DsRNA targeting expression of pxakh / pxakhr gene in plutella xylostella and application thereof

Abstract

The application discloses a kind of dsRNA for targeting in vivo of plutella xylostella PxAKH / PxAKHR Gene expression and its application, belong to the technical field of plant protection, for solving the problem of plutella xylostella sensitivity decline caused by long-term use of bacillus thuringiensis.The dsRNA is ds PxAKH1 , ds PxAKH2 Or ds PxAKHR The nucleotide sequence of the sense strand thereof is shown in SEQ ID NO:20-22, respectively, which is prepared by in vitro transcription and annealing of primer amplification product with T7 promoter; at least one of them is used as an active ingredient to make a medicament, or combined with Bt suspension to form a combination drug independently packaged. After 24-48 h of 500 ng dsRNA administration to three instar larvae, Bt treatment is carried out again, which can increase the sensitivity of plutella xylostella to Bt by 5.72-13.97 times, and is used for enhancing the insecticidal activity of Bt and reducing the dosage of Bt.

Description

Technical Field

[0001] This invention relates to the field of plant protection technology, specifically to a method for targeting the diamondback moth. PxAKH / PxAKHR dsRNA for gene expression and its applications. Background Technology

[0002] Bacillus thuringiensis (Bt) Bacillus thuringiensis Bt is a class of biological pesticides widely used in agricultural production. Due to its advantages such as high efficiency, strong specificity, and environmental friendliness, it is widely used for the control of lepidopteran pests, especially the diamondback moth. Plutella xylostella It has played an important role in controlling major agricultural pests such as [unspecified pests]. However, with the long-term use of Bt preparations alone, pest resistance has gradually intensified, leading to a decline in its control efficacy, which has become one of the important factors restricting the further promotion and application of Bt.

[0003] To enhance the insecticidal efficacy of Bt and delay the development of resistance, current technologies primarily focus on synergistic effects through combined use with other biological or chemical agents. For example, this can be achieved by combining it with chemical pesticides, using it in conjunction with insect pathogenic fungi or viruses, or by expressing Bt toxins in transgenic crops. Furthermore, with the development of RNA interference technology in recent years, studies have attempted to enhance Bt efficacy by targeting key insect genes. For instance, some studies have reported that interfering with insect immune-related genes or energy metabolism-related genes (such as arginine kinases) can significantly increase the toxicity of Bt to pests. These studies indicate that regulating insect physiological processes can enhance the insecticidal effect of Bt to some extent.

[0004] However, current technologies mainly focus on immune regulation or intervention at the level of single metabolic enzymes, lacking systematic research from the perspective of the overall energy metabolism regulatory network in insects. In particular, the regulatory role of the insect neuroendocrine system in the action of biopesticides (Bt) remains under-researched. Adipokines (AKH), ​​as important energy mobilization hormones in insects, play a central role in regulating lipid and carbohydrate metabolism. They participate in the adaptation of insects to environmental stresses by mediating signal transduction with their receptor AKHR. Previous studies have shown that the AKH signaling pathway plays an important role in insect physiological processes such as starvation and flight, but its function in biopesticide stress, especially in the action of Bt, has not been systematically reported.

[0005] Therefore, current technologies lack a technique for enhancing Bt insecticidal activity based on insect neuroendocrine regulation mechanisms, particularly targeting the AKH / AKHR signaling pathway. Developing novel Bt-enhancing strategies from the perspective of insect energy metabolism regulation is crucial for improving biopesticide utilization efficiency, reducing dosage, and delaying resistance development. Summary of the Invention

[0006] The purpose of this invention is to provide a targeted method for the diamondback moth. PxAKH / PxAKHR dsRNA for gene expression and its applications: The present invention develops a targeted RNA for diamondback moth in vivo. PxAKH / PxAKHR dsRNA of gene expression interferes with the life of diamondback moth PxAKH / PxAKHR By regulating gene expression and its energy metabolism processes, the toxicity of Bt to diamondback moth can be significantly increased, reducing the dosage of Bt and improving control efficiency, thereby solving the following problems: 1. The long-term use of Bacillus thuringiensis (Bt) alone has led to a decrease in the sensitivity of pests such as diamondback moth to it, and the control effect has gradually decreased. There is an urgent need to develop new Bt enhancement technologies. 2. Existing Bt enhancement technologies mostly rely on chemical pesticide combinations or targeted immunity and single metabolic gene regulation, lacking technical means to systematically intervene at the level of overall energy metabolism regulation in insects; 3. Insects can enhance their adaptability and reduce Bt toxicity by regulating energy metabolism-related pathways under Bt stress. However, the target discovery and application of such regulatory mechanisms are still unclear, which limits the further development of biopesticide enhancement strategies.

