Odor binding protein OBP7 gene and application of dsRNA thereof in prevention and treatment of solenopsis invicta

Abstract

The invention discloses an odorant binding protein OBP7 gene and application of dsRNA of the odorant binding protein OBP7 gene in prevention and treatment of solenopsis invicta. The invention proves that SiOPB7 can identify undecanal so as to trigger the corpse stacking behavior of solenopsis invicta, specifically, the corpse stacking behavior of solenopsis invicta triggered by undecanal is inhibited by feeding worker solenopsis invicta with dsOBP7, the pathogen transmission risk in a solenopsis invicta nest is increased, the social sexual behavior is interfered, the group adaptability is reduced, and the effect of controlling solenopsis invicta is achieved. The invention provides a theoretical basis for developing a preparation for preventing and controlling the solenopsis invicta by taking the SiOBP7 gene / protein as a target, and has important significance on green prevention and control of the solenopsis invicta.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to the application of the odor-binding protein OBP7 gene and its dsRNA in the control of red imported fire ants. Background Technology

[0002] Red imported fire ants Solenopsis invicta Red imported fire ants (Phnom Penh ants) are a globally invasive species native to South America and are among the most dangerous ants in the world. Their high aggressiveness, reproductive capacity, and rapid spread make them difficult to control. They spread through nuptial flights, water currents, and human activities, and have already invaded 534 counties in 12 provinces of China, causing annual economic losses of 160 billion yuan. Red imported fire ants are highly aggressive; their bites can cause skin ulcers, anaphylactic shock, and even death. They prey on crop seeds and seedlings, reducing the yields of crops such as sugarcane and corn. They damage local ecosystems, leading to the decline of invertebrate populations and ant diversity. They also gnaw on cables and damage circuits, causing infrastructure safety hazards and posing a serious threat to agriculture, ecology, and public safety.

[0003] In red imported fire ant nests, the enclosed and damp environment makes corpses highly susceptible to the growth of various pathogens and rapid decomposition, affecting the health of the nest. Worker ants can keenly identify the unique "death signals" on the corpses—oleic acid and linoleic acid—and quickly remove them from the nest, a behavior known as corpse-moving behavior (also called corpse-piling behavior). This behavior effectively reduces the risk of pathogen transmission, increases the survival rate of the ant colony, and thus maintains the normal operation of the colony's social behavior and the integrity of its social structure. Simultaneously, fungal infections can cause the rapid accumulation of fatty acids on the surface of corpses, further accelerating the corpse-moving behavior of worker ants. Studies have also shown that worker ants accurately identify and quickly remove dead individuals from the colony by recognizing life-related chemical substances, rather than the accumulation of fatty acids.

[0004] In recent years, numerous studies have reported that the social behavior of social insects is regulated by a series of olfactory system-related proteins. For example, in the Western honeybee (Honeybee... A. mellifera In studies of red imported fire ants, CSP3 is highly expressed in the antennae of drones and may be involved in the detection of queen sex pheromones. OR11 specifically responds to the queen pheromone 9-ODA and regulates worker bee aggregation and brood rearing behavior. In studies of red imported fire ants, IR75 may be involved in recognizing decaying food signals and regulating cleaning behavior. Currently, in studies of red imported fire ant carcass-carrying behavior, the key odor-binding proteins that respond to carcass piles are still unclear.

[0005] Red imported fire ants' high reproductive rate and aggressive characteristics enable them to rapidly invade and spread, increasing the risk of pathogen transmission within the nest, interfering with social behavior, and reducing colony adaptability. This is one way to effectively reduce harm. In natural environments, adding liquid bait containing dsRNA makes it easier for the bait to be transmitted throughout the ant colony through the feeding behavior of social insects, thereby reducing worker ant littering and increasing the risk of pathogen transmission, thus achieving the goal of pest control. Summary of the Invention

[0006] Based on the above-mentioned shortcomings and defects, the present invention aims to provide the application of the odor-binding protein OBP7 gene and its dsRNA in the control of red imported fire ants.

