Application of knockout or inhibition of insect Kir2A gene in insect ovicidal

Abstract

The invention discloses application of knockout or inhibition of an insect Kir2A gene in insect ovicidal, and belongs to the technical field of pesticide science. The invention provides an application of a gene for knocking out or inhibiting an insect Kir2A channel in insect ovicidal. By inhibiting the channel activity of the Kir2A or interfering the expression of the Kir2A gene, the egg abortion of the plutella xylostella is caused, and a new target is provided for developing an ovicide with a brand new action mechanism.

Description

Technical Field

[0001] This invention relates to the technical field of pesticide science, specifically to the application of knocking out or inhibiting the insect Kir2A gene in insect ovicidal activity. Background Technology

[0002] Diamondback moth ( Plutella xylostella Diamondback moth (Linnaeus) is a pest belonging to the family Plutellidae in the order Lepidoptera. It is widely distributed throughout the world and primarily damages cruciferous vegetables. Due to the long-term irrational use of chemical pesticides, the diamondback moth has developed high levels of resistance to many pesticides. Therefore, developing pesticides with novel mechanisms of action remains a top priority for diamondback moth control.

[0003] Insecticide development has gone through three stages: early utilization of natural substances, modification and upgrading of active compounds, and the development of new pesticides based on targets. Targets are the molecular sites on which insecticides act, such as insect enzymes, receptors, and ion channels. Identifying targets is a prerequisite for designing highly effective insecticides and is the starting point for current new insecticide development. Currently, about 80% of commercially available insecticides act on the ion channels of pests, such as sodium ion channels (targets of pyrethroid insecticides), ryanodine receptors (targets of diamide insecticides), and chloride ion channels (targets of abamectin). In addition to these channels, nicotinic acetylcholine receptors and transient receptor potential (TRP) ion channels are also popular targets for pesticide development. Although these ion channel targets were the first choice for insecticide development at the time, promoting the research and utilization of new pesticides, significant patent barriers exist in this field, hindering new entrants.

[0004] Potassium channels are the largest class of ion channels in insects, and currently, there are relatively few insecticides that target potassium channels. Inwardly rectified potassium (Kir) channels primarily promote potassium ion inflow into cells, playing a crucial role in maintaining resting membrane potential, regulating potassium homeostasis, and participating in cellular metabolism. Insect Kir channels are widely distributed in various physiological systems, including the excretory, digestive, nervous, and muscular systems, and are believed to participate in the regulation of these organs and physiological functions. The Kir2 subfamily is the most evolutionarily complex Kir family; for example, Lepidoptera have two subtypes, Kir2A and Kir2B, while Hemiptera have only one Kir2 subtype. Interestingly, among Diptera, Aedes aegypti has multiple Kir2 subtypes, while Drosophila has only one. Through RNA interference and inhibitor bioassays, researchers found that inhibiting Kir2B in Aedes aegypti reduces urine production and disrupts potassium balance. Inhibiting Kir2B in Rice stem borer reduces egg production. Suppressing Kir2 in cotton aphids reduces saliva secretion, thus affecting feeding. Kir2 mutations in fruit flies affect wing development. These findings suggest the important value of the Kir2A channel in the development of new pesticides.

[0005] Currently, research on the electrophysiological and pharmacological properties of the Kir2 channel in insects is relatively limited. Due to a lack of effective technical means, the electrophysiological characteristics and physiological functions of Kir2A in lepidopteran insects remain largely unexplored. Its specific functions in insect growth and development are not yet clear, and it has not been developed as an insecticide target. Summary of the Invention

[0006] The purpose of this invention is to overcome the above-mentioned technical deficiencies and provide an application of the inward rectifying potassium ion channel Kir2A in the preparation of diamondback moth ovicidal agents, thereby solving the technical problems of insufficient targets and severe resistance of traditional agents in existing diamondback moth ovicidal agents.

[0007] To achieve the above-mentioned technical objectives, the present invention provides an application of knocking out or inhibiting the insect Kir2A channel gene in insect ovicidal activity.

[0008] In any embodiment, inhibiting the channel activity of Kir2A or interfering with the expression of the Kir2A gene leads to insect egg abortion.

[0009] In any embodiment, the insect is the diamondback moth.