[0007] To achieve the above objectives, the technical solution designed by the present invention is as follows: This invention provides a method for targeting the body of diamondback moth. PxAKH / PxAKHR dsRNA of gene expression, wherein the dsRNA is ds PxAKH1 ds PxAKH2 and ds PxAKHR Any one of them; PxAKH / PxAKHR Genes of diamondback moth PxAKH1 , PxAKH2 and PxAKHR Genes; among which, The ds PxAKH1 The nucleotide sequence of the positive strand is shown in SEQ ID NO.20, which is the diamondback moth. PxAKH1 Continuous nucleotide segments in a gene, and PxAKH1 The nucleotide sequence of the gene is shown in SEQ ID NO.1; The ds PxAKH2 The nucleotide sequence of the positive strand is shown in SEQ ID NO.21, and its diamondback moth... PxAKH2 Continuous nucleotide segments in a gene, and PxAKH2 The nucleotide sequence of the gene is shown in SEQ ID NO.2; The ds PxAKHR The nucleotide sequence of the positive strand is shown in SEQ ID NO.22, which is the diamondback moth. PxAKHRContinuous nucleotide segments in a gene, and PxAKHR The nucleotide sequence of the gene is shown in SEQ ID NO.3.

[0008] Furthermore, the ds PxAKH1 Using the sequence shown in SEQ ID NO.1 as a template, and through primer pair ds PxAKH1 -F / R amplification yields DNA fragments that are transcribed and annealed in vitro, and primer pairs ds PxAKH1 -F / R are respectively: ds PxAKH1 -F: taatacgactcactataggGTTTGGTGATCGTCAGTGAGG, as shown in SEQ ID NO.12; ds PxAKH1 -R: taatacgactcactataggCATGATTTCTGGCATAGAAGAAGTCT, as shown in SEQ IDNO.13; The ds PxAKH2 Using the sequence shown in SEQ ID NO.2 as a template, and through primer pair ds PxAKH2 -F / R amplification yields DNA fragments that are transcribed and annealed in vitro, and primer pairs ds PxAKH2 -F / R are respectively: ds PxAKH2 -F: taatacgactcactataggATGTTGGTGTTGGCAGACG, as shown in SEQ ID NO.14; ds PxAKH2 -R: taatacgactcactataggTCACTTCTGCTGGCAGGC, as shown in SEQ ID NO.15; The d sPxAKHR Using the sequence shown in SEQ ID NO.3 as a template, and through primer pair ds PxAKHR -F / R amplification yields DNA fragments that are transcribed and annealed in vitro, and primer pairs ds PxAKHR -F / R are respectively: ds PxAKHR- F: taatacgactcactataggAGCTGTCGCCGATCTTATGG, as shown in SEQ ID NO.16; ds PxAKHR- R: taatacgactcactataggGCCGTAGGATACGCATTGGA, as shown in SEQ ID NO.17; The present invention also provides the above-mentioned target moth inside the body. PxAKH / PxAKHRA method for preparing dsRNA for gene expression includes the following steps: (1) Using diamondback moths respectively PxAKH1, PxAKH2 or PxAKHR Using the cDNA sequence of the gene as a template, primer pairs with the T7 promoter sequence added to the 5′ end were used. PxAKH1 -F / R, ds PxAKH2 -F / R or ds PxAKHR -F / R was used for PCR amplification to obtain a DNA template with T7 promoter sequences at both ends; (2) The DNA template obtained in step (1) was used for in vitro transcription to obtain the sense strand and the antisense strand, respectively; (3) The sense and antisense strands obtained in step (2) are annealed to form double-stranded RNA, which is then purified and dissolved in sterile DEPC water to obtain ds PxAKH1 ds PxAKH2 or ds PxAKHR .

[0009] The present invention also provides an interference PxAKH / PxAKHR A gene-based pesticide for controlling diamondback moth, wherein the active ingredient of the pesticide is at least one of the aforementioned dsRNAs.

[0010] Furthermore, in the pharmaceutical preparation, the active ingredient is ds PxAKH1 With ds PxAKHR A mixture, or an active ingredient dsPxAKH2 With ds PxAKHR The mixture contains two dsRNAs in a 1:1 mass ratio.

[0011] Furthermore, the agent also contains at least one of a surfactant, a thickener, and a stabilizer.

[0012] The present invention also provides an application of the above-mentioned agent in enhancing the insecticidal activity of Bacillus thuringiensis against diamondback moth.

[0013] The present invention also provides a combination drug that enhances the insecticidal activity of Bacillus thuringiensis against diamondback moth. The combination drug comprises a first component and a second component packaged separately. The first component is the aforementioned agent, and the second component is a Bacillus thuringiensis suspension.

[0014] Furthermore, the viable cell concentration of the Bacillus thuringiensis suspension is 1×10⁻⁶. 5 cfu / mL to 1×10 9 cfu / mL.

[0015] The present invention also provides an application of the above-mentioned combination drug in the control of diamondback moth. The application method is to apply 500 ng of the dsRNA to each third instar larva of diamondback moth, and then treat the third instar larvae of diamondback moth with Bacillus thuringiensis suspension 24h to 48h after application.

[0016] The beneficial effects of this invention are: 1. Innovative target selection: It provides a Bt enhancement pathway targeting the AKH / AKHR signaling pathway, expanding the selectable targets of Bt enhancers from the perspective of insect neuroendocrine regulation.

[0017] 2. Significant synergistic effect: through interference PxAKH1, PxAKH2 and PxAKHR The gene increased the sensitivity of diamondback moth to Bt by 5.72 times, 9.14 times, and 10.74 times, respectively; co-interference further increased the sensitivity by 11.59 times and 13.97 times.

