CN114573676B

CN114573676B - Application of neuropeptide Gm-Crz and receptor thereof in prevention and treatment of oriental fruit moth

Info

Publication number: CN114573676B
Application number: CN202210128457.5A
Authority: CN
Inventors: 刘小侠; 程杰; 张松斗; 李贞�
Original assignee: China Agricultural University
Current assignee: China Agricultural University
Priority date: 2022-02-11
Filing date: 2022-02-11
Publication date: 2023-04-18
Anticipated expiration: 2042-02-11
Also published as: CN114573676A

Abstract

The application provides grapholitha molesta neuropeptide Gm-Crz, a neuropeptide receptor Gm-CrzR and a gene thereof, wherein the corresponding amino acid and nucleotide sequence are SEQ ID NO.25-28; the application also provides application of the grapholitha molesta neuropeptide or the gene thereof, and an antagonist or an interfering agent of the grapholitha molesta neuropeptide receptor or the gene thereof in the control of the grapholitha molesta. The neuropeptide and the neuropeptide receptor have good application prospect in the aspect of preparing novel specific pollution-free insecticides.

Description

Application of neuropeptide Gm-Crz and receptor thereof in prevention and treatment of oriental fruit moth

Technical Field

The application belongs to the field of biotechnology and the field of agricultural pest control, and particularly provides grapholitha molesta neuropeptides Gm-Crz and receptors thereof, and application of the grapholitha molesta neuropeptides to control.

Background

The oriental fruit moth is a worldwide important fruit tree pest. Because the fruit eaten by the young insects of the insect species is hidden as a pest, the control effect of the pesticide is not ideal, and the adult period is an excellent control period. The Grapholitha molesta adults have strong fertility and have important research significance in preventing and controlling the pests by preventing the normal reproduction of the Grapholitha molesta.

Therefore, the development of the study on the nerve regulation and control of the reproductive capacity of the oriental fruit moth imagoes is not only beneficial to the research and development of a specific control agent for preventing and controlling the oriental fruit moth imagoes, but also has very important application prospect for preventing and controlling orchard pests.

Disclosure of Invention

The neuropeptide Gm-Crz of the oriental fruit borer and the receptor Gm-CrzR thereof are found in the oriental fruit borer for the first time, and a specific tissue expression mode is determined; the grapholitha molesta neuropeptide Gm-Crz can be combined with a receptor Gm-CrzR in the grapholitha molesta, and the neuropeptide Gm-Crz and the receptor Gm-CrzR can also regulate and control the generation of sperm protein of the grapholitha molesta male worms, so that the fertility is regulated and controlled.

In one aspect, the application provides grapholitha molesta neuropeptide Gm-Crz with an amino acid sequence of SEQ ID No.28.

On the other hand, the application provides the grapholitha molesta neuropeptide Gm-Crz gene, and the nucleotide sequence of the gene is SEQ ID NO.27.

On the other hand, the application provides a grapholitha molesta neuropeptide receptor Gm-CrzR, and the amino acid sequence of the grapholitha molesta neuropeptide receptor Gm-CrzR is SEQ ID NO.26.

On the other hand, the application provides a grapholitha molesta neuropeptide receptor Gm-CrzR gene, and the nucleotide sequence of the gene is SEQ ID NO.25.

On the other hand, the application provides the application of the grapholitha molesta neuropeptide or the gene thereof, and the antagonist or the interference agent of the grapholitha molesta neuropeptide receptor or the gene thereof in preparing a medicament for preventing and treating the grapholitha molesta.

In another aspect, the present application provides a method of controlling grapholitha molesta comprising administering the above-described grapholitha molesta neuropeptides, or genes thereof, antagonists or interferents of the grapholitha molesta neuropeptide receptors, or genes thereof.

Further, the antagonist or the interfering agent is an RNA interfering agent.

Further, the RNA interference reagent is dsRNA, and the template sequences for synthesizing the dsRNA are SEQ ID NO.29 and SEQ ID NO.30.

Further, the medicament or method reduces the content of seminal protein in the androgenic gland of the oriental fruit moth.

Further, the agent or method reduces the egg laying rate and hatchability of mated females of oriental fruit moth.

