Genes for increasing insecticide sensitivity and uses thereof

Abstract

The present application relates to an insect gene UGT 2B20-like, the expression of the gene is inhibited, the sensitivity of the insect to the insecticide can be increased by more than 10%. Therefore, the double-stranded RNA synthesized based on the gene can be used for the reduced application of chemical pesticides. The present application also discloses the mechanism of action of down-regulating the expression of the gene to improve the sensitivity of the insecticide.

Description

Technical Field

[0001] This invention belongs to the field of insecticides in biotechnology; more specifically, this invention relates to improving insecticide sensitivity by utilizing the antioxidant-related genes of insects, which can be used to improve the application effect of existing insecticides in pest control. Background Technology

[0002] In the continuous interaction between the insect gut and its microorganisms, the production of reactive oxygen species (ROS) mediated by dual oxidases (DUOX) is a major mechanism regulating the homeostasis of the insect gut microbiota. Under normal circumstances, the organism maintains a stable balance between ROS production and clearance, with relatively low intracellular ROS levels. However, when this balance is disrupted, it leads to excessive ROS production or accumulation, causing damage to the organism. Enzymes such as POD, SOD, and CAT are important components of the antioxidant system and can clear excess ROS. Silencing the UGT gene in *Apis cerana cerana* inhibits the expression of oxidation-related genes, reduces the activity of antioxidant enzymes, and increases malondialdehyde concentration, thereby increasing oxidative stress. However, in *Helicoverpa armigera*, the influence of gut microbiota on UGT expression and the regulation of oxidative stress by UGT remain unclear.

[0003] Most UGT proteins are located in the endoplasmic reticulum lumen and consist of two functional domains: an N-terminal domain and a C-terminal domain. The N-terminus is responsible for binding to glycosidic ligands, while the C-terminus is responsible for binding to and catalyzing substrates. UGTs are widely distributed in all living organisms, including plants, animals, bacteria, and viruses. Compared to P450s, CarEs, and GSTs, UGTs have been less studied in insects. Current research indicates that UGTs are involved in tactile responses, antioxidant responses, and the degradation of endogenous and exogenous harmful substances. In studies on the beet armyworm (Spodoptera litura), UGTs were found to be highly expressed in the antennae. Furthermore, UGT transcription was regulated to varying degrees after exposure to sex pheromones, plants, or pyrethroid insecticides. Overexpression of NlUGT386F2D contributes to the resistance of the brown planthopper (Nilaparvata lugens) to thiamethoxam. Further experiments revealed that reactive oxygen species inhibitors (N-acetylcysteine) can reduce the expression of NlUGT386F2, thereby enhancing the sensitivity of brown planthoppers to thiamethoxam. Furthermore, silencing Duox to reduce the production of reactive oxygen species can significantly improve the sensitivity of brown planthoppers to thiamethoxam. Summary of the Invention

[0004] The present invention first provides the use of a UGT 2B20-like protein, its coding sequence, a nucleic acid construct containing the coding sequence, a cell expressing the protein, or a cell containing the coding sequence or a nucleic acid construct in screening candidate substances that can modulate the sensitivity of insects to pesticides.

[0005] This invention provides the use of UGT 2B20-like protein or its coding sequence in screening candidate substances that can regulate insect sensitivity to pesticides.

[0006] In one or more embodiments, the amino acid sequence of the UGT 2B20-like protein is as shown in SEQ ID NO:2 or a sequence having at least 80% identity with it.

[0007] In one or more embodiments, the coding sequence of the UGT 2B20-like protein comprises or has at least 80% identity with the sequence described in SEQ ID NO:1.

[0008] This invention also provides a method for screening candidate substances that can enhance insect sensitivity to pesticides, including...

[0009] (1) Contact the candidate material with a system containing a UGT 2B20-like protein or its coding sequence, and

[0010] (2) Detect the expression or activity of UGT 2B20-like protein and compare it with the control group. If the expression or activity of UGT 2B20-like protein is downregulated, the substance is a candidate substance for enhancing the sensitivity of insects to pesticides.

[0011] In one or more embodiments, the control is the same system that does not contain the substance.