[0010] In any implementation, the Kir2A channel gene is any one of the following a1)-a3); a1) Genes with nucleotide sequences as shown in SEQ ID No. 1; a2) A protein encoded by the nucleotide shown in SEQ ID No. 1 having the amino acid sequence shown in SEQ ID No. 2; a3) Genes or proteins that have more than 80% homology with the nucleotide or amino acid sequences shown in SEQ ID No. 1 and SEQ ID No. 2, are derived from diamondback moth, and have the same biological function.

[0011] In addition, the present invention also proposes an RNA that targets the Kir2A channel gene of diamondback moth, wherein the RNA is a single-stranded or double-stranded RNA sequence obtained using the Kir2A channel gene sequence as a template; Furthermore, the RNA is a single-stranded or double-stranded RNA sequence with a continuous 20 nt or more of 100% homologous region as shown in SEQ ID No.1.

[0012] In addition, the present invention also proposes an RNA expression vector for the Kir2A channel of the diamondback moth, which can express the above-mentioned single-stranded or double-stranded RNA in insect cells or individual insects.

[0013] Furthermore, this invention also proposes the application of the above-mentioned RNA or the above-mentioned RNA expression vector in the preparation of RNA pesticides for controlling diamondback moth.

[0014] Furthermore, this invention also proposes the application of the diamondback moth Kir2A channel inhibitor in the preparation of diamondback moth ovicidal agents.

[0015] In any embodiment, the inhibitor includes one or more of VU590, VU625, gliclazide, quinidine, veratrine, scopolamine, flonicamid, and bromfenac.

[0016] Compared with the prior art, the beneficial effects of the present invention include: the present invention proposes the application of knocking out or inhibiting the insect Kir2A channel gene in insect ovicidal activity, the present invention shows that knocking out the Kir2A gene or inhibiting the Kir2A channel can lead to the abortion of diamondback moth eggs, demonstrating that the diamondback moth Kir2A gene and its encoded Kir2A channel are new targets for the development of diamondback moth ovicidal agents. Attached Figure Description

[0017] Figure 1 This study describes the CRISPR knockout of the Kir2A gene in the diamondback moth and its expression in embryos, as shown in Example 1. (a) Schematic diagram of the diamondback moth CRISPR / Cas9 knockout system. Gray dashed boxes indicate cross-generational genotypic selection. Gray represents autosomes, and orange indicates mutation events. (b) Types of mutations induced by CRISPR / Cas9 in exon 2 of Kir2A. Representative sequencing chromatograms of wild-type (WT, top) and homozygous mutants (bottom); target sites are marked in yellow boxes. (c) Images of late embryonic development: wild-type (top) and Kir2A knockout (bottom). (d) Relative Kir2A expression levels at 1 hour, 12 hours, 24 hours, 48 ​​hours, 60 hours post-oviposition and in the first instar larvae. Data were analyzed using one-way ANOVA followed by Tukey post-hoc tests (n=3).

[0018] Figure 2 The following are the electrophysiological results of the Kir2A channel in Example 2; (a) Kir2A channel current recorded in Sf9 cells. Voltage pulses ranged from -120 mV to +20 mV in 10 mV increments. (b) Current-voltage relationship of Kir2A in Sf9 cells. Normalized current-voltage relationships (n = 6-8) are shown for Kir2A-expressing cells in the presence and absence of Ba²⁺.

[0019] Figure 3 This section presents the results of the inhibitory effects of various compounds on the Kir2A channel of the diamondback moth in Example 2. The effects of various compounds on Kir2A current were evaluated using whole-cell patch-clamp techniques at a membrane potential of –120 mV. Specifically, Example 2 evaluated the effects of (a) small molecule inhibitors targeting the Kir channel, (b) bioactive plant-derived compounds, and (c) neuroactive pesticides, each at a concentration of 20 μM, on Kir2A channel activity. Furthermore, Figure (d) shows the concentration-response relationships of VU590, VU625, and chlorantraniliprole. VU625 exhibited the strongest inhibitory effect and was used as a positive control; while chlorantraniliprole, targeting the ryanodine receptor, showed almost no inhibitory effect (2.44 ± 1.50%) and was used as a negative control. Data were analyzed using one-way ANOVA followed by Tukey post-hoc tests. n = 6–8.

