CN116287316B - Molecular markers of malathion resistance in Bactrocera dorsalis and their applications and detection methods - Google Patents

Abstract

The present invention discloses a molecular marker for malathion resistance of the oriental fruit fly and its application in malathion content detection. The molecular marker is the oriental fruit fly odor-binding protein BdorOBP28a-2 gene. Also disclosed are a primer pair, a kit, and a detection method for detecting the molecular marker: using an oriental fruit fly pesticide-sensitive population as a control, detecting the expression level of the OBP28a-2 gene of the oriental fruit fly to be tested; if the expression level of the OBP28a-2 gene of the oriental fruit fly to be tested is significantly higher than that of the sensitive population, it can be determined that the oriental fruit fly to be tested is a malathion-resistant population; if there is no significant difference in the expression level of the OBP28a-2 gene between the oriental fruit fly to be tested and the sensitive population, it cannot be determined that the oriental fruit fly to be tested is a malathion-resistant population. The present invention identifies and verifies for the first time that the oriental fruit fly odor-binding protein gene OBP28a-2 regulates the oriental fruit fly's resistance to malathion.

Description

Molecular marker of resistance of bactrocera dorsalis to malathion, application and detection method thereof

Technical Field

The invention belongs to the technical field of molecular biology, and relates to a molecular marker, in particular to a molecular marker of resistance of bactrocera dorsalis to malathion, and application and a detection method thereof.

Background

Insect odor binding proteins (odorant-bindingproteins, OBPs) are a hydrophilic carrier protein, and the transport of hydrophobic odor molecules to olfactory receptor neurons via hydrophilic lymph is a very important protein in insect olfactory perception. Bactrocera dorsalis Bactrocera dorsalis (Hendel) is an important agricultural pest, and has the characteristics of strong environmental adaptability, wide host range, rapid invasion and diffusion and the like, thereby causing great economic loss to agricultural production. In recent years, due to the wide and large-scale use of chemical pesticides, the drug resistance of the bactrocera dorsalis is obviously enhanced, and great difficulty is brought to the control of the bactrocera dorsalis. At present, the research on the drug resistance mechanism of bactrocera dorsalis is mainly focused on metabolic resistance and target resistance, and three detoxified metabolic enzymes are mainly used, but recently, reports have found that the odor binding protein and other proteins participating in the olfactory function of insects have special effects in regulating the drug resistance of insects to drugs.

Disclosure of Invention

The invention aims at providing a molecular marker of resistance of bactrocera dorsalis to malathion, and application and detection method thereof.

In order to achieve the purpose, the invention adopts the following technical scheme:

a molecular marker of the resistance of the bactrocera dorsalis to malathion is provided, wherein the molecular marker is a bactrocera dorsalis odor binding protein BdorOBP a-2 gene, and the nucleotide sequence of the molecular marker is shown as SEQ ID No. 23.

The invention provides application of the molecular marker in malathion content detection.

In the application technical scheme, a plurality of malathion standard products with known concentrations are used for detecting the affinities of the OBP28a-2 protein and the standard products, an affinity curve is fitted by taking Ma Laliu concentration as an abscissa and an affinity detection fluorescence value as an ordinate, the affinities of a to-be-detected product containing malathion and the OBP28a-2 protein are detected, and the malathion concentration of the to-be-detected sample is obtained according to the affinity curve of the standard products;

preferably, a micro thermal surge technology is adopted to detect the affinity of the OBP28a-2 protein and a malathion sample;

Preferably, the malathion concentration of the sample to be measured is 1M-100 nM.

The invention also provides application of the molecular marker in classifying the malathion sensitive population and the resistant population of the bactrocera dorsalis.

The invention also provides a primer pair for detecting the molecular marker, the primer sequence of the primer pair is that an upstream primer is OBP28a-2qPCR F:5'-GGTGCTGCTGACTCTGACAT-3',

The downstream primer is OBP28a-2qPCR R5 '-GCCTCGCAATGATCATCTGG-3'.

The invention also provides a kit for detecting the molecular marker, which comprises the primer pair.

Preferably, the kit further comprises a total RNA extraction reagent, an RNA reverse transcription reagent and a real-time fluorescent quantitative PCR amplification reagent.

The invention finally provides a detection method of the molecular marker, which takes the sensitive population of the bactrocera dorsalis medicament as a contrast, detects the expression quantity of the OBP28a-2 gene of the bactrocera dorsalis to be detected, if the expression quantity of the OBP28a-2 gene of the bactrocera dorsalis to be detected is obviously higher than that of the sensitive population, the bactrocera dorsalis to be detected can be judged as a resistance population to malathion, and if the expression quantity of the OBP28a-2 gene of the bactrocera dorsalis to be detected and that of the sensitive population have no obvious difference, the bactrocera dorsalis to be detected cannot be judged as the resistance population to malathion.

