A dsRNA targeting the lethal gene Phospholipase B1 in the planthopper and its application

Abstract

This invention discloses a dsRNA targeting the lethal gene PLB1 of the planthopper and its application, belonging to the field of agricultural biotechnology. The dsRNA targeting the PLB1 gene described in this invention refers to a dsRNA synthesized based on the nucleic acid sequence of the planthopper PLB1 gene as shown in SEQ ID No. 1. The sequence of the dsRNA targeting the PLB1 gene is shown in SEQ ID No. 6. Treatment of planthoppers with the dsRNA targeting the PLB1 gene can significantly downregulate the expression of the PLB1 gene, effectively inducing planthopper mortality and thus reducing its damage to rice and other plants. This invention provides a new target for the development of RNAi-based planthopper control technology.

Description

Technical Field

[0001] This invention belongs to the field of agricultural biotechnology, specifically relating to a dsRNA targeting the lethal gene phospholipase B1 (PLB1) in the planthopper and its application. Background Technology

[0002] Grey planthopper ( Laodelphax striatellus The rice planthopper (Gynostemma pentaphyllum) is a major agricultural pest, primarily damaging grain crops such as rice, wheat, and corn. It feeds directly on plant sap using its piercing-sucking mouthparts, causing stunted growth, reduced tillering, and yellowing leaves. When plant populations are high, affected plants rapidly dehydrate and die, leading to a condition known as "planthopper burn." Furthermore, the planthopper transmits various plant viral diseases, including rice stripe leaf blight, rice black-streaked dwarf virus, and corn rough dwarf virus, resulting in severe yield losses.

[0003] Chemical control is an important means of controlling planthoppers. However, long-term use of chemical pesticides has led to high levels of resistance in planthoppers to many commonly used agents, significantly reducing the effectiveness of field control. Farmers have had to increase the frequency and concentration of pesticide applications, further exacerbating the development of resistance and the risk of environmental pollution. In recent years, novel biological control strategies based on RNA interference have provided new ideas for planthopper control. Existing research has identified some lethal genes that can cause mortality in planthoppers, such as trehalase, Ribosomal protein L19, and ATPase, but the number of currently available highly effective lethal target genes is still limited, and there is an urgent need to discover new target genes with significant lethal effects. To this end, this invention provides a dsRNA targeting the lethal gene PLB1 in planthoppers. Applying this dsRNA to planthoppers can produce a significant lethal effect, providing a new target for the development of RNAi-based planthopper control technology. Summary of the Invention

[0004] In response to the above research background, this invention provides a dsRNA targeting the lethal gene PLB1 of the planthopper and its application.

[0005] The main steps of PLB1 dsRNA synthesis include: (1) Primer design. Primers are designed based on the PLB1 sequence of the planthopper as shown in SEQ ID No. 1, and a T7 promoter sequence for dsRNA synthesis is added before the primer sequence to obtain upstream and downstream primers, which are then sent to a primer synthesis company for synthesis. (2) PLB1 gene fragment amplification. PLB1 gene fragments of 200-600 bp in length are obtained by PCR amplification using planthopper cDNA as a template and sequenced for confirmation. (3) dsRNA synthesis. The PCR product is purified and dsRNA targeting the PLB1 gene is synthesized using an in vitro dsRNA synthesis kit.

[0006] Applying the target lethal gene PLB1 dsRNA of the planthopper described in this invention to planthoppers via microinjection or other methods can cause the planthoppers to die, thereby protecting rice and other plants from damage.

[0007] The beneficial effects of this technical solution include: (1) the dsRNA targeting the lethal gene PLB1 of the planthopper can effectively silence the expression of the planthopper PLB1 gene and has a significant lethal effect on the planthopper; (2) after treating planthoppers with the lethal gene PLB1 dsRNA, the damage of planthoppers to rice and other plants can be reduced; (3) the dsRNA of this invention is designed based on the planthopper PLB1 gene and has the advantages of high specificity, high safety and environmental friendliness, which can provide a target for the development of precision control technology for planthoppers. Attached Figure Description

[0008] Figure 1 Changes in PLB1 gene expression in the planthopper after PLB1 dsRNA treatment

[0009] Figure 2 Mortality rate of planthoppers after PLB1 dsRNA treatment

[0010] Figure 3 Symptoms of rice planthopper damage after PLB1 dsRNA treatment Detailed Implementation

[0011] To further illustrate the technical means and beneficial effects of the present invention, the technical solution of the present invention will be further described below by way of embodiments.