[0020] Figure 4Example 2: Egg mortality rates of diamondback moths after treatment with various Kir2A channel inhibitors. Egg mortality was assessed after treatment with different inhibitor concentrations (0, 1, and 2 mg / mL). Eggs that did not hatch within 48 hours after treatment were considered dead. Data were analyzed using a two-tailed unpaired t-test. *P<0.05, **P<0.01, ***P<0.001; ns indicates no statistically significant difference. Detailed Implementation

[0021] The "range" disclosed in this application is defined by a lower limit and an upper limit. A given range is defined by selecting a lower limit and an upper limit, which define the boundaries of a particular range. Ranges defined in this way can include or exclude endpoints and can be arbitrarily combined; that is, any lower limit can be combined with any upper limit to form a range. For example, if ranges of 60~120 and 80~110 are listed for a specific parameter, it is also expected that ranges of 60~110 and 80~120 are also included. Furthermore, if minimum range values ​​of 1 and 2 are listed, and if maximum range values ​​of 3, 4, and 5 are listed, then the following ranges are all expected: 1~3, 1~4, 1~5, 2~3, 2~4, and 2~5. In this application, unless otherwise stated, the numerical range "a~b" represents a shortened representation of any combination of real numbers between a and b, where a and b are real numbers. For example, the numerical range "0~5" indicates that all real numbers between "0~5" have been listed in this article; "0~5" is simply a shortened representation of these numerical combinations. Furthermore, when a parameter is stated as an integer ≥2, it is equivalent to disclosing that the parameter is, for example, an integer such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, etc.

[0022] Unless otherwise specified, the terms "comprising" and "including" as used in this application can be open-ended or closed-ended. For example, "comprising" and "including" can mean that other components not listed may also be included, or that only the listed components may be included.

[0023] Unless otherwise specified, the term "or" is inclusive in this application. For example, the phrase "A or B" means "A, B, or both A and B". More specifically, the condition "A or B" is satisfied by any of the following conditions: A is true (or exists) and B is false (or does not exist); A is false (or does not exist) and B is true (or exists); or both A and B are true (or exist).

[0024] This invention successfully studied the electrophysiological and pharmacological properties of Kir2A in the diamondback moth using an insect expression system. Simultaneously, through gene editing-mediated gene knockout combined with toxicological assays, it was discovered that Kir2A plays a crucial role in egg development. This provides a novel target for the development of new ovicides and contributes to advancing the research and development of ovicides for lepidopteran pests.

[0025] This specific embodiment provides an application of knocking out or inhibiting the insect Kir2A channel gene in insect ovicidal processes. Furthermore, it inhibits the channel activity of Kir2A or interferes with the expression of the Kir2A gene, leading to insect egg abortion.

[0026] In some embodiments, the insect is a diamondback moth.

[0027] In some embodiments, the Kir2A channel gene is any one of the following a1)-a3); a1) Genes with nucleotide sequences as shown in SEQ ID No. 1; a2) A protein encoded by the nucleotide shown in SEQ ID No. 1 having the amino acid sequence shown in SEQ ID No. 2; a3) Genes or proteins that have more than 80% homology with the nucleotide or amino acid sequences shown in SEQ ID No. 1 and SEQ ID No. 2, are derived from diamondback moth, and have the same biological function.

[0028] In addition, this specific embodiment also proposes an RNA that targets the Kir2A channel gene of the diamondback moth, wherein the RNA is a single-stranded or double-stranded RNA sequence obtained using the Kir2A channel gene sequence as a template; Furthermore, the RNA is a single-stranded or double-stranded RNA sequence with a continuous 20 nt or more of 100% homologous region as shown in SEQ ID No.1.

[0029] Furthermore, this specific embodiment also proposes an RNA expression vector for the Kir2A channel of the diamondback moth, which can express the above-mentioned single-stranded or double-stranded RNA in insect cells or individual insects.

[0030] This specific embodiment also proposes the application of the above-mentioned RNA or the above-mentioned RNA expression vector in the preparation of RNA pesticides for controlling diamondback moth.

[0031] This specific embodiment also proposes the application of Kir2A channel inhibitors of diamondback moth in the preparation of ovicidal agents for diamondback moth. The inhibitors include one or more of VU590, VU625, gliclazide, quinidine, veratrine, scopolamine, flonicamid, and bromfenac.

[0032] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0033] In this invention, the terms "some embodiments," "this embodiment," and examples are used to describe a subset of all possible embodiments. However, it is understood that "some embodiments" can be the same subset or different subsets of all possible embodiments and can be combined with each other without conflict.