[0018] 3. Reduced dosage: This method can significantly reduce the dosage of Bt, thereby reducing the use of chemical pesticides, lowering production costs, and having significant economic and ecological value.

[0019] 4. Delaying the development of pesticide resistance: By using a strategy of simultaneous interference at multiple targets, the control effect of Bt can be maintained more stably, which helps to delay the development of pesticide resistance in pests.

[0020] 5. Technological integrity: The method encompasses a complete technical system, from gene expression detection and RNA interference system establishment to bioactivity assessment, providing a systematic scientific basis for the development of Bt synergists. Attached Figure Description

[0021] Figure 1 for PxAKH1 , PxAKH2 and PxAKHR Expression level map of diamondback moth at different developmental stages; Where A is PxAKH1 A map of relative gene expression levels during the egg stage, 1st to 4th instar larval stages (L1 to L4), pupal stage, and adult stage; B is PxAKH2 Map of relative gene expression levels at each of the above developmental stages; C is PxAKHR Map of relative gene expression levels at each of the above developmental stages; In the figure, different lowercase letters above each column indicate significant differences between developmental stages (P<0.05), and the error bar represents the mean ± standard error.

[0022] Figure 2 After treatment of Bt suspensions of different concentrations PxAKH1, PxAKH2 andPxAKHR Dynamic graph of gene expression in third instar larvae of diamondback moth; In the figure, CK is the control group without Bt, and Bt is the original concentration (1×10⁻⁶). 8 The Bt suspension treatment group (cfu / mL) consisted of Bt5, Bt10, and Bt20, which were diluted 5-fold, 10-fold, and 20-fold, respectively. A is PxAKH1 Bar chart showing the relative expression levels of genes in each treatment group at 12 h, 24 h, 48 h, and 72 h after treatment; B is PxAKH2 Bar chart showing the relative expression levels of genes in each treatment group at each of the above time points; C is PxAKHR Bar chart showing the relative expression levels of genes in each treatment group at each of the above time points; D is PxAKH1 A heatmap of relative gene expression levels, with the horizontal axis representing treatment time and the vertical axis representing treatment groups. The color scale on the right, from blue to red, indicates that the expression level increases from low to high. E is PxAKH2 Heatmap of relative gene expression levels F is PxAKHR Heatmap of relative gene expression levels Above the pillars A~C , , , The values ​​P<0.05, P<0.01, P<0.001, and P<0.0001 represent the statistical significance of the difference compared with the control group (CK) at the same time point.

[0023] Figure 3 The relative expression levels of each target gene at 24 h, 48 h, and 72 h after injection of a single dsRNA (used to characterize the RNA interference efficiency of drugs 1-3). Where A is the injection ds PxAKH1 Groups and ds EGFP In the negative control group PxAKH1 The relative expression levels of genes at the above time points; B is for injecting ds PxAKH2 In the group and the dsEGFP negative control group PxAKH2 The relative expression levels of genes at the above time points; C represents the injection of ds PxAKHR Groups and ds EGFP In the negative control group PxAKHR The relative expression levels of genes at the above time points; In the picture, Indicates the same time point ds EGFPThe difference was significant compared to the negative control group (P<0.05), ns indicates no significant difference.

[0024] Figure 4 The cumulative mortality curve of the effect of genes interfering with the AKH signaling pathway on the susceptibility of diamondback moth to Bt. In the figure, the horizontal axis represents the number of days (1d~7d) after Bt suspension treatment. The vertical axis represents the cumulative mortality rate (%), and the four curves from bottom to top are ds EGFP Negative control group, ds PxAKH1 Processing group, ds PxAKH2 Processing group and ds PxAKHR The error bar for the treatment group is the mean ± standard error.

[0025] Figure 5 The graph shows the relative expression levels of related genes in diamondback moth after co-interference of AKH ligand genes and receptor genes (used to characterize the RNA interference efficiency of agents 4-5). Where A is ds PxAKH1 +ds PxAKHR The co-interference group at 24 h, 48 h, and 72 h post-injection PxAKH1 and PxAKHR Relative gene expression level graph; B is ds PxAKH2 +ds PxAKHR The co-interference group at 24 h, 48 h, and 72 h post-injection PxAKH2 and PxAKHR Relative gene expression level graph; In the picture, This indicates the common interference group and the same time point ds EGFP The difference was significant compared to the negative control group (P<0.05). Detailed Implementation

[0026] The present invention will now be described in further detail with reference to specific embodiments, so that those skilled in the art can understand it.

[0027] Example 1: Inside the diamondback moth PxAKH / PxAKHR Spatiotemporal expression characteristics analysis of genes To verify the key gene ( PxAKH1 , PxAKH2 and PxAKHR To investigate the functional relevance of these genes in the life cycle of the diamondback moth, this embodiment used qRT-PCR technology to detect the expression levels of these genes at different developmental stages of the diamondback moth (including the egg stage, 1st-4th instar larval stage, pupal stage, and adult stage).

