DsRNA targeting saSMCT1 gene of sitobion avenae and application thereof in preventing and treating sitobion avenae

Abstract

This invention belongs to the fields of biotechnology and biopesticides, and specifically relates to a gene targeting the wheat aphid. SaSMCT1 This invention describes the in vitro synthesis of dsRNA and its application in controlling wheat aphids. SaSMCT1 Indoor bioassays were conducted, and ds were found SaSMCT1 Compared with the control, the aqueous solution treatment showed that the larvae of the wheat aphid began to die rapidly on the second day, and the population reduction rate reached 57.56±5.45% after 7 days, with a corrected control efficacy of 52.39±7.36%; ds SaSMCT1 Evenly spraying the entire stem and leaves of wheat during the waxy ripening stage can effectively silence the spores. SaSMCT1 Genes, 3 days after drug administration, experimental drug treatment group ds SaSMCT1 SaSMCT1 The control efficacy was 82.79%, while the control efficacy of the control agent was 69.2%. This application provides a new target and technical solution for the green and efficient control of wheat aphid, which has good application prospects and is of great significance for the green control of wheat aphid.

Description

Technical Field

[0001] This invention belongs to the fields of biotechnology and biopesticides, and specifically relates to the application of dsRNA in the control of wheat aphid. Background Technology

[0002] RNA interference (RNAi) was discovered by Fire et al. in 1998. Their research demonstrated that double-stranded RNA (dsRNA) can silence the expression of target genes, and they were awarded the Nobel Prize in 2006. Since then, RNAi technology has rapidly developed into an important tool for gene function research, especially in plants and animals where genetic manipulation tools are still underdeveloped. In addition, it can also serve as a valuable tool for targeted pesticide and pharmaceutical development.

[0003] In the field of agricultural pest and disease control, two studies in 2002 found that silencing specific target genes could lead to abnormal insect development, embryonic malformation, and even death. This marked the beginning of the application of RNAi technology in entomological research and application. A 2007 study confirmed that expressing insect dsRNA in transgenic plants could achieve insecticidal effects, providing strong evidence for the application of RNAi technology in pest control. RNA biopesticides (or nucleic acid pesticides, RNA pesticides, or RNA interference agents) developed based on this technology have polynucleotides as their core component, which can specifically bind to the mRNA transcribed from the target gene in the target organism. RNA biopesticides synthesize specific dsRNA fragments of the target gene in vitro, introduce them into the target species, and inhibit the expression of the target gene, thereby hindering gene function and ultimately affecting the growth and development of the target species, even leading to death. RNA biopesticides possess characteristics such as high target species specificity, convenient target development, and easy degradation, essentially covering all the functional characteristics required for green pesticides. Therefore, they are hailed by the industry as the third revolution in pesticide production history and have attracted significant attention from numerous international pesticide companies. Multinational corporations such as Bayer-Monsanto, Dow AgroSciences, and Syngenta have invested heavily in pesticide research and development in this field, and related products have reportedly already been launched or are about to be launched. For example, Chinese patent publication number CN111328345A discloses several dsRNA molecules that are toxic to hemiptera insects (including stink bugs). Currently, screening for highly efficient and specific insecticidal target genes is a core step in the development of RNA biopesticides.

[0004] Wheat aphid Sitobion avenae[Fabricius] belongs to the order Hemiptera, family Aphididae. It is widely distributed in wheat-producing areas of China, as well as in Asia, Europe, and North America. Its core hosts are wheat, barley, oats, and other cereal crops, but it also damages rice, corn, and sugarcane in southern regions. In the past three years, the annual affected area of ​​wheat aphids in my country has remained stable at around 200 million mu (approximately 13.3 million hectares), making it a major pest of wheat production. Studies have shown a highly significant negative correlation between the number of aphids per 100 plants and the thousand-grain weight during the early to mid-grain-filling stage. For example, when the number of aphids per 100 plants reaches 1000, it may lead to a loss of approximately 3.3% to 3.8% in thousand-grain weight. If not controlled in time, the cumulative losses will increase exponentially, and severe outbreaks can lead to wheat yield reductions of more than 50%, with some severely affected areas even facing the risk of total crop failure. Therefore, the discovery and utilization of novel aphid-resistant target genes, and the cultivation of new aphid-resistant wheat germplasm or the development of biological pesticides through transgenic technology or direct application of dsRNA, are of great significance for ensuring my country's food security and promoting the development of green agriculture.