The technical scheme of the method comprises the following steps of extracting total RNA of the bactrocera dorsalis to be detected, reversing the total RNA into cDNA, carrying out qPCR amplification by using the cDNA as a template and using the primer pair, and measuring the expression level of the OBP28a-2 gene in an object to be detected by using the bactrocera dorsalis pesticide sensitive population as a contrast, wherein when the expression level of the OBP28a-2 gene of the object to be detected is obviously higher than that of the sensitive population, the bactrocera dorsalis to be detected can be judged to be a malathion resistant population.

Preferably, in the technical scheme of the method, the qPCR amplification reaction system comprises NovoStart SYBR qPCR S mu perMix Pl mu s 5.0 mu L,10 mu M of upstream/downstream primer 0.3 mu L, 0.5 mu L of cDNA template and no ribozyme water to 10 mu L, wherein the qPCR amplification conditions comprise 95 ℃ pre-denaturation for 2min, 95 ℃ denaturation for 30s,60 ℃ annealing extension for 30s and 40 cycles, and 60 ℃ 30s and 95 ℃ 15s to form a melting curve.

Early studies in the laboratory by the inventors identified the odor binding protein genes of bactrocera dorsalis, including OBP28a-2, OBP49a, OBP56g, OBP57e, and the like. The invention adopts an RT-qPCR method to detect the expression level of the high-expression OBPs of the bactrocera dorsalis foot under different populations and malathion induction modes. The results indicate that OBP28a-2 expression is significantly up-regulated in malathion resistant populations and that OBP28a-2 expression is also significantly increased after malathion induction. The OBP28a-2 ^-/- mutant produced by CRISPR/Cas9 increased the sensitivity of adults to malathion. Subsequently, we tested the in vitro affinity of OBP28a-2 protein with pesticides, and experimental results show that OBP28a-2 has a strong affinity with malathion, which indicates that OBP28a-2 participates in regulating and controlling the sensitivity of bactrocera dorsalis to malathion. According to the research, the odor binding protein OBP28a-2 of the bactrocera dorsalis is used for regulating and controlling the resistance of the bactrocera dorsalis to malathion, meanwhile, the OBP28a-2 can be used as an indicator gene for monitoring the resistance of the bactrocera dorsalis to malathion, and meanwhile, the in vitro synthesized protein can be used for detecting the content of malathion.

The method has the beneficial effects that the resistance of the bactrocera dorsalis to malathion is regulated and controlled by the bactrocera dorsalis smell binding protein gene OBP28a-2 through first identification verification, the function cognition of the bactrocera dorsalis smell binding protein is widened, and a search idea is provided for identification of other medicament related smell binding proteins. Upregulated expression of the odor binding protein OBP28a-2 was used as an indicator of increased malathion resistance, and differences in resistance were indicated by fold change in upregulation. The novel method for detecting the malathion content in trace is provided by utilizing the OBP28a-2 protein in vitro and an affinity test method, and can be applied as a novel method for detecting malathion residues of agricultural products, crops and the like.

The invention establishes the molecular marker for rapidly detecting the resistance of the bactrocera dorsalis to malathion, has simple operation, strong detection specificity and high sensitivity, can simultaneously detect a large number of samples, and can rapidly and effectively detect whether the field population of the bactrocera dorsalis has resistance to malathion. Is beneficial to early monitoring of drug resistance of field bactrocera dorsalis and provides a new action target for developing new pesticides.

Drawings

FIG. 1 is a graph showing the expression pattern of odor binding proteins under different conditions.

FIG. 2 is an electrophoresis result of the OBP28a-2 ^-/- mutant strain.

FIG. 3 shows the results of malathion bioassays for OBP28a-2 ^-/- mutant lines.

FIG. 4 shows the result of SDS-PAGE and western blot of OBP28a-2 protein.

FIG. 5 shows the results of an affinity test for OBP28a-2 protein with malathion agents.

Detailed Description

The invention is further illustrated, but is not limited, by the following examples.

The experimental methods in the following examples are all conventional methods unless otherwise specified, and the chemical and biological reagents used, unless otherwise specified, are all conventional chemical and biological reagents in the art and are commercially available.

Malathion, a solid standard available from LGC technologies (Shanghai) Inc., 99.4% purity, chemical formula C ₁₀H₁₉O₆PS₂, CAS registry number 121-75-5.

EXAMPLE 1 determination of malathion LC based on drug film method ₅₀

Malathion raw material (purity 99.4%) is dissolved in acetone as raw liquid, 0.1g raw material is taken and dissolved in 10mL acetone to obtain raw liquid with concentration of 10000 mg/L. Stock solutions were diluted to 6 different concentrations (125 mg/L,100mg/L,75mg/L,50mg/L,25mg/L,12.5mg /) and then 0.8mL of each dilution was placed in 6 100mL glass bottles. Immediately, the vial was rolled for 5min to uniformly distribute the diluent residue on the inner surface of the vial, leaving the vial mouth open and stationary for 2h, allowing the acetone to evaporate, and then covering the vial mouth with cotton. The insecticide-containing vials were then used for toxicity tests and vials treated with acetone were used as controls.

