Application of SfCPR1 and SfCPR91 genes and dsRNA in controlling Spodoptera frugiperda

Abstract

The application discloses SfCPR1 and SfCPR91 The application discloses a gene and a dsRNA and application of the gene and the dsRNA in prevention and treatment of Spodoptera exigua. The application obtains 30 candidate cuticle proteins based on phenotype, screens 8 RNA biological pesticides target effectiveness verification through space-time expression characteristics, finally obtains 2 RNA biological pesticides targets which have significant inhibition on the weight of the Spodoptera exigua larvae and the pupation rate, and through a formula of 'dsRNA+chemical pesticide+nanometer delivery', the dsRNA is delivered by using a nanometer material SPc, the toxicity and the prevention and treatment effect of various insecticides are effectively improved. The application provides a technical method for green prevention and treatment and resistance management of the Spodoptera exigua.

Description

Technical Field

[0001] This invention belongs to the field of research and application technology of RNA biopesticides for harmful organisms, specifically involving SfCPR1 and SfCPR91 Application of genes and their dsRNA in the control of fall armyworm. Background Technology

[0002] The fall armyworm, originating from the Americas, is an invasive pest that poses a serious threat to my country's food security. Its migratory and reproductive capabilities are extremely strong, making it very difficult to control. Since 2020, the Ministry of Agriculture and Rural Affairs has consistently included it in the "List of Class A Crop Diseases and Pests." As a major grain crop in my country, effective control of the fall armyworm is crucial for protecting grain production from pests and ensuring a bumper and stable corn yield. However, the fall armyworm has developed significant resistance, and there are few effective pesticides available, leading to a serious threat to corn production in my country. Therefore, there is an urgent need to develop new, green control technologies.

[0003] RNA biopesticides deliver specific double-stranded RNA (dsRNA) to target and silence key genes in pests, interfering with their normal growth and development. They offer numerous advantages, including ease of creation, short development cycles, precise targeting, non-toxicity, no residue, and strong environmental compatibility, and are hailed as the "third revolution in pesticide history." In 2017, Monsanto launched MON87411, a transgenic corn variety expressing dsSnf7, marking the first commercial application of RNA interference (RNAi) technology. In 2023, Calantha™, the world's first sprayable RNA biopesticide, was approved for marketing in the United States, signifying that RNAi-based biopesticides have officially moved from the laboratory to field applications. Lepidoptera insects possess a large number of nucleases that rapidly degrade dsRNA, and their alkaline midgut environment makes the phosphodiester bonds of dsRNA unstable, resulting in low RNAi efficiency in lepidoptera. Studies have confirmed that nanomaterials SPc encapsulating dsRNA can effectively improve gene silencing efficiency in lepidoptera pests. For example, in the Asian corn borer, encapsulating dsRNA with SPc nanomaterials effectively protects dsRNA from nuclease degradation and efficiently delivers it into the insect's body. Feeding the insect with SPc / dsRNA can then efficiently silence the expression of the target gene. Therefore, the cross-integration of RNAi technology and nanotechnology effectively promotes the development of RNA biopesticides for lepidopteran pests based on SIGS technology.

[0004] The creation of RNA biopesticides based on insect epidermal proteins offers the potential for direct pest control through growth- and developmental lethality, or for effective mitigation of resistance by mixing with existing insecticides to increase penetration. These biopesticides represent ideal targets for RNA biopesticides. Epidermal proteins are crucial components of the insect's cuticle and periphyte, providing essential physical protection and participating in various physiological processes such as growth, development, and resistance formation. Pan et al. discovered that the brown planthopper (… Nilaparvata question disable The epidermal protein gene CP21.92 of *Triplophysa gracilis* is highly expressed primarily in the epidermis, and decreased expression levels lead to abnormal cell morphology and insect death. Jasrapuria et al. found that silencing the expression of *Triplophysa gracilis*... Tribolium castaneum The epidermal protein genes CPAP1-C, CPAP1-H, and CPAP1-J cause stunted development from pupa to adult, incomplete development of the epidermis and elytra, and ultimately, complete death. Jan et al. found that the silent beet armyworm (… Spodoptera exigua The epidermal protein gene CPG316 causes pupal death in test insects; silencing CPG860 causes blackening and hardening of the intersegmental membrane epidermis in larvae, leading to larval death before molting; silencing CPG4855 causes complete blackening and hardening of the larval epidermis, also resulting in larval death. Insect epidermal proteins also participate in the formation of resistance to chemical pesticides in pests. Studies by Cai et al. have shown that, compared with the sensitive German cockroach (… Blattella germanica Compared to other insecticides, pesticides have lower penetration into the epidermis of resistant German cockroaches. Silencing the epidermal protein CPLCP1 significantly thinned the epidermis of German cockroaches and significantly reduced their resistance levels. Simma et al. used RNAi technology to silence Anopheles africanus (…). Anopheles arabiensis After three epidermal protein genes were upregulated in the cypermethrin-resistant temperate bedbug strain and the CpCPR63 and CpCPR47 epidermal protein genes were highly expressed in the deltamethrin-resistant Culex pipiens quinquefolius strain, the resistance levels of both pests showed a significant downward trend.

