Use of hearNPV-miR-hr5b for prevention and treatment of cotton bollworm

Abstract

This application belongs to the field of biopesticides and provides the application of HearNPV-miR-hr5b in the control of cotton bollworm. The nucleotide sequence of HearNPV-miR-hr5b is CGGGCAAAUUUCAAAUAAA. HearNPV-miR-hr5b of this application can enhance the replication and accumulation of HearNPV in the host cotton bollworm, accelerate the larval mortality rate, and increase the rate of resistant cotton bollworm control. It can be used to develop HearNPV synergists and has good application prospects in the biological control of cotton bollworm.

Description

Technical Field

[0001] This application belongs to the field of biological pesticides, specifically, this application provides the application of HearNPV-miR-hr5b in controlling cotton bollworm. Background Technology

[0002] The cotton bollworm, *Helicoverpa armigera* (Hübner), belonging to the family Noctuidae in the order Lepidoptera, is a major global agricultural pest. It has an extremely wide host range, damaging over 200 plant species from more than 30 families, causing severe economic losses worldwide. Since the 1990s, Bt transgenic insect-resistant cotton, due to its ability to continuously express insecticidal proteins, has shown excellent control effects against target pests such as the cotton bollworm, leading to its widespread global cultivation. However, with the long-term large-scale promotion of Bt cotton, the problem of cotton bollworm resistance has gradually become prominent. Since the beginning of the 21st century, field resistance cases have been reported in many parts of the world. For example, in cotton-growing areas of northern China, due to the long-term large-scale planting of Cry1Ac Bt cotton and the lack of effective non-Bt cotton shelters, the frequency of Cry1Ac resistance alleles in field cotton bollworm populations has significantly increased. Currently, the resistance of cotton bollworm to Bt crops has become a significant challenge to the sustainable development of global agriculture, prompting countries to continuously optimize their resistance management strategies.

[0003] HearNPV, a baculovirus, is an insect pathogenic microorganism with the advantages of high specificity and environmental friendliness, and is widely used in pest control.

[0004] However, HearNPV works slowly, so it is necessary to develop adjuvants that can improve the insecticidal speed and efficiency of HearNPV, while also enhancing its control effect against resistant bollworms. Summary of the Invention

[0005] This study identified a HearNPV-derived microRNA, HearNPV-miR-hr5b, through transcriptome microRNA sequencing. This microRNA plays a significant role in promoting HearNPV infection of host cotton bollworm larvae, improving insecticidal efficiency, and enhancing the lethality of resistant cotton bollworms, thus demonstrating its potential application value in improving the biocontrol efficacy of HearNPV against cotton bollworms.

[0006] On the one hand, this application provides the application of HearNPV-miR-hr5b in controlling cotton bollworm, wherein the nucleotide sequence of HearNPV-miR-hr5b is CGGGCAAATTTCAAATAAA.

[0007] On the other hand, this application provides the application of HearNPV-miR-hr5b's agomir for controlling cotton bollworm, wherein the nucleotide sequence of the positive strand of the HearNPV-miR-hr5b's agomir or mimics is CGGGCAAATTTCAAATAAA.

[0008] Furthermore, the agomir of HearNPV-miR-hr5b includes one or more of the following modifications: 3' end cholesterol modification; 5' end thiosclerosis modification; 3' end thiosclerosis modification; full-chain methoxy modification.

[0009] Furthermore, the HearNPV-miR-hr5b, or the agomir of the HearNPV-miR-hr5b, is administered via a formulation comprising microcapsules or a formulation comprising nanoparticles, in combination with cotton bollworm nucleopolyhedrovirus.

[0010] On the other hand, this application provides a biological control pesticide for cotton bollworm, which contains HearNPV-miR-hr5b with the nucleotide sequence CGGGCAAAUUUCAAAUAAA.

[0011] On the other hand, this application provides a biological control pesticide for cotton bollworm, which contains an agomir of HearNPV-miR-hr5b, wherein the nucleotide sequence of the positive strand of the agomir of HearNPV-miR-hr5b is CGGGCAAAUUUCAAAUAAA.

[0012] Furthermore, the agomir positive chain of HearNPV-miR-hr5b includes one or more of the following modifications: 3' end cholesterol modification; 5' end thiosclerosis modification; 3' end thiosclerosis modification; full-chain methoxy modification.

