A gene derived from Spodoptera exigua with antiviral effect and its application

Abstract

The present invention provides a gene derived from Spodoptera exigua with antiviral effect and its application. The amino acid sequence of the encoded protein is SEQ ID NO:1, and the nucleotide sequence is SEQ ID NO:2. The present invention also provides a method for increasing the sensitivity of Spodoptera exigua to Spodoptera exigua nuclear polyhedrosis virus, which is to reduce the expression level of the gene in Spodoptera exigua. The present invention has screened a gene with antiviral effect from the important agricultural pest Spodoptera exigua, analyzed its sequence structure and spatio-temporal expression pattern, and at the same time used overexpression and RNA interference techniques to prove that the gene has an effect against Spodoptera exigua nuclear polyhedrosis virus, providing a new target for better controlling Spodoptera exigua with viruses in production and having good application prospects.

Description

Technical Field

[0001] The invention belongs to the technical field of functional gene screening and application, and specifically relates to a gene with antiviral effect derived from beet armyworm and application thereof. Background Art

[0002] The beet armyworm (Spodoptera exigua) belongs to the order Lepidoptera, family Noctuidae. It is a globally widespread agricultural pest with strong migratory ability, severe damage, and easy outbreaks, which greatly threatens the safe production of corn, soybeans and other grain and oil crops and vegetables in my country. The long-term irrational use of chemical pesticides has led to a rapid increase in the resistance of the beet armyworm and caused pollution to the ecological environment. Therefore, biological control of the beet armyworm is imminent.

[0003] Beet armyworm nuclear polyhedrosis virus (SeMNPV) is a double-stranded, circular DNA baculovirus with strong host specificity, non-toxicity to humans and animals, environmental friendliness, and natural transmission. It has attracted widespread attention and is an important biological control agent for beet armyworm. However, in practical applications, due to the powerful innate immune system of insects, baculovirus still has outstanding problems of low virulence and efficacy that need to be solved urgently. Therefore, how to suppress the immune system of host insects and enhance the pathogenicity of viruses has become a current research hotspot.

[0004] In the innate immune response of insects, pattern recognition proteins first recognize and bind to pathogen-related molecular patterns unique to the surface of pathogens, and then initiate processes such as signal transduction pathways and effector mechanisms to complete the killing and removal of pathogens. Peptidoglycan recognition protein (PGRP) is an important pattern recognition protein that plays an important role in resisting pathogen invasion. As one of the main types of the PGRP family, the PGRP-S1 protein, current research is mainly focused on the host's immune response to fungi and bacteria, and its function in the host's antiviral immunity is little known. The present invention cloned the PGRP-S1 gene from the beet armyworm, used overexpression and RNAi technology to study its anti-SeMNPV function, and clarified the antiviral effect of PGRP-S1 in the beet armyworm. The present invention will provide a new target based on the immune regulation pathway for green pest control, provide theoretical support for effectively improving the control effect of baculovirus biopesticides, and promote the use of baculovirus insecticides in biological control practices with higher effects. Summary of the invention

[0005] The object of the present invention is to provide a gene with antiviral effect derived from Spodoptera exigua and its application, that is, a gene derived from Spodoptera exigua and resistant to Spodoptera exigua nuclear polyhedrosis virus (SeMNPV).

[0006] The present invention first provides a Spodoptera exigua gene protein with antiviral effects. The protein is named SePGRP-S1, and the SePGRP-S1 protein comprises:

[0007] 1) a protein with the amino acid sequence of SEQ ID NO:1;

[0008] 2) a protein derived from 1) by substituting, deleting or adding one or several amino acids on 1);

[0009] The sequence of SEQ ID NO:1 is specifically as follows:

[0010] MELFFKVFVLFATLVAVKGDCDVVSKKQWDGLNPVHVEYLARPVDLVIIQHTVTRTCNTDAACAE IVRNIQENHMDNLNYWDIGSSFVIGGNGKVYEGAGWLHVGAHTYGYNRRSYGITFIGNYNNDTPTQAS LDALKALLRCGVERGHLTANYHIVGHRQLIATQSPGRRLYNEIRRWPNWLEDVSSLKN;

[0011] The gene encoding the above protein has a specific nucleotide sequence as follows:

[0012] Atggagttattttttaaagttttcgttttatttgctactttagtagctgtgaaaggggattgtga

[0013] cgtagtgtctaaaaaacaatgggatggcttgaatccagtccacgtggagtacctggcgaggccagtag

[0014] acctggtcatcatccagcataccgtcactcgaacttgcaacacggacgctgcttgtgctgagatcgtg

[0015] aggaatatacaagagaaccatatggacaacttgaactattgggacattgggtcctcgttcgtgattgg

[0016] tggtaatggcaaagtgtacgagggtgctggctggttgcatgttggtgcacacacatatggctacaaca

[0017] ggagatcttatggcattacttttattggtaactataacaatgacacgccaacccaagcgtcgttggat

[0018] gcattgaaggctttgctccgttgcggcgtggaacgaggtcacttgacggccaactaccatattgttgg

[0019] tcatcgtcagctcattgccactcagagtcctggaagaaggctctacaatgagatcagaagatggccca

[0020] attggcttgaagatgtcagttcacttaagaactaa(SEQ ID NO:2)

[0021] The present invention also provides a recombinant expression vector, in which the nucleotide fragment encoding the above gene is inserted.

[0022] The present invention also provides a method for reducing the resistance of Spodoptera exigua to SeMNPV, which is to reduce the expression level of the SePGRP-S1 gene in Spodoptera exigua;

[0023] As a specific record of an embodiment, reducing the expression level of the SePGRP-S1 gene in Spodoptera exigua is to use RNA interference method to reduce the expression level of the SePGRP-S1 gene in cells;

[0024] Among them, for the RNA interference method, a sequence of the dsRNA used is as follows (SEQ ID NO:3):

[0025] Agcugugaaaggggauugugacguagugucuaaaaaacaaugggauggcuugaauccaguccacguggaguaccuggcgaggccaguagaccuggucaucauccagcauaccgucacucgaacuugcaacacggacgcugcuugugcugagaucgugaggaauauacaagagaaccauauggacaacuugaacuauugggacauuggguccucguucgugauuggugguaauggcaaaguguacgagggugcuggcugguugcauguuggugcacacacauauggcuacaacaggagaucuuauggcauuacuuuuauugguaacuauaacaaugacacgccaacccaagcgucguuggaugcauugaaggcuuugcuccguu。

[0026] The present invention screened a gene with antiviral effect from the important agricultural pest Spodoptera exigua, analyzed its sequence structure and spatio-temporal expression pattern, and at the same time used overexpression and RNA interference techniques to prove that this gene has the effect of resisting Spodoptera exigua multiple nucleopolyhedrovirus (SeMNPV), providing a new target for better controlling Spodoptera exigua with virus in production and having good application prospects. Brief Description of the Drawings

[0027] Figure 1 : Diagram showing the expression of SePGRP-S1 of the present invention in different tissues of the 5th instar larvae of Spodoptera exigua.

[0028] Figure 2 : Diagram showing the expression of SePGRP-S1 of the present invention at different developmental stages of Spodoptera exigua.

[0029] Figure 3 : Diagram showing the inhibition of SeMNPV proliferation by overexpression of SePGRP-S1 of the present invention, where A is the transcriptional level of SePGRP-S1 in SePGRP-S1 overexpressing cells, B and C are the infection rates of SeMNPV on SePGRP-S1 cells. D is the ODV yield of SeMNPV at different times after SeMNPV infects cells, E is the BV titer of SeMNPV at different times after SeMNPV infects cells, and F is the OBs yield of SeMNPV at 96 h after SeMNPV infects cells.

[0030] Figure 4 : Gel electrophoresis detection diagram of dsRNA of the present invention.

[0031] Figure 5 : Diagram showing that the interference of SePGRP-S1 of the present invention promotes the proliferation of SeMNPV. Among them, in A, after the expression of SePGRP-S1 is down-regulated by dsRNA interfering with Se-3 cells, in B and C, the infection rate of SeMNPV on cells after dsRNA interference, in D, the ODV yield of SeMNPV at different times after dsRNA interference, in E, the BV titer of SeMNPV at different times after dsRNA interference, and in F, the OBs yield of SeMNPV at 96 h after dsRNA interference.

