Method and kit for detecting drug resistance of plutella xylostella to emamectin benzoate based on CYP9G2 gene

Abstract

The invention discloses a real-time fluorescent quantitative PCR (Polymerase Chain Reaction) detection method based on a cytochrome P450 gene CYP9G2 and a matched kit, which are used for rapidly and accurately detecting the drug resistance of plutella xylostella to emamectin benzoate. According to the method, whether a plutella xylostella individual or population has resistance to emamectin benzoate or not is judged by quantitatively analyzing the mRNA expression level of a CYP9G2 gene. The kit comprises a specific primer pair, and the sequences of the specific primer pair are respectively SEQ ID NO.1 (forward) and SEQ ID NO.2 (reverse). The method is easy and convenient to operate and rapid in detection, sample treatment to result analysis can be completed within 3-4 hours, and the method is remarkably superior to a traditional biological determination method. The application of the method can realize early discovery and dynamic monitoring of the drug resistance of field plutella xylostella, provides a direct basis for scientific use and resistance treatment of emamectin benzoate in vegetable production, is beneficial to reduction of the pesticide use amount, reduction of the control cost and guarantee of the vegetable quality safety, and has important practical popularization value in the aspects of agricultural green production and pesticide application reduction.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and relates to a method for detecting resistance of Plutella xylostella to emamectin benzoate based on a molecular biology method and a special primer pair thereof. BACKGROUND

[0002] Plutella xylostella belongs to Lepidoptera and Yponomeutidae, and is one of the important pests of cruciferous vegetables worldwide, causing economic losses and control costs of up to 4-5 billion US dollars per year. At present, chemical control is still the main means to control P. xylostella, but due to long-term unreasonable use of pesticides and the biological characteristics of the pest itself, P. xylostella has developed resistance to almost all commonly used insecticides. According to the Arthropod Pesticide Resistance Database (APRD, 2025), P. xylostella has shown varying degrees of resistance to 104 active ingredients.

[0003] China has a vast territory, and the climate differs significantly due to differences in latitude and longitude. The occurrence and generation of P. xylostella also differ significantly in different regions. The occurrence period is the earliest in Hainan and Guangdong, generally from February to March every year, and the number of generations per year is the highest, reaching more than 20 generations. With the increase of latitude, the occurrence period is delayed, and the number of generations is gradually reduced. In northern regions, the occurrence of P. xylostella has obvious seasonality, such as in Northeast China, the peak occurrence period is from June to July, and the number of generations per year is 2-3. Since the 1990s, P. xylostella has continued to outbreak in China, especially in the main vegetable production areas of South China and Southwest China, causing serious damage. According to statistics, the total area of vegetable planting in China is about 300 million mu per year, and China has become the largest vegetable producer in the world, which provides sufficient host conditions for P. xylostella. The annual reduction in vegetable yield and control costs due to P. xylostella is up to 770 million US dollars (Li et al., 2016). The rapid development of resistance of P. xylostella, combined with unreasonable use of pesticides, often leads to excessive pesticide residues in vegetables, seriously threatening the yield and quality safety of agricultural products. Therefore, the management of resistance of P. xylostella still faces severe challenges.

[0004] The avermectins include abamectin, emamectin benzoate (emamectin), and ivermectin. Abamectin is a group of sixteen-membered macrolides produced by fermentation of Streptomyces avermitilis, which can be further divided into four major components A1a, A2a, B1a, B2a and four minor components A1b, A2b, B1b, B2b. Due to its excellent insecticidal and insect repellent activity, it is widely used in the prevention and control of agricultural pests and parasites. Ivermectin is a broad-spectrum anti-parasitic drug commonly used to treat parasitic infections in humans and animals. Emamectin is a derivative synthesized by introducing a methylamino group and a benzoic acid group based on the structure of abamectin B1, which significantly improves its insecticidal activity, especially against lepidopteran pests. However, there are currently many agricultural pests that have developed serious resistance to emamectin, including whitefly, cabbage aphid, cotton stink bug, western flower thrips, cotton bollworm, beet armyworm, Spodoptera frugiperda, Spodoptera litura, and thrips (APRD, 2025).

[0005] The mechanisms of action of avermectin agents are similar, and they are all classified as allosteric modulators of glutamate-gated chloride channels (GluCl). Therefore, related research in this field has mainly focused on the correlation between GluCl channels as targets of insecticides and insect resistance, especially the macrolide insecticides abamectin and ivermectin. In contrast, the mechanism of action of emamectin is limited, and its specific mechanism needs to be further elucidated. Studies have found that the resistance of western flower thrips to emamectin is mainly related to the overexpression of mutant GluCl in the cuticle. Among the three predicted GluCl genes, FoGluClc has four key amino acid changes, and its transcription levels in the head and cuticle of the emamectin-resistant strain are 3.8 times and 31 times higher than those of the sensitive strain, respectively. Backcross analysis and RNA interference experiments have shown that the amino acid substitution and increased expression of FoGluClc are key factors leading to emamectin resistance in western flower thrips (Gao et al., 2022). In Spodoptera frugiperda, knocking down SfGluCl through RNAi reduces the sensitivity of larvae to emamectin, and subsequent molecular docking shows that emamectin binds to the classic neurotransmitter site of the large amino-terminal extracellular structure of SfGluCl, so SfGluCl is likely to be the target of emamectin, but further experiments are needed to confirm (Wang et al., 2023). In addition, a study in Spodoptera exigua used Bulked Segregant Analysis (BSA) and CRISPR / Cas9 technology to find that the F116V point mutation in the CYP9A186 gene is associated with emamectin resistance (Zuo et al., 2021).

