Method for identifying mutation of glutamate-gated chloride channel gene of plutella xylostella and primer

Abstract

The present application relates to the field of biotechnology, and particularly relates to a method for identifying mutation of a gene of glutamate-gated chloride channel of plutella xylostella and primers. The present application provides a protein mutant related to resistance to avermectin insecticide, wherein a valine at position 263 of a glutamate-gated chloride channel protein is mutated into isoleucine. The present application can identify the resistance of insects to avermectin insecticide.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to a method for identifying mutation of Plutella xylostella glutamate-gated chloride channel gene and primers. BACKGROUND

[0002] Avermectin (AVMs) is a sixteen-membered macrocyclic lactone compound with miticidal, nematicidal and insecticidal activity produced by fermentation of Streptomyces avermitilis. It mainly has stomach toxicity and also has contact toxicity. Avermectin insecticides can cause insects to lose coordination and paralysis, and have a significant effect on the prevention and control of Plutella xylostella. At present, it has become one of the leading agents for preventing and controlling Plutella xylostella in vegetable production.

[0003] Plutella xylostella belongs to Lepidoptera and Plutellidae, and is a widely distributed worldwide pest, mainly damaging Brassica oleracea, radish, Chinese cabbage and other cruciferous plants. It has the characteristics of short growth cycle, serious overlapping generations, strong reproductive capacity and long-distance migration ability, and is prone to outbreak under suitable climatic conditions.

[0004] At present, the prevention and control of Plutella xylostella mainly relies on chemical methods. Due to long-term, large-scale and unreasonable use of chemical pesticides, Plutella xylostella has developed resistance to almost all kinds of insecticides such as organophosphorus, organochlorine, carbamate, pyrethroid and Bacillus thuringiensis. Some field Plutella xylostella has developed rapidly resistance to avermectin.

[0005] Glucls is an important target of avermectin insecticides. Glucls was first discovered in locust muscle and plays an important role in locust swallowing, movement and perception. Avermectin insecticides act on Glucls to activate chloride channels, causing inward chloride flow, hyperpolarizing nerve membranes, inhibiting the generation of excitatory action potentials, and ultimately causing insect death. Target mutation is one of the important reasons for the development of Plutella xylostella resistance to avermectin. It has been reported that mutation of alanine (Ala) at position 309 of Plutella xylostella Glucls to valine (Val) (A309V) is related to its resistance to avermectin.

[0006] In the prior art, there are very few methods for identifying mutation of Plutella xylostella glutamate-gated chloride channel gene, and there is a lack of a new molecular marker that can be used to detect the resistance of field Plutella xylostella to avermectin. SUMMARY

[0007] The application provides a method for identifying mutation of a Plutella xylostella glutamate-gated chloride channel gene and primers, which are used to solve the defect that there is no new type of molecular marker for detecting resistance of Plutella xylostella to abamectin in the prior art, and realize detection of resistance of Plutella xylostella to abamectin.

[0008] The application provides a protein mutant related to abamectin resistance, wherein a valine at position 263 of a glutamate-gated chloride channel protein is mutated into isoleucine.

[0009] The application further provides a nucleic acid encoding the protein mutant.

[0010] The nucleic acid according to the application has any one of the following DNA sequences:

[0011] 1) a sequence as shown in SEQ ID NO. 5;

[0012] 2) a DNA molecule hybridizing with the DNA sequence defined in 1) and encoding a protein having the same function;

[0013] 3) a DNA molecule having more than 90% homology with the DNA sequence in 1) or 2) and encoding a protein having the same function.

[0014] The application further provides a glutamate-gated chloride channel gene, which has any one of the following DNA sequences:

[0015] 1) a sequence as shown in SEQ ID NO. 3;

[0016] 2) a DNA molecule hybridizing with the DNA sequence defined in 1) and encoding a protein having the same function;

[0017] 3) a DNA molecule having more than 90% homology with the DNA sequence in 1) or 2) and encoding a protein having the same function. The sequence is a gene fragment near a mutation point of the Plutella xylostella glutamate-gated chloride channel gene before mutation. The difference between the sequence as shown in SEQ ID NO. 3 and the sequence as shown in SEQ ID NO. 5 is that the base at position 145 is different, G in SEQ ID NO. 3 and A in SEQ ID NO. 5.

