Inhibitor for suppressing expression of antibacterial peptide in brown planthopper and application thereof

Abstract

To provide an inhibitor for suppressing expression of an antibacterial peptide in brown planthopper and an application thereof.SOLUTION: A dsNlPCE4 inhibitor designed based on the NlPCE4 gene of the brown planthopper is obtained. The inhibitor, when applied to brown planthoppers inoculated on their body surface with Metarhizium anisopliae, significantly increases the mortality of the brown planthopper. The present invention combines two technical strategies for pest control, biocontrol fungi and RNA interference (RNAi), to provide a new target for brown planthopper control technology based on cooperative mediation of RNAi.SELECTED DRAWING: Figure 5

Description

[Technical Field]

[0001] The present invention relates to the field of genetic engineering, and in particular to an inhibitor that inhibits the expression of antimicrobial peptides in the brown planthopper and its application. [Background technology]

[0002] The brown planthopper (Nilaparvata lugens) is a serious rice pest in China, causing damage by sucking rice sap, laying eggs, and transmitting viral diseases, severely impacting rice quality and yield. Currently, the primary method for controlling the brown planthopper is the use of chemical insecticides. However, the continued and unreasonable use of chemical insecticides can lead to serious "3R" problems (drug resistance, re-infestation, and pesticide residues). While microbial control is recommended for the biological control of brown planthoppers, biocontrol fungi offer distinct advantages in controlling this piercing-sucking pest due to their unique body wall contact infection pattern. Metarhizium anisopliae, a typical representative of biocontrol fungi, has demonstrated significant potential for use in controlling brown planthoppers.

[0003] Under natural conditions, the host infection process by Metarhizium anisopliae begins with the attachment of conidia to the host body wall, germinating and penetrating the host's body wall to invade the hemocoel. After breaching the host's immune defense system, the conidia rapidly multiply until nutrients in the hemocoel are depleted, ultimately resulting in the host's death. Clearly, overcoming the host insect's immune defense response is a key prerequisite for the insecticidal efficacy of Metarhizium anisopliae. Existing research has shown that artificially suppressing the insect's immune system can significantly improve the insecticidal efficacy of biocontrol fungi against target pests. The discovery and utilization of host immune regulation-related genes in fungal-insect interactions is an important foundation and link for the development of efficient fungal insecticides for the brown planthopper.

[0004] RNAi, a technology that uses dsRNA to efficiently and specifically inhibit the expression of target genes, has been widely used in pest control research. Targeting and silencing key genes that affect pest growth and development or key behaviors using synthetic dsRNA can effectively reduce insect populations and achieve pest prevention and control. Combining biocontrol fungi with RNAi technology can achieve synergistic effects in the control of targeted pests. The core of these combined technologies is efficient target gene screening.

[0005] Proclotting enzyme (PCE) is a type of serine protease with diverse functions that contains a Clip domain and is widely distributed in insects and other arthropods. After activation, PCE mediates proteolytic cascade reactions, thereby regulating many physiological processes, such as insect growth and development and immune defense, making it a potential target for effective pest control. The present invention solves this problem by combining two pest control strategies, biological control fungi and RNAi, to significantly increase the mortality rate of brown planthoppers. Summary of the Invention [Problem to be solved by the invention]

[0006] In order to solve the deficiencies of existing technologies, the object of the present invention is to provide an inhibitor that inhibits the expression of antimicrobial peptides in the brown planthopper and its application. The present invention provides a dsNlPCE4 inhibitor designed based on the NlPCE4 gene of the brown planthopper, which acts on the brown planthopper surface-inoculated with Metarhizium anisopliae, thereby significantly increasing the mortality rate of the brown planthopper. [Means for solving the problem]

[0007] To achieve the above objectives, the present invention adopts the following technical solutions: The inhibitor inhibits the expression of antimicrobial peptides in the brown planthopper, and is a double-stranded RNA designed based on the NlPCE4 gene of the brown planthopper. The cDNA sequence of the NlPCE4 gene of the brown planthopper is SEQ No. ID: 01, and the encoded protein sequence is SEQ No. ID: 02.