[0006] The existing detection method is usually a traditional biological detection technology, which needs to collect test insects and breed to a certain scale before detection, so the detection period is long; at the same time, the standardization requirement of the traditional biological detection technology for test insects leads to a large feeding pressure of test insects, and the error and individual difference of insect bodies have a great influence on the result. The detection speed, accuracy, sample demand and sensitivity need to be improved. At present, the research on the resistance mechanism of the diamondback moth to emamectin benzoate is still limited, and the genetic basis and key resistance genes of the resistance have not been determined, so there is a lack of fast and accurate early diagnosis technology for resistance in actual production.

[0007] Therefore, it is of great significance to scientifically guide field pesticide application, reduce pesticide abuse and improve the quality and safety level of vegetables by deeply exploring the resistance mechanism of the diamondback moth to emamectin benzoate, identifying the resistance-related genes and developing a practical resistance detection kit on this basis. SUMMARY

[0008] The present application aims to solve the problems of low sensitivity, long period and high requirement for experimental materials in the existing detection method of the resistance of the diamondback moth to emamectin benzoate, and provides the application of the cytochrome P450 gene CYP9G2 of the diamondback moth or a substance for detecting the expression amount of the cytochrome P450 gene CYP9G2 of the diamondback moth in identifying or assisting in identifying the resistance of the diamondback moth to emamectin benzoate, and a resistance detection method based on molecular biology technology and a corresponding primer pair.

[0009] The present application locates the resistance gene of the emamectin benzoate-resistant strain (TH-EB) of the diamondback moth in the 1-2 Mb interval of chromosome 17 through bulk segregant analysis (BSA), finds that the up-regulation of the expression of the P450 gene CYP9G2 (GenBank accession number: LOC105383605) is closely related to the emamectin benzoate resistance through gene screening, transcriptome and qPCR analysis, and verifies that the gene has metabolic activity on emamectin benzoate through cell expression and in-vitro metabolism experiments, and constructs an overexpression strain by using a piggyBac transposition system, and verifies that it can cause emamectin benzoate resistance. Based on the above findings, the present application establishes a method for judging the emamectin benzoate resistance of the diamondback moth by detecting the expression amount of the CYP9G2 gene.

[0010] The object of the present application can be achieved by the following technical solutions:

[0011] The first object of the present application is to provide the application of the cytochrome P450 gene CYP9G2 of the diamondback moth or a substance for detecting the expression amount of the cytochrome P450 gene CYP9G2 of the diamondback moth in identifying or assisting in identifying the resistance of the diamondback moth to emamectin benzoate.

[0012] Further, if the gene CYP9G2 of the to-be-detected diamondback moth sample is overexpressed, the sample is resistant to emamectin benzoate.

[0013] The cytochrome P450 gene CYP9G2 is located at about 1.4 Mb of chromosome 17 of the diamondback moth, and up-regulation of the expression of the gene can result in enhanced metabolic activity of the diamondback moth to emamectin benzoate, weaken the insecticidal activity of the pesticide, and thus produce resistance, significantly reducing the insecticidal effect of the pesticide.

[0014] Further, the substance for detecting the expression amount of the cytochrome P450 gene CYP9G2 of the diamondback moth is a specific primer for amplifying the gene CYP9G2, or a reagent or kit comprising the specific primer.

[0015] Further, the specific primer is a forward primer shown in SEQ ID NO. 1, and a reverse primer shown in SEQ ID NO. 2.

[0016] A second object of the present application is to provide a substance for detecting the resistance of the diamondback moth to emamectin benzoate, which is a specific primer for amplifying the gene CYP9G2, or a reagent or kit comprising the specific primer, and the specific primer is a forward primer shown in SEQ ID NO. 1, and a reverse primer shown in SEQ ID NO. 2.

[0017] The kit comprises various commonly used experimental components, such as reverse transcriptase, DNase, RNase inhibitor, dNTP mixture, specific primer, MgCl2 solution, hot-start Taq DNA polymerase, fluorescence quantitative reaction solution, buffer solution, and RNase-free water, etc. These components are conventional reagents in the field of molecular biology, and the experimenters can select and prepare them according to the specific experimental requirements.

[0018] A third object of the present application is to provide a quantitative PCR detection method for detecting the resistance of the diamondback moth to emamectin benzoate, which comprises the following steps:

[0019] (1) extracting total RNA of the diamondback moth sample to be tested, and reverse transcribing it into cDNA;

[0020] (2) mixing the forward primer shown in SEQ ID NO. 1 and the reverse primer shown in SEQ ID NO. 2 with the cDNA obtained in step (1) as a template, and adding common reaction components to obtain a PCR reaction system;

[0021] (3) performing real-time fluorescence quantitative PCR reaction on the PCR reaction system obtained in step (2), recording the Ct value (threshold cycle number) obtained after the test sample is subjected to the quantitative PCR reaction; and performing melting curve analysis after the real-time fluorescence quantitative PCR process is completed;

[0022] If the test sample produces a typical "S" type amplification curve and a single-peak melting curve, and the difference (i.e., ΔCt) between the Ct value of the target gene CYP9G2 and the Ct value of the internal reference gene β-actin is ≤ 6, it indicates that the test diamondback moth sample has developed resistance to emamectin benzoate; if the ΔCt value is > 6, it indicates that the test diamondback moth sample has not developed obvious resistance to emamectin benzoate.

[0023] Further, the real-time fluorescent quantitative PCR reaction system in step (3) comprises 2×ChamQ Universal SYBR qPCR Master Mix 10 μL, diamondback moth sample cDNA template 2 μL, 10 μM of primers each 1 μL, and double-distilled water to a total reaction volume of 25 μL; the amplification procedure of the real-time fluorescent quantitative PCR reaction is as follows: 95℃ pre-denaturation for 30 sec, 95℃ denaturation for 10 sec, 60℃ annealing for 30 sec, and 40 cycles are set.

[0024] Further, the melting curve procedure in step (3) is as follows: 65℃ for 5 s, then 65℃ to 95℃ at a rate of 0.5℃ / s, and finally 95℃ for 5 s.