[0005] Sodium-coupled monocarboxylate transporter 1-like (SMCT1) is composed of... SLC5A8 The gene encodes a transmembrane transport protein belonging to solute carrier family 5. It primarily functions as an electrosensitive sodium ion (Na+) transporter. + ) and chloride ions (Cl - The SLC5A8 is a lactate-dependent cotransporter responsible for the synergistic transport of various monocarboxylate salts (such as L-lactic acid, D-lactic acid, and pyruvate), short-chain fatty acids (such as acetic acid, propionic acid, butyric acid, and valeric acid), and ketone bodies (such as β-D-hydroxybutyric acid and acetoacetic acid) into cells with sodium ions. Its function is tissue-specific: in the kidneys, it acts as a high-affinity lactate transporter, responsible for reabsorbing lactate to maintain stable blood glucose levels; in the thyroid gland, it mediates the transport of iodide ions from thyroid cells to the follicular lumen. Crucially, the human SLC5A8 gene is a tumor suppressor gene that is silenced in approximately 60% of primary colorectal cancers due to promoter methylation. Loss of its function blocks butyrate (an inhibitor of histone deacetylase produced by gut microbiota) from entering colon cells, thereby affecting histone acetylation status and gene expression, which is considered the core mechanism of its tumor suppressor function. Furthermore, this gene is involved in neuronal energy metabolism and nicotinamide biosynthesis, and its dysfunction is also associated with various cancers.

[0006] However, no such technology has been seen in the present invention. SMCT1 While there are reports on the application of this gene in the control of wheat aphids, whether it can serve as an effective target for RNA interference remains a blank. Therefore, exploring whether it can be used as an RNAi target gene is of significant theoretical and practical value for filling the gap in novel green control targets for wheat aphids and promoting the precision application of nucleic acid pesticides. Summary of the Invention

[0007] To address the aforementioned technical problems, this invention proposes a target gene for controlling wheat aphids. SaSMCT1 dsRNA and its application in the control of wheat aphid.

[0008] The technical solution of this invention is implemented as follows:

[0009] On the one hand, this invention provides a target gene that is lethal to the wheat aphid based on RNA interference technology. SaSMCT1 .

[0010] Secondly, this invention also provides a gene targeting the wheat aphid. SaSMCT1 The dsRNA, comprising the nucleotide sequence shown in SEQ ID No. 2 and its reverse complementary nucleotide sequence, is the *Aphidus longiflora* gene. SaSMCT1 The nucleotide sequence is shown in SEQ ID NO:1.

[0011] Thirdly, this invention applies to protect a biological pesticide formulation, wherein the active ingredient of the biological pesticide formulation is the aforementioned dsRNA.

[0012] Preferably, the concentration of dsRNA in the above preparation is 50~1000 μg / mL.

[0013] The aforementioned biological pesticide formulations may also contain the dsRNA protectant described in the invention patent "dsRNA Protectant and its Preparation Method" with application number 202610136781.X, to protect against dsRNA contamination. SaSMCT1 Protect and prepare the formula, and control wheat aphids.

[0014] Fourthly, the present invention also seeks to protect a primer pair for in vitro synthesis of the above-mentioned dsRNA, the primer pair comprising a forward primer with the sequence shown in SEQ ID No. 3 and a reverse primer with the sequence shown in SEQ ID No. 4.

[0015] Fifthly, this invention applies to protect the use of the above-mentioned dsRNA and the above-mentioned biological pesticide preparation in the control of wheat aphid.