Has the advantages that:

the invention shows that the silent neuropeptide Crz and the receptor CrzR can obviously reduce the secretion of the seminal protein of the oriental fruit moth males, thereby reducing the genital force. The ligand polypeptide of the grapholitha molesta neuropeptide Crz and a receptor CrzR thereof have good binding capacity, and the ligand provided by the invention has in vitro activity, and the provided ligand polypeptide and DNA encoding the ligand polypeptide can be used for developing a grapholitha molesta specificity control agent aiming at the GPCR. According to the expression system of the recombinant GPCR protein and the ligand-receptor binding activity determination system, mature peptide analogues with the specificity of the grapholitha molesta neuropeptide Crz can be screened, agonists or antagonists are screened by blocking or over-stimulating through exogenous ligands, and the normal reproduction of the grapholitha molesta is interrupted, so that the grapholitha molesta is used for preventing and treating the grapholitha molesta and has potential application prospects in the aspect of preparing novel specific pollution-free insecticides.

Drawings

FIG. 1 is a diagram showing a structure of a precursor sequence of the Gm-Crz gene; wherein the signal peptide is underlined; the predicted mature peptide sequence is shaded in grey; font boxed indicates amidation signal; bold font indicates the site of the break.

FIG. 2 is a diagram showing the structure of ORF sequence of the Gm-CrzR gene; wherein the grey shading indicates 7 transmembrane domains.

FIG. 3 is a gene expression pattern diagram of Grapholitha molesta Gm-Crz and Gm-CrzR; wherein A: gm-Crz gene expression pattern; b: a Gm-CrzR gene expression pattern; c: anatomical map of male reproductive system of Grapholitha molesta Busck; d: expression pattern of Gm-CrzR gene in male reproductive system. Data in the figure represent mean ± sem, different letters represent significant differences between different groups (P < 0.05).

FIG. 4 shows the Ca in CHO cells transfected with pcDNA3.1 (+)/Gm-CrzR plasmid stimulated by different gradient concentrations of Gm-Crz and Gm-AKH (control) ²⁺ The effect of concentration.

FIG. 5 shows the effect of RNA interference on Gm-Crz and Gm-CrzR genes; wherein A is the RNA interference efficiency of the Gm-Crz gene; b is the RNA interference efficiency of the Gm-CrzR gene. .

FIG. 6 shows the effect of Gm-Crz and Gm-CrzR genes on the adnexal seminal protein of Grapholitha molesta.

FIG. 7 shows the effect of Gm-Crz and Gm-CrzR genes on the fertility of Grapholitha molesta males; the statistical indexes are the egg laying amount (A) and the egg hatching amount (B) of the female mating with the statistical indexes in 7 days.

Detailed Description

Experimental materials:

adults of oriental fruit moth, a population artificially bred in an IPM laboratory of an insect line from the university of Chinese agriculture, university plant protection institute, have been bred continuously for many generations. The adult oriental fruit moth with strong vitality is selected for use on the same day. Adult oriental fruit moths are bred in a disposable transparent plastic box, honey water with the concentration of 5 percent is fed, female moths lay eggs on the inner wall of the plastic box, then the female moths are cut into egg cards and placed in a new transparent plastic box filled with apples, and the adult oriental fruit moths are bred in the plastic box with the set photoperiod of 14 illumination: 10 dark, at a temperature of 25. + -. 2 ℃ and a relative humidity of 70%.

The statistical analysis method comprises the following steps:

all statistics were analyzed using GraphPad Prism 7.00 software. Multiple data comparison experiments Using the Turkey's HSD multiple comparison test of ANOVA, the different lower case letters indicated significant differences (P;)<0.05). Effective medium concentrations were calculated using a non-linear curve fit (50% effective concentrations, EC) ₅₀ ) The value is obtained. Differential statistics of interference efficiency Using Two-tailed Student's T test (P ×)<0.01). Comparison of egg production with egg hatchability Using the Mann-Whitney U test (P. Star.)<0.05；P**<0.01)。

Example 1 acquisition and analysis of Gene sequences of Grapholitha molesta Gm-Crz and Gm-CrzR

Obtaining ORF sequences of the oriental fruit moth Gm-Crz and Gm-CrzR genes:

the picrocardia pyricularis central nervous system transcriptome database (NCBI SRA accession number: SRR 10762445) constructed based on the experiment searches files annotated by the database through keywords, finds unigene sequences and compares the unigene sequences on an NCBI website, and screens to obtain suspected Gm-Crz and Gm-CrzR gene sequences.

Cloning and sequence analysis of Grapholitha molesta Gm-Crz and Gm-CrzR genes:

and designing primers for amplifying ORF according to the obtained suspected Gm-Crz and Gm-CrzR gene sequences for PCR verification. The primer information is shown in Table 1.