[0005] Currently, there is a lack of reports on the function of epidermal proteins in the fall armyworm and their targeting potential as RNA biopesticides. Summary of the Invention

[0006] The purpose of this invention is to provide SfCPR1 and SfCPR91 Application of genes and their dsRNA in the control of fall armyworm Fall armyworm epidermal protein SfCPR1 and SfCPR91 The genes, whose nucleotide sequences are shown in SEQ ID NO:1 and SEQ ID NO:2, respectively.

[0007] Targeted SfCPR1 and SfCPR91The dsRNA of the gene has nucleotide sequences as shown in SEQ ID NO:3 and SEQ ID NO:4, respectively.

[0008] The target SfCPR1 The primers for dsRNA synthesis of the gene are shown in SEQ ID NO: 5 and SEQ ID NO: 6.

[0009] The target SfCPR91 The primers for dsRNA synthesis of the gene are shown in SEQ ID NO: 7 and SEQ ID NO: 8.

[0010] A biological pesticide comprising the targeted SfCPR1 and SfCPR91 dsRNA of genes.

[0011] Preferably, the biopesticide further comprises SPc nanomaterials.

[0012] Preferably, the biological pesticide also includes a chemical pesticide. The biological pesticide is chlorantraniliprole, chlorpyrifos, indoxacarb, emamectin benzoate, or chlorfenapyr.

[0013] The beneficial effects of this invention are as follows: Through research on the function of epidermal proteins in the fall armyworm, this application screened and obtained two epidermal proteins that significantly affect its growth, development, and pupation rate. Based on these two target sites, an RNA biopesticide formulation of "dsRNA + SPc delivery + chemical pesticide" was developed. Studies have found that dsCPR1 and dsCPR91 can significantly enhance the toxicity of various insecticides and significantly improve field control efficacy, effectively reducing the use of chemical insecticides and representing a green pest control technology. The "dsRNA + SPc delivery + chemical pesticide" RNA biopesticide formulation researched in this invention can delay the lifespan of insecticides that have already developed resistance. Attached Figure Description

[0014] Picture 1 The effects of ribavirin treatment on the molting of fall armyworm are shown in Figure A, where symptoms of ribavirin poisoning in fall armyworms are shown in Figure B, and symptoms of ribavirin poisoning in fall armyworms 24 hours after ingestion. CPR1 and CPR91 Expression levels were significantly suppressed.

[0015] Picture 2 For silence CPR1 and CPR91 The effects of SPc-delivered dsCPR1 on the growth and development of fall armyworm, where A represents the effect of SPc-delivered dsCPR1 on fall armyworm growth and development. CPR1 The silencing efficiency of dsCPR91 delivered by SPc against fall armyworm. CPR91 The silencing efficiency, C is the silencing efficiency. CPR1 Effect on the weight of fall armyworm larvae, D represents silence. CPR91 Effect on the weight of fall armyworm larvae, E represents silence. CPR1 and CPR91 The effect on the pupation rate of fall armyworm, F represents silence. CPR1 and CPR91 The effect on the emergence rate of fall armyworm was investigated, with CK as the control and dsGFP as the green fluorescent protein double-stranded RNA control. Detailed Implementation

[0016] To facilitate understanding of the present invention, a more comprehensive description will be given below. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the present invention.

[0017] The SPc nanomaterials described in the following examples were prepared according to the method disclosed in "Nanomaterial inactivates environmental virus and enhances plant immunity for controlling tobacco mosaic virus disease, Nature Communications, Qinhong Jiang, Yonghui Xie, Bingcheng Zhou, Zhijiang Wang, Dekai Ning, Hongming Li, Junzheng Zhang, Meizhen Yin, JieShen 1 & Shuo Yan".

[0018] Example 1: Ribavirin inhibits epidermal protein expression and interferes with normal molting in fall armyworm. Experimental materials: Ribavirin, 3rd instar larvae of the fall armyworm.

[0019] Experimental methods: Artificial feed containing ribavirin was prepared. The effect of ribavirin on the growth and development of fall armyworm was determined by the poisoned feed method. After 24 h of treatment, test insects were collected for RNA extraction and epidermal protein expression level determination.

[0020] Picture 1 The effect of ribavirin treatment on the molting of the fall armyworm. Picture 1 A represents the symptoms of poisoning caused by the fall armyworm ingesting ribavirin. Picture 1 B was treated with ribavirin for 24 hours. CPR1 and CPR91 Expression levels were significantly suppressed. From Picture 1 It can be inferred from the middle CPR1 and CPR91It plays an important role in the epidermal structure of the fall armyworm, and its expression level inhibition will lead to molting obstruction in the fall armyworm, making it a potential target site for RNA biopesticides.

[0021] Example 2: Effects of silencing epidermal proteins on the growth and development of fall armyworm. Experimental materials: Fall armyworm larvae, dsRNA, SPc Experimental Methods: The epidermal protein gene-specific sequence was ligated into the L4440 vector with a dual T7 promoter using the HT115-L4440 expression system, resulting in high-level expression of dsRNA in HT115. The dsRNA was mixed with an equal mass of nanomaterial SPc (final concentration 5 μg / ml). Second-instar fall armyworm larvae were continuously fed this nucleic acid solution using a feeding method, and silencing was measured. CPR1 and CPR91 The impact on the growth and development of the fall armyworm.