[0028] 1. Sample collection and RNA extraction Samples of diamondback moths at different developmental stages were collected, and different tissue samples were isolated and collected. Total RNA was extracted according to the operating instructions of the TaKaRa RNAiso Plus animal and plant tissue total RNA extraction reagent.

[0029] 2. cDNA Synthesis The first strand of cDNA was synthesized according to the operating procedures of the Evo M-MLV reverse transcription kit (Hunan Aike Rui Biotechnology Co., Ltd.). This cDNA was used as a template for subsequent qPCR analysis.

[0030] 3. qRT-PCR detection Targeting the diamondback moth PxAKH1, PxAKH2 and PxAKHR qPCR primers were designed based on the gene sequences (e.g., SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3), and the sequences are shown in Table 1. Quantitative real-time PCR was performed using the SYBR Green Pro Taq HS premixed qPCR kit (Hunan Aike Rui Biotechnology Co., Ltd.).

[0031] Table 1 Primer Sequences for Quantitative PCR The results are as follows Figure 1 As shown: PxAKH1 , PxAKH2 and PxAKHR The genes exhibited specific expression patterns throughout the diamondback moth's development, demonstrating highly consistent spatiotemporal expression regularities. Specifically, the relative expression levels of these three genes remained at low basal levels during the larval (L1-L4) and pupal stages. However, as the diamondback moth developed into an adult, PxAKH1 , PxAKH2 and PxAKHR The transcriptional levels of all components were significantly upregulated. PxAKH1 Expression levels peaked during the adult stage, significantly higher than in all other developmental stages; similarly, PxAKH2 and PxAKHR The expression abundance in the adult stage was also significantly higher than that in the larval and pupal stages.

[0032] Example 2: Diamondback moth induced by Bt treatment Px AKH / Px Analysis of changes in AKHR expression To investigate the gene described in this invention ( PxAKH1 , PxAKH2 and PxAKHR To investigate the response mechanism of diamondback moth to Bt stress, diamondback moth larvae were treated with different concentrations of Bt suspension, and changes in gene expression levels at different time points (12 h, 24 h, 48 h, 72 h) after treatment were detected.

[0033] 1. Experimental design and sample collection and processing To investigate the effects of different concentrations of Bt suspension stress on key genes in the AKH / AKHR signaling pathway of diamondback moth (Prunella vulgaris) PxAKH1 , PxAKH2 and PxAKHR To investigate the effects of Bt toxin on the expression of these genes, this study treated third-instar larvae with gradient concentrations of Bt toxin and examined the transcriptional dynamics of these genes.

[0034] The experiment used healthy third-instar diamondback moth larvae of uniform growth and set up four experimental groups: each group was fed with the original concentration (1×10⁻⁶). 8 Artificial diets containing Bt suspensions at concentrations of 100 cfu / mL, 5-fold dilution, 10-fold dilution, and 20-fold dilution were prepared, with an equal volume of Bt-free artificial diet serving as a control. Larval samples were collected at 12 h, 24 h, 48 h, and 72 h after treatment. Each concentration-time combination had three biological replicates, with at least 20 larvae per replicate.

[0035] 2. Gene expression detection Total RNA was extracted using TaKaRa's RNAiso Plus total RNA extraction reagent from animal and plant tissues, and then reverse transcribed to synthesize the first strand of cDNA. RpL32 This is an internal reference gene, detected by qRT-PCR. PxAKH1 , PxAKH2 and PxAKHR The relative expression levels of the genes were determined. The qRT-PCR reaction system (10 μL) contained 5 μL of 2 × SYBR Green Pro Taq HS Premix, 0.5 μL each of forward and reverse primers, 0.5 μL of cDNA template, and 3.5 μL of ddH2O; the reaction program was 95℃ pre-denaturation for 30 s, followed by 40 cycles (95℃ denaturation for 5 s, 60℃ annealing and extension for 30 s). The relative expression levels of the genes were determined according to a 2... ΔΔCt Calculation by method.

[0036] Experimental results are as follows Figure 2 As shown, PxAKH1 , PxAKH2 and PxAKHR Gene expression levels showed a significant concentration- and time-dependent induction effect on Bt treatment.

[0037] Time-effect analysis: In the early stages after Bt treatment (12 h and 24 h), the expression levels of the target gene in each experimental group were not significantly different from those in the control group (CK). However, with the extension of treatment time, especially after 48 h, the expression level of the target gene began to show an upward trend; at 72 h, this induction effect reached a highly significant level. Heatmap analysis (Figure DF) visually shows that the 72 h treatment group exhibited significantly high expression, indicating that prolonged Bt stress strongly activated the AKH signaling pathway.

[0038] Concentration effect analysis: Over 72 h, the concentration of Bt suspension increased (from Bt5 to Bt20). PxAKH1 , PxAKH2 and PxAKHR The relative expression levels of these substances showed a significant dose-dependent increase. For example, in the high-concentration Bt treatment groups (Bt10, Bt20, and Bt), PxAKH1 and PxAKHR The expression level was increased by several to dozens of times compared with the control group.

