Nilaparvata lugens NlCPSF73 gene, encoding protein thereof and application of dsRNA thereof in prevention and treatment of Nilaparvata lugens

Abstract

The invention relates to the technical field of biology, in particular to a brown planthopper NlCPSF73 gene, encoding protein of the brown planthopper NlCPSF73 gene and application of dsRNA of the brown planthopper NlCPSF73 gene in prevention and treatment of brown planthopper. The invention discloses an NlCPSF73 gene. The function of the gene is inhibited, so that the survival rate of brown planthoppers is reduced; after the dsRNA of the NlCPSF73 synthesized in vitro is used for silencing the NlCPSF73 gene in a microinjection manner, the survival rate of the brown planthopper is obviously reduced. The invention has a good application prospect, and is of great significance to prevention and treatment of brown planthopper.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to the brown planthopper. NlCPSF73 Application of genes, their encoded proteins, and their dsRNAs in the control of brown planthoppers. Background Technology

[0002] As one of my country's most important food crops, increasing rice yield is a top priority for agricultural development. Brown planthopper ( Nilaparvata lugens Brown planthoppers (Stål) have long posed a serious threat to rice yields and food security. Currently, the main control measures rely on chemical pesticides, but these present problems such as environmental pollution and the evolution of pesticide resistance. Therefore, there is an urgent need to find and develop efficient, safe, and sustainable control strategies. RNAi technology, or RNA inference, is a technique that uses double-stranded RNA (dsRNA) to induce the silencing of target gene mRNA. RNAi is an effective means of studying insect gene function, confirming gene function by exploring phenotypic changes in insects before and after gene silencing. However, the genomic information of the brown planthopper is vast, and whether RNAi technology can achieve good control effects depends on which gene is selected as the silencing target.

[0003] In eukaryotes, the processing of the 3′ end of mRNA is a crucial step in gene expression regulation. This process relies on the cleavage and polyadenylation specificity factor (CPSF), a complex of various protein factors. CPSF73 (a 73 kDa subunit of the cleavage and polyadenylation specificity factor) is a core component, possessing metal-dependent endonuclease activity and serving as a key enzyme for cleaving precursor mRNA at specific sites. CPSF73 is highly conserved throughout evolution and is widely found in yeast, plants, insects, and mammals. Its loss of function typically leads to abnormal mRNA maturation and stabilization, resulting in cell death or developmental abnormalities. Current research indicates that CPSF73 plays a role not only in the processing of coding gene mRNA but also in the processing of non-coding RNAs such as small nuclear RNA and histone mRNA, thus playing an irreplaceable role in maintaining normal development and survival. Reports have shown that silencing CPSF73 in some species leads to death, demonstrating the gene's critical importance for survival. Although the function of CPSF73 in model organisms has been extensively studied, research on CPSF73 in the brown planthopper has not yet been reported. Therefore, this invention focuses on the study of CPSF73 in the brown planthopper. NlCPSF73 Based on in-depth research into gene function, a method for utilizing... NlCPSF73 A novel control strategy targeting interference. By silencing this gene via RNAi, the survival and development of the brown planthopper can be significantly inhibited, thus providing a new technical approach for the green control of rice pests. Summary of the Invention

[0004] The purpose of this invention is to provide brown planthoppers NlCPSF73 The application of genes, their encoded proteins, and their dsRNAs in the control of brown planthoppers aims to address the problems raised in the background art.

[0005] This invention provides brown planthoppers NlCPSF73 The nucleotide sequence of the gene is shown in the sequence listing SEQ ID NO:1.

[0006] This invention provides brown planthoppers NlCPSF73 The protein encoded by the gene has the amino acid sequence shown in SEQ ID NO:2 in the sequence listing.

[0007] This invention provides brown planthoppers NlCPSF73 The gene dsRNA sequence is shown in SEQ ID NO:3 in the sequence listing.

[0008] Furthermore, the brown planthopper NlCPSF73 The preparation method of gene dsRNA is as follows: NlCPSF73 Genes are used as targets, and primers are designed for amplification and purification. NlCPSF73 Gene dsRNA.