[0018] All the above are related to abamectin resistance.

[0019] In some embodiments, the DNA molecule in 2) above hybridizes to the DNA sequence defined in 1) under stringent conditions and encodes a protein having the same function. Preferably, the stringent conditions can be hybridization in a solution of 6xSSC, 0.5% SDS at 65°C, followed by washing the membrane once with 2xSSC, 0.1% SDS and 1xSSC, 0.1% SDS, respectively.

[0020] The present application also provides primers for detecting the mutant protein.

[0021] The primers according to the present application have the sequences shown in SEQ ID NO. 1-2. The primers can be used to amplify the glutamate-gated chloride channel gene and determine whether the gene is mutated. The primers can also identify or assist in identifying the resistance of the diamondback moth to the avermectin insecticide.

[0022] The present application also provides a kit comprising the primers described above.

[0023] The present application also provides a method for identifying the V263I mutant of the glutamate-gated chloride channel gene, using the primers described above to amplify the glutamate-gated chloride channel gene of the insect to be tested, and determining the nucleotide at position 787 of the sequence shown in SEQ ID NO. 4. SEQ ID NO. 4 is the full-length of the diamondback moth GluCl receptor and is used to locate and describe the position of the mutation in the receptor (nucleotide 787), which corresponds to position 145 of the amplified fragment in SEQ ID NO. 3.

[0024] The method for identifying the V263I mutant of the glutamate-gated chloride channel gene according to the present application amplifies the glutamate-gated chloride channel gene of the insect to be tested and identifies the peak of the nucleotide at position 787 of the sequence shown in SEQ ID NO. 4.

[0025] If it is a single-peak G, it is the GG genotype, if it is a double-peak G / A, it is the GA genotype, and if it is a single-peak A, it is the AA genotype.

[0026] In some embodiments, a method for identifying the genotype of Plutella xylostella against V263I mutation comprises the following steps: taking the genomic DNA of the Plutella xylostella to be tested as a template, performing ordinary PCR amplification with a single-stranded DNA molecule shown as SEQ ID NO. 1, SEQ ID NO. 2, gel electrophoresis detection can obtain a bright DNA band at 500 bp position, sequencing the single DNA band, and detecting whether the 5' end 787th nucleotide is GG genotype, GA genotype or AA genotype according to the DNA peak graph. If the 787th nucleotide is single-peak G, it is GG genotype, and the Plutella xylostella to be tested is suspected to be sensitive to avermectin insecticides; if the 787th nucleotide is double-peak G / A, it is GA genotype, and the Plutella xylostella to be tested is suspected to be resistant to avermectin insecticides; if the 787th nucleotide is single-peak A, it is AA genotype, and the Plutella xylostella to be tested is suspected to be resistant to avermectin insecticides.

[0027] The application also provides a method for identifying the resistance of insects to avermectin insecticides, amplifying the glutamate-gated chloride channel gene of the insect to be tested, and identifying the 787th nucleotide in the sequence shown as SEQ ID NO. 4.

[0028] If it is GG genotype, the insect to be tested is sensitive to avermectin insecticides; if it is GA genotype, the insect to be tested is resistant to avermectin insecticides; and if it is AA genotype, the insect to be tested is resistant to avermectin insecticides.

[0029] According to the method for identifying the resistance of insects to avermectin insecticides, the glutamate-gated chloride channel gene of the insect to be tested is amplified, and the peak of the 787th nucleotide in the sequence shown as SEQ ID NO. 4 is identified.

