Beet armyworm-derived gene with antiviral effect and application of beet armyworm-derived gene

Abstract

The invention provides a beet armyworm-derived gene with an antiviral effect and application thereof, the amino acid sequence of the encoded protein is SEQ ID NO: 1, and the nucleotide sequence of the encoded protein is SEQ ID NO: 2. The invention also provides a method for increasing the sensitivity of the beet armyworm to the beet armyworm nuclear polyhedrosis virus, which is used for reducing the expression quantity of the gene in the beet armyworm. According to the invention, a gene with an antiviral effect is screened from an important agricultural pest beet armyworm. The sequence structure and spatio-temporal expression mode of the gene are analyzed, incremental expression and RNA interference technologies are utilized to prove that the gene has an effect of resisting beet armyworm nuclear polyhedrosis virus, a new target is provided for better prevention and control of beet armyworms by utilizing the virus in production, and the gene has a very good application prospect.

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 beet armyworm gene protein with antiviral effect, the protein is named SePGRP-S1, and the SePGRP-S1 protein comprises:

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

[0008] 2) Substituting, deleting or adding one or more amino acids in 1) to produce a protein derived from 1);

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

[0010] MELFFKVFVLFATLVAVKGDCDVVSKKQWDGLNPVHVEYLARPVDLVIIQHTVTRTCNTDAACAE IVRNIQENHMDNLNYWDIGSSFVIGGNGKVYEGAGWLHVGAHTYGYNRRSYGITFIGNYNNDTPTQAS LDALKALLRCGVERGHLTANYHIVGHRQLIATQSPGRRLYNEIRRWPNWLEDVSSLKN;

[0011] A specific nucleotide sequence of the gene encoding the above protein is as follows:

[0012] Atggagttattttttaaagttttcgttttatttgctactttagtagctgtgaaaggggattgtga

[0013] cgtagtgtctaaaaaaacaatgggatggcttgaatccagtccacgtggagtacctggcgaggccagtag

[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, into which the nucleotide fragment of the above-mentioned coding gene is inserted.

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

[0023] The reduction of the expression of SePGRP-S1 gene in Spodoptera exigua is described as a specific example of reducing the expression of SePGRP-S1 gene in cells by RNA interference method;

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

[0025] Agcugugaaaggggauugugacguagugucuaaaaaacaaugggauggcuugaauccaguccacguggaguaccuggcgaggccaguagaccuggucauccagcauaccgucacucgaacuugcaacacggacgcugcuuggcugagaucgugaggaauauacaagagaaccauauggacaacuugaa cuauugggacauuggguccucguucgugauuggugguaauggcaaaaguguacgagggugcuggcugguugcauguuggugcacacacauauggcuacaacaggagaaucuuauggcauuacuuuauugguaacuauaacaaugacacgccaacccaagcgucguuggaugcauugaaggcuuugcuccguu.

[0026] The present invention screened and obtained a gene with antiviral effect from the important agricultural pest beet armyworm. The sequence structure and spatiotemporal expression pattern were analyzed, and the incremental expression and RNA interference technology were used to prove that the gene has the effect of resisting beet armyworm nuclear polyhedrosis virus (SeMNPV), which provides a new target for better prevention and control of beet armyworm by using viruses in production, and has a good application prospect. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0028] Figure 2 : Expression diagram of SePGRP-S1 of the present invention in different developmental stages of Spodoptera exigua.

[0029] Figure 3 : The figure of the inhibition of SeMNPV proliferation by the incremental expression of SePGRP-S1 of the present invention, wherein A is the transcription level of SePGRP-S1 in cells with incremental expression of SePGRP-S1, B and C are the infection rate of SeMNPV to SePGRP-S1 cells. D is the ODV production of SeMNPV at different times of SeMNPV infection in cells, E is the BV titer of SeMNPV at different times of SeMNPV infection in cells, and F is the OBs production of SeMNPV at 96h of SeMNPV infection in cells.