[0025] Further, the primers for amplifying the internal reference gene β-actin in step (3) are as shown in SEQ ID NO. 3 and SEQ ID NO. 4.

[0026] Further, the test diamondback moth sample is a single-head sample or a mixed sample, and the diamondback moth is a fourth instar larva.

[0027] The fluorescent quantitative PCR reaction product in the above steps is subjected to fluorescent quantitative detection, and whether the gene is up-regulated is judged according to the Ct value difference (i.e., ΔCt) between the target gene CYP9G2 and the internal reference gene β-actin of each sample in the fluorescent detection. If the test sample produces a typical "S" type amplification curve and a single-peak melting curve, and the difference (i.e., ΔCt) between the Ct value of the target gene CYP9G2 and the Ct value of the internal reference gene β-actin is ≤ 5, it indicates that the test diamondback moth sample has developed resistance to emamectin benzoate; if the ΔCt value is ≥ 6, it indicates that the test diamondback moth sample has not developed obvious resistance to emamectin benzoate.

[0028] Beneficial effects

[0029] The present application provides the correlation between CYP9G2 and emamectin benzoate resistance, the up-regulated expression of the diamondback moth cytochrome P450 gene CYP9G2 is closely linked to the emamectin benzoate resistance phenotype, and the functional verification analysis is carried out through in vitro metabolism experiment, and the molecular mechanism that the diamondback moth enhances the metabolism ability of emamectin benzoate through CYP9G2 overexpression to produce drug resistance is clarified.

[0030] Further, the application establishes a molecular detection method for resistance of Plutella xylostella to emamectin benzoate.

[0031] Compared with the conventional technology (biological assay) for pest resistance determination, the application has the following significant advantages and positive effects:

[0032] (1) Fast detection speed: Traditional biological assay technology (such as resistance level determination, diagnostic dose analysis, etc.) requires collection of test insects and propagation to a certain scale before detection, and the whole process takes at least 3 weeks. The application can directly detect individual Plutella xylostella in the field, and the detection result is obtained only in 3-4 hours from sample extraction, greatly shortening the detection period.

[0033] (2) Accurate and reliable results: Traditional biological assay technology requires standardization of test insects, usually using 3rd instar larvae, and sampling error and individual differences of insects have a great influence on the results, leading to unstable detection results. The application is based on the principle of nucleotide amplification, and the amplification threshold is directly read by a quantitative PCR instrument, improving the accuracy of detection.

[0034] (3) Small sample requirement: In traditional biological assay technology, at least 200-300 standard test insects are required to determine a standard curve, which requires a lot of manpower and material resources for rearing. The application can complete the detection of a population with only 20-30 test insects, or even detect a single sample to determine the individual.

[0035] (4) High sensitivity: Traditional biological assay technology is a relatively rough resistance level determination method, which can only detect the overall toxicity response of 200-300 insects at different concentrations, and individual survival does not affect the results of biological assay, and cannot detect early emamectin benzoate resistance or low-frequency resistance individuals. The application is based on the expression changes of emamectin benzoate resistance-related genes in Plutella xylostella, and the Ct value obtained after quantitative PCR reaction with specific primers is used to determine whether the population or individual has resistance to emamectin benzoate, realizing the detection of a single individual and effectively improving the detection sensitivity.

[0036] In summary, the application is superior to traditional biological assay technology in terms of detection speed, accuracy, sample requirement and sensitivity, and provides more efficient and accurate technical support for Plutella xylostella resistance monitoring and management. The accurate detection result based on quantitative PCR can quickly guide the selection of drugs for Plutella xylostella in the field, and can provide timely and reliable technical support for resistance management and rational use of drugs in the field. BRIEF DESCRIPTION OF DRAWINGS

[0037] Figure 1 Emamectin benzoate resistance gene mapping of Plutella xylostella, wherein:

[0038] A is the resistance gene mapping based on the absolute value of allele frequency difference Δ (SNP-index);

[0039] B is the resistance gene mapping based on the G' value of genome sliding window statistical test.

[0040] Figure 2 Transcriptome data analysis of the resistant strain of Plutella xylostella to emamectin benzoate, wherein:

[0041] A is the number of differentially expressed genes between the sensitive strain and the emamectin benzoate-resistant strain of Plutella xylostella.

[0042] B is the volcano plot of differentially expressed genes between the sensitive strain and the emamectin benzoate-resistant strain of Plutella xylostella.

[0043] Figure 3 Schematic diagram of the construction of the CYP9G2 overexpression strain of Plutella xylostella, wherein:

[0044] A is a schematic diagram of the insertion position of the introduced CYP9G2 gene in the Plutella xylostella genome.

[0045] B is the genotyping of transgenic individuals based on PCR products of specific primers.

[0046] Figure 4 CYP9G2 gene-specific primer amplification sequence of Plutella xylostella.

[0047] Figure 5 From top to bottom, they are the amplification curve, amplification efficiency and melting curve of the CYP9G2 gene of Plutella xylostella.

[0048] Figure 6 β-actin gene-specific primer amplification sequence of Plutella xylostella.

[0049] Figure 7 From top to bottom, they are the amplification curve, amplification efficiency and melting curve of the β-actin gene of Plutella xylostella.

[0050] Figure 8 Amplification curve (left) and melting curve (right) of CYP9G2 and β-actin genes of Plutella xylostella SY population.

[0051] Figure 9 Amplification curve (left) and melting curve (right) of CYP9G2 and β-actin genes of Plutella xylostella CZ population.

[0052] Figure 10 Amplification curve (left) and melting curve (right) of CYP9G2 and β-actin genes of Plutella xylostella YX population. DETAILED DESCRIPTION

[0053] The present application is further explained in conjunction with the following examples, which do not purport to be of any form of limitation to the present application.

[0054] The experimental methods in the following examples are all conventional methods unless otherwise specified.

[0055] The materials, reagents and the like used in the following examples can be obtained from commercial channels unless otherwise specified.

