Molecular detection method for pyrethroid drug resistance of odontothrips bubali

Abstract

The molecular detection method for the pyrethroid drug resistance of the odontothrips bubali comprises the following contents: a sodium ion channel alpha subunit of the odontothrips bubali has G533S and V538I double mutation sites, and the nucleotide sequence of the sodium ion channel alpha subunit of the odontothrips bubali is shown as SEQ ID NO: 1; the mutation site at the 533rd amino acid part of the sodium ion channel alpha subunit is GGC mutated into AGC, glycine Gly at the 533rd position is changed into serine Ser, the mutation site at the 538th amino acid part of the sodium ion channel alpha subunit is GTC mutated into ATC, and valine Val at the 538th position is changed into isoleucine Ile; the method comprises the following steps: extracting DNA (Deoxyribonucleic Acid), carrying out PCR (Polymerase Chain Reaction) amplification, sequencing a PCR product if a 382bp band appears in agarose gel electrophoresis of the PCR product, comparing sequences obtained by sequencing, and if double mutation appears, determining that the thrips are pyrethroid drug resistant thrips. The method is high in accuracy, low in detection condition requirement, short in time consumption and few in required insect source.

Description

Technical Field

[0001] The invention belongs to the technical field of gene mutation site detection and application, and particularly relates to a molecular detection method for pyrethroid resistance of Cyprinus cyprinid. Background Art

[0002] Thrips are a widespread and serious agricultural pest that can damage the entire crop plant and spread a variety of plant viruses. Odontothrips loti ) is an oligophagous pest that takes leguminous plants as its host. It is particularly serious on alfalfa. During the peak period of summer, the number of thrips can reach more than 80% of the total number of thrips in the field. In North China and Northwest my country, the rate of plant damage is as high as 70% to 100%. The thrips use rasping mouthparts to feed on the stems, leaves, flowers and young pods of the plants they damage. They use their mouthparts to scratch the surface of the plants, enter the vascular bundles and suck the phloem juice of the plants, causing the leaves to curl and shrink, the growing points to turn yellow and wither and cannot continue to grow, resulting in the falling of flowers and pods, reducing crop yields, and even causing crop failure in severe cases.

[0003] At present, spraying insecticides is still the main means of controlling thrips in the field. Commonly used agents include pyrethroids and neonicotinoids. Due to the strong reproductive capacity of thrips, overlapping generations in the field, and short life cycle, their field resistance develops very rapidly. It has been reported that thrips in the field of leguminous crops have developed moderate or above resistance to a variety of insecticides, among which they have developed high resistance to pyrethroid insecticides. Pyrethroid insecticides are a type of chemical pesticide derived from natural pyrethrins. They are widely used in the control of vegetable pests, fruit tree pests, and grassland pests because of their high insecticidal activity, fast knockdown rate, broad insecticide spectrum, low toxicity to mammals, effectiveness against organophosphate and carbamate resistant strains, and easy degradation in the environment. It is also widely used in agricultural pest management. The resistance of thrips to pyrethroid insecticides seriously affects the control of field pests in leguminous crops and has become an important factor restricting production.

[0004] Chinese patent application CN118773326A discloses a method for rapid detection of T929I mutation site and drug resistance of the α-subunit of the sodium ion channel of flower thrips. Resistance is detected by determining whether the agarose gel electrophoresis band amplified after constructing an enzyme cutting system encodes a resistant amino acid. Its special primers are only effective for flower thrips. Chinese patent application CN112342283A discloses a method for rapid detection of I904S mutation of the sodium ion channel of cowpea thrips and special primers. Mutation and resistance are detected by sequencing after amplification. Its special primers are only effective for cowpea thrips. Chinese patent application CN107130051A discloses a molecular detection method for identifying the resistance of western flower thrips to spinosad. The resistance level is evaluated by constructing a cloning vector and calculating the mutant ratio according to the number of positive clones after transformation. Its special primers are only effective for western flower thrips.

[0005] However, in the prior art, there are no reports on the detection or rapid diagnosis of pyrethroid resistance in B. cerana. The resistance assessment of B. cerana still uses indoor bioassay methods. This method has the problems of cumbersome detection operation, long time consumption, high detection conditions, the need to collect a large number of insect sources and unclear source of resistance.

