CN108129559B

CN108129559B - Diamondback moth neuropeptide Px-sNPF and receptor thereof and application of neuropeptide Px-sNPF in regulating trehalose content in diamondback moth body

Info

Publication number: CN108129559B
Application number: CN201711242448.4A
Authority: CN
Inventors: 陈学新; 王泽华; 邹佳妮; 黄健华; 时敏
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: CN108129559A

Abstract

The invention discloses plutella xylostella neuropeptide Px-sNPF, a receptor thereof and application of the plutella xylostella neuropeptide Px-sNPF in regulating and controlling trehalose content in bodies of plutella xylostella. The invention discovers plutella xylostella neuropeptide Px-sNPF and receptor Px-sNPFR thereof in plutella xylostella for the first time; the plutella xylostella neuropeptide Px-sNPF not only can be combined with a receptor Px-sNPFR in a plutella xylostella body cavity, but also can regulate and control the content of trehalose in the plutella xylostella body.

Description

Diamondback moth neuropeptide Px-sNPF and receptor thereof and application of neuropeptide Px-sNPF in regulating trehalose content in diamondback moth body

Technical Field

The invention relates to the technical field of molecular biology and genetic engineering, in particular to plutella xylostella neuropeptide Px-sNPF, a receptor thereof and application thereof in regulating and controlling trehalose content in plutella xylostella.

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 neurooptide 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.

The Plutella xylostella (Lepidoptera) is an important vegetable pest, the problem of drug resistance is prominent, and the problem of how to effectively control the Plutella xylostella is always concerned. Therefore, the research on the function of the neuropeptide of the plutella xylostella is helpful for exploring novel insecticidal active macromolecules and developing novel insect growth regulators.

Disclosure of Invention

The invention provides plutella xylostella neuropeptide Px-sNPF, a receptor thereof and application of the plutella xylostella neuropeptide Px-sNPF in regulating the trehalose content in plutella xylostella bodies.

The specific contents are as follows:

the invention provides plutella xylostella neuropeptide Px-sNPF, and the amino acid sequence 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 plutella xylostella neuropeptide Px-sNPF in regulating and controlling the trehalose content in plutella xylostella bodies.

Specifically, the application comprises the following steps: RNA interference is carried out on the gene coding the plutella xylostella neuropeptide Px-sNPF.

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

The invention also provides a precursor sequence of plutella xylostella neuropeptide Px-sNPF, wherein the amino acid sequence of the precursor sequence is shown as SEQ ID No.2, the precursor sequence is obtained by encoding an Open Reading Frame (ORF) of a Px-sNPF gene, and the nucleotide sequence of the Open Reading Frame (ORF) of the Px-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 plutella xylostella neuropeptide Px-sNPF, wherein the receptor is Px-sNPFR, the amino acid sequence 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 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:

in the invention, the plutella xylostella neuropeptide Px-sNPF and the receptor Px-sNPFR thereof are found in a plutella xylostella coilia-plutella xylostella parasitic system for the first time; the plutella xylostella neuropeptide Px-sNPF not only can be combined with a receptor Px-sNPFR in a plutella xylostella body cavity, but also can regulate and control the content of trehalose in the plutella xylostella body.

Drawings

FIG. 1 shows the precursor sequence of the Px-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 Px-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 Px-sNPF; cAMP Level represents the concentration of cAMP.

FIG. 4 shows the Px-sNPF stimulation pairs transfected with pcDNA3.1(+)Ca in HEK293 cells of the/Px-sNPFR plasmid²⁺The effect of concentration.

FIG. 5 shows the effect of RNA interference on the Px-sNPF gene in example 4;

wherein A is RNA interference efficiency; b is the change in the level of trehalose in the blood lymph of the diamondback moth after the interference (this result is an experimental result, n is 7); relative Transcript Level denotes the Relative Transcript Level; trehalose inhemolymph represents the content of Trehalose in hemolymph; dsgfp represents double stranded RNA of GFP; dspx-sNPF represents double-stranded RNA of Px-sNPF; all data are mean ± sem; significance analysis was performed using SPSS 19 and Student's Test tests were performed between groups at significance levels P <0.05, P <0.01, P < 0.001.

Detailed Description

Example 1

1. Acquisition of ORF sequences of plutella xylostella Px-sNPF gene and plutella xylostella Px-sNPFR gene

(1) 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 by TRIzol method, and then using kit SuperScript^TMII ReverseTranscriptase (Invitrogen) first strand cDNA was synthesized.

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

2. cloning and sequence analysis of plutella xylostella Px-sNPF gene and plutella xylostella Px-sNPFR gene

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

TABLE 1 primers for PCR amplification of Px-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 Px-sNPF gene is 543bp (the nucleotide sequence is shown as SEQ ID NO. 1), 180 amino acids (namely a precursor sequence, the amino acid sequence is shown as SEQ ID NO. 2) are coded, and the predicted molecular weight is 19.6 kDa.