[0034] If the application documents contain similar descriptions such as "first / second", the following explanation shall be added: In the following description, the terms "first / second / third" are used only to distinguish similar objects and do not represent a specific ordering of objects. It is understood that "first / second / third" may be interchanged in a specific order or sequence where permitted, so that the embodiments described herein can be implemented in an order other than that illustrated or described herein.

[0035] In this embodiment, the term "and / or" is merely a description of the relationship between related objects, indicating that there can be three relationships. For example, object A and / or object B can represent three situations: object A exists alone, object A and object B exist simultaneously, and object B exists alone.

[0036] The following describes embodiments of this application. The embodiments described below are exemplary and are only used to explain this application, and should not be construed as limiting this application. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Reagents or instruments used, unless otherwise specified, are all conventional products that can be obtained commercially.

[0037] Example 1: Knocking out the Kir2A channel in diamondback moth causes egg abortion 1.1 Design and preparation of diamondback moth sgRNA Kir2A gene knockout was performed using CRISPR / Cas9 technology. First, the guider RNA target sequence was screened using the online analysis tool Cas-Designer, with the sequence 5'-ATAGAGACGCAGCACACGATCGG-3'. Then, specific primers CRISPR-C1-F and universal reverse primer CRISPR-R were synthesized. Their sequences are: CRISPR-C1-F: 5'-GAAATTAATACGACTCACTATAGGATAGAGACGCAGCACACGATGTTTTAGAGCTAGAAATAGC-3'; and CRISPR-R: 5'-AAAAGCACCGACTCGGTGCCACTTTTTCAAGTTGATAACGGACTAGCCTTATTTTAACTTGCTATTTCTAGCTCTAAAAC-3'.

[0038] Full-length sgDNA template was synthesized via overlap PCR. PCR amplification conditions were: pre-denaturation at 95°C for 3 min; 35 cycles of 95°C for 15 s, 70°C for 15 s, and 72°C for 30 s; followed by a final extension at 72°C for 10 min. After purification and quantification using Nanodrop 2000, sgRNA was synthesized via in vitro transcription according to the T7 High Yield RNA Transcription Kit instructions. The obtained sgRNA samples were used immediately or aliquoted and stored at –80°C.

[0039] 1.2 Microinjection of diamondback moth eggs and detection of mutants Fifteen to twenty pairs of adult diamondback moths were anesthetized with ethyl acetate or CO2 and placed in a 15×10×6cm plastic box with ventilation holes for mating and egg laying. Embryos laid within one hour were collected from a dry microscope slide (24×50 mm) covered with a sealing film and placed in the egg-laying box for microinjection. Glass needles for microinjection were prepared using a Narisge PC-10 needle puller and their tips were ground using a Narisge EG-401 needle grinder for easier puncture. A mixture of Cas9 (300 ng / µl) and sgRNA (150 ng / µL) was injected into the collected eggs at a dose of 1 nL per egg using a pneumatically controlled microinjection system. After injection, the embryos were transferred to a plastic culture box containing moistened absorbent paper (approximately 200 µL of sterile water) to prevent dehydration. They were then incubated under normal culture conditions for 3 days until hatching.

[0040] Following microinjection of embryos, the first generation of adults was defined as generation zero (G0). G0 adults were mated individually with wild-type adults to produce the G1 generation. After oviposition, the G0 adults were sequenced, and G1 generations with gene-edited maternal lineage were selected for further rearing. Similarly, G2 generations with a nonsense mutation in the maternal lineage were backcrossed with wild-type adults, and G3 generations with a nonsense mutation in the maternal lineage were backcrossed with wild-type adults to obtain G4 generations with the Kir2A nonsense mutation for subsequent processing.

[0041] The gDNA fragment of PxKir2A (297 bp) was amplified using specific primers PxKir2A-C9tF (CCTTCATCCTGTCCTGGCTCG) and PxKir2A-C9tR (TCGCAAACACTATGCCCACC). PCR amplification was performed using 2×Rapid Taq MasterMix under the following conditions: 95℃ for 3 min; 35 cycles of 95℃ for 15 s, 58℃ for 15 s, 72℃ for 10 s; and a 72℃ extension for 5 min. The PCR products were directly sequenced to determine the Kir2A mutant type.

