Helicoverpa armigera olfaction receptors OR48 and OR75 and application and function verification method thereof

Abstract

The invention relates to cotton bollworm olfaction receptors OR48 and OR75 as well as an application and a function verification method thereof, belongs to the technical field of biological gene engineering, obtains cotton bollworm genes OR48 and OR75 through cloning for the first time, verifies the regulation effect of ecdysone and insulin in a cotton bollworm body on the ecdysone and the insulin, and finds that the ecdysone up-regulates the expression of OR48 through a nuclear receptor EcR of the ecdysone, so that the cotton bollworm gene OR48 and OR75 is obtained. Insulin up-regulates the expression of OR75 through an insulin receptor, and the research of the molecular mechanism provides a new target for pest control.

Description

Technical Field

[0001] The invention relates to the technical field of biological genetic engineering, in particular to cotton bollworm olfactory receptors OR48 and OR75 and their application and function verification method. Background Art

[0002] Lepidoptera is the second largest order of Insecta, including butterflies and moths, as well as many agricultural pests. Cotton bollworm is one of the Lepidoptera pests, which seriously damages cotton and other crops. The study of the molecular mechanisms and physiological processes of cotton bollworm development remains very important. The cotton bollworm completes the egg, six larval stages, pupa and adult stages in its life, among which ecdysone 20E (20-hydroxyecdysone) and insulin play a vital role. The larval stage is divided into the feeding stage and the molting and metamorphosis stage. The larvae pose the greatest threat to crops during the feeding stage, feeding on a variety of crops, including cotton, corn, soybeans, peanuts, etc. Therefore, studying the differential expression of genes in the feeding and non-feeding stages of cotton bollworm is of great significance for pest control.

[0003] Olfactory receptors (ORs) are an important part of the olfactory system, responsible for detecting odors and initiating the sense of smell. These receptors belong to the G protein-coupled receptor family and are characterized by having seven hydrophobic membrane-spanning regions. ORs play a vital role in olfactory recognition because they can recognize and distinguish a wide range of odor molecules. But the function of ORs is not limited to the recognition of odor molecules. Studies in recent years have also revealed the role of olfactory receptors in glucose metabolism. Olfactory receptors play an important role in maintaining blood glucose homeostasis by regulating hormone secretion, fat metabolism, insulin sensitivity, etc.

[0004] In cotton bollworm, multiple odorant binding proteins (OBPs) and olfactory receptors have been identified, which play a role in different physiological processes. However, all ORs in the genome have not been systematically and completely analyzed. ORs in cotton bollworm not only play a role in olfactory perception in adults, but also play a vital role in food selection and avoidance mechanisms in larvae. For example, Gr180 is a bitter taste receptor that is highly expressed in the upper proboscis of larvae and responds to plant secondary metabolites such as coumarins, which are feeding inhibitors in many plants. In addition, some ORs are also involved in the migratory behavior of cotton bollworm. For example, OBP3 / OBP6 supports flight activity by interacting with lipid molecules. This finding reveals the potential role of cotton bollworm ORs in insect energy metabolism and movement. However, the exploration of other functions of cotton bollworm ORs is not very in-depth, and the perception of endogenous hormones in the body is not very clear. Human sex hormones and insect ecdysone are both steroid hormones. Studies have shown that sex hormones play an important role in odor perception. However, the underlying mechanism has not been explored in depth. This is the inadequacy of existing technologies. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide two cotton bollworm olfactory receptors OR48 and OR75 and their application and function verification methods in view of the deficiencies in the prior art.

[0006] This scheme is achieved through the following technical measures: the technical scheme of the present invention is to first identify all the olfactory receptors in the genome of cotton bollworm, screen out the olfactory receptors that are highly expressed in the feeding period and the molting and metamorphosis period respectively through transcriptome database analysis, conduct insect hormone stimulation verification, and clone the full-length cDNA of OR48 and OR75 genes from the cotton bollworm for functional verification.

[0007] The names of the cotton bollworm olfactory receptor genes provided by the present invention are OR48 and OR75, and the nucleotide sequence of the OR48 gene cDNA is shown in Sequence Table 1:

[0008]

[0009] Sequence Listing 1

[0010] The nucleotide sequence of the OR75 gene cDNA is shown in Sequence Table 2:

[0011]

[0012] Sequence Listing 2

[0013] The present invention also provides the amino acid sequences of the above-mentioned olfactory receptor genes OR48 and OR75, and the encoded protein of the OR48 is shown in Sequence Table 3:

[0014]

[0015] The encoded protein of OR75 is shown in Sequence Table 4:

[0016]

[0017] The present invention also provides cotton bollworm overexpression vectors PIEX-OR48-GFP-His and PIEX-OR75-GFP-His containing the cotton bollworm OR48 and OR75 genes.

