CN107840875B

CN107840875B - Plutella xylostella cotesia ruber neuropeptide Cv-sNPF and receptor thereof and application of plutella xylostella cotesia ruber neuropeptide Cv-sNPF in increasing trehalose content in plutella xylostella

Info

Publication number: CN107840875B
Application number: CN201711243966.8A
Authority: CN
Inventors: 陈学新; 邹佳妮; 王泽华; 时敏; 黄健华
Original assignee: Zhejiang University ZJU
Current assignee: Zhejiang University ZJU
Priority date: 2017-11-30
Filing date: 2017-11-30
Publication date: 2020-09-08
Anticipated expiration: 2037-11-30
Also published as: CN107840875A

Abstract

The invention discloses plutella xylostella cotesia neuropeptide Cv-sNPF, a receptor thereof and application of the plutella xylostella cotesia neuropeptide Cv-sNPF in improving the content of trehalose in bodies of plutella xylostella. The invention discovers plutella xylostella coilform neuropeptide Cv-sNPF and a receptor Px-sNPFR in a plutella xylostella coilform-plutella xylostella parasitic system for the first time; the plutella xylostella cotesia elatus neuropeptide Cv-sNPF not only can be combined with a receptor Px-sNPFR in a plutella xylostella body, but also can improve the content of trehalose in the plutella xylostella body.

Description

Plutella xylostella cotesia ruber neuropeptide Cv-sNPF and receptor thereof and application of plutella xylostella cotesia ruber neuropeptide Cv-sNPF in increasing trehalose content in plutella xylostella

Technical Field

The invention relates to the technical field of molecular biology and genetic engineering, in particular to plutella xylostella cocoon neuropeptide Cv-sNPF, a receptor thereof and application thereof in improving the trehalose content in diamondback moth bodies.

Background

Neuropeptides are important small molecule information-transmitting substances that affect almost all physiological processes in the body, exist as neuromodulators in the central nervous system, and play a role as neurohormones in the blood.

short neuropeptide F (sNPF) is an important peptide substance in invertebrates and participates in various physiological processes such as regulation and control of food intake, growth and development, exercise, clock rhythm, learning, memory and the like. It is a direct homologous protein of vertebrate neuropeptide Y (NPY), has sequence similarity, and activates downstream effector to play a role in regulation by binding G protein-coupled receptor (GPCR).

The sNPF is widely distributed in insects and crustaceans, and the C terminal of the mature peptide has a conserved receptor activating sequence of xPxLRL Famide (x refers to any amino acid). The copy of the sNPF gene occurs in the gene, i.e., from 1 to many copies are contained in a precursor sequence, and the copy number is different among different species. Among insects, higher metamorphotic insects (coleoptera, diptera, lepidoptera) tend to have longer peptide precursor sequences and higher copy numbers.

Currently, the study of sNPF is mainly performed around model insects, mainly Drosophila melanogaster, and the study of non-model insect deficiency system. At present, the function analysis of sNPF is not comprehensive, and the existing research mainly aims at regulating and controlling feeding, growth and development; meanwhile, the function of the sNPF is not completely consistent among different species and different developmental stages of the same species.

Parasitic wasps are agriculturally important natural enemy insects, and part of life history of the parasitic wasps depends on hosts. Parasitic wasps utilize a variety of means to control their hosts and create superior growth environments for their own or later generations of development. An interaction exists between the parasitic wasp and the host in the development process, and once the parasitic wasp is separated, the parasitic wasp can cause dysplasia and even death. In the long-term evolution process, parasitic wasps master the strategy of dealing with and utilizing hosts, can inhibit the immune system of hosts, and change the hormone level and other physiological processes of hosts.

Taking the parasitic wasp as an example, the parasitic wasp injects parasitic factors such as venom and virus particles into the host body while spawning, destroys the immune system of the host and regulates and controls some physiological behaviors of the host. In addition, malformed cells formed by the release of serosal layer and grown young bees can secrete substances to the host body to play a regulating role in the hatching process of bee eggs.

