Inhibitor of brown planthopper RNA binding protein NlRBP gene and application thereof

Abstract

The present application provides a kind of brown planthopper RNA binding protein NlRBP gene inhibitor and application, the NlRBP gene inhibitor is the dsRNA capable of inhibiting brown planthopper NlRBP gene expression, the nucleotide sequence of the dsRNA is as shown in SEQ ID NO.3.The present application also provides the application of the above-mentioned brown planthopper RNA binding protein NlRBP gene inhibitor in the prevention and treatment of brown planthopper.The prevention and treatment of brown planthopper is to inhibit the survival rate of brown planthopper.The survival rate of brown planthopper is realized by inhibiting the expression of brown planthopper RNA binding protein NlRBP gene.The present application also provides the application of the above-mentioned dsRNA in preparation product.The present application provides a new prevention and treatment strategy and target for brown planthopper prevention and treatment, establishes the technology based on RNAi green control brown planthopper, provides foundation and reference for the precision, sustained pest control of brown planthopper, and has good application prospect.

Description

Technical Field

[0001] This invention relates to the field of biological control technology, and in particular to a repressor of the NlRBP gene, an RNA-binding protein of brown planthopper, and its application. Background Technology

[0002] Brown planthopper ( Nilaparvata lugens Brown planthoppers (Hemiptera: Delphacidae) belong to the family Delphacidae in the order Hemiptera and are a significant migratory pest of rice in many countries. Currently, chemical methods are widely used to control brown planthoppers. However, the large-scale and long-term use of pesticides increases production costs, kills natural enemies of brown planthoppers, and can lead to pesticide resistance, causing them to re-emerge. Therefore, researching and developing precise, green, and sustainable methods and technologies for controlling brown planthoppers is a trend and a necessity for sustainable agricultural development.

[0003] RNA interference (RNAi) refers to gene silencing mediated by double-stranded RNA (dsRNA) assembled from sense and antisense RNA. The discovery of RNAi has not only advanced research into insect gene function and regulation, but also, as a valuable reverse genetics tool for studying insect gene function, opened a new avenue for pest control. RNAi technology is currently recognized as the most promising technology for developing next-generation green and environmentally friendly insecticides.

[0004] Brown planthoppers congregate at the base of rice plants, directly harming the rice by sucking phloem sap and laying eggs, which injure stem and leaf tissues. They also indirectly harm rice by transmitting rice viral diseases such as herbaceous dwarf disease and toothed leaf dwarf disease, and by exacerbating diseases like rice sheath blight. Recent studies have shown that the components of saliva secreted by brown planthoppers during feeding are crucial to their feeding behavior and population reproduction. RNA-binding proteins are a component abundant in brown planthopper saliva; therefore, exploring the gene function of these RNA-binding proteins and using them as precise control targets to develop green control technologies based on RNAi is a technical problem that needs to be solved by those skilled in the art. Summary of the Invention

[0005] The purpose of this invention is to provide a suppressor of the NlRBP gene, an RNA-binding protein of brown planthopper, and its application, so as to achieve pest control of brown planthopper based on RNAi technology.

[0006] The technical problem solved by this invention is achieved by the following technical solution:

[0007] A repressor of the brown planthopper RNA-binding protein NlRBP gene, wherein the repressor of the NlRBP gene is a dsRNA capable of inhibiting the expression of the brown planthopper NlRBP gene, and the nucleotide sequence of the dsRNA is shown in SEQ ID NO.3.

[0008] The full-length cDNA sequence of the brown planthopper RNA-binding protein NlRBP gene is shown in SEQ ID NO.1.

[0009] The amino acid sequence of the protein encoded by the brown planthopper RNA-binding protein NlRBP gene is shown in SEQ ID NO.2.

[0010] This invention provides the application of the inhibitor of the above-mentioned brown planthopper RNA-binding protein NlRBP gene in the control of brown planthoppers.

[0011] The control of brown planthoppers refers to suppressing their survival rate.

