Use of circular RNA circLOCMI02072 (1, 2) as a locust behavior regulator

Abstract

This invention discloses circular RNA circLOCMI02072 (1, 2） Its application as a behavior regulator in locusts falls under the field of agricultural biotechnology. This invention discovers circular RNA. circLOCMI02072 (1, 2） The expression of this circular RNA differs between gregarious and solitary locusts, and its expression level is negatively correlated with locust swarming behavior. Increasing the expression level of this circular RNA in locusts can significantly inhibit locust swarming behavior. This invention provides a novel, green, and pollution-free locust control technology that is highly efficient, specific, and environmentally friendly, and has important application value for locust control.

Description

Technical Field

[0001] This invention belongs to the field of agricultural biotechnology, and more specifically, relates to circular RNA. circLOCMI02072 (1,2) Application as a behavior regulator for locusts. Background Technology

[0002] Locust plagues are one of the three major natural disasters in human history, posing a serious threat to agriculture, animal husbandry, the environment, and the economy. The long-term use of chemical pesticides to control locusts has led to a series of problems, including pesticide residues, environmental pollution, locust resistance, and food safety issues. These problems with chemical control technologies have forced researchers to seek new, green, and pollution-free locust control technologies.

[0003] Biological control, as an environmentally friendly control strategy, has received widespread attention in recent years. Among its applications, regulating locust swarming behavior is considered an effective way to control locust plagues. Locusts exhibit two ecotypes: solitary and gregarious, and the transition from solitary to gregarious behavior is a prerequisite for locust outbreaks. Currently, some studies have explored the regulatory mechanisms of locust swarming behavior, such as the roles of neurotransmitters like dopamine and serotonin, as well as some small RNA molecules. However, these regulatory factors lack specificity and suffer from problems such as poor stability and easy degradation in practical applications.

[0004] Circular RNAs (RNAs) are a novel class of non-coding RNA molecules with a covalently closed circular structure, making them less susceptible to degradation by exonucleases and thus more stable than linear RNAs. In recent years, the regulatory role of circular RNAs in the development and progression of mammalian diseases has been extensively studied, but research in the field of insect behavioral regulation is relatively limited. Currently, there are no systematic reports on the role of circular RNAs in regulating locust swarming behavior, and there is a lack of circular RNA molecules that specifically regulate locust swarming behavior.

[0005] Current technologies for controlling locust swarming behavior primarily rely on traditional methods such as chemical pheromones and neurotransmitters. These methods suffer from low specificity, poor environmental stability, and susceptibility to resistance. Therefore, developing novel, green, and efficient locust control technologies has become an urgent problem to be solved. Summary of the Invention

[0006] The purpose of this invention is to provide circular RNA. circLOCMI02072 (1,2) As a behavior regulator of locusts, this circular RNA is used to suppress locust swarming behavior by increasing its expression level in locusts.

[0007] To achieve the above-mentioned objectives, the present invention adopts the following technical solution: This invention provides circular RNA circLOCMI02072 (1,2) Application in the preparation of agents that inhibit locust swarming behavior, the circular RNA circLOCMI02072 (1,2) The nucleotide sequence is shown in SEQ ID NO.1.

[0008] Furthermore, the formulation increases the level of circular RNA in locusts. circLOCMI02072 (1,2) The expression level was used to suppress locust swarming behavior.

[0009] Furthermore, the increase in circular RNA in locusts circLOCMI02072 (1,2) The expression level was achieved by directly introducing circular RNA into the locust. circLOCMI02072 (1,2) .

[0010] Furthermore, the introduction of circular RNA into the locust's body... circLOCMI02072 (1,2) The method is to use circular RNA circLOCMI02072 (1,2) The lipid nanoparticles were encapsulated and injected into the brain of the locust via microinjection.

[0011] Furthermore, the circular RNA circLOCMI02072 (1,2) The injection concentration was 4 μg / μL, and the injection dose for each fourth-instar locust was 69 nL.