[0016] Using ds SaSMCT1After spraying the aqueous solution of the aphid, the larvae of the aphid began to die rapidly on the second day. The population reduction rate reached 57.56±5.45% after 7 days, and the corrected control efficacy was 52.39±7.36%. Using the protectant in the patent "dsRNA protectant and its preparation method" (patent application number: 202610136781.X) to protect the dsRNA, the control effect reached 82.79% after 3 days, indicating that the target gene has a good control effect on wheat aphids.

[0017] Sixthly, this invention claims a method for controlling wheat aphids, which involves spraying wheat plants with the aforementioned biological pesticide formulation.

[0018] Preferably, the concentration of dsRNA in the above reagent is 50~1000 μg / mL, and the dosage is 1.0 g / mu.

[0019] Preferably, the above-mentioned spraying treatment involves uniformly spraying the entire stem and leaves of the wheat plant, which is in the waxy maturity stage of its growth.

[0020] Preferably, the pressure of the spray is 0.15-0.4 MPa and the flow rate is ≥0.5 L / min.

[0021] The present invention has the following beneficial effects:

[0022] This invention provides a target gene that is lethal to the wheat aphid based on RNA interference technology. SaSMCT1 This invention uses genes SaSMCT1 Specific dsRNAs were designed and obtained as targets. Indoor bioassays showed that, compared to the control, dsRNAs showed superior performance in dsRNAs. SaSMCT1 After treatment, the larvae of the wheat aphid began to die rapidly on the second day, and the population reduction rate reached 57.56±5.45% after 7 days, with a corrected control efficacy of 52.39±7.36%. A biopesticide formulation was prepared using this dsRNA as the active ingredient. Applying 1.0 g of the dsRNA formulation as a uniform spray to the entire stem and leaf of wheat during the waxy ripening stage effectively silenced the wheat aphid. SaSMCT1 Genes, experimental drug group ds SaSMCT1 The control efficacy was 21.36%, while the control efficacy of the chemical agent group was 29.53%; 3 days after application, the control efficacy of the experimental agent group was [data missing]. SaSMCT1 The control efficacy was 82.79%, while the control efficacy of the chemical agent was 69.2%; 5 days after application, the control efficacy of the experimental agent-treated group was [data missing]. SaSMCT1 The control efficacy was 63.67%, while the control efficacy of the chemical agent group was 90.94%, indicating that the target gene has a good control effect on wheat aphid. This application provides a novel target and technical solution for the green and efficient control of wheat aphid, with good application prospects and great significance for the green control of wheat aphid. Attached Figure Description

[0023] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0024] Figure 1 For spraying ds SaSMCT1 The population decline rate of wheat aphid within 7 days.

[0025] Figure 2 For spraying ds SaSMCT1 The control effect of wheat aphid within 7 days. Detailed Implementation

[0026] The technical solution 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.

[0027] Unless otherwise specified, the experimental methods used in the following experimental examples are conventional methods; the materials and reagents used are commercially available unless otherwise specified.

[0028] The efficacy calculation method used in this invention:

[0029] Insect population reduction rate (%) = 100 × (number of live insects before treatment - number of live insects after treatment) / number of live insects before treatment;

[0030] Corrected control efficacy (%) = 100 × (population decline rate in the treatment area - population decline rate in the control group) / (100 - population decline rate in the control group).

[0031] Statistical analysis methods used in this invention:

[0032] The Owo-way ANOVA function in IBM SPSS Statistical software (v. 26.0) was used for analysis, including LSD, SNK, Tuki, Tuki Sb, and Duncan tests (with Duncan analysis as the primary method and the others as auxiliary methods). The Mean ± SEM error display method was used to express differences between groups. The significance of differences between data was analyzed using the T-test. The results were obtained by calculating the mean and the standard deviation (SD) between the data.