[0056] Example 1 Discovery of the correlation between CYP9G2 and resistance to emamectin benzoate

[0057] This example illustrates the process of locating the emamectin benzoate resistance locus of Plutella xylostella and identifying the resistance candidate gene CYP9G2, and confirms the causal relationship between the up-regulation of the expression of the gene and the resistance to emamectin benzoate by over-expressing CYP9G2 in a sensitive strain of Plutella xylostella through transgenic experiments. The specific implementation process is as follows:

[0058] (1) Location of the emamectin benzoate resistance locus of Plutella xylostella:

[0059] ① Construction of a genetic mapping population by bulk segregant analysis (BSA)

[0060] The 3rd instar larvae of Plutella xylostella collected in Yunnan (TH22) were screened using 1 mg / mL emamectin benzoate, and the survivors were reared to adults. The adults were mated with the sensitive strain IPP-S in a single pair, and the F1 generation larvae were mated with each other after eclosion, and the operation was continued to the F6 generation (denoted as the TH-EB strain). The F6 generation 3rd instar larvae were treated with high-dose (1 mg / L) and low-dose (0.002 mg / L) emamectin benzoate, respectively. The individuals that survived under high-dose treatment constituted the resistant population, while the individuals that died under low-dose treatment constituted the sensitive population.

[0061] ② Extraction of gDNA from parent individuals, resistant population and sensitive population

[0062] In this experiment, the genomic DNA was extracted by the phenol-chloroform method, and the specific experimental steps are as follows:

[0063] The whole Plutella xylostella was placed in a 2 mL sterilized EP tube, a steel ball was added, and 500 μL of lysis solution (Tris 6.057 g, EDTA 9.305 g, NaCl 5.844 g, SDS 5 g, pH = 8.0) was added, and the sample was ground in a grinder for 5 min at 30 Hz;

[0064] The sample was taken out and placed in a 56°C water bath for 10 min;

[0065] 500 μL of DNA extraction reagent (phenol:chloroform:isopropyl alcohol = 25:24:1, pH > 7.8) was added to the EP tube, mixed well by inverting, and then left to stand for 3 min, and centrifuged at 14000 x g for 10 min;

[0066] Take the supernatant into a new 2mL EP tube, add 500μL DNA extraction reagent, mix well by inverting, stand for 3min, centrifuge at 14000xg for 10min;

[0067] Transfer the supernatant to a new 2mL EP tube, and add 500μL chloroform isoamyl alcohol mixture (chloroform: isoamyl alcohol = 24:1) to it, mix well by inverting, stand for 3min, centrifuge at 14000xg for 10min;

[0068] Transfer the supernatant to a new 1.5mL EP tube, add 400μL isopropanol, and put it in the -20℃ refrigerator for precipitation for at least 30min;

[0069] Take out the sample and put it in the 4℃ centrifuge, centrifuge at 14000xg for 10min, at this time the white precipitate can be seen at the bottom of the tube;

[0070] Discard the supernatant, wash the precipitate with pre-cooled 75% alcohol twice, centrifuge at 4℃, 14000xg for 10min;

[0071] Air dry the precipitate at room temperature, add 50μL Eluent to dissolve the DNA. Add 1μL RNase to it, incubate at 37℃ for 1h to completely degrade the RNA in the sample;

[0072] The DNA sample is detected by OneDrop spectrophotometer for concentration and absorbance value.

[0073] ③BSA sequencing and data processing

[0074] The qualified DNA samples are sent to Beijing Nuoweziyuan Technology Co., Ltd. for whole genome resequencing. Among them, the parent individuals are separately constructed, the F6 generation resistant population and the sensitive population are mixed with equal amount of DNA respectively, and the resistance pool and the sensitive pool are constructed, and then the PCR-free library construction is carried out. Use Illumina NovaSeq X Plus-PE150 sequencing platform for sequencing, the sequencing depth of parent is 30x, and the sequencing depth of mixed pool of offspring is ensured to have at least 1x coverage for each individual.

[0075] After sequencing, raw reads are obtained, which contain reads with adapters and low quality. In order to improve the accuracy and quality of data analysis, raw reads need to be screened according to specific standards to obtain clean reads. The final effective data is aligned to the reference genome of Plutella xylostella PxLV.1 (GCA_019096205.1).

[0076] ④SNP marker development and resistance locus determination

[0077] According to the parent detection results, two SNP sites with differences between the two parents and in homozygous state were screened and used as polymorphic markers. The genome of one parent was selected as a reference, and the SNP-index of the two offspring pools at the marker sites was calculated respectively. If the SNP-index is completely consistent with the reference genome, the value is 0; if it is completely inconsistent, the value is 1. Then, the Δ(SNP-index) of the two offspring pools was calculated, and the sliding window method was used to count the Δ(SNP-index) of each marker site in each window. 1000 permutation tests were performed, and a 95% confidence level was used as the screening threshold. Finally, those windows with Δ(SNP-index) greater than the threshold at a 95% confidence level were selected as candidate intervals, and the G value was used to identify and evaluate the statistical significance of QTL.

[0078] The results showed that only one locus was identified to be associated with the selection of methidathall-resistant P. xylostella, which was located near 1.4 Mb on chromosome 17 ( Figure 1 ), both of which exceeded the whole genome significance threshold (P<0.05). Therefore, this study selected 1-2 Mb as the candidate region of the resistance gene.

[0079] (2) Identification of resistance candidate gene CYP9G2

[0080] ① Transcriptome sequencing

[0081] After obtaining the candidate interval of the resistance gene, in order to determine whether there are differentially expressed genes in the candidate interval, 30 4th instar larvae of TH-EB strain were randomly selected, and IPP-S strain was used as the control group, each with three biological replicates. The insect body was ground and broken using liquid nitrogen, then Trizol reagent was added and frozen in liquid nitrogen, and stored in a -80°C refrigerator.