[0012] In one or more embodiments, the system is a solution system, a cell system, a tissue system, a plant, or an animal, such as the insect.

[0013] In one or more embodiments, the insect is a noctuid moth, preferably a bollworm, and more preferably a cotton bollworm (Helicoverpa armigera).

[0014] In one or more embodiments, the insecticide includes organophosphates, carbamates, pyrethroids, organochlorines, neonicotinoids, insect growth regulators, etc., preferably including one or more selected from the following: imidacloprid, acetamiprid, thiamethoxam, dinotefuran, diflubenzuron, diflubenzuron, tebufenozide, and tebufenozide.

[0015] This invention provides a nucleic acid construct comprising

[0016] (1) Nucleic acid sequence encoding UGT 2B20-like protein

[0017] (2) Nucleic acid sequences that specifically interfere with the transcription and / or expression of UGT 2B20-like genes.

[0018] In one or more embodiments, the nucleic acid sequence of the UGT 2B20-like protein comprises the sequence described in SEQ ID NO:1 or has at least 80% identity with it.

[0019] In one or more embodiments, the amino acid sequence of the UGT 2B20-like protein is as shown in SEQ ID NO:2 or a sequence having at least 80% identity with it.

[0020] In one or more embodiments, the nucleic acid that specifically interferes with the transcription and / or expression of the UGT 2B20-like gene comprises the sequence described in SEQ ID NO:3 or has at least 80% identity with it.

[0021] This invention provides a genetically engineered host cell, which

[0022] (1) Expression, inclusion, or secretion of UGT 2B20-like protein, or

[0023] (2) Contains a nucleic acid sequence encoding the Hsp70-3 protein, or

[0024] (3) Includes the nucleic acid constructs described in any of the embodiments described herein.

[0025] The present invention provides a method for regulating the sensitivity of insects to pesticides, the method comprising: downregulating the expression or activity of UGT2B20-like protein in insects to increase the sensitivity of insects to pesticides, or upregulating the expression or activity of UGT2B20-like protein in insects to decrease the sensitivity of insects to pesticides.

[0026] In one or more embodiments, the insect is a noctuid moth, preferably a bollworm, and more preferably a cotton bollworm (Helicoverpa armigera).

[0027] In one or more embodiments, the insecticide includes organophosphates, carbamates, pyrethroids, organochlorines, neonicotinoids, insect growth regulators, etc., preferably including one or more selected from the following: imidacloprid, acetamiprid, thiamethoxam, dinotefuran, diflubenzuron, diflubenzuron, tebufenozide, and tebufenozide.

[0028] In one or more embodiments, downregulating UGT 2B20-like protein expression in insects includes:

[0029] (1) Specific interference with the transcription and / or expression of UGT 2B20-like genes.

[0030] (2) Downregulates the activity of UGT 2B20-like protein.

[0031] (3) Express a reduced-activity UGT 2B20-like protein variant or its coding sequence in insects.

[0032] In one or more embodiments, (1) the interference is interference with gene transcription or translation of its transcripts.

[0033] In one or more embodiments, (2) the downregulation includes in insects:

[0034] (i) Expressing specific antibodies or ligands that can downregulate protein activity, or

[0035] (ii) Import the nucleic acid sequence encoding (i) and / or a nucleic acid construct that can express (i).

[0036] In one or more embodiments, the nucleic acid that specifically interferes with the transcription and / or expression of the UGT 2B20-like gene is selected from the group consisting of (1) antisense nucleic acids, microRNA, siRNA, RNAi, dsRNA, sgRNA or combinations thereof, and (2) nucleic acid constructs that can express or form (1).

[0037] In one or more embodiments, the nucleic acid that specifically interferes with the transcription and / or expression of the UGT 2B20-like gene comprises the sequence described in SEQ ID NO:3 or has at least 80% identity with it.

[0038] In one or more embodiments, the upregulation of UGT 2B20-like protein expression in insects includes: transferring the coding sequence of the UGT 2B20-like protein into insects to obtain transformed insects.