[0007] The first objective of this invention is to provide the application of the red imported fire ant odor-binding protein SiOBP7 gene in the control of red imported fire ants, wherein the nucleotide sequence of the gene encoding the odor-binding protein SiOBP7 is shown in SEQ ID NO.1.

[0008] Preferably, the application involves downregulating the expression level of the red imported fire ant odor-binding protein SiOBP7 gene, thereby inhibiting the red imported fire ant's recognition of substances that trigger carcass-piling behavior.

[0009] Preferably, the substance that triggers the pile-up behavior is undecylaldehyde.

[0010] A second objective of this invention is to provide the dsRNA of the red imported fire ant odor-binding protein SiOBP7, the nucleotide sequence of which is shown in SEQ ID NO.2.

[0011] A third objective of this invention is to provide a primer pair for preparing dsRNA of the red imported fire ant odor-binding protein SiOBP7, wherein the nucleotide sequence of the upstream primer of the primer pair is shown in SEQ ID NO.7 and the nucleotide sequence of the downstream primer of the primer pair is shown in SEQ ID NO.8.

[0012] The fourth objective of this invention is to provide a formulation for controlling red imported fire ants, wherein the active ingredient is dsRNA containing the red imported fire ant odor-binding protein SiOBP7.

[0013] The fifth objective of this invention is to provide the application of the dsRNA of the red imported fire ant odor-binding protein SiOBP7 or the preparation thereof in the control of red imported fire ants.

[0014] Preferably, the application includes the following steps: using the dsRNA of the red imported fire ant odor-binding protein SiOBP7 or the preparation described above as a bait to control red imported fire ants.

[0015] Preferably, the red imported fire ants are worker red imported fire ants.

[0016] The beneficial effects of this invention are: This invention identifies a protein that regulates the corpse-piling behavior of worker red imported fire ants. After downregulating the expression of the SiOBP7 gene in worker ants, the corpse-piling behavior significantly decreases. Over time, the worker ants also exhibit behaviors such as escaping and moving away from the corpses, thus altering their social behavior. This protein can be combined with highly efficient, targeted, controlled-release, and environmentally friendly nanomaterials to achieve the goal of controlling red imported fire ants. When used in conjunction with red imported fire ant control agents, the effectiveness of the control can be further improved. Attached Figure Description

[0017] Figure 1 The graph shows the mRNA expression levels of SiOBP7 in different tissues of red imported fire ant workers of different castes and adult red imported fire ant workers; where A is the mRNA expression level of OBP7 in different castes of red imported fire ants, and B is the mRNA expression level of OBP7 in different tissues of red imported fire ant workers; different letters indicate significant differences between different individuals or different tissues (p<0.05).

[0018] Figure 2 This is a diagram illustrating the behavior of red imported fire ant worker ants piling up their bodies after being fed dsOBP7.

[0019] Figure 3 This is a statistical chart showing the weight of the corpses of red imported fire ant worker ants within 90 minutes after being fed dsOBP7; **** indicates a significant difference, p<0.0001. Detailed Implementation

[0020] The following embodiments are further illustrations of the present invention, but not limitations thereof.

[0021] The following examples use the SiOBP7 (Odorant-binding Protein 7) gene of red imported fire ants as the target to explore its application in the control of red imported fire ants.

[0022] Example 1 Artificial ant nests were constructed, and the test red imported fire ant nests were placed in plastic boxes (40 cm × 20 cm × 15 cm) for rearing. The inner walls were coated with talcum powder. 50 mL centrifuge tubes were filled with water, stuffed with cotton, and placed in the ant nests as water test tubes. Food was fed on time every day.

[0023] Total RNA extraction: Red imported fire ant eggs, 4th instar worker larvae, early-stage worker pupae, late-stage worker pupae, and adult worker ants were collected. Different tissues of adult worker ants (antennae, head without antennae, thorax, ovary, and midgut) were collected under a microscope. Total RNA was extracted using the SteadyPure RNA Extraction Kit (Aikerui, China; Catalog No.: AG21024), with three replicates for each sample. 1 μg of total RNA was reverse transcribed into cDNA using the Evo M-mLV reverse transcription kit (Aikerui, China; Catalog No.: AG21024).