At present, researches on physiological regulation in a parasitic system mainly focus on the aspects of immunity, growth and development and nutrition metabolism, and other researches on neuroendocrine aspects are only sporadically reported, for example, the expressions of some neuropeptide genes of hosts, hypopharynx (allatostatin), prothymus (pth) and the like can be obviously changed before and after parasitic wasp parasitism, but the regulation mechanism is not involved.

Therefore, there is a need for further exploration of the function of sNPF in non-model insects, especially in neuroendocrine studies of parasitic wasps.

Disclosure of Invention

The invention provides plutella xylostella cotesia neuropeptide Cv-sNPF, a receptor thereof and application thereof in improving the trehalose content in plutella xylostella.

The specific contents are as follows:

the invention provides a plutella xylostella cocoon bee neuropeptide Cv-sNPF, the amino acid sequence of which is shown in SEQ ID No. 6. The neuropeptide is mature peptide and is a peptide segment formed by cutting a precursor sequence by protease.

The invention finds the application of the neuropeptide Cv-sNPF of the plutella xylostella cotesia nitidum in improving the trehalose content in bodies of plutella xylostella.

Specifically, the application comprises the following steps: injecting the plutella xylostella cotesia ruber neuropeptide Cv-sNPF into the body of the plutella xylostella.

Specifically, the trehalose is located in plasma of plutella xylostella.

The invention also provides a precursor sequence of the neuropeptide Cv-sNPF of the plutella xylostella cocoon bee, wherein the amino acid sequence of the precursor sequence is shown as SEQ ID NO.2, the precursor sequence is obtained by encoding the Open Reading Frame (ORF) of the Cv-sNPF gene, and the nucleotide sequence of the Open Reading Frame (ORF) of the Cv-sNPF gene is shown as SEQ ID NO. 1.

The precursor sequence can also be used for increasing the content of trehalose in bodies of plutella xylostella; namely: injecting the precursor sequence into the body of the diamondback moth.

The invention also provides a receptor of the neuropeptide Cv-sNPF of the plutella xylostella cocoon bee according to claim 1, wherein the receptor is Px-sNPFR, the amino acid sequence of the receptor is shown as SEQ ID No.4, and the nucleotide sequence of the gene for coding the receptor is shown as SEQ ID No. 3.

The invention also provides an application of the Px-sNPFR as a plutella xylostella cocoon neuropeptide Cv-sNPF receptor; the amino acid sequence of the Px-sNPFR is shown in SEQ ID NO. 4.

The invention also provides application of the receptor in improving the trehalose content in the diamondback moth body.

Compared with the prior art, the invention has the following beneficial effects:

the invention discovers plutella xylostella coilform neuropeptide Cv-sNPF and a receptor Px-sNPFR in a plutella xylostella coilform-plutella xylostella parasitic system for the first time; the plutella xylostella cotesia elatus neuropeptide Cv-sNPF not only can be combined with a receptor Px-sNPFR in a plutella xylostella body, but also can improve the content of trehalose in the plutella xylostella body.

Drawings

FIG. 1 shows the precursor sequence of the Cv-sNPF gene in example 1;

wherein the signal peptide is underlined; bold indicates predicted mature peptide sequence; light grey indicates the site of cleavage; dark grey indicates amidation signal.

FIG. 2 is the ORF sequence of the Px-sNPFR gene in example 1;

wherein, the ORF sequence of the Px-sNPFR gene has 7 typical transmembrane domains and belongs to GPCR family; the 7 transmembrane domains are underlined.

FIG. 3 is a graph showing the effect of different gradient concentrations of Cv-sNPF stimulation on cAMP concentration in HEK293 cells transfected with pcDNA3.1(+)/Px-sNPFR plasmid in example 3;

wherein Log Ligand represents the concentration of Cv-sNPF; cAMP Level represents the concentration of cAMP.

FIG. 4 shows the stimulation of Ca in HEK293 cells transfected with pcDNA3.1(+)/Px-sNPFR plasmid by Cv-sNPF at different gradient concentrations in example 3²⁺The effect of concentration.