[0039] The present invention also provides a method for controlling insects, comprising the steps of:

[0040] (1) Downregulates the expression or activity of UGT 2B20-like proteins in insects, and

[0041] (2) Expose insects to pesticides.

[0042] In one or more embodiments, the insect is a noctuid moth, preferably a bollworm, and more preferably a cotton bollworm (Helicoverpa armigera).

[0043] In one or more embodiments, the insecticide includes organophosphates, carbamates, pyrethroids, organochlorines, neonicotinoids, insect growth regulators, etc., preferably including one or more selected from the following: imidacloprid, acetamiprid, thiamethoxam, dinotefuran, diflubenzuron, diflubenzuron, tebufenozide, and tebufenozide.

[0044] In one or more embodiments, downregulating the expression or activity of UGT 2B20-like proteins in insects includes:

[0045] (1) Specific interference with the transcription and / or expression of UGT 2B20-like genes.

[0046] (2) Downregulates the activity of UGT 2B20-like protein.

[0047] (3) Express a reduced-activity UGT 2B20-like protein variant or its coding sequence in insects.

[0048] In one or more embodiments, (1) the interference is interference with gene transcription or translation of its transcripts.

[0049] In one or more embodiments, (2) the downregulation includes in insects:

[0050] (i) Expressing specific antibodies or ligands that can downregulate protein activity, or

[0051] (ii) Import the nucleic acid sequence encoding (i) and / or a nucleic acid construct that can express (i).

[0052] In one or more embodiments, the nucleic acid that specifically interferes with the transcription and / or expression of the UGT 2B20-like gene is selected from the group consisting of (1) antisense nucleic acids, microRNA, siRNA, RNAi, dsRNA, sgRNA or combinations thereof, and (2) nucleic acid constructs that can express or form (1).

[0053] In one or more embodiments, the nucleic acid that specifically interferes with the transcription and / or expression of the UGT 2B20-like gene comprises the sequence described in SEQ ID NO:3 or has at least 80% identity with it.

[0054] In one or more embodiments, (1) the downregulation comprises contacting the insect with an inhibitor that downregulates the expression or activity of the UGT 2B20-like protein in the insect. In one or more embodiments, the inhibitor is selected from the group consisting of (i) antisense nucleic acids, microRNAs, siRNAs, shRNAs, dsRNAs, sgRNAs, small molecule compounds, or combinations thereof, and (ii) nucleic acid constructs capable of expressing or forming (i). In one or more embodiments, the inhibitor is a nucleic acid that specifically interferes with the transcription and / or expression of the UGT 2B20-like gene. In one or more embodiments, the nucleic acid that specifically interferes with the transcription and / or expression of the UGT 2B20-like gene comprises the sequence described in SEQ ID NO:3 or has at least 80% identity with it. Preferably, the nucleic acid is a double-stranded DNA, wherein one strand has the sequence shown in SEQ ID NO:3 or has at least 80% identity with it. Alternatively, the nucleic acid is a double-stranded RNA, wherein one strand has an RNA counterpart of the sequence shown in SEQ ID NO:3 or has at least 80% identity with it.

[0055] The present invention also provides the use of substances that regulate the expression or activity of UGT 2B20-like proteins in insects in regulating the sensitivity of insects to pesticides.

[0056] In one or more embodiments, the insect is a noctuid moth, preferably a bollworm, and more preferably a cotton bollworm (Helicoverpa armigera).

[0057] In one or more embodiments, the insecticide includes organophosphates, carbamates, pyrethroids, organochlorines, neonicotinoids, insect growth regulators, etc., preferably including one or more selected from the following: imidacloprid, acetamiprid, thiamethoxam, dinotefuran, diflubenzuron, diflubenzuron, tebufenozide, and tebufenozide.

[0058] In one or more embodiments, the substance is an inhibitor of UGT 2B20-like protein expression or activity, and the regulation is to downregulate UGT 2B20-like protein expression or activity in insects, thereby increasing the insects' sensitivity to insecticides.

[0059] In one or more embodiments, the inhibitor is selected from:

[0060] (1) Inhibitors that specifically interfere with the transcription and / or expression of UGT 2B20-like genes.