[0030] If it is single-peak G, the insect to be tested is suspected to be sensitive to avermectin insecticides; if it is double-peak G / A, the insect to be tested is suspected to be resistant to avermectin insecticides; and if it is single-peak A, the insect to be tested is suspected to be resistant to avermectin insecticides.

[0031] In some embodiments, a method for assisting in identifying the resistance of Plutella xylostella to avermectin insecticides comprises the following steps: using the genomic DNA of the Plutella xylostella to be tested as a template, performing ordinary PCR amplification with a single-stranded DNA molecule as shown in sequences SEQ ID NO. 1 and SEQ ID NO. 2, performing gel electrophoresis to detect a bright DNA band at a position of 500 bp, performing sequencing on a single DNA band, and detecting whether the 5' end 787th nucleotide is a GG genotype, a GA genotype, or an AA genotype according to the DNA peak graph. If the 787th nucleotide is a single-peak G, it is a GG genotype, and the Plutella xylostella to be tested is suspected to be sensitive to avermectin insecticides; if the 787th nucleotide is a double-peak G / A, it is a GA genotype, and the Plutella xylostella to be tested is suspected to be resistant to avermectin insecticides; and if the 787th nucleotide is a single-peak A, it is an AA genotype, and the Plutella xylostella to be tested is suspected to be resistant to avermectin insecticides.

[0032] The application further provides the use of the above-mentioned protein mutant, the above-mentioned nucleic acid, the above-mentioned primer, the above-mentioned kit, and the above-mentioned method in any one of the following:

[0033] 1. Identifying whether a glutamate-gated chloride channel gene is mutated;

[0034] 2. Identifying the phenotype of a glutamate-gated chloride channel protein of an insect;

[0035] 3. Identifying or assisting in identifying the resistance of an insect to avermectin insecticides.

[0036] According to the above use, the insect is a Lepidoptera pest, preferably a Plutellidae insect, further preferably a Plutella insect, and more preferably Plutella xylostella.

[0037] According to the above use, the avermectin insecticide comprises one or more of abamectin and emamectin benzoate.

[0038] According to the above use, the application can be used to identify whether the Plutella xylostella has a V263I mutation of a glutamate-gated chloride channel gene. The V263I mutation of the glutamate-gated chloride channel gene refers to that the open reading frame of the glutamate-gated chloride channel gene is mutated from G to A at the 787th nucleotide from the 5' end (the open reading frame of the glutamate-gated chloride channel gene of the Plutella xylostella is a DNA molecule as shown in sequence SEQ ID NO. 4 from the 1st to 1344th nucleotide from the 5' end).

[0039] The application has the following beneficial effects:

[0040] (1) The inventors found that there is a new mutation V263I in Plutella xylostella Glucl, and the mutation can cause more than 100 times higher level of resistance to abamectin when introduced into a sensitive population, thus can be used as a new molecular marker for detection, and the detection results are of great significance for guiding rational drug use and delaying resistance development.

[0041] (2) The primers, kits and methods provided by the application can identify whether V263I mutation of the glutamate-gated chloride channel associated with abamectin resistance in Plutella xylostella occurs, and can be used for detecting the resistance of Plutella xylostella to abamectin insecticides.

[0042] (3) The Plutella xylostella with AA genotype at the 787th nucleotide of the 5' end of the glutamate-gated chloride channel gene has higher resistance to abamectin insecticides (abamectin insecticides) than the Plutella xylostella with GA genotype and the Plutella xylostella with GG genotype. The application and method can be used for identifying the resistance of Plutella xylostella to abamectin insecticides (abamectin insecticides).

[0043] (4) The primers and kits provided by the application can effectively identify whether V263I mutation of the glutamate-gated chloride channel associated with abamectin resistance in Plutella xylostella occurs, so as to judge the resistance or sensitivity of Plutella xylostella to abamectin insecticides, and have the advantages of being fast and effective and high in sensitivity, which is of great significance for monitoring the resistance gene frequency and resistance development level of Plutella xylostella to abamectin insecticides, timely adjusting the Plutella xylostella control strategy, guiding rational drug use and delaying resistance development. BRIEF DESCRIPTION OF DRAWINGS

[0044] In order to more clearly illustrate the technical solutions in the present application or prior art, the following will briefly introduce the drawings needed to be used in the embodiments or prior art description. Obviously, the drawings in the following description are some embodiments of the present application, and other drawings can also be obtained by those skilled in the art without creative labor.