[0006] In order to solve the problems in existing indoor bioassay methods for detecting resistance of Botrytis cinerea, such as complicated detection operation, long time consumption, high detection condition requirements, need to collect a large number of insect sources, and unclear resistance, the present invention provides a molecular detection method for resistance of Botrytis cinerea to pyrethroid agents. Summary of the invention

[0007] The purpose of the present invention is to overcome the shortcomings of the prior art and provide a molecular detection method for pyrethroid resistance of Botrytis cinerea, which has the advantages of simple detection operation, short time consumption, low detection condition requirements, no need to collect a large number of insect sources, and high accuracy.

[0008] The objective of the present invention is achieved through the following technical scheme: a molecular detection method for the resistance of thrips to pyrethroid agents, comprising the following contents: there are double mutation sites G533S and V538I in the sodium ion channel alpha subunit of the thrips, the nucleotide sequence of the sodium ion channel alpha subunit of the thrips is as shown in SEQ ID NO: 1, the mutation site at the 533rd amino acid position of the sodium ion channel alpha subunit is GGC mutated to AGC, the glycine Gly at the 533rd position is changed to serine Ser, the mutation site at the 538th amino acid position of the sodium ion channel alpha subunit is GTC mutated to ATC, and the valine Val at the 538th position is changed to isoleucine Ile; if the double mutations G533S and V538I appear in the molecular detection, the thrips is a thrips resistant to pyrethroid agents.

[0009] The molecular detection method comprises the following steps: 1) Extraction of genomic DNA from adult thrips; 2) Establish a PCR amplification system for target sequence DNA fragments; 3) Perform PCR amplification; 4) Perform agarose gel electrophoresis and observe the electrophoresis bands on a gel imaging system; if a 382 bp band appears, sequence the PCR product and test it in one direction; 5) Compare the sequence obtained by sequencing with the 382bp target fragment sequence to obtain the result.

[0010] Furthermore, the PCR amplification system in step 2) includes Premix Taq TM , target fragment DNA, ddH2O and a pair of specific primers; The specific primers are as follows: OlNaF: 5'-CACGAGCTCCTCAGCAACATC-3', SEQ ID NO: 2; OlNaR: 5'-CCCAGTTGAAGTAGCTGCCA-3', SEQ ID NO: 3.

[0011] Furthermore, the PCR amplification reaction conditions in step 2) are: pre-denaturation at 94°C for 2 min; denaturation at 98°C for 10 sec, annealing at 56°C for 30 sec, extension at 68°C for 1 min, for 30 cycles; final extension at 72°C for 5 min; and storage at 4°C.

[0012] Furthermore, in the step 2), agarose gel electrophoresis is performed using 1% agarose gel electrophoresis at a voltage of 120 V for 20 min.

[0013] The present invention identifies that a double mutation site exists in a fragment of the sodium channel protein alpha subunit of a population of pyrethroid-resistant thrips, such as that shown in SEQ ID NO: 1. A target fragment is amplified by PCR using a pair of specific primers that can detect the mutation fragment. After sequencing, whether the double mutation occurs is compared, so as to quickly detect whether the thrips are resistant to pyrethroids.

[0014] The beneficial effects of the present invention are as follows: the present invention diagnoses the double mutation of the sodium channel alpha subunit G533S and V538I of the horn flower tooth thrips, and quickly determines whether the thrips are resistant to pyrethroid agents according to the double mutation. The present invention has high accuracy and is simple to operate. It only needs conventional molecular experimental conditions to perform the diagnosis, and the diagnosis result can be obtained within 48 hours, which is short in time. The method of the present invention requires a small number of insect sources. The conventional bioassay resistance detection method requires more than 200 insect sources, while the present invention only needs 10 thrips to achieve rapid detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 This is the agarose gel electrophoresis image of the PCR amplification product of the DNA of the female adult of Thrips tatarinowii. DETAILED DESCRIPTION

[0016] The present invention is described in detail below in conjunction with the accompanying drawings.

[0017] The whole genome of pyrethroid-resistant and sensitive populations of Thrips taurus was resequenced, and then single nucleotide mutation sites (SNPs) were mined at the genome level of the two populations. SNPs that were significantly differentiated between the two populations and strongly selected in the resistant population and had a moderate or above effect on gene function were screened. The genes were determined based on the SNP sites, and then the genes with significant differences in expression levels before and after insecticide treatment between the pyrethroid-resistant and sensitive populations were further screened. Finally, the gene shown in SEQ ID NO: 1 was screened and its mutation site was identified. It was found that Thrips taurus, which was resistant to pyrethroid agents, had double mutations of G533S and V538I in the α subunit of the sodium ion channel of Thrips taurus. Specifically, there are double mutation sites of G533S and V538I in the α subunit of the sodium ion channel of B. cerana var. cerana. The nucleotide sequence of the α subunit of the sodium ion channel of B. cerana var. cerana is shown in SEQ ID NO: 1. The mutation site at the 533rd amino acid position of the sodium ion channel α subunit is GGC mutated to AGC, and the glycine Gly at the 533rd position is changed to serine Ser. The mutation site at the 538th amino acid position of the sodium ion channel α subunit is GTC mutated to ATC, and the valine Val at the 538th position is changed to isoleucine Ile.