[0082] The prepared samples were sent to Beijing Novogene Bioinformatics Technology Co., Ltd. for transcriptome sequencing. After constructing the standard transcriptome library, Illumina HiSeq sequencing platform was used to perform double-end sequencing on these libraries. The amount of sequencing data for each sample was 6G. The raw sequences obtained by sequencing include sequences with adapters and low quality. In order to ensure the quality of analysis, it is necessary to filter the raw reads to obtain clean reads. All subsequent analyses will be based on clean reads. The distribution of clean reads after alignment with the reference genome was statistically analyzed, and the location regions included exons (Exon), introns (Intron) and intergenic regions (Intergenic). By comparing the FPKM density plots and violin plots of all genes in the sensitive and resistant strains, the differences in gene expression levels between the two strains can be directly obtained.

[0083] ii. Transcriptome data analysis

[0084] RNA-seq analysis results showed that a total of 931 genes were differentially expressed in the sensitive and resistant lines. Among them, 628 genes were up-regulated, and 303 genes were down-regulated Figure 2 A). The P450 gene CYP9G2 was found to be up-regulated in the 1-2 Mb region of chromosome 17 Figure 2 B), with a log2FC of 2.81. This indicates that this gene may be involved in the formation of TH-EB resistant Plutella xylostella to emamectin benzoate. The CYP9G2 gene is the target gene of the present case.

[0085] (3) CYP9G2 overexpression leads to emamectin benzoate resistance in Plutella xylostella

[0086] To verify the causal relationship between CYP9G2 gene overexpression and emamectin benzoate resistance in Plutella xylostella, a piggyBac transgenic system was used to successfully construct a Plutella xylostella transgenic overexpression line expressing PxCYP9G2 gene, and the contribution of the gene to emamectin benzoate resistance in Plutella xylostella was determined.

[0087] i. Construction of transgenic lines

[0088] The mRNA (400 ng / μL) expressing transposase and the transgenic recombinant plasmid (500 ng / μL) were injected into Plutella xylostella eggs using a microinjector. After microinjection, the hatched larvae were fed to the adult stage. Subsequently, all G0 individuals were mated in groups. After reaching the 2nd instar, the G1 larvae were screened for green fluorescent positive individuals using a Nikon SMZ25 body fluorescence microscope (Nikon, Japan), and then transferred to artificial feed for normal feeding. The green fluorescent G1 positive individuals were mated with the background line IPP-S in a single pair after feeding to adulthood. After the offspring were laid, the genomic DNA of the positive parent individuals was extracted. The qualified DNA was sent to Beijing Novogene Biotechnology Co., Ltd. for whole genome resequencing. The resequencing alignment results showed that PxCYP9G2 was inserted into the 5.4 Mb region of chromosome 3 of Plutella xylostella Figure 3 A).

[0089] The 43 green fluorescent marker larvae of the single pair G2 generation were fed to adulthood and mated in groups to produce the G3 generation. The green fluorescent marker adults of the G3 generation were mated in a single pair to obtain 26 effective single pairs. After the offspring were collected, 48 parents were subjected to PCR amplification, and the expected 5.3 kb insertion was genotyped, of which 8 heads of parents of 4 single pairs were 5.3 kb insertion homozygotes Figure 3B). The offspring were pooled and reared to form transgenic line PxCYP9G2.

[0090] 2. Transgenic line target gene expression level and its emamectin benzoate resistance level.

[0091] The mRNA level of PxCYP9G2 gene in the 4th instar larvae of the transgenic line of diamondback moth was determined by RT-qPCR. Compared with the background line IPP-S, the expression level of PxCYP9G2 in the PxCYP9G2 line increased by 55 times. The results of the bioassay carried out simultaneously showed that the PxCYP9G2 line produced 15.5 times of resistance to emamectin benzoate. Therefore, this part of the functional experiment not only verifies the causal relationship between the overexpression of CYP9G2 gene and emamectin benzoate resistance found in the previous part, but also through the same genetic background material, it is clear that the contribution of the single genetic factor (i.e. CYP9G2 overexpression) to emamectin benzoate resistance.

[0092] Example 2 Design of CYP9G2 detection method

[0093] In this example, the indoor sensitive line IPP-S and the resistant line TH-EB obtained by emamectin benzoate selection were subjected to bioassay, and the expression levels of the target genes of the two lines were detected by using the preferred technology of the present application. The toxic reaction of the above-mentioned lines to emamectin benzoate was determined by the feed coating method, and the specific data are shown in the following table.

[0094]

[0095] Note: The IPP-S line is a sensitive material that has been reared in the laboratory for many years, and its median lethal concentration (LC 50 value) to emamectin benzoate in this experiment is used as a control baseline to determine the resistance level of other insects.

[0096] The specific implementation steps of the molecular detection technology include:

[0097] This example sets 6 biological replicates, each containing 5 4th instar diamondback moth larvae, and the RNA extraction steps are as follows:

[0098] (1) Put the sample into a 2 mL RNase-free EP tube, add 1 steel ball and 1 mL Trizol reagent to it, and use a grinder to grind it thoroughly (30 Hz, 200 s);

[0099] (2) Add 200 μL of chloroform and mix thoroughly by oscillation, and stand at room temperature for 5 min;

[0100] (3) Centrifuge at 13000 x g at 4°C for 15 min. After centrifugation, the solution is divided into three layers, and the RNA is in the colorless upper layer;

[0101] (4) Carefully transfer the supernatant to another 1.5 mL RNase-free tube, then add an equal volume of isopropanol to the supernatant, mix gently until a flocculent precipitate appears. Then, let it stand at room temperature for 10 minutes;

[0102] (5) 13000 x g centrifugation at 4°C for 15 min. After centrifugation, a white precipitate can be seen at the bottom of the EP tube;

[0103] (6) Discard the supernatant, add 1 mL of 75% ethanol to the tube, mix gently to resuspend the precipitate, and centrifuge at 13000 x g at 4°C for 5 min;

[0104] (7) Repeat step 6, discard the supernatant, and centrifuge again for 1 min to remove the residual ethanol on the tube wall;

[0105] (8) Use an RNase-free gun tip to absorb the ethanol and dry it on a clean bench, then add 50 μL of RNase-free water to dissolve the RNA;

[0106] (9) After complete dissolution, take part of the sample to detect the concentration and absorbance value using OneDrop, and verify the RNA integrity by electrophoresis.