TABLE 1 primers for PCR amplification of Gm-Crz and Gm-CrzR genes

After the PCR product is connected through a T Vector of a pClone007 Simple Vector (TSINGKE) kit, the connecting product is transformed into Escherichia coli, and the specific method comprises the following steps:

a) -80 ℃ in a freezer, the competent cells (DH 5. Alpha.) were immediately placed on ice and thawed for about 10min;

b) Adding 4 μ L of the ligation product or plasmid into 50 μ L of competent cells, gently stirring, and performing ice bath for 30 min;

c) Water bath at 42 deg.C, heat shock for 45s, taking out, and incubating on ice for 2min;

d) Adding an SOC culture medium to a final volume of 1mL, placing in a 37 ℃ shaking table, shaking at 225rpm for 1h;

e) Aspirate 100. Mu.L of LB solid medium. The mixture was placed upside down in a 37 ℃ incubator in the dark and incubated overnight.

f) And picking positive single colonies for detection.

The positive clones were sent to the company for sequencing to obtain gene sequences, which were analyzed as follows: identification of the ORF using the NCBI ORF Finder; signal peptide was predicted using SignalP v4.0 Server; using NeuroPred to predict the mature peptide cleavage site; transmembrane region was predicted using TMHMM server v 2.0.

Sequence analysis showed that: the ORF size of the Gm-Crz gene is 318bp, and 105 amino acids are coded in total, namely a precursor sequence. The amino terminus of the Gm-Crz gene encoding the Gm-Crz peptide precursor predicts 1 signal peptide followed by the mature peptide, the amino terminus has an amidation signal and "KR" is a binary cleavage site (fig. 1). The ORF size of the Gm-CrzR gene is 1284bp, and 427 amino acids are coded in total. The Gm-CrzR gene belongs to a typical GPCR, with 7 typical transmembrane domains (TM 1-7, as shown in FIG. 2).

Example 2 analysis of tissue expression patterns of Grapholitha molesta Gm-Crz and Gm-CrzR genes

Sampling male oriental fruit moth, dissecting several tissues (CNS, fat body FB, intestinal tract Gut, ma tube MT, reproductive system MRS, other residual tissues, and whole worm), and finely dissecting reproductive system to obtain main tissues (spermary TE, adynaud AG, ejaculatory duct DED + uniejaculatory duct)DES, seminal vesicle SmV + vas deferens VD + seminal vesicle SV). Immediately after collection, the cells were placed in liquid nitrogen. Three biological replicates were set up for each experiment. The expression level of the Gm-Crz and Gm-CrzR genes in the tissues is detected by using fluorescent quantitative PCR. The total RNA of each sample collected above was extracted with TRIzol reagent (Invitrogen), and the concentration and purity thereof were examined with NanoDrop 2000 (Invitrogen). First strand cDNA was synthesized using the Tiangen first strand synthesis kit. Primers for the Gm-Crz and Gm-CrzR genes and the reference genes actin and GAPDH were designed using Primer 5 software (Table 2). The PCR efficiency of each gene primer was examined by diluting the synthesized cDNA 5-fold. qRT-PCR mixture according to Takara

Instructions for Premix Ex TaqTM (TliRNase H Plus). This reaction system was operated with a fluorescent quantitative PCR StepOne thermocycler (ABI, USA) under the conditions of 94 ℃ for 2min, followed by 40 cycles of 95 ℃ for 15s, 60 ℃ for 30s and 60 ℃ for 1min, heating at 95 ℃ for 30s and cooling at 60 ℃ for 15s for melting curve detection. qRT-PCR data 2 ^-ΔΔCT The method is carried out.

TABLE 2.QPCR primers

The expression modes of Gm-Crz and Gm-CrzR genes in different tissues of the oriental fruit moth are researched by fluorescent quantitative PCR. The Gm-Crz gene is highly expressed only in the CNS (fig. 3A); the Gm-CrzR gene expression sites are various, with the highest expression in CNS and FB and higher expression in MRS (FIG. 3B). By fine dissection of the male reproductive system (FIG. 3C), the Gm-CrzR gene was found to be expressed in the highest amount, particularly in testis TE and accessory gland AG (FIG. 3D).

Example 3 detection of the binding Capricorn fruit Gm-Crz mature peptide to its receptor Gm-CrzR

Construction of Grapholitha molesta Gm-CrzR expression plasmid:

a Gm-CrzR gene segment with an enzyme cutting site is amplified by PCR by using a primer (table 3) designed as follows, and the Gm-CrzR is connected to a eukaryotic expression vector pcDNA3.1 by a conventional vector construction method such as double enzyme cutting, connection, transformation and the like.