FIG. 5 is the variation of trehalose content in the Plutella xylostella haemolymph after injection of Cv-sNPF in example 4;

wherein Trehalose in hemolymph represents the content of Trehalose in hemolymph; all data are mean ± sem; significance analysis was performed using SPSS 19 and one-way anova between groups (Tukey's test) with significance levels of P <0.05, P <0.01, P < 0.001.

Detailed Description

Example 1

1. Acquisition of ORF sequences of Chorista discoreans Cv-sNPF gene and diamondback moth Px-sNPFR gene

(1) Collecting young diamondback moth cocoon bee and diamondback moth larvae: taking the diamondback moth after 5-6 days, dissecting in an insect culture medium under a microscope. Tearing one opening to facilitate the young bee to slide out, adding a new culture medium, cleaning for several times, discarding the cleaning solution, adding precooled 1ml TRIzol, grinding and placing at-80 ℃ for later use; 3 plutella xylostella larvae growing to 4 years old are taken, washed by alcohol, placed in 1ml of TRIzol, ground and placed at-80 ℃ for later use.

(2) Extracting total RNA of diamondback moth cocoon bee and diamondback moth by TRIzol method, and then utilizing kit SuperScript^TMII Reverse Transcriptase (Invitrogen) first strand cDNA was synthesized.

(3) According to the transcriptome (SAMN03406711) data of the plutella xylostella cocoon bee and the genome database DBM (http:// iae.fafu.edu.cn/DBM /), sequences of suspected Cv-sNPF and Px-sNPFR genes are obtained by screening through a bioinformatics method.

2. Cloning and sequence analysis of cabbage moth cocoon bee Cv-sNPF gene and diamondback moth Px-sNPFR gene

And designing primers for amplifying ORF according to the obtained suspected Cv-sNPF and Px-sNPFR gene sequences for PCR verification. The primer information is shown in Table 1.

TABLE 1 primers for PCR amplification of Cv-sNPF and Px-sNPFR

After the PCR product is connected by a pGEM-Teasy (Promega) kit, the connection product is transformed into escherichia coli by a heat shock method, and the specific method is as follows:

1) taking out the competent cells from a refrigerator at the temperature of minus 80 ℃, immediately placing the competent cells on ice, and thawing the competent cells for about 10 min;

2) adding 10 μ l of the ligation product or plasmid into 100 μ l of competent cells, gently stirring, and performing 15min ice bath;

3) heating in 42 deg.C water bath for 1min, taking out, and incubating on ice for 10 min;

4) adding 600 μ l of antibiotic-free LB culture medium, placing in a 37 deg.C shaking table, and activating at 180rpm for 45-60 min;

5) spreading appropriate volume of transformed cells on LB plate with corresponding resistance, and culturing at 37 deg.C for 10-11 hr;

6) and picking positive single colonies for detection.

Positive clones were sent to the company for sequencing to obtain the gene sequence.

Sequence analysis showed that:

the ORF size of the Cv-sNPF gene is 327bp (the nucleotide sequence is shown as SEQ ID NO. 1), 108 amino acids are coded (namely a precursor sequence, the amino acid sequence is shown as SEQ ID NO. 2), the predicted molecular weight is 12.5kDa, and the isoelectric point is 9.304.

The amino-terminal of the Cv-sNPF peptide precursor sequence encoded by the Cv-sNPF gene predicts 1 signal peptide (i.e. mature peptide) flanked by a single or binary cleavage site with only one amidation signal (as shown in FIG. 1).

The size of the ORF of the Px-sNPFR gene is 1320bp (the nucleotide sequence is shown as SEQ ID NO. 3), 439 amino acids are coded (namely a precursor sequence, the amino acid sequence is shown as SEQ ID NO. 4), and the predicted protein molecular weight is 50.3 kDa.

The Px-sNPFR gene belongs to GPCRs and has 7 typical transmembrane domains (TM 1-7, shown in figure 2).