The amino terminus of the encoded Px-sNPF peptide precursor of the Px-sNPF gene is predicted to have 1 signal peptide, 3 amidation signals, and a single or double cleavage site (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 Px-sNPF polypeptide 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. Localization of Px-sNPF in the Plutella xylostella nervous System

2.1 dissect different tissues

Taking 10-20 heads of diamondback moths of 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, neuronal cells secreting Px-sNPF were localized in the plutella xylostella nervous system by immunohistochemistry.

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 show that Px-sNPF-secreting cells are predominantly in the diamondback moth brain. The terminal diamondback moth larva of 4 years old has abundant neural secretory cells Px-sNPF, and each half brain has about 40.

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 Lipofectaminereagent2000(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 the stably transfected cells with 90% confluence, adding a proper amount of 0.025% trypsin

Digesting EDTA mixed solution, collecting cells, centrifuging at 2000rpm for 1min, discarding the old culture medium, adding 1ml of new culture medium, and blowing gently to form single cells;

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

In HEK293 cell line expressing the fusion plasmid pcDNA3.1(+)/Px-sNPFR, synthetic Px-sNPF mature peptides (shown as SEQ ID NO. 6) of 7 different gradient concentrations (1 μ M, 100nM, 10nM, 1nM, 100pM, 10pM, 1pM) were stimulated with 10 μ M Forskolin as positive control.

The results show that Px-sNPF can cause the decrease of cAMP concentration in cells, EC₅₀6.16nM (FIG. 3); the synthetic short peptide Px-sNPF can be combined with the receptor Px-sNPFR, thereby causing the decrease 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 are prepared by Hanks buffer solution, which are Px-sNPF polypeptide solutions of 1 mu M, 10nM and 100pM respectively.

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 Fura 2-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 Fura 2-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 mul of cell sample to be detected into each black 96-well plate, treating the cell sample by the Px-sNPF polypeptide solution with different concentrations prepared above, detecting fluorescence simultaneously, detecting the luminous intensity of Fura 2-AM by using excitation light of 340nm and 380nm of a fluorescence spectrophotometer, thereby calculating Ca²⁺Concentration, and output the data in chart format.

As shown in FIG. 4, HEK293 cell line expressing the fusion plasmid pcDNA3.1(+)/Px-sNPFR was stimulated with three different concentrations (1. mu.M, 10nM, 100pM) of synthetic Px-sNPF mature peptide. The results show that Px-sNPF causes intracellular Ca²⁺The concentration increases and the higher the ligand concentration, the stronger the reaction.

Example 4

1. RNA interference diamondback moth Px-sNPF gene

dsRNA was synthesized according to the (promega) kit using the primers shown in Table 4 below.

TABLE 4 primers for the synthesis of double-stranded RNA

Diamondback moths were divided into 2 groups, one group was injected with dsRNA of synthetic Px-sNPF, and the other group was injected with dsRNA of GFP gene (which is a green fluorescent protein gene used as a negative control) as a 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. Detection of RNA interference Effect

Two days after RNA interference, 3 plutella xylostella per group were ground with 1ml Trizol, and total RNA was extracted. And carrying out reverse transcription on the extracted RNA by using a kit to synthesize cDNA. Subsequently, the expression level of Px-sNPF after RNA interference was determined by quantitative PCR using the primers shown in Table 5 below.

TABLE 5qPCR primers

Quantitative PCR results showed that the Px-sNPF transcript levels were significantly down-regulated after 2 days of interfering with the Px-sNPF gene compared to the dsGFP-injected control (fig. 5A), indicating that RNA interference with Px-sNPF was effective. 3. 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.

4. Effect of Px-sNPF on the level of trehalose in Plutella xylostella haemolymph

Two days after the interference, trehalose concentrations in the diamond back moth haemolymph were measured and found to be reduced compared to the dsGFP-injected control (figure 5B). This indicates that interfering with transcription of this gene will cause a change in the trehalose concentration in the blood cavity of plutella xylostella.

Sequence listing

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<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>6

Ser Val Arg Ser Pro Ser Arg Arg Leu Arg Phe

1 5 10

<210>7

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>7

ccgtcaaggc ggctgagatt 20

<210>8

<211>25

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>8

aggggtatat atttgaatgt tgtcg 25

<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 neuropeptide Px-sNPF is characterized in that the amino acid sequence is shown as SEQ ID NO. 6.

2. The use of the plutella xylostella neuropeptide Px-sNPF of claim 1 for regulating the trehalose content in plutella xylostella.

3. The application of the receptor of plutella xylostella neuropeptide Px-sNPF in regulating the trehalose content in plutella xylostella according to claim 1, wherein the receptor is Px-sNPFR, and the amino acid sequence is shown as SEQ ID No. 4.