[0042] Analysis of 13 surviving G0 generation adults revealed that 3 of them had a 731T insertion mutation in the coding sequence, which disrupts protein function (Figures 1a and 1b). To reduce off-target gene editing effects, heterozygous offspring containing the 731T insertion mutation were repeatedly backcrossed with wild-type individuals. Sequencing analysis showed that only maternal offspring containing nonsense mutations were retained for further culture in each generation.

[0043] 1.3 Self-pollination of heterozygotes indicates that Kir2A knockout in the diamondback moth is lethal. Starting from generation G4, homozygous Kir2A mutants were identified through self-crossing and cloning sequencing. The genotype ratio of the offspring from self-crossing heterozygotes was equal to that of wild-type Kir2A. + / + Kir2A + / - =1:2 (Table 1). According to Mendel's laws of inheritance, the expected ratio should be Kir2A. + / + Kir2A + / - Kir2A - / - =1:2:1, which indicates that Kir2A - / - The genotype may be lethal. To test this hypothesis, individual genotyping was performed on 56 embryos from the sixth pair of offspring of the G7 generation heterozygous self-crosses. The results showed 17 wild-type, 25 heterozygous, and 14 homozygous, in a ratio close to 1:2:1 (Table 1), consistent with Mendel's laws of inheritance. This indicates that the Kir2A-KO homozygous embryos ceased development late in embryonic development, with no offspring surviving. Figure 1 a, c).

[0044] The nucleotide sequence of SEQ ID No. 1 (nucleotide sequence of the Kir2A coding region of diamondback moth, 1566 bp) is shown below: 2. The amino acid sequence of SEQ ID No. 2 (Kir2A amino acid sequence of diamondback moth) is as follows: MNEINSTKCSQRHIHSKTQLYHSKNLSPQMKGEGTYEMKIREEGQNGDAKNRPLLSSGKIFYNPGADEETDETSAFESSVVESPMLSGSGLLRPPRNHTSCSLHRVPTYSSSIGQFTIHRNDTCRSRSHR HGVTRRTRRRAILKNGECNILKSRISQRRLRFLQDMFTTLVDAQWRWTLLVFTLSFILSWLGFGLIWWLISFTHGDLEEEHLPPLQEANNWKPCVFNIFGFTSCFLFSIETQHTIGYGARTTTEECPEAI FIMCFQSIVGVMIQAFMVGIVFAKMTRPKHRTQTLLFSKYAVICQRDGELCLMFRVGDLRKSHIIGASVRAQLIRSRTTKEGEQLAHYQTELELHADGCDSNLFFIWPITMVHRINAESPFYGVSAADIL QERFEIVVILEGTIESTGQTTQARSSYTTSELMWGHRFVSLVSYNRERQGYEVDYSRFEETAQVDTPLCSAKELDEFYGSQADRRSLEVGEPLMLKMPSPGDPPPPQPPLSPQTPPPPVAEQNDGDFTVTL To further confirm the crucial role of Kir2A in oocyte development, its expression pattern during the oocyte stage was analyzed. The results showed that its relative expression level remained low during the first 24 hours of embryonic development, then increased significantly by approximately 182-fold at 48 hours (Figure 1d), demonstrating the important role of this gene in the later stages of oocyte development.

[0045] Table 1 Genotype distribution of PxKir2A+ / - self-crossed offspring

[0046] Example 2: Kir2A inhibitors of diamondback moth exhibit ovicidal activity. 2.1 Electrophysiological characteristics of Kir2A moth Whole-cell patch-clamp recording was employed. Glass electrodes were fabricated from borosilicate capillaries (1.5 mm outer diameter, 0.86 mm inner diameter) using a Narisge PC-10 drawing apparatus and thermally polished using a Narisge MF-900, achieving a tip resistance of 3–5 megohms. The electrode internal solution (mM) consisted of 135 KCl, 2 MgCl2, 1 EGTA, 10 HEPES, and 2 Na2ATP, adjusted to pH 6.4 with KOH and osmotic pressure of 370 mOsm / kg with sucrose. The cell bath solution (mM) consisted of 90 NaCl, 50 KCl, 2 CaCl2, 1 MgCl2, 5 glucose, and 10 HEPES, adjusted to pH 6.4 with NaOH and osmotic pressure of 370 mOsm / kg with sucrose. Recording was performed using an Axon MultiClamp 700B amplifier, a Digidata 1500B converter, and Clampfit 10.6 software (Molecular Devices LLC).