[0012] Example 1: Obtaining a gene fragment for synthesizing PLB1 dsRNA from the planthopper. The method for obtaining the PLB1 gene fragment for PLB1 dsRNA synthesis includes the following steps: designing primers for amplifying the PLB1 gene fragment, extracting total RNA from the planthopper, synthesizing cDNA, PCR amplification, and purifying the gene fragment for PLB1 dsRNA synthesis.

[0013] (1) Design primers to amplify the PLB1 gene fragment.

[0014] Based on the nucleic acid sequence of the lethal gene PLB1 from the planthopper shown in SEQ ID No. 1, primers for amplifying gene fragments of 200–600 bp for PLB1 dsRNA synthesis were designed using Primer Primer 6.0 software. The upstream primer dsPLB1-F sequence is shown in SEQ ID No. 2, and the downstream primer dsPLB1-R sequence is shown in SEQ ID No. 3. To further analyze the effectiveness of PLB1 dsRNA, primers for amplifying gene fragments used to synthesize control GFP dsRNA were also designed. The upstream primer dsGFP-F sequence is shown in SEQ ID No. 4, and the downstream primer dsGFP-R sequence is shown in SEQ ID No. 5. The designed primers were then sent to a primer synthesis company for synthesis.

[0015] (2) Extract total RNA from the planthopper.

[0016] Take 10-20 adult or nymphal planthoppers and place them in a 1.5 mL centrifuge tube. Freeze in liquid nitrogen, then grind with a grinding rod. Add 200 µL of Trizol reagent and vortex thoroughly. Add 40 μL of chloroform, vortex vigorously for 15 seconds, and let stand at room temperature for 5 minutes. Centrifuge at 4°C and 12,000 × g for 15 minutes. Carefully aspirate the colorless aqueous supernatant and transfer it to a new RNase-free centrifuge tube. Add an equal volume of pre-chilled isopropanol, gently invert to mix, and let stand at room temperature for 10 minutes. Then, precipitate at -20°C for 30 minutes. Centrifuge at 4°C and 12,000 × g for 10 minutes and discard the supernatant. Wash the precipitate with 1 mL of 75% ethanol prepared with DEPC water, centrifuge at 4°C and 12,000 × g for 5 minutes, and discard the supernatant. Repeat this washing step once. After a brief centrifugation, aspirate the remaining liquid and air-dry the precipitate in a clean bench. Finally, the RNA precipitate was dissolved in 20 μL of RNase-free water, and its concentration and purity were measured before storage at -80℃ for later use.

[0017] (3) Synthesis of cDNA in gray planthoppers.

[0018] Using the extracted total RNA as a template, cDNA from the planthopper was synthesized using a commercial reverse transcription kit. In this example, the ABScript III RT Master Mix for qPCR kit was used for reverse transcription. The reaction mixture was as follows: 1 µg total RNA, 4 µL 5× ABScript III RT Mix, and RNase-free water to a final volume of 20 µL. The following program was run on the PCR instrument: incubation at 55°C for 15 minutes, followed by heating at 85°C for 5 minutes to inactivate the reverse transcriptase. The resulting cDNA was stored at -20°C for subsequent PCR amplification.

[0019] (4) PCR amplification and purification of gene fragments used for PLB1 dsRNA synthesis.

[0020] Using cDNA from the planthopper as a template, PCR was performed with upstream primer dsPLB1-F and downstream primer dsPLB1-R to amplify the PLB1 gene fragment. Simultaneously, using a plasmid containing the GFP gene as a template, PCR was performed with primers dsGFP-F and dsGFP-R to amplify the GFP gene fragment as a control. The 20 µL PCR reaction mixture contained: 10 µL 10×Taq PCR Master Mix, 1 µL cDNA template, 0.5 µL each of upstream and downstream primers, and 8 µL ddH2O. The PCR reaction program was as follows: 95℃ pre-denaturation for 3 minutes; 94℃ denaturation for 30 seconds, 55℃ annealing for 30 seconds, 72℃ extension for 30 seconds, 32 cycles; 72℃ extension for 10 minutes. After amplification, the PCR products were separated by electrophoresis on a 1% agarose gel. Gel blocks containing the target band were excised under UV light and purified using a DNA gel extraction kit. The purified product was sent to a sequencing company for sequencing. After sequencing verification, the PLB1 gene fragment for dsRNA synthesis was obtained, as shown in SEQ ID No. 6.

[0021] Example 2 Synthesis and purification of dsRNA of the lethal gene PLB1 from the planthopper

[0022] (1) Synthesis of dsRNA.