[0045] Figure 1 In order to use the individual of the sensitive strain of Plutella xylostella as a template, the primers shown in sequences SEQ ID NO. 1 and SEQ ID NO. 2 are used for PCR amplification, and the agarose gel electrophoresis diagram of the PCR amplification product is shown in the following figure.

[0046] Figure 2 In order to use the individual of the 2020 Boluo population of Plutella xylostella as a template, the primers shown in sequences SEQ ID NO. 1 and SEQ ID NO. 2 are used for PCR amplification, and the agarose gel electrophoresis diagram of the PCR amplification product is shown in the following figure.

[0047] Figure 3For taking the individual of the sensitive strain of Plutella xylostella as a template, the primer shown in sequence SEQ ID NO. 1 and SEQ ID NO. 2 is used for PCR amplification, and the DNA sequencing peak graph of the PCR amplification product.

[0048] Figure 4 For taking the individual of the 2020 Boluo population of Plutella xylostella as a template, the primer shown in sequence SEQ ID NO. 1 and SEQ ID NO. 2 is used for PCR amplification, and the DNA sequencing peak graph of the PCR amplification product. DETAILED DESCRIPTION

[0049] In order to make the purpose, technical scheme and advantages of the present application clearer, the technical scheme in the present application will be described clearly and completely below in combination with the drawings in the present application. Obviously, the described embodiments are part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative labor belong to the scope of protection of the present application. In the following examples, the experimental methods are conventional methods unless otherwise specified. In the following examples, the test materials used are commercially available from conventional biochemical reagent stores unless otherwise specified. In the following examples, the quantitative tests are set up with three repeated experiments, and the average value is taken as the result.

[0050] Example 1

[0051] The glutamate-gated chloride channel gene of Plutella xylostella individuals with different resistance to avermectin insecticides is analyzed, so as to find and verify the relationship between the glutamate-gated chloride channel gene V263I mutation and the resistance.

[0052] PCR amplification and sequencing verification of the glutamate-gated chloride channel of 50 Plutella xylostella individuals collected from Boluo City, Guangdong Province in 2019 are carried out. The sequencing results show that there is a SNP (single nucleotide polymorphism) in the glutamate-gated chloride channel gene of Plutella xylostella, i.e. the open reading frame of the glutamate-gated chloride channel gene of Plutella xylostella resistant to avermectin insecticides is mutated from G to A at the 787th nucleotide from the 5' end, resulting in a mutation of glutamate-gated chloride channel from valine (V) to isoleucine (I) at the 263rd amino acid residue from the N terminus, i.e. V263I mutation. The glutamate-gated chloride channel gene of Plutella xylostella is shown in sequence SEQ ID NO. 4, and its open reading frame is sequence SEQ ID NO. 4 from the 1st to 1344th nucleotide from the 5' end.

[0053] The glutamate-gated chloride channel gene of 30 randomly sampled susceptible strains of Plutella xylostella and 30 individuals of abamectin-resistant strain of Plutella xylostella collected from Boluo City, Guangdong Province in 2020 were sequenced, and genotyped according to the V263I mutation. The individuals of the susceptible strain were all susceptible homozygous individuals (SS), and the individuals of the resistant strain included susceptible homozygous individuals (SS), resistant heterozygous individuals (RS) and resistant homozygous individuals (RR).