[0012] The suppression of brown planthopper survival rate is achieved by inhibiting the expression of the brown planthopper RNA-binding protein NlRBP gene.

[0013] This invention provides the application of the above-mentioned dsRNA in the preparation of a product, which is an insecticide for the control of brown planthoppers.

[0014] Beneficial effects:

[0015] This invention uses the NlRBP gene, a glycine-rich RNA-binding protein from the brown planthopper, as the target gene for interference. Through both feeding and microinjection of the brown planthopper, the transcriptional expression of the target gene can be effectively silenced. After NlRBP gene expression is suppressed, the mortality rate of the brown planthopper increases significantly, thereby controlling its population. This invention provides a new control strategy and target for brown planthopper control, and lays the foundation and reference for establishing RNAi-based green control technology for brown planthoppers, enabling precise and sustainable pest control. It has promising application prospects. Attached Figure Description

[0016] Figure 1 A schematic diagram of the agarose gel electrophoresis results of the full-length cDNA of the NlRBP gene in the brown planthopper.

[0017] Figure 2 This is a schematic diagram of the dsRNA agarose gel electrophoresis results;

[0018] Figure 3 The survival rate of brown planthoppers after being fed dsRNA is shown in the figure.

[0019] Figure 4 The survival rate of brown planthoppers after in vivo microinjection of dsRNA is shown in the figure.

[0020] Figure 5 The silencing effect of the NlRBP gene on day 10 after feeding dsRNA is shown in the figure.

[0021] Figure 6 The silencing effect of NlRBP gene after in vivo microinjection of dsRNA is shown in the figure. Detailed Implementation

[0022] To facilitate understanding of the present invention, a more complete description will be given below with reference to the accompanying drawings. Several embodiments of the invention are illustrated in the drawings. However, the invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.

[0023] In the following examples, the brown planthopper, the test insect, was collected from the rice fields of Jiangxi Agricultural University and then raised indoors in an artificial light incubator with rice. The rearing temperature was 27±2℃, the relative humidity was 70±5%, and the light intensity was 14:10h (light:dark). Unless otherwise specified, the experimental methods used were conventional methods. Unless otherwise specified, the materials and reagents used in the examples were commercially available. The instruments used in the experiments were all conventional instruments used in molecular biology laboratories.

[0024] Example 1: Obtaining the full-length cDNA sequence of the glycine-rich RNA-binding protein gene NlRBP from the brown planthopper.

[0025] 1. Extracting total RNA from brown planthoppers

[0026] Total RNA from insects was extracted using TRIzol® RNA separation and extraction reagent (TIANGEN) under RNase-free conditions. The specific extraction steps are as follows:

[0027] (1) After washing and drying the mortar and pestle, wrap it tightly with tin foil and then put it into a high-temperature dry heat sterilizer for sterilization. After sterilization, take it out and cool it to room temperature. Then soak it in chloroform for 20 minutes and then dry it in a fume hood.

[0028] (2) Take 8-10 fresh brown planthoppers, put them into a 1.5 mL centrifuge tube without RNase in a clean bench, and quickly add 500 μL of Trizol® reagent into a glass homogenizer and grind them thoroughly.

[0029] (3) Add 100 μL of chloroform to the well-ground homogenate, shake to mix, and then centrifuge at 12000 rpm for 15 min in a centrifuge at 4℃.

[0030] (4) Transfer the upper aqueous phase after centrifugation in step (3) to a new treated 1.5 mL centrifuge tube, add 2 times the volume of anhydrous ethanol, mix by inversion, and place at -80℃ for 1 h.

[0031] (5) Centrifuge the centrifuge tubes after low-temperature precipitation at 12,000 rpm for 15 min in a centrifuge at 4℃;

[0032] (6) Discard the supernatant after centrifugation in step (5), wash the RNA precipitate once with 75% ethanol (first add 750 μL of anhydrous ethanol, then add 250 μL of RNAase-fee H2O), and centrifuge at 12000 rpm for 10 min in a centrifuge at 4℃.