[0018] The invention also provides the use of cotton bollworm olfactory receptors OR48 and OR75 in cultivating insect-resistant plants.

[0019] The present invention also provides a method for verifying the functions of cotton bollworm olfactory receptors OR48 and OR75, comprising the following steps:

[0020] (1) Analysis of the olfactory receptor gene family in cotton bollworm;

[0021] (2) Cloning of OR48 and OR75 gene cDNA sequences and construction of cell line overexpression vectors;

[0022] (3) Expression analysis of OR48 and OR75 genes under ecdysone 20E and insulin stimulation;

[0023] (4) Acquisition of dsRNA and RNA interference in insects;

[0024] (5) Phenotypic identification of knockdown of OR48 and OR75 in insects;

[0025] (6) Study on the mechanism of regulation of OR48 and OR75 by 20E and insulin.

[0026] Preferably, in step (1), the gene family domain hmmer hidden Markov model file is downloaded, and all protein sequences in the genome are searched using the hmmsearch software, and genes with e-Value<1e-5 are screened as candidate gene family genes; the gene family domains are screened in the SMART, CDD and Pfam databases as candidate gene family genes; 81 genes of the cotton bollworm olfactory receptor gene family are finally determined; based on the ID information of the identified gene family genes, protein sequences are extracted to draw an evolutionary tree; first, multiple sequence alignment is performed using the muscle software, and then the phylogenetic tree is constructed using the Mega11 software NJ method.

[0027] Preferably, step (2) includes:

[0028] (2-1) Reverse transcription to synthesize cDNA: Gene-specific primers were designed based on the OR48 and OR75 sequences in the genome, total RNA of cotton bollworm was extracted, and reverse transcribed into cDNA using a reverse transcription kit;

[0029] (2-2) The sequence of the primer is:

[0030] OR48F: 5′-TCGGCGCGCCTGCAGGTCGACATGTCCGAGTCAACAATA-3′,

[0031] OR48R: 5′-CTTCGAACCGGTACCGTCGACATCATCACTGTACATGGT-3′, OR75F: 5′-TCGGCGCGCCTGCAGGTCGACATGGAATTGGATTTTAAC-3′, OR75R: 5′-CTTCGAACCGGTACCGTCGACATTCCACATACAATTCAT-3′;

[0032] (2-3) PCR amplification using the synthesized cDNA as a template;

[0033] (2-4) PCR amplification products were detected by 1% agarose gel electrophoresis;

[0034] (2-5) Recovery of the amplified fragment and seamless cloning link with the pIEx-4-GFP-His vector;

[0035] (2-6) Cloning and sequencing of recovered fragments.

[0036] Preferably, step (3) includes:

[0037] (3-1) 20E was dissolved in DMSO to 10 mg / mL, and insulin was diluted in PBS to 100 ng / μL. Then different concentrations of 20E and insulin were injected into the sixth instar larvae at 6 h, and DMSO and PBS were used as controls, respectively.

[0038] (3-2) Extracting total RNA from cotton bollworm using Trizol reagent;

[0039] (3-3) Synthesize the first-strand cDNA using ChamQ Universal SYBR qPCR Master Mix Kit;

[0040] (3-4) RT-qPCR reaction and electrophoresis.

[0041] Preferably, step (4) includes:

[0042] The T7 promoter sequence was added to the 5' end of the primer sequence. The primer sequences were:

[0043] dsOR48F:5'-GCGTAATACGACTCACTATAGGGAACAAGCGAAACGGGAA AT-3',

[0044] dsOR48R:5'-GCGTAATACGACTCACTATAGGTCAGTAGCTGACGGAACAC G-3';

[0045] dsOR75F:5'-GCGTAATACGACTCACTATAGGTACCATTGGGTCCGATAT-3',dsOR75R:5'-GCGTAATACGACTCACTATAGGAGGTTTGAGTGCGTTCTG-3;

[0046] Using cDNA as a template, the corresponding target gene fragment is amplified, and then the target gene fragment with a T7 promoter is used as a template to synthesize dsRNA.

