CN118006622A - Bai Beifei lice SfDpp gene, dsRNA and application thereof in pest control - Google Patents

Abstract

The present invention relates to the field of genetic engineering technology, and specifically to a white-backed planthopper SfDpp gene, dsRNA and its application in pest control, wherein the sequence of the white-backed planthopper SfDpp gene contains: 1) the gene coding region sequence shown in sequence 1 in the sequence table; the dsRNA is an RNA composed of nucleotides shown in sequence 2 in the sequence table; the present invention also provides the application of the above-mentioned white-backed planthopper SfDpp gene or the above-mentioned dsRNA in any of the following a1)-a4): a1) controlling white-backed planthopper; a2) inhibiting the development of white-backed planthopper wings; a3) inhibiting the maturation and extension of white-backed planthopper wings; a4) inhibiting the proliferation of white-backed planthopper compound eyes. The present invention finds that the SfDpp gene plays an important role in regulating the wing development process of white-backed planthoppers, and its expression can inhibit the maturation and extension of white-backed planthopper wings, and inhibit the proliferation of white-backed planthopper compound eyes, thereby providing a reference for the sustainable management of white-backed planthoppers.

Description

SfDpp gene, dsRNA of white-backed planthopper and its application in pest control

Technical field:

The invention belongs to the technical field of genetic engineering, and specifically relates to a white-backed planthopper SfDpp gene, dsRNA and application thereof in pest control.

Background technique:

The white-backed planthopper Sogatella furcifera (Horvath) is one of the important migratory pests on rice in Asia. Both its adults and nymphs can directly suck the phloem sap of rice, causing the plants to turn yellow or even die. In addition, they can also spread viruses and induce a variety of rice diseases during feeding, such as South rice black-streaked dwarf virus (SRBSDV), which can lead to reduced rice yields or even crop failure when the disease is severe. As a migratory pest, the complete development of wings is of great significance for successful migration.

The wing polymorphism and development mechanism of insects have been studied very thoroughly in general, and the wing type and development mechanism of rice planthoppers have also been fully analyzed. Many differentially expressed genes that regulate wing extension have also been identified one after another, but there are few reports on wing development genes in the important rice pest white-backed planthopper. It needs further confirmation to know which genes in the white-backed planthopper can participate in regulating its wing development process.

Summary of the invention:

In view of the deficiencies in the prior art, the present invention provides a white-backed planthopper SfDpp gene, dsRNA and application thereof in pest control.

Specifically, the first aspect of the present invention provides a white-backed planthopper SfDpp gene, wherein the sequence of the white-backed planthopper SfDpp gene contains:

1) The gene coding region sequence shown in Sequence 1 in the sequence listing, or

2) A nucleotide sequence derived from 2) which has the same or different activity as the sequence shown in Sequence 1 in the sequence listing, wherein one or more nucleotides are substituted, deleted or added.

The present invention also provides a dsRNA of the above-mentioned white-backed planthopper SfDpp gene, wherein the dsRNA is an RNA composed of nucleotides shown in sequence 2 in the sequence table.

The present invention also provides an expression box, a recombinant vector, a recombinant bacterium or a transgenic cell line containing the white-backed planthopper SfDpp gene.

The present invention also provides the use of the above-mentioned white-backed planthopper SfDpp gene or the above-mentioned dsRNA or the above-mentioned expression cassette, recombinant vector, recombinant bacteria or transgenic cell line in any one of the following a1)-a4):

a1) Control of white-backed planthopper;

a2) inhibiting the wing development of white-backed planthoppers;

a3) inhibiting the maturation and extension of the wings of white-backed planthoppers;

a4) Inhibit the proliferation of compound eyes of white-backed planthopper.

The present invention also provides a method for inhibiting the wing development of white-backed planthoppers, wherein a substance that inhibits the expression of the wing development gene SfDpp of white-backed planthoppers is introduced into the white-backed planthoppers, thereby inhibiting the wing development of the white-backed planthoppers.

Furthermore, the substance that inhibits the expression of the white-backed planthopper wing development gene SfDpp is the above-mentioned dsRNA.