[0024] Quantitative PCR detection: using HRbio TM The PCR SYBR® Green Master Mix kit (Horror, China; catalog number: HRF0032) was used for qPCR detection of SiOBP7. The primers for quantitative amplification of the SiOBP7 gene and the internal reference gene EF-Beta are as follows: OBP7-qF: 5′-GCTCCGTGCTAAATTGCCACC-3′, SEQ ID NO.3; OBP7-qR: 5′-GCATTTGAACACTTTGCCTCCT-3′, SEQ ID NO.4; EF-Beta-qF: 5′-CCTGAAGACCGATAAGGGCAT-3′, SEQ ID NO.5; EF-Beta-qR: 5′-GATTGTTGTGTTGGTGGTTTCC-3′, SEQ ID NO. 6.

[0025] The results showed that the red imported fire ant odor-binding protein 7 gene was specifically expressed in adult red imported fire ant worker ants. Figure 1 A in the text is not explicitly stated, but it is significantly highly expressed in the antennae and head of adult red imported fire ant worker ants. Figure 1 (B in the middle).

[0026] Example 2 The SiOBP7 gene sequence of the red imported fire ant worker ant was downloaded (GenBank accession number: LOC105201278, its CDS nucleotide sequence is shown in SEQ ID NO.1). The target gene sequence (215 bp, its nucleotide sequence is shown in SEQ ID NO.2) was selected from conserved regions and major functional regions to design and synthesize dsOBP7 primers for this gene. The dsEGFP primer sequence was designed using the enhanced green fluorescent protein (EGFP) gene sequence (GenBank accession number: U57609.1). The specific dsRNA primer sequences are as follows: dsOBP7-F: 5′-ATCGGTGCTTGCCATTTCTTT-3′, SEQ ID NO.7; dsOBP7-R: 5′-TCCGCATTCATAATTCCGAT-3′, SEQ ID NO.8; dsEGFP-F: 5′-GCCTGAAGTTCATCTGCACCA-3′, SEQ ID NO.9; dsEGFP-R: 5′-GTGTTCTGCTGGTAGTGGTCG-3′, SEQ ID NO. 10.

[0027] Using the cDNA of adult red imported fire ants from Example 1 as a template, Hieff Clone was used. TM The Zero TOPO-BluntSimple Cloning Kit (Yisheng, China; Catalog No.: 10910ES20) was used to amplify the target fragment. Following transformation, plating, bacterial picking, and shaking, positive clones were selected and sent to the company for sequencing. The returned plasmid with the correct sequence was used as a template for subsequent experiments. The specific primers for amplifying the target fragment are as follows: OBP7-F: 5′-ATGAAAGCAATCATTATCGTGC-3′, SEQ ID NO.11; OBP7-R: 5′-TTATGATAAAAGATGAAAATGTTTGTTGT-3′, SEQ ID NO. 12.

[0028] Using a plasmid as a template, the target gene was amplified by PCR using forward and reverse primers with a 5′ T7 promoter ligated to both ends. This yielded a target gene fragment with T7 promoter sequences ligated to both ends, which was then used as a template for dsRNA synthesis. The specific primer sequences are as follows: T7-OBP7-F: 5′-TAATACGACTCACTATAGGGATCGTGCTTGCCATTTCTTT-3′, SEQ IDNO.13; T7-OBP7-R: 5′-TAATACGACTCACTATAGGGTCCGCATTCATAATTCCGAT-3′, SEQ IDNO.14; T7-EGFP-F: 5′-TAATACGACTCACTATAGGGGCCTGAAGTTCATCTGCACCA-3′, SEQ IDNO.15; T7-EGFP-R: 5′-TAATACGACTCACTATAGGGGTGTTCTGCTGGTAGTGGTCG-3′, SEQ ID NO. 16.

[0029] According to Promega's T7 RiboMAX TM The instructions for the press RNAi System (catalog number: P1700) state that dsRNA can be synthesized to obtain dsOBP7 and dsEGFP.