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

[0031] Figure 5 : The figure shows that the interference of SePGRP-S1 of the present invention promotes the proliferation of SeMNPV, wherein A shows the down-regulation of SePGRP-S1 expression after dsRNA interference in Se-3 cells, B and C show the infection rate of SeMNPV to cells after dsRNA interference, D shows the ODV production of SeMNPV at different times after dsRNA interference, E shows the BV titer of SeMNPV at different times after dsRNA interference, and F shows the OBs production of 96hSeMNPV after dsRNA interference.

[0032] Figure 6 : The down-regulation of SePGRP-S1 expression in the present invention affects the survival rate of beet armyworm larvae infected with SeMNPV, wherein A is the down-regulation of SePGRP-S1 expression after dsRNA is injected into beet armyworm larvae, and B is the survival rate of beet armyworm larvae infected with SeMNPV injected with dsPGRP-S1 or dsGFP.

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

[0034] Figure 8 : The down-regulation of SePGRP-S1 expression in the present invention affects the survival rate of beet armyworm larvae infected with SeMNPV, wherein A represents the down-regulation of SePGRP-S1 expression after beet armyworm larvae are fed with bacteria expressing dsRNA, and B represents the survival rate of beet armyworm larvae infected with SeMNPV and fed with bacteria expressing dsPGRP-S1 or bacteria expressing dsGFP. DETAILED DESCRIPTION

[0035] The present invention screens and obtains a beet armyworm gene protein with antiviral effect from the beet armyworm genome by analyzing the genome, and the protein is named SePGRP-S1, and the amino acid sequence of SePGRP-S1 protein is SEQ ID NO: 1. However, a person skilled in the art can replace, delete, add one or more amino acids based on SEQ ID NO: 1 by conventional methods to obtain a protein derived from 1); the derived protein should also have an effect of resisting beet armyworm nuclear polyhedrosis virus (SeMNPV).

[0036] The SePGRP-S1 recombinant expression vector was constructed, and a beet armyworm cell line with stable and incremental expression of SePGRP-S1 was obtained by liposome transfection.

[0037] The expression of SePGRP-S1 gene in cells was increased by incremental expression, and then the virus was infected to detect whether the virus proliferation was inhibited. Both qPCR and microscopic observations showed that the proliferation of SeMNPV was significantly inhibited; the results confirmed that increasing the expression of SePGRP-S1 can significantly reduce the sensitivity of Spodoptera exigua cells to SeMNPV.

[0038] The present invention is described in detail below in conjunction with embodiments and drawings.

[0039] Example 1: Cloning and spatiotemporal expression analysis of SePGRP-S1 gene

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

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

[0042] The total RNA of beet armyworm eggs, 1st to 5th instar larvae, male pupae and female pupae was extracted by Trizol method. The total RNA of female antennae, male antennae, head, salivary glands, ganglia, female glands, male glands, female genitalia, male genitalia, hemolymph, fat body, epidermis, Malpighian tubules and midgut tissues of 5th instar larvae were extracted. Then the RNA was sent to Shenzhen BGI Co., Ltd. for transcriptome sequencing. The expression results of SePGRP-S1 in different tissues of beet armyworm are shown in Figure 2. Figure 1 The results show that SePGRP-S1 is expressed at the highest level in the fat body. Figure 2 As shown, the results showed that SePGRP-S1 was expressed at the highest level in the 5th instar larvae of Spodoptera exigua.