Example 2

1. Synthesis of Cv-sNPF mature peptide and preparation of specific antibody

The hydrophilicity and secondary structure analysis of the peptide precursor protein sequence were analyzed by the Protean software, and a specific 10-20aa fragment was selected for synthesis (shown in SEQ ID NO.5), the synthetic sequences are shown in Table 2. The synthesized short peptide is synthesized by Shanghai bio-engineering company, 5mg is synthesized in total, and the purity is more than 90%. The antibody is prepared by using the synthesized polypeptide (such as SEQ ID NO.5) as an antigen.

Table 2 design of synthetic short peptide sequences

2. Cv-sNPF location in the Neplutella xylostella cocoon bee larva nervous system

2.1 dissect different tissues

Taking 10-20 heads of the plutella xylostella cocoon bee larvae with the same age and consistent growth and development, dissecting brain tissues under a stereoscopic microscope, and putting the dissected brain tissues into cold PBS for later use.

2.2 tissue localization

Using the antibody prepared in step 1 of this example, the Cv-sNPF-secreting neuronal cells were localized in the juvenile bee nervous system by immunohistochemical methods.

Immunohistochemistry was as follows:

1) preparing 100ml of 1 XPBTTX (100ml of PBS + 100. mu.l of Triton-X + 50. mu.l of Tween) solution;

2) dissecting a sample in 1 XPBS buffer solution, picking a required tissue, washing for several times, and placing the tissue in a 1.5ml centrifuge tube containing PBS;

3) adding 16% paraformaldehyde (Thermo Scientific), diluting to 4%, and rotating and mixing at 360 deg.C for 20min at room temperature in dark;

4) slightly sucking away the solution containing paraformaldehyde, adding a proper amount of PBTX, uniformly mixing for 5min at 360 degrees, removing the washing solution, and repeatedly washing for 3 times;

5) blocking with 1% BSA for 2 hr;

6) removing the blocking solution, adding PBTX solution containing primary antibody with the dilution ratio of 1:50 or 1:100, and incubating overnight at 4 ℃;

7) collecting primary anti-incubation liquid in other centrifuge tubes, and synchronously washing for 5min for 3 times in the step 4);

8) adding secondary antibody incubation solution, diluting at a ratio of 1:1000, and incubating at room temperature for 2 hr;

9) washing with PBTX for 3 times, each for 10 min;

dissecting the target tissue in PBS under dark conditions, placing on a glass slide with a trace amount of PBS, placing, gently sucking away the excess PBS with filter paper, coating a cover slip with DAPI mounting solution (Thermo scientific), inverting on the glass slide, mounting at-20 deg.C overnight.

2.3 Picture acquisition and processing

The mounted sample is observed in a Zeiss LSM 780 laser confocal microscope. The nucleus was observed with a 405nm laser and the red fluorescence was observed with a 561nm laser. The images were processed using a software ZEN 2009 digital image processing system.

2.4 tissue localization results analysis

Immunohistochemical results showed that there was a signal for binding of Cv-sNPF antibody in the brains of young 2-year-old bees. Two clusters of Cv-sNPF nerve secretory cells which are symmetrically distributed are obviously arranged in the central position of the front end of the brain of the young bee, and each cluster comprises 7 cells.

Example 3

1. Selection of stably transfected cell lines

1.1 construction of eukaryotic expression vectors

The Px-sNPFR gene fragment with the enzyme cutting sites is amplified by PCR by using the primers (table 3) designed as follows, and is connected to the eukaryotic expression vector pcDNA3.1 by the conventional vector construction methods of double enzyme cutting, connection, transformation and the like.

