A method and system for controlling blattella germanica based on nanoparticle delivery of lethal gene dsrna

Abstract

The application belongs to but is not limited to the technical field of Blattella germanica prevention and treatment, and discloses a Blattella germanica prevention and treatment method and system based on nanoparticle delivery of lethal gene dsRNA, which comprises the following steps: Blattella germanica lethal gene full-length cloning; plasmid construction and dsRNA expression and purification; and RNAi feeding. The application first confirms an efficient RNAi lethal target point for Blattella germanica, ensures the efficiency of the technical scheme, realizes low-cost and large-scale preparation of an active ingredient of an RNAi insecticide, breaks through the cost bottleneck of commercial application, creatively constructs a nanocomposite delivery system, efficiently overcomes the technical bottleneck of oral delivery of RNAi, and provides an environment-friendly practical prevention and treatment scheme, and has important application value and prospect.

Description

Technical Field

[0001] This invention belongs to, but is not limited to, the field of German cockroach control technology, and particularly relates to a method and system for controlling German cockroaches based on the delivery of lethal gene dsRNA using nanoparticles. Background Technology

[0002] The German cockroach (Blattella germanica) is recognized worldwide as one of the most important urban sanitation pests. Due to its strong environmental adaptability, astonishing reproductive capacity, and secretive lifestyle, this pest is widely distributed in indoor spaces where people live and work, such as residential kitchens, restaurants, hospitals, and food processing plants. The German cockroach not only contaminates food, water sources, and object surfaces through feeding and activity, but also acts as a mechanical vector for various pathogenic microorganisms (such as Salmonella and Shigella), posing a serious threat to public health. Furthermore, its secretions, excrement, and carcass debris are potent allergens, significantly contributing to the triggering and exacerbation of diseases such as asthma and allergic rhinitis.

[0003] Currently, the main method of controlling German cockroaches is still chemical insecticides, such as the widespread use of sprays, poison baits or powders containing effective ingredients such as pyrethroids, organophosphates, and neonicotinoids. However, existing chemical control technologies have the following insurmountable defects: (1) Increasingly serious resistance problem: Due to the short reproductive cycle and high genetic diversity of German cockroaches, under long-term and high-intensity selection pressure of chemical agents, their population has developed high resistance to many mainstream insecticides, resulting in a sharp reduction in the effectiveness of conventional dosages of agents, or even complete ineffectiveness, making control work increasingly difficult. (2) Environmental and health safety hazards: The residues of chemical insecticides in the indoor environment pose a potential threat to human health, especially to sensitive groups such as children and the elderly. At the same time, their production and use processes may also cause harm to non-target organisms, which does not meet the current social requirements for green and sustainable development. (3) Continuously rising control costs: In order to cope with pest resistance, it is often necessary to increase the dosage of pesticides, increase the frequency of application, or replace them with more expensive new agents, which directly leads to a significant increase in control costs.

[0004] To address the technical challenges posed by the aforementioned chemical control methods, researchers in this field have begun seeking alternative technologies with novel mechanisms of action. RNA interference (RNAi) technology, a biological mechanism that precisely "shuts down" the expression of specific genes at the post-transcriptional level by specifically inducing the degradation of target messenger RNA (mRNA) through double-stranded RNA (dsRNA) molecules, has shown great potential as a next-generation green pest control technology. Its advantages include flexible target selection, strong species specificity, and no risk of cross-resistance with traditional insecticides.

[0005] Despite the promising prospects of RNAi technology, its development into a practically applicable product for controlling German cockroaches still faces the following key technical bottlenecks: (1) Low stability and delivery efficiency of dsRNA: Naked dsRNA molecules are easily degraded by nucleases in the environment. In particular, the digestive tract of German cockroaches contains highly active nucleases. Most of the dsRNA ingested orally is digested and decomposed before reaching the target tissue cells, failing to effectively exert gene silencing effects and resulting in extremely low bioavailability. (2) High production cost of dsRNA: The cost of preparing dsRNA through in vitro chemical synthesis or enzymatic reactions is extremely high, making it completely unfeasible for large-scale economic production as an agricultural or sanitary insecticide. Although bio-fermentation using engineered strains (such as Escherichia coli HT115) is an effective way to reduce the production cost of dsRNA, it does not solve the aforementioned problems of delivery efficiency and stability.

