RNAi target gene for efficiently preventing and controlling wheat pests, and screening method and application thereof

Abstract

This invention discloses an RNAi target gene for highly efficient control of wheat pests, its screening method, and its application, belonging to the field of agricultural biotechnology. Firstly, this invention provides a method for screening RNAi target genes. By selecting aromatic amino acid hydroxylase-related genes involved in the dopamine synthesis pathway in the wheat aphid as candidate targets, dsRNA is synthesized and delivered using a wheat seedling soaking method. The expression level of the target gene and the aphid mortality rate are detected to screen for highly efficient target genes. The dopamine decarboxylase gene of the wheat aphid was obtained through this method. SmDDC As an RNAi target, this study further provides dsRNA molecules that specifically silence this target, RNAi agents, and their application methods. Experiments have shown that the wheat aphid feeds via... SmDDC After wheat seedlings were treated with dsRNA, the expression of the target gene was significantly suppressed, and the mortality rate of aphids was significantly increased. This invention provides core targets and technical support for the development of green and efficient nucleic acid pesticides, and has significant agricultural application value.

Description

Technical Field

[0001] This invention belongs to the field of agricultural biotechnology and pest control, specifically relating to an RNAi target gene for the efficient control of wheat pests, its screening method, and its application. Background Technology

[0002] Wheat aphids (especially the wheat long-tubed aphid) are major pests affecting wheat production. Long-term reliance on chemical pesticides for control has led to problems such as increased pesticide resistance, the elimination of natural enemies, pesticide residues, and environmental pollution. There is an urgent need to develop green and efficient new control technologies. RNA interference (RNAi), as a novel biological control technology, can inhibit the growth and development of pests by specifically silencing key genes, and is hailed as the "third pesticide revolution." RNAi-based nucleic acid pesticides were selected as one of the top ten scientific breakthroughs in *Science* magazine in 2024. However, in the control of wheat aphids, there is still a lack of highly efficient and specific target genes and a simple and reliable delivery system.

[0003] This invention addresses the aforementioned technical bottlenecks by using the wheat aphid (Aphis hymenoptera) Sitobion miscanthi Taking key enzyme genes involved in the dopamine synthesis pathway in vivo as a starting point, we screened and verified the dopa decarboxylase gene. SmDDC The feasibility of using this as an RNAi target. This involves synthesizing the target in vitro. SmDDC The dsRNA was delivered using the wheat seedling soaking method, achieving efficient silencing of the target gene and significantly improving the mortality rate of aphids. This provides a core target and technical support for the development of aphid nucleic acid pesticides based on RNAi technology. Summary of the Invention

[0004] This invention aims to provide a method for screening RNAi target genes for the efficient control of wheat pests, to screen out key target genes that can be used to control wheat aphid, and to provide dsRNA molecules, RNAi preparations and their application methods based on these targets.

[0005] The technical solution of the present invention to solve the above-mentioned technical problems is as follows: This invention provides a method for screening RNAi target genes for efficient control of wheat pests, comprising the following steps: S1: From the wheat aphid ( Sitobion miscanthi Genes related to aromatic amino acid hydroxylases involved in the dopamine synthesis pathway were selected as candidate targets. S2: Synthesize the dsRNA of the candidate target gene; S3: dsRNA was delivered to the wheat aphid using the wheat seedling soaking method; S4: Detect the expression levels of candidate target genes in aphids and the mortality rate of aphids; The screening criteria were: genes that could significantly inhibit the expression of candidate target genes (p<0.05) and significantly increase the mortality rate of aphids were identified as RNAi target genes for the efficient control of wheat pests.

[0006] Preferably, the candidate target gene is the wheat aphid dopa decarboxylase gene. SmDDC Its nucleotide sequence is shown in SEQ ID NO:1.

[0007] Secondly, the present invention provides the application of RNAi target genes obtained by the above screening method in the control of wheat pests.

[0008] Thirdly, this invention provides an RNAi target gene for controlling the wheat pest, the wheat aphid, wherein the target gene is the wheat aphid dopa decarboxylase gene. SmDDC Its nucleotide sequence is shown in SEQ ID NO:1.

[0009] Fourthly, the present invention provides a dsRNA molecule for specifically silencing the target gene, wherein the targeting fragment of the dsRNA molecule is selected from the nucleotide sequence shown in SEQ ID NO:2.