[0012] Furthermore, the locust in question is the East Asian migratory locust.

[0013] This invention provides a method for suppressing locust swarming behavior, the method comprising introducing circular RNA into the locust. circLOCMI02072 (1,2) To improve the circular RNA circLOCMI02072 (1,2) The expression level in locusts can be reduced, thereby inhibiting locust swarming behavior. Beneficial effects

[0014] (1) The circular RNA provided by the present invention circLOCMI02072 (1,2) It can specifically inhibit locust swarming behavior, providing a new approach and method for locust control.

[0015] (2) Compared with traditional chemical control methods, the method of the present invention is highly efficient, specific and environmentally friendly, and is a new type of green and pollution-free locust control technology. Attached Figure Description

[0016] Figure 1 To verify circular RNA circLOCMI02072 (1,2) The resulting diagram of the ring structure.

[0017] Figure 2 For injecting circular RNA circLOCMI02072 (1,2) The results of real-time fluorescence quantitative PCR were then used to detect its expression level. The P-value was calculated using t-test, and ****P < 0.0001.

[0018] Figure 3 For injecting circular RNA circLOCMI02072 (1,2)The expression of the linear transcripts of the source gene was then determined, and the P-value was calculated using a t-test. ****P < 0.0001.

[0019] Figure 4 For injecting circular RNA circLOCMI02072 (1,2) The expression of miR-9a regulated by it was then analyzed, and the P-value was calculated using a t-test. ****P < 0.0001.

[0020] Figure 5 To overexpress circular RNA circLOCMI02072 (1,2) The results of the locust behavior trend measurement show that the horizontal axis represents the degree of gregariousness of locust individuals, with 0 indicating absolutely solitary behavior and 1 indicating absolutely gregarious behavior; the vertical axis represents the proportion of locusts in a certain stage; the median is P. greg The median value is indicated by a red arrow.

[0021] Figure 6 For injecting circular RNA circLOCMI02072 (1,2) The attraction coefficient of the locust was determined, and the P-value was calculated using a t-test. ****P < 0.0001.

[0022] Figure 7 For injecting circular RNA circLOCMI02072 (1,2) The total distance traveled by the locusts was measured, and the P-value was calculated using a t-test. ****P < 0.0001.

[0023] Figure 8 For injecting circular RNA circLOCMI02072 (1,2) The results of the determination of the total duration of locust movement were obtained, and the P-value was calculated using t-test. ****P < 0.0001. Detailed Implementation

[0024] To enable those skilled in the art to better understand the technical solutions of this invention, the present application will be further described in detail below with reference to embodiments. Example

[0025] Based on a locust transcriptome database, bioinformatics methods were used to analyze the locust transcriptome. LOCMI02072 Genes are searched, and after sequence analysis and alignment, the results are obtained. LOCMI02072 Gene sequence (SEQ ID NO.1).

[0026] according to LOCMI02072 Gene sequences were obtained, and specific primers were designed using Primer Premier 5.0 software (Table 1). All primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0027] Table 1 Specific primer information

[0028] Total RNA was extracted from tissue samples of locusts in their fourth instar. 2 µg of total RNA was taken and reverse transcribed according to the standard procedure of the M-MLV reverse transcriptase kit (Promega, Madison, USA) to obtain cDNA.

[0029] Using cDNA derived from the reverse transcription of total RNA extracted from locusts as a template, PCR amplification was performed to obtain locusts. circLOCMI02072 （1，2） After the PCR product of the gene at the trans adapter was confirmed to be the correct size by agarose gel electrophoresis, it was ligated into the Blunt Zero vector, transformed into Trans1-T1 competent cells, and after colony PCR was confirmed to be the correct size, it was sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The sequencing results were compared with the transcriptome search results to verify the sequence correctness of the circular RNA trans adapter. Figure 1 ).