[0023] Using the purified PLB1 or GFP gene fragment from Example 1 as a template, dsRNA was synthesized in vitro using a dsRNA in vitro reverse transcription kit. The synthesis steps were performed according to the kit's instructions. In this example, dsRNA was synthesized using the Thermo Scientific TranscriptAid T7 High Yield Transcription in vitro transcription kit. The specific steps were as follows: 5× TranscriptAid was added to centrifuge tubes...TM Reaction Buffer 4 μL, ATP / CTP / GTP / UTP mix 8 μL, purified PLB1 gene fragment 1 μg, Transcript Aid TM Add 2 μL of EnzymeMix and DEPC-treated water to a final volume of 20 µL. Mix thoroughly, centrifuge briefly, and incubate overnight at 37°C. Add 2 μL of DNase I and mix well. Incubate at 37°C for 15 minutes. Then add 2 μL of 0.5M EDTA (pH 8.0) and incubate at 65°C for 10 minutes to obtain the dsRNA of the lethal gene PLB1 from the planthopper and the GFP dsRNA as a control.

[0024] (2) dsRNA purification.

[0025] Add 115 μL of DEPC-treated water and 15 μL of 3M pH 5.2 sodium acetate solution to 20 μL of dsRNA synthesis medium and mix well. Add an equal volume of chloroform, mix well, and centrifuge at 12,000×g for 15 minutes at 4°C. Transfer the upper aqueous phase to a new DEPC-treated centrifuge tube, add 2 volumes of anhydrous ethanol, mix well, and incubate at -20°C for 30 minutes to precipitate. Then, centrifuge at 12,000 rpm for 10 minutes at 4°C and discard the supernatant. Wash the precipitate once with 1 mL of pre-chilled 70% ethanol, centrifuge, discard the supernatant, and dry at room temperature. Finally, dissolve the precipitate in 20 μL of DEPC-treated water to obtain purified dsRNA.

[0026] Example 3: Injecting the dsRNA of the lethal gene PLB1 into the planthopper

[0027] (1) Take 3-4 instar nymphs of the planthopper, freeze them on ice, and use them for injection after they are in a coma.

[0028] (2) PLB1 dsRNA was mixed with microinjection buffer containing an indicator and added to a glass capillary tube using a micropipette tip. Microinjection was performed using a Nanoject III microinjector, with 30 ng of PLB1 dsRNA injected into each planthopper nymph. 130 ng of GFP dsRNA was injected simultaneously as a control. Each treatment was administered in triplicate, with each replicate containing 30 planthoppers.

[0029] (3) After injecting dsRNA, the planthoppers were placed on healthy rice seedlings for rearing. Three planthoppers treated with PLB1 dsRNA or GFP dsRNA were placed on each rice plant. The number of dead planthoppers was counted daily, and the growth status of the rice was observed after 7 days.

[0030] Three days after injection, the expression level of the PLB1 gene in the planthopper was analyzed by RT-qPCR. The results showed that, compared with the control group injected with GFP dsRNA, the expression level of the PLB1 gene in the planthopper injected with PLB1 dsRNA was significantly reduced, only 43.5% of the control level. Figure 1 This indicates that PLB1 dsRNA treatment can significantly inhibit the expression of the lethal gene PLB1.

[0031] Mortality statistics showed that on day 11 post-injection, the mortality rate of the control GFP dsRNA treatment was 14.6%, while the mortality rate of the PLB1 dsRNA treatment was 77.53%. Figure 2 This indicates that PLB1 dsRNA treatment significantly increased the mortality rate of planthoppers.

[0032] Planthopper nymphs were treated with PLB1 dsRNA and GFP dsRNA, then placed on rice seedlings to feed. After 7 days of feeding, the growth status of the rice plants treated with the two methods was compared. Figure 3 The results showed that rice plants treated with PLB1 dsRNA exhibited significantly better growth than the GFP dsRNA control group. These results indicate that the lethal gene PLB1 dsRNA of the rice planthopper can be used to control the planthopper and thus reduce its damage to rice.

[0033] The specific embodiments described above illustrate the technical means and beneficial effects of the present invention in detail. Those skilled in the art can make modifications or improvements based on the present invention; any modifications or improvements made without departing from the spirit and scope of the present invention are within the scope of protection of the present invention.

Claims

1. A dsRNA targeting the lethal gene PLB1 in the planthopper, characterized in that, The dsRNA targeting the PLB1 gene is a dsRNA synthesized based on the nucleic acid sequence of the PLB1 gene of the planthopper as shown in SEQ ID No.

1.

2. A dsRNA targeting the lethal gene PLB1 in the planthopper, characterized in that, The dsRNA sequence targeting the PLB1 gene is shown in SEQ ID No.

6.

3. The application of the dsRNA targeting the lethal gene PLB1 as described in any one of claims 1 to 2 in the control of planthoppers, characterized in that, PLB1 dsRNA was administered to the planthopper via microinjection and other methods, causing its death.

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