To test for toxicity, 5 day old adult bactrocera dorsalis was frozen and anesthetized for 2min, then 15-20 bactrocera dorsalis were placed in each vial, the mouth was plugged with cotton to prevent the adults from escaping, and each treatment was repeated 3 times. The glass flask containing the fly for the biological test was placed in an incubator (temperature: 27.+ -. 0.5 ℃ C.; relative humidity: 70.+ -. 5% and photoperiod: 14 hours light/10 hours dark). In each case, bactrocera dorsalis was exposed to insecticide for 2 hours, then removed and placed in a clean cage for 24 hours, and then the mortality was counted. When the bactrocera dorsalis is touched with a soft brush, it is considered to have died when either none or only one of the legs moves. Mortality after insecticide treatment was considered to have occurred when the mortality rate of the control group was 10%. The bioassay results are shown in Table 1, and the concentration in death was 44.44mg/L.

TABLE 1 biological assay results of malathion

EXAMPLE 2 expression patterns of OBPs at different developmental stages

Extracting total RNA of 2 whole insects, and detecting the expression profile of OBPs genes in a resistant population and a sensitive population. Sensitive populations refer to the passage of feed without any pesticide contact after 2009 collection to the laboratory. After the resistant population is collected from 2008 to a laboratory, the concentration of malathion in each generation is continuously increased by treating and screening malathion medicament in each generation, the resistance multiple of the currently used resistant population is 50 times that of the sensitive population, and the malathion LC ₅₀ value reaches 6600mg/L. Extracting total RNA of bactrocera dorsalis by using a TRIzol reagent, putting 2 whole insects into a 1.5mL coreless enzyme centrifuge tube, treating with liquid nitrogen, adding 500 mu LTRIzol, grinding the whole insects into homogenate, incubating for 5-10min at room temperature, then adding 200 mu L chloroform, vortex shaking for 15s, incubating for 2-3 min at room temperature, incubating for 4 ℃ and centrifuging for 15min at 12000g, sucking out supernatant of the layering liquid, adding 500 mu L isopropanol, gently shaking and mixing, standing and incubating for 10min at room temperature, centrifuging for 4 ℃ and 12000g for 10min, discarding the supernatant, retaining white precipitate, adding 1mL of 75% ethanol to wash the white precipitate, centrifuging for 5min at 4 ℃ and 7600g, discarding the supernatant, drying the precipitate for 5-10min at room temperature, adding 30-100 mu L of coreless enzyme water, dissolving the precipitate, sucking and mixing. 1.5 mu LRNA was assayed for concentration and purity using a Nanodrop ONE (ThermoFisher, USA), 5 mu L was subjected to agarose gel electrophoresis to detect RNA integrity, and the qualified RNA was stored in a-80℃refrigerator for use.

Treating a qualified RNA sample with RQI DNase kit (Promega Co., USA), removing genomic DNA from the sample, and obtaining a purified RNA sample according toRT REAGENT KIT instructions of kit (Takara Co., japan), the first strand cDNA was synthesized by reverse transcription.

To determine whether OBPs could be expressed by malathion, total RNA from 4 adult feet after 2h induction with acetone and LC ₅₀ malathion (44.44 mg/L) was extracted (induction method was the same as in the bioassay method of example 1: i.e., the adults were induced in glass flasks with a drug film on the inner wall). There were 3 biological replicates per treatment. RNA extraction and cDNA acquisition methods were as above.

All primers for OBPs were designed via the on-line site Primer3 (http:// Primer3.Ut. Ee /). RT-qPCR experiments and results were performed with bactrocera dorsalis RPS3 (GenBank: XM_ 011212815) and alpha-tublin (GenBank: GU 269902) as reference genes. The detected OBPs include OBP28a-2, OBP49a, OBP56g, OBP57e. The names of the genes corresponding to the primers and the sequences of the primers are given in Table 2:

TABLE 2 sequence listing

Using the synthesized cDNA as a template, rps3 and alpha-tubulin were used as reference genes, the reaction system was NovoStart SYBR qPCR S. Mu. perMix Pl. Mu.s (brand: near shore, china) 5.0. Mu.L, each of the upstream and downstream primers (10. Mu.M) 0.3. Mu.L, the cDNA template 0.5. Mu.L, and the nuclease-free water was made up to 10. Mu.L.

PCR procedure 95℃pre-denaturation 2min, 95℃denaturation 30s,60℃annealing extension 30s,40 cycles, 60℃30s,95℃15s (forming melting curve).