TABLE 3 primers for construction of eukaryotic expression vectors with restriction sites

Cell culture and transfection:

CHO cell (Chinese hamster ovary cell) culture Medium containing the following components was used: DMEM/F-12, bovine serum (total bovine serum) and antibiotics (Penicillin/streptomycin). 5% CO at 37 deg.C ₂ The incubator cultures the cells. The cells are passaged in a sterilization table, the original culture medium is firstly sucked away, 1mL of sterile PBS buffer solution is added into the culture bottle for cleaning, and then a proper amount of 0.025 percent trypsin is added into the culture bottle, so that the digestive juice is sucked out after all the cells are covered. And (3) putting the culture bottle into an incubator at 37 ℃ for incubation for 5min, performing microscopic examination, adding 1mL of culture medium after the cells are found to be detached, and gently blowing and uniformly mixing to resuspend the cells. The collected medium was put into a 1.5mL sterile centrifuge tube, centrifuged at 1000rpm for 1min and then removed. The supernatant was aspirated, 1mL of medium was added, the mixture was aspirated and mixed, and the cells were passaged according to 1.

Selecting a culture flask with a good cell state, and performing cell transfection by adopting the following method: (1) adding 5 mu g of the constructed Gm-CrzR expression vector plasmid and 5 mu g of Aequorin (Aequorin) plasmid into a DMEM/F-12 minimal medium; (2) according to the plasmid: transfection reagent =1, 3 adding transfection reagent Lipofectamine 2000 (Invitrogen), gently mixing by a pipette, and standing at room temperature for 15min; (3) adding the above transfection mixture to the cells prepared in advance by spot-adding, at 37 deg.C, 5% CO ₂ Culturing for 24h under the condition.

Intracellular Ca ²⁺ And (3) detection:

(1) the transfected cells were collected, resuspended in minimal medium, and then added to a storage solution of Coelenterazine (Coelenterazine) (final concentration 500. Mu.M) protected from lightStirring for 3h, and finally quantifying until the cell concentration is 10 ⁶ –10 ⁷ Per mL; (2) ligand to be tested (polypeptide sequence: pQTFQYSRGWTN-NH) ₂ ) With a control ligand (polypeptide sequence: pQLTFTSSWGG-NH ₂ ) From 10 ^-6 To 10 ¹ Mu M dilution with 8 concentrations in a total gradient; (3) detection of intracellular Ca ²⁺ Changing: the cells are placed in an opaque 96-hole enzyme label plate, and chemiluminescence values are detected by adopting a SpectraMax i3x multifunctional enzyme label instrument under the stimulation of ligands with different concentrations. DMEM/F-12 minimal medium was used as a negative control, and 100. Mu.M ATP was used as a positive control. The reaction contained 50. Mu.L of ligand and 50. Mu.L of cells. All tested at least 3 biological replicates.

As shown in FIG. 4, eight different concentrations of the synthetic Gm-Crz and Gm-AKH mature peptides were used to stimulate the CHO cell line expressing the fusion plasmid pcDNA3.1 (+)/Gm-CrzR. The results show that only Gm-Crz can cause intracellular Ca ²⁺ Increased concentration and higher ligand concentration, stronger reaction, calculated EC ₅₀ The value was 86.76nM.

Example 4 Effect of Haemophilus littoralis Gm-Crz and Gm-CrzR Gene silencing on males

Synthesis and injection of dsRNA:

dsRNA was synthesized according to T7 RiboMAX TM Express RNAi System (Promega) kit using the primers shown in Table 4 below. After the concentration of the synthesized dsRNA was determined, it was diluted with DEPC water to a final concentration of 5. Mu.g/. Mu.L and placed at-80 ℃ until use.

TABLE 4 primers for the Synthesis of double-stranded RNA

The Grapholitha molesta was divided into 3 groups, the first group was injected with dsRNA of synthesized Gm-Crz, the second group was injected with dsRNA of synthesized Gm-CrzR, and the last group was injected with dsRNA of GFP gene (this gene is green fluorescent protein gene, used as negative control) as control. During injection, carbon dioxide is used for getting rid of vigna nivea Graham's moth, and the precordial part of the nivea Graham's moth is selected as the injection part. The injected oriental fruit moth is put in a light cycle of 14:10 and a humidity of 70% and a temperature of 25 ℃.

Detection of RNA interference Effect:

after 3 days of RNA interference, 5 litchis each group were taken, and total RNA of litchis was extracted using an RNA extraction kit. And carrying out reverse transcription on the extracted RNA by using a kit to synthesize cDNA. Then, the expression levels of the Gm-Crz and Gm-CrzR genes after RNA interference were detected by quantitative PCR using the primers shown in Table 2.