[0047] Preferably, step (5) includes:

[0048] dsRNA was diluted to 200 ng / μL with 1×PBS, and sixth-instar 6-hour-old larvae were placed on ice for about 15 min until the larvae were immobile. 1 μg dsRNA was injected into the larvae from the third ventral foot forward in the abdomen of the larvae using a microinjector. The injection was performed once every 24 h. For the third injection, 1 μg dsRNA and 500 ng 20E were injected into the larvae together. Each experimental group and control group had no less than 30 larvae, and three biological replicates were performed. An equal amount of dsGfp was used as the control group.

[0049] Preferably, step (6) includes:

[0050] (6-1) After knocking down the 20E nuclear receptor EcR and the insulin receptor InR, the expression changes of OR48 and OR75 were detected respectively;

[0051] (6-2) Chromatin immunoprecipitation (CHIP) was used to detect the binding of EcR and OR48.

[0052] Compared with the prior art, the technical scheme of the present invention has the following technical effects: the present invention cloned the cotton bollworm genes OR48 and OR75 for the first time, verified the regulatory effects of ecdysone and insulin on the two genes in the cotton bollworm, and found that ecdysone upregulated the expression of OR48 through its nuclear receptor EcR, and insulin upregulated the expression of OR75 through the insulin receptor. The study of this molecular mechanism provides a new target for pest control.

[0053] It can be seen that compared with the prior art, the present invention has outstanding substantive features and significant progress, and the beneficial effects of its implementation are also obvious. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] Figure 1 Phylogenetic analysis of the olfactory receptor gene family of cotton bollworm, where the red circles represent the olfactory receptors HarmORs of cotton bollworm and the green triangles represent the olfactory receptors BmorORs of silkworm.

[0055] Figure 2 Expression patterns of OR48 and OR75 in different developmental stages of cotton bollworm and expression analysis under hormone stimulation. (AB) RT-qPCR detection of the relative expression levels of OR48 and OR75 in antennae, epidermis, midgut and fat body; 5F, 5th instar feeding larvae; 5M, 5th instar molting larvae; 6th-6h-120h, 6th instar larvae at different ages; P2 d~P8 d, 0-8 days old pupal stage; Adult, adult; F, feeding stage; M, molting stage; MM, molting metamorphosis stage; P, pupal stage; (CD) Changes in the relative expression level of OR75 after injection of different concentrations and time gradients of insulin; (EF) Changes in the relative expression level of OR48 after injection of different concentrations and time gradients of 20E.

[0056] Figure 3 To knock down OR75 by RNA interference and cause the larvae to pupate earlier, (A) the relative expression level of OR75 was analyzed by RT-qPCR (the second injection was performed 24 hours after the first injection of dsGfp or dsOR75, 3 μg dsRNA / larva); (B) the phenotype after injection of dsGfp and dsOR75; (C) the phenotypic ratio after OR75 knockdown; (D) the average pupation time after knocking down Gfp and OR75; (E) the midgut morphology; (F-Hi) the morphological changes of the midgut and fat body after knocking down Gfp and OR75 were observed by HE staining, TUNEL staining and electron microscopy.

[0057] Figure 4 Knockdown of OR48 by RNA interference delayed pupation; (A) Relative expression of OR48 was analyzed by RT-qPCR (the second injection was performed 24 hours after the first injection of dsGfp or dsOR48, 3 μg dsRNA / larva); (B) Phenotype after injection of dsGfp and dsOR48; (C) Phenotypic ratio after OR48 knockout; (D) Average pupation time after knockdown of Gfp and OR48; (E) Midgut morphology; (F-Hi) Morphological changes of midgut and fat body after knockdown of Gfp and OR48 were observed by HE staining, TUNEL staining and electron microscopy.

[0058] Figure 5 20E promotes the expression of OR48 through EcR, and insulin promotes the expression of OR75 through InR; (A) The EcRE site on the OR48 promoter was predicted using the JASPAR transcription factor database; (B) After EcR knockdown, the expression levels of EcR and OR48 were detected; (C) ChIP experiment verified that 20E promoted the expression of OR48 by binding to EcR; IgG was used as a negative control; (D) After InR knockdown, RT-qPCR was used to detect the expression levels of InR and OR75. DETAILED DESCRIPTION

[0059] In order to clearly illustrate the technical features of this solution, this solution is described below through specific implementation methods and in conjunction with the accompanying drawings.