[0033] (7) Discard the supernatant after centrifugation in step (6), dry the tube wall on a clean bench (about 5 min), add 20 μL RNAase-fee H2O to the centrifuge tube to dissolve the precipitate and obtain an RNA solution;

[0034] (8) Take 1 μL of the RNA solution obtained in step (7) and dilute it to 10 μL. Use 5 μL to detect the integrity of the RNA extraction obtained in step (7) by agarose gel electrophoresis, use 5 μL to detect the quality of the RNA extraction obtained in step (7) by NanoDrop instrument, and use the remaining RNA for the synthesis of full-length cDNA.

[0035] 2. Synthesis of first-strand cDNA

[0036] The first-strand cDNA of brown planthopper was synthesized according to the instructions of the Takara SMARTer® RACE 5' / 3' kit, and the synthesized 5'-RACE-Ready cDNA or 3'-RACE-Ready cDNA of brown planthopper was stored at -20°C.

[0037] 3. Rapid amplification of cDNA ends (RACE)

[0038] (a) Specific primers for the NlRBP gene were designed. The specific primer sequences for the first round of 5'-RACE-Ready cDNA amplification are shown in SEQ ID NO.4, and the specific primer sequences for the first round of 3'-RACE-Ready cDNA amplification are shown in SEQ ID NO.5. The specific primer sequences for the second round of 5'-RACE-Ready cDNA amplification are shown in SEQ ID NO.6, and the specific primer sequences for the second round of 3'-RACE-Ready cDNA amplification are shown in SEQ ID NO.7.

[0039] (b) Using the first-strand cDNA obtained in step 2 as a template, PCR amplification was performed using the gene-specific primers described in step (a) for the first round of amplification. The reagents shown in Table 1 were added to two centrifuge tubes respectively.

[0040] Table 1

[0041]

[0042] After mixing thoroughly, centrifuge briefly. The PCR program was as follows: 98 ℃, 30 s; 98 ℃, 10 s; 58 ℃, 5 s; 72 ℃, 5 s (30 cycles in total); 72 ℃, 1 min. The PCR products were then detected by agarose gel electrophoresis.

[0043] (c) Using a 10-fold dilution of the first-round amplification product as the template for the second-round amplification, PCR amplification was performed using the gene-specific primers for the second-round amplification described in step (3). The reagents shown in Table 2 were added to two centrifuge tubes respectively.

[0044] Table 2

[0045]

[0046] After mixing thoroughly, centrifuge briefly. The PCR program was as follows: 98 ℃, 30 s; 98 ℃, 10 s; 58 ℃, 5 s; 72 ℃, 5 s (30 cycles in total); 72 ℃, 1 min. The PCR products were then detected by agarose gel electrophoresis.

[0047] (d) Purify the PCR product obtained in step (4), recover the target fragment, and then ligate the purified PCR product into the pClone007 cloning vector to obtain the recombinant vector. Finally, transform the recombinant vector into Trelief-5α competent cells and screen for positive clones containing the target fragment.

[0048] (e) The positive clones containing the target fragment screened in step (d) were sequenced. After sequence assembly, the full-length cDNA sequence of the NlRBP gene was obtained, and finally, the sequence was verified by PCR amplification. The agarose gel electrophoresis results of the full-length cDNA of the NlRBP gene in the brown planthopper are shown below. Figure 1 As shown, Figure 1 In the middle, the leftmost lane: DNA marker; the rightmost lane: NlRBP gene full-length cDNA agarose gel electrophoresis image.

[0049] Example 2: Preparation and in vitro synthesis of dsRNA template for NlRBP gene in brown planthopper

[0050] 1. Preparation of dsRNA template for NlRBP gene in brown planthopper

[0051] (1) Based on the full-length cDNA sequence of the NlRBP gene of the brown planthopper, specific primers with the T7 promoter were designed using the online software NCBI_primer. The forward primer is shown in SEQ ID NO.8 and the reverse primer is shown in SEQ ID NO.9.