[0107] Then, cDNA synthesis was performed using HiScript III RT SuperMix for qPCR (+gDNA wiper) (Novogene), with the following specific steps:

[0108] (1) Genomic DNA removal: Prepare the following reaction in a 200 μL RNase-free centrifuge tube:

[0109]

[0110] Mix by pipetting, and incubate at 42°C for 2 min.

[0111] (2) Prepare the reverse transcription system: directly add 4 μL of 5x HiScript III qRT SuperMix to the system from step (1) after the reaction is complete, and mix by pipetting.

[0112] (3) Reverse transcription reaction conditions: incubate at 37°C for 15 min, and terminate the reaction at 85°C for 5 s. The reaction solution after completion is stored in a -20°C refrigerator for standby.

[0113] Dilute the cDNA 15-fold, and perform qPCR detection using ChamQ Universal SYBR qPCR Master Mix (Novogene). Prepare the following mixed system in a 96-well plate:

[0114]

[0115]

[0116] The specific primers for amplifying the Plutella xylostella cytochrome P450 gene CYP9G2 are as follows:

[0117] CYP9G2-F: 5'-ACGCTTCTCGACTGATTCGT-3' (SEQ ID NO. 1);

[0118] CYP9G2-R: 5'-TGGTCGTTCGATGTTGTTGT-3' (SEQ ID NO. 2);

[0119] The Plutella xylostella cytochrome P450 gene CYP9G2 gene is amplified by PCR, and the cDNA length is 284 bp. Figure 4 The figure shows the amplified sequence of the Plutella xylostella CYP9G2 gene conserved fragment, and the upstream CYP9G2-F is SEQ ID NO. 1, and the downstream CYP9G2-R is SEQ ID NO. 2.

[0120] The PCR reaction system is prepared, including 2xChamQ Universal SYBR qPCR Master Mix 10 μL, gradient diluted cDNA template extracted from the Plutella xylostella sensitive strain IPP-S 2 μL, 10 μM CYP9G2 primers each 1 μL, and double distilled water to a total reaction volume of 25 μL. The reaction system is placed in a CFX Connect real-time fluorescent quantitative PCR instrument (Bio-Rad), the emission wavelength is 450-580 nm, the amplification program is: 95°C pre-denaturation for 30 sec, 95°C denaturation for 10 sec, 60°C annealing for 30 sec, and 40 cycles are set. After the cycle is completed, the melting curve program is run, that is, 65°C for 5 s, then the temperature is raised from 65°C to 95°C at a rate of 0.5°C / s, and finally 95°C for 5 s.

[0121] The results are shown in Figure 5 The amplification product of the specific primer pair used in the present case shows a clear S-shaped curve, the baseline is straight, and there is no obvious upward trend; the curve inflection point is clear, the exponential phase slope is proportional to the amplification efficiency Figure 5 ), the amplification curve slope is -3.26 Figure 5 ), the amplification efficiency is 103%, which meets the detection requirements. By observing the melting curve of the reaction, it is found that there is only one main peak at 83°C Figure 5 ), which indicates that the primer has good specificity and no primer dimer or other non-target products appear.

[0122] The specific primers for amplifying the reference gene β-actin of the diamondback moth are as follows:

[0123] β-actin-F: 5'-CGACTTGACCGACTACCT-3' (SEQ ID NO. 3);

[0124] β-actin-R: 5'-AGCAGAGCTTCTCCTTGA-3' (SEQ ID NO. 4);

[0125] The reference gene β-actin is amplified by PCR, and the length of the cDNA is 104 bp. Figure 6 The figure shows the amplified sequence of the most commonly used reference gene β-actin in the art, and the upstream of the β-actin-F is SEQ ID NO. 3, and the downstream of the β-actin-R is SEQ ID NO. 4.

[0126] The PCR reaction system is prepared, including 2xChamQ Universal SYBR qPCR Master Mix 10 μL, gradient-diluted cDNA template extracted from the sensitive strain IPP-S of the diamondback moth 2 μL, 10 μM of β-actin primers each 1 μL, and double-distilled water to a total reaction volume of 25 μL; the amplification procedure of the real-time fluorescent quantitative PCR reaction is as follows: 95°C pre-denaturation for 30 sec, 95°C denaturation for 10 sec, 60°C annealing for 30 sec, and 40 cycles are set. After the cycle, the melting curve program is run, that is, 65°C for 5 s, then the temperature is increased from 65°C to 95°C at a rate of 0.5°C / s, and finally 95°C for 5 s.

[0127] The results are shown in Figure 7 The amplification product of the specific primer pair used in the present case shows a clear S-shaped curve, the baseline period shows a straight line without obvious upward trend; the curve inflection point is clear, the exponential period slope is proportional to the amplification efficiency Figure 7 (the upper figure), the amplification curve slope is -3.27 Figure 7 (the middle figure), the amplification efficiency is 102%, which meets the detection requirements. By observing the melting curve of the reaction, it is found that there is only one main peak at 83°C Figure 7 (the lower figure), which indicates that the primer has good specificity and no primer dimer or other non-target products are present.

[0128] Further, the DNA extracted from the aforementioned sensitive strain IPP-S and emamectin benzoate-resistant material TH-EB is used as a template to obtain cDNA products, and the reaction system is placed into a CFX Connect real-time fluorescent quantitative PCR instrument (Bio-Rad) for quantitative PCR amplification.