[0032] Figure 6 : Diagram showing that the down-regulation of SePGRP-S1 expression of the present invention affects the survival rate of Spodoptera exigua larvae infected with SeMNPV. Among them, in A, the expression of SePGRP-S1 is down-regulated after dsRNA is injected into Spodoptera exigua larvae, and in B, the survival rate of Spodoptera exigua larvae injected with dsPGRP-S1 or dsGFP infected with SeMNPV.

[0033] Figure 7 : Gel detection diagram of bacterial expression of dsRNA of the present invention.

[0034] Figure 8 : Diagram showing that the down-regulation of SePGRP-S1 expression of the present invention affects the survival rate of Spodoptera exigua larvae infected with SeMNPV. Among them, in A, the expression of SePGRP-S1 is down-regulated after feeding Spodoptera exigua larvae with bacterial expression of dsRNA, and in B, the survival rate of Spodoptera exigua larvae fed with bacterial expression of dsPGRP-S1 or bacterial expression of dsGFP infected with SeMNPV. Detailed implementation mode

[0035] In the present invention, from the genome of Spodoptera exigua, by analyzing the genome, a Spodoptera exigua gene protein with antiviral effect is screened and obtained. The protein is named SePGRP-S1, and the amino acid sequence of SePGRP-S1 protein is SEQ ID NO:1. However, those of ordinary skill in the art can, on the basis of SEQ ID NO:1, by conventional methods, replace, delete, or add one or several amino acids to obtain a protein derived from 1); the derived protein should also have the effect of resisting Spodoptera exigua multiple nucleopolyhedrovirus (SeMNPV).

[0036] Construct a SePGRP-S1 recombinant expression vector, and obtain a Spodoptera exigua cell line with stable increased expression of SePGRP-S1 through liposome transfection.

[0037] Increase the expression level of the SePGRP-S1 gene in cells by means of incremental expression, then infect with the virus, and detect whether the virus proliferation is inhibited. Both qPCR and microscopic observation showed that the proliferation of SeMNPV was significantly inhibited; the results confirmed that increasing the expression level of SePGRP-S1 could significantly reduce the sensitivity of Spodoptera exigua cells to SeMNPV.

[0038] The present invention will be described in detail below with reference to the embodiments and the accompanying drawings.

[0039] Example 1: Cloning and spatio-temporal expression analysis of the SePGRP-S1 gene

[0040] By analyzing the genome of Spodoptera exigua, specific primers were designed. The forward primer sequence was 5′-ATGGAGTTATTTTTTAAAGTTTTCG-3′, and the reverse primer sequence was 5′-TTAGTTCTTAAGTGAACTGACATC-3′.

[0041] Using the cDNA of Spodoptera exigua larvae as a template for PCR amplification. The PCR reaction conditions were: pre-denaturation at 94°C for 4 min; denaturation at 94°C for 40 s, annealing at 48°C for 40 s, extension at 72°C for 40 s, for a total of 35 cycles; extension at 72°C for 10 min, and 10 min at 4°C. The PCR product was identified by agarose gel electrophoresis and recovered, then ligated to the pMD19-T vector. Under the action of T4 DNA ligase, ligation was carried out at room temperature for 1 h, and then transformed into DH5α competent cells. After obtaining positive clones, they were sent to Qingdao Branch of Beijing Tsingke Biotechnology Co., Ltd. for sequencing. The sequencing results are shown in SEQ ID NO:2, and this sequence was named the SePGRP-S1 gene.

[0042] The total RNA of Spodoptera exigua eggs, 1st to 5th instar larvae, male pupae and female pupae was extracted using the Trizol method. At the same time, the total RNA of the female antennae, male antennae of Spodoptera exigua, the heads, salivary glands, ganglia, female glands, male glands, female genitalia, male genitalia, hemolymph, fat body, epidermis, Malpighian tubules and midgut tissues of 5th instar larvae was extracted. Subsequently, the RNA was sent to BGI-Shenzhen Co., Ltd. for transcriptome sequencing. The analysis of the expression results of SePGRP-S1 in different tissues of Spodoptera exigua is as Figure 1 shown, and the results showed that the expression level of SePGRP-S1 was the highest in the fat body. The expression results of SePGRP-S1 in different developmental stages of Spodoptera exigua are as Figure 2 shown, and the results showed that the expression level of SePGRP-S1 was the highest in the 5th instar larvae of Spodoptera exigua.