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

[0044] In order to study the function of SePGRP-S1 gene, the expression of SePGRP-S1 in Spodoptera exigua Se-3 cells was increased by incremental expression, and then the cells were observed to be infected by virus under a microscope, and the changes in virus proliferation were detected by qPCR to prove the function of the gene. The specific steps are as follows:

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

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

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

[0048] SeMNPV was used to infect SePGRP-S1 and SeGFP cells at the 12th generation. Cells and culture medium were collected at 24 h, 48 h, 72 h and 96 h after infection, respectively. The DNA of SeMNPV inclusion-derived virions (ODV) in cells and SeMNPV budded virions (BV) in culture medium were extracted using a viral DNA / RNA extraction kit (Yisheng Biotechnology Shanghai Co., Ltd., China). The specific primer set of SeMNPV GP41 gene was used, wherein the primer set contained:

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

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

[0051] The viral content was quantitatively detected by qPCR. The preparation of the quantitative detection standard curve was based on the method in the embodiment of the incremental expression of SePGRP-S1. The results are shown in Figure 3As shown in D and E. The results of qPCR detection showed that cells with incremental expression of SePGRP-S1 could significantly inhibit the proliferation of viruses ODV and BV. At 48h, 72h and 96h 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 cells was only 0.27%, 0.01% and 0.23% of that in the control.

[0052] When the cells were infected with SeMNPV for 96 h, the virus infection symptoms on the cells were observed and recorded under a microscope, and the virus infection rate and the content of inclusion bodies (OBs) in the cells were calculated. Figure 3 As shown in B and C. The results showed that the virus infection rate of cells expressing SePGRP-S1 was significantly lower than that of the control, only 50.6% of the control; Figure 3 As shown in F, the results showed that the content of OBs in SePGRP-S1 cells was also significantly lower than that in the control, only 13.42% of the control, confirming that the incremental 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] The correctly sequenced SePGRP-S1 plasmid (plasmid with SEQ ID NO: 2 sequence) was used as a template, and dsRNA primers with T7 promoter sequence were used for PCR amplification. The PCR reaction conditions were: 94°C pre-denaturation for 4 min; 94°C denaturation for 40 s, 60°C annealing for 40 s, 72°C extension for 30 s, for a total of 35 cycles; 72°C extension for 7 min, 4°C for 10 min. The PCR product was identified and recovered by agarose gel electrophoresis, wherein the primer set included:

[0055] dsPGRP-S1-F1:taatacgactcactataggggAGCTGTGAAAGGGGATTGTG,

[0056] dsPGRP-S1-R1:taatacgactcactataggggAACGGAGCAAAGCCTTCAAT;

[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 simultaneously detected using a micro-volume UV spectrophotometer and agarose gel. The detection gel image of dsPGRP-S1 is shown in FIG. Figure 4 The results showed that the dsRNA fragment dsPGRP-S1 had a clear band at about 300 bp and could be used for subsequent experiments.

[0058] Reference II. The reagent instructions were followed. dsPGRP-S1 was transfected into Spodoptera exigua Se-3 cells by liposomes to perform RNA interference of SePGRP-S1 gene. At the same time, dsGFP was transfected into Spodoptera exigua Se-3 cells as a control. After RNA interference, the results were as follows Figure 5 As shown in A, 24h, 48h and 72h 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. The expression level of SePGRP-S1 was significantly reduced compared with the control. 24h after RNA interference, cells were infected with SeMNPV, and cells and cell culture fluid were taken at 24h, 48h, 72h and 96h after virus infection, respectively. The DNA of SeMNPV inclusion-derived virus particles (ODV) in cells and SeMNPV budded virus particles (BV) in cell culture fluid were extracted using the method in Example 2. SeMNPV ODV and BV were detected by qPCR using the specific primers GP41qF and GF41qR of SeMNPV virus GP41 gene. The results are shown in Figure 5 As shown in D and E, 48h, 72h and 96h after virus infection, the contents of ODV and BV in the cells interfered with dsPGRP-S1 were significantly higher than those in the control. The contents of ODV in the cells interfered with dsPGRP-S1 were 2.40, 2.51 and 3.30 times that of the control, respectively, and the contents of BV in the cells interfered with dsPGRP-S1 were 4.14, 2.19 and 2.43 times that of the control, respectively.