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

The present invention finds that the SfDpp gene plays an important role in regulating the wing development process of white-backed planthoppers. Its expression can inhibit the maturation and extension of white-backed planthopper wings and inhibit the proliferation of white-backed planthopper compound eyes, thereby providing a reference for the sustainable management of white-backed planthoppers.

Description of the drawings:

Figure 1 shows the SfDpp gene sequence and deduced amino acid sequence of the white-backed planthopper, with the underline indicating the start codon and the stop codon.

Figure 2 shows the expression pattern of the SfDpp gene in different developmental stages of white-backed planthoppers, and the error bars in the figure represent standard errors. eggs: eggs; N1-1d-N5-3d: 1st instar 1-day nymphs, 5th instar 3-day nymphs, AN: newly emerged adults, A20-60: adults that have emerged for 20min-60min, A12 h-48h: adults that have emerged for 12h-48h. Different lowercase letters on the bar graph indicate significant differences (α=0.05).

Figure 3 shows the expression pattern of SfDpp gene in different tissues of white-backed planthopper, and the error bars in the figure represent standard errors. Different lowercase letters on the bar graph represent significant differences (α=0.05).

Figure 4 shows the Dpp gene interference efficiency and phenotype of white-backed planthopper, and the error bars in the figure represent standard errors. Different lowercase letters on the bar graph represent significant differences (α=0.05).

Detailed ways:

The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

In order to study the regulatory effect of key genes of the Hedgehog signaling pathway on the wingspan of white-backed planthoppers, the present invention cloned the Sogatella furcifera Decapentaplegic (SfDpp) gene, analyzed its spatiotemporal expression pattern in white-backed planthoppers, and studied its role in regulating the wingspan of white-backed planthoppers through RNAi technology.

Example 1 Obtaining dsRNA of the SfDpp gene of the white-backed planthopper

1.1 Test insects

The white-backed planthopper used in the present invention is collected from the rice field of the teaching experimental field of Guizhou University in Huaxi, Guiyang, and is placed in an artificial climate box. It is raised and reproduced for multiple generations using TN1 rice under the conditions of 16:8 (L:D), temperature 25±1°C, and humidity 75%±5 for future use.

1.2 Main instruments and reagents

1.2.1 Main instruments

1.2.2 Main reagents

1.3 Test methods

1.3.1 Cultivation of TN 1 rice seedlings

1) Seed soaking and germination: Take out an appropriate amount of TN1 rice seeds and put them into a plastic box with an inner diameter of 32cm×24cm×12cm, add water to immerse the seeds, remove the shriveled seeds floating on the water surface, pour out the excess water, and then place them in an artificial climate box for germination. Wash and replace the water every 24 hours until the rice seeds begin to turn white.

2) Seedling cultivation: Spread a layer of humus soil about 5 cm thick in a 45cm×30cm×10cm stainless steel tray, sprinkle water on it, and then evenly spread the white rice seeds on the humus soil, and then cover it with a layer of humus soil about 1 cm thick. Cover the tray with plastic wrap to retain moisture and prevent evaporation too quickly. After the seedlings emerge from the soil, remove the plastic wrap, fill with water, and observe and water them every day until transplanting.

3) Transplanting: When the rice seedlings in the tray grow to about 20 cm, they can be transplanted into a plastic bucket half filled with soil. Apply compound fertilizer once after they turn green, observe and water them in time every day, and use them when they reach the peak tillering period.

1.3.2 Rearing of white-backed planthoppers

1) Insect cage breeding: Move the rice seedlings in the peak tillering stage into a 70cm×70cm×70cm nylon mesh insect cage, and place white-backed planthoppers in the insect cage, 50-100 in each cage, and allow them to reproduce freely. When the nymphs hatch and grow to the 5th instar, pat them into a plastic basin, suck them into the insect tube for breeding, and wait for them to emerge.