[0047] 2 μg of pIE2-PxKir2A-EGFP plasmid expressing the PxKir2A and EGFP fusion protein was mixed with 4 μL of Lipofectamine 2000 and transfected into Sf9 cells in 35 mm diameter dishes. After transfection, the cells were sputtered at 0.8 × 10⁻⁶ ppm. 5 Cells were reseeded in fresh cell culture dishes. After 24 hours, the culture medium was discarded, and 1 ml of cell bath buffer was added. Cells expressing green fluorescent protein were selected for testing using a fluorescence microscope. Current recording was performed by clamping cells at -20 mV for 40 ms, then stepping from -120 mV to +20 mV in 10 mV increments over 120 ms, followed by holding at -20 mV for 40 ms. Data was sampled at 10 kHz with series resistance compensation and a 2 kHz low-pass filter. The current-voltage (IV) relationship was plotted as membrane potential (mV) against current density (pA / pF). All experiments were performed at room temperature (25°C). For inhibitor assays, after achieving stable recording at -120 mV, 0.1 ml of the test compound was added to 0.9 mL of cell bath buffer. The current was recorded after stabilization, and the inhibition rate was calculated based on changes in current amplitude. The inhibitor concentration-response curve was fitted using the Boltzmann equation.

[0048] 2.2 Pharmacological properties of the Kir2A channel Whole-cell patch-clamp recordings showed that Sf9 cells transfected with the PxKir2A plasmid exhibited significant inward currents (Fig. 2a). Furthermore, 5 mM Ba²⁺... + It can almost completely suppress the inward current mediated by PxKir2A, and its IV curve exhibits typical Kir channel characteristics (Figure 2b).

[0049] Several Kir1 channel inhibitors—including small-molecule potassium channel blockers, plant-derived compounds, and common neurotoxic pesticides—were tested for their inhibitory effects on PxKir2A channels at a concentration of 20 μM. The results showed significant differences in their effects. VU625 exhibited the strongest inhibitory activity (97.55 ± 0.38%) and was used as a positive control; VU590 also showed strong inhibitory activity against PxKir2 channels, with an inhibition rate of 89.7% ± 3.53%. Carvedilol, ML297, and glibenclamide also showed significant inhibitory effects on PxKir2A, with inhibition rates of 49.02% ± 3.29, 48.80% ± 3.16, and 45.47% ± 3.77, respectively. Other compounds showed weaker inhibitory effects, ranging from 16.64% to 33.63% (Figure 3a). Among the plant-derived compounds, veratridine showed the highest inhibition rate, at 47.62% ± 5.22%. Evodiaein, scopolamine, artemisinin, arecoline, hesperidin, and oxymatrine exhibited moderate inhibitory effects, ranging from 31.90% to 42.76%. Rotenone and azadirachtin showed lower inhibitory effects, not exceeding 27.11% (Figure 3b). Among the tested pesticides, chlorfenapyr showed the strongest inhibitory effect, at 83.01% ± 3.28%, followed by flonicamid at 45.34% ± 1.01%. Pesticides targeting sodium ion channels (indoxacarb, tetrafluoromethrin, and lambda-cyhalothrin) and pesticides targeting nicotinic acetylcholine receptors (imidacloprid and thiamethoxam) showed weak but stable inhibitory effects on PxKir2A, ranging from 21.0% to 31.94%. Lufenuron and tebufenozide showed weaker inhibitory effects (<20%). Chlorantraniliprole, which targets the ryanodine receptor, showed the least inhibitory effect (2.44±1.50%) and was used as a negative control. Meanwhile, avermectin, which targets the chloride ion channel, also showed no significant inhibitory effect on PxKir2A (Figure 3c).

[0050] The dose-dependent curves of VU625, VU590, and bromuconazole on the PxKir2A channel were evaluated. The half-maximal inhibitory concentration (IC50) of VU625 was 0.2673 μM (R²=0.9556; 95% confidence interval: 0.2286–0.3106 μM), the IC50 of VU590 was 0.6647 μM (R²=0.9760; 95% confidence interval: 0.5452–0.8082 μM), and the IC50 of bromuconazole was 6.056 μM (R²=0.9474; 95% confidence interval: 5.266–6.940 μM) (Figure 3d).