[0059] When SeMNPV infected Spodoptera exigua cells for 96 hours, the virus infection symptoms on the cells were observed and recorded under a microscope, and the virus infection rate was calculated. The results are as follows: Figure 5 As shown in Figures B and C, the results show that SePGRP-S1 interference significantly increased the virus infection rate to cells, which was 1.32 times that of the control GFP interference. Figure 5As shown in F, the content of OBs in SePGRP-S1 interfered 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 beet armyworm cells to SeMNPV.

[0060] Example 4 Injection of dsPGRP-S1 inhibits the expression of SePGRP-S1 gene in beet armyworm larvae and increases the mortality rate of SeMNPV to beet armyworm

[0061] By microinjection, the dsPGRP-S1 in Example 3 was injected into the third-instar larvae of Spodoptera exigua to interfere with SePGRP-S1, and dsGFP was injected into the third-instar larvae of Spodoptera exigua as a control. The interference efficiency of the target gene was detected by taking insect bodies 1d, 2d, 3d, 4d, 5d, 6d, 7d and 8d after interference. The results are shown in Figure 2. Figure 6 As shown in middle A, at 3d, 4d, 5d, 6d, 7d and 8d after dsRNA interference, the expression level of SePGRP-S1 in the dsPGRP-S1 interfered larvae was lower than that in 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 worms were taken after dsPGRP-S1 and dsGFP injection for 1 day, and each worm was fed with 2 μL 4×10 5 PIBs / mL of SeMNPV (the virus was added to the diet of Spodoptera exigua) and the survival rate of larvae was counted every day until they pupated. Figure 6 As shown in B, the survival rate of larvae after dsPGRP-S1 interference was significantly lower than that of the control. The results showed that SePGRP-S1 interference reduced the survival rate of the virus to beet armyworm larvae and significantly increased the sensitivity of beet armyworm to SeMNPV.

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

[0064] Two restriction sites were selected on the L4440 plasmid, namely SpeⅠ (ACTAGT) and HindⅢ (AAGCTT), and the correctly sequenced SePGRP-S1 plasmid (the plasmid with the sequence of SEQ ID NO: 2) was used as a template. PCR amplification was performed using dsPGRP-S1 specific primers with corresponding restriction sites and protective bases. The amplification conditions and method were the same as those in Example 3. The PCR product was subjected to agarose gel detection and gel recovery to obtain gel recovery product 1, wherein the primer set comprises:

[0065] dsPGRP-S1-F2:ATTactagtAGCTGTGAAAGGGGATTGTG

[0066] dsPGRP-S1-R2:CGGaagcttAACGGAGCAAAGCCTTCAAT

[0067] According to the instruction manual of Takara endonuclease, the gel recovery product 1 was double-digested with SpeⅠ and HindⅢ (Takara), and the large fragments in the gel were recovered by gel electrophoresis to obtain gel recovery product 2. At the same time, the vector plasmid of L4440 was double-digested with SpeⅠ and HindⅢ, and the large fragments in the gel were recovered by gel electrophoresis to obtain gel recovery product 3. Gel recovery products 2 and 3 were ligated overnight at 4°C using T4 DNA ligase (Takara), and the ligation products were transformed into DH5α competent medium. After obtaining positive clones, they were sent to Qingdao Branch of Beijing Qingke Biotechnology Co., Ltd. for sequencing to obtain the recombinant vector L4440-SePGRP-S1.

[0068] The plasmid of the positive clone of L4440-SePGRP-S1 was extracted using Omega's plasmid mini-extraction kit and transformed into HT115 competent cells to screen for positive clones that successfully expressed the dsRNA of the beet armyworm PGRP-S1 gene. The clones expressing the recombinant vector L4440-SePGRP-S1 were cultured in a shaking incubator at 37°C overnight and inoculated into LB medium containing ampicillin (100 μg / mL) and tetracycline (10 μg / mL) at a ratio of 1:50. The culture was shaken at 37°C for 2-3 h until the OD 600 When the concentration of dsRNA reached 0.4-0.6, IPTG (final concentration 0.8 mM) was added to induce dsRNA production, and the culture was continued for about 4.5 hours and the bacterial solution was collected for use. RNA was extracted using the Trizol (Themo) method, and dsRNA was detected by 1.5% agarose gel electrophoresis and the dsRNA concentration was detected using a Nanodrop 2000 spectrophotometer. Figure 7 As shown, the results showed successful induction of dsPGRP-S1.