[0060] The nucleotide sequence of the gene cDNA of the cotton bollworm olfactory receptor OR48 is shown in Sequence Table 1;

[0061]

[0062] Sequence Listing 1

[0063] The nucleotide sequence of the gene cDNA of the cotton bollworm olfactory receptor OR75 is shown in Sequence Table 2:

[0064]

[0065] Sequence Listing 2

[0066] The application of cotton bollworm olfactory receptors OR48 and OR75 in cultivating insect-resistant plants and the method for verifying their functions include the following steps:

[0067] (1) Analysis of the olfactory receptor gene family in cotton bollworm;

[0068] (2) Cloning of OR48 and OR75 gene cDNA sequences and construction of cell line overexpression vectors;

[0069] (3) Expression analysis of OR48 and OR75 genes under 20E and pancreatic hormone stimulation;

[0070] (4) Obtaining dsRNA (double-stranded RNA) and RNA interference in insects;

[0071] (5) Phenotypic identification of knockdown of OR48 and OR75 in insects;

[0072] (6) Study on the mechanism of regulation of OR48 and OR75 by 20E and pancreatic hormones.

[0073] Specifically, step (1) analyzes the olfactory receptor gene family of cotton bollworm.

[0074] First, download the gene family domain hmmer hidden Markov model file, use hmmsearch software to search all protein sequences in the genome, and select e-Value <1e-5 as candidate gene family genes; select gene family domains in SMART, CDD and Pfam databases as candidate gene family genes; finally determine 81 genes in the cotton bollworm olfactory receptor gene family. According to the ID information of the identified gene family genes, extract protein sequences and draw evolutionary trees; first use muscle software for multiple sequence alignment, and then use Megacacc11 software NJ method to construct a phylogenetic tree. The results are as follows: Figure 1 shown.

[0075] Step (2) Cloning of OR48 and OR75 gene cDNA sequences and construction of cell line overexpression vectors.

[0076] (2-1) Reverse transcription to synthesize cDNA:

[0077] Gene-specific primers were designed based on the OR48 and OR75 sequences in the genome, and RNA was extracted from sesame leaves. The RNA was reverse transcribed into cDNA using a 5×All-In-One RT MasterMix reverse transcription kit (abm). The reaction system was:

[0078] RNA 2 μg

[0079] AccuRT Reaction Mix (4×) 2 μL

[0080] Add 8 μL of RNAase-free water

[0081] Mix well and incubate at 42℃ for 2min

[0082] AccuRT Reaction Stopper(5×)2μL

[0083] 5×All-In One RT MasterMix4μL

[0084] RNAase-free Water 6μL

[0085] Incubate at 25°C for 10 min, 42°C for 30 min, 85°C for 5 min, and store at -20°C.

[0086] (2-2) Primer sequence:

[0087] OR48F: 5′-TCGGCGCGCCTGCAGGTCGACATGTCCGAGTCAACAATA-3′,

[0088] OR48R: 5′-CTTCGAACCGGTACCGTCGACATCATCACTGTACATGGT-3′, OR75F: 5′-TCGGCGCGCCTGCAGGTCGACATGGAATTGGATTTTAAC-3′, OR75R: 5′-CTTCGAACCGGTACCGTCGACATTCCACATACAATTCAT-3′.

[0089] (2-3) PCR amplification was performed using the synthesized cDNA as a template. The reaction system (50 μL) was:

[0090]

[0091]

[0092] (2-4) The PCR reaction procedure is:

[0093] 95℃, 3min; 95℃, 45sec, 58℃, 1min, 72℃, 1min, cycle 35 times; 72℃, 5min.

[0094] (2-5) 1% agarose gel electrophoresis:

[0095] The PCR amplified products were detected by 1% agarose gel electrophoresis and the target bands were found at 1197 bp for OR48 and 1182 bp for OR75.

[0096] (2-6) Recovery of amplified fragments and seamless cloning and linking with pIEx-4-GFP-His vector:

[0097] The amplified bands were recovered using the agarose gel recovery kit from Novagen, and the steps were carried out according to the instructions. The PCR product and the vector were connected using the Novagen seamless cloning kit, and the connection system was:

[0098] System: 20μL

[0099] Insert fragment 6μL

[0100] pIEx-4-GFP-His vector 3 μL

[0101] 5×CE II Buffer 4μL

[0102] Exnase II 2 μL

[0103] ddH2O 5μL

[0104] 1) Gently mix the reconstituted solution and centrifuge briefly to collect the reaction solution.