[0043] Example 2: Incremental expression of SePGRP-S1 inhibits the proliferation of SeMNPV

[0044] To study the function of the SePGRP-S1 gene, the expression of SePGRP-S1 in Spodoptera exigua Se-3 cells was increased by an incremental expression method, and then the cell infection by the virus was observed under a microscope, and the change in virus proliferation was detected by qPCR to prove the function of this gene. The specific steps are as follows:

[0045] Using the full-length CDS sequence of the SePGRP-S1 gene as the target, primers for incremental expression were designed. The upstream primer was: 5′-ATTaagcttATGGAGTTATTTTTTAAAGTTTTCG-3′, and the downstream primer was: 5′-CCGtctagaGTTCTTAAGTGAACTGACATC-3′ (lowercase letters are restriction sites). PCR amplification was performed using the cloned SePGRP-S1 gene as a template. The PCR reaction conditions were: pre-denaturation at 94°C for 4 min; denaturation at 94°C for 40 s, annealing at 48°C for 40 s, extension at 72°C for 40 s, for a total of 35 cycles; extension at 72°C for 10 min, and 10 min at 4°C. The amplified target band was detected, gel-extracted, and sequenced.

[0046] The PCR gel-extracted product verified by sequencing in the previous step was double-digested with HindⅢ and XbaⅠ, identified by agarose gel electrophoresis and recovered to obtain the SePGRP-S1 digested fragment; at the same time, the pIZ / V5-GFP-His vector was double-digested with HindⅢ and XbaⅠ, identified by agarose gel electrophoresis and recovered to obtain the digested fragment of pIZ / V5-GFP-His. Under the action of T4 DNA ligase, the SePGRP-S1 and pIZ / V5-GFP-His digested fragments were ligated together, transformed into DH5α competent cells, and positive clones were screened to obtain the incremental expression vector pIZ / V5-SePGRP-S1-GFP-His.

[0047] Refer to Ⅱ Reagent Description: The plasmids of pIZ / V5-SePGRP-S1-GFP-His and the control pIZ / V5-GFP-His were transfected into Spodoptera exigua Se-3 cells respectively to obtain SePGRP-S1 cells and Se-GFP cells. The cells were subcultured using TNM-FH cell culture medium (containing 10% fetal bovine serum). RNA of the 12th generation of SePGRP-S1 and SeGFP cells was extracted and reverse transcribed into cDNA. The specific fluorescence quantitative primers for the SePGRP-S1 gene, SePGRP-S1-qF: 5’-TCACTTGACGGCCAACTACC-3’ and SePGRP-S1-qR: 5’-ATTGGGCCATCTTCTGATCTC-3’, were used for absolute quantitative detection of the transcription of this gene. The detection method is as follows: Using the SePGRP-S1 plasmid as a template, PCR amplification was performed with SePGRP-S1-qF and SePGRP-S1-qR. The PCR product was ligated to the pMD-19T vector using T4 DNA ligase. Positive clones were selected and propagated, and plasmid SePGRP-S1q was extracted. SePGRP-S1q was diluted in a 10-fold gradient. Using it as a template, fluorescence quantitative PCR was performed with SePGRP-S1-qF and SePGRP-S1-qR. A standard curve for absolute quantitative detection was made using the Bio-Rad CFX MaestroV2.2 software. The detection results are as Figure 3 shown in A in the figure. The results showed that the expression level of SePGRP-S1 in SePGRP-S1 cells was 558.6 times that of the control cells, which was extremely significantly higher than the control.

