Application of sltrypsin1 and sltrypsin7 genes and dsrna in controlling spodoptera litura

Abstract

The application discloses SlTrypsin1 and SlTrypsin7 The application discloses the application of genes and dsRNA in the prevention and treatment of Spodoptera litura. Through functional screening, two RNA biological pesticide targets for significantly inhibiting the body weight and pupation rate of Spodoptera litura larvae are obtained from seven Spodoptera litura trypsin. Through the protection and delivery of nano material SPc, a compound formula of 'dsRNA + chemical pesticide + SPc' is developed, which effectively improves the toxicity of dsRNA to various pesticides. SlTrypsin1 and SlTrypsin7 The application provides a technical method for the green prevention and control and resistance management of Spodoptera litura.

Description

Technical Field

[0001] This invention belongs to the field of research and application technology of RNA biopesticides for harmful organisms, specifically involving SlTrypsin1 and SlTrypsin7 Application of genes and their dsRNA in the control of beet armyworm. Background Technology

[0002] Spodoptera litura ( Spodoptera litura ) belongs to the order Lepidoptera ( Lepidoptera Noctuidae ( Noctuidae The beet armyworm (Spodoptera litura) is a globally distributed omnivorous agricultural pest with a wide host range, damaging over 290 species of crops from 99 families, including tobacco, cotton, cabbage, kale, soybeans, and cruciferous vegetables. This insect reproduces rapidly, has many generations, its larvae are voracious eaters, and it possesses strong migratory abilities, easily causing severe economic losses. Currently, pesticides used to control the beet armyworm mainly include two types: chemical insecticides and biological pesticides. However, due to the long-term and large-scale use of chemical pesticides, the beet armyworm has developed varying degrees of resistance to traditional and new agents such as organophosphates, pyrethroids, carbamates, macrolides, and diamides, seriously affecting the effectiveness of field control. Therefore, the development of green and novel control technologies is urgently needed.

[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 an expression-based biopesticide. dsSnf7 The transgenic maize variety MON87411 represents 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, marking the formal transition of RNAi-based biopesticides from the laboratory to field application. Lepidoptera insects possess numerous nucleases that rapidly degrade dsRNA, and their alkaline midgut environment leads to instability of the phosphodiester bonds in dsRNA, resulting in low RNAi efficiency. Studies have confirmed that encapsulating dsRNA with the nanomaterial SPc can effectively improve gene silencing efficiency in lepidopteran pests. For example, in the Asian corn borer, encapsulating dsRNA with SPc nanomaterials effectively protects it from nuclease degradation and efficiently delivers it into the insect's body. Feeding the insect SPc / dsRNA effectively silences the expression of the target gene. Therefore, the cross-integration of RNAi and nanotechnology effectively promotes the development of RNA biopesticides for lepidopteran pests based on SIGS technology.

[0004] Trypsin, a key enzyme in the insect digestive system, plays an irreplaceable role in maintaining insect growth and development. Belonging to the serine protease superfamily, its active form contains a highly conserved catalytic triplet composed of histidine, aspartic acid, and serine, which can specifically recognize and cleave the carboxyl terminus of lysine or arginine residues in peptide chains. In insects, trypsin is synthesized by midgut wall cells and secreted into the midgut lumen, participating in the digestion and breakdown of proteins in food—its core physiological function. Studies have shown that inhibiting trypsin activity in insects can effectively control pests. Regarding trypsin inhibitors, Mcmanus et al. found that 50 mg / L STI inhibited trypsin activity in Spodoptera litura larvae by up to 88%. In 2024, Lü Dongbiao et al. used RNA interference technology to conduct a functional study on the trypsin gene in the midgut of the pear fruit moth. The results showed that silencing this target gene significantly reduced the activity of both trypsin and chymotrypsin in the larval midgut, while also significantly prolonging the larval stage, decreasing pupal weight, and severely inhibiting growth and development. In 2025, Liang Wenkai et al. found that feeding the fall armyworm with serine protease inhibitors significantly suppressed the activity of midgut trypsin, leading to a significant reduction in the weight of both larvae and pupae. These results indicate that trypsin plays a crucial physiological role in the life cycle of pests, and targeted control based on trypsin activity inhibition has promising application potential and research prospects. However, there are currently no reports on the development of RNA biopesticides targeting the trypsin of the fall armyworm. Summary of the Invention

[0005] The purpose of this invention is to provide SlTrypsin1 and SlTrypsin7 Application of genes and their dsRNA in the control of beet armyworm.