[0006] Based on the above analysis, the urgent technical problems that need to be solved in the existing technology are:

[0007] In existing technologies, on the one hand, chemical control faces severe resistance challenges and environmental safety pressures; on the other hand, RNAi technology, as an alternative, has not yet developed mature and efficient commercial products due to its two major bottlenecks: "delivery efficiency" and "production cost," especially how to protect dsRNA to effectively resist the digestive tract barrier of German cockroaches.

[0008] Therefore, there is an urgent need in this field for a novel biological control technology that combines low-cost dsRNA production technology with an efficient and stable delivery system to achieve highly effective eradication of German cockroaches via oral administration. Summary of the Invention

[0009] To address the problems existing in the prior art, this invention provides a method for controlling German cockroaches based on the delivery of lethal gene dsRNA using nanoparticles.

[0010] This invention is implemented as follows: a method for controlling German cockroaches based on the delivery of the lethal gene dsRNA using nanoparticles, characterized in that the method specifically includes:

[0011] S1: Full-length clone of the lethal gene of the German cockroach;

[0012] S2: Plasmid construction and dsRNA expression and purification;

[0013] S3: Feed RNAi.

[0014] Further, in step S1, the target gene sequence of the German cockroach is obtained and verified, and the steps are as follows:

[0015] (1) Total RNA was extracted from the tissue of German cockroaches using the TransZol Up Total RNA Isolation Kit (TransGen Biotech, Beijing, China) and the total RNA was reverse transcribed into the first strand of cDNA using the TransScript® One-Step gDNA Removal and cDNA Synthesis SuperMix (Transgen, Beijing, China) reverse transcription kit as a template.

[0016] (2) Using the cDNA as a template, polymerase chain reaction (PCR) amplification was performed using specific primers designed based on the transcriptome data of the German cockroach. The PCR reaction system contained cDNA, 2×Taq Plus Master Mix II, forward and reverse primers, and ddH2O was added to make up the volume. The reaction program was set as follows: pre-denaturation at 95℃ for 2 minutes; followed by 40 amplification cycles (denaturation at 95℃ for 30 seconds, annealing at 62℃ for 20 seconds, extension at 72℃ for 1 minute); and finally, terminal extension at 72℃ for 10 minutes.

[0017] (3) The amplified products were detected by 1% agarose gel electrophoresis. After confirming the target band, they were purified using FastPure® Gel DNA Extraction Mini Kit (Vazyme, Nanjing, China).

[0018] (4) The purified and recovered DNA fragments were ligated into pEASY®-T5 Zero Cloning Kit (TransGenBiotech, Beijing, China), and the ligation product was transformed into Trans-T1 competent cells (Transgene, Beijing, China). The transformed bacterial culture was plated on LB solid medium containing 100 mg / mL ampicillin and cultured overnight to screen for positive clones;

[0019] (5) Selected positive single colonies were sent to Chongqing Qingke Biotechnology Co., Ltd. for sequence verification, and the sequencing results were compared with the NCBI database to ensure the accuracy of the obtained gene fragments;

[0020] (6) Extract plasmids containing the target gene from the verified strains according to the FastPure® Plasmid Mini Kit (Vazyme, Nanjing, China) for subsequent experiments.