[0039] In summary, the experimental data indicate that the AKH signaling pathway is a key response system for diamondback moth to Bt toxin stress. Bt infection leads to a sharp increase in energy metabolism demand or tissue damage in diamondback moths, thereby specifically inducing high expression of AKH and its receptor genes to mobilize energy reserves.

[0040] Example 3: Targeting the diamondback moth PxAKH / PxAKHR dsRNA synthesis and preparation for gene expression Diamondback moth obtained from the GenBank database on the NCBI website. PxAKH1 , PxAKH2 and PxAKHR The cDNA sequence of the gene was used to design and synthesize primer sequences (see Table 2 for the primer list) and corresponding specific double-stranded RNA (dsRNA). A T7 promoter sequence was added to the 5' end of both the upstream and downstream primers. When designing primers, care was taken to avoid complete overlap between the amplification region of the quantitative PCR primer and the product region of the dsRNA. Additionally, dsRNA targeting the Enhanced Green Fluorescent Protein (EGFP) gene was used as a negative control. All dsRNAs were generated using T7RiboMA. TM The RNAi System kit (Promega) was synthesized, purified, and dissolved in sterile DEPC water for later use; among which, ds PxAKH1 ds PxAKH2 and ds PxAKHRThe nucleotide sequences of the positive strand are shown in SEQ ID NO:20~SEQ ID NO:22.

[0041] Table 2 dsRNA synthesis primer sequences Example 4 Interference PxAKH / PxAKHR Formulation of gene-based pesticides for controlling diamondback moth 1. Active ingredients of the drug The active ingredient of the pharmaceutical preparation is the ds obtained in Example 3. PxAKH1 ds PxAKH2 and ds PxAKHR At least one of them. When the active ingredient is a mixture of two dsRNAs, dsRNA is selected. PxAKH1 With ds PxAKHR A mixture, or ds PxAKH2 With ds PxAKHR The mixture contains two dsRNAs in a 1:1 mass ratio, meaning that under the same total dosage, the ligand gene dsRNA and the receptor gene dsRNA each account for half.

[0042] 2. Selection of adjuvants To facilitate field application and prolong the effective retention time of dsRNA at the application interface, the agent may also contain at least one of a surfactant, a thickener, and a stabilizer: (1) Surfactant: Selected from one or two of Tween-20, Tween-80 or organosilicon spreading agents, with a dosage of 0.01%~0.1% (w / v), preferably 0.05% (w / v). Its function is to reduce the surface tension of the liquid and improve the wetting and spreading of the liquid on the waxy leaf surface of cabbage and other insect bodies.

[0043] (2) Thickener: Selected from one or two of xanthan gum and sodium carboxymethyl cellulose, with a dosage of 0.05%~0.5% (w / v), preferably 0.1% (w / v). Its function is to increase the viscosity of the solution and reduce the loss of the solution on the leaf surface.

[0044] (3) Stabilizer: Selected from one or more of trehalose (2%~5%, w / v), EDTA (0.1~1.0 mmol / L) and sodium citrate buffer (5~20 mmol / L, pH 6.5~7.5). Among them, EDTA is used to chelate metal ions, and trehalose and buffer system are used to maintain the integrity of dsRNA during preparation and storage.

[0045] (4) The remainder is RNase-free water. The preparation process should be carried out under RNase-free conditions. The prepared reagent should be stored at 4°C for a short period or at [other conditions]. Store at 20℃ for extended periods.

[0046] 3. Formula interference PxAKH / PxAKHR Formula 1 for gene-based control of diamondback moth: dsPxAKHR 1.0 mg / mL, balance: RNase-free water Depending on the specific implementation, other substances may be added: Tween-20: 0.05% (w / v), xanthan gum: 0.1% (w / v), trehalose: 3% (w / v), EDTA: 0.5 mmol / L, sodium citrate buffer: 10 mmol / L (pH 7.0).

[0047] interference PxAKH / PxAKHR Gene-based pesticide formulation for diamondback moth control: ds PxAKHR It can also be replaced with an equal amount dsPxAKH1 .

[0048] interference PxAKH / PxAKHR Gene-based pesticide formulation for diamondback moth control: ds PxAKHR It can also be replaced with an equal amount dsPxAKH2 .

[0049] interference PxAKH / PxAKHR Gene-based pesticide formulation for controlling diamondback moth (Prunella vulgaris): dsPxAKH1 0.5 mg / mL and dsPxAKHR: 0.5 mg / mL (mass ratio 1:1), the other components and dosages are the same as those in formulation 1.

[0050] interference PxAKH / PxAKHR Gene-based pesticide formulation for diamondback moth control (Formula 5): ds PxAKH2 0.5 mg / mL and ds PxAKHR : 0.5 mg / mL (mass ratio 1:1), the remaining components and dosages are the same as those in drug 1.

[0051] The above-mentioned agents can be diluted with RNase-free water to the required application concentration before use. When using micro-injection, surfactants and thickeners can be omitted, and the agent can be prepared using only sterile DEPC water or a buffer system containing stabilizers to avoid additional mechanical or chemical irritation to the larvae.