[0030] Example 2: Circular RNA circLOCMI02072 (1,2) Impact on locust swarming behavior 1. Circular RNA circLOCMI02072 (1,2) injection Synthesized circular RNA circLOCMI02072 (1,2) Dilute with nuclear-free water to 4 µg / µL and incubate with an equal volume of lipid nanoparticles. After 15 minutes, aspirate 69 nL of circular RNA using a microsyringe. circLOCMI02072 (1,2) Fourth-instar locusts were injected. Sampling or behavioral testing was performed 48 hours after injection.

[0031] Treated with the same lipid nanoparticles for injection GFP The fourth-instar locust was used as a control.

[0032] 2. Circular RNA circLOCMI02072 (1,2) Detection of expression levels Collection injection GFP and circLOCMI02072 (1,2) Fourth-instar migratory locusts were used. Total RNA was extracted using the Trizol method, and first-strand cDNA was obtained by reverse transcription using the M-MLV reverse transcriptase kit (Promega, Madison, USA). Real-time quantitative PCR was then performed to detect the cDNA.

[0033] The results showed that, compared with the control group, injection of circular RNA... circLOCMI02072 (1,2) Afterwards, the RNA of the treatment group circLOCMI02072 (1,2) The expression of was significantly increased ( Figure 2 ).

[0034] Simultaneously targeting RNA circLOCMI02072 (1,2) Regulated miRNAs and circLOCMI02072 (1,2) The source gene was detected. The results showed that... circLOCMI02072 (1,2) The source gene showed no significant change, indicating that the injected RNA... circLOCMI02072 (1,2) Only specifically improved circLOCMI02072 (1,2) The expression ( Figure 3 ), miR-9a The increase in expression levels indicates circular RNA circLOCMI02072 (1,2) Possibly through with miR-9a These interactions regulate the swarming behavior of locusts. Figure 4 ).

[0035] 3. Behavioral testing Injecting circular RNA circLOCMI02072 (1,2) Behavioral tests were conducted on locusts, including indicators such as swarming behavior, attraction coefficient, total distance traveled, and total duration of movement.

[0036] The results showed that injecting circular RNA circLOCMI02072 (1,2) Afterwards, the swarming behavior of locusts significantly decreased. Figure 5 The attraction coefficient decreased ( Figure 6 The total distance and total duration of exercise decreased ( Figure 7 and Figure 8 This indicates that locusts have shifted from a gregarious to a solitary lifestyle.

Claims

1. Circular RNA circLOCMI02072 (1,2) Its application in the preparation of agents that inhibit locust swarming behavior is characterized by, The circular RNA circLOCMI02072 (1,2) The nucleotide sequence is shown in SEQ ID NO.

1.

2. The application according to claim 1, characterized in that, The formulation increases the level of circular RNA in locusts. circLOCMI02072 (1,2) The expression level was used to suppress locust swarming behavior.

3. The application according to claim 2, characterized in that, The increase in circular RNA in locusts circLOCMI02072 （1，2） The expression level was achieved by directly introducing circular RNA into the locust. circLOCMI02072 (1,2) .

4. The application according to claim 3, characterized in that, The introduction of circular RNA into the locust's body circLOCMI02072 (1,2) The method is to use circular RNA circLOCMI02072 (1,2) The lipid nanoparticles were encapsulated and injected into the brain of the locust via microinjection.

5. The application according to claim 4, characterized in that, The circular RNA circLOCMI02072 (1,2) The injection concentration was 4 μg / μL, and the injection dose for each fourth-instar locust was 69 nL.

6. The application according to claim 5, characterized in that, The locust in question is the East Asian migratory locust.

7. A method for suppressing locust swarming behavior, characterized in that, The method includes introducing circular RNA into the locust's body. circLOCMI02072 (1,2) To improve the circular RNA circLOCMI02072 (1,2) The expression level in locusts can be reduced, thereby inhibiting locust swarming behavior.

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