[0009] This invention provides brown planthoppers NlCPSF73 Application of gene dsRNA in the control of brown planthopper.

[0010] Furthermore, the application method is as follows: [The text abruptly ends here, so the translation stops as well.] NlCPSF73 Gene dsRNA was injected into brown planthoppers or an expression structure was constructed. NlCPSF73 Transgenic insect-resistant crops containing the dsRNA gene, or crops sprayed with the dsRNA.

[0011] Compared with the prior art, the beneficial effects achieved by the present invention are: the present invention discloses the brown planthopper NlCPSF73 Its application as a target in the control of brown planthoppers, by silencing the brown planthoppers. NlCPSF73 Genes can reduce the survival rate of brown planthoppers, thereby reducing the impact of brown planthoppers' direct feeding on rice and achieving the goal of brown planthopper control. Detailed Implementation

[0012] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0013] Materials and sources used in this invention: The tested brown planthoppers were biotype 1 brown planthoppers (TN1 population) that feed on sensitive rice TN1. The rearing temperature was 26±2°C, the humidity was 70%, and the photoperiod was 16L:8D. The Escherichia coli was a commercially available DH5α strain. All other materials were commercially available products.

[0014] Prior to the specific embodiments of the present invention, the inventors studied the brown planthopper. NlCPSF73 The spatiotemporal expression patterns of genes were analyzed relatively quantitatively.

[0015] The specific procedure for analyzing expression patterns at different developmental stages was as follows: Total RNA was extracted from insects at different developmental stages, including samples from 1st to 5th instar nymphs and male and female adults 72 hours and 168 hours after emergence. Each group contained 10 samples, and all samples were replicated three times.

[0016] Total RNA was extracted using the RNAiso Plus kit (Takara, Dalian, China) according to the manufacturer's instructions. After confirming the RNA was free of degradation by agarose gel electrophoresis, its concentration and purity were determined using a NanoDrop 2000 (Thermo Fisher Scientific, USA). Using 1 μg of total RNA as a template, cDNA was synthesized according to the PrimeScript™ RTreagent Kit with gDNAEraser (Perfect Real Time) (TaKaRa) kit instructions.

[0017] The reaction was carried out in two steps using a 20 μL reaction system: First, remove genomic DNA; Second, it is reverse transcribed into cDNA.

[0018] Specifically, it includes the following steps: Step 1: 1 μg total RNA, 2 μL 5×g DNAEraser Buffer, 1 μL g DNAEraser, and RNase-free dH2O to a final volume of 10 μL. The reaction conditions were 42℃ for 2 min, then terminated at 4℃.

[0019] Step 2: 10 μL of the reaction solution from Step 1, 1 μL of PrimeScript RT Enzyme Mix I, 1 μL of RT Primer Mix, 4 μL of 5×PrimeScript Buffer 2 (for Real Time), and RNase-free dH2O were added to a total volume of 20 μL. Reaction conditions: 37℃ for 15 min, 85℃ for 50 s to terminate the reaction. The entire experiment was performed on a clean bench, all consumables were RNase-free, and all reaction solutions were prepared on ice. The synthesized cDNA was stored at -20℃ for subsequent real-time quantitative PCR.

[0020] The expression differences of the NIPSF73 gene at different ages were detected using a CFX96 real-time quantitative PCR instrument (Bio-Rad Laboratories, USA). Following the instructions for the 2×M5 HiPerSYBR Premix EsTaq (Aggregat), a 10 μL PCR reaction system was selected, including 5 μL of SYBR, 2 μL of cDNA template, 0.4 μL of primers (mixed), and 2.6 μL of ddH2O.

[0021] Real-time quantitative PCR amplification program: 95℃ pre-denaturation for 60 s; 95℃ denaturation for 10 s, 60℃ annealing for 60 s, 40 cycles. Using brown planthoppers... 18S ribosomal RNA ( Nl18s The JN662398 gene was used as an internal reference, and the relative expression level of the target gene was measured using a 2-1 ratio. -ΔΔCt The calculation was performed using the method described in Table 1. Table 1. As shown in Table 1, NlCPSF73 The gene is expressed in all life stages of the brown planthopper, with significantly higher expression in the first instar nymphs than in other instars.