[0061] (2) Inhibitors that downregulate the activity of UGT 2B20-like proteins,

[0062] (3) UGT 2B20-like protein variants or their coding sequences that have been downregulated.

[0063] In one or more embodiments, the inhibitor of item (1) is selected from the group consisting of (i) antisense nucleic acids, microRNA, siRNA, shRNA, dsRNA, sgRNA, small molecule compounds or combinations thereof, and (ii) nucleic acid constructs that can express or form (i).

[0064] In one or more embodiments, the inhibitor of item (1) is a nucleic acid that specifically interferes with the transcription and / or expression of the UGT 2B20-like gene.

[0065] In one or more embodiments, the inhibitor of item (2) is selected from the group consisting of (i) specific antibodies or ligands of UGT 2B20-like proteins, and (ii) nucleic acid sequences encoding (i) and / or nucleic acid constructs capable of expressing (i).

[0066] In one or more embodiments, the nucleic acid that specifically interferes with the transcription and / or expression of the UGT 2B20-like gene comprises the sequence described in SEQ ID NO:3 or has at least 80% identity with it. Preferably, the nucleic acid is double-stranded DNA, wherein one strand has the sequence shown in SEQ ID NO:3 or has at least 80% identity with it. Alternatively, the nucleic acid is double-stranded RNA, wherein one strand has the RNA counterpart of the sequence shown in SEQ ID NO:3 or has at least 80% identity with it.

[0067] In one or more embodiments, the substance is a promoter of UGT 2B20-like protein expression or activity, and the regulation is to upregulate the expression or activity of UGT 2B20-like protein in insects, thereby reducing the insects' sensitivity to insecticides.

[0068] In one or more embodiments, the promoter is a UGT 2B20-like protein or its coding sequence.

[0069] The present invention also provides a method for regulating the antioxidant capacity of insects, the method comprising: downregulating the expression or activity of UGT2B20-like protein in insects, thereby reducing the antioxidant capacity of insects, and upregulating the expression or activity of UGT2B20-like protein in insects, thereby increasing the antioxidant capacity of insects.

[0070] In one or more embodiments, the insect is a noctuid moth, preferably a bollworm, and more preferably a cotton bollworm (Helicoverpa armigera).

[0071] In one or more embodiments, downregulating UGT 2B20-like protein expression in insects includes:

[0072] (1) Specific interference with the transcription and / or expression of UGT 2B20-like genes.

[0073] (2) Downregulates the activity of UGT 2B20-like protein.

[0074] (3) Express a reduced-activity UGT 2B20-like protein variant or its coding sequence in insects.

[0075] In one or more embodiments, (1) the interference is interference with gene transcription or translation of its transcripts.

[0076] In one or more embodiments, (2) the downregulation includes in insects:

[0077] (i) Expressing specific antibodies or ligands that can downregulate protein activity, or

[0078] (ii) Import the nucleic acid sequence encoding (i) and / or a nucleic acid construct that can express (i).

[0079] In one or more embodiments, the nucleic acid that specifically interferes with the transcription and / or expression of the UGT 2B20-like gene is selected from the group consisting of (1) antisense nucleic acids, microRNA, siRNA, RNAi, dsRNA, sgRNA or combinations thereof, and (2) nucleic acid constructs that can express or form (1).

[0080] In one or more embodiments, the nucleic acid that specifically interferes with the transcription and / or expression of the UGT 2B20-like gene comprises the sequence shown in SEQ ID NO:3 or has at least 80% identity with it.

[0081] In one or more embodiments, the upregulation of UGT 2B20-like protein expression in insects includes: transferring the coding sequence of the UGT 2B20-like protein into insects to obtain transformed insects.

[0082] The present invention also provides reagents and insecticides for downregulating the expression or activity of UGT 2B20-like proteins in the control of insects.

[0083] In one or more embodiments, the insect is a noctuid moth, preferably a bollworm, and more preferably a cotton bollworm (Helicoverpa armigera).