[0013] Furthermore, the bollworm biological control pesticide is a formulation comprising microcapsules or nanoparticles. It also contains acceptable excipients for pesticide use.

[0014] Furthermore, the bollworm biological control pesticide also includes a preparation containing bollworm nucleopolyhedrovirus.

[0015] The HearmiR-hr5b of this application can be prepared into various formulations acceptable in the pesticide field, including but not limited to nanoparticle-based formulations, such as liposome nanoparticles and polymer nanoparticles; microcapsule-based formulations, such as polyamide or sodium alginate microcapsules; and can also be prepared into solutions or suspensions directly using water, oil or organic solvents after adding various excipients such as RNase inhibitors, stabilizers, and penetration enhancers.

[0016] For reasons of stability and permeability, HearmiR-hr5b can be modified or analogs can be prepared, including but not limited to cholesterol modification, thiosclerosis modification, and full-chain methoxy modification of the 3' end of miRNA mimics or the agomir used in the examples. Based on the known basic sequence, those skilled in the art can routinely try various modification methods and verify their effects.

[0017] Preparations containing Helicobacter pylori nucleopolyhedrovirus can be liquid or solid preparations prepared by those skilled in the art using conventional methods, and can be administered sequentially or simultaneously with HearNPV-miR-hr5b, agomir, or mimics.

[0018] Beneficial effects:

[0019] This invention provides a miRNA encoding HearNPV (HearNPV-miR-hr5b) for the cotton bollworm nucleopolyhedrovirus (SEQ ID NO.1), which can enhance the replication and accumulation of HearNPV in the host cotton bollworm, accelerate the larval mortality rate, and increase the mortality height of infected larvae, thereby facilitating virus spread. This invention improves the insecticidal effect of HearNPV against wild-type and Cry1AC-resistant cotton bollworms by mimicking endogenous HearNPV-miR-hr5b using agomir. The HearNPV-miR-hr5b sequence provided by this invention can be used to develop cotton bollworm nucleopolyhedrovirus enhancers, showing potential application prospects in the biological control of cotton bollworm. Attached Figure Description

[0020] Figure 1 This shows the location of HearNPV-miR-hr5b on the viral genome.

[0021] Figure 2 This section shows the effects of HearNPV-miR-hr5b on viral replication and larval infection. Part A: Relative content of HearNPV-miR-hr5b at 24 and 48 h after injection with Agomir; Part B: Expression level of viral polyhedrin gene at 24, 48, 72, and 96 h after injection with Agomir.

[0022] Figure 3The effects of HearNPV-miR-hr5b on viral replication and larval infection were shown. Part AB: Viral infection status of Ha-8 cells 48 h after transfection with HearNPV-miR-hr5b via Agomir and subsequent infection treatment. Part CD: Viral infection status of larvae 48 h after injection of HearNPV-miR-hr5b via Agomir.

[0023] Figure 4 This shows the effect of HearNPV-miR-hr5b agomir injection on mortality of infected larvae; Note: Survival curves of infected larvae after HearNPV-miR-hr5b agomir injection compared to NC-agomir injection. Data represent the mean ± standard error of three biological replicates and were analyzed by Log-rank test. *0.01 < P < 0.05; ***P < 0.001.

[0024] Figure 5 This shows the effect of HearNPV-miR-hr5b agomir injection on mortality rates in Cry1Ac-infected larvae; Note: Daily survival curves of larvae after HearNPV-miR-hr5b agomir injection compared to NC-agomir injection. Data are presented as mean ± standard error of three biological replicates, analyzed by Log-rank test. **0.001 < P < 0.01. Detailed Implementation