Influence of Gm-Crz and Gm-CrzR on the sperm protein of Grapholitha molesta Busck:

the dsRNA-injected males, after emerging for 2 days (reaching sexual maturity), were dissected in PBS to obtain accessory gland tissues and transferred to protein buffer. The homogenate was centrifuged for 15min at 10 000g at 60s,4 ℃ to separate into seminal protein (supernatant) and sperm (pellet). Protein concentration was measured according to the BCA method protein detection kit (Beyotime) instructions. Influence of Gm-Crz and Gm-CrzR on the fecundity of Grapholitha molesta males:

the male worms injected by the dsRNA are subjected to 1 part pairing with untreated female worms after emerging for 2 days (reaching sexual maturity), the male worms are removed after the pairing is finished, the female worms successfully mated continue to be normally raised, the egg laying amount within 7 days is counted, and the egg hatching rate is counted.

Quantitative PCR results show that after the Gm-Crz and Gm-CrzR genes are interfered for 3 days, the transcription levels of the Gm-Crz and the Gm-CrzR genes are obviously reduced compared with a control injected with dsGFP (figure 5), and the RNA interference on the Gm-Crz and Gm-CrzR genes is effective.

As shown in fig. 6, the seminal protein content in the accessory glands after injection of GmCrz and GmCrzR dsRNA was significantly reduced compared to the injection of GFP dsRNA.

The influence of the Gm-Crz and Gm-CrzR genes on the fertility of males was studied by mating dsGm-Crz and dsGm-CrzR knockdown males with untreated females. The results show that the 7-day egg laying amount of the female mated with the male in the treated group is remarkably reduced (figure 7A), and the egg hatchability is also remarkably reduced (figure 7B).

The application relates to other sequences as follows:

Gm-CrzR nucleotide (SEQ ID NO. 25)

ATGGGAAGTTCAGGCAACAATACCACATCATATGACGATGGGTACCTTAGTTCCCCAGGCAATCTAGAAATTCTACCACCATTCGCAATATGGCCAATAGATAAATGCATAAGATTAGCTATAATAGAAAATAATACAGATCAATCAAAAGTGACGGACACTGACTTTTTGTACAATGAGACTCGTGTAAAGTGTTGGGACCATTCACCAGTTTTGGAAAATGGAACGTTAATAAAAGCTAGTGTACTCTCAGTGATAGCAGTATTATCATTTTTTGGGAATATAGCTACTATAATAAGTATAAAAAGAGGCAAGAGGAGCAGAGGTCGAGCAAGGCCATCTTGGACGGCGATATATAGCCTGATATTTCAGCTGAGCATCGCTGATTTGCTTGTAACTGTATTTTGTATTGGTGGAGAAGCCGCCTGGATGATCACCGTAGAGTGGTACGCTGGGAACATAGGATGCAAGTTATTTAAGTTTGTGCAAATGTTCTCATTGTATCTGAGCACATTCATATTGGTGTTGATTGGAGTAGACCGGTGGTTGGCTGTCAAATATCCTATGAAAAGTATAGCGACCGCAACGAGAAGTGGACGACTAGTCATTATAGCATGGATATCAAGTTTCTTCTTGAGTATACCGCAGGCAATCGTGTTTCGTGTCGCAAAAGGCCCATTTGTTGAAGAGTTTTATCAATGTGTCACACACGGGTATTACACGGAGAAGTGGCAAGAGCAGCTATATACGACAGTGACCTTGATTTTCATGTTTATCCTGCCACTTCTGATTCTTGTGTCCACTTATGTTTCTACTGTTCGGACTATTGCTCAAAGCGAAAAAGTTTTTAAGCCAGAAGTAATAAGACATGAAAAGTACCTCACCCCGGACTTGAACCGGAGACGACTGATCGACCGAGCCAAGATGAAATCTTTACGGATGTCTGTCGTCATTGTGGCGGCGTTTTTAATATGCTGGACACCGTATTATGTCATGATGGTTATATTCACATTTCTCGATCCTGATAAAAATTTAAGTGAGGATCTTCGGAATGCAATATTCTGCTTCGGGATGTCCAATAGCCTGGTAAATCCAGTAATATACGGCGCCTTCCATTTGTGGCCTCGGAAGAAACCCGTGCACAGACATAGTGACAGAGACTCTGGACACCACGCCTCCCTCCTCAGGCGTGGAGACAATACCTCATCAGTGAGACTGACTACCATTAGATCTATCAGATCCTCAAACAAATATACTAACGGAAACAATGTTAGCTTGTTATAA

Gm-CrzR amino acid (SEQ ID NO. 26)