[0105] 2) After reacting at 37°C for 30 min, immediately cool on ice.

[0106] (2-7) Cloning and sequencing of recovered fragments:

[0107] 1) Cloning and transformation of target fragments

[0108] ① Take out 1 tube (100 μL) of E. coli DH5a strain (Novozyme) competent cells from the -80℃ freezer and slowly thaw on ice for 30 minutes;

[0109] ② Add 10 μL of the recombinant product to the competent cells, flick the tube wall to mix, and place on ice for 30 minutes;

[0110] ③Heat shock in a 42℃ water bath for 90 seconds, then immediately place on ice for 2-3 minutes;

[0111] ④ Add 900 μL of LLB liquid culture medium (without antibiotics) to the centrifuge tube on a clean bench, mix well, and culture at 37°C with shaking for 1 hour (220 rpm);

[0112] ⑤ Preheat the LB solid medium plate containing Amp resistance in a 37°C incubator, centrifuge at 5000rpm for 6 minutes at room temperature, discard 800μL of supernatant, resuspend the bacteria in the remaining supernatant, apply it evenly to the prepared plate with a spreader, and leave it for 30 minutes;

[0113] ⑥ Invert the plate and place it in a 37°C constant temperature incubator overnight for 12-16 hours;

[0114] ⑦ Use a sterilized toothpick to pick up a single colony and place it in a test tube containing 10 mL of LB liquid culture medium (containing 50 μg / mL Amp) and shake the culture at 37°C overnight.

[0115] 2) PCR identification and sequencing of recombinant plasmids

[0116] Design the upstream primer located on the vector and the downstream primer located on the target gene to amplify the inserted fragment and identify the recombinant plasmid;

[0117] ①PCR program: 95℃, 3min; 95℃, 45sec, 58℃, 1min, 72℃, 1min, 35 cycles; 72℃, 5min;

[0118] ②PCR amplification products were detected by 1% agarose electrophoresis, and the bacterial solution of positive clones was sent to the company for sequencing. The sequencing results are shown in SEQ ID No.1 and SEQ ID No.2. The nucleic acid sequences SEQ ID No.1 and SEQ ID No.2 obtained by sequencing were translated into corresponding amino acid sequences using DNAman software or NCBI online software, and the results are shown in SEQ ID No.1.1 and SEQ ID No.2.1.

[0119] Step (3) Analysis of the expression of OR48 and OR75 genes under the stimulation of 20E and insulin hormones.

[0120] (3-1) Material processing:

[0121] 20E was dissolved in DMSO to 10 mg / mL, insulin (P3376-400 IU, Bio-Tech) was diluted in PBS (phosphate buffered saline) to 100 ng / μL, and then different concentrations of 20E and insulin were injected into the sixth instar larvae at 6 hours old, with DMSO (dimethyl sulfoxide) and PBS as controls, respectively. The concentration gradient of 20E was set to 100, 200 and 500 ng, and the concentration gradient of insulin was set to 1, 2 and 5 μg. 500 ng 20E or 5 μg insulin were injected at 1, 3, 6, 12 and 24 hours, respectively.

[0122] (3-2) Extraction of Total RNA from cotton bollworm using Trizol reagent (Novozyme):

[0123] The specific steps are carried out according to the instructions.

[0124] (3-3) Synthesis of the first-strand cDNA:

[0125] ChamQ Universal SYBR qPCR Master Mix Kit (Norvastatin) was used according to the instructions.

[0126] (3-4) RT-qPCR reaction and electrophoresis:

[0127] 1) RT-qPCR reaction was performed using cotton bollworm cDNA as a template. The primers are as follows:

[0128] HaAct-F: 5′-CCTGGTATTGCTGACCGTATGC-3′

[0129] HaAct-R: 5′-CTGTTGGAAGGTGGAGAGGGAA-3′

[0130] OR48-RTF: 5′-AACAAGCGAAACGGGAAAT-3′

[0131] OR48-RTR: 5′-GCCGCAGACGGACACTATG-3′

[0132] OR75-RTF: 5′-TGTACCATTGGGTCCGATAT-3′

[0133] OR75-RTR: 5′-TGTACCATTGGGTCCCGATAT-3′

[0134] 2) RT-qPCR system

[0135] 2×ChamQ Universal SYBR qPCR Master Mix5μL

[0136]

[0137] 3) Reaction procedure

[0138] 95℃, 30s; 95℃, 5s, 60℃, 30s, 40s, 40 cycles; 94℃, 15s, 60℃, 60s, 95℃, 15s. qPCR results are as follows Figure 2 .