[0052] (2) Using a plasmid containing the full-length cDNA of the brown planthopper NlRBP gene as a template, PCR amplification was performed using high-fidelity DNA polymerase. The PCR reaction system was as follows (total volume 25 μL): 1 μL of template with a concentration of 100 ng / μL, 0.5 μL each of upstream and downstream primers with a concentration of 10 µM, 12.5 μL of 2X BioRun Pfu PCR Mix, and 10.5 μL of nuclease-free water;

[0053] The PCR reaction conditions were as follows: pre-denaturation at 95℃ for 5 min; denaturation at 95℃ for 15 s, annealing at 58℃ for 15 s, extension at 72℃ for 30 s, for a total of 30 cycles; extension at 72℃ for 5 min, and storage at 4℃ to obtain the PCR product.

[0054] The PCR product obtained in step (2) was detected by 1% agarose gel electrophoresis, and a single bright band was obtained. The PCR product was then purified using a DNA product purification kit. The specific purification process was carried out according to the kit instructions. The purified PCR product (concentration not less than 200 ng / μL) was used directly as a template for dsRNA synthesis.

[0055] 2. In vitro synthesis of dsRNA

[0056] The DNA recovered and purified in step 1 was transcribed in vitro to synthesize dsRNA (hereinafter referred to as NlRBP-dsRNA) using the T7 RiboMAX™ Express RNAi System kit (Promega). The nucleotide sequence of NlRBP-dsRNA is shown in SEQ ID NO.3. The specific steps are as follows:

[0057] (1) In a clean bench, add the reagents shown in Table 3 to 200 μL centrifuge tubes respectively:

[0058] Table 3

[0059]

[0060] After gently mixing, incubate in a PCR instrument at 37 ℃ for 90 min; then at 70 ℃ for 10 min.

[0061] (2) Remove DNA and ssRNA. Add 1 μL DNAase I and 1 μL RNase A (diluted at a ratio of 1:200) to the centrifuge tube from step (1), mix gently, and incubate at 37 °C for 30 min in a PCR instrument.

[0062] (3) Add 0.1 volume of sodium acetate (3M, pH 5.2) and 2.5 volume of 95% ethanol to the reaction system in step (2), place on ice for 5 min, and centrifuge at maximum speed for 10 min.

[0063] (4) Discard the supernatant after centrifugation in step (3), add 1 mL of pre-cooled 70% ethanol to wash the precipitate, 12,000 g, 4°C, 5 minutes;

[0064] (5) Discard the supernatant after centrifugation in step (4), dry it at room temperature, and add 2-5 times the amount of reaction system (30~50 µL) of Nuclease-free water to dissolve the product;

[0065] (6) Take 1 µL of the dsRNA solution obtained in step (5) and dilute it to 10 µL. Use 5 µL to detect the mass of the dsRNA obtained in step (5) by agarose gel electrophoresis, and use 5 µL to detect the concentration of the dsRNA extracted in step (5) by NanoDrop instrument. Adjust the concentration to 5000 ng / µL, aliquot 10 µL into each tube, and store at -80 °C.

[0066] (7) Simultaneously, a dsRNA solution of GFP (Green Fluorescent Protein) was synthesized and purified as described above as a negative control (hereinafter referred to as GFP-dsRNA). The upstream and downstream primer sequences used for GFP fragment PCR amplification were dsGFP-T7F (as shown in SEQ ID NO.10) and dsGFP-T7R (as shown in SEQ ID NO.11), respectively. The agarose gel electrophoresis results of the synthesized NlRBP-dsRNA and GFP-dsRNA are as follows: Figure 2 As shown, Figure 2 In the middle, the leftmost lane: DNA marker; lanes 1-3: GFP-dsRNA electrophoresis; lanes 4-6: NlRBP-dsRNA electrophoresis.

[0067] Example 3: Experiment on inhibiting the survival of brown planthoppers

[0068] 1. Feeding brown planthoppers with NlRBP gene dsRNA and calculating survival rate.