[0033] This invention provides a method for treating wheat aphids ( Sitobion avenae The lethal RNAi target gene sodium-coupled monocarboxylate transporter 1-like ( SMCT1 The study also covers RNA biopesticides formulated using the double-stranded RNA of this gene and their control effects on wheat aphids, with the specific effects as follows:

[0034] Example: Acquisition of target gene sequences and dsRNA synthesis

[0035] 1. Extraction of total RNA from the wheat aphid

[0036] Using the wheat aphid as material, the Trizol method was used for extraction, and the samples were purified using conventional methods and treated with DNase to obtain total RNA samples with a concentration ≥300 ng / μL, a total amount ≥6 μg, and an OD260 / 280 of 1.8~2.2.

[0037] 2. Isolation of mRNA and synthesis of cDNA

[0038] mRNA containing polyA was isolated using magnetic beads with oligo-dT, and then the first strand of cDNA was synthesized using random 6-mers and Invitrogen's Superscript II reverse transcriptase kit.

[0039] 3. Gene amplification and sequencing

[0040] Using Table 1 SaSMCT1 Gene-specific primers were used for amplification. The obtained gene fragment was purified, ligated into the PMD-18 vector (manufactured by Takara), transformed into Top10 strains, screened using blue-white screening, and sequenced for positive strains. Primers for amplifying this target gene are shown in Table 1.

[0041] 4. dsRNA synthesis

[0042] The dsRNA was synthesized using the UltraClean T7 RNA Transcription Kit (SJ001B) from Shanghai Zhisheng Yougu Biotechnology Co., Ltd. See the kit for details.

[0043] Table 1 Primer sequences involved in this patent

[0044]

[0045] Table 2 Gene sequences involved in this patent

[0046]

[0047]

[0048] Note: The underlined sequences are the primer sequences used to design dsRNA.

[0049] This invention provides a target gene that is lethal to the wheat aphid based on RNA interference technology. SaSMCT1 The primer sequences for amplifying the gene and synthesizing its dsRNA are shown in Table 1. The full-length cDNA sequence and the synthesized dsRNA sequence of the gene are shown in Table 2.

[0050] Example 1: Indoor pest control effect test

[0051] This invention will synthesize the good SaSMCT1 The dsRNA of the gene was directly dissolved in water to make a water-soluble RNA biopesticide, and then the control effect of this preparation on wheat aphid was tested.

[0052] 1. Selection of test subjects, crops and varieties

[0053] After the wheat has grown to the point of having two leaves, it is moved to an insect cage at 23±1℃ and 50±10% humidity to infect and feed the wheat aphid population.

[0054] 2. Experimental Methods

[0055] The experiment was conducted with a final concentration of 1000 μg per leaf. Each experiment with one agent used 20 test insects as a biological replicate, and there were a total of 3 biological replicates for one agent. Data (number of survivors, number of dead insects, and abnormal phenotypes and their quantities) were recorded in detail for each group from 1 to 7 days after the experiment.

[0056] The control effect of RNA biopesticides on wheat aphids:

[0057] Indoor bioassay results showed that, compared with the control, in ds SaSMCT1 After treatment, the larvae of the wheat aphid began to die rapidly on the second day, and the population reduction rate reached 57.56±5.45% after 7 days, with a corrected control efficacy of 52.39±7.36%. Figure 1 , Figure 2 ).

[0058] Example 2 of implementation results: Field control effect test

[0059] 1. Selection of test subjects, crops and varieties

[0060] Experimental subjects: Aphids that use wheat as a host, with the dominant species being the wheat aphid (Aphis leptospira). Sitobion avenae ).

[0061] Crop: Wheat, variety "Yangmai" series.

[0062] 2. Environmental or cultivation conditions

[0063] The experiment was conducted at the Shanghai Nongmao Grain Professional Cooperative in Yuejin Village, Pujiang Town, Minhang District. The previous crop was rice. The wheat was machine-sown on November 17, 2025. At the time of the experiment, the wheat was in the waxy maturity stage and growing well. The wheat aphid infestation in the field was relatively severe. When the pesticide was applied, the aphid damage was about 800 aphids per 100 plants, and the aphids were present at all ages.