[0022] The specific procedures for analyzing expression patterns in different tissues were as follows: Samples were collected 72 hours after female emergence, including samples from the legs (n=50), intestine (n=20), salivary glands (n=50), fat body (n=20), and ovary (n=20), as well as testicular samples from males 72 hours after emergence (n=50). 'n' represents the number of samples in each group, and all samples were replicated in triplicate. Subsequent analysis of expression patterns at the same age was performed, and the results are shown in Table 2.

[0023] Table 2. As shown in Table 2, NlCPSF73 The gene is expressed in all tissues of the brown planthopper, with significantly higher expression levels in the ovary than in other tissues.

[0024] Example 1: Brown planthopper NlCPSF73 Gene cloning Step 1: Place 4 brown planthoppers in a 1.5 mL RNase-free centrifuge tube and extract total RNA from the brown planthoppers using the TRIzol method. The specific procedure is as follows: (1) Add two steel balls and 500 μL of RNAisoPlus reagent to the RNase-free centrifuge tube containing brown planthoppers, tighten the cap of the centrifuge tube, grind thoroughly on a grinder, centrifuge briefly, and let stand on ice for 5 min. (2) Add 73 μL of chloroform to the centrifuge tube, invert it, and let it stand on ice to separate the layers; centrifuge at 12000 rpm for 15 min at 4℃; (3) Take all the supernatant into a new 1.5 mL RNase-free centrifuge tube, add 73 μL of isopropanol, invert the tube, place it on ice and let it stand for 1 h, centrifuge at 4°C and 12000 rpm for 10 min; (4) Discard the supernatant, add 500 μL of 75% anhydrous ethanol (prepared fresh), invert, wash the precipitate, centrifuge at 7500 rpm for 5 min at 4°C, carefully aspirate the supernatant, dry the RNA precipitate with a constant temperature metal bath, open the centrifuge tube cap, and disperse the residual alcohol to avoid affecting the quality of RNA. (5) Add 15 μL of RNase-free H2O to dissolve the RNA; (6) RNA quality was detected by agarose gel electrophoresis. 1 μL was diluted 10-fold, and the RNA concentration and purity were detected using a micro-spectrophotometer. Step 2: Synthesis of brown planthopper cDNA was performed according to the instructions of the TaKaRa PrimeScript RT reagent with gDNA Erase kit, using it as a PCR amplification template. The specific operation method is as follows: (1) PCR primers were designed based on the NCBI predicted sequences. The primer sequences are as follows: NlCPSF73 -1F:5'-ATGTCAGCTAAGAGAAAACC-3' NlCPSF73 -1R:5'-TTATGATGCGGCAGGGTCCA-3' PCR amplification was performed using the primers described above, and the system is as follows: 10×Buffer 5μL, dNTP 4μL, Ex Taq 0.25μL, PCR primers 1μL each, cDNA 1μL, add ddH2O to 50μL.

[0025] The PCR reaction steps were as follows: 95°C pre-denaturation for 5 min; 95°C denaturation for 30 s; 52°C annealing for 30 s; 72°C extension for 43 s; 35 cycles; and a final extension at 72°C for 10 min.

[0026] (2) The accuracy of PCR band positions was detected by 1% agarose gel electrophoresis. PCR products with correctly positioned bands were ligated into the pMD18-T vector and transformed into *E. coli*. Selection was performed using LB medium containing ampicillin, followed by bacterial culture PCR identification. Finally, the bacterial cultures with correctly positioned bands were sent for sequencing, and the sequencing results were analyzed to obtain... NlCPSF73 The nucleotide sequence of the brown planthopper is shown in SEQ ID NO:1. NlCPSF73 Encoded by genes NlCPSF73 The protein, with its amino acid sequence shown in SEQ ID NO. 2.