[0054] Example 2, primer design

[0055] Based on the findings of Example 1, allele-specific PCR primers were designed according to the sequence of the glutamate-gated chloride channel gene of Plutella xylostella, as shown in Table 1:

[0056] Table 1 Nucleotide sequence of each primer

[0057]

[0058]

[0059] Example 3, application of primers

[0060] I. Detection of resistance of Plutella xylostella to abamectin

[0061] The method for detecting the resistance of Plutella xylostella to abamectin used the leaf membrane method, as follows:

[0062] Abamectin technical material with a purity of 96.8% (Hebei Weiyuan Biological Chemical Co., Ltd.) was dissolved in acetone to prepare a stock solution of 20,000 mg / L (concentration based on active ingredient). A series of working solutions with concentration gradients were prepared by diluting the stock solution of step 1 with 0.1% Triton X-100 aqueous solution. Cabbage leaves with main veins removed were cut into square leaves with a side length of 5 cm x 5 cm, and a toothbrush was used to gently brush the leaves to remove the surface wax and wash them clean. After drying, the leaves were soaked in each concentration of pesticide for 20 seconds, then taken out and air-dried, and then placed in a culture dish (diameter 9 cm) lined with filter paper, and 15 three-day-old first instar larvae hatched on the same day were introduced. Three replicates were set up for each concentration. Leaves soaked in 0.1% Triton X-100 aqueous solution were used as controls. The results were checked after 72 hours, and a fine brush was used to touch the insect bodies without reaction was considered dead. The data obtained were analyzed using PoloPlus 2.0 software (LeOra Software Inc., California, USA) to calculate the slope value (Slope), LC 50 (median lethal concentration) value and its 95% confidence interval (CI), chi-square and degrees of freedom [χ 2(df) and resistance ratio (RR). The above bioassays were performed on the susceptible strain and the Plutella xylostella population in Boluo, respectively. The bioassay results are shown in Table 1.

[0063] Table 2 Bioassay results of the susceptible strain and the Boluo population against abamectin

[0064]

[0065] As can be seen from Table 2, the LC 50 value of the Boluo population in 2020 was significantly higher than that of the susceptible Plutella xylostella population. The LC 50 value of the susceptible Plutella xylostella population was set to 1, and the ratio RR of the LC 50 values between the Boluo population and the susceptible population was calculated. That is, the resistance index RR of the Boluo population was 292.0. Generally, RR < 10 indicates low-level resistance; 10 < RR < 100 indicates medium-level resistance; RR > 100 indicates high-level resistance. That is, the Boluo population has high-level resistance to abamectin.

[0066] II. Application of primers

[0067] 30 individuals were randomly selected from the susceptible strain population of Plutella xylostella to abamectin-like insecticides, and at the same time, 30 individuals were randomly selected from the resistant strain of Plutella xylostella to abamectin-like insecticides, that is, the Boluo population in 2020 for the following detections: Genomic DNA of the individuals was extracted, and genomic DNA was used as a template, and common PCR amplification was performed using the primers shown in SEQ ID NO.1 and SEQ ID NO.2, and then the PCR amplification products were subjected to agarose gel electrophoresis.

[0068] The reaction system (20 μl) for PCR amplification using the susceptible primer pair is shown in Table 3:

[0069] Table 3 PCR amplification system

[0070] DNA template 1 μl 2 x Taq Master Mix 10 μl 263-F 0.5 μl 263-R 0.5 μl dd H2O 8 μl Total system 20 μl

[0071] The conditions for common PCR amplification of the above system are shown in Table 4:

[0072] Table 4 Common PCR amplification conditions

[0073]

[0074] Using the individuals of the susceptible strain of Plutella xylostella as a template, common PCR amplification was performed using the primers according to the above conditions. The agarose gel electrophoresis diagram of the PCR amplification products is shown in Figure 1 , and a specific DNA band of 500 bp can be obtained.

[0075] The individual of the Boro race of Plutella xylostella in 2020 is used as a template, and the primer is amplified by general PCR according to the above conditions, and the agarose gel electrophoresis diagram of the PCR amplification product is shown in Figure 2 A specific DNA band of 500 bp can be obtained.