2) Test tube rearing: Pull out the rice seedlings in the peak tillering period, wash the soil on the roots with clean water, cut off the leaves at the top, and then soak and wrap the roots with absorbent cotton, put them into a 3cm×30cm two-way rolled glass test tube, and then absorb the white-backed planthoppers that have emerged for about 24 hours and put them into the insect tube, 10-30 heads per tube, cover them with nylon gauze, and then tighten them with rubber bands, and place them in a climate box to allow them to reproduce. Fresh rice seedlings are replaced every 2-3 days. When the rice seedlings are replaced with rice seedlings, the white-backed planthoppers are first sucked into the new insect tube with a homemade insect sucker, and fresh rice seedlings are replaced, and then placed in an artificial climate box for rearing. The replaced rice seedlings are then put back into the original test tube, sealed with gauze, and placed in an artificial climate box until the nymphs hatch. During this period, they are checked every 24 hours. If nymphs float out, they are promptly absorbed into the new insect tube and reared with TN1 rice seedlings, and fresh rice seedlings are replaced every 3-4 days. When they grow to 3-5 years old, put them in plastic pots and carry out relevant tests according to their age.

1.3.3 Treatment of white-backed planthopper samples

A mixed sample of 4th and 5th instar nymphs and adults 1-3 days after emergence was selected from the white-backed plant hoppers reared in a test tube, pipetted into a 1.5 mL centrifuge tube (EP tube), quickly frozen with liquid nitrogen, and stored in a -80°C refrigerator for later use.

1.3.4 Total RNA extraction from white-backed planthopper

The RNA extraction method of white-backed planthopper was carried out according to the HP Total RNA Kit. The specific operation process is as follows:

1) Place an appropriate amount of magnetic beads in a grinding tube and add 10-20 white-backed plant hoppers into the tube;

2) Pipette 500 μL buffer GTC into the sample grinding tube and add 10 μL β-Mercaptoethanol to each tube;

3) Cover the sample tube with GTC and β-Mercaptoethanol added and put it into the automatic sample grinder for grinding;

4) Take out the ground sample and centrifuge at 14000×g for 5 min at room temperature;

5) Place the gDNA centrifuge column in a collection tube, transfer the supernatant from step 4 to the gDNA column, centrifuge at 14,000 × g for 2 min at room temperature, and transfer the flow-through to a new 1.5 ml centrifuge tube;

6) Add 250ul of anhydrous ethanol to the centrifuge tube and shake to mix;

7) Take out RNA Mini Column is placed in a collection tube, and the mixed solution in the centrifuge tube is aspirated The RNA column was centrifuged at 10,000 × g for 60 s at room temperature, and the flow-through was discarded;

8) Add 300 μL RNA Wash Buffer I, centrifuge at 10,000 × g for 60 s at room temperature, and discard the flow-through;

9) Add 400 μL RNA Wash Buffer I, let stand for 5 min, centrifuge at 10,000 × g for 60 s at room temperature, and discard the flow-through;

10) Add 500 μL RNA Wash Buffer II, centrifuge at 10,000 × g for 60 s at room temperature, and discard the flow-through;

11) Centrifuge at 12000×g for 2 min to remove residual Buffer II, and leave the tube open for 2 min;

12) Transfer the centrifuge column to a new 1.5 mL centrifuge tube, add 30 μL of DEPC-treated water to the center of the column, let stand for 2 minutes, and centrifuge at 10,000 × g for 1 minute;

13) Take 1 μL and 2 μL respectively to detect the concentration and purity; store at -20℃ for later use and at -80℃.

1.3.5 Synthesis of the first strand of cDNA of white-backed planthopper

The extracted total RNA of white-backed planthopper was used as a template and the RNA was reverse transcribed into cDNA using HiFiScript cDNA First-Strand Synthesis Kit. The reverse transcription reaction system was as follows:

Vortex to mix, centrifuge briefly to collect the solution on the tube wall to the bottom of the tube. Incubate at 42℃ for 50 minutes and 85℃ for 5 minutes. After the reaction is completed, centrifuge briefly and cool on ice. Store at -20℃ for a long time.

1.3.6 Primer design

Based on the genome and transcriptome information of white-backed planthopper, the present invention screened the SfDpp gene (Gene ID: Scaffold-34.12), exported the nucleotide sequence of the gene from the Meiji Biocloud system, and performed BLAST in the NCBI database. After comparing and verifying the correctness of the sequence, RNAi and RT-qPCR primers were designed from the coding region of the SfDpp gene using Primer Premier 6.0 software (Table 1.3.6), and sent to Shanghai Shenggong Biological Co., Ltd. for synthesis.