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

1.2 cell culture, transfection and selection

The HEK293 cells were cultured in a DMEM high-glucose medium manufactured by Invitrogen, supplemented with inactivated 10% Fetal Bovine Serum (FBS). Cells were cultured at 37 ℃ in a 5% CO2 incubator (Thermoscientific). Cell passage was performed in a sterile table, uv-sterilized for more than 20min before starting. During operation, the table top is disinfected by alcohol, the original culture medium is absorbed in the disinfection table, 1ml of sterile PBS buffer solution is added to the wall of the disinfection table to clean the culture dish, and residual calcium and magnesium plasma is removed. Immediately, a proper amount of 0.025% trypsin-EDTA mixture was added to the dish to ensure coverage of all cells and then the digestive juice was aspirated. And (3) putting the culture dish into an incubator at 37 ℃ for incubation for 2min, performing microscopic examination, adding 1ml of culture solution after cell retraction becomes round and is detached from the wall, slightly blowing and beating the mixed cell suspension, and blowing and beating the mixed cell suspension to a single cell state as much as possible. The collected medium was put into a 1.5ml sterile centrifuge tube, centrifuged at 2000rpm for 1min and then removed. The supernatant was aspirated, 1ml of culture medium was added, the mixture was blown up and mixed, and the cells were passaged at a ratio of 1: 6.

Selecting a culture dish with a good cell state, and performing cell transfection by adopting the following method when the cell confluence is 70-90 percent:

1) 18. mu.l of the transfection Reagent Lipofectamine Reagent2000(Invitrogen) was diluted with 300. mu.l of serum-free medium Opti-MEM (1X);

2) mu.g of transfection plasmid DNA was diluted with 500. mu.l of Opti-MEM (1X);

3) take 300. mu.l of the solution diluted in 2) and the reagent in 1), 1:1, mixing, and incubating for 5min at room temperature;

4) the old medium was aspirated off, washed once with sterile PBS, and the wash solution was discarded;

5) adding appropriate amount of Opti-MEM (1X) to cover the cells, adding the mixture of 3), mixing, incubating for 6hr, sucking out, and culturing in common culture medium containing serum.

The next day, the cells were screened with G418(0.8mg/ml), and the G418 concentration was increased as needed at the later stage. Two weeks after selection, stably transfected cell lines were obtained.

2. Measurement of cAMP

2.1 adherent cell culture

1) Taking stably transfected cells with the confluence degree of 90%, adding a proper amount of 0.025% trypsin-EDTA mixed solution for digestion, collecting the cells, centrifuging at 2000rpm for 1min, discarding the old culture medium, adding 1ml of new culture medium, and gently blowing to form a single cell state;

2) cells were diluted as required for the experiment to ensure that approximately 50,000 cells per well and 100 μ l per well were added to a 96-well plate and used for measurement of cAMP content 24 hours after adherent growth.

2.2 measurement of cAMP content

Solutions of the mature peptide of Cv-sNPF (shown as SEQ ID NO. 6) at concentrations of 1. mu.M, 100nM, 10nM, 1nM, 100pM, 10pM, 1pM, and 0M were prepared, and the adherent cells were treated with these Cv-sNPF mature peptide solutions at different concentrations for 15min, followed by the use of cAMP-Glo produced by Promega corporation^TMKit, according to the instructionsMeasurement of cAMP content was performed and the data were analyzed.

The results showed that stimulation of the cell line with synthetic short peptide Cv-sNPF at various gradient concentrations caused a decrease in intracellular cAMP concentration with an EC50 of 1.47nM (fig. 3); the synthetic mature peptide Cv-sNPF is proved to be capable of binding to the receptor Px-sNPFR, and further causing the reduction of the cAMP concentration in cells.

3、Ca²⁺Measurement of

Ca was carried out by the method described below²⁺And (4) measuring the concentration.

3.1 preparation work

Preparing 0.02% EDTA PBS solution; 3 gradient ligand solutions, namely Cv-sNPF mature peptide solutions (shown as SEQ ID NO. 6) of 1 mu M, 10nM and 100pM are prepared by using Hanks buffer solution. 3.2 cell sample preparation

1) Digesting the cells: rinsing with PBS, treating with 0.02% EDTA PBS solution, covering cells, incubating in a 37 deg.C CO2 incubator for 5min until the cells fall off, gently blowing, mixing, and sucking into 1.5ml centrifuge tube;

2) centrifuging at 3500rpm for 2min, resuspending with Hanks solution, and washing for 2 times (gently sucking liquid to reduce cell loss during washing);

3) adding 1ml Hanks for resuspension, adding Fura2-AM 5. mu.l mother liquor (0.5mmol/L), incubating in a CO2 incubator at 37 deg.C in the dark for 30min, and mixing 20 times by reversing every 5 min;

4) centrifuging at 3500rpm for 2min, resuspending with Hanks solution, washing for 2 times, and sufficiently removing residual Fura2-AM working solution;

5) adding 600 plus 700 mul Hanks buffer solution, resuspending, incubating for 10min, ensuring the complete de-esterification of AM in the cells, and keeping out of the sun for testing.