The final relative expression level was calculated by the method of 2 ^-ΔΔCt. The expression patterns of 4 genes under different populations and under the induction of malathion are detected, the quantitative results of the expression patterns of the genes of the OBP28a-2 in the malathion long-term resistant population and the malathion sensitive population are shown in figure 1A, the expression quantity of the OBP28a-2 in the resistant population is obviously up-regulated according to statistical analysis and is 6 times higher than that of the sensitive population, the expression pattern of the OBP28a-2 under the short-term malathion medicament stress is shown in figure 1B, the expression quantity of the genes can be obviously up-regulated under the medicament stress, and compared with the control treatment of acetone, the expression quantity of the OBP28a-2 is improved by 1 time. The results of this example show that OBP28a-2 is highly expressed in resistant populations and that OBP28a-2 also exhibits significant upregulation in response to induction by malathion after induction.

EXAMPLE 3 malathion bioassay of OBP28a-2 ^-/- mutant lines

1. Embryo injection

Based on the bactrocera dorsalis genome, the exons of OBP28a-2 were predicted by https:// i5k.nal.usda.gov/and two gRNA targets were selected on the first and second exons. The corresponding gRNA was synthesized in vitro and purified using GeneArt Precision GRNA SYNTHESIS KIT kit (Invitrogen, USA) with reference to the instructions.

The gRNA sequence is:

gRNAF4(SEQ ID NO.13):

5’-TAATACGACTCACTATATAGACTAGAGCGGAGGAGTGCCG-3’,

gRNAR4(SEQ ID NO.14):

5’-TTCTAGCTCTAAAACCGGCACTCCTCCGCTCTAGT-3’,

gRNAF8(SEQ ID NO.15):

5’-TAATACGACTCACTATATAGGTACCAGCGTCCAGTAAGGA-3’,

gRNAR8(SEQ ID NO.16):

5’-TTCTAGCTCTAAAACTCCTTACTGGACGCTGGTAC-3’。

and diluting and uniformly mixing the synthesized gRNA and Cas9 protein on ice according to the detected concentration, wherein the final concentration of the gRNA and Cas9 enzyme is 500-600 ng/mu L, and storing the mixture at-80 ℃ for standby.

The orange fly embryo injection method comprises the steps of utilizing orange juice to prepare an egg-attracting device to attract eggs of orange fly in an egg laying period for 20min, collecting eggs laid for 20min, soaking eggs in a mixed solution of sodium hypochlorite and ultrapure water in a ratio of 1:2 for 90s, repeatedly rinsing with ultrapure water, soaking the treated eggs in the ultrapure water, picking 10-20 eggs with a writing brush, neatly arranging the eggs on a clean glass slide, drying for 2-3min in a drying box, dripping halogenated hydrocarbon oil to keep the embryos moist, injecting a mixed solution of gRNA and Cas9 into a capillary glass needle with a liquid transfer device, and mounting the glass needle on a microinjection instrument for embryo injection.

The injected G0 embryo is placed in an incubator for normal condition and is bred, the hatched larva is picked into larva feed after 48 hours, and is bred in the incubator (temperature: 27+/-0.5 ℃, relative humidity: 70+/-5%, photoperiod, 14 hours illumination/10 hours darkness).

2. Mutant screening

A total of 120 eggs were injected, 8 of which were successfully developed into adults. After the breeding of the strain is 9 days old, 8 parent adults are hybridized with wild type, 200-300 child eggs are collected, genome DNA of the parent adults is extracted by using TIANamp Genomic DNAKit kit (Tiangen Co., germany) and PCR amplification is carried out, and G ₀ heritable mutation condition is detected. Specific operations are described with reference to the specification. Designing mutant screening primers before and after the position of the gRNA target point, and carrying out PCR (polymerase chain reaction) amplification by taking genomic DNA as a template, wherein the sequences of the primers are as follows:

KO-Jc F2(SEQ ID NO.17)：5'-ATGGCCAAATTCATTCTATTCG-3',KO-Jc R2(SEQ ID NO.18)：5'-GTACTGCAATATCGGCGCA-3'.

The PCR reaction system was as follows, T3 Super PCR Mix (Beijing engine biosciences Co., ltd.) 12.5. Mu.L, 10. Mu.M each of the upstream and downstream primers 1. Mu.L, DNA template 5. Mu.L, and the mixture was supplemented with nuclease-free water to 25. Mu.L. The PCR procedure was as follows, 98℃pre-denaturation for 3min, 98℃denaturation for 15s,60℃annealing for 15s,72℃extension for 15s,35 cycles, 72℃extension for 5min,12℃storage. The PCR products were used for band detection using 1% agarose gel electrophoresis, and the polymorphism of the PCR products was used to determine the gene editing status of individuals of generation G ₀. As a result, it was found that 3 adults showed multiple bands, possibly mutants.

The corresponding sub-generation individuals are then bred to the adult stage. The genomic DNA of the fruit fly hind paw was extracted using INSTAGENETM MATRIX (Bio-rad, USA) kit, and the specific procedure was as follows, cutting the fruit fly adult hind paw and placing it in a 200. Mu.L centrifuge tube. Add 20. Mu. L INSTAGENETM Matrix to centrifuge tube and incubate at 56℃for 1h. The sample was rapidly shaken for 30s, centrifuged for 30s, and incubated at 100℃for 10min. Rapidly shaking the sample for 15s, centrifuging for 15s, and sucking the supernatant as a DNA template for storage. The PCR amplification and mutant detection methods are the same as those of the parent generation. The same is true for the detection method of the subsequent offspring.