[0018] Example Molecular Detection Method for Pyrethroid Resistance of Thrips tadpoles A molecular detection method for resistance of Cypripedium cyprinid to pyrethroids comprises the following steps: 1) Extract genomic DNA from 10 female adults of Thrips tatarinus; 2) Establish a PCR amplification system for the target sequence DNA fragment, as shown in Table 1, including Premix Taq TM, target fragment DNA, ddH2O and a pair of specific primers (synthesized by Sangon Biotechnology (Shanghai) Co., Ltd.); The specific primers are as follows: OlNaF: 5'-CACGAGCTCCTCAGCAACATC-3', SEQ ID NO: 2; OlNaR: 5'-CCCAGTTGAAGTAGCTGCCA-3', SEQ ID NO: 3;

[0019] 3) Perform PCR amplification, the reaction conditions are shown in Table 2;

[0020] 4) Use 1% agarose gel, run electrophoresis at 120V for 20min, and observe the electrophoresis bands on a gel imaging system; Figure 1 As shown, if a 382 bp band appears, the PCR product and primers are sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing, one-way sequencing; 5) Compare the sequence obtained by sequencing with the 382bp target fragment sequence. If double mutations of G533S and V538I are found, it means that the thrips is resistant to pyrethroids.

[0021] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, rather than to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions of the technical solution of the present invention by ordinary technicians in this field do not deviate from the essence and scope of the technical solution of the present invention.

Claims

1. A molecular detection method for resistance of pyrethroids to thrips, characterized in that The invention comprises the following contents: a sodium ion channel alpha subunit of Botrytis cinerea has double mutation sites of G533S and V538I, the nucleotide sequence of the sodium ion channel alpha subunit of Botrytis cinerea is as shown in SEQ ID NO: 1, the mutation site at the 533rd amino acid position of the sodium ion channel alpha subunit is GGC mutated to AGC, the glycine Gly at the 533rd position is changed to serine Ser, the mutation site at the 538th amino acid position of the sodium ion channel alpha subunit is GTC mutated to ATC, the valine Val at the 538th position is changed to isoleucine Ile; if the double mutations of G533S and V538I are found in molecular detection, the Botrytis cinerea is a thrips resistant to pyrethroid agents.

2. The molecular detection method of pyrethroid resistance of Cynoglossum sphaerocephalus according to claim 1, characterized in that: The molecular detection method comprises the following steps: 1) Extraction of genomic DNA from adult thrips; 2) Establish a PCR amplification system for target sequence DNA fragments; 3) Perform PCR amplification; 4) Perform agarose gel electrophoresis and observe the electrophoresis bands on a gel imaging system; if a 382 bp band appears, sequence the PCR product and test it in one direction; 5) Compare the sequence obtained by sequencing with the 382bp target fragment sequence to obtain the result.

3. The molecular detection method of pyrethroid resistance of Cynoglossum sphaerocephalus according to claim 2, characterized in that: The PCR amplification system in step 2) includes Premix Taq TM , target fragment DNA, ddH2O and a pair of specific primers; The specific primers are as follows: OlNaF: 5'-CACGAGCTCCTCAGCAACATC-3', SEQ ID NO: 2; OlNaR: 5'-CCCAGTTGAAGTAGCTGCCA-3', SEQ ID NO:

3.

4. The molecular detection method of pyrethroid resistance of Cyprinus cyprinid according to claim 2, characterized in that: The PCR amplification reaction conditions in step 2) are: pre-denaturation at 94°C for 2 min; denaturation at 98°C for 10 sec, annealing at 56°C for 30 sec, extension at 68°C for 1 min, 30 cycles; final extension at 72°C for 5 min; and storage at 4°C.

5. The molecular detection method of pyrethroid resistance of Cynoglossum sphaerocephalus according to claim 2, characterized in that: In the step 2), the agarose gel electrophoresis is performed using 1% agarose gel electrophoresis at a voltage of 120 V for 20 min.

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