[0006] Spodoptera litura trypsin SlTrypsin1 and SlTrypsin7 The genes, whose nucleotide sequences are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively.

[0007] Targeted SlTrypsin1 and SlTrypsin7 The dsRNA of the gene has nucleotide sequences as shown in SEQ ID NO:3 and SEQ ID NO:4, respectively.

[0008] The target SlTrypsin1 The primers for dsRNA synthesis of the gene are shown in SEQ ID NO: 5 and SEQ ID NO: 6.

[0009] The target SlTrypsin7 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 SlTrypsin1 and SlTrypsin7 dsRNA of genes.

[0011] Preferably, it also contains SPc nanomaterials.

[0012] Preferably, it also includes chemical pesticides.

[0013] The chemical pesticide is one or more of the following: lufenuron, chlorantraniliprole, chlorantraniliprole, abamectin, spinosad, cypermethrin, indoxacarb, dinotefuran, and chlorpyrifos.

[0014] The beneficial effects of this invention are as follows: Through research on the function of trypsin in the beet armyworm, this application screened and obtained two trypsin inhibitors 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, which can significantly improve the toxicity of various insecticides, effectively reduce the use of chemical insecticides, and is a green pest control technology. Attached Figure Description

[0015] Figure 1 Schematic diagram of the L4440 vector and determination of its silencing effect; A: Schematic diagram of the L4440 vector structure, with key elements labeled (T7 promoter, multiple cloning site, terminator, and antibiotic resistance gene, etc.); B: Relative expression levels of Trypsin1 gene after 24 h and 48 h of dsTrypsin1 treatment; C: Relative expression levels of Trypsin2 gene after 24 h and 48 h of dsTrypsin2 treatment; D: Relative expression levels of Trypsin3 gene after 24 h and 48 h of dsTrypsin3 treatment; E: Relative expression levels of Trypsin4 gene after 24 h and 48 h of dsTrypsin4 treatment; F: Relative expression levels of Trypsin5 gene after 24 h and 48 h of dsTrypsin5 treatment; G: Relative expression levels of Trypsin6 gene after 24 h and 48 h of dsTrypsin6 treatment; H: Relative expression levels of Trypsin7 gene after 24 h and 48 h of dsTrypsin7 treatment. The relative expression level of the Trypsin7 gene after h.

[0016] Figure 2The following data were used to measure the weight changes of Spodoptera litura larvae over 7 days after feeding them with seven different diets containing a mixture of dsTrypsin / SPc: A: Weight change of larvae over 7 days after feeding with dsTrypsin1; B: Weight change of larvae over 7 days after feeding with dsTrypsin2; C: Weight change of larvae over 7 days after feeding with dsTrypsin3; D: Weight change of larvae over 7 days after feeding with dsTrypsin4; E: Weight change of larvae over 7 days after feeding with dsTrypsin5; F: Weight change of larvae over 7 days after feeding with dsTrypsin6; G: Weight change of larvae over 7 days after feeding with dsTrypsin7; H: Weight change of larvae over 7 days after a combined treatment of dsTrypsin1 and dsTrypsin7. Larval weight was recorded every 24 hours during the experiment to assess the effects of different Trypsin gene silencing treatments on the growth and development of Spodoptera litura larvae.

[0017] Figure 3 The effects of dsTrypsin gene silencing on relevant indicators of the pupal stage of *Spodoptera litura* were investigated. A: Prepupa to larva ratio: The ratio of prepupae to larvae in each treatment group on day 8. B: Pupation rate: The percentage of individuals that successfully pupated in each treatment group out of the total number of tested insects. C: Pupal development duration: The number of days from pupation to emergence in each treatment group. D: Pupal deformity rate: The percentage of deformed pupae with abnormal morphology or incomplete development in each treatment group out of the total number of pupae. E: Pupal weight: The weight of a single pupa in each treatment group was measured.