[0025] Example 1: Test insects, cell lines, and viruses

[0026] Cotton bollworms were artificially reared in the Integrated Pest Management Laboratory of China Agricultural University. Rearing conditions included a temperature of (27±1)℃, relative humidity of 75%±10%, and a photoperiod of 14L:10D. Larvae were fed artificial feed, while adults were fed a 10% honey solution. After hatching, 3-5 newly hatched larvae were placed in a 25 mL feed container with artificial feed. When the larvae reached approximately the fourth instar, they were reared individually to prevent cannibalism. After pupation, the larvae were removed and allowed to emerge as adults. After adult emergence, 15-20 pairs of adults were placed in a plastic box with a suitable gauze pad placed on top for egg laying. They were fed a 10% honey solution. After egg laying, the gauze was removed, and the larvae were placed in a resealable bag for hatching. Cry1Ac-resistant cotton bollworm larvae were obtained from the laboratory of Liang Gemei at the Institute of Plant Protection, Chinese Academy of Agricultural Sciences. The Ha-8 cell line used in the experiment was obtained from the laboratory of Zhang Huan at the Institute of Zoology, Chinese Academy of Sciences.

[0027] The wild-type cotton bollworm nucleopolyhedrovirus (HearNPV) stock powder used in the experiment was 5 × 10⁻⁶.11 The PIB / g HearNPV recombinant virus with fluorescent tag was constructed using the HearNPV recombinant virus construction system developed by Hu Zhihong of the Wuhan Institute of Virology and stored in the IPM laboratory.

[0028] Example 2: Identification of HearNPV encoding HearNPV-miR-hr5b

[0029] We used central nervous system transcriptome sequencing data from healthy and infected cotton bollworms at three time points: 48, 72, and 96 hours. Sequences of 10-30 nt in length were aligned and analyzed. After removing duplicates, we screened for microRNAs expressed only in infected larvae but not in healthy larvae. These microRNAs were then aligned using Bowtie with the cotton bollworm genome (GenBank: GCA_002156985.1) and the HearNPV genome (AF271059.2). Sequences that aligned only with the HearNPV genome but not with the cotton bollworm genome were selected as final candidate sequences for further experimental analysis.

[0030] Further detection and verification of the candidate microRNA using qPCR clarified its viral origin; specifically, the sequence was detected and confirmed only in infected larvae, but not in healthy larvae. The final identification revealed the virus encoding HearNPV-miR-hr5b (sequence: CGGGCAAAUUUCAAAUAAA, SEQ ID NO.1). Figure 1 ).

[0031] Example 3: Effects of HearNPV-miR-hr5b on viral replication and infection severity in infected larvae

[0032] (1) The method of infecting wild virus is as follows: the wild virus raw powder is prepared into 2×10 by double distilled water. 8 A PIB / mL suspension was used to culture 3rd instar molting larvae of uniform growth in finger tubes. The larvae were starved overnight. The next day, the larvae molted to the early 4th instar and were then fed the virus. The bollworm diet was cut into small rectangular pieces approximately 8 mm × 4 mm × 1 mm in size. 5 μL of virus suspension was added to each piece, and each larva was fed one piece of virus-infected feed. A healthy control group was given an equal volume of double-distilled water. Most larvae consumed the virus-infected feed within 12 hours, resulting in a final virus concentration of 10. 6 OBs / insects were considered to have been exposed to the virus 0 h after consuming the feed. Larvae that had consumed the virus-infected feed were selected for later experiments.

[0033] (2) Method for infecting cotton bollworm cells with fluorescent virus: Add 1×10⁻⁶ fluorescent virus to Ha-8 cells that have been seeded in 96-well plates the night before. 8 Infection was performed using 2 μL of fluorescent virus at a PIB / mL concentration, and the infection status was recorded under a fluorescence microscope after 72 h.

[0034] (3) Method for infecting larvae with fluorescent virus: Infect larvae with a concentration of 1×10⁻⁶ 8 2 μL of fluorescent virus liquid at a concentration of PIB / mL was injected into the cotton bollworm larvae via the tip of the second abdominal leg using a microsyringe. The infection was recorded under a stereofluorescence microscope 48 h later.

[0035] (4) Sample collection: At 0 h post-exposure, larvae of uniform size were injected with HearNPV-miR-hr5b agomir (synthesized by Shanghai Jima Pharmaceutical Technology Co., Ltd., sequence: CGGGCAAATTTCAAATAAA, SEQ ID NO.1; agomir is modified in the antisense strand, with cholesterol modification at the 3' end, two thiosclerotic modifications at the 5' end, four thiosclerotic modifications at the 3' end, and full-chain methoxy modification). The injection volume was 1000 nM x 2 μL / larva, and the control was injected with the same amount corresponding to NC. Head samples from cotton bollworms were collected at 24 and 48 h post-exposure for microRNA content detection; head samples were collected at 24, 48, 72, and 96 h post-exposure for virus content detection. Three biological replicates were used for each sample group.