[0010] Furthermore, the dsRNA molecule is synthesized via in vitro transcription using a primer pair containing a T7 promoter, the primer pair sequence being: Upstream primer: GATCACTAATACGACTCACTATAGGGCCGATTGAAGGGCAACAACG Downstream primer: GATCACTAATACGACTCACTATAGGGGACTTGACCACGATGGGGTGT.

[0011] Fifthly, the present invention provides the application of the above-mentioned dsRNA molecule in the preparation of RNAi formulations for the control of wheat aphid.

[0012] In a sixth aspect, the present invention provides an RNAi formulation for controlling wheat aphids, comprising an effective amount of the aforementioned dsRNA molecules and an agriculturally acceptable vector. Preferably, the concentration of the dsRNA molecules in the formulation is 500 ng / μL.

[0013] In a seventh aspect, the present invention provides a method for controlling wheat aphids, wherein the wheat aphids feed on wheat seedlings treated with the aforementioned RNAi preparation. Preferably, the treatment involves soaking the wheat seedlings in the RNAi preparation, drying them, and then feeding them to the wheat aphids; the wheat aphids are fourth-instar nymphs, and the feeding treatment time is 48 hours.

[0014] Eighthly, the present invention provides the application of the above-mentioned target gene, dsRNA molecule or RNAi preparation in the preparation of nucleic acid pesticides for the control of wheat aphids.

[0015] The present invention has the following beneficial effects: The RNAi target gene screening method provided by this invention can efficiently screen key genes that can significantly silence target gene expression and lead to a significant increase in aphid mortality from aromatic amino acid hydroxylase-related genes involved in the dopamine synthesis pathway in the wheat aphid. The dopamine decarboxylase gene obtained by this screening method is... SmDDC It exhibits high specificity as an RNAi target. Experiments have shown that delivering dsSmDDC via wheat seedling soaking significantly inhibits the expression of the target gene in aphids, while the aphid mortality rate is significantly higher than that of the control group.

[0016] This invention exhibits strong targeting and safety for non-target organisms; it is simple to operate, requiring no complex equipment; it leaves no chemical pesticide residues, making it environmentally friendly; and it can effectively reduce aphid populations and delay the development of pesticide resistance. This invention provides a core target and reliable technical support for the development of RNAi-based nucleic acid pesticides, and has broad application prospects in the field of green pest control in agriculture. Attached Figure Description

[0017] Figure 1 Effect of silencing the dopa decarboxylase gene on gene expression levels in the wheat aphid (qPCR results).

[0018] Figure 2 The effect of silencing the dopa decarboxylase gene on the mortality rate of wheat aphid (bioassay results). Detailed Implementation

[0019] The principles and features of the present invention are described below with reference to the accompanying drawings. The examples given are only for explaining the present invention and are not intended to limit the scope of the present invention.

[0020] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be described in detail below with reference to specific embodiments. The following embodiments are for illustrative purposes only and should not be considered as limitations on this invention. Experimental methods not specifying specific conditions in the embodiments are generally performed under conventional conditions or according to the conditions recommended by the reagent manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0021] From the wheat aphid ( Sitobion miscanthi Genes related to aromatic amino acid hydroxylases involved in the dopamine synthesis pathway were screened from transcriptome databases. After sequence alignment and functional annotation, the dopamine decarboxylase gene (named...) was obtained. SmDDCThe nucleotide sequence of the coding region of this gene is shown in SEQ ID NO:1. Dopa decarboxylase is a key rate-limiting enzyme in the dopamine synthesis pathway, widely involved in physiological processes such as insect epidermal formation, neural regulation, immune defense, and reproductive development. It is highly conserved in aphids and has little impact on non-target organisms, therefore it was selected as a candidate target gene for RNAi.