[0051] 2.3 Lethal Activity of Kir2A Channel Inhibitors on Diamondback Moth Eggs Bioassays were performed using the oocyte immersion method. Nine compounds with potential Kir channel regulatory activity (VU625, VU590, bromfenac, flonicamid, scopolamine hydrobromide, veratrine, carvedilol, gliclazide, and quinidine) were selected and assayed at concentrations of 1 mg / mL and 2 mg / mL. The results showed that these compounds exhibited dose-dependent ovicidal activity against embryos in late developmental stages (>48 h). Figure 4 ).

[0052] Among potassium channel inhibitors, the small molecules VU590 and VU625 showed significant concentration-dependent oocyte-killing effects. VU625 resulted in an oocyte mortality rate of 46.77 ± 3.70% at a concentration of 1 mg / mL and 87.87 ± 2.37% at 2 mg / mL. For VU590, the mortality rates were 49.25 ± 7.28% and 73.85 ± 4.11%, respectively. Gliclazide acted as a blocker of ATP-sensitive potassium (KATP) currents throughout β-cells. Its potency was relatively low, with a mortality rate of 52.24 ± 1.37% at a concentration of 2 mg / mL. Quinidine showed no significant effect at 1 mg / mL (P > 0.05), but the mortality rate increased to 40.67 ± 3.50% (P < 0.05) at 2 mg / mL (Figure 4a).

[0053] Among plant-derived compounds, scopolamine exhibited strong ovicidal activity, with egg mortality rates of 84.24±4.97% and 92.55±2.47% at two different concentrations. Veratril showed weaker activity, with a mortality rate of 34.39±2.61% at 1 mg / mL and 58.28±3.17% at 2 mg / mL. Flupyradifurone and chlorantraniliprole, insecticides targeting the insect nervous system, showed strong ovicidal effects, with mortality rates of 77.92±5.07% and 72.63±5.92% at 1 mg / mL, respectively, increasing to 91.58±5.29% and 89.76±8.51% at 2 mg / mL (Figure 4b).

[0054] Compared with the prior art, the beneficial effects of the present invention are: 1. This invention discovers the key role of the Kir2A gene in the egg development of the diamondback moth through gene knockout, providing a target for the development of RNA pesticides using RNAi technology.

[0055] 2. This invention discovers highly effective inhibitors targeting the Kir2A channel of diamondback moth, such as VU625, VU590, and bromonitrile, which provides a reference for the development and utilization of Kir2A inhibitors.

[0056] 3. This invention discovers that Kir2A inhibitors also have ovicidal activity, providing a new target for the development of ovicidal agents with novel mechanisms of action.

[0057] The specific embodiments of the present invention described above do not constitute a limitation on the scope of protection of the present invention. Any other corresponding changes and modifications made in accordance with the technical concept of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. The application of knocking out or inhibiting the insect Kir2A channel gene in insect ovicidal activity.

2. The application according to claim 1, characterized in that, Inhibiting the channel activity of Kir2A or interfering with the expression of the Kir2A gene can lead to insect egg abortion.

3. The application according to claim 1 or 2, characterized in that, The insect in question is the diamondback moth.

4. The application according to claim 1, characterized in that, The Kir2A channel gene is any one of the following a1)-a3); a1) Genes with nucleotide sequences as shown in SEQ ID No. 1; a2) A protein encoded by the nucleotide shown in SEQ ID No. 1 having the amino acid sequence shown in SEQ ID No. 2; a3) Genes or proteins that have more than 80% homology with the nucleotide or amino acid sequences shown in SEQ ID No. 1 and SEQ ID No. 2, are derived from diamondback moth, and have the same biological function.

5. An RNA targeting the Kir2A channel gene in the diamondback moth, characterized in that, The RNA is a single-stranded or double-stranded RNA sequence obtained using the Kir2A channel gene sequence as a template. Furthermore, the RNA is a single-stranded or double-stranded RNA sequence with a continuous 20 nt or more of 100% homologous region as shown in SEQ ID No.

1.

6. An RNA expression vector for the Kir2A channel of the diamondback moth, characterized in that, The expression vector is capable of expressing the single-stranded or double-stranded RNA of claim 5 in insect cells or individual insects.

7. The use of the RNA of claim 5 or the RNA expression vector of claim 6 in the preparation of RNA pesticides for controlling diamondback moth.

8. Application of Kir2A channel inhibitors in the preparation of ovicidal agents for diamondback moth.

9. The application according to claim 8, characterized in that, The inhibitors include one or more of VU590, VU625, gliclazide, quinidine, veratrine, scopolamine, flonicamid, and bromfenac.

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