[0008] The inhibitor that inhibits the expression of the antimicrobial peptide of the brown planthopper is obtained by gene cloning to obtain the cDNA sequence of the NlPCE4 gene of the brown planthopper, and the primers for cloning the NlPCE4 gene are cPCE4-F SEQ No. ID:4 and cPCE4-R SEQ No. ID:5.

[0009] The inhibitor that inhibits the expression of the above-mentioned antimicrobial peptides of the brown planthopper is dsNlPCE4, which can significantly reduce the expression levels of the genes encoding the four antimicrobial peptides of the brown planthopper: DefensinA, DefensinB, LugensinA, and LugensinB.

[0010] The inhibitor dsNlPCE4, which inhibits the expression of the antimicrobial peptide of the brown planthopper, is synthesized by designing a dsRNA synthesis primer from the cDNA sequence of NlPCE4.

[0011] The dsRNA synthesis primers are dsNlPCE4-F SEQ No ID:10 and dsNlPCE4-R SEQ No ID:11.

[0012] The sequence of the inhibitor dsNlPCE4 that inhibits the expression of the antimicrobial peptide of the brown planthopper is SEQ ID No: 03.

[0013] This study relates to the application of an inhibitor that inhibits the expression of antimicrobial peptides in the brown planthopper, which is used to reduce the survival rate of the brown planthopper. The inhibitor is a double-stranded RNA designed based on the NlPCE4 gene of the brown planthopper. The cDNA sequence of the NlPCE4 gene of the brown planthopper is SEQ No. ID:01, and the encoded protein sequence is SEQ No. ID:02.

[0014] The present invention relates to the application of an inhibitor that inhibits the expression of antimicrobial peptides in the brown planthopper, and is used to improve the insecticidal efficiency of biological control fungi against the brown planthopper. There are no particular limitations on the type of biological control fungus, and as long as it is a fungus that can parasitize the brown planthopper or other pests, all of them are within the scope of protection of the present invention and have been inspired by the present invention.

[0015] The present invention relates to the application of inhibitors that inhibit the expression of antimicrobial peptides in the brown planthopper, and is also applicable to inhibitors that act on brown planthoppers whose body surface has been inoculated with a biological control bacterium.

[0016] This relates to the application of an inhibitor that inhibits the expression of antimicrobial peptides in the above-mentioned brown planthopper, and the biological control fungus is Metarhizium anisopliae strain Ma456. [Effects of the Invention]

[0017] The effects of the present invention are as follows. This invention cloned the NlPCE4 gene and synthesized double-stranded RNA (dsRNA) to prepare an inhibitor. This inhibitor acts on brown planthoppers inoculated with Metarhizium anisopliae, significantly increasing the mortality rate of the brown planthoppers. This combines two pest control strategies, biological control fungi and RNAi, to solve the pest problem.

[0018] The present invention can significantly reduce the resistance of brown planthoppers to Metarhizium anisopliae infection by inhibiting the expression of NlPCE4 via RNAi.

[0019] After interference with NlPCE4 inhibitors, the expression levels of genes encoding four antimicrobial peptides (DefensinA, DefensinB, LugensinA, and LugensinB) in the brown planthopper were significantly reduced.