[0030] Example 3 Several cylindrical cups were prepared, their inner walls coated with talcum powder. 0.5 g of red imported fire ant worker ants were weighed and placed in each cup. After 12 h of starvation, each worker ant was fed 120 µL of dsOBP7, averaging 1600 ng of dsOBP7 per ant. An equal amount of synthetic dsEGFP was fed as a control. After 36 h, the time it took for the red imported fire ant worker ants to accumulate carcasses and the weight of the accumulated carcasses were tested.

[0031] The prepared petri dish apparatus (structure as follows) Figure 2 The culture dish was divided into a treatment area and a control area. Filter paper (20×30 mm) was placed in each area. In the treatment area, 20 µL of 10 µg / µL undecylaldehyde solution (solvent: n-hexane) was dropped onto the filter paper. In the control area, 20 µL of n-hexane was dropped onto the filter paper. Both the treatment and control areas were allowed to evaporate for 20 seconds. In the remaining area, 0.1 g of red imported fire ant worker carcasses and 0.5 g of worker ants fed with dsRNA (dsOBP7 or dsEGFP) were placed. A transparent glass plate was placed on the culture dish to prevent worker ants from escaping. After 3 h, the weight of the carcasses in the treatment and control areas was weighed. SPSS test was used to compare the significance of the differences in the weight of the carcasses carried by the red imported fire ant worker ants.

[0032] The results showed that in the behavioral selection experiment, red imported fire ant workers fed dsEGFP were significantly affected by undecylaldehyde, triggering the red imported fire ant's corpse-piling behavior. After 90 minutes of treatment, they piled almost all the red imported fire ant worker corpses on filter paper dripped with undecylaldehyde, while workers fed dsOBP7 did not pile their corpses on filter paper dripped with undecylaldehyde after 90 minutes of treatment. Figure 2 ), and there were also significant differences in the weight of the piled-up corpses ( Figure 3 This study demonstrated that downregulating OBP7 expression could inhibit the triggering of corpse-piling behavior in red imported fire ant workers by undecaldehyde, significantly reducing the corpse-piling behavior of red imported fire ant workers.

[0033] The aforementioned dsOBP7 can be combined with highly efficient, targeted, controlled-release, and environmentally friendly nanomaterials to achieve the purpose of controlling red imported fire ants. When used in conjunction with red imported fire ant control agents, the effect of controlling red imported fire ants can be further improved.

Claims

1. The application of the SiOBP7 gene, a odor-binding protein in red imported fire ants, in the control of red imported fire ants, characterized in that... The nucleotide sequence of the gene encoding the odor-binding protein SiOBP7 is shown in SEQ ID NO.

1.

2. The application according to claim 1, characterized in that, This study aimed to downregulate the expression level of the SiOBP7 gene, a odor-binding protein in red imported fire ants, thereby inhibiting their recognition of substances that trigger carcass-piling behavior.

3. The application according to claim 2, characterized in that, The substance that triggers the pile-up of corpses is undecylaldehyde.

4. The dsRNA of the red imported fire ant odor-binding protein SiOBP7, characterized in that, Its nucleotide sequence is shown in SEQ ID NO.

2.

5. A primer pair for preparing dsRNA of the red imported fire ant odor-binding protein SiOBP7 as described in claim 4, characterized in that, The nucleotide sequence of the upstream primer of the primer pair is shown in SEQ ID NO.7, and the nucleotide sequence of the downstream primer of the primer pair is shown in SEQ ID NO.

8.

6. A preparation for controlling red imported fire ants, characterized in that, The active ingredient is dsRNA containing the red imported fire ant odor-binding protein SiOBP7 as described in claim 4.

7. The use of the dsRNA of the red imported fire ant odor-binding protein SiOBP7 as described in claim 4 or the preparation as described in claim 6 in the control of red imported fire ants.

8. The application according to claim 7, characterized in that, Includes the following steps: The dsRNA of the red imported fire ant odor-binding protein SiOBP7 or the preparations described herein can be used as bait to control red imported fire ants.

9. The application according to claim 7, characterized in that, The red imported fire ants mentioned are worker red imported fire ants.

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