[0018] Figure 4 The changes in feed conversion efficiency (FCE) of Spodoptera litura larvae after feeding them with seven different dsTrypsin / SPc mixtures (days 3-7) are shown below: A: FCE of the dsTrypsin1 treatment group; B: FCE of the dsTrypsin2 treatment group; C: FCE of the dsTrypsin3 treatment group; D: FCE of the dsTrypsin4 treatment group; E: FCE of the dsTrypsin5 treatment group; F: FCE of the dsTrypsin6 treatment group; G: FCE of the dsTrypsin7 treatment group; H: FCE of the combined treatment group of dsTrypsin1 and dsTrypsin7 (dsTrypsin1+dsTrypsin7). Feed conversion efficiency (FCE) was obtained by calculating the ratio of larval weight gain to leaf ingestion.

[0019] Figure 5Effects of feeding on soybean leaves sprayed with a mixture of dsTrypsin1 / SPc or dsTrypsin7 / SPc on the growth and development of *Spodoptera litura* larvae; A: Soybean leaf consumption weight: The amount of soybean leaves consumed by different treatment groups during the experimental period (day 1 to day 7); B: Weight gain of *Spodoptera litura* larvae after feeding on soybeans: The change in larval weight over days in different treatment groups; C: Larval to pupa ratio: The ratio of *Spodoptera litura* larvae to pupated larvae after 8 days; D: Pupation rate: The pupation rate of *Spodoptera litura* larvae under different treatments; E: Pupal malformation rate: The malformation rate of pupae under different treatment groups; F: Pupal weight: The pupal weight of normal pupae in different treatment groups; G: Emergence rate: The emergence rate of *Spodoptera litura* pupae in different treatment groups.

[0020] Figure 6 The effects of feeding on maize leaves sprayed with a mixture of dsTrypsin1 / SPc or dsTrypsin7 / SPc on the growth and development of *Spodoptera litura* larvae; A. Weight of maize leaves consumed: the amount of maize leaves consumed by different treatment groups during the experimental period (day 1 to day 7); B. Weight of maize larvae consumed: the change in larval weight with the number of days in different treatment groups; C. Larval to pupa ratio: the ratio of *Spodoptera litura* larvae to pupae after 8 days; D. Pupation rate: the pupation rate of *Spodoptera litura* larvae under different treatments; E. Pupal malformation rate: the malformation rate of pupae under different treatment groups; F. Pupal weight: the pupal weight of normal pupae in different treatment groups; G. Emergence rate: the emergence rate of *Spodoptera litura* pupae in different treatment groups. Detailed Implementation

[0021] 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.

[0022] 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".

[0023] Example 1: Effects of silencing trypsin on the growth and development of Spodoptera litura. Experimental materials: Spodoptera litura larvae, dsRNA, SPc Spodoptera litura trypsin SlTrypsin1 and SlTrypsin7 The genes, whose nucleotide sequences are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively. Targeting SlTrypsin1 and SlTrypsin7 The dsRNA of the gene, with nucleotide sequences shown in SEQ ID NO:3 and SEQ ID NO:4, respectively. The targeted... SlTryp1 The primers for dsRNA synthesis of the gene are shown in SEQ ID NO: 5 and SEQ ID NO: 6. The targeted... SlTryp7 The primers for dsRNA synthesis of the gene are shown in SEQ ID NO: 7 and SEQ ID NO: 8.

[0024] Experimental Methods: Using the HT115-L4440 expression system, a trypsin gene-specific sequence was ligated into the L4440 vector with a dual T7 promoter, resulting in high-level expression of dsRNA in HT115. The dsRNA was mixed with an equal mass of nanomaterial SPc, and feed was soaked in the above nucleic acid solution. Third-instar Spodoptera litura larvae were continuously fed using a feeding method to determine the effects of silencing Trypsin1 and Trypsin7 on the growth and development of Spodoptera litura.