[0084] In one or more embodiments, the insecticide includes organophosphates, carbamates, pyrethroids, organochlorines, neonicotinoids, insect growth regulators, etc., preferably including one or more selected from the following: imidacloprid, acetamiprid, thiamethoxam, dinotefuran, diflubenzuron, diflubenzuron, tebufenozide, and tebufenozide.

[0085] In one or more embodiments, the agent for downregulating UGT 2B20-like protein expression or activity is an inhibitor of UGT2B20-like protein expression or activity, said inhibitor being selected from:

[0086] (1) Inhibitors that specifically interfere with the transcription and / or expression of UGT 2B20-like genes.

[0087] (2) Inhibitors that downregulate the activity of UGT 2B20-like proteins,

[0088] (3) UGT 2B20-like protein variants or their coding sequences that have been downregulated.

[0089] In one or more embodiments, the inhibitor of item (1) is selected from the group consisting of (i) antisense nucleic acids, microRNAs, siRNAs, shRNAs, dsRNAs, sgRNAs, small molecule compounds, or combinations thereof, and (ii) nucleic acid constructs capable of expressing or forming (i). In one or more embodiments, the inhibitor of item (1) is a nucleic acid that specifically interferes with the transcription and / or expression of UGT 2B20-like genes.

[0090] In one or more embodiments, the inhibitor of item (2) is selected from the group consisting of (i) specific antibodies or ligands of UGT 2B20-like proteins, and (ii) nucleic acid sequences encoding (i) and / or nucleic acid constructs capable of expressing (i).

[0091] In one or more embodiments, the nucleic acid that specifically interferes with the transcription and / or expression of the UGT 2B20-like gene comprises the sequence shown in SEQ ID NO:3 or has at least 80% identity with it.

[0092] The present invention also provides a composition or kit for controlling insects, comprising a reagent and an insecticide that downregulates the expression or activity of UGT 2B20-like protein.

[0093] In one or more embodiments, the insect is a noctuid moth, preferably a bollworm, and more preferably a cotton bollworm (Helicoverpa armigera).

[0094] In one or more embodiments, the insecticide includes organophosphates, carbamates, pyrethroids, organochlorines, neonicotinoids, insect growth regulators, etc., preferably including one or more selected from the following: imidacloprid, acetamiprid, thiamethoxam, dinotefuran, diflubenzuron, diflubenzuron, tebufenozide, and tebufenozide.

[0095] In one or more embodiments, the agent for downregulating UGT 2B20-like protein expression or activity is an inhibitor of UGT2B20-like protein expression or activity, said inhibitor being selected from:

[0096] (1) Inhibitors that specifically interfere with the transcription and / or expression of UGT 2B20-like genes.

[0097] (2) Inhibitors that downregulate the activity of UGT 2B20-like proteins,

[0098] (3) UGT 2B20-like protein variants or their coding sequences that have been downregulated.

[0099] In one or more embodiments, the inhibitor of item (1) is selected from the group consisting of (i) antisense nucleic acids, microRNAs, siRNAs, shRNAs, dsRNAs, sgRNAs, small molecule compounds, or combinations thereof, and (ii) nucleic acid constructs capable of expressing or forming (i). In one or more embodiments, the inhibitor of item (1) is a nucleic acid that specifically interferes with the transcription and / or expression of UGT 2B20-like genes.

[0100] In one or more embodiments, the inhibitor of item (2) is selected from the group consisting of (i) specific antibodies or ligands of UGT 2B20-like proteins, and (ii) nucleic acid sequences encoding (i) and / or nucleic acid constructs capable of expressing (i).

[0101] In one or more embodiments, the nucleic acid that specifically interferes with the transcription and / or expression of the UGT 2B20-like gene comprises the sequence shown in SEQ ID NO:3 or has at least 80% identity with it. Preferably, the nucleic acid is double-stranded DNA, wherein one strand has the sequence shown in SEQ ID NO:3 or has at least 80% identity with it. Alternatively, the nucleic acid is double-stranded RNA, wherein one strand has the RNA counterpart of the sequence shown in SEQ ID NO:3 or has at least 80% identity with it.