[0139] Step (4) obtaining dsRNA and RNA interference in insects.

[0140] The T7 promoter sequence was added to the 5′ end of the primer sequence. The primer sequences were:

[0141] dsOR48F:5′-GCGTAATACGACTCACTATAGGGAACAAGCGAAACGGGAA AT-3′,

[0142] dsOR48R:5′-GCGTAATACGACTCACTATAGGTCAGTAGCTGACGGAACA CG-3′,

[0143] dsOR75F:5′-GCGTAATACGACTCACTATAGGTACCATTGGGTCCGATAT-3′,

[0144] dsOR75R:5′-GCGTAATACGACTCACTATAGGAGGTTTGAGTGCGTTCTG-3′,

[0145] Using cDNA as a template, the corresponding target gene fragment is amplified, and then the target gene fragment with a T7 promoter is used as a template to synthesize dsRNA. 80U T7 RNA polymerase, 2.4μL 10mM ATP / UTP / CTP / GTP and 120U RNasin (TaKaRa) were added to 2μg cDNA template, and the mixture was made up to 50μL with RNase-free water. The mixture was incubated in a 37°C water bath overnight. 10μL DNaseI and 10μL DNaseI Buffer were added, and the mixture was made up to a total volume of 100μL with RNase-free water. The mixture was incubated in a 37°C water bath for 1h to degrade DNA. RNA was extracted with phenol / chloroform, and the mixture was made up to a total volume of 200μL with RNase-free water. The same volume of phenol / chloroform-saturated water was added, and the mixture was centrifuged at 9,000×g at 4°C for 15min. The upper aqueous phase was transferred to a new centrifuge tube containing an equal volume of chloroform, and the mixture was centrifuged again. The supernatant was transferred to another new centrifuge tube, and 2.5 volumes of anhydrous ethanol and 1 / 10 volume of 3M sodium acetate (pH 4.0), mixed at -20℃, collected the precipitate, washed the dsRNA precipitate with 75% ethanol prepared with DEPC water, centrifuged at 7,500rpm at 4℃ for 5min, discarded the supernatant, air-dried to completely remove the residual ethanol, and finally added 50μL of RNase-free water to dissolve. The concentration of dsRNA was detected by Infinite M200PRONanoQuant spectrophotometer, and the quality of dsRNA was detected by 1% agarose gel electrophoresis.

[0146] Step (5) Phenotypic identification of knockdown of OR48 and OR75 in insects.

[0147] Dilute dsRNA to 200ng / μL with 1×PBS, take the sixth instar 6h old larvae and place them on ice for about 15min until the worm body stops moving, use a microinjector to inject 1μg dsRNA into the larvae from the third ventral foot of the larvae's abdomen to the front (be careful not to touch the midgut), inject once every 24h, and for the third injection, inject 1μg dsRNA and 500ng 20E into the larvae together. Each experimental group and control group had no less than 30 larvae, and three biological replicates were performed, with an equal amount of dsGfp as the control group. The phenotype was obtained 120h after the first injection of dsRNA for statistical analysis, and the pupation time required for the development of the sixth instar 0h larvae to pupation was statistically analyzed; the morphology of the midgut was observed 60h after the first injection of dsRNA; the HE staining of the midgut and fat body after OR knockdown was observed 60h after the first injection of dsRNA (results are shown in Figure 3 FG and 4F-G) and Tunel staining and electron microscopy were used to observe apoptosis and autophagy in fat bodies (the results are shown in Figure 3 GH and 4G-H).

[0148] Step (6) Study on the mechanism of regulation of OR48 and OR75 by 20E and insulin.

[0149] (6-1) After knocking down the 20E nuclear receptor EcR (ecdysone receptor) and the insulin receptor InR (insulin receptor), the expression levels of OR48 and OR75 were detected after knocking down EcR and InR in the worms (the results are shown in Figure 5 B and 5D):

[0150] The primers are:

[0151] dsEcRF:5′-GAGTAATACGACTCACTATAGGGACGCTGGTATAACAACGGAGGA-3′,

[0152] dsEcRR:5′-GAGTAATACGACTCACTATAGGGAAGCTGGAGCAACTCCTC ACG-3′;

[0153] dsInRF:5′-GCGTAATACGACTCACTATAGGCTTTCAACACCTTCCGCACT A-3′,

[0154] dsInRR:5′-GCGTAATACGACTCACTATAGGCACCCAGGACTAAGAACATTATCAT-3′.