[0027] Example 2: Synthesis of dsRNA Step 1: Take 73 μL of the correctly sequenced bacterial culture from Example 1, add 2 mL of LB liquid medium containing ampicillin, and incubate overnight at 37°C in a shaker. Plasmid extraction was performed using the M5 plasmid miniprep plus kit from Beijing Polymer Technology Co., Ltd. dsRNA primers with the T7 promoter were synthesized; the primer sequences are as follows: dsNlCPSF73 -F:5'- TAATACGACTCACTATAGGGGCTGTGGAGAAGCCTAAA-3' dsNlCPSF73 -R:5'- TAATACGACTCACTATAGGGCAGAATGGCAGTGATAAT-3' Step 2: Amplify the plasmid DNA template containing the T7 promoter sequence, and then purify the amplified product using the MiniBEST Agarose Gel DNA Extraction Kit Ver. 4.0 (TaKaRa). Once the purified DNA template is of usable concentration, it becomes the dsRNA template. The synthesis of dsRNA follows the instructions of the MEGAscript™ T7 Transcription Kit (Thermo). Brown planthopper NlCPSF73 The dsRNA sequence of the gene is shown in the sequence listing SEQ ID NO:3.

[0028] Comparative Example 1: The sample was synthesized and purified according to the method described in Example 2 above. GFP (Green fluorescent protein) dsRNA was used as a negative control. GFP The upstream and downstream primer sequences used in fragment PCR amplification are as follows: ds GFP -F:5'-TAATACGACTCACTATAGGGAGAACGTAAACGGCCACAAGTTC-3' ds GFP -R:5'-TAATACGACTCACTATAGGGAGATGTTCTGCTGGTAGTGGTCG-3' The remaining specific steps are the same as in Example 2, for the preparation of dsRNA.

[0029] The dsRNA prepared in Example 2 was named ds NlCPSF73 This was used as the experimental group, and the dsRNA prepared in Comparative Example 1 was named dsRNA. GFP And used it as the control group. The experimental group's ds NlCPSF73 and control group ds GFP Microinjection was performed on brown planthopper samples to test its effect on brown planthopper ds NlCPSF73 Gene silencing effect, survival rate of brown planthopper, and changes in morphological characteristics of brown planthopper.

[0030] Experiment 1: dsRNA on brown planthoppers NlCPSF73 Silence effect test Third instar nymphs of the brown planthopper were selected, and 40 ng ds was injected into the middle part of the midcoxel and hind coxel of the thorax using a microinjector. GFP and ds NlCPSF73 Samples were taken at 24, 48, and 72 hours later, and the silencing effect was verified by qRT-PCR, with four biological replicates per replicate containing four worms. The effect of dsRNA on silencing in the experimental and control groups was detected. NlCPSF73 The effect of gene silencing. The experimental results are shown in Table 3 below. Table 3. Experiment 2: Verification of the effects of RNAi on brown planthoppers Third-instar nymphs of brown planthoppers were selected, and 40 ng ds was injected into their thorax using a microinjector. NlCPSF73 and ds GFP Three biological replicates were performed, with 40–60 planthoppers per replicate. The microinjected brown planthopper samples were placed on TN1 rice plants, and the survival rate of the planthoppers was counted and calculated from day 1 to day 10. The results are shown in Table 4 below.

[0031] Table 4. Compared with the control group, the survival rate of brown planthoppers on rice was significantly reduced after microinjection of dsRNA into the experimental groups.

[0032] It should be noted that, in this document, relational terms such as "first" and "second" are used only to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0033] Finally, it should be noted that the above descriptions are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. Brown planthopper NlCPSF73 Genes are characterized by: The nucleotide sequence of this gene is shown in SEQ ID NO:1 in the sequence listing.

2. The brown planthopper according to claim 1 NlCPSF73 The protein encoded by the gene is characterized by: Brown planthopper NlCPSF73 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO:2 in the sequence listing.

3. The brown planthopper according to claim 1 NlCPSF73 The dsRNA of a gene is characterized by: Brown planthopper NlCPSF73 The dsRNA sequence of the gene is shown in the sequence listing SEQ ID NO:

3.

4. The brown planthopper according to claim 3 NlCPSF73 Application of dsRNA of genes in the control of brown planthoppers.

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