[0048] The 12th generation of SePGRP-S1 and SeGFP cells were infected with SeMNPV. Cells and culture supernatants were collected at 24 h, 48 h, 72 h, and 96 h after infection respectively. The DNA of the occlusion-derived virus particles (ODV) of SeMNPV in the cells and the budded virus particles (BV) of SeMNPV in the culture supernatant were extracted using a virus DNA / RNA extraction kit (Yeasen Biotechnology Co., Ltd., Shanghai, China). The specific primer set for the SeMNPV GP41 gene was used, and the primer set included:

[0049] GP41qF: 5’-ACGTCATCGACGATCTTTTCG-3’;

[0050] GP41qF: 5’-CGGTGCCATGTAGCTAATGTTT-3’;

[0051] qPCR quantitative detection of the virus content was performed. The method for making the quantitative detection standard curve was referred to the method in the example of increased expression of SePGRP-S1. The results are as Figure 3As shown in D and E in the figure. The qPCR detection results showed that cells with increased expression of SePGRP-S1 could significantly inhibit the proliferation of virus ODV and BV. At 48 h, 72 h, and 96 h after virus infection: the content of ODV in SePGRP-S1 cells was only 0.47%, 0.26%, and 4.63% of that in the control; the content of BV in the cells was only 0.27%, 0.01%, and 0.23% of that in the control.

[0052] At 96 h after SeMNPV-infected cells, the infection symptoms of the virus on the cells were observed and recorded under a microscope, and the infection rate of the virus and the content of inclusion bodies (OBs) in the cells were calculated, as Figure 3 shown in B and C in the figure. The results showed that the infection rate of the virus on cells with increased expression of SePGRP-S1 was significantly lower than that of the control, only 50.6% of that of the control; as Figure 3 shown in F in the figure, the results showed that the content of OBs in SePGRP-S1 cells was also significantly lower than that of the control, only 13.42% of that of the control, confirming that increased expression of SePGRP-S1 significantly reduced the sensitivity of Spodoptera exigua cells to SeMNPV.

[0053] Example 3 RNA interference of SePGRP-S1 gene inhibits the expression of PGRP-S1 gene in Spodoptera exigua cells and promotes the proliferation of SeMNPV

[0054] Using the correctly sequenced SePGRP-S1 plasmid (the plasmid with the sequence of SEQ ID NO:2) as a template, PCR amplification was carried out using dsRNA primers with T7 promoter sequence. The PCR reaction conditions were: pre-denaturation at 94 °C for 4 min; denaturation at 94 °C for 40 s, annealing at 60 °C for 40 s, extension at 72 °C for 30 s, for a total of 35 cycles; extension at 72 °C for 7 min, 4 °C for 10 min. The PCR products were identified by agarose gel electrophoresis and recovered. The primer set included:

[0055] dsPGRP-S1-F1: taatacgactcactatagggAGCTGTGAAAGGGGATTGTG,

[0056] dsPGRP-S1-R1: taatacgactcactatagggAACGGAGCAAAGCCTTCAAT;

[0057] The double-stranded RNA dsPGRP-S1 (SEQ ID NO: 3) of the SePGRP-S1 gene was synthesized and purified using the TranscriptAid T7 high yield transcription kit (Thermo, Waltham, MA, USA) reagent according to the instructions. The quality and concentration of dsPGRP-S1 were detected simultaneously using a micro ultraviolet spectrophotometer and agarose gel. The detection gel image of dsPGRP-S1 is as shown in Figure 4 . The results showed that there was a clear band of the dsRNA fragment dsPGRP-S1 at around 300 bp, which could be used for subsequent experiments.

[0058] Refer to the operation instructions of Reagent Ⅱ. dsPGRP-S1 was transfected into Spodoptera exigua Se-3 cells through liposomes for RNA interference of the SePGRP-S1 gene. At the same time, dsGFP was transfected into Spodoptera exigua Se-3 cells as a control. After RNA interference, the results are as shown in Figure 5 A. At 24 h, 48 h, and 72 h after RNA interference, the expression levels of SePGRP-S1 in dsPGRP-S1-interfered cells were 49.96%, 45.97%, and 33.92% of the control, respectively. Compared with the control, the expression level of SePGRP-S1 was significantly decreased. At 24 h after RNA interference, the cells were infected with SeMNPV. At 24 h, 48 h, 72 h, and 96 h after virus infection, the cells and cell culture supernatants were taken. The DNA of the occlusion-derived virus particles (ODV) of SeMNPV in the cells and the budded virus particles (BV) of SeMNPV in the cell culture supernatants were extracted using the method in Example 2. The ODV and BV of SeMNPV were detected by qPCR using the specific primers GP41qF and GF41qR of the SeMNPV virus GP41 gene. The results are as shown in Figure 5 D and E. At 48 h, 72 h, and 96 h after virus infection, the contents of ODV and BV in dsPGRP-S1-interfered cells were significantly higher than those in the control. The contents of ODV in dsPGRP-S1-interfered cells were 2.40, 2.51, and 3.30 times that of the control, respectively. The contents of BV in dsPGRP-S1-interfered cells were 4.14, 2.19, and 2.43 times that of the control, respectively.