[0025] Figure 1 A schematic diagram of the L4440 vector and the determination of its silencing effect are shown. The silencing efficiency test results indicate that, through SPc delivery of dsTrypsin, the expression levels of all seven trypsin genes were significantly inhibited within 24 h, with silencing efficiencies ranging from 33.4% to 54.6%. This demonstrates that SPc nanomaterials can effectively deliver and protect dsRNA, thus effectively silencing trypsin.

[0026] Figure 2 The study investigated the changes in body weight of *Spodoptera litura* larvae over seven days after feeding them with seven different diets containing a mixture of dsTrypsin / SPc. The dsTrypsin1 / SPc and dsTrypsin7 / SPc treatment groups showed significantly lower larval weights on days 2, 3, 4, 5, 6, and 7 compared to the dsGFP and CK treatment groups. The dsTrypsin5 / SPc treatment group showed significantly lower larval weights on days 4, 5, 6, and 7 compared to the dsGFP treatment group.

[0027] Figure 3This study investigated the effects of Trypsin gene silencing on relevant indicators during the pupal stage of *Spodoptera litura*. Feeding larvae dipped in a mixture of dsTrypsin1 / SPc, dsTrypsin5 / SPc, dsTrypsin6 / SPc, dsTrypsin7 / SPc, and dsTrypsin1+7 / SPc resulted in similar prepupal proportions, but significantly lower than the control group. The pupation rate was significantly reduced in the dsTrypsin1 / SPc, dsTrypsin5 / SPc, dsTrypsin7 / SPc, and dsTrypsin1+7 / SPc treatment groups. The pupal malformation rate was significantly higher in the dsTrypsin1 / SPc, dsTrypsin2 / SPc, dsTrypsin7 / SPc, and dsTrypsin1+7 / SPc treatment groups than in the dsGFP / SPc treatment group. In addition, feeding dsTrypsin1 / SPc and dsTrypsin7 / SPc significantly reduces pupal weight.

[0028] Figure 4 The study investigated the changes in feed conversion efficiency (FCE) of *Spodoptera litura* larvae after feeding them with seven different dsTrypsin / SPc mixtures. Feeding larvae dipped in mixtures of dsTrypsin1 / SPc, dsTrypsin5 / SPc, and dsTrypsin7 / SPc significantly reduced FCE. The FCE of the dsTrypsin1 / SPc treatment group was significantly lower than that of the dsGFP treatment group on days 3, 4, 5, 6, and 7; similarly, the FCE of the dsTrypsin7 / SPc treatment group was significantly lower than that of the dsGFP treatment group on days 3, 4, 6, and 7.

[0029] In conclusion, silencing the Trypsin1 and Trypsin7 genes has a significant impact on the growth and development of Spodoptera litura larvae, including inhibiting larval weight, prolonging the developmental period, reducing pupal weight and pupation rate, and increasing pupal malformation rate, making them promising target sites for RNA biopesticides.

[0030] Example 2: Field simulation of the control efficacy of dsTrypsin1 / SPc and dsTrypsin7 / SPc Experimental materials: Spodoptera litura larvae, dsRNA, SPc, soybean plants, and corn plants.

[0031] Experimental method: The dsTrypsin / SPc mixture was sprayed onto the leaves of detached plants using a spraying method. The leaves were then placed in 12-well boxes. Finally, third-instar Spodoptera litura larvae were introduced into the 12-well boxes to determine the effects of silencing Trypsin1 and Trypsin7 on the feeding and growth of Spodoptera litura, thus simulating the field spraying efficacy.

[0032] Figure 5The effects of feeding on soybean leaves sprayed with a mixture of dsTrypsin1 / SPc or dsTrypsin7 / SPc on the growth and development of the beet armyworm larvae were investigated. The results showed that after feeding on soybean leaves sprayed with dsTrypsin1 / SPc or dsTrypsin7 / SPc, the larval body weight significantly decreased on days 3, 4, 5, 6, and 7. Simultaneously, the amount of soybean leaves consumed by larvae significantly decreased on days 4, 6, and 7. On day 8, the larval / pupa ratio significantly increased, the pupation rate significantly decreased, the pupal weight significantly decreased, and the pupal deformity rate significantly increased.