Through electrophoresis detection, a plurality of obvious bands are found, and the bands are sent to the Optimu family company for sequencing detection, and the occurrence of a hetero peak at a target point is found. The heterozygote individuals with obvious multiple bands are selected for hybridization with the heterozygote individuals, offspring are bred to adults, the genotypes of the offspring are detected, and large-fragment knockout homozygous individuals can be obtained, and the electrophoresis result of the OBP28a-2 ^-/- mutant strain is shown in FIG. 2. The sequencing results are as follows:

Wild type nucleotide sequence (SEQ ID NO.19)：5'-AATTTTATGACTAGAGCGGAGGAGTGCCGTGGCGAAGTGGGTGCTGCTGACTGT GAGTATAAGAATCACTATAATTCAATATTGATGAATTATACCTAGCGTTATCTAAACCGCAGCTGACATTCAGGACATAGTCGCAAAAGTACCAGCGTCCAGTAAGGAAGGCAAATGCTTGCGCTCCTGTTTGATGAAAAAATATGGCGCGGTAAGTATTTATTTTACATGATAATATTTTATGTCATTACTACCAACATATTTTATTTGACGTTCCTCAGATGGAT-3';

Homozygous nucleotide sequence (SEQ ID NO.20)：5'-AATTTTATGACTAGAGCGGAGGAGTGGGAAGGCAAATGCTTGCGCTCCTGTTTGA TGAAAAAATATGGCGCGGTAAGTATTTATTTTACATGATAATATTTTATGTCATTACTAC CAACATATTTTATTTGACGTTCCTCAGATGGAT-3'.

3. Mutant bioassays

To determine whether the sensitivity of the mutant bactrocera dorsalis to malathion was altered, the mortality was determined after LC ₅₀ (44.44 mg/L) exposure to malathion for 2h,24h following the bioassay procedure in example 1. As shown in FIG. 3, the survival rates of OBP28a-2 ^-/+ (heterozygous mutant with the OBP28a-2 gene knocked out) and OBP28a-2 ^-/- (homozygous mutant with the OBP28a-2 gene knocked out) mutant bactrocera dorsalis exposed to malathion were 62% and 80%, respectively. The mortality rate is significantly improved by 22% and 40% compared to wild-type bactrocera dorsalis treated with the same malathion. Example 4 heterologous expression of OBP28a-2 protein

The full-length primer is designed and amplified to obtain the full-length sequence by obtaining the open reading frame sequence information of the OBP28a-2 from an internal database (a Bactrian) of a laboratory where the inventor is located. The full-length amplification primers were as follows:

OBP28a-2F2(SEQ ID NO.21):5’-ATGGCCAAATTCATTCTATT-3’,

OBP28a-2R2(SEQ ID NO.22):5’-TCAGTCAAACTTTTGCATACCGT-3’。

The target gene was amplified using the foot cDNA of bactrocera dorsalis as a template in a reaction system of 12.5. Mu.L 2X PRIMESTAR MAX PREMIX (TaKaRa, japan), 9.5. Mu.L of nuclease-free water, 1. Mu.L of forward and reverse primers, and 2. Mu.L of cDNA, in total, 25. Mu.L. The reaction conditions were 98℃pre-denaturation for 3min, 98℃denaturation for 15s, 60℃annealing for 15s, 72℃extension for 30s, 35 cycles, and finally 72℃extension for 5min,12℃storage.

Sequencing to obtain ORF sequence of BdorOBP a-2 gene (SEQ ID NO.23)：5'-ATGGCCAAATTCATTCTATTCGCCGCCTTATGTATTTTGAGTGCCGCTGTCTCCAAC GCTGCTTTCAACAAAGAAGAGGCTATAAAAAATTTTATGACTAGAGCGGAGGAGTGCCGTGGCGAAGTGGGTGCTGCTGACTCTGACATTCAGGACATAGTCGCAAAAGTACCAGCGTCCAGTAAGGAAGGCAAATGCTTGCGCTCCTGTTTGATGAAAAAATATGGCGCGATGGATAGTAATGGCAAGTTTGTTAAGTCGGTCGTCGATCAGCATGCACAGGACTTCACTGACGGTGATGCAGATAAATTGAAGACAGCTCGTGAAATTATCGACGCTTGCGCCGATATTGCAGTACCAGATGATCATTGCGAGGCAACTGAAGTGTATGGCAAATGCTTTATGGATCAAGCGAAAGCTCACGGTATGCAAAAGTTTGACTTTTGA-3'.