[0155] The same insect RNAi method as above was used for knockdown, and RT-qPCR detection of OR48 and OR75 was performed.

[0156] (6-2) Chromatin immunoprecipitation (CHIP) to detect the binding of EcR and OR48:

[0157] The epidermal cells of cotton bollworm were cultured in a six-well plate until the cells grew to a density of about 80%, and then the pIEx-4-EcR-RFP-His plasmid was transfected. After 48 hours, 20E was added to treat the cells for 6 hours, and DMSO was used as a control. Chromatin immunoprecipitation (ChIP) kit was used for detection. First, cross-link the cells with 1% formaldehyde, incubate in a 37℃ incubator in the dark for 10 minutes, add 0.125M glycine, incubate for 10 minutes, terminate cross-linking, wash the cells twice with PBS, add SDS lysis buffer (1% SDS, 10mMEDTA, 50mM Tris-HCl, pH 8.1) to resuspend the cells, shear the DNA into 200-1000bp fragments with Bioruptor Pico ultrasonic crusher, centrifuge at 4℃ for 15 minutes, add 50μL washed protein A agarose beads to the supernatant, incubate at 4℃ for 1h to remove non-specific binding of protein A, centrifuge and take an appropriate amount of supernatant and place at 4℃ as the sample supernatant in RT-qPCR (Real-time quantitative reverse transcription PCR), and incubate the rest of the supernatant with anti-GFP monoclonal mouse antibody (IgG mouse antibody as negative control) at 4℃ overnight. Add 50μL washed protein A agarose beads and incubate at 4℃ for 2h. The mixture was washed once with low-salt wash buffer (0.1% SDS, 1.0% Triton X-100, 2mM EDTA, 200mM Tris-HCl, pH 8.0, 150mM NaCl), then washed once with high-salt wash buffer (0.1% SDS, 1.0% Triton X-100, 2mM EDTA, 20mM Tris-HCl, pH 8.0, 500mM NaCl), washed once with LiCl wash buffer (10mM Tris-HCl, pH 8.1, 0.25M LiCl, 1mM EDTA, 1% NP-40, 1% bile salt), washed twice with TE buffer (10mM Tris-HCl, pH 8.1, 1mM EDTA), and finally eluted with elution buffer (1% SDS, 0.1M NaHCO3). The DNA-protein complex was incubated in a 65°C water bath overnight, and then subjected to RNase A and proteinase K treatment, DNA was purified by phenol / chloroform method, and then RT-qPCR detection (results are shown in Figure 5 C).

[0158] The present invention cloned the cotton bollworm genes OR48 and OR75 for the first time, and verified the regulatory effects of ecdysone and insulin on the two genes in cotton bollworm. It was found that ecdysone upregulated the expression of OR48 through its nuclear receptor EcR, and insulin upregulated the expression of OR75 through the insulin receptor. The study of this molecular mechanism provides a new target for pest control.

[0159] Technical features not described in the present invention can be achieved by or by using existing technologies and will not be described in detail herein. Of course, the above description is not a limitation of the present invention, and the present invention is not limited to the above implementation modes. Changes, modifications, additions or substitutions made by ordinary technicians in this field within the essential scope of the present invention should also fall within the scope of protection of the present invention.

Claims

1. Olfactory receptors OR48 and OR75 of cotton bollworm, characterized in that: The nucleotide sequence of the gene cDNA of the cotton bollworm olfactory receptor OR48 is shown in Sequence Table 1; the nucleotide sequence of the gene cDNA of the cotton bollworm olfactory receptor OR75 is shown in Sequence Table 2.

2. Application of cotton bollworm olfactory receptors OR48 and OR75 in cultivating insect-resistant plants.

3. A method for verifying the function of cotton bollworm olfactory receptors OR48 and OR75, characterized in that: The steps include: (1) Analysis of the olfactory receptor gene family in cotton bollworm; (2) Cloning of OR48 and OR75 gene cDNA sequences and construction of cell line overexpression vectors; (3) Expression analysis of OR48 and OR75 genes under ecdysone 20E and insulin stimulation; (4) Acquisition of dsRNA and RNA interference in insects; (5) Phenotypic identification of knockdown of OR48 and OR75 in insects; (6) Study on the mechanism of regulation of OR48 and OR75 by ecdysone 20E and insulin.

