amplification reaction, and sequencing the amplified product; the primer set comprises a forward primer having a nucleotide sequence as shown in SEQ ID No.1 and a reverse primer as shown in SEQ ID No.2; When the sequencing results show that the amplification products of the test population are all T at the 191bp position of the sequence shown in SEQ ID No. 3, the test population is a sensitive population; When the sequencing results show that the amplified product of the test population has C at the 191 bp position of the sequence shown in SEQ ID No. 3, the test population is a resistant population.
2. The method according to claim 1, characterized in that: The number of the test group is not less than 30.
3. The method according to claim 1, characterized in that: The PCR amplification system, based on 25 μL, includes: 5.00 μL of reaction buffer, 1.25 μL of 10 μM forward primer, 1.25 μL of 10 μM reverse primer, 0.50 μL of 10 mM dNTPs, 0.25 μL of DNA polymerase, 1.00 μL of genomic DNA and the balance of ddH2O.
4. The method according to claim 1 or 3, characterized in that: The procedure of the PCR amplification reaction includes: pre-denaturation at 98°C for 30s; denaturation at 98°C for 10s, annealing at 57°C for 30s, extension at 72°C for 10s, 35 cycles, and finally extension at 72°C for 2min.