[0052] Example 5: Target gene interference efficiency of the above-mentioned pesticides 1-3 for controlling diamondback moth and their synergistic effect on the insecticidal activity of Bacillus thuringiensis. 1. Application schemes and treatment designs for agents 1-3 Third-instar diamondback moth larvae of uniform development were selected and randomly divided into ds PxAKH1 Processing group, ds PxAKH2 Processing group, ds PxAKHR Processing group and ds EGFPThe negative control group consisted of three biological replicates per group. 500 ng of the corresponding dsRNA was injected into the larvae of each treatment group using a microsyringe. a.ds PxAKHR Treatment group: Injection of agent 1 (agent 1 contains 500 ng of...) into the larvae dsPxAKHR ); b.ds PxAKH1 Treatment group: Injection of agent 2 (agent 2 containing 500 ng) into the larvae dsPxAKH1 ); c.ds PxAKH2 Treatment group: Injection of agent 3 (agent 3 containing 500 ng) into the larvae dsPxAKH2 ); d.ds EGFP Negative control group: injected with an equal volume of ds EGFP .

[0053] After injection, all treated larvae were placed in an artificial climate chamber under standard conditions for continued rearing.

[0054] 2. Determination of the interference efficiency of drugs 1-3 on target genes Larval samples were collected from each group at 24 h, 48 h, and 72 h post-injection, and were flash-frozen in liquid nitrogen and then stored. An 80℃ refrigerator was used for subsequent total RNA extraction from diamondback moth and detection of target gene interference efficiency.

[0055] The results are shown in Figure 3, compared with the control group ( dsEGFP Compared to other treatments, all dsRNA treatment groups significantly reduced the mRNA expression levels of the corresponding target genes, achieving significant inhibition at both 48 h and 72 h, with the highest interference efficiency at 48 h. This result confirms that the RNAi system of pesticides used to control diamondback moth can efficiently and specifically silence the AKH signaling pathway genes in diamondback moth.

[0056] 3. Determination of the synergistic effect of pesticides 1-3 on the insecticidal activity of Bt 3.1 Determination of cumulative mortality rate in each treatment group at the diagnostic dose of Bt To assess the impact of AKH signaling pathway gene silencing on Bt sensitivity, surviving larvae from each treatment group were selected 24 h after drug injection and treated with Bt suspension. Mortality was monitored for 7 consecutive days. Details are as follows: Transfer the larvae to a location containing 1×10 8The larvae were fed on cabbage leaves treated with a Bt suspension at cfu / mL, with cabbage leaves without Bt serving as a control. Larval mortality was recorded for 7 consecutive days after the start of Bt treatment, and the cumulative mortality rate was calculated. Changes in larval feeding, growth, and development were also observed.

[0057] The results are as follows Figure 4 As shown, with ds EGFP In comparison, all gene interference groups (ds PxAKH1 ds PxAKH2 and ds PxAKHR The mortality rates of the control group and the interference group were significantly higher than those of the control group. PxAKH1 , PxAKH2 and PxAKHR All genes significantly increased the susceptibility of diamondback moth to Bt. Specifically, after Bt treatment, ds EGFP The mortality rate in the control group gradually increased over time, reaching approximately 90% at day 7. The interference groups exhibited higher mortality rates in the early stages (days 1-5), but after day 5, the mortality rates in all groups approached 100%. This indicates that silencing the AKH signaling pathway gene accelerates Bt-induced death in diamondback moths. Inhibiting this pathway via RNAi can effectively enhance the insecticidal efficiency of Bt toxins and shorten the lethal time, providing strong experimental evidence for the development of RNAi-based Bt synergists.

[0058] 3.2 LC-12 of each treatment group under Bt gradient concentrations 50 Measurement To further evaluate the effect of gene interference with the AKH signaling pathway on the susceptibility of diamondback moth to Bt, diamondback moth larvae were treated with Bt suspensions of varying concentrations after interference to determine the median lethal concentration (LC50) of Bt in each treatment group. 50 ), as detailed below: In the bioassay, in addition to using the Bt diagnostic dose, concentrations of 1×10⁻⁶ were also prepared. 5 1×10 6 1×10 7 1×10 8 and 1×10 9 A Bt suspension of CFU / mL was used to determine the gradient toxicity of larvae in each treatment group. After the experiment, the median lethal concentration (LC50) of Bt for each treatment group was calculated using probabilistic unit analysis. 50 The study analyzed the relevant toxicity parameters and compared the differences in Bt sensitivity among different treatment groups to conduct an analysis. PxAKH1 , PxAKH2 and PxAKHR Function of genes in the diamondback moth Bt response process.

[0059] The results are shown in Table 3. Compared with the negative control group, the interference ds PxAKH1ds PxAKH2 and ds PxAKHR All genes significantly reduced the LC of diamondback moth to Bt. 50 The values, and their 95% confidence intervals are consistent with ds EGFP The fact that none of them overlap indicates that the differences are significant. Specifically, ds EGFP LC50 of the control group of diamondback moth 50 The value is 6.09 × 10 7 cfu / mL, while ds PxAKH1 ds PxAKH2 and ds PxAKHR LC of the processing group 50 The values ​​decreased to 1.06 × 10⁻⁶. 7 6.67×10 6 and 5.67×10 6 cfu / mL. Corresponding relative lethal concentration (LCR) 50 The figures were 5.72, 9.14, and 10.74 times respectively, indicating that silence... PxAKH1 , PxAKH2 and PxAKHR The genes increased the diamondback moth's susceptibility to Bt by 5.72 times, 9.14 times, and 10.74 times, respectively.