3.3 fluorescence detection

1) Starting a multifunctional microplate reader TECAN F200PRO, setting the temperature to be 37 ℃, and setting a detection program;

2) controlling the injector, washing the injector for several times by using sterilized deionized water, changing A, B channels into Hanks buffer solution, and rinsing for 1 time respectively;

adding 90 μ l of cell sample to be detected into each black 96-well plate, and using the different concentrations prepared aboveThe cell sample is treated by Cv-sNPF mature peptide solution, fluorescence is detected, and the luminous intensity of Fura2-AM is detected by using 340nm and 380nm excitation light of a fluorescence spectrophotometer, so that Ca is calculated²⁺Concentration, and output the data in chart format.

As shown in FIG. 4, the HEK293 cell line expressing the fusion plasmid pcDNA3.1/Px-sNPFR was stimulated with three different concentrations (1. mu.M, 10nM, 100pM) of the polypeptide Cv-sNPF, which caused intracellular Ca²⁺The concentration increases instantaneously and the higher the ligand concentration, the stronger the reaction.

Example 4

1. Microinjection of Cv-sNPF mature peptide

Diamondback moths were divided into 4 groups, each of which was injected daily with 0.5, 0.05, 0.005ng of Cv-sNPF mature peptide (as shown in SEQ ID No. 6), and the other was injected with PBS as a negative control. During injection, the diamondback moth is fixed, and an internode membrane at 4-5 sections of the reciprocal abdomen of a diamondback moth larva is selected as an injection part. The injected plutella xylostella is placed in a light cycle of 16: 9 and a humidity of 60% and a temperature of 25 ℃.

2. Extraction of plutella xylostella hemolymph and determination of trehalose content

The body surface was washed with 75% alcohol, washed with PBS pH 7.4, and water was blotted with filter paper. Dissected on a clean Parafilm membrane. The larval body surface is torn open by dissecting forceps under a body type microscope, and the hemolymph is sucked by a capillary (Eppendorf) and then added into a 0.2ml centrifuge tube containing a certain volume of mineral oil. Centrifuge at 1000g for 10min at 4 ℃ and aspirate hemolymph into a volume of water for dilution. The content of trehalose in hemolymph after dilution was determined by a conventional anthrone method.

3. Effect of Cv-sNPF on the level of trehalose in Plutella xylostella haemolymph

When 0.5, 0.05 and 0.005 ng/day Cv-sNPF are respectively injected into hemolymph of 3-instar-middle diamondback moth larvae without parasitism, the hemolymph trehalose level of the diamondback moth is obviously increased to 25-30 nmol/mu l from the original 20 nmol/mu l after continuous injection for 3 days compared with a control injected with PBS (figure 5).

Sequence listing

<110> Zhejiang university

<120> plutella xylostella coilia neuropeptide Cv-sNPF and receptor thereof and application of neuropeptide Cv-sNPF in increasing trehalose content in plutella xylostella

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<213> diamondback moth cocoon bee (Cotesia vestalis)

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gatgcggaga gagagagaaa aagacgcaca aataggatgc taatagcgat ggtggcgata 780

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Pro Ile Glu Asp Lys Ala Ile Gln Ala Ala Phe Cys Thr Ala Tyr Thr

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Ile Ile Phe Val Val Gly Ile Phe Gly Asn Ala Leu Val Cys Tyr Ala

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Val Ile Arg Asn Arg Ala Met Gln Thr Val Thr Asn Leu Phe Ile Thr

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Asn Leu Ala Leu Ser Asp Ile Leu Leu Cys Val Phe Ala Val Pro Phe