Primers were then designed based on the signal peptide predictions, which had removed the self signal peptide and had two cleavage sites, kpnI and XhoI:

Kpn I OBP28a-2F(SEQ ID NO.24):5’-CGGGGTACCGCTTTCAACAAAGAAGAGG-3’,Xho I OBP28a-2R(SEQ ID NO.25):

5’-CCGCTCGAGAAAGTCAAACTTTTGCATACCGT-3’。

The target gene was amplified using 2X PrimeSTARMax Premix foot cDNA as a template, and the reaction was the same as above. After electrophoresis detection, the product was sent to the sequencing of the Optimago company.

Its post sequence for removing signal peptide (SEQ ID NO.26)：5'-GCTTTCAACAAAGAAGAGGCTATAAAAAATTTTATGACTAGAGCGGAGGAGTGCC GTGGCGAAGTGGGTGCTGCTGACTCTGACATTCAGGACATAGTCGCAAAAGTACCAGCGTCCAGTAAGGAAGGCAAATGCTTGCGCTCCTGTTTGATGAAAAAATATGGCGCGATGGATAGTAATGGCAAGTTTGTTAAGTCGGTCGTCGATCAGCATGCACAGGACTTCACTGACGGTGATGCAGATAAATTGAAGACAGCTCGTGAAATTATCGACGCTTGCGCCGATATTGCAGTACCAGATGATCATTGCGAGGCAACTGAAGTGTATGGCAAATGCTTTATGGATCAAGCGAAAGCTCACGGTATGCAAAAGTTTGACTTTTGA-3'.

The size of the target band was determined to be correct by 1% agarose gel electrophoresis, a PCR sample containing a single target band was subjected to liquid recovery according to the instructions of TIANGEN universaL DNAPurification Kit kit (Tiangen), and the concentration and quality of the recovered product were determined by using a nucleic acid concentration meter.

And (3) enzyme digestion, namely respectively carrying out double enzyme digestion on the pure target gene fragment obtained by recovering the product stock solution of the PCR reaction and a vector plasmid pET30a (Sieimer), wherein the system is as follows, 10X Quickcutsmart Buffer mu L, kpnI1 mu L, xhoI1 mu L, OBP28a-2 sequence fragment/pET 30a vector 1 mu g, and no nuclease water is added to 50 mu L. The procedure was set to 37 ℃ for 8h, after which the product was all run on a 1% agarose gel and recovered. The cleavage product was ligated to vector pET30a, and the reaction system was 2X Ligtion Buffer. Mu.L, 3. Mu.L of the target gene, 1. Mu.L of PET30a, and 1. Mu.L of nuclease-free water.

The well mixed reaction solution was incubated overnight at 16℃in a PCR apparatus, followed by transformation with Trans5α CHEMICALLY COMPETENT CELL competence (Beijing full gold) according to the instructions. The transformed bacterial liquid was spread on LB (containing 50. Mu.g/mL kanamycin) solid medium and cultured at 37℃with shaking at 200 rpm. The specific mode of bacterial detection PCR is the same as above, the positive PCR product is sent to be detected, and bacterial liquid with qualified sequencing is subjected to propagation, plasmid extraction and bacterial liquid preservation. Recombinant plasmid pET30a-BdorOBP a-2 was obtained, the recombinant plasmid sequence (partial vector sequence (underlined) +OBP28 sequence (de-signal peptide)) was as follows (SEQ ID NO.27)：5'-TTCTTCTGGTCTGGTGCCACGCGGTTCTGGTATGAAAGAAACCGCTGCTGCTAAATTCGAACGCCAGCACATGGACAGCCCAGATCTGGGTACCGCTTTCAACAAAGAAGAGGCTATAAAAAATTTTATGACTAGAGCGGAGGAGTGCCGTGGCGAAGTGGGTGCTGCTGACTCTGACATTCAGGACATAGTCGCAAAAGTACCAGCGTCCAGTAAGGAAGGCAAATGCTTGCGCTCCTGTTTGATGAAAAAATATGGCGCGATGGATAGTAATGGCAAGTTTGTTAAGTCGGTCGTCGATCAGCATGCACAGGACTTCACTGACGGTGATGCAGATAAATTGAAGACAGCTCGTGAAATTATCGACGCTTGCGCCGATATTGCAGTACCAGATGATCATTGCGAGGCAACTGAAGTGTATGGCAAATGCTTTATGGATCAAGCGAAAGCTCACGGTATGCAAAAGTTTGACTTTCTCGAGCACCACCACCACCACCACTGAGATCCGGCTGCTAACAAAGCCCGAAAGGAAGCTGAGTTGGCTGCTGCCACCGCTGAGCAATAACTAGCATAACCCCTTGG-3'.