[0069] Brown planthopper nymphs of uniform size and health were selected and divided into three groups: a control group fed only with artificial feed (without dsRNA), a control group fed with GFP-dsRNA, and an experimental group fed with NlRBP-dsRNA. The brown planthopper rearing apparatus consisted of a double-walled glass tube (3*12cm). One end of the tube contained a feed chamber formed by two layers of Parafilm membrane with feed wrapped in between. After introducing the planthoppers, gauze was tied to the other end of the tube with a rubber band, and the entire rearing apparatus was covered with a damp black cloth, leaving only the feed-containing end exposed and facing the light. Rearing conditions were: temperature 27±2℃, relative humidity 70±5%, and light intensity 14:10 h (L:D). Before the feeding experiment, the planthoppers were allowed a two-day acclimatization period with only artificial feed. Then, on the first day, they were fed sucrose water containing 250 ng / μL dsRNA, and on the second day, they were fed only artificial feed. This alternating feeding pattern was repeated for 10 days. The number of surviving larvae in each incubator was observed and recorded for 10 consecutive days. Each incubator contained 25 third-instar brown planthopper nymphs, with at least three replicates per group. Figure 3 As shown, CK was the unfed control group, dsGFP was the control group fed with GFP-dsRNA, and dsNlRBP was the experimental group fed with NlRBP-dsRNA; on day 5, for the CK group and the dsNlRBP group, P = 0.033, for the dsGFP group and the dsNlRBP group, P = 0.019; On day 6, for the CK group and the dsNlRBP group, P = 0.042, for the dsGFP group and the dsNlRBP group, P = 0.055; On day 7, for the CK group and the dsNlRBP group, P = 0.037, for the dsGFP group and the dsNlRBP group, P = 0.105; On day 8, for the CK group and the dsNlRBP group, P = 0.008, for the dsGFP group and the dsNlRBP group, P = 0.009; On day 9, for the CK group and the dsNlRBP group, P <0.001, for the dsGFP group and the dsNlRBP group, P = 0.001; On day 10, for the CK group and the dsNlRBP group, P <0.001, for the dsGFP group and the dsNlRBP group, P <0.001.

[0070] from Figure 3It can be seen that, compared with the control group, the survival rate of brown planthoppers in the dsNlRBP group was significantly suppressed from the 5th day of continuous alternating feeding (survival rate of approximately 65.6%). On the 10th day of continuous alternating feeding, the survival rate of brown planthoppers in the dsNlRBP group was significantly reduced to approximately 45.8%.

[0071] 2. Microinjection of NlRBP gene dsRNA from brown planthopper into the body and statistical analysis of survival rate.

[0072] Brown planthopper nymphs of uniform size and health were selected and divided into three groups: a control group (no injection), a control group injected with GFP-dsRNA, and an experimental group injected with NlRBP-dsRNA. Before injection, the nymphs were anesthetized with ether for 60 seconds. The anesthetized nymphs were then transferred to a 1.5% agar plate, abdomen facing upwards. Using a microinjector (WPI, Nanoliter 2020), in vitro synthesized dsRNA was injected into the ventral depression between the mesothorax and metathorax of the nymph. The injection volume was 46 nL, and the concentration was 5 μg / μL. After injection, the microinjected nymphs were transferred to plastic petri dishes containing moistened filter paper and young rice leaves to recover for 0.5 h. The recovered nymphs were then transferred to TN1 rice plants at the five-leaf stage to feed. Starting from the day of injection, the number of surviving nymphs on each rice plant was observed and recorded for 10 consecutive days. Each group was injected with 25 nymphs, and at least 9 biological replicates were established. Figure 4 As shown, CK was the blank group without microinjection, dsGFP was the control group microinjected with GFP-dsRNA, and dsNlRBP was the experimental group microinjected with NlRBP-dsRNA; on day 2, for the CK group and the dsNlRBP group, P = 0.001, for the dsGFP group and the dsNlRBP group, P = 0.201; On day 3, for the CK group and the dsNlRBP group, P <0.001, for the dsGFP group and the dsNlRBP group, P = 0.001; On day 4, for the CK group and the dsNlRBP group, P <0.001, for the dsGFP group and the dsNlRBP group, P <0.001; On day 5, for both the CK group and the dsNlRBP group, P <0.001, for the dsGFP group and the dsNlRBP group, P <0.001; On day 6, for both the CK group and the dsNlRBP group, P <0.001, for the dsGFP group and the dsNlRBP group, P <0.001; On day 7, for both the CK group and the dsNlRBP group, P<0.001, for the dsGFP group and the dsNlRBP group, P <0.001; On day 8, for both the CK group and the dsNlRBP group, P <0.001, for the dsGFP group and the dsNlRBP group, P = 0.001; On day 9, for the CK group and the dsNlRBP group, P <0.001, for the dsGFP group and the dsNlRBP group, P = 0.001; On day 10, for the CK group and the dsNlRBP group, P <0.001, for the dsGFP group and the dsNlRBP group, P =0.001.