MGSSGNNTTSYDDGYLSSPGNLEILPPFAIWPIDKCIRLAIIENNTDQSKVTDTDFLYNETRVKCWDHSPVLENGTLIKASVLSVIAVLSFFGNIATIISIKRGKRSRGRARPSWTAIYSLIFQLSIADLLVTVFCIGGEAAWMITVEWYAGNIGCKLFKFVQMFSLYLSTFILVLIGVDRWLAVKYPMKSIATATRSGRLVIIAWISSFFLSIPQAIVFRVAKGPFVEEFYQCVTHGYYTEKWQEQLYTTVTLIFMFILPLLILVSTYVSTVRTIAQSEKVFKPEVIRHEKYLTPDLNRRRLIDRAKMKSLRMSVVIVAAFLICWTPYYVMMVIFTFLDPDKNLSEDLRNAIFCFGMSNSLVNPVIYGAFHLWPRKKPVHRHSDRDSGHHASLLRRGDNTSSVRLTTIRSIRSSNKYTNGNNVSLL

Gm-Crz nucleotide (SEQ ID NO. 27)

ATGGGGTCCAACATCAAGGCCTCAACTCTGCTCCTAATCTTCGTGACCTTCACCACCGTGGCTGCCCAGACCTTCCAGTACTCCCGGGGATGGACCAACGGCAAGAGGGCCCACAAGAGGGACGAGGCTGTGGCTGGGGTGGCTGGAAACCTGGAGCGGATCTTGAGCCCATGCCAGATGAGCAAGCTTAAGTATGTGCTGGAAGGAAAGCCTTCAAGCGAGCGGTTACTTCTACCATGTGACTACTCGGAAGAGGACGAAGCCCCCAAACGTTACAAGAACGAACGCCAAGATCCACTTTTTGACAGCTTTCTTTGA

Gm-Crz amino acid (SEQ ID NO. 28)

MGSNIKASTLLLIFVTFTTVAAQTFQYSRGWTNGKRAHKRDEAVAGVAGNLERILSPCQMSKLKYVLEGKPSSERLLLPCDYSEEDEAPKRYKNERQDPLFDSFL

dsRNA template:

dsGm-CrzR(SEQ ID NO.29)

GCAAGAGGAGCAGAGGTCGAGCAAGGCCATCTTGGACGGCGATATATAGCCTGATATTTCAGCTGAGCATCGCTGATTTGCTTGTAACTGTATTTTGTATTGGTGGAGAAGCCGCCTGGATGATCACCGTAGAGTGGTACGCTGGGAACATAGGATGCAAGTTATTTAAGTTTGTGCAAATGTTCTCATTGTATCTGAGCACATTCATATTGGTGTTGATTGGAGTAGACCGGTGGTTGGCTGTCAAATATCCTATGAAAAGTATAGCGACCGCAACGAGAAGTGGACGACTAGTCATTATAGCATGGATATCAAGTTTCTTCTTGAGTATACCGCAGGCAATCGTGTTTCGTGTCGCAAAAGGCCCATTTGTTGAAGAGTTTTATCAATGTGTCACACACGGGTATTACACGGAGAAGTGGCAAGAGCAGCTATATACGACAGTGACCTTGATTTTCATGTTTATCCTGCCACTTCTGATTCTTGTGTCCACTTATGTTTCTACTGTTCGGACTATTGCTC

dsGm-Crz(SEQ ID NO.30)

GGGGTCCAACATCAAGGCCTCAACTCTGCTCCTAATCTTCGTGACCTTCACCACCGTGGCTGCCCAGACCTTCCAGTACTCCCGGGGATGGACCAACGGCAAGAGGGCCCACAAGAGGGACGAGGCTGTGGCTGGGGTGGCTGGAAACCTGGAGCGGATCTTGAGCCCATGCCAGATGAGCAAGCTTAAGTATGTGCTGGAAGGAAAGCCTTCAAGCGAGCGGTTACTTC

SEQUENCE LISTING

<110> university of agriculture in China

<120> neuropeptide Gm-Crz and application of receptor thereof in prevention and treatment of oriental fruit moth