[0060] Table 3. Effects of RNA interference AKH signaling pathway genes on the susceptibility of diamondback moth to Bt. Example 6: The co-interference efficiency of the target genes of the above-mentioned pesticides 4-5 for controlling diamondback moth and their synergistic effect on the insecticidal activity of Bacillus thuringiensis. 1. Application plan and treatment design for agents 4-5 To further verify the effect of AKH ligand-receptor synergistic silencing on enhancing the sensitivity of diamondback moth to Bacillus thuringiensis (Bt), a co-interference experiment was conducted using agents 4-5 for controlling diamondback moth, based on the RNA interference system established in Example 5. The changes in target gene expression and Bt bioactivity after co-interference were then detected.

[0061] Healthy third-instar diamondback moth larvae of uniform development were selected and randomly divided into ds PxAKH1 +ds PxAKHR Common interference group, ds PxAKH2 +ds PxAKHR Common interference group and ds EGFP The negative control group had three biological replicates per group. a.ds PxAKH1 +ds PxAKHR Co-interference group: Injection of agent 4 (agent 4 contains 500 ng of dsRNA) into larvae , wherein, dsPxAKH1 and ds PxAKHR 250 ng each); b.ds PxAKH2 +ds PxAKHR Co-interference group: Injection of agent 5 (containing 500 ng of dsRNA) into larvae , wherein, ds PxAKH2 and ds PxAKHR 250 ng each); c.ds EGFP Negative control group: injected with an equal volume of ds EGFP .

[0062] 2. Determination of the co-interference efficiency of the drug on 4-5 pairs of target genes: After injection, larvae in each treatment group were placed in an artificial climate chamber under standard conditions for further rearing. Larval samples were collected at 24 h, 48 h, and 72 h post-injection, and then flash-frozen in liquid nitrogen and stored. An 80℃ freezer was used for total RNA extraction and common interference efficiency detection. Total RNA was extracted using TaKaRa's RNAiso Plus animal and plant tissue total RNA extraction reagent, and cDNA first strand was synthesized by reverse transcription. RpL32 As an internal reference gene, it was detected using the qRT-PCR primers listed in Table 1. PxAKH1 , PxAKH2 and PxAKHR The relative expression level. The relative gene expression level is calculated in 2... ΔΔCt The expression changes of each target gene in the co-interference group and the single-gene interference group were compared and analyzed. PxAKH and its receptors PxAKHR The interference effect of co-silencing and its impact on the expression of pathway-related genes.

[0063] The results are as follows Figure 5 As shown, with ds EGFP Compared with the negative control group, the transcription levels of the corresponding target genes in the co-interference group were significantly reduced, indicating that the established co-RNA interference system can effectively silence AKH and its receptor genes simultaneously.

[0064] Among them, in ds PxAKH1 +ds PxAKHR In the common interference group, PxAKH1 and PxAKHR The expression levels of [a specific substance] were significantly downregulated in both the control and control groups from 24 to 72 hours post-injection; in ds PxAKH2 +ds PxAKHR In the common interference group, PxAKH2 and PxAKHRThe expression levels of AKH signaling pathway-related genes were significantly downregulated between 24 and 72 hours post-injection compared to the control group. Further comparison revealed that the co-interference group, compared to the corresponding single-gene interference group, exhibited either a further enhanced inhibitory effect on AKH signaling pathway-related genes or maintained at a lower expression level. This indicates that by simultaneously inhibiting both the AKH ligand and its receptor, the AKH signaling pathway's response to Bt stress can be more stably weakened.

[0065] 3. Determination of the synergistic effect of pesticides 4-5 on the insecticidal activity of Bt Based on the RNA co-interference efficiency test results, 48 ​​h post-injection was the time point with the most significant target gene silencing effect. Therefore, larvae surviving 48 h post-injection were selected for Bt bioactivity assays. In the bioassay, concentrations of 1×10⁻⁶ were prepared. 5 1×10 6 1×10 7 1×10 8 and 1×10 9 A Bt suspension of CFU / mL was used to determine the gradient toxicity of larvae in each treatment group. After the experiment, the median lethal concentration (LC50) of Bt for each treatment group was calculated using probabilistic unit analysis. 50 The study investigated the Bt sensitivity of the diamondback moth and related virulence parameters, and compared the differences in Bt sensitivity between the co-interference group, the single-gene interference group, and the negative control group. Px AKH and its receptor Px The potential of AKHR combined with silence in Bt enhancement.

[0066] The results are shown in Table 4: ds PxAKH1 +ds PxAKHR Common interference group and ds PxAKH2 +ds PxAKHR LC of the common interference group 50 They are 4.67×10 6 cfu / mL and 3.88×10 6 cfu / mL, corresponding relative lethal concentration (LCR) 50 The increases were 11.59 times and 13.97 times, respectively. These results indicate that through joint interference... PxAKH1 or PxAKH2 and PxAKHR The gene can further enhance or stabilize the effect of improving the sensitivity to Bt in diamondback moth, providing further experimental evidence for Bt enhancement technology based on the AKH signaling pathway.

[0067] Table 4. Effects of combined interference from AKH1 / AKH2 and AKHR on the Bt susceptibility of diamondback moth. All other parts not described in detail are existing technologies. Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, not all embodiments. Those skilled in the art can obtain other embodiments based on these embodiments without creative effort.

Claims

1. A method for targeting the diamondback moth's body PxAKH / PxAKHR dsRNA for gene expression is characterized by: The dsRNA is ds PxAKH1 ds PxAKH2 and ds PxAKHR Any one of them; The ds PxAKH1 The nucleotide sequence of the positive strand is shown in SEQ ID NO.20; The ds PxAKH2 The nucleotide sequence of the positive strand is shown in SEQ ID NO.21; The ds PxAKHR The nucleotide sequence of the positive strand is shown in SEQ ID NO.

22.

2. The dsRNA according to claim 1, characterized in that: The ds PxAKH1 Using the sequence shown in SEQ ID NO.1 as a template, and through primer pair ds PxAKH1 The DNA fragment obtained by -F / R amplification is transcribed and annealed in vitro, and the primer pair dsPxAKH1-F / R is as follows: ds PxAKH1 -F: taatacgactcactataggGTTTGGTGATCGTCAGTGAGG, as shown in SEQ ID NO.12; ds PxAKH1 -R: taatacgactcactataggCATGATTTCTGGCATAGAAGAAGTCT, as shown in SEQ ID NO.13; The ds PxAKH2 Using the sequence shown in SEQ ID NO.2 as a template, and through primer pair ds PxAKH2 -F / R amplification yields DNA fragments that are transcribed and annealed in vitro, and primer pairs ds PxAKH2 -F / R are respectively: ds PxAKH2 -F: taatacgactcactataggATGTTGGTGTTGGCAGACG, as shown in SEQ ID NO.14; ds PxAKH2 -R: taatacgactcactataggTCACTTCTGCTGGCAGGC, as shown in SEQ ID NO.15; The d sPxAKHR Using the sequence shown in SEQ ID NO.3 as a template, and through primer pair ds PxAKHR -F / R amplification yields DNA fragments that are transcribed and annealed in vitro, and primer pairs ds PxAKHR -F / R are respectively: ds PxAKHR- F: taatacgactcactataggAGCTGTCGCCGATCTTATGG, as shown in SEQ ID NO.16; ds PxAKHR- R: taatacgactcactataggGCCGTAGGATACGCATTGGA, as shown in SEQ ID NO.

17.

3. The method described in claim 2 for targeting the diamondback moth's body PxAKH / PxAKHR A method for preparing dsRNA for gene expression, characterized in that: Includes the following steps: (1) Using diamondback moths respectively PxAKH1, PxAKH2 or PxAKHR Using the cDNA sequence of the gene as a template, PCR amplification was performed using primer pairs dsPxAKH1-F / R, dsPxAKH2-F / R, or dsPxAKHR-F / R with the T7 promoter sequence added to the 5′ end, to obtain a DNA template with the T7 promoter sequence at both ends. (2) The DNA template obtained in step (1) was used for in vitro transcription to obtain the sense strand and the antisense strand, respectively; (3) The sense and antisense strands obtained in step (2) are annealed to form double-stranded RNA, which is then purified and dissolved in sterile DEPC water to obtain ds PxAKH1 ds PxAKH2 or ds PxAKHR .

4. A type of interference PxAKH / PxAKHR A gene-based pesticide for controlling the diamondback moth, characterized by: The active ingredient of the pharmaceutical preparation is at least one of the dsRNAs described in claim 1.

5. The pharmaceutical preparation according to claim 4, characterized in that: In the aforementioned pharmaceutical preparation, the active ingredient is ds PxAKH1 With ds PxAKHR A mixture, or an active ingredient dsPxAKH2 With ds PxAKHR The mixture contains two dsRNAs in a 1:1 mass ratio.

6. The pharmaceutical agent according to claim 4 or 5, characterized in that: The agent also contains at least one of surfactants, thickeners, and stabilizers.

7. The application of the agent according to any one of claims 4 to 6 in enhancing the insecticidal activity of Bacillus thuringiensis against diamondback moth.

8. A combination drug that enhances the insecticidal activity of Bacillus thuringiensis against diamondback moth, characterized in that: The combined drug comprises a first component and a second component packaged separately, wherein the first component is the agent as described in claim 4, and the second component is a Bacillus thuringiensis suspension.

9. The combination drug according to claim 8, characterized in that, The viable cell concentration of the Bacillus thuringiensis suspension is 1×10⁻⁶. 5 cfu / mL to 1×10 9 cfu / mL.

10. The application of the combination drug as described in claim 8 or 9 in the control of diamondback moth, characterized in that: The application method involves administering 500 ng of the dsRNA to each third-instar larva of the diamondback moth, followed by treatment with Bacillus thuringiensis suspension for 24-48 hours after administration.

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