[0022] Picture 2 For silence CPR1 and CPR91 Effects on the growth and development of the fall armyworm. The silencing efficiency assay results showed that delivery of dsCPR1 and dsCPR91 via SPc... CPR1 and CPR91 Expression levels were significantly suppressed at both 24 and 48 h, with a silencing efficiency as high as 58.63%. Picture 2 A, 2B). The weight gain of second-instar fall armyworm larvae fed on dsCPR1 and dsCPR91 larvae was significantly inhibited, with weight gain inhibition rates of 41.18% and 37.88% respectively by day 8 (i.e., the end of the 6th instar). Picture 2 C, 2D). Silence. CPR1 and CPR91 The pupation rates of larvae decreased by 42.86% and 23.80%, respectively. Picture 2 E), the emergence rates decreased by 21.73% and 39.13% respectively. Picture 2 F). The above results indicate that... CPR1 and CPR91 It has a significant impact on the growth and development of the fall armyworm, and silences it. CPR1 and CPR91 It can effectively suppress the population reproduction of fall armyworm, with comprehensive control efficacy reaching 55.28% and 53.62% respectively, making it a good target site for RNA biopesticides.

[0023] Example 3: Effect of Silencing Epidermal Proteins on Indoor Toxicity of Insecticides Experimental materials: fall armyworm larvae, dsRNA, SPc, chlorantraniliprole, chlorpyrifos, indoxacarb, abamectin, chlorfenapyr.

[0024] Experimental methods: dsRNA was administered via feed dipping method, and insecticide was administered via droplet method. Silencing was determined. CPR1 and CPR91 Effects of insecticide toxicity indoors for 48 hours.

[0025] Table 1 lists the Silent Grassland Fall Armyworm. CPR1 and CPR91 Effects on indoor toxicity of insecticides. Results showed that silence... CPR1 and CPR91 The fall armyworm showed significantly increased susceptibility to chlorantraniliprole, chlorpyrifos, abamectin, and chlorfenapyr. The median lethal dose (LD50) of chlorantraniliprole decreased from 8.42 μg / worm to 5.24 μg / worm and 4.39 μg / worm, respectively.

[0026] Table 1. Effects of the silent fall armyworm CPR1 and CPR91 on insecticide toxicity.

[0027] RR represents the change in resistance ratio. Example 4: Effect of silencing epidermal proteins on the field efficacy of insecticides Experimental materials: fall armyworm larvae, dsRNA, SPc, chlorantraniliprole, chlorpyrifos.

[0028] Experimental Methods: A pesticide solution was prepared using a combination of dsRNA, SPc delivery, and chemical pesticide. Second-instar fall armyworm larvae were inoculated onto corn plants, and silencing was determined using a spraying method. CPR1 and CPR91 Effects of chlorantraniliprole and chlorpyrifos on field efficacy over 72 h.

[0029] Table 2 lists the Silent Grassland Fall Armyworm. CPR1 and CPR91 The effect on the field efficacy of insecticides. Results showed that silencing... CPR1 and CPR91 Chlorantraniliprole increased the control efficacy against fall armyworm by 27.45% and 21.56%, respectively, and against chlorpyrifos by 21.76% and 73.95%, respectively. These results indicate that dsCPR1 and dsCPR91 significantly improved both the indoor toxicity and field efficacy of insecticides. Using a combination formulation of "dsRNA + SPc delivery + chemical pesticides" can significantly reduce the amount of chemical insecticides used and extend the lifespan of resistant insecticides. This represents a green control technology with significant development and application potential.

[0030] Table 2. Effects of CPR1 and CPR91 on the field control efficacy of insecticides against the silent fall armyworm.

[0031] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. 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 all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. Epidermal protein of the fall armyworm SfCPR1 and SfCPR91 Genes, characterized by, Their nucleotide sequences are shown in SEQ ID NO:1 and SEQ ID NO:2, respectively.

2. Targeted SfCPR1 and SfCPR91 The dsRNA of a gene is characterized by, Their nucleotide sequences are shown in SEQ ID NO:3 and SEQ ID NO:4, respectively.

3. The target according to claim 2 SfCPR1 and SfCPR91 The dsRNA of a gene is characterized by, The target SfCPR1 The primers for dsRNA synthesis of the gene are shown in SEQ ID NO: 5 and SEQ ID NO:

6.

4. The target according to claim 2 SfCPR1 and SfCPR91 The dsRNA of a gene is characterized by, The target SfCPR91 The primers for dsRNA synthesis of the gene are shown in SEQ ID NO: 7 and SEQ ID NO:

8.

5. A biological pesticide, characterized in that, Includes the target as described in claim 2 SfCPR1 and SfCPR91 dsRNA of genes.

6. The biological pesticide according to claim 5, characterized in that, It also contains SPc nanomaterials.

7. The biological pesticide according to claim 5, characterized in that, It also includes chemical pesticides.

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