<160> 30

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acggagaagt ggcaagagca gctatatacg acagtgacct tgattttcat gtttatcctg 780

ccacttctga ttcttgtgtc cacttatgtt tctactgttc ggactattgc tcaaagcgaa 840

aaagttttta agccagaagt aataagacat gaaaagtacc tcaccccgga cttgaaccgg 900

agacgactga tcgaccgagc caagatgaaa tctttacgga tgtctgtcgt cattgtggcg 960

gcgtttttaa tatgctggac accgtattat gtcatgatgg ttatattcac atttctcgat 1020

cctgataaaa atttaagtga ggatcttcgg aatgcaatat tctgcttcgg gatgtccaat 1080

agcctggtaa atccagtaat atacggcgcc ttccatttgt ggcctcggaa gaaacccgtg 1140

cacagacata gtgacagaga ctctggacac cacgcctccc tcctcaggcg tggagacaat 1200

acctcatcag tgagactgac taccattaga tctatcagat cctcaaacaa atatactaac 1260

ggaaacaatg ttagcttgtt ataa 1284

<210> 26

<211> 427

<212> PRT

<213> Grapholita molesta

<400> 26

Met Gly Ser Ser Gly Asn Asn Thr Thr Ser Tyr Asp Asp Gly Tyr Leu

1 5 10 15

Ser Ser Pro Gly Asn Leu Glu Ile Leu Pro Pro Phe Ala Ile Trp Pro

20 25 30

Ile Asp Lys Cys Ile Arg Leu Ala Ile Ile Glu Asn Asn Thr Asp Gln

35 40 45

Ser Lys Val Thr Asp Thr Asp Phe Leu Tyr Asn Glu Thr Arg Val Lys

50 55 60

Cys Trp Asp His Ser Pro Val Leu Glu Asn Gly Thr Leu Ile Lys Ala

65 70 75 80

Ser Val Leu Ser Val Ile Ala Val Leu Ser Phe Phe Gly Asn Ile Ala

85 90 95

Thr Ile Ile Ser Ile Lys Arg Gly Lys Arg Ser Arg Gly Arg Ala Arg

100 105 110

Pro Ser Trp Thr Ala Ile Tyr Ser Leu Ile Phe Gln Leu Ser Ile Ala

115 120 125

Asp Leu Leu Val Thr Val Phe Cys Ile Gly Gly Glu Ala Ala Trp Met

130 135 140

Ile Thr Val Glu Trp Tyr Ala Gly Asn Ile Gly Cys Lys Leu Phe Lys

145 150 155 160

Phe Val Gln Met Phe Ser Leu Tyr Leu Ser Thr Phe Ile Leu Val Leu

165 170 175

Ile Gly Val Asp Arg Trp Leu Ala Val Lys Tyr Pro Met Lys Ser Ile

180 185 190

Ala Thr Ala Thr Arg Ser Gly Arg Leu Val Ile Ile Ala Trp Ile Ser

195 200 205

Ser Phe Phe Leu Ser Ile Pro Gln Ala Ile Val Phe Arg Val Ala Lys

210 215 220

Gly Pro Phe Val Glu Glu Phe Tyr Gln Cys Val Thr His Gly Tyr Tyr

225 230 235 240

Thr Glu Lys Trp Gln Glu Gln Leu Tyr Thr Thr Val Thr Leu Ile Phe

245 250 255

Met Phe Ile Leu Pro Leu Leu Ile Leu Val Ser Thr Tyr Val Ser Thr

260 265 270

Val Arg Thr Ile Ala Gln Ser Glu Lys Val Phe Lys Pro Glu Val Ile

275 280 285

Arg His Glu Lys Tyr Leu Thr Pro Asp Leu Asn Arg Arg Arg Leu Ile

290 295 300

Asp Arg Ala Lys Met Lys Ser Leu Arg Met Ser Val Val Ile Val Ala

305 310 315 320

Ala Phe Leu Ile Cys Trp Thr Pro Tyr Tyr Val Met Met Val Ile Phe

325 330 335

Thr Phe Leu Asp Pro Asp Lys Asn Leu Ser Glu Asp Leu Arg Asn Ala

340 345 350

Ile Phe Cys Phe Gly Met Ser Asn Ser Leu Val Asn Pro Val Ile Tyr

355 360 365

Gly Ala Phe His Leu Trp Pro Arg Lys Lys Pro Val His Arg His Ser

370 375 380

Asp Arg Asp Ser Gly His His Ala Ser Leu Leu Arg Arg Gly Asp Asn

385 390 395 400

Thr Ser Ser Val Arg Leu Thr Thr Ile Arg Ser Ile Arg Ser Ser Asn

405 410 415

Lys Tyr Thr Asn Gly Asn Asn Val Ser Leu Leu

420 425

<210> 27

<211> 318

<212> DNA

<213> Grapholita molesta

<400> 27

atggggtcca acatcaaggc ctcaactctg ctcctaatct tcgtgacctt caccaccgtg 60

gctgcccaga ccttccagta ctcccgggga tggaccaacg gcaagagggc ccacaagagg 120

gacgaggctg tggctggggt ggctggaaac ctggagcgga tcttgagccc atgccagatg 180

agcaagctta agtatgtgct ggaaggaaag ccttcaagcg agcggttact tctaccatgt 240

gactactcgg aagaggacga agcccccaaa cgttacaaga acgaacgcca agatccactt 300