[0064] 3. The medications used are shown in Table 3:

[0065] Table 3. Details of pesticides used in field experiments

[0066]

[0067] 4. Community Arrangements

[0068] The experiment consisted of three treatment plots with no replicates, arranged in a randomized block design. Each plot had an area of ​​135 square meters, for a total of 405 square meters.

[0069] 5. Application method

[0070] The entire plant and its foliage were sprayed evenly using a backpack electric sprayer (model 3WBD-20, Taizhou Xieyou, China) for different treatment areas. The pressure was 0.15-0.4 MPa and the flow rate was ≥0.5 L / min. The water consumption per area was 20 L.

[0071] 6. Survey time and number of times

[0072] Before applying the pesticide, the initial population of aphids was investigated. After applying the pesticide, the number of live aphids was investigated 1, 3, and 5 days (May 19, 21, and 23).

[0073] 7. Survey Methods

[0074] A five-point sampling method was used, with 20 ears marked at each point and 100 ears surveyed in each plot. The total number of live aphids was recorded and compared with the control area to calculate the control efficacy.

[0075] Table 4. Results of field efficacy trials of different pesticides for controlling wheat aphids (May 2026)

[0076]

[0077] Subsequently, we applied the dsRNA protectant from the patent "dsRNA Protectant and Preparation Method Thereof" (patent application number: 202610136781.X) to dsRNA. SaSMCT1Protection and formulation were carried out for the control of wheat aphids. The experimental results are shown in Table 4. One day after application, the ds of the experimental pesticide group... SaSMCT1 The control efficacy was 21.36%; the control efficacy of the chemical agent group was 29.53%. Three days after application, the control efficacy of the experimental agent-treated group was [data missing]. SaSMCT1 The control efficacy was 82.79%; the control efficacy of the chemical agent was 69.2%. Five days after application, the control group treated with the experimental agent showed [data missing]. SaSMCT1 The control efficacy was 63.67%; the control efficacy of the chemical agent group was 90.94%. This indicates that the target gene's ds SaSMCT1 It has a good control effect on wheat aphid.

[0078] 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 gene targeting the wheat aphid SaSMCT1 The dsRNA is characterized by: The dsRNA comprises a nucleotide sequence as shown in SEQ ID No. 2 and a nucleotide sequence that is inversely complementary to it.

2. The gene targeting the wheat aphid as described in claim 1 SaSMCT1 The dsRNA is characterized by: The wheat aphid gene SaSMCT1 The nucleotide sequence is shown in SEQ ID NO:

1.

3. A biological pesticide formulation, characterized in that: The active ingredient of the biological pesticide formulation is the dsRNA described in claim 2.

4. The biological pesticide formulation according to claim 3, characterized in that: The concentration of dsRNA in the formulation is 50-1000 μg / mL.

5. Primer pair for in vitro synthesis of the dsRNA of claim 2, characterized in that: The primer pair includes a forward primer with a sequence as shown in SEQ ID No. 3 and a reverse primer with a sequence as shown in SEQ ID No.

4.

6. The application of the dsRNA according to claim 1 or 2 and the biological pesticide formulation according to claim 3 or 4 in the control of wheat aphid.

7. A method for controlling wheat aphids, characterized in that: The biological pesticide formulation described in claim 4 is used to spray wheat plants.

8. The method for controlling wheat aphids according to claim 7, characterized in that: The concentration of dsRNA in the reagent is 50-1000 μg / mL, and the dosage is 1.0 g / mu.

9. The method for controlling wheat aphids according to claim 8, characterized in that: The spraying treatment involves uniformly spraying the entire stem and leaves of the wheat plant, which is in the waxy maturity stage of its growth.

10. The method for controlling wheat aphids according to claim 9, characterized in that: The spray pressure is 0.15-0.4 MPa and the flow rate is ≥0.5 L / min.

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