[0076] III. Result verification

[0077] Each specific band in step two is recovered and sequenced for verification.

[0078] The sequencing results show that, using the genomic DNA of 30 individuals of the sensitive race of Plutella xylostella as a template, and using the primers shown in sequences SEQ ID NO. 1 and SEQ ID NO. 2, the PCR amplification product obtained is 500 bp, and the DNA peak diagram obtained by sequencing is shown in the accompanying drawings Figure 3 , that is, no V263I mutation occurs.

[0079] The sequencing results show that, using the genomic DNA of 30 individuals of the Boro race of Plutella xylostella in 2020 as a template, and using the primers shown in sequences SEQ ID NO. 1 and SEQ ID NO. 2, the PCR amplification product obtained is 500 bp, and the DNA peak diagram obtained by sequencing is shown in the accompanying drawings Figure 4 , that is, part of the V263I mutation occurs.

[0080] The mutation frequency of V263I of the sensitive race and the Boro race in 2020 is counted, as shown in Table 5:

[0081] Table 5 Mutation frequency of V263I of the sensitive race and the Boro race in 2020

[0082]

[0083] The results show that the primers shown in sequences SEQ ID NO. 1 and SEQ ID NO. 2 can specifically amplify the genomic DNA of the sensitive race and the Boro race of Plutella xylostella, and obtain a specific fragment of 500 bp in length, and the genotype of the 787th nucleotide can be judged by sequencing. If the sequence obtained by sequencing includes the GA genotype or the AA genotype, it can be identified as a Plutella xylostella resistant to avermectin insecticides. The primers provided by the present application can specifically detect whether the mutation of V263I of the glutamate-gated chloride channel gene of Plutella xylostella occurs, and further judge the resistance of Plutella xylostella to avermectin insecticides.

[0084] It should be pointed out finally that the above embodiments are only used to illustrate the technical solutions of the present application, but not to limit the same; and although the present application has been described in detail with reference to the foregoing embodiments, it should be appreciated by those skilled in the art that the technical solutions recorded in the foregoing embodiments can be modified, or some technical features thereof can be replaced equivalently; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present application.

Claims

1. A mutant of a protein associated with resistance to avermectin insecticides, characterized in that, The protein mutant is based on glutamate-gated chloride channel protein, and the valine at position 263 is mutated into isoleucine. The nucleotide sequence of the coding gene of the glutamate-gated chloride channel protein is shown in SEQ ID NO.

4.

2. Nucleic acid encoding the protein mutant of claim 1.

3. The nucleic acid of claim 2, wherein the nucleotide sequence comprises the sequence shown in SEQ ID NO.

5.

4. A method for identifying the V263I mutant of the glutamate-gated chloride channel gene, characterized in that, The glutamate-gated chloride channel gene of the insect to be tested is amplified using primers with sequences shown in SEQ ID NO. 1-2, and the nucleotide at position 787 in the sequence shown in SEQ ID NO. 4 is determined.

5. A method of identifying resistance of an insect to an avermectin insecticide, characterized by, The glutamate-gated chloride channel gene of the insect to be tested is amplified, and the nucleotide at position 787 in the sequence shown in SEQ ID NO. 4 is identified. If the genotype is GG, the insect to be tested is sensitive to avermectin insecticides; if the genotype is GA, the insect to be tested is resistant to avermectin insecticides; and if the genotype is AA, the insect to be tested is resistant to avermectin insecticides.

6. Use of the protein mutant of claim 1, the nucleic acid of claim 2 or 3, or the method of claim 4 or 5 in any of the following: 1) identifying whether the glutamate-gated chloride channel gene is mutated; 2) identifying the phenotype of the glutamate-gated chloride channel protein of the insect; 3) identifying or assisting in identifying the resistance of the insect to avermectin insecticides.

7. Use according to claim 6, characterized in that, The avermectin insecticides include one or more of abamectin, emamectin benzoate.

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