Table 1.3.6 Cloning of RNAi fragments of SfHh and SfDpp genes of white-backed planthopper and RT-qPCR primers

1.3.7 Cloning and PCR amplification of the SfDpp gene of white-backed planthopper

The cDNA obtained by reverse transcription was used as a template and PCR amplification was performed using Taq PCR Master Mix. The PCR reaction system was as follows:

Centrifuge for 30 seconds and place in a PCR instrument to perform the following reaction:

1.3.8 Rubber recycling

Glue recycling steps reference Follow the instructions of QuiControl Gel Extraction kit.

1). Prepare 1% agarose gel electrophoresis plate;

2). Detect the PCR product in an electrophoresis instrument at 120V for 30min;

3). Assemble the rubber cutting blade and burn it on an alcohol lamp until the blade turns red to sterilize it;

4). Weigh the empty centrifuge tube, write a label and record it, turn on the water bath and set the water temperature to 55℃;

5). Place the run gel plate in the gel imaging system and cut the target band with the prepared blade;

6). Weigh the centrifuge tube containing the target band again and calculate the weight of the gel block.

7) Add 3 times the volume of GSB solution to the centrifuge tube containing the target band (100 mg of gel can be regarded as 100 ul, and so on), place it in a 55°C water bath to melt the gel for 6 to 10 minutes (the gel block in the tube is completely melted), invert and mix once every 2 minutes to make it fully melted, take it out and let it stand;

8) Take out the centrifuge column and place it in a collection tube, write a label, wait for the melted gel solution to cool to room temperature, then absorb it into the centrifuge column, let it stand for 1 minute, centrifuge at 12500r for 1 minute, and discard the effluent;

9). Add 650ul WB, let stand for 1min, centrifuge at 12500r for 1min, and discard the flow-through;

10). Centrifuge at 12500r for 2 minutes to remove residual WB; take out the centrifuge column, place it in a new centrifuge tube, open the cover and let it stand for 2 minutes to allow the alcohol to evaporate;

11). Add 30ul of EB to the centrifuge column (preheating at 55℃ in advance will have a better effect), let it stand for 2min; centrifuge at 12500r for 1min, discard the centrifuge column, and store the effluent at -20℃ for later use.

1.3.9 Connection

The gel-recovered product with the target fragment was connected to the pMD-18T vector. The system is as follows:

After thorough mixing, the ligation was carried out at 16°C overnight.

1.3.10 Conversion

The ligation product was transformed into E. coli DH5α competent cells. The transformation steps are as follows:

1) Prepare liquid and solid culture media according to the following system;

Liquid culture medium preparation method:

Solid culture medium preparation method:

2) Pipette the ligation product into 250 μl of liquid culture medium without ampicillin (Amp-), place it in a shaker, and shake at 180 r/min for 1-2 hours;

1.3.11TA cloning and sequencing

1). Briefly centrifuge the bacterial solution at 8000g for 30 seconds, discard the supernatant, and transfer it to an ultra-clean workbench;

2). Use a pipette to draw the strain at the bottom of the tube onto a solid culture medium with ampicillin (Amp ⁺ ) added, light the alcohol lamp, and use a bacterial smear stick to evenly spread the bacterial solution on the solid culture medium next to the burning alcohol lamp;

3). After coating, seal with sealing film, write a label, and invert in a 37℃ incubator for 8-12h;

4). After the strain to be cultured grows colonies, take them to the clean bench, use a 10μl pipette to pick a single colony and dissolve it in 10μl DEPC-treated water, and use universal primers M13-47 and M13-48 to perform PCR to verify the correctness of the clone;

5). Aspirate the strain that produced the target band in PCR into 800 μl of Amp ⁺ liquid culture medium and culture it in a 37°C incubator with shaking overnight.

6). Send the overnight cultured bacterial solution to Shanghai Shenggong Bioengineering Co., Ltd. for sequencing.