Example 5 ex vivo affinity test of OBP28a-2 protein and malathion Agents

1. Recombinant protein purification

The recombinant plasmid pET30a-BdorOBP a-2 obtained in example 4 was transformed into E.coli BL21 (DE 3), and the transformation and bacterial detection method was the same as in example 4. Adding positive clone bacteria into 400mL LB (containing 50 mug/mL kanamycin) liquid culture medium, culturing at 37 ℃ under shaking at 200rpm until bacterial liquid OD600 = 0.6-1.0, adding isopropyl-beta-D-thiogalactoside (IsopropyL-beta-D-thiogaLactopyranoside, IPTG) to a final concentration of 0.5mM, culturing at 37 ℃ under shaking at 200rpm for about 16 hours to induce bacterial strain to express protein, taking the induced bacterial liquid at 4 ℃, centrifuging at 4000 Xg for 15min, discarding the supernatant, re-suspending bacterial precipitation by using 25mL of 1 XPBS buffer, crushing the re-suspended escherichia coli cells by using an ultrasonic cell disrupter (pulse 5s, interval 3s, total time 15 min), centrifuging at 4 ℃ and 12000 Xg for 15min, and collecting the supernatant and the precipitate for detecting protein expression by SDS-PAGE electrophoresis.

After the buffer in the Ni-NTA column (Siemens flier) was completely dropped, 2mL of 20% alcohol and 1 XPBS were added into the column, then 5mL of supernatant containing recombinant protein was added into the Ni-NTA column each time until the supernatant was completely dropped, the target protein was eluted with 1 XPBS buffer containing imidazole (40 mM, 80mM, 160mM, 250 mM) at different concentrations, and the solution containing the target protein was collected for SDS-PAGE electrophoresis to detect the molecular weight of the protein, and the specific procedure was set using 150v,30min electrophoresis conditions according to the instructions (Berle) of SDS-PAGE pre-gel. The Ni-NTA column was then equilibrated with 1 XPBS, 20% alcohol and stored at 4 ℃.

Transferring SDS-PAGE gel onto polyvinylidene fluoride (PVDF) membrane immersed in transfer buffer (10 mL transfer buffer,10mL absolute ethanol, 30mL pure water, and mixed) for 2min, transferring to membrane under 25V, 200mA condition for 3min, washing PVDF membrane with 1 XTBE (Tris-HCL and Tween-20 mixed solution), blocking with blocking solution (50 mL 1 XTBE+2.5 g skim milk powder) at 37 ℃ for 1h, immersing PVDF membrane with His-tag Antibody (His-tag Antibody), incubating at 4 ℃ overnight, recovering primary Antibody, washing PVDF membrane with 1 XTBE, incubating PVDF membrane in goat anti-mouse IgG serum (Biyun) for 2h, recovering secondary Antibody, washing PVDF membrane with 1 XTBE, mixing solution A and solution B1:1 using super-sensitive ECL chemiluminescent substrate kit, dripping onto PVDF membrane, reacting for 2-3min, and photographing under imaging system. As shown in FIG. 4, FIG. 4A shows SDS-PAGE results, M pore canal represents protein Marker, pore canal shown in a is purified protein, pore canal shown in B is empty protein, only pore canal a has obvious band at the predicted size of 15-20kDa, protein specific antibody mark through WB (FIG. 4B) shows that specific band only exists at 15-20kDa, and OBP28a-2 recombinant protein is successfully obtained. The purified protein was subjected to BCA protein concentration assay kit (bi yun) and recombinant protein concentration was measured by referring to the protocol.

2. Recombinant protein and pesticide binding assays

Recombinant proteins were labeled with reference to RED-tris-NTA protein labeling kit instructions. And (5) analyzing the affinity of the recombinant protein to pesticides. Dissolving 2 μL of 5 μM fluorescent dye solution in 98 μL of 1 XPBST buffer to make the fluorescent dye concentration 100nM, diluting recombinant protein to 200nM with 1 XPBST buffer to 100 μL, mixing the fluorescent dye and recombinant protein solution uniformly, and incubating for 30min at room temperature in dark place. The insecticide was dissolved in acetone and prepared as a 10mM stock solution for use. The pesticide includes malathion, avermectin (94% of original drug, jiangsu Feng source organism), fenpropathrin (20% of emulsifiable concentrate, wildd chemical industry) and deltamethrin (97% of original drug, schrad organism).

10MM insecticide was graded at a 1:1 ratio, diluted to 16 gradients (10mM,5mM,2.5mM,mM,1.25mM,625μM,312.5μM,156.25μM,78.13μM,39.06μM,19.53μM,9765nM,4883nM,2441nM,1221nM,610nM,305nM). with PBST, and the labeled recombinant protein solution was mixed with insecticide dilutions in equal volumes (10. Mu.L each) and incubated for 5-10min. The sample was siphoned using capillary MonolithTMNT.115series CAPILLARIES (NANO TEMPER TECHNOLOGY), and the binding capacity of the recombinant protein to the fluorescent dye was measured on a micropulse MonolithNT.115 (Nano Temper Technology) device, with parameters set to expression power 60, MST power 40, temperature constant 25 ℃, MST test time 20s. The recombinant proteins were analyzed for affinity Kd to dye using mo. Affinityanalysis (version 2.2.4) software.