[0033] Figure 6 The effects of feeding corn leaves sprayed with a mixture of dsTrypsin1 / SPc or dsTrypsin7 / SPc on the growth and development of Spodoptera litura larvae. After feeding corn leaves sprayed with the mixture of dsTrypsin1 / SPc or dsTrypsin7 / SPc, the larval body weight decreased significantly on days 3, 4, 5, 6, and 7; simultaneously, the amount of corn leaves consumed by larvae decreased significantly on days 4, 6, and 7; and on day 8, the larval / pupa ratio increased significantly, the pupation rate decreased significantly, the pupal weight decreased significantly, and the pupal malformation rate increased significantly.

[0034] The results of the above-mentioned field efficacy simulation experiments were basically consistent with the indoor results. Specifically, spraying corn or soybean leaves with dsTrypsin1 / SPc and dsTrypsin7 / SPc respectively resulted in a 37.1-42.3% decrease in the normal pupation rate of Spodoptera litura and a 12.7-30.4% decrease in the emergence rate. The overall field efficacy was between 48.1% and 57.4%, demonstrating that SlTrypsin1 and SlTrypsin7 dsRNA have good performance in the field control of Spodoptera litura.

[0035] Example 3: Effect of “dsTrypsin1 / 7 + chemical pesticide + SPc” on the indoor toxicity of insecticides Experimental materials: Spodoptera litura larvae, dsRNA, SPc, lufenuron, chlorantraniliprole, chlorantraniliprole, abamectin, spinosad, cypermethrin, indoxacarb, dinotefuran, chlorpyrifos.

[0036] Experimental methods: dsRNA was administered via feed dipping method, and insecticides were administered via feed mixing method. The effects of silencing Trypsin1 and Trypsin7 on the toxicity of insecticides in the room for 48 h were determined.

[0037] Table 1 shows the effects of silenced beet armyworm Trypsin1 or Trypsin7 on the indoor toxicity of insecticides. The results indicated that feeding beet armyworm larvae with a mixture of dsTrypsin1 / SPc significantly increased their sensitivity to lufenuron, chlorantraniliprole, abamectin, spinosad, and cypermethrin, with increases of 1.96, 1.67, 1.52, 1.57, and 1.55 times, respectively. Feeding beet armyworm larvae with a mixture of dsTrypsin7 / SPc significantly increased their sensitivity to chlorantraniliprole and lufenuron, with increases of 1.76 and 1.50 times, respectively.

[0038] The above results indicate that silencing Trypsin1 and Trypsin7 can significantly enhance the toxicity of most insecticides against the beet armyworm. Considering the inhibitory effect of silencing Trypsin1 and Trypsin7 alone on the pupation and emergence rates of the beet armyworm population, and the ability to enhance its sensitivity when used in combination with chemical insecticides, the "dsTrypsin1 / 7 + chemical pesticide + SPc" model can be clearly adopted. This model can ensure the efficacy of RNA biopesticides, reduce the amount of chemical insecticides used, and extend the lifespan of resistant insecticides. It is a green control technology with significant development and application prospects.

[0039] Table 1. Effects of Trypsin1 and Trypsin7 on insecticide sensitivity in the silent beet armyworm.

[0040] Note: +G, +1, and +7 refer to the relevant mixed feed being dipped in a mixture of dsGFP / SPc, dsTrypsin1 / SPc, and dsTrypsin7 / SPc, respectively.

[0041] 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. Trypsin from Spodoptera litura (beet armyworm) SlTrypsin1 and SlTrypsin7 Genes, characterized by, Their nucleotide sequences are shown in SEQ ID NO:1 and SEQ ID NO:2, respectively.

2. Targeted SlTrypsin1 and SlTrypsin7 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 SlTrypsin1 and SlTrypsin7 The dsRNA of a gene is characterized by, The target SlTrypsin1 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 SlTrypsin1 and SlTrypsin7 The dsRNA of a gene is characterized by, The target SlTrypsin7 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 SlTrypsin1 and SlTrypsin7 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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