[0036] (5) Total RNA extraction: Add 400 μL of Trizol reagent (TaKaRa) to the centrifuge tube containing the collected samples, grind three times at 50 Hz for 60 s each time using a tissue homogenizer, incubate on ice for 5 min, and then add 600 μL of Trizol reagent. Add 200 μL of chloroform, vortex to mix, and incubate on ice for 10 min. Centrifuge at 12000 rpm for 15 min at 4°C. After centrifugation, carefully remove the tube and transfer 300 μL of supernatant to a new enzyme-free 1.5 mL centrifuge tube. Add 300 μL of isopropanol, gently invert to mix, and incubate at -20°C for 10 min. Centrifuge at 12000 rpm for 15 min at 4°C, discard the supernatant, and use a pipette to remove as much of the remaining supernatant as possible. Add 800 μL of 75% ethanol solution prepared with DEPC-treated water, invert the container, and centrifuge at 12000 rpm for 8 min at 4°C. Discard the supernatant. Add 200 μL of 75% ethanol solution prepared with DEPC-treated water, invert the container, and centrifuge at 12000 rpm for 8 min at 4°C. Aspirate the supernatant using a 200 μL pipette tip, and then carefully aspirate as much of the remaining supernatant as possible using a 10 μL pipette tip. Centrifuge at 12000 rpm for 1 min at 4°C, aspirate the remaining liquid, and air dry in a clean bench. Dissolve the sample thoroughly in 20 μL of DEPC-treated water, and determine the sample concentration and purity using a NanoDrop 2000 (Thermo Fisher) instrument.

[0037] (6) cDNA synthesis: Total RNA was subjected to gDNA removal and reverse transcription using the PrimeScript RT reagent Kit with gDNA Eraser (TaKaRa). The gDNA removal reaction system consisted of 2.0 μL of 5×gDNA Eraser Buffer, 1.0 μL of gDNA Eraser, 2.0 μL of Total RNA, and 5.0 μL of RNase-Free ddH2O. The reaction was carried out at 42°C for 2 min. The reverse transcription reaction system consisted of 10.0 μL of the previous reaction solution, 1.0 μL of PrimeScript RT Enzyme Mix I, 1.0 μL of RT Primer Mix, 4.0 μL of 5×PrimeScript Buffer 2 (for Real Time), and 4.0 μL of RNase-Free dH2O. The reaction was carried out at 37°C for 15 min, followed by a reaction at 85°C for 5 s.

[0038] (7) Quantitative PCR: qRT-PCR was performed using Taq Pro Universal SYBR qPCR Master Mix (Novizan) and a CFX Connect quantitative PCR instrument (BIO-RAD) according to the instructions. The reaction system consisted of 10.0 μL of 2 × Taq Pro Universal SYBR qPCR Master Mix, 0.4 μL of Primer 1 (10 µM), 0.4 μL of Primer 2 (10 µM), 1.0 μL of cDNA, and 8.2 μL of ddH2O. The reaction program was 40 cycles of 95°C for 30 s, 95°C for 10 s, and 60°C for 30 s. The instrument's default melting curve acquisition program was used (the relative expression level of the target gene in qRT-PCR is expressed as 2 × 10⁻⁶). -ΔΔCT (Method calculation).

[0039] Table 1. qPCR primers

[0040]

[0041] result:

[0042] Using the synthetically produced HearNPV-miR-hr5b agomir to mimic endogenous miRNA, the level of HearNPV-miR-hr5b in infected larvae was upregulated via injection. Larvae injected with NC-agomir served as a control group. QPCR analysis of larval HearNPV-miR-hr5b levels at 24 and 48 hours after injection showed a significant upregulation, indicating the effectiveness of HearNPV-miR-hr5b injection. To ensure the sustained effect of HearNPV-miR-hr5b agomir injection in larvae, a second injection was administered at 48 hours after initial infection (0 hours after infection). The copy number of the HearNPV polyhedrin gene (representing viral replication) at 24, 48, 72, and 96 hours after injection showed that HearNPV-miR-hr5b agomir injection significantly promoted viral replication in larvae (Figure 2).