The protein of the OBP28a-2 is obtained in an isolated mode, the micro-thermal surge technology is utilized to detect the affinity of the OBP28a-2 with various medicaments, the result is shown in figure 5, the OBP28a-2 and malathion can be well fitted to an affinity curve, the affinity is strong, the Kd value is 62.39nM, the OBP28a-2 participates in regulating and controlling the sensitivity of the bactrocera dorsalis to malathion, and the research finds that the odor binding protein OBP28a-2 of the bactrocera dorsalis regulates and controls the resistance of the bactrocera dorsalis to malathion, and meanwhile, the OBP28a-2 can be used as an indicator gene of the bactrocera dorsalis to malathion resistance.

Example 6 detection of malathion content in samples

A method for detecting malathion content in a sample comprises the steps of using 16 malathion standards of known concentrations in example 5, detecting the affinity of OBP28a-2 protein to the standards, and fitting an affinity curve with Ma Laliu concentration on the abscissa and affinity detection fluorescence value on the ordinate. The affinity of the malathion-containing test sample with the OBP28a-2 protein was detected, the OBP28a-2 protein was labeled in the labeling manner as in example 5, the test sample was diluted 100-fold with PBST after extraction with acetone, and the labeled recombinant protein solution was mixed with the test sample in equal volumes (10. Mu.L each) and incubated for 5-10min. The labeled recombinant protein solution was mixed with an equal volume of PBST as a detection reference. Specific detection methods are described in example 5. Substituting the fluorescence detection result of the sample to be detected into an affinity curve obtained by fitting to obtain the malathion concentration of the sample to be detected.

Wherein, the affinity of the OBP28a-2 protein and the malathion sample is detected by adopting a micro thermal surge technology.

Claims (6)

1. Application of the BdorOBP28a-2 protein in malathion content detection, characterized in that: a plurality of malathion standards of known concentration are used to detect the affinity of the BdorOBP28a-2 protein and the standards; an affinity curve is fitted with the malathion concentration as the abscissa and the affinity detection fluorescence value as the ordinate; the affinity between the test sample containing malathion and the BdorOBP28a-2 protein is detected; and the malathion concentration of the test sample is obtained based on the affinity curve of the standards; the nucleotide sequence of the gene encoding the BdorOBP28a-2 protein is shown in SEQ ID NO. 23; and the malathion concentration of the test sample is between 305 nM and 1.25 mM. 2. The use according to claim 1, characterized in that the affinity of BdorOBP28a-2 protein to malathion sample is detected by microcalorimetry. 3. The use of a reagent for detecting the gene expression level of a molecular marker of the resistance of the oriental fruit fly to malathion in the classification of oriental fruit fly populations sensitive to malathion and populations resistant to malathion, wherein the molecular marker is the BdorOBP28a-2 gene encoding the oriental fruit fly odor-binding protein, and its nucleotide sequence is shown in SEQ ID NO.23. 4. The use according to claim 3, characterized in that: the expression level of the BdorOBP28a-2 gene of the tested fruit fly is detected using a malathion-sensitive population of the fruit fly as a control; if the expression level of the BdorOBP28a-2 gene of the tested fruit fly is significantly higher than that of the sensitive population, it can be determined that the tested fruit fly is a malathion-resistant population; if there is no significant difference in the expression level of the BdorOBP28a-2 gene between the tested fruit fly and the sensitive population, it cannot be determined that the tested fruit fly is a malathion-resistant population. 5. The use according to claim 4, characterized in that it comprises the following steps: extracting total RNA of the to-be-tested Bactrocera dorsalis, converting it into cDNA, using the cDNA as a template, performing qPCR amplification with a primer pair, and using a malathion-sensitive population of Bactrocera dorsalis as a control, determining the expression level of the BdorOBP28a-2 gene in the to-be-tested object; when the expression level of the BdorOBP28a-2 gene in the to-be-tested object is significantly higher than that in the sensitive population, it can be determined that the to-be-tested Bactrocera dorsalis is a malathion-resistant population; the primer sequence of the primer pair is: Upstream primer: OBP28a-2 qPCR F: 5’-GGTGCTGCTGACTCTGACAT-3’, Downstream primer: OBP28a-2 qPCR R: 5’-GCCTCGCAATGATCATCTGG-3’. 6. The use according to claim 5, characterized in that the qPCR amplification reaction system comprises: 5.0 μL of NovoStart SYBRqPCR SuperMix Plus, 0.3 μL each of 10 μM upstream/downstream primers, 0.5 μL of cDNA template, and nuclease-free water to 10 μL; the qPCR amplification conditions are: pre-denaturation at 95°C for 2 min; 40 cycles of denaturation at 95°C for 30 s, annealing and extension at 60°C for 30 s; and melting curve formation at 60°C for 30 s and 95°C for 15 s.