[0020] The present invention provides a new target for brown planthopper control technology based on the cooperative mediation of RNA interference (RNAi) and biocontrol fungi. [Brief explanation of the drawings]

[0021] [Figure 1] 1 shows the induction expression pattern of NIPCE4 after infection of fifth-instar larvae of the brown planthopper with Metahisium anisopliae in the present invention. [Figure 2] 1 shows the relative expression level of NIPCE4 in fifth instar larvae of the brown planthopper after injection of dsRNA in the present invention. [Figure 3] 1 shows the survival rate of fifth instar larvae of the brown planthopper after injection of dsRNA in the present invention. [Figure 4] 1 shows the corrected mortality rate (A) and half-lethal time (B) of fifth-instar nymphs of the brown planthopper after combined treatment with dsRNA injection and inoculation with Metarhizium anisopliae according to the present invention. [Figure 5] 1 shows the relative expression levels of antimicrobial peptide genes of the brown planthopper after injection of dsRNA in the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0022] The present invention will now be more particularly described with reference to the accompanying drawings and specific embodiments. The technical effect is verified by the following experiment. Preparation process of dsNlPCE4 inhibitor:

[0023] 1. Materials and Methods 1.1 Test insects and pathogenic fungi The test brown planthopper population was reared in a climate chamber with a rearing temperature of (24±1)°C, a relative humidity of 70%±5%, and a photoperiod of 16h:8h. The rice cultivar used was TN1. The pathogenic fungus to be tested was Metarhizium anisopliae Ma456 strain, which was cultured and subcultured on PDA slant medium at 25°C.

[0024] 1.2 Cloning and sequencing of the NlPCE4 gene Using the silkworm coagulase protein sequence (XP_037867888) as the search source, a homologous sequence search was performed with blastP in the brown planthopper genome database to obtain a predicted protein, which was named NlPCE4. To verify the cloning, we first extracted total RNA from adult brown planthoppers using the MiniBEST Universal RNA Extraction Kit (TaKaRa, Japan), and then used the qualified RNA as a template to run the PCR using PrimeScript. TM cDNA was synthesized using a First Strand cDNA Synthesis Kit (TaKaRa, Japan). PCR amplification was performed using this as a template. Primer Premier 5 was used to design NlPCE4 amplification primers, cPCE4-F and cPCE4-R (Table 1). The total volume of the PCR reaction was 50 μL, containing 25 μL of 2x Taq Premix, 1 μL each of upstream and downstream primers (10 μmol / L), 2 μL of cDNA template, and 21 μL of ddH2O. The PCR reaction conditions were 35 cycles of pre-denaturation at 94°C for 5 minutes, denaturation at 94°C for 30 seconds, renaturation at 55°C for 30 seconds, and extension at 72°C for 1.0 minute, followed by extension at 72°C for 10 minutes. The PCR products were electrophoresed on a 1.5% agarose gel and extracted using a gel recovery kit. The recovered product was ligated into the pMD19-T cloning vector and then transformed into Escherichia coli DH5α competent cells, and positive transformants were selected and sent to Zhejiang Youkang Biotechnology Co., Ltd. for bidirectional sequencing.

[0025] 1.3 Analysis of the expression pattern of the NlPCE4 gene by biocontrol bacteria Conidia of the biological control bacterium Metarhizium anisopliae were added to the soil in a 0.02% Tween 80 solution at a concentration of 1 × 10 8 The suspension was adjusted to a concentration of 10 ... (R) Premix Ex Taq TM qRT-PCR detection was performed using the II kit (TaKaRa, Japan). The total volume of the qRT-PCR reaction was 20 μL, which included 10 μL of 2× SYBR (R) The kit contained Premix Ex TaqTMII, 1 μL each of upstream and downstream primers (10 μmol / L), 2 μL of cDNA template, and 6 μL of ddH2O. The reaction conditions were 40 cycles of pre-denaturation at 95°C for 30 seconds, denaturation at 95°C for 5 seconds, and annealing at 60°C for 30 seconds. The 18S rRNA of the brown planthopper was used as the internal reference gene. -ΔΔCt The relative expression level of NlPCE4 gene was calculated using the method.