[0021] Further, in step S2, to achieve large-scale expression of dsRNA, 12 gene fragments that had previously been correctly sequenced and verified, namely the Bgpp-α fragment (570 bp), Bginr-a fragment (502 bp), Bghsc70-3 fragment (524 bp), Bggw fragment (583 bp), Bgα-SNAP fragment (521 bp), Bgcact fragment (618 bp), Bgsrp54k fragment (433 bp), Bgrop fragment (624 bp), Bgshi fragment (576 bp), Bgrpt3a fragment (324 bp), Bgrpt3b fragment (440 bp), and Bgrpn7 fragment (460 bp), were cloned into the pET-2P dsRNA expression vector. This process was performed using a seamless assembly cloning kit (CloneSmarter, Irvine). A recombinant vector was constructed in the USA and first transformed into DH5α cells for verification. After confirming positive clones, plasmids were extracted, and the recombinant plasmid containing the target gene was then transformed into HT115 competent cells. Engineered strains capable of successfully expressing the target dsRNA were obtained by screening on LB agar plates containing 50 mg / mL kanamycin and 12.5 mg / mL tetracycline hydrochloride. The obtained engineered strains were then transferred to LB liquid medium containing the corresponding antibiotics and cultured at 37°C and 250 rpm for approximately 3 hours until the bacterial culture reached OD500. 600The concentration reached 1; subsequently, IPTG was added to a final concentration of 500 mM for induction of expression, and cultured for another 10-12 hours under the same conditions; after culture, dsRNA was extracted using the phenol-chloroform method. Specifically, bacterial cells were collected by centrifugation at 10,000 g for 2 minutes at 4°C, and the bacterial pellet was resuspended in STE buffer (Solarbio, Shanghai, China) at a ratio of 100:1, and an equal volume of phenol / chloroform RNA extraction buffer (25:24:1, pH<5, Zoman Biotechnology, Beijing, China) was added; after vigorous vortexing for 3 minutes, centrifugation was performed at 15,000 g for 15 minutes at 4°C. Collect the supernatant, add an equal volume of isopropanol, mix well, and incubate at room temperature for 10 minutes to precipitate RNA. Then, centrifuge at 12,000 g for 10 minutes at 4°C to collect the precipitate. Wash the precipitate twice with 75% ethanol (centrifuge at 10,000 g for 2 minutes each time at 4°C), dry at room temperature for 5 minutes, and dissolve in RNase-free water. Incubate the resulting solution at 37°C for 15 minutes, and then heat-treat it in a water bath at 72°C for 10 minutes. Finally, analyze the product on a 1% agarose gel and quantify the band intensity using ImageJ v1.80 (NIH, Maryland, USA) software to determine the dsRNA concentration. Store the prepared sample at -80°C for later use.

[0022] Furthermore, step S3 includes:

[0023] (1) The lethal gene dsRNA obtained from the above bacterial culture expression and dsGFP as a control were combined with a star-shaped cationic polymer nanomaterial SPc (a gift from China Agricultural University, with a concentration of 58.57 mg / mL) according to the electrophoresis gray-scale quantitative results. The SPc to dsRNA mass ratio was 1.5:1 to prepare a 50 µL complex, so that the final concentration of dsRNA reached 1000 ng / µL and the final concentration of SPc reached 1500 ng / µL.

[0024] (2) The nanocomposite preparation was used in a feeding experiment on 1st-instar German cockroach nymphs.

[0025] Furthermore, the nanocomposite preparation was used in a feeding experiment on first-instar German cockroach nymphs. The specific procedure was as follows: 40 mg of rat food was soaked in 50 µL of the above mixture. After the liquid was completely absorbed, it was transferred as poisoned bait into a glass tube (80 cm long × 30 cm wide × 40 cm high) for feeding the nymphs. The experiment set up multiple treatment groups with dsGFP / SPc as the control group. Each treatment group contained 20 nymphs, and 6 replicates were performed. During the experimental period, the rat food containing fresh compound was replaced for the nymphs every day. The experimental results were evaluated in two ways. Three replicates were used to measure and record lethal effects every 24 hours over 40 days. The remaining three replicates were used after 20 days of continuous feeding. Total RNA was extracted from surviving nymphs, and qPCR was performed using the PerfectStar® Green qPCR SuperMix kit (Transgene, Beijing, China) with Elongation factor-1-alpha (Efla) and Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a reference to assess gene silencing efficiency. The qPCR reaction volume was 10 µL, containing 5 µL of 2×PerfectStart® Green qPCR SuperMix, 2 µL of cDNA, 0.2 µL each of forward and reverse primers (10 µM), with the remaining volume made up by ddH2O. The reaction was performed on a Bio-iQ5 qPCR instrument (Bio-Rad, USA) under the following conditions: 94℃ for 30 seconds; followed by 50 cycles (94℃ for 5 seconds, 60℃ for 30 seconds); finally, the temperature was increased to 55℃. The steps take 60 seconds.