[0069] Example 6 Feeding the bacteria expressing dsPGRP-S1 inhibits the expression of PGRP-S1 gene in Spodoptera exigua and increases the mortality rate of SeMNPV to Spodoptera exigua

[0070] The bacterial solution expressing dsPGRP-S1 in Example 5 was centrifuged at 8000 rpm / min for 5 minutes, and the bacterial bodies were collected. 5 mL of DEPC water was added to each 50 mL of bacterial solution, and the mixture was mixed and stored at -80°C for later use. The concentrated bacterial bodies containing dsPGRP-S1 were added to the artificial diet of beet armyworm, stirred evenly, and fed to beet armyworm larvae.

[0071] Healthy third-instar larvae of Spodoptera exigua were selected and fed with the above feed, with the same concentration of HT115 bacterial solution containing dsGFP as a control. Each insect was raised individually in a 15 mL plastic box, with the same volume of feed in each box, and fresh feed containing dsRNA was replaced every day. After feeding dsRNA, insects were taken every day to test the interference efficiency of dsRNA (3 insects per replicate, 3 replicates per treatment). The test results are shown in the figure below. Figure 8 As shown in A, at 4d, 5d, 6d, 7d, 8d and 9d after the insects were fed with the bacterially expressed dsRNA, the transcription levels of PGRP-S1 in the beet armyworm treated with dsPGRP-S1 were lower than those in the dsGFP treated group (control), which were 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 with by the bacterially expressed dsPGRP-S1.

[0072] The beet armyworm was fed with dsRNA expressed by bacteria every day according to the above method. Each treatment had 125 insects. Only on the second day of feeding dsRNA, each insect was fed 2 μL of 4×10 5 PIBs / mL of SeMNPV (the virus was added to the diet of beet armyworms) and the survival of insects was counted every day until the larvae pupated. Figure 8 As shown in B, the results showed that the interference of bacterially expressed dsPGRP-S1 with the SePGRP-S1 gene increased the mortality rate of SeMNPV against beet armyworm larvae and significantly increased the sensitivity of beet armyworm to SeMNPV.

Claims

1. A beet armyworm gene protein having an antiviral effect, characterized in that: The protein comprises: 1) a protein having an amino acid sequence of SEQ ID NO: 1; 2) Substituting, deleting or adding one or more amino acids in 1) to produce a protein derived from 1).

2. A gene, characterized in that The gene encodes the protein according to claim 1.

3. The gene according to claim 2, characterized in that The nucleotide sequence of the gene is SEQ ID NO:

2.

4. A recombinant expression vector, characterized in that: The nucleic acid fragment of the gene according to claim 2 is inserted into the recombinant expression vector.

5. A dsRNA, characterized in that The dsRNA can reduce the gene expression level of the protein according to claim 1.

6. The dsRNA according to claim 5, characterized in that The sequence of the dsRNA is SEQ ID NO:

3.

7. A method for reducing resistance of Spodoptera exigua to Spodoptera exigua nuclear polyhedrosis virus, characterized in that: The method is to reduce the activity of the protein described in claim 1 or the expression level of the encoding gene.

8. The method according to claim 7, characterized in that The method is to use the dsRNA according to claim 5 to reduce the expression level of the coding gene.

9. The method according to claim 8, characterized in that The dsRNA is provided by the expression amount of the recombinant engineering strain.

10. A product for reducing resistance of beet armyworm to beet armyworm nuclear polyhedrosis virus, characterized in that: The preparation contains an engineered strain for recombinantly expressing the dsRNA according to claim 5.

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