[0026] 1.4 NlPCE4 gene RNAi analysis Based on the cDNA sequences of the NlPCE4 and green fluorescent protein (GFP) genes, dsRNA (double-stranded RNA) synthesis primers were designed (Table 1). TMFollowing the instructions in the Express RNAi System (Promega, USA) kit, dsNlPCE4 and dsGFP were synthesized, respectively. Their quality and concentration were tested and qualified using a NanoDrop ND-2000 and 1.5% agarose gel electrophoresis. After that, they were diluted with ddH2O to a final concentration of 2500 ng / µL and stored at -80°C for later use.

[0027] dsNlPCE4 sequence: GCTATAGTTCGGCAAGCAGGCTGGGATCCAACGAGCCGTTCCAGAATGTGTCGAGAAGCTATCGGCAGGGGGGGGCCTCGGGCGCTGCCCCTGGCTACACCATGATGAGTGGGCCATTTCCAAGC AGGATAACTCCAGCAGGGGATCGTTCCAGATCGTCATTGATGTTTGACGGTGGATACGATCTCAGACGCCCCCAGTGGGCTCAAGCGCCATCTTCCGACTACCGACCGAGGCGAGCCTACTCTCA GAGTGACGTCACACTGTGGCATGATGCGGGGCGCAGCAATCGCAACTCCAAGATCGCACCCGAGTTCCTTGCGCCCAACCAGCGCCCAGGGGCGGACGATGAGTCGGGGCGCCTGCTTTCCAGTG ACACCATCGCTACCATTTCGGAGACATTGGGTGCTATAAACACTGTCGGCAGGTATTTGGTCAACTACACCCGGGCCAGCACTGCACCTGGCGATAGGGTCGATGCTGTTCATACACCGCCAGTG SEQ ID No:03.

[0028] Age-matched fifth-instar nymphs of brown planthoppers were selected and injected with 20 nL of dsNlPCE4 between the base of the mid- and hind legs using a microinjector (an equal amount of dsGFP was injected as a control). Four sets of experiments were set up in parallel.

[0029] (1) The first group is used to determine the interference efficiency of dsNlPCE4 against the target gene. Live brown planthoppers were collected 1, 3, 5, and 7 days after injection of dsNlPCE4 and dsGFP, respectively, and the expression levels of NlPCE4 at each time point were detected according to the qRT-PCR detection method described in section 1.3. Three biological replicates were set for each treatment, with 10 test insects per replicate.

[0030] (2) The second group was used to detect the survival rate of brown planthoppers after NlPCE4 knockdown. The number of dead brown planthopper insects was counted daily within 10 days after injection of dsNlPCE4 or dsGFP. Three biological replicates were set up for each treatment, with 40 test insects per replicate.

[0031] (3) The fourth group was used to determine the mortality rate of brown planthoppers after combined treatment with RNAi and biocontrol fungi. After injecting dsNlPCE4 or dsGFP into the test insects, 1 mL of a 1 × 10 Metarhizium anisopliae conidial suspension prepared in 0.02% Tween 80 water solution was immediately injected. 8 The insects were inoculated onto the body surface at a concentration of 1000 cells / mL. A 0.02% Tween 80 solution was used as a blank control. Three biological replicates were performed for each treatment, with 40 test insects per replicate. The number of dead insects was counted regularly daily for 10 consecutive days, and the corrected mortality rate was calculated. Corrected mortality rate (%) = [(mortality rate of the pathogenic fungus treatment group - mortality rate of the blank control group) / (1 - mortality rate of the blank control group)] × 100%.

[0032] (4) The fifth group will be used to determine the expression levels of antimicrobial peptide genes in the brown planthopper after RNAi. Live brown planthoppers were collected 1, 3, 5, and 7 days after injection of dsNlPCE4 and dsGFP, respectively, and the expression levels of immune effector antimicrobial peptide genes in the brown planthoppers were detected at each time point using the qRT-PCR detection method described in Section 1.3. The qRT-PCR detection primers are listed in Table 1. Three biological replicates were set up for each treatment, with 10 test insects per replicate.