[0026] Based on the above technical solutions and the technical problems solved, the advantages and positive effects of the technical solution to be protected by this invention are as follows:

[0027] First, this invention is the first to confirm highly effective RNAi lethal targets against the German cockroach, ensuring the high efficiency of the technical solution. This invention systematically evaluated the lethal effects of 12 candidate genes as RNAi targets against the German cockroach via oral delivery, and screened and confirmed four highly effective lethal gene targets: Bgsrp54k, Bgrpt3a, Bgrpt3b, and Bgrpn7. This provides a validated, highly lethal target sequence for the development of RNAi biopesticides against the German cockroach, solving the technical problems of unclear targets or uncertain effects in existing technologies, and is the fundamental guarantee of the high efficiency of the technical solution of this invention.

[0028] This invention achieves low-cost, large-scale preparation of RNAi insecticide active ingredients, overcoming the cost bottleneck for commercial applications. The invention employs genetic engineering methods to construct the sequence of a highly efficient gene target into a high-expression vector (pET2p), and utilizes the *E. coli* HT115 expression system for fermentation production. This system can express high-quality dsRNA in large quantities and stably. Compared with existing methods relying on chemical synthesis or in vitro transcription kits, the biosynthesis method of this invention has a simplified operation process, shorter production cycle, and higher safety. More importantly, its production cost is significantly reduced, successfully solving the cost problem that restricts the large-scale application of RNAi technology.

[0029] This invention innovatively constructs a nanocomposite delivery system, efficiently overcoming the technical bottleneck of oral RNAi delivery. This invention combines bacterial expression-derived dsRNA with SPc nanomaterials to form a stable nano-formulation. The SPc nanomaterials, acting as a delivery carrier, provide an effective physical protective barrier for the internal dsRNA active ingredient, resisting degradation by highly active nucleases in the digestive tract of the German cockroach, significantly improving the stability and bioavailability of the dsRNA. This delivery system ensures that the active ingredient reaches the target cells intact and exerts its gene-silencing effect, thereby greatly improving the insecticidal efficiency of oral RNAi and solving the core problem of easy inactivation of naked dsRNA in existing technologies.

[0030] This invention provides an environmentally friendly and practical pest control solution with significant application value and promising prospects. The resulting nanocomposite formulation can be prepared as poison bait by soaking rodent food, and can be directly administered to control German cockroaches. This application method closely aligns with conventional pest control practices, making it simple and easy to implement. More importantly, this technical solution is based on the highly species-specific principle of RNAi, ensuring safety for humans, livestock, and non-target organisms. Furthermore, the active substance, dsRNA, can naturally degrade in the environment without leaving any residual pollution. Therefore, this invention not only provides a novel technological solution to the problem of German cockroach resistance but also offers the market a green, safe, and sustainable new option for pest management, with broad application prospects.

[0031] Secondly, the technical solution of this invention fills a technological gap in the industry both domestically and internationally:

[0032] (1) Filling the gap in highly efficient oral RNAi technology for cockroaches: Globally, how to achieve highly efficient RNAi for pests with strong digestive abilities, such as the German cockroach, via oral administration has been a technological gap that has not been effectively solved. This invention utilizes the SPc nanodelivery system to successfully achieve highly efficient oral delivery and lethality of dsRNA to the German cockroach for the first time, filling the gap in this key application area.

[0033] (2) Filling the gap in the lethal target gene library of German cockroach RNAi: This invention systematically screened and verified for the first time that four genes, Bgsrp54k, Bgrpt3a, Bgrpt3b, and Bgrpn7, are oral lethal targets of German cockroach. These highly effective targets, verified by in vivo oral experiments, are the basis for developing any related RNAi products and greatly enrich the core target gene library in this field.

