Application of Osa-miR160e-3p in prevention and treatment of brown planthopper

Abstract

The invention belongs to the technical field of pest control, discloses application of Osa-miR160e-3p in brown planthopper control, and particularly discloses application of Osa-miR160e-3p in pest control and / or preparation of products for pest control. According to the application, it is found for the first time that after an artificially synthesized Osa-miR160e-3p mimics is injected into brown planthoppers, compared with a control group injected with DEPC water, the survival rate of nymphs of the brown planthoppers can be remarkably reduced, and it is proved that the Osa-miR160e-3p can be used for preventing and treating the brown planthoppers. Therefore, the Osa-miR160e-3p can be directly applied to field spraying and transgenic plants, and has a relatively good application value.

Description

Technical Field

[0001] The invention belongs to the technical field of pest control, and particularly relates to application of Osa-miR160e-3p in the control of brown planthopper. Background Art

[0002] Brown planthopper is a serious pest that harms the rice planting industry in East Asia and South Asia. Large-scale invasion of brown planthopper can cause huge economic losses in the affected areas. Brown planthopper is one of the newly released Chinese Class I crop pests and diseases list in 2023. At present, the main means of controlling brown planthoppers in agriculture is still spraying chemical pesticides, mainly including organophosphates, carbamates, pyrethroids, neonicotinoids and other pesticides, but the long-term use of chemical pesticides has caused serious ecological pollution and increased pest resistance. Therefore, there is an urgent need to develop new, efficient and environmentally friendly pest control methods.

[0003] RNAi is a conserved post-transcriptional gene silencing mechanism that exists in eukaryotes. Pesticides based on this mechanism can interfere with the normal growth, development, feeding, immunity, reproduction and other behaviors of pests by inhibiting gene expression, thereby achieving the purpose of pest control. Therefore, RNAi pesticides have also become a new type of pesticide recognized in the field of agriculture and forestry as being highly efficient, specific and environmentally friendly. Exploring sources of nucleic acid components that are more effective against insects, safer for humans and animals, and more environmentally friendly will be beneficial to the application of RNAi pesticides. Screening miRNAs from plants that humans and animals eat daily for pest control has the advantage of being more environmentally friendly than traditional artificially synthesized dsRNA or insect-derived miRNAs. However, there are currently very few studies on the use of plant-derived miRNAs for pest control. Among brown planthoppers, only Shen et al. found that rice-derived miR162a caused a decrease in adult fertility by acting on the TOR gene of brown planthoppers. And there is no research reporting that Osa-miR160e-3p can be used for pest control. Summary of the invention

[0004] The purpose of the first aspect of the present invention is to provide the use of Osa-miR160e-3p and related biological materials in pest control and / or the preparation of pest control products.

[0005] The second aspect of the present invention aims to provide a product.

[0006] The third aspect of the present invention aims to provide a method for controlling pests.

[0007] In order to achieve the above object, the technical solution adopted by the present invention is:

[0008] The first aspect of the present invention provides the use of any one of the following substances (1) to (5) in pest control and / or in the preparation of pest control products;

[0009] (1) Osa-miR160e-3p;

[0010] (2) Osa-miR160e-3p precursor;

[0011] (3) Osa-miR160e-3p mimic;

[0012] (4) a nucleic acid molecule encoding the Osa-miR160e-3p described in (1) or the precursor of the Osa-miR160e-3p described in (2);

[0013] (5) An expression cassette, recombinant vector or transgenic cell containing the nucleic acid molecule described in (4).

[0014] In some embodiments of the present invention, the nucleotide sequence of Osa-miR160e-3p is shown in SEQ ID NO:1.

[0015] In some embodiments of the present invention, miRNA mimics are mimics of endogenous miRNA in organisms, synthesized by chemical synthesis, and can enhance the function of endogenous miRNA. Specifically, Osa-miR160e-3pmimics, i.e., mimics of Osa-miR160e-3p, are small double-stranded miRNAs designed and synthesized for the mature form of Osa-miR160e-3p, and have the same function as the mature Osa-miR160e-3p in vivo, and can upregulate the content of the corresponding Osa-miR160e-3p in cells and enhance the function of endogenous Osa-miR160e-3p. The mimics of Osa-miR160e-3p contain the sequence shown in SEQ ID NO: 1 and / or its complementary sequence.

[0016] In some embodiments of the present invention, the Osa-miR160e-3p of the present invention includes functional equivalents of the constituent nucleic acid molecules, i.e., variants, which show the same functions as the complete Osa-miR160e-3p nucleic acid molecules, which may be mutated by deletion, substitution or insertion of nucleotide residues.