[0059] At 96 h after SeMNPV infected Spodoptera exigua cells, the infection symptoms of the virus on the cells were observed and recorded using a microscope, and the infection rate of the virus was calculated. The results are as shown in Figure 5 B and C. The results showed that the interference of SePGRP-S1 significantly increased the infection rate of the virus on the cells, which was 1.32 times that of the control GFP interference. The results are as shown in Figure 5As shown in F, the content of OBs in SePGRP-S1 interfering cells was also significantly higher than that of the control, 1.63 times that of the control, confirming that silencing the SePGRP-S1 gene significantly increased the sensitivity of Spodoptera exigua cells to SeMNPV.

[0060] Example 4 Injection of dsPGRP-S1 inhibits the expression of SePGRP-S1 gene in Spodoptera exigua larvae and increases the lethality of SeMNPV to Spodoptera exigua

[0061] Using the method of microinjection, dsPGRP-S1 in Example 3 was injected into the 3rd instar larvae of Spodoptera exigua to interfere with SePGRP-S1. At the same time, dsGFP was injected into the 3rd instar larvae of Spodoptera exigua as a control. At 1d, 2d, 3d, 4d, 5d, 6d, 7d and 8d after interference, the interference efficiency of the target gene was detected in the insect bodies. The results are as Figure 6 As shown in A, at 3d, 4d, 5d, 6d, 7d and 8d after dsRNA interference, the expression level of SePGRP-S1 in the larvae interfered with dsPGRP-S1 was lower than that of the control, which were 75.41%, 28.78%, 58.46%, 52.48%, 50.76% and 53.15% of the control respectively. The results showed that the SePGRP-S1 gene was successfully interfered.

[0062] 110 insects injected with dsPGRP-S1 and dsGFP for 1 day were taken respectively, and each insect was fed 2 μL of 4×10 5 PIBs / mL of SeMNPV (the virus was added to the diet of Spodoptera exigua), and the survival rate of the larvae was counted every day until they pupated. The results are as Figure 6 As shown in B, the survival rate of the larvae interfered with dsPGRP-S1 was significantly lower than that of the control. The results showed that the interference of SePGRP-S1 reduced the survival rate of the virus to Spodoptera exigua larvae and significantly increased the sensitivity of Spodoptera exigua to SeMNPV.

[0063] Example 5 Preparation of bacterial expression bacterial liquid expressing dsRNA of Spodoptera exigua PGRP-S1 gene

[0064] Two restriction enzyme sites were selected on the L4440 plasmid, namely SpeⅠ (ACTAGT) and HindⅢ (AAGCTT). Using the correctly sequenced SePGRP-S1 plasmid (the plasmid with the sequence of SEQ ID NO:2) as a template, PCR amplification was carried out with dsPGRP-S1 specific primers with corresponding restriction enzyme sites and protection bases. The amplification conditions and methods were the same as those in Example 3. The PCR products were detected by agarose gel and gel recovery was carried out to obtain gel recovery product 1. The primer set included:

[0065] dsPGRP-S1-F2: ATTactagtAGCTGTGAAAGGGGATTGTG

[0066] dsPGRP-S1-R2: CGGaagcttAACGGAGCAAAGCCTTCAAT

[0067] Referring to the instruction manual of Takara restriction enzymes, the gel-extracted product 1 was double-digested with SpeⅠ and HindⅢ (Takara). After detection by agarose gel electrophoresis, the large fragments in the gel were gel-extracted to obtain gel-extracted product 2. At the same time, the vector plasmid of L4440 was double-digested with SpeⅠ and HindⅢ, detected by agarose gel electrophoresis, and the large fragments in the gel were gel-extracted to obtain gel-extracted product 3. T4 DNA ligase (Takara) was used to ligate gel-extracted product 2 and 3 overnight at 4°C. The ligation product was transformed into DH5α competent cells. After obtaining positive clones, they were sent to Qingdao Branch of Beijing Tsingke Biotechnology Co., Ltd. for sequencing to obtain the recombinant vector L4440-SePGRP-S1.