[0034] (3) It fills the gap in the complete solution of RNAi insecticide from production to application: existing research focuses on a single point of target screening or delivery system. This invention integrates "low-cost biomanufacturing of dsRNA + high-efficiency nanodelivery system + poison bait application formulation" for the first time, providing a complete and industrializable technical solution from source to end, filling the gap in systematic application solutions in this field. Attached Figure Description

[0035] Figure 1 This is a flowchart of the German cockroach control method based on nanoparticle delivery of lethal gene dsRNA provided in an embodiment of the present invention;

[0036] Figure 2 This is a schematic diagram of plasmid construction and dsRNA expression and purification provided in the embodiments of the present invention;

[0037] Figure 3 This is a schematic diagram showing the survival rate, molting rate, and weight of German cockroaches after feeding treatment, provided in an embodiment of the present invention.

[0038] Figure 4 This is a schematic diagram of the relative gene expression levels after 20 days of feeding treatment provided in an embodiment of the present invention. Detailed Implementation

[0039] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0040] In the existing German cockroach control system, chemical pesticides have long dominated, with their mechanisms of action primarily relying on neurotoxins or metabolic inhibitors to disrupt the pest's life processes. However, the industrial application of these pesticides has gradually revealed a series of technical challenges, including excessive residues, accumulation of resistance, and increased ecological risks to non-target organisms. Particularly in residential areas, restaurants, and hospitals, the use of chemical pesticides is facing increasing policy and public scrutiny, directly limiting the industrial promotion and sustainable application of traditional methods.

[0041] To address this bottleneck, RNA interference technology offers a novel approach: silencing target genes specifically with exogenous double-stranded RNA to inhibit key physiological functions at the molecular level. However, in practical applications, naked double-stranded RNA is highly susceptible to degradation by nucleases in the environment and lacks stability in the insect digestive tract, leading to a significant decrease in silencing efficiency and hindering large-scale deployment. This issue has become a core obstacle to the industrialization of RNAi strategies.

[0042] To this end, this invention utilizes nanoparticles as a delivery carrier to form a stable complex with lethal double-stranded RNA and cationic polymer nanomaterials. Its physicochemical properties effectively encapsulate and protect the double-stranded RNA molecules, preventing their degradation during storage and ingestion. Simultaneously, the surface charge of the nanoparticles enhances their interaction with the cell membrane, enabling the double-stranded RNA to enter the intestinal epithelial cells of the German cockroach more efficiently, thus improving the efficiency of silencing the target gene.

[0043] The target gene was cloned and transformed into a bacterial expression system, yielding a large amount of double-stranded RNA through induction. After nanocompositing, these molecules remained active for an extended period within the poison bait carrier. When German cockroach nymphs ingested the bait, the complex was gradually released in the midgut environment. The double-stranded RNA was absorbed by the cell and entered the RNA-induced silencing complex, thereby triggering the specific degradation of the lethal gene mRNA. This mechanism disrupts essential physiological pathways for insect life at the molecular level, resulting in highly efficient lethality.

[0044] Unlike traditional chemical agents, this method's targeting and designability allow for combined attacks on multiple genes, thus avoiding the rapid development of resistance to single targets. Furthermore, double-stranded RNA poses lower non-specific risks to mammals and the environment, offering high safety and sustainability for industrial applications. The process primarily involves bacterial expression and routine purification, making it relatively simple to operate and feasible for large-scale production.

[0045] Therefore, this method solves the core problem that existing technologies struggle to overcome in industrial applications: how to maintain high-efficiency pest control while simultaneously ensuring environmental safety and resistance management. By achieving stable, efficient, and controllable in vivo release of double-stranded RNA through a nanoparticle delivery platform, this invention not only provides a novel pest control pathway but also offers reliable technical support and theoretical basis for the future development of green pest control products.

[0046] like Figure 1 As shown, this embodiment of the invention provides a method for controlling German cockroaches based on the delivery of lethal gene dsRNA using nanoparticles. The method specifically includes:

[0047] S1: Full-length clone of the lethal gene of the German cockroach;

[0048] S2: Plasmid construction and dsRNA expression and purification;

[0049] S3: Feed RNAi.