[0017] It is well known to those skilled in the art that in order to ensure the stability of miRNA, protective bases such as TT can be added to one or both ends of miRNA; miRNA bases can also be modified, but the function of miRNA is not affected. Therefore, it is well known to those skilled in the art that under the condition that the function of Osa-miR160e-3p is not affected, the sequence obtained by base modification of Osa-miR160e-3p or adding bases at both ends is also included in the protection scope of the present invention, that is, the Osa-miR160e-3p can be any one or more ribonucleotides in the sequence shown in SEQ ID NO: 1 are modified.

[0018] In some embodiments of the present invention, the Osa-miR160e-3p is a mature miRNA, a precursor miRNA or a primary transcript.

[0019] In some embodiments of the present invention, the pests include insects of the order Homoptera.

[0020] In some embodiments of the present invention, the pests are one or more of leafhoppers, leafhoppers, treehoppers, planthoppers, psyllids, whiteflies, aphids and scale insects.

[0021] In some embodiments of the present invention, the pests are planthoppers, including but not limited to brown planthoppers, gray planthoppers, and white-backed planthoppers.

[0022] In some embodiments of the present invention, the product is a pesticide or insecticide.

[0023] In some embodiments of the present invention, the product can be prepared into conventional dosage forms for use, such as dry powder, wettable powder, emulsifiable concentrate, microemulsion, paste, granules or suspensions.

[0024] In some embodiments of the present invention, the application is to kill pests.

[0025] The rice-derived miRNA (Osa-miR160e-3p) has a control effect on the rice pest brown planthopper. Since Osa-miR160e-3p is derived from rice, a major crop, it is safe for humans and can be used as a new type of insecticide for safe and effective control of brown planthoppers. It can be used directly and in the construction of transgenic plants; therefore, Osa-miR160e-3p transgenic plants (i.e. plants overexpressing Osa-miR160e-3p) for brown planthopper control will have practical application value.

[0026] The second aspect of the present invention provides a product, comprising any one of the following substances (1) to (5);

[0027] (1) Osa-miR160e-3p;

[0028] (2) Osa-miR160e-3p precursor;

[0029] (3) Osa-miR160e-3p mimic;

[0030] (4) a nucleic acid molecule encoding the Osa-miR160e-3p described in (1) or the precursor of the Osa-miR160e-3p described in (2);

[0031] (5) An expression cassette, recombinant vector or transgenic cell containing the nucleic acid molecule described in (4).

[0032] In some embodiments of the present invention, the nucleotide sequence of Osa-miR160e-3p is shown in SEQ ID NO:1.

[0033] In some embodiments of the present invention, the product includes at least one of a reagent, a medicine, and a pesticide.

[0034] In some embodiments of the present invention, the product further comprises a pharmaceutically acceptable carrier, including but not limited to: a diluent, a buffer, a suspension, an emulsion, a granule, an encapsulation, an excipient, a filler, an adhesive, a spray, a transdermal absorbent, a wetting agent, a disintegrant, an absorption enhancer, a surfactant, a colorant, a flavoring agent or an adsorption carrier.

[0035] The third aspect of the present invention provides a method for controlling pests, comprising applying the substance described in the first aspect of the present invention or the product described in the second aspect of the present invention to pests or pest habitats.

[0036] In some embodiments of the invention, the method comprises treating pests, pest food (such as rice), pest habitat (soil, area, material or environment where pests are growing or can grow, or materials, cultivated plants, plant propagation materials (such as seeds), soil, surfaces to be protected from pest attack or infestation) with the substance or product.

[0037] In some embodiments of the present invention, an effective dose of the substance described in the first aspect of the present invention or the product of the second aspect of the present invention is applied to pests or pest habitats.

[0038] In general, "effective amount" means the amount of active ingredient required to achieve an observable effect on growth, including necrosis, death, retardation, prevention and removal, destruction or reduction of the presence and activity of the target organism. For Osa-miR160e-3p used in the present invention, the effective amount can vary. The effective amount of Osa-miR160e-3p also varies according to the prevailing conditions, such as the desired insecticidal effect and duration, climate, target species, location, mode of application, etc.

[0039] In some embodiments of the present invention, the pests include insects of the order Homoptera.

[0040] In some embodiments of the present invention, the pests are one or more of leafhoppers, leafhoppers, treehoppers, planthoppers, psyllids, whiteflies, aphids and scale insects.

[0041] In some embodiments of the present invention, the pests are planthoppers, including but not limited to brown planthoppers, gray planthoppers, and white-backed planthoppers.