[0073] from Figure 4 It can be seen that, compared with the control group, the survival rate of brown planthoppers in the dsNlRBP group was significantly inhibited from day 3 after microinjection (survival rate was approximately 55.4%). On day 10 after microinjection, the survival rate of brown planthoppers in the dsNlRBP group was significantly reduced to approximately 20.0%.

[0074] 3. Detection of the silencing effect of the NlRBP gene in brown planthopper

[0075] On day 10 of the NlRBP-dsRNA feeding experiment, four brown planthoppers were collected from each group, with three biological replicates. Total RNA was extracted from the brown planthoppers and reverse transcribed into cDNA. The silencing effect of NlRBP gene after NlRBP-dsRNA feeding was detected using qRT-PCR. Figure 5 As shown, compared with the dsGFP group, continuous alternating feeding of dsNlRBP on day 10 significantly inhibited the expression of the NlRBP gene (inhibition rate of approximately 48.2%). Figure 5 In the text, different letters represent significant differences ( P <0.05). During the 10-day in vivo microinjection of NlRBP-dsRNA experiment, brown planthoppers were sampled every 48 hours to detect the gene silencing effect. Four planthoppers were collected from both the control and experimental groups, with three biological replicates. Figure 6 As shown, compared with the dsGFP group, in vivo microinjection of dsNlRBP significantly inhibited NlRBP gene expression from day 2 (inhibition rate of approximately 81.6%), and the inhibitory effect lasted until day 10 post-injection. Figure 6 In the text, different letters represent significant differences ( P<0.05). In this embodiment, β-actin was used as an internal reference gene to correct differences in sample cDNA. The primer sequences used are shown in SEQ ID NO.12, SEQ ID NO.13, SEQ ID NO.14, and SEQ ID NO.15, respectively.

[0076] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims.

Claims

1. A suppressor of the Nilaparvata lugens RNA-binding protein NlRBP gene, characterized by, The inhibitor of the NlRBP gene is a dsRNA capable of inhibiting the expression of the NlRBP gene of the brown planthopper, and the nucleotide sequence of the dsRNA is shown as SEQ ID NO.

3.

2. The suppressor of claim 1, wherein, The full-length cDNA sequence of the RNA binding protein NlRBP gene of the brown planthopper is shown as SEQ ID NO.

1.

3. The suppressor of claim 1, wherein, The amino acid sequence of the protein encoded by the RNA binding protein NlRBP gene of the brown planthopper is shown as SEQ ID NO.

2.

4. The use of the inhibitor of the RNA binding protein NlRBP gene of the brown planthopper according to claim 1 in the control of the brown planthopper.

5. Use according to claim 4, characterized in that, The control of the brown planthopper is to inhibit the survival rate of the brown planthopper.

6. Use according to claim 5, characterized in that, The inhibition of the survival rate of the brown planthopper is achieved by inhibiting the expression of the RNA binding protein NlRBP gene of the brown planthopper.

7. Use of the dsRNA according to claim 1 for the manufacture of a product, characterized in that, The product is an insecticide for the control of the brown planthopper.

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