[0050] In step S1, to obtain and verify the target gene sequence of the German cockroach, total RNA was first extracted from German cockroach tissue using the TransZol Up TotalRNA Isolation Kit (TransGen Biotech, Beijing, China). The total RNA was then reverse transcribed into cDNA first strand using the TransScript® One-Step gDNA Removal and cDNA Synthesis SuperMix kit (Transgen, Beijing, China) as a template. Subsequently, using this cDNA as a template, polymerase chain reaction (PCR) amplification was performed using specific primers designed based on German cockroach transcriptome data. The PCR reaction system contained cDNA, 2×Taq Plus Master Mix II, forward and reverse primers, and ddH2O was used to make up the volume.

[0051] The reaction program was set as follows: pre-denaturation at 95℃ for 2 minutes; followed by 40 amplification cycles (denaturation at 95℃ for 30 seconds, annealing at 62℃ for 20 seconds, extension at 72℃ for 1 minute); and finally, a terminal extension at 72℃ for 10 minutes. The amplified products were detected by 1% agarose gel electrophoresis. After confirming the target band, purification was performed using the FastPure® Gel DNA Extraction Mini Kit (Vazyme, Nanjing, China). The purified DNA fragments were ligated into the pEASY®-T5 ZeroCloning Kit (TransGen Biotech, Beijing, China), and the ligation product was transformed into Trans-T1 competent cells (Transgene, Beijing, China). The transformed bacterial culture was plated on LB agar containing 100 mg / mL ampicillin and cultured overnight to screen for positive clones. Selected positive single colonies were sent to Chongqing Qingke Biotechnology Co., Ltd. for sequence verification, and the sequencing results were compared with the NCBI database to ensure the accuracy of the obtained gene fragments. Finally, plasmids containing the target gene were extracted from the validated strains using the FastPure® Plasmid Mini Kit (Vazyme, Nanjing, China) for subsequent experiments.

[0052] In step S2, to achieve large-scale expression of dsRNA, 12 gene fragments that were previously verified by sequencing, namely Bgpp-α (570 bp), Bginr-a (502 bp), Bghsc70-3 (524 bp), Bggw (583 bp), Bgα-SNAP (521 bp), Bgcact (618 bp), Bgsrp54k (433 bp), Bgrop (624 bp), Bgshi (576 bp), Bgrpt3a (324 bp), Bgrpt3b (440 bp), and Bgrpn7 (460 bp), were cloned into the pET-2P dsRNA expression vector. The procedure used a seamless assembly cloning kit (CloneSmarter, Irvine, USA) to construct recombinant vectors, which were first transformed into DH5α cells for verification. After confirming positive clones, plasmids were extracted, and the recombinant plasmids containing the target gene were then transformed into HT115 competent cells. Engineered strains capable of successfully expressing the target dsRNA were obtained by screening on LB agar plates containing 50 mg / mL kanamycin and 12.5 mg / mL tetracycline hydrochloride. The obtained engineered strains were then transferred to LB liquid medium containing the corresponding antibiotics and cultured at 37°C and 250 rpm for approximately 3 hours until the bacterial culture reached OD. 600The value reached 1. Subsequently, IPTG was added to a final concentration of 500 mM for induction of expression, and cultured for another 10-12 hours under the same conditions. After culture, dsRNA was extracted using the phenol-chloroform method. Specifically, bacterial cells were collected by centrifugation at 10,000 g for 2 minutes at 4°C, and the bacterial pellet was resuspended in STE buffer (Solarbio, Shanghai, China) at a ratio of 100:1, with an equal volume of phenol / chloroform RNA extraction buffer (25:24:1, pH<5, Zoman Biotechnology, Beijing, China). After vigorous vortexing for 3 minutes, the pellet was centrifuged at 15,000 g for 15 minutes at 4°C. The supernatant was collected, an equal volume of isopropanol was added, and the mixture was incubated at room temperature for 10 minutes to precipitate RNA, followed by centrifugation at 12,000 g for 10 minutes at 4°C to collect the pellet. The precipitate was washed twice with 75% ethanol (centrifuged at 10,000 g for 2 minutes each time at 4°C), dried at room temperature for 5 minutes, and then dissolved in RNase-free water. The resulting solution was incubated at 37°C for 15 minutes, followed by heat treatment in a 72°C water bath for 10 minutes. Finally, the product was analyzed on a 1% agarose gel, and the band intensity was quantified using ImageJ v1.80 (NIH, Maryland, USA) software to determine the dsRNA concentration. The prepared samples were stored at -80°C for later use.