[0042] The beneficial effects of the present invention are:

[0043] The present invention first discovered that after injecting artificially synthesized Osa-miR160e-3p mimics into brown planthoppers, the survival rate of brown planthopper nymphs can be significantly reduced compared with the control group injected with DEPC water, which proves that Osa-miR160e-3p can be used for the prevention and control of brown planthoppers. At the same time, studies have shown that Osa-miR160e-3p is beneficial to plants (such as rice), therefore, Osa-miR160e-3p can be directly used for field spraying and transgenic plants, and has good application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 The figure shows the flow chart of screening of plant-derived miRNAs.

[0045] Figure 2 Statistical diagram of miRNA targeting brown planthopper.

[0046] Figure 3 The proportion of rice-derived insect-resistant miRNAs targeting brown planthopper insect-resistant target genes.

[0047] Figure 4 The survival of brown planthoppers after microinjection of Osa-miR160e-3p (left) and the phenotype of brown planthoppers (right). The scale bar in the figure is 0.5 mm, and the scale bar in the enlarged image is 0.2 mm. DETAILED DESCRIPTION

[0048] The present invention is further described in detail below through specific examples.

[0049] It should be understood that these embodiments are only used to illustrate the present invention and are not used to limit the scope of the present invention.

[0050] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0051] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0052] Example 1

[0053] In order to mine miRNAs with the function of controlling brown planthoppers in the host plants of brown planthoppers, the present embodiment collects the identified mature miRNAs of rice and other major crops through the miRBase database (https: / / mirbase.orgmirbase_new / wsgi.py / ), and compares them with the CDS region and 3'UTR region of the whole mRNA of brown planthoppers (GenBank No. GCA_014356525.1) for target gene prediction analysis (target gene prediction table is shown in Table 1), and preliminarily screens the miRNAs according to the screening principle of more reported lethal target genes (Table 2) (screening process is shown in Table 2). Figure 1 As shown, the results are Figure 2 ), and found that rice-derived miRNAs have more targeted transcription factors, detoxification enzymes, and some genes in neural pathways ( Figure 3 ). Among them, Osa-miR160e-3p has more target genes. Bioinformatics predicts that the main target genes of Osa-miR160e-3p are chitin synthase ch-2 isoform, neuronal acetylcholine receptor subunit α-7-like, ecdysone receptor, cuticle protein, transcriptional egg inhibitor, transcriptional inhibitor and other genes.

[0054] The nucleotide sequence of Osa-miR160e-3p is 5′-GCGUGCGAGGUGCCAAGCAUG-3′ (SEQ ID NO: 1).

[0055] Table 1 Target gene prediction table

[0056]

[0057]

[0058] Table 2 Proven lethal genes

[0059]

[0060]

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[0100] Example 2

[0101] miRNA can act on multiple target mRNAs at the same time. It is still unclear how exogenous miRNA chooses which targets to act on after entering the body. Therefore, it is necessary to conduct in vivo testing on Osa-miR160e-3p to verify its control effect on brown planthoppers. The specific process is as follows:

[0102] Experimental methods

[0103] 1. Test insects

[0104] The brown planthopper strain was obtained from the Institute of Plant Protection, Guangdong Academy of Agricultural Sciences. From March to November each year, it was raised at room temperature in the laboratory and fed with Taichung Native 1 (TN1) rice. In winter, it was raised in an artificial climate chamber under the conditions of 27°C, 14 hours of light and 10 hours of darkness in every 24 hours, and 80% relative humidity.

[0105] 2. Plant Culture

[0106] Rice line (TN1) was planted outdoors in plastic pots. When the seedlings grew to the early stage of tillering, they were transplanted into plastic cups with a diameter of 10 cm and a height of 15 cm, with 4 to 5 seedlings per cup. During the experiment, they were covered with a plastic cover with a diameter of 9.5 cm and a height of 60 cm containing air holes.

[0107] 3. Injection of synthetic miRNA mimics into brown planthopper nymphs

[0108] 1) Dilute miRNA mimics to a concentration of 1 ng / mL with DEPC water and place on ice for later use;

[0109] 2) Collect 5-year-old brown planthopper nymphs into a clean 50 mL centrifuge tube, plug the tube mouth with cotton wool, insert a CO2 ventilation gun and inject CO2 at a flow rate of 200 mL / min for 30 seconds to anesthetize the nymphs;

[0110] 3) Pour the anesthetized nymphs onto an agarose gel plate and inject the connection between the second and third pairs of appendages of the nymphs with a microsyringe under a stereomicroscope. The injection volume for each nymph is 100 nL.