[0068] The plasmid of the L4440-SePGRP-S1 positive clone was extracted using the plasmid miniprep kit of Omega and transformed into HT115 competent cells. Positive clones successfully expressing dsRNA of the Spodoptera exigua PGRP-S1 gene were screened. The clones expressing the recombinant vector L4440-SePGRP-S1 were cultured overnight in a shaker at 37°C and inoculated into LB medium containing ampicillin (100 μg / mL) and tetracycline (10 μg / mL) at a ratio of 1:50. They were cultured at 37°C for 2 - 3 h until the OD 600 reached 0.4 - 0.6. IPTG (final concentration 0.8 mM) was added to induce the production of dsRNA, and the culture was continued for about 4.5 h to collect the bacterial liquid for standby. RNA was extracted using the Trizol (Themo) method, and dsRNA was detected by 1.5% agarose gel electrophoresis and the concentration of dsRNA was detected using a Nanodrop2000 spectrophotometer. As Figure 7 shown, the results showed that dsPGRP-S1 was successfully induced.

[0069] Example 6 Feeding the Bacterial Liquid Expressing dsPGRP-S1 Inhibits the Expression of PGRP-S1 Gene in Spodoptera exigua and Increases the Lethality of SeMNPV to Spodoptera exigua

[0070] The bacterial solution expressing dsPGRP-S1 in Example 5 was centrifuged at 8,000 rpm for 5 minutes to collect the bacterial cells. For every 50 mL of the bacterial solution, 5 mL of DEPC water was added to the collected bacterial cells. After mixing, it was stored at -80 °C for later use. The concentrated bacterial cells containing dsPGRP-S1 were added to the artificial diet of Spodoptera exigua, stirred evenly, and then used to feed the larvae of Spodoptera exigua.

[0071] Healthy 3rd instar larvae of Spodoptera exigua were selected and fed with the above diet, with the HT115 bacterial solution containing dsGFP at the same concentration as the control. Each insect was individually reared in a 15 mL plastic box, and the volume of the diet in each box was the same. Fresh diet containing dsRNA was replaced every day. After feeding with dsRNA, the interference efficiency of dsRNA was detected in the insects every day (3 insects per replicate, 3 replicates for each treatment). The test results are as Figure 8 shown in A. At 4 d, 5 d, 6 d, 7 d, 8 d, and 9 d after feeding the insects with the dsRNA expressed by bacteria, the transcriptional level of PGRP-S1 in Spodoptera exigua treated with dsPGRP-S1 was lower than that in the dsGFP treatment group (control), being 36.13%, 49.61%, 29.47%, 32.89%, 85.96%, and 75.73% of the control respectively. The results showed that the SePGRP-S1 gene was successfully interfered by the dsPGRP-S1 expressed by bacteria.

[0072] The larvae of Spodoptera exigua were fed with the dsRNA expressed by bacteria every day according to the above method. For each treatment, there were 125 insects. Only on the second day of feeding with dsRNA, each insect was fed with 2 μL of 4×10 5 PIBs / mL of SeMNPV (the virus was added to the diet of Spodoptera exigua). The survival of the insects was counted every day until the larvae pupated. The test results are as Figure 8 shown in B. The results showed that the interference of the SePGRP-S1 gene by the dsPGRP-S1 expressed by bacteria increased the lethality of SeMNPV to the larvae of Spodoptera exigua, and significantly increased the sensitivity of Spodoptera exigua to SeMNPV.

Claims

1. A method for reducing the resistance of Spodoptera exigua to Spodoptera exigua nuclear polyhedrosis virus, characterized in that: The method is to reduce the activity of the protein with the amino acid sequence of SEQ ID NO: 1 or the expression level of the coding gene.

2. The method according to claim 1, characterized in that The method is to use dsRNA to reduce the expression level of the coding gene, and the dsRNA sequence is SEQ ID NO:

3.

3. The method according to claim 2, characterized in that The dsRNA is prepared by expression using a recombinant engineering strain.

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