[0043] Using the synthetic HearNPV-miR-hr5b agomir to mimic endogenous miRNA, 500 nM microRNA was transfected into Ha-8 cells using Roche's X-tremeGENE transfection reagent to upregulate the HearNPV-miR-hr5b content in Ha-8 cells. Cells were then inoculated with the virus, with Ha-8 cells transfected with NC-agomir serving as a control group. The average fluorescence intensity of cells 48 h after infection was observed under a stereofluorescence microscope as an indicator of viral infection. Results showed that HearNPV-miR-hr5b transfection significantly increased the fluorescence intensity of infected cells, indicating a significant promotion of viral infection. Similarly, HearNPV-miR-hr5b agomir was injected into larvae infected with fluorescent virus for 0 h, and the degree of viral infection was observed under a stereofluorescence microscope 48 h later. Results showed that HearNPV-miR-hr5b agomir injection significantly promoted viral infection in larvae. Figure 3 ).

[0044] Example 4: The effect of HearNPV-miR-hr5b on mortality rate from drug exposure

[0045] Cotton bollworm larvae exposed to the virus for 0 h were injected with microRNA agomir, followed by a booster injection at 36 h. Larval survival was then recorded every 12 h until death or pupation. Student's-test was performed using SPSS software for differential analysis. Mortality rates were expressed as survival curves, and differences were analyzed using the Log-rank test.

[0046] result:

[0047] The effects of HearNPV-miR-hr5b agomir injection on the climbing height and mortality rate of infected larvae were investigated. The results showed that HearNPV-miR-hr5b agomir injection significantly promoted the climbing height of infected larvae before death and significantly accelerated the mortality rate of infected larvae. Figure 4 ).

[0048] The effect of HearNPV-miR-hr5b agomir injection on the mortality rate of Cry1Ac-resistant larvae was investigated. The results showed that HearNPV-miR-hr5b agomir injection significantly accelerated the mortality rate of Cry1Ac-resistant larvae. Figure 5 ).

Claims

1. The application of HearNPV-miR-hr5b in controlling cotton bollworm, characterized in that, The nucleotide sequence of HearNPV-miR-hr5b is CGGGCAAAUUUCAAAUAAA.

2. The application of HearNPV-miR-hr5b's agomir for controlling cotton bollworm, characterized by, The nucleotide sequence of the agomir positive strand of HearNPV-miR-hr5b is CGGGCAAAUUUCAAAUAAA.

3. In the application according to claim 2, the agomir positive chain of HearNPV-miR-hr5b includes one or more of the following modifications: 3' end cholesterol modification; 5' end thiosclerosis modification; 3' end thiosclerosis modification; full-chain methoxy modification.

4. The application according to any one of claims 1-3, wherein the HearNPV-miR-hr5b agomir is administered via a formulation comprising microcapsules or a formulation comprising nanoparticles.

5. The application according to any one of claims 1-4, further comprising the application of a preparation containing bollworm nucleopolyhedrovirus.

6. A biological control pesticide for cotton bollworm, characterized in that, The bollworm biological control pesticide contains HearNPV-miR-hr5b with the nucleotide sequence CGGGCAAAUUUCAAAUAAA.

7. A biological control pesticide for cotton bollworm, characterized in that, The bollworm biological control pesticide contains the agomir nucleotide sequence CGGGCAAAUUUCAAAUAAA of HearNPV-miR-hr5b.

8. The bollworm biological control pesticide according to claim 7, wherein the agomir positive chain of HearNPV-miR-hr5b includes one or more of the following modifications: 3' end cholesterol modification; 5' end thiosclerosis modification; 3' end thiosclerosis modification; full-chain methoxy modification.

9. The bollworm biological control pesticide according to any one of claims 6-8, wherein the bollworm biological control pesticide is a formulation comprising microcapsules or a formulation comprising nanoparticles.

10. The bollworm biological control pesticide according to any one of claims 6-9, wherein the bollworm biological control pesticide further comprises a preparation containing bollworm nucleopolyhedrovirus.

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