[0111] 4) Place the injected nymphs in a clean 50 mL centrifuge tube and observe their recovery status;

[0112] 5) Transfer the successfully revived nymphs to a plastic cup with air holes and feed them with fresh rice;

[0113] 6) The survival rate and phenotypic changes of the nymphs were counted every 24 hours after injection until the end of the experiment at 168 hours.

[0114] Mimics are mimics of endogenous miRNA in organisms, synthesized by chemical synthesis, and can enhance the function of endogenous miRNA. Specifically, Osa-miR160e-3p mimics, i.e., mimics of Osa-miR160e-3p, are small double-stranded miRNAs designed and synthesized for the mature form of Osa-miR160e-3p, and have the same function as the mature Osa-miR160e-3p in vivo, and can upregulate the content of the corresponding Osa-miR160e-3p in cells and enhance the function of endogenous Osa-miR160e-3p. Osa-miR160e-3p mimics contain the sequence shown in SEQ ID NO: 1 and / or its complementary sequence.

[0115] The nucleotide sequence of NC mimics (ie, nonsense fragment sequence) is as follows: 5'-UUCUCCGAACGUGUCACGUTT-3' (SEQ ID NO: 2).

[0116] result

[0117] After injecting DEPC water and Osa-miR160e-3p mimics (about 0.1 μg per worm) into the 5th instar nymphs of brown planthopper, the mortality of brown planthopper was observed within 0 to 168 hours. Figure 4 As shown in the figure, the survival rate of nymphs injected with Osa-miR160e-3p mimics from the host plant was significantly lower than that of the control group injected with DEPC water and NC. The survival rate of nymphs injected with Osa-miR160e-3pmimics was 31.84%, and the phenotype of molting failure (i.e., failure to molt in the next instar) appeared, accounting for 22.4% of the dead nymphs. The results of three batches of repeated experiments showed that Osa-miR160e-3pmimics significantly reduced the survival rate of 4th to 5th instar nymphs of brown planthoppers, indicating that Osa-miR160e-3p from the host plant of brown planthoppers has a good insect-resistant effect on brown planthoppers. Therefore, Osa-miR160e-3p can be directly used for field spraying or transgenic plants for pest control, and has certain application value.

[0118] The embodiments of the present invention are described in detail above in conjunction with the accompanying drawings, but the present invention is not limited to the above embodiments. Various changes can be made within the knowledge of ordinary technicians in the relevant technical field without departing from the purpose of the present invention. In addition, the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

Claims

1. Use of any of the following substances (1) to (5) in pest control and / or preparation of pest control products; (1) Osa-miR160e-3p; (2) Osa-miR160e-3p precursor; (3) Osa-miR160e-3p mimic; (4) a nucleic acid molecule encoding the Osa-miR160e-3p described in (1) or the precursor of the Osa-miR160e-3p described in (2); (5) An expression cassette, recombinant vector or transgenic cell containing the nucleic acid molecule described in (4).

2. The use according to claim 1, characterized in that: The nucleotide sequence of Osa-miR160e-3p is shown in SEQ ID NO:

1.

3. The use according to claim 1, characterized in that: The pests include insects of the order Homoptera; The pests are preferably one or more of leafhoppers, leafhoppers, treehoppers, planthoppers, psyllids, whiteflies, aphids and scale insects.

4. The use according to any one of claims 1 to 3, characterized in that: The application is to kill pests.

5. A product comprising any one of the following substances (1) to (5); (1) Osa-miR160e-3p; (2) Osa-miR160e-3p precursor; (3) Osa-miR160e-3p mimic; (4) a nucleic acid molecule encoding the Osa-miR160e-3p described in (1) or the precursor of the Osa-miR160e-3p described in (2); (5) an expression cassette, a recombinant vector or a transgenic cell containing the nucleic acid molecule described in (4); Preferably, the nucleotide sequence of Osa-miR160e-3p is as shown in SEQ ID NO:

1.

6. The product according to claim 5, characterized in that The product includes at least one of a reagent, a medicine, and a pesticide.

7. A method for controlling pests, comprising applying the substance according to claim 1 or the product according to claim 5 to the pests or the habitat of the pests.

8. The method according to claim 7, characterized in that The method comprises treating the pests, the food of the pests, or the habitat of the pests with the substance or product.

9. The method according to claim 7 or 8, characterized in that: The pests include insects of the order Homoptera.

10. The method according to claim 9, characterized in that The pests are one or more of leafhoppers, leafhoppers, treehoppers, planthoppers, psyllids, whiteflies, aphids and scale insects.

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