tttgacagct ttctttga 318

<210> 28

<211> 105

<212> PRT

<213> Grapholita molesta

<400> 28

Met Gly Ser Asn Ile Lys Ala Ser Thr Leu Leu Leu Ile Phe Val Thr

1 5 10 15

Phe Thr Thr Val Ala Ala Gln Thr Phe Gln Tyr Ser Arg Gly Trp Thr

20 25 30

Asn Gly Lys Arg Ala His Lys Arg Asp Glu Ala Val Ala Gly Val Ala

35 40 45

Gly Asn Leu Glu Arg Ile Leu Ser Pro Cys Gln Met Ser Lys Leu Lys

50 55 60

Tyr Val Leu Glu Gly Lys Pro Ser Ser Glu Arg Leu Leu Leu Pro Cys

65 70 75 80

Asp Tyr Ser Glu Glu Asp Glu Ala Pro Lys Arg Tyr Lys Asn Glu Arg

85 90 95

Gln Asp Pro Leu Phe Asp Ser Phe Leu

100 105

<210> 29

<211> 522

<212> DNA

<213> Artificial

<400> 29

gcaagaggag cagaggtcga gcaaggccat cttggacggc gatatatagc ctgatatttc 60

agctgagcat cgctgatttg cttgtaactg tattttgtat tggtggagaa gccgcctgga 120

tgatcaccgt agagtggtac gctgggaaca taggatgcaa gttatttaag tttgtgcaaa 180

tgttctcatt gtatctgagc acattcatat tggtgttgat tggagtagac cggtggttgg 240

ctgtcaaata tcctatgaaa agtatagcga ccgcaacgag aagtggacga ctagtcatta 300

tagcatggat atcaagtttc ttcttgagta taccgcaggc aatcgtgttt cgtgtcgcaa 360

aaggcccatt tgttgaagag ttttatcaat gtgtcacaca cgggtattac acggagaagt 420

ggcaagagca gctatatacg acagtgacct tgattttcat gtttatcctg ccacttctga 480

ttcttgtgtc cacttatgtt tctactgttc ggactattgc tc 522

<210> 30

<211> 230

<212> DNA

<213> Artificial

<400> 30

ggggtccaac atcaaggcct caactctgct cctaatcttc gtgaccttca ccaccgtggc 60

tgcccagacc ttccagtact cccggggatg gaccaacggc aagagggccc acaagaggga 120

cgaggctgtg gctggggtgg ctggaaacct ggagcggatc ttgagcccat gccagatgag 180

caagcttaag tatgtgctgg aaggaaagcc ttcaagcgag cggttacttc 230

Claims

1. The application of an antagonist or an interference agent of the grapholitha molesta neuropeptide Gm-Crz gene and/or the grapholitha molesta neuropeptide receptor Gm-CrzR gene in the preparation of a medicament for reducing the content of seminal protein in the adnexal gland of the grapholitha molesta androgenic gland; the antagonist or the interference agent is dsRNA, and the synthetic template sequence of the dsRNA is SEQ ID NO.29 and/or SEQ ID NO.30.

2. The application of antagonists or interference agents of the grapholitha molesta neuropeptide Gm-Crz gene and/or the grapholitha molesta neuropeptide receptor Gm-CrzR gene in the preparation of medicaments for reducing the egg laying rate and the hatching rate of mated grapholitha molesta female; the antagonist or the interference agent is dsRNA, and the synthetic template sequence of the dsRNA is SEQ ID NO.29 and/or SEQ ID NO.30.

3. A method of reducing the content of seminal fluid proteins in the adglands of grapholitha molesta, said method comprising administering an antagonist or an interferent of the grapholitha molesta neuropeptide Gm-Crz gene and/or the grapholitha molesta neuropeptide receptor Gm-CrzR gene; the antagonist or the interference agent is dsRNA, and the synthetic template sequence of the dsRNA is SEQ ID NO.29 and/or SEQ ID NO.30.

4. A method of reducing egg laying and hatchability of mated female grapholita molesta comprising administering an antagonist or an interferent of the grapholita molesta neuropeptide Gm-Crz gene and/or the grapholita molesta neuropeptide receptor Gm-CrzR gene; the antagonist or the interference agent is dsRNA, and the synthetic template sequence of the dsRNA is SEQ ID NO.29 and/or SEQ ID NO.30.