[0033] [Table 1]

[0034] 2. Verification and analysis of technical effects 2.1 Cloning and identification of the NlPCE4 gene The sequence of the product amplified using the brown planthopper cDNA as a template was determined and analyzed. As a result, it was found that the cDNA sequence of the brown planthopper NlPCE4 gene was 1608 bp long and encoded 535 amino acids.

[0035] The cDNA sequence is

[0036] The encoded protein sequence is MDRLRNLLESPLTWMDELNTYKFNQIADFEKCTFQFHMFFIRDWTSYSSASRLGSNEPFQNVSRSYRQGGTSGAAPGYTMMSGPFPSRITPAGDRSRSLMFDGGYDLRRPQWAQAPSSDYRPRRAYSQSDVT LWHDAGRSNRNSKIAPEFLAPNQRPGADDESGRLLSSDTIATISETLGAINTVGRYLVNYTRASTAPGDRVDAVHTPPVVSSGEDLPGAIYTISKNVLGRNVTDSIAPLVRGLPPLVGSTGKTQAAAVGDANAA SGGPRPCTTPAGAAGVCDDLSNCPQLLLNLSNLRQSICFKSLFVPGVCCPKRGDTFNVGDYSTESSIPVIHTTTAVTTTRRPPIISATSVPLHLVTKPPTTTFAPFPAFSKEEKSHPKQIVFEGTTLIKAQVRP PTTSDESKSKSSEESEEEFDDDDDDESAADDFSPEIQFYEQRHKSLKNIKKMPRGRPTYAVPVVEEETMECGQPEVAKFRVVGGEEALPGRWPWMAAIFLHGPRRTEFWCGGSFIGPKHILTAAHCTRDTRQRP It is SEQ ID No:02.

[0037] 2.3 Expression pattern of NlPCE4 after infection with Metarhizium anisopliae Ma456 A conidial suspension of Metarhizium anisopliae Ma456 was surface-inoculated onto fifth-instar larvae of the brown planthopper (Nilaparvata lugens), and the expression of NlPCE4 in the brown planthopper was detected after different induction times. The results are shown in Figure 1. Compared to the control group (unvaccinated), NlPCE4 expression showed a significant upward trend within four days of Ma456 induction (P<0.05). Three days after Ma456 inoculation, NlPCE4 expression was highest, 5.9-fold higher than the control. Two and four days after induction, the expression levels were 3.3- and 3.7-fold higher than the control, respectively, and the difference from the control reached a significant level (P<0.05).

[0038] 2.4 Interference efficiency of the NlPCE4 gene qRT-PCR analysis showed that 1, 3, 5, and 7 days after microinjection of dsNlPCE4, the expression levels of NlPCE4 in fifth-instar larvae of the brown planthopper were significantly reduced compared with those in the control group injected with dsGFP, by 76.9%, 84.3%, 74.2%, and 69.4%, respectively, all of which reached significant levels (P<0.05) (Fig. 2).

[0039] 2.5 Survival rate of brown planthoppers after NlPCE4 interference Figure 3 shows the survival rates of brown planthoppers over 10 consecutive days after NlPCE4 interference. Compared with the control group (dsGFP-injected), the survival rates of brown planthoppers in the interference group gradually decreased significantly from day 3 (P < 0.05). On days 5 and 7 after NlPCE4 interference, the survival rates of brown planthoppers decreased to 73.3% and 59.2%, respectively, whereas the survival rate of the control group remained high at over 92.5%.