[0022] Primer design and PCR amplification: Based on SmDDC Gene sequences were analyzed, and T7 promoter sequences (GATCACTAATACGACTCACTATAGGG) were added to both ends of the target fragment. Specific primers were designed accordingly. Green fluorescent protein (GFP) genes were used as negative controls, and corresponding T7 primers were designed. Primer sequences are as follows: T7-dsSmDDC-F GATCACTAATACGACTCACTATAGGGCCGATTGAAGGGCAACAACG T7-dsSmDDC-R GATCACTAATACGACTCACTATAGGGGACTTGACCACGATGGGTGT T7-GFP-F GATCACTAATACGACTCACTATAGGGGGATCCTAATACGACTCACT T7-GFP-R GATCACTAATACGACTCACTATAGGGTGGATCCTAATACGACTCAC Using cDNA from the wheat aphid as a template, PCR amplification was performed using the primers described above to obtain the target fragment containing the T7 promoter. The PCR product was verified to be of the correct size by 1.5% agarose gel electrophoresis and then used for subsequent purification.

[0023] PCR product purification: PCR products were purified using the phenol / chloroform / isoamyl alcohol extraction method: 50 μL of PCR product was added, and ddH2O was added to a final volume of 100 μL. An equal volume of DNA extraction buffer (phenol / chloroform / isoamyl alcohol = 25:24:1) was added, and the mixture was thoroughly vortexed and centrifuged at 10,000 rpm for 10 min at 4 °C. The upper aqueous phase was transferred to a new centrifuge tube, and 1 / 10 volume of 3M sodium acetate (pH 5.2) and 2 volumes of anhydrous ethanol were added. The mixture was gently mixed and incubated at -20 °C for at least 30 min to precipitate the DNA. Subsequently, the mixture was centrifuged at 12,000 rpm for 10 min at 4 °C, the supernatant was discarded, and the precipitate was washed with 1 mL of pre-chilled 75% ethanol. The precipitate was centrifuged at 10,000 rpm for 2 min, the ethanol was discarded, and the mixture was air-dried at room temperature. An appropriate amount of ddH2O was added to dissolve the precipitate, and the concentration was determined for later use.

[0024] dsRNA in vitro synthesis: The procedure was performed according to the T7 RNAi transcription kit (Nanjing Novizan Biotechnology). The purified PCR product and T7 enzyme mixture were added to a 20 μL transcription reaction system and incubated overnight at 37°C. After the reaction, 160 μL of RNase-free H2O and 20 μL of 3M sodium acetate (pH 5.2) were added to 40 μL of the final reaction solution and mixed thoroughly. An equal volume of RNA extraction buffer (phenol / chloroform mixture, 1:1) was added for extraction once, followed by two extractions with an equal volume of chloroform, collecting the supernatant phase each time. Two volumes of anhydrous ethanol were added, and the mixture was precipitated at -20°C for at least 30 min. The RNA precipitate was collected by centrifugation at 12000 rpm for 10 min at 4°C. The precipitate was washed once with 500 μL of pre-chilled 75% ethanol, centrifuged to discard the ethanol, and air-dried. 40 μL of RNase-free H2O was added to dissolve the precipitate.

[0025] dsRNA quality assessment: dsRNA concentration was measured using a nanospectrophotometer. Simultaneously, a suitable sample was subjected to 1.5% agarose gel electrophoresis to verify that the dsRNA bands were uniform in size and showed no significant degradation. Qualified samples were aliquoted and stored at -80℃ for later use. GFP gene dsRNA was used as a negative control for subsequent experiments.

[0026] Aphid rearing: The tested wheat aphids were obtained from a non-toxic population that had been reared in our laboratory for a long time, and were subcultured in an artificial climate chamber using infected wheat seedlings. Culture conditions: temperature 24±1℃, relative humidity 60%±5%, photoperiod 16L:8D. Healthy 5-day-old (approximately fourth instar) nymphs of uniform development were selected for the experiment.

[0027] dsRNA treatment of wheat seedlings: The roots of fresh wheat seedlings (approximately 8-10 cm tall) were immersed in 1.5 mL centrifuge tubes containing 500 ng / μL dsSmDDC solution (with dsGFP as a negative control) for 3 h. After immersion, the seedlings were removed and air-dried at room temperature. Then, the seedlings were placed in 1.5 mL centrifuge tubes with sterile, enzyme-free water added.

[0028] Aphid feeding and mortality statistics: Each treatment had 3 biological replicates, with 20 fourth-instar nymphs inoculated into each replicate. The replicates were sealed with plastic wrap but with holes for ventilation and placed under the same climatic conditions as described above. Aphid survival was observed 48 hours after treatment; death was defined as the absence of noticeable spontaneous movement when lightly touched with a brush. The number of dead aphids in each treatment group was recorded, and the corrected mortality rate was calculated.