[0129] The Ct values read by the real-time fluorescence quantitative PCR instrument were analyzed as shown in the following table. The average Ct value of the β-actin gene of the indoor sensitive IPP-S strain was 17.8, the average Ct value of the target gene CYP9G2 was 23.84, and the ΔCt value was 6.65. The average Ct value of the TH-EB strain artificially selected for resistance to emamectin benzoate was 18.61, the average Ct value of the target gene CYP9G2 was 20.66, and the ΔCt value was 2.05.

[0130]

[0131] Explanation of ΔCt value determination standard: The sensitive strain used in this case is IPP-S, which has been cultured indoors for more than 15 years and has not been exposed to any pesticides. It is highly sensitive to a variety of insecticides, including emamectin benzoate. The ΔCt value obtained from testing this strain is 6.65. Rounding to the nearest whole number, i.e., using 6 as the determination standard, facilitates practical operation and uniform comparison. Based on common sense in the field of pest resistance and the occurrence of diamondback moths in the field, theoretically, the ΔCt of other populations with different levels of resistance will be between 0 and 6. Therefore, the determination standard for resistant diamondback moths in this case is ΔCt ≤ 6.

[0132] According to the aforementioned resistance determination rule based on ΔCt value, i.e., the difference (ΔCt) between the Ct value of the target gene CYP9G2 and the Ct value of the internal reference gene β-actin ≤ 6, it indicates that the TH-EB strain of diamondback moth tested has developed resistance to emamectin benzoate, which is consistent with the 155-fold resistance level obtained from biological assays. The ΔCt value of the IPP-S strain is > 6, indicating that this strain has not developed significant resistance to emamectin benzoate, which is consistent with the toxicological characteristics of this strain being highly sensitive to pesticides. Therefore, the results obtained from the detection method for emamectin benzoate resistance in diamondback moths based on the CYP9G2 gene are accurate and reliable.

[0133] Example 3

[0134] To further test whether CYP9G2 is involved in the evolution of emamectin benzoate resistance in diamondback moth populations in the field, we collected diamondback moth populations from Sanya (SY) in Hainan, Changzhou (CZ) in Jiangsu, and Yuxi (YX) in Yunnan, respectively. The leaf dipping method was used to measure the median lethal concentration of emamectin benzoate for each population, which was 0.568 mg / L, 0.149 mg / L, and 0.256 mg / L, respectively. Compared with the indoor sensitive strain (0.002 mg / L), the three geographic populations had resistance to emamectin benzoate by 284-fold, 74.5-fold, and 128-fold, respectively.

[0135] DNA was extracted from the above three populations, with 5 fourth instar diamondback moth larvae mixed as one sample, and 6 biological replicates were set. The specific operation steps include:

[0136] (1) Take various population samples into 2 mL RNase-free EP tubes, add 1 steel ball and 1 mL Trizol reagent to each, and use a grinder to grind thoroughly (30 Hz, 200 s);

[0137] (2) Add 200 μL chloroform, mix thoroughly by oscillation, and stand at room temperature for 5 min;

[0138] (3) Centrifuge at 13000 x g at 4°C for 15 min. After centrifugation, the solution is divided into three layers, with RNA in the colorless upper layer;

[0139] (4) Carefully transfer the supernatant to another 1.5 mL RNase-free tube, then add an equal volume of isopropanol to the supernatant, mix gently by inversion until a flocculent precipitate appears. Then, stand at room temperature for 10 min;

[0140] (5) Centrifuge at 4°C, 13000 x g for 15 min. After centrifugation, a white precipitate is formed at the bottom of the EP tube;

[0141] (6) Discard the supernatant, add 1 mL of 75% ethanol to the tube, mix gently to resuspend the precipitate, centrifuge at 4°C, 13000 x g for 5 min;

[0142] (7) Repeat step 6, discard the supernatant, and centrifuge again for 1 min to remove the residual ethanol on the tube wall;

[0143] (8) Use an RNase-free gun tip to absorb the ethanol, and dry in a clean bench. Add 50 μL RNase-free water to dissolve the RNA;

[0144] (9) After complete dissolution, take part of the sample to detect the concentration and absorbance value using OneDrop, and verify the RNA integrity by electrophoresis.

[0145] Then, cDNA synthesis was performed using HiScript III RT SuperMix for qPCR(+gDNA wiper) (Novogene), with the following specific steps:

[0146] (1) Genomic DNA removal. Prepare the following reaction in a 200 μL RNase-free centrifuge tube:

[0147]

[0148]

[0149] Mix by pipetting, incubate at 42°C for 2 min.

[0150] (2) Preparation of reverse transcription system: directly add 4 μL 5x HiScript III qRT SuperMix to the reaction system of step (1) and mix well with a pipette.

[0151] (3) Reverse transcription reaction conditions: incubate at 37°C for 15 min, terminate the reaction at 85°C for 5 s. The reaction solution after completion is stored in a refrigerator at -20°C for standby. Dilute the cDNA 15-fold and use ChamQ Universal SYBR qPCR Master Mix (Novozyme) for qPCR detection. Prepare the following mixed system in a 96-well plate:

[0152]

[0153] Prepare the PCR reaction system, including 2x ChamQ Universal SYBR qPCR Master Mix 10 μL, gradient-diluted cDNA templates of various populations of Plutella xylostella 2 μL, 10 μM CYP9G2 primers or β-actin primers each 1 μL, and double-distilled water to a total reaction volume of 25 μL. Place the reaction system in a CFX Connect real-time fluorescent quantitative PCR instrument (Bio-Rad), with an emission wavelength of 450-580 nm, and an amplification program of 95°C pre-denaturation for 30 sec, 95°C denaturation for 10 sec, 60°C annealing for 30 sec, with 40 cycles. After the cycles, run the melting curve program, i.e. 65°C for 5 s, then increase from 65°C to 95°C at a rate of 0.5°C / s, and finally 95°C for 5 s.