1.3.12.dsRNA synthesis

In order to accurately synthesize the dsRNA of the target gene, the template used to synthesize the dsRNA needs to be sequenced. PCR amplification is performed by designing and synthesizing dsRNA primers (Table 1.3.6), and then TA cloning is performed. The bacterial solution with correct sequencing is expanded and the plasmid is extracted for further experiments. PrimerSTAR Max DNA Polymerase is used for PCR reaction. The specific reaction system is as follows:

Add the above reagents into the PCR tube in sequence, flick to mix, centrifuge briefly, and then place on the PCR instrument for reaction. PCR amplification conditions are:

The annealing temperature refers to the Tm value of the primer synthesis report returned by the company. The PCR product was tested for the correctness of the amplified fragment by 1% agarose gel electrophoresis, and then TA cloning was performed and sent to the company for sequencing.

Add the reserved bacterial solution with correct sequencing to 5 mL of LB liquid medium containing Amp, and culture overnight on a shaker at 37°C and 180 rpm. Plasmid MiniPrep Kit was used to extract plasmids. The specific operation steps were carried out according to the instructions. The extracted plasmids were used as templates and the primers in the primer list were used for PCR amplification. The reaction system and conditions were the same as those for the gene cloning PCR amplification mentioned above. The PCR products were tested for the correctness of the amplified fragments by 1% agarose gel electrophoresis, and then the amplified fragments were analyzed by / > The PCR product was recovered and purified using QuiControl Gel Extraction Kit, and the concentration of the purified product was detected using Nanodrop 2000 nucleic acid concentration analyzer to ensure that the concentration of the purified product reached at least 300 ng/μL.

The recovered high-concentration dsDNA was used as a template to synthesize dsRNA in vitro using the TranscriptAid T7 High Yield Transcription Kit. Before use, the 5X TranscriptAid Reaction Buffer needs to be thawed at room temperature, and other reagents need to be thawed on ice. After all reagents are thawed, they need to be flicked to mix and centrifuged briefly. Samples are added according to the following reaction system:

Add the above solutions to a 200 μL PCR tube in sequence, flick to mix, centrifuge briefly, and then place it in a PCR instrument and incubate at 37°C overnight.

After the reaction is completed, add 2 μL DNase I to the reaction system, flick to mix, centrifuge briefly, and then place it in a PCR instrument and incubate at 37°C for 30 min. Then add 2 μL EDTA to the reaction system, flick to mix, centrifuge briefly, and then place it in a PCR instrument and incubate at 37°C for 30 min to terminate the reaction.

1.3.13 dsRNA purification

The dsRNA was purified using the GeneJET RNA Purification Kit. The specific steps are as follows:

1) Transfer the synthesized dsRNA to a 1.5 mL centrifuge tube without enzyme, dilute to 100 μL with DEPC water, add 300 μL Lysis Buffer, and mix well with a pipette;

2) Add 180 μL of anhydrous ethanol and mix by pipetting;

3) Transfer the mixed solution to the adsorption column, centrifuge at 12000g for 1 min at room temperature, discard the waste liquid, and transfer the adsorption column to a new 2 mL collection tube;

4) Add 700 μL Wash buffer 1, centrifuge at 12000 g for 1 min at room temperature, and discard the waste liquid;

5) Add 600 μL Wash buffer 2, centrifuge at 12000 g for 1 min at room temperature, and discard the waste liquid;

6) Add 250 μL Wash buffer 2, centrifuge at 12000 g for 1 min at room temperature, and discard the waste liquid; 7) Centrifuge the empty tube at 12500 g for 2 min at room temperature to remove the residual liquid, and transfer the adsorption column to a 1.5 mL enzyme-free centrifuge tube;

8) Add 50-100 μL of DEPC water to the central filter membrane of the adsorption column, centrifuge at 12000 g for 1 min at room temperature, measure the concentration of the purified dsRNA using a Nanodrop 2000 nucleic acid concentration analyzer, and store at -80°C.