[0029] Forty-eight hours after feeding, surviving aphids were collected from each treatment group (at least five per replicate), immediately flash-frozen in liquid nitrogen, and stored at -80°C for later use.

[0030] Total RNA was extracted from aphids using the TaKaRa MiniBEST Universal RNA Extraction Kit (TaKaRa, Dalian). RNA integrity and concentration were assessed by agarose gel electrophoresis and nanospectrophotometer. Reverse transcription was performed using the PrimeScript™ 1st Strand cDN ASynthesis Kit (TaKaRa, Dalian) with total RNA as a template and OligodT as primers, following the kit instructions.

[0031] Using cDNA obtained from reverse transcription as a template, qRT-PCR was performed using the SYBR Green method. SmDDC Relative gene expression levels. The housekeeping gene of wheat aphid (EF1α) was used as an internal control, with three technical replicates for each sample. The reaction was performed on a real-time quantitative PCR instrument, with the following amplification program: 95℃ pre-denaturation for 30 s; 95℃ denaturation for 5 s; 60℃ annealing and extension for 30 s, for a total of 40 cycles; finally, melting curve analysis was performed.

[0032] Using 2^ −ΔΔCt The relative gene expression levels were calculated using a method. The results showed ( Figure 1 Compared with the dsGFP control group, wheat aphids that ingested dsSmDDC had significantly higher levels of [something]. SmDDC Gene expression was significantly suppressed (p<0.01), indicating that the dsRNA synthesized in this invention can be effectively delivered through wheat seedlings and produce a specific RNA interference effect in aphids.

[0033] Bioassay data shows that ( Figure 2 The mortality rate of aphids feeding on wheat seedlings treated with dsSmDDC reached 46.8% within 48 hours. Statistical analysis showed that the mortality rate of the dsSmDDC treatment group was significantly higher than that of the control group (p<0.01).

[0034] The above results confirm that the dopa decarboxylase gene of the wheat aphid is silenced. SmDDC It can significantly inhibit the expression of target genes and cause mass mortality in aphids. Therefore, SmDDC Genes can serve as key RNAi targets for the efficient control of wheat aphids, and have significant application potential in the development of RNAi-based nucleic acid pesticides and the realization of green and long-term control of aphid populations.

[0035] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. 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. A method for screening RNAi target genes for efficient control of wheat pests, characterized in that, include: S1: Genes related to aromatic amino acid hydroxylases involved in the dopamine synthesis pathway were selected from wheat aphid as candidate targets. S2: Synthesize the dsRNA of the candidate target gene; S3: dsRNA was delivered to the wheat aphid using the wheat seedling soaking method; S4: Detect the expression levels of candidate target genes in aphids and the mortality rate of aphids; The screening criteria were: genes that could significantly inhibit the expression of candidate target genes (p<0.05) and significantly increase the mortality rate of aphids were identified as RNAi target genes for the efficient control of wheat pests.

2. The screening method according to claim 1, characterized in that, The candidate target gene is the wheat aphid dopa decarboxylase gene. SmDDC Its nucleotide sequence is shown in SEQ ID NO:

1.

3. The application of RNAi target genes screened by the screening method according to claim 1 or 2 in the control of wheat pests.

4. An RNAi target gene for controlling the wheat aphid, a wheat pest, characterized in that, The target gene is the dopa decarboxylase gene of the wheat long-tube aphid. SmDDC Its nucleotide sequence is shown in SEQ ID NO:

1.

5. A dsRNA molecule for specifically silencing the target gene of claim 4, characterized in that, The target fragment of the dsRNA molecule is selected from the nucleotide sequence shown in SEQ ID NO:

2.

6. The dsRNA molecule according to claim 5, characterized in that, The dsRNA molecule was synthesized via in vitro transcription using a primer pair containing a T7 promoter. The primer pair sequence is as follows: Upstream primer: GATCACTAATACGACTCACTATAGGGCCGATTGAAGGGCAACAACG; Downstream primer: GATCACTAATACGACTCACTATAGGGGACTTGACCACGATGGGGTGT.

7. The use of the dsRNA molecule of claim 5 or 6 in the preparation of an RNAi formulation for the control of wheat aphid.

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