[0053] S3, for oral RNAi bioassay, firstly, the lethal gene dsRNAs obtained from the aforementioned bacterial culture expression and dsGFP as a control were combined with a star-shaped cationic polymer nanomaterial SPc (a gift from China Agricultural University, concentration 58.57 mg / mL) based on the electrophoretic gray-scale quantification results. A 50 µL complex was prepared at a SPc:dsRNA mass ratio of 1.5:1, resulting in a final dsRNA concentration of 1000 ng / µL and a final SPc concentration of 1500 ng / µL. This nanocomposite preparation was used in a feeding experiment on first-instar German cockroach nymphs. Specifically, 40 mg of rat food was soaked in 50 µL of the above mixture. After complete absorption, the mixture was transferred as poisoned bait to glass tubes (80 cm long × 30 cm wide × 40 cm high) used to rear the nymphs. Multiple treatment groups were set up, with dsGFP / SPc as the control group. Each treatment group contained 20 nymphs, and six replicates were performed. During the experimental period, the nymphs were fed a fresh compound-containing rat diet daily. The experimental results were evaluated in two ways. Three replicates were used to measure and record lethal effects every 24 hours over 40 days. For the remaining three replicates, after 20 days of continuous feeding, total RNA was extracted from surviving nymphs, and qPCR was performed using the PerfectStar® Green qPCR SuperMix kit (Transgene, Beijing, China) with elongation factor-1-alpha (Efla) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as controls to assess gene silencing efficiency. The qPCR reaction volume was 10 µL, containing 5 µL of 2×PerfectStart® GreenqPCR SuperMix, 2 µL of cDNA, 0.2 µL each of forward and reverse primers (10 µM), with the remaining volume made up by ddH2O. The reaction was performed on a Bio-iQ5 qPCR instrument (Bio-Rad, USA) under the following conditions: 94℃ for 30 seconds; followed by 50 cycles (94℃ for 5 seconds, 60℃ for 30 seconds); and finally, a step of 55℃ for 60 seconds was added.

[0054] The effectiveness of this invention was verified through bioassays on German cockroaches. In a 40-day oral feeding experiment, the negative control group fed with the dsGFP / SPc complex showed a survival rate as high as 96.77% and a molting rate of 93%, demonstrating that the SPc nanodelivery system itself has no adverse effect on the growth and development of German cockroaches, thus ensuring the reliability of the experimental system.

[0055] In contrast, all dsRNA treatments targeting the 12 endogenous lethal genes showed significant lethal effects and developmental inhibition. The four treatments targeting Bgsrp54k, Bgrpt3a, Bgrpt3b, and Bgrpn7 were the most effective, achieving 100% mortality within 40 days. The molting rates of nymphs were also severely inhibited to 62%, 33%, 32%, and 25%, respectively, indicating severe disruption to their survival and development. Furthermore, the formulations targeting Bghsc70-3 and Bgpp-α also exhibited strong lethal activity, with survival rates of only 5.00% and 6.67% after 40 days, respectively. The remaining treatments also resulted in varying degrees of mortality and decreased molting rates.