[0040] 2.6 Survival of brown planthoppers after combined NlPCE4 interference and biocontrol fungal infection Bioassay experiments showed that inhibition of NlPCE4 expression by RNAi could significantly reduce the resistance of brown planthoppers to Metarhizium anisopliae infection. The corrected mortality rates of fifth-instar larvae of brown planthoppers in the dsNlPCE4-treated group 4 and 7 days after Metarhizium anisopliae infection were 51.4% and 78.7%, respectively, significantly higher than the 25.9% and 59.6% of the dsGFP control (Figure 4A). The median time to lethal (LT) of Metarhizium anisopliae against brown planthoppers in the dsNlPCE4-treated group was 1.2% and 1.2%, respectively. 50 ) at 3.8 days, which was 29.6% lower than that of the dsGFP-injected control group (5.4 days), reaching a significant level (P < 0.05) (Figure 4B).

[0041] 2.7 Expression levels of antimicrobial peptide genes after NlPCE4 interference The qRT-PCR analysis results are shown in Figure 5. Compared to the control group (dsGFP injection), the interference group (dsNlPCE4 injection) showed a significant decrease in the expression of genes encoding four antimicrobial peptides (DefensinA, DefensinB, LugensinA, and LugensinB). For example, three days after microinjection of dsNlPCE4, the expression levels of DefensinA, DefensinB, LugensinA, and LugensinB in fifth-instar larvae of the brown planthopper were significantly reduced by 75.0%, 63.5%, 56.6%, and 53.0%, respectively, compared to the control group (P<0.05).

[0042] In this invention, an inhibitor was created by cloning the NlPCE4 gene and synthesizing double-stranded RNA (dsRNA). This inhibitor acts on brown planthoppers that have been inoculated with Metarhizium anisopliae, significantly increasing the mortality rate and reducing the hatching rate of the brown planthoppers. By combining two pest control strategies, biological control fungi and RNAi, the problems of pest and disease damage are solved. This invention provides a new target for brown planthopper control technology based on the cooperative mediation of RNA interference (RNAi) and biological control fungi.

[0043] The basic principles, main features and advantages of the present invention have been shown and described above. Those skilled in the art should understand that the above embodiments do not limit the present invention in any way, and any technical solutions obtained by equivalent substitution or equivalent transformation fall within the protection scope of the present invention.

Claims

1. a double-stranded RNA designed according to the brown planthopper N1PCE4 gene, An inhibitor that inhibits the expression of an antimicrobial peptide of the brown planthopper, wherein the cDNA sequence of the brown planthopper N1PCE4 gene is SEQ No. ID: 01 and the encoded protein sequence is SEQ No. ID:

02.

2. The cDNA sequence of the brown planthopper NIPCE4 gene is obtained by gene cloning, and the primers for cloning the NIPCE4 gene are cPCE4-F SEQ No. ID: 4 and cPCE4-R SEQ No. ID: 5; The inhibitor is dsNlPCE4, which can significantly reduce the expression levels of DefensinA, DefensinB, LugensinA, and LugensinB genes encoding four antimicrobial peptides of the brown planthopper; The dsNlPCE4 was synthesized using a dsRNA synthesis primer designed based on the cDNA sequence of NlPCE4; The inhibitor of claim 1, which inhibits the expression of an antimicrobial peptide of the brown planthopper, wherein the dsRNA synthesis primers are dsNlPCE4-F SEQ No. ID: 10 and dsNlPCE4-R SEQ No. ID: 11, and the dsNlPCE4 sequence is SEQ ID No:

03.

3. 1. An application of an inhibitor that inhibits the expression of an antimicrobial peptide of a brown planthopper, which is used to reduce the survival rate of the brown planthopper, The inhibitor is a double-stranded RNA designed according to the N1PCE4 gene of the brown planthopper, The cDNA sequence of the brown planthopper N1PCE4 gene is SEQ No. ID: 01, and the encoded protein sequence is SEQ No. ID:

02.

4. The inhibitor is used to improve the insecticidal efficiency of a biological control fungus against the brown planthopper, or to act on the brown planthopper surface-inoculated with a biological control fungus; The use of an inhibitor that inhibits the expression of antimicrobial peptides in brown planthoppers according to claim 3, wherein the biocontrol fungus is Metarhizium anisopliae Ma456 strain.

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