Example 2 Application of SfDpp gene in inhibiting wing development of white-backed planthopper

2.1 Microinjection

The 5th instar day 1 and 3rd instar day 1 nymphs of white-backed planthopper were selected and placed in a test tube. They were treated with _CO2 for about 90s to temporarily stun them, and the test insects were placed on a 3% agarose gel plate with grooves. The dsRNA was injected into the nymphs of white-backed planthoppers using an IM-31 microsyringe. Before use, a 1μL standard capillary glass tube was used to quantify the volume pumped out each time. Each test insect was injected with about 0.1μL, and an equal volume of GFP gene dsRNA was injected as a negative control. Three biological replicates were set for each treatment, and 50 insects were injected in each replicate. After the injection, the test insects were placed in a two-way glass test tube containing fresh TN1 rice seedlings and placed in an artificial climate box with a temperature of 25±1℃, a relative humidity of 70±5%, and a photoperiod of 16L:8D until they emerged.

2.2 Interference effect RT-qPCR detection

24 hours after the injection of the target gene dsRNA, surviving insects (10) were randomly selected from each treatment group, quick-frozen in liquid nitrogen, RNA was extracted, and reverse transcribed into cDNA to detect changes in the expression of the target gene.

2.3 Phenotypic photography observation

24 hours after emergence, the wing extension of the white-backed planthoppers in each treatment group was observed, and the adults were sucked out, quickly frozen in liquid nitrogen, and photographed using a Nikon SMZ25 stereo microscope.

2.4 Data Analysis

Bioinformatics analysis of the SfDpp gene sequence of the white-backed planthopper was performed by using SeqMan software to proofread and assemble the sequencing results. The obtained gene sequence was edited and the amino acid sequence was deduced using DNAMAN 7.0 software. The BLAST tool was used for sequence homology comparison, and the ORF Finder tool was used to find the open reading frame (ORF). ProtParam was used to predict the amino acid molecular composition, relative molecular mass, isoelectric point and other physical and chemical properties of the encoded protein; SignalP 4.1Server was used to predict the signal peptide; SMART was used for domain analysis; the neighbor-joining method (NJ) in MEGA 6.06 was used to construct a phylogenetic tree, and amino acid sequence cluster analysis was performed using 1000 repeated samplings for each branch.

The data obtained in this section were first organized using Microsoft Excel 2019, and then statistically analyzed using SPSS 22.0 software. The 2 ^-ΔΔCt method was used to analyze the relative expression of the SfDpp gene in different instars and tissues of white-backed planthoppers. Real-time fluorescence quantitative PCR data (RT-qPCR) were expressed as mean ± standard error, and one-way analysis of variance (ANOVA) and Duncan's multiple range test were used for multiple comparison analysis (α = 0.05) between treatments.

2.5 Results and Analysis

2.5.1 Cloning and sequence analysis of the SfDpp gene of the white-backed planthopper

A 1034 bp sequence was cloned and identified by NCBI Blast alignment, which confirmed that the gene fragment was a decapentaplegic gene named SfDpp. The results of Open Reading Frame (ORF) analysis (Figure 1) showed that the SfDpp gene contained an open reading frame (ORF) of 954 bp in length, encoding 317 amino acids.

2.5.2 Spatial and temporal expression analysis of the SfDpp gene in white-backed planthopper

The relative expression level of SfDpp was detected by RT-qPCR using the primers described in Primer List 1. From the relative expression results of 19 developmental stages of white-backed planthoppers collected, including egg stage, 1-5 instar nymphs and 0-48 hours of adults, SfDpp is expressed in all developmental stages of white-backed planthoppers. Compared with 5-instar nymphs and adults, the SfDpp mRNA level of young nymphs (1-3 instar nymphs) is relatively low. The expression of SfDpp begins to be upregulated from the second day of the 4th instar nymph, and then a significantly increased expression level is observed after molting, reaching a peak at 40min (2/3 hours) of adults, and then the transcriptional abundance gradually decreases (Figure 2). The present invention observed that white-backed planthoppers usually complete the extension process within 40min, which indicates that this gene plays an indispensable role in the wing extension development stage.

In order to detect the expression level of the SfDpp gene in different tissues of the white-backed planthopper, the present invention dissected the head, thorax, abdomen, wings, legs and epidermis of the 24-hour-emergence adult, and detected the mRNA level of SfDpp in these 6 tissues by RT-qPCR. The results showed (Figure 3) that the expression level of this gene in different tissues was different. The SfDpp gene showed high transcriptional abundance in the head and wings, while the transcription level was low in the thorax, feet and epidermis, but the lowest level was observed in the abdomen. This shows that it is mainly expressed in the head and wings and plays an important role in the development of eyes and wings.