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Thr Pro Leu Tyr Thr Phe Leu Gly Arg Trp Val Phe Gly Gly Pro Leu

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Cys His Ile Met Pro Tyr Ala Gln Gly Cys Ser Val Tyr Ile Ser Thr

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Leu Thr Leu Thr Ser Ile Ala Ile Asp Arg Phe Phe Val Ile Ile Tyr

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Pro Phe Lys Pro Arg Met Gln Leu Lys Thr Cys Ile Gly Leu Ile Ile

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Phe Ile Trp Val Phe Ala Leu Ser Val Thr Phe Pro Tyr Gly Tyr Tyr

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Met Ala Leu Glu Asp Val Tyr Cys Ala Glu Lys Trp Pro Ser Asp His

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Pro Phe Ile Val Met Ala Phe Cys Tyr Thr Cys Val Ser Ile Lys Leu

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Asn Asp Arg Leu Lys Ser Arg Pro Gly Ser Lys Asn Thr Arg Lys Glu

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Asp Ala Glu Arg Glu Arg Lys Arg Arg Thr Asn Arg Met Leu Ile Ala

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Met Val Ala Ile Phe Gly Leu Ser Trp Leu Pro Leu Asn Leu Ile Asn

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Ile Ser Ser Asp Phe Tyr Ser Phe Ala Glu Asp Trp Arg Tyr Tyr Met

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Val Leu Phe Phe Ala Ala His Phe Ile Ala Met Ser Ser Thr Cys Tyr

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Asn Pro Phe Leu Tyr Ala Trp Leu Asn Glu Asn Phe Arg Lys Glu Phe

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Lys Gln Ile Leu Pro Cys Leu Gly Ala Leu Val Thr Lys Lys Ser Lys

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Arg Lys Phe Asn Gln Ser Glu Arg Thr Asp Met Phe Arg Ser Glu Lys

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Thr Cys Asn Gly Asn Glu Gln Thr Val Gln Glu Ser Leu Leu Thr Ser

355 360 365

Ala Val His Lys Pro Ser Val Asn Tyr Arg Leu Asp Leu Asn Glu Val

370 375 380

Tyr Asn Glu Ser Asn Val Lys Val Cys Glu Asn Glu Glu Asn Asn Ile

385 390 395 400

Ser Pro Asp Glu Lys Pro Glu Glu Asn Arg Ser Pro Ser Asp Asp Gly

405 410 415

Val Asn Leu Tyr Met Phe Ala Asp Lys Ser Met Thr Ser Ser Asp Lys

420 425 430

Glu Pro Ile Val Ser Ala Leu

435

<210>5

<211>16

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>5

Ala Glu Asn Tyr Leu Asp Tyr Gly Glu Glu Asn Ala Asp Arg Asn Ile

1 5 10 15

<210>6

<211>11

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>6

Ser Gln Arg Ser Pro Ser Leu Arg Leu Arg Phe

1 5 10

<210>7

<211>21

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>7

atgaggagtt acagatgcgc t 21

<210>8

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>8

tcaattgtcc tcaaaagatg 20

<210>9

<211>22

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>9

atgccgacgg aaactaccaa at 22

<210>10

<211>23

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>10

ctagagcgcg gacacgatgg gct 23

<210>11

<211>34

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>11

cgcggatcca tgccgacgga aactacaaat ggat 34

<210>12

<211>34

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>12

ccgctcgagc tagagcgcgg acacgatggg ctcc 34

Claims

1. A plutella xylostella cocoon bee neuropeptide Cv-sNPF is characterized in that the amino acid sequence is shown as SEQ ID NO. 6.

2. The use of the neuropeptide Cv-sNPF of diamondback moth cocoon bee according to claim 1 for increasing the fucose content of diamondback moth in vivo.

3. The application of the receptor of the neuropeptide Cv-sNPF of the plutella xylostella cocoon as defined in claim 1 in increasing the trehalose content in the plutella xylostella, wherein the receptor is Px-sNPFR in the plutella xylostella and has an amino acid sequence shown as SEQ ID No. 4.