[0154] The results are shown in Figures 8-10 The amplification products of the CYP9G2 primers or β-actin primers used in the present case all showed obvious S-shaped curves in the three test populations Figures 8-10 , which were flat in the amplification baseline period without obvious upward trend; the amplification curve inflection point was clear, and an obvious plateau appeared in the late PCR reaction, indicating that it met the detection requirements. In the three Plutella xylostella populations, the melting curves of CYP9G2 primers and β-actin met the requirements of quantitative PCR detection, with a single main peak appearing near 80°C, indicating that the primers had good specificity and no non-specific amplification occurred.

[0155] The Ct values read by the real-time fluorescence quantitative PCR instrument are analyzed, and it is found that the Ct average of the beta-actin gene of the Sanya (SY) population in Hainan is 16.63, the Ct average of the target gene CYP9G2 is 17.05, and the ΔCt value is 0.42; it is found that the Ct average of the beta-actin gene of the Changzhou (CZ) population in Jiangsu is 17.02, the Ct average of the target gene CYP9G2 is 17.49, and the ΔCt value is 0.47; it is found that the Ct average of the beta-actin gene of the Yuxi (YX) population in Yunnan is 16.55, the Ct average of the target gene CYP9G2 is 17.05, and the ΔCt value is 0.50.

[0156]

[0157] The analysis of the experimental data shows that the ΔCt between the target gene CYP9G2 and the internal reference gene beta-actin of the Sanya (SY), Changzhou (CZ) and Yuxi (YX) populations in Hainan, Jiangsu and Yunnan is significantly less than 6. According to the aforementioned resistance determination rule based on the ΔCt value, it can be known that the Sanya (SY), Changzhou (CZ) and Yuxi (YX) populations collected in the field all produce obvious resistance to emamectin benzoate, which is consistent with the result of the bioassay, indicating that the result obtained by the detection technology described in the case is accurate and reliable, and is suitable for the rapid detection of emamectin benzoate resistance of field populations. The target gene CYP9G2 described in the application is a molecular marker closely linked to the emamectin benzoate resistance of the diamondback moth, and can be used for the molecular detection of the emamectin benzoate resistance of the diamondback moth.

[0158] The above only describes the preferred embodiments of the present application, and it should be noted that, for those skilled in the art, some improvements and refinements can be made without departing from the principles of the present application, and these improvements and refinements should also be considered as the protection scope of the present application.

Claims

1. Application of the diamondback moth cytochrome P450 gene CYP9G2 or substances for detecting the expression level of the diamondback moth cytochrome P450 gene CYP9G2 in the identification or auxiliary identification of diamondback moth resistance to emamectin benzoate.

2. The application according to claim 1, characterized in that, If the diamondback moth sample being tested has overexpression of the CYP9G2 gene, it is a sample that has developed resistance to emamectin benzoate.

3. The application according to claim 1, characterized in that, The substance used to detect the expression level of the diamondback moth cytochrome P450 gene CYP9G2 is a specific primer for amplifying the CYP9G2 gene, or a reagent or kit containing the specific primer.

4. The application according to claim 3, characterized in that, The specific primers are the forward primer shown in SEQ ID NO.1 and the reverse primer shown in SEQ ID NO.

2.

5. A substance for detecting resistance of diamondback moth to emamectin benzoate, characterized in that, The substance is a specific primer for amplifying the gene CYP9G2, or a reagent or kit containing the specific primer, wherein the specific primer is the forward primer shown in SEQ ID NO.1 and the reverse primer shown in SEQ ID NO.

2.

6. A quantitative PCR detection method for detecting resistance of diamondback moth to emamectin benzoate, characterized in that, The method includes the following steps: (1) Total RNA was extracted from the diamondback moth samples to be tested and reverse transcribed into cDNA; (2) Mix the forward primer shown in SEQ ID NO.1 and the reverse primer shown in SEQ ID NO.2 with the cDNA obtained in step (1) as a template, and add common reaction components to obtain a PCR reaction system; (3) Perform real-time quantitative PCR on the PCR reaction system obtained in step (2), and record the Ct value (threshold cycle number) obtained after the quantitative PCR reaction of the test sample; analyze the melting curve after the real-time quantitative PCR process is completed. If the test sample produces a typical "S"-shaped amplification curve and a single-peak melting curve, and the difference between the Ct value of the target gene CYP9G2 and the Ct value of the internal reference gene β-actin (i.e., ΔCt) is ≤6, it indicates that the tested diamondback moth sample has developed resistance to emamectin benzoate; if the ΔCt value is >6, it indicates that the tested diamondback moth sample has not developed significant resistance to emamectin benzoate.

7. The quantitative PCR detection method according to claim 6, characterized in that, The real-time quantitative PCR reaction system described in step (3) includes 10 μL of 2×ChamQ Universal SYBR qPCR Master Mix, 2 μL of diamondback moth cDNA template, 1 μL of 10 μM primers, and double-distilled water to a total reaction volume of 25 μL. The amplification program for the real-time quantitative PCR reaction is as follows: 95℃ pre-denaturation for 30 sec, 95℃ denaturation for 10 sec, 60℃ annealing for 30 sec, for 40 cycles.

8. The quantitative PCR detection method according to claim 6, characterized in that, The melting curve program in step (3) is to run at 65°C for 5 seconds, then increase the temperature from 65°C to 95°C at a rate of 0.5°C / s, and finally run at 95°C for 5 seconds.

9. The quantitative PCR detection method according to claim 6, characterized in that, The primer pairs used in step (3) to amplify the internal reference gene β-actin are shown in SEQ ID NO.3 and SEQ ID NO.

4.

10. The quantitative PCR detection method according to claim 6, characterized in that, The diamondback moth samples to be tested are either single-headed or mixed samples, and the diamondback moths are fourth-instar larvae.

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