2.5.3 Functional analysis of the SfDpp gene in regulating wing extension in white-backed planthoppers

After the dsRNA of SfDpp was injected into the 4th instar 1-day-old nymphs, samples were taken every 24 hours to detect the interference efficiency. The results showed (Figure 4A) that the expression of SfDpp decreased by 70% 24 hours after the interference, and the interference efficiency reached 91.3% at 48 hours. The results of detecting the relative transcription level of SfDpp gene mRNA in the successfully eclosing white-backed planthoppers with different phenotypes after SfDpp interference showed (Figure 4B) that the expression of SfDpp gene mRNA in the white-backed planthoppers with wing deformities and deaths was significantly lower than that in the control, while the expression level of SfDpp gene in the white-backed planthoppers with normal phenotypes was not significantly different from that in the control. In the control group, except for the dead individuals, 75% of the nymphs could successfully eclode into normal adults (Figure 4C). After the SfDpp gene interference, the dead individuals and those who failed to eclode accounted for 29.2% of the total; 74.1% of the individuals had completely wrinkled wings, curled or bent wings at the ends, and the wings did not spread (Figure 4D).

In summary, the expression level of SfDpp is different in different developmental stages and different tissues of white-backed planthoppers. The expression pattern of the SfDpp gene of white-backed planthoppers shows that it is related to the extension of wings. The present invention compares the expression levels of SfDpp at different ages and finds that the expression level is the highest during the wing extension period, which indicates that Dpp is involved in regulating the maturation and extension of wings. For newly emerged adults, their wings are folded into a ball on the back, so they need to be smoothly extended in order to obtain the ability to fly to carry out physiological activities such as migration and foraging. The present invention found that white-backed planthoppers generally complete the extension of wings in 40 minutes, and the relative transcription level of SfDpp is also the highest during this period. After 1 hour after the emergence of adults, the wings have been unfolded to completely cover their abdomen, and the expression level of SfDpp also decreases accordingly. In the present invention, the expression level of SfDpp in different tissues is the highest in the head and wings, which indicates that SfDpp plays a vital role in regulating the proliferation and pattern of compound eyes.

It should be noted that, in this article, terms such as "comprises", "includes" or any other variations thereof are intended to cover non-exclusive inclusion, so that a process, method, article or apparatus that includes a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or apparatus.

Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims (6)

1. The plant hopper SfDpp gene is characterized in that the sequence of the Bai Beifei plant hopper SfDpp gene comprises the following components:

1) A gene coding region sequence shown in a sequence 1 in a sequence table, or

2) The nucleotide sequence derived from 2) with equivalent or differential activity is obtained by substituting, deleting or adding one or more nucleotides in the sequence shown in the sequence 1 in the sequence table.

2. A dsRNA of Bai Beifei louse SfDpp gene according to claim 1, wherein said dsRNA is an RNA consisting of nucleotides represented by sequence 2 in the sequence listing.

3. An expression cassette, recombinant vector, recombinant bacterium or transgenic cell line comprising the Bai Beifei th lice SfDpp gene of claim 1.

4. Use of the sogatella furcifera SfDpp gene of claim 1 or the dsRNA of claim 2 or the expression cassette, recombinant vector, recombinant bacterium or transgenic cell line of claim 3 in any of the following a 1) -a 4):

a1 Preventing and controlling the sogatella furcifera;

a2 Inhibiting development of wings of the sogatella furcifera;

a3 Inhibiting maturation and stretching of the wings of the sogatella furcifera;

a4 Inhibit proliferation of the brown eye of the sogatella furcifera.

5. A method for inhibiting the development of the wings of the sogatella furcifera comprises the step of introducing a substance for inhibiting the expression of the sogatella furcifera wing development gene SfDpp into the sogatella furcifera to inhibit the development of the sogatella furcifera wings.

6. The method according to claim 5, wherein: the substance for inhibiting the expression of the wing development gene SfDpp of the sogatella furcifera is the dsRNA of claim 2.