[0102] In one or more embodiments, the composition or kit further comprises at least one component selected from the group consisting of carrier agents, surfactants, cationic lipids, organosilicons, organosilicon surfactants, polynucleotide herbicidal molecules, non-polynucleotide herbicidal molecules, non-polynucleotide pesticides, safeners and insect growth regulators.

[0103] In one or more embodiments, the composition is in the form of at least one selected from the group consisting of solids, liquids, powders, suspensions, emulsions, sprays, encapsulating agents, microbeads, carrier microparticles, thin films, matrices, seed treatment agents, soil drenching agents, implantable formulations and furrow irrigation formulations. Description of Drawings

[0104] Figure 1 , the expression pattern of UGT 2B20-like in Helicoverpa armigera. The expression levels of UGT 2B20-like in different developmental stages of Helicoverpa armigera. L1: first-instar larvae; L2: second-instar larvae; L3: third-instar larvae; L4: fourth-instar larvae; L5: fifth-instar larvae; L6: sixth-instar larvae. (A): Midgut; head: head; epipdrmis: epidermis; body: whole worm. (B): egg: egg; L1: first-instar larvae; L2: second-instar larvae; L3: third-instar larvae; L4: fourth-instar larvae; L5: fifth-instar larvae; L6: sixth-instar larvae; pupa: pupa.

[0105] Figure 2 , down-regulating the expression of UGT 2B20-like gene increases the sensitivity of Helicoverpa armigera to insecticides. (A) Relative expression level of UGT 2B20-like after injection of dsUGT 2B20-like; (B) Larval survival rate after injection of dsUGT 2B20-like; (C) Effect of injection of dsUGT 2B20-like on larval survival rate after imidacloprid treatment; (D) Effect of injection of dsUGT 2B20-like on larval survival rate after diflubenzuron treatment. Differences between data were analyzed by t-test, 0.01 < P ≤ 0.05 is marked as *, 0.001 < P ≤ 0.01 is marked as **, and p ≤ 0.001 is marked as ***.

[0106] Figure 3 , test of the role of UGT 2B20-like in oxidative defense, note: effect of silencing UGT 2B20-like on POD activity (A), CAT activity (B), SOD activity (C), H2O2 content (D) and MDA content (E). Differences between data were analyzed by t-test, 0.01 < P ≤ 0.05 is marked as *, 0.001 < P ≤ 0.01 is marked as **, and p ≤ 0.001 is marked as ***.

[0107] Figure 4Effects of down-regulation of UGT 2B20-like on ROS-related genes and antioxidant-related genes. Note: differences between data are analyzed by t-test, 0.01 < P ≤ 0.05 is marked as *, 0.001 < P ≤ 0.01 is marked as **, P ≤ 0.001 is marked as ***. Detailed Description of Embodiments

[0108] It should be understood that within the scope of the present invention, the above technical features of the present invention and the technical features specifically described in the following (e.g., embodiments) can be combined with each other to form preferred technical solutions.

[0109] In the present invention, the inventors explored the effect of UGT 2B20-like on insect susceptibility to insecticides. Further studies found that after feeding with B. cereus (Ba 6), the contents of ROS and MDA increased, and the expression of antioxidant genes such as POD decreased. Similar results were also obtained after silencing UGT 2B20-like. In conclusion, the present invention clarifies the relationship among Helicoverpa armigera symbionts, UGT and antioxidant activity. The present invention not only analyzes the molecular mechanism of insect gut microorganisms indirectly regulating insecticide resistance, but also broadens the methods for pest control. Through the insect's own symbiotic bacteria, the use of pesticides can be greatly reduced, thereby protecting agricultural ecosystems.

[0110] UGT 2B20-like protein and promoters and inhibitors thereof

[0111] Herein, the amino acid sequence of the UGT 2B20-like protein is shown as SEQ ID NO: 2 or a sequence having at least 80% identity thereto, and the coding sequence of the UGT 2B20-like protein comprises the sequence shown as SEQ ID NO: 1 or a sequence having at least 80% identity thereto.

[0112] The present invention relates to promoters and inhibitors of UGT 2B20-like protein expression or activity.

[0113] Any