4. The method for functional verification of the cotton bollworm olfactory receptors OR48 and OR75 according to claim 1, characterized in that: In step (1), the gene family domain hmmer hidden Markov model file was downloaded, and all protein sequences in the genome were searched using the hmmsearch software, and genes with e-Value < 1e-5 were selected as candidate gene family genes; the gene family domains were selected as candidate gene family genes in the SMART, CDD and Pfam databases; 81 genes of the cotton bollworm olfactory receptor gene family were finally determined; based on the ID information of the identified gene family genes, protein sequences were extracted to draw an evolutionary tree; first, multiple sequence alignment was performed using the muscle software, and then the phylogenetic tree was constructed using the NJ method of the Mega11 software.

5. The method for functional verification of the cotton bollworm olfactory receptors OR48 and OR75 according to claim 4, characterized in that: Step (2) includes: (2-1) Reverse transcription and cDNA synthesis: Gene-specific primers were designed based on the OR48 and OR75 sequences in the genome, total RNA of cotton bollworm was extracted, and reverse transcription was performed into cDNA using a reverse transcription kit; (2-2) The sequence of the primer is: OR48F: 5′-TCGGCGCGCCTGCAGGTCGACATGTCCGAGTCAACAATA-3′, OR48R: 5′-CTTCGAACCGGTACCGTCGACATCATCACTGTACATGGT-3′, OR75F: 5′-TCGGCGCGCCTGCAGGTCGACATGGAATTGGATTTTAAC-3′, OR75R: 5′-CTTCGAACCGGTACCGTCGACATTCCACATACAATTCAT-3′; (2-3) PCR amplification using the synthesized cDNA as a template; (2-4) PCR amplification products were detected by 1% agarose gel electrophoresis; (2-5) Recovery of the amplified fragment and seamless cloning link with the pIEx-4-GFP-His vector; (2-6) Cloning and sequencing of recovered fragments.

6. The method for functional verification of the cotton bollworm olfactory receptors OR48 and OR75 according to claim 5, characterized in that: Step (3) includes: (3-1) 20E was dissolved in DMSO to 10 mg / mL, and insulin was diluted in PBS to 100 ng / μL. Then different concentrations of 20E and insulin were injected into the sixth instar larvae at 6 h, and DMSO and PBS were used as controls, respectively. (3-2) Extracting total RNA from cotton bollworm using Trizol reagent; (3-3) Synthesize the first-strand cDNA using ChamQ Universal SYBR qPCR Master Mix Kit; (3-4) RT-qPCR reaction and electrophoresis.

7. The method for functional verification of the cotton bollworm olfactory receptors OR48 and OR75 according to claim 6, characterized in that: Step (4) includes: The T7 promoter sequence was added to the 5' end of the primer sequence. The primer sequences were: dsOR48F:5'-GCGTAATACGACTCACTATAGGGAACAAGCGAAACGGGAA AT-3', dsOR48R:5'-GCGTAATACGACTCACTATAGGTCAGTAGCTGACGGAACAC G-3'; dsOR75F:5'-GCGTAATACGACTCACTATAGGTACCATTGGGTCCGATAT-3', dsOR75R:5'-GCGTAATACGACTCACTATAGGAGGTTTGAGTGCGTTCTG-3; Using cDNA as a template, the corresponding target gene fragment is amplified, and then the target gene fragment with a T7 promoter is used as a template to synthesize dsRNA.

8. The method for functional verification of the cotton bollworm olfactory receptors OR48 and OR75 according to claim 7, characterized in that: Step (5) includes: dsRNA was diluted to 200 ng / μL with 1×PBS, and sixth-instar 6-hour-old larvae were placed on ice for about 15 min until the larvae were immobile. 1 μg dsRNA was injected into the larvae from the third ventral foot forward in the abdomen of the larvae using a microinjector. The injection was performed once every 24 h. For the third injection, 1 μg dsRNA and 500 ng 20E were injected into the larvae together. Each experimental group and control group had no less than 30 larvae, and three biological replicates were performed. An equal amount of dsGfp was used as the control group.

9. The method for functional verification of the cotton bollworm olfactory receptors OR48 and OR75 according to claim 8, characterized in that: Step (6) includes: (6-1) After knocking down the 20E nuclear receptor EcR and the insulin receptor InR, the expression changes of OR48 and OR75 were detected respectively; (6-2) Chromatin immunoprecipitation (CHIP) was used to detect the binding of EcR and OR48.

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