[0056] The efficacy of this invention was also confirmed at the physiological and molecular levels. After 20 days of continuous feeding, the average weight of nymphs in all treatment groups was significantly lower than that in the control group (3.8 mg), exhibiting strong growth inhibition, with the lowest weight being only 1.8 mg (dsBghsc70-3 group). Simultaneous qPCR results fundamentally confirmed the mechanism of action: the relative expression levels of target gene mRNA in all treatment groups were significantly downregulated (p < 0.01), confirming highly efficient gene silencing. Particularly noteworthy is the extremely high gene silencing efficiency of the dsBghsc70-3, dsBgsrp54k, and dsBgrpt3a treatment groups, which suppressed the relative expression levels of the target genes to only 0.0011-0.0041. Furthermore, all other treatment groups also showed excellent silencing effects; even in the treatment group with the lowest effect, the target gene expression level decreased to 0.1734, demonstrating the universally high efficiency of this technique.

[0057] In summary, the experimental data comprehensively demonstrate that the dsRNA-SPc nanocomposite formulation constructed in this invention can induce highly efficient and long-lasting gene silencing in German cockroaches via oral administration, thereby causing developmental arrest, growth inhibition, and ultimately achieving a high mortality rate, thus verifying the feasibility and efficiency of this oral delivery platform.

[0058] Table 1. German cockroach homologs identified as essential genes in the red-faced beetle.

[0059]

[0060] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any modifications, equivalent substitutions, and improvements made by those skilled in the art within the scope of the technology disclosed in the present invention, and within the spirit and principles of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A method for controlling German cockroaches based on the delivery of lethal double-stranded RNA using nanoparticles, characterized in that, Includes the following steps: S1. Full-length cloning of the lethal gene of the German cockroach; S2. Construct a plasmid containing a lethal gene and express and purify the double-stranded RNA; S3. The double-stranded RNA is delivered via nanoparticles and fed to the patient.

2. The method as described in claim 1, characterized in that, The steps of S1 include: extracting total RNA from German cockroach tissue, reverse transcribing it into cDNA, using the cDNA as a template for specific polymerase chain reaction amplification, detecting and recovering the amplification product by agarose gel electrophoresis, ligating the recovered product into a cloning vector and transforming competent cells, screening positive clones and verifying their correctness by sequencing to obtain a plasmid containing the target gene.

3. The method as described in claim 1, characterized in that, The steps of S2 include: cloning the verified gene fragments into double-stranded RNA expression vectors, transforming them with bacteria and screening for positive clones, inducing double-stranded RNA expression using isopropyl thiogalactoside, extracting the double-stranded RNA using the phenol-chloroform method and purifying it by isopropanol precipitation and ethanol washing, and finally dissolving and heat-treating the double-stranded RNA sample.

4. The method as described in claim 1, characterized in that, Step S3 includes: combining the double-stranded RNA with a cationic polymer star-shaped nanomaterial, wherein the composite ratio is 1.5:1 by mass of cationic polymer to double-stranded RNA, to prepare a complex with a final concentration of 1000 nanograms per microliter of double-stranded RNA and a final concentration of 1500 nanograms per microliter of cationic polymer.

5. The method as described in claim 1, characterized in that, The further step of S3 is as follows: the nanocomposite is adsorbed onto rat food to form a poisoned bait for German cockroach nymphs to eat. Fresh poisoned bait is replaced daily, the mortality rate is recorded, and RNA of surviving individuals is extracted. The silencing effect of the target gene is detected by real-time quantitative polymerase chain reaction.

6. A double-stranded RNA delivery system for the lethal gene of the German cockroach used to implement the method of claim 1, characterized in that, include: Double-stranded RNA preparation unit, used to obtain and purify double-stranded RNA of the lethal gene of German cockroach; Nanocomposite units are used to combine the double-stranded RNA with cationic polymer nanomaterials to form a composite formulation; Feeding exposure unit for applying compound preparations to individual German cockroach nymphs.

7. The delivery system as claimed in claim 6, characterized in that, The double-stranded RNA preparation unit includes a plasmid construction module, a bacterial expression module, and an RNA extraction module. The RNA extraction module obtains double-stranded RNA through phenol-chloroform extraction, isopropanol precipitation, and ethanol washing.

8. The delivery system as claimed in claim 6, characterized in that, The nanocomposite unit can control the mass ratio of double-stranded RNA to cationic polymer nanomaterials and ensure that the final concentration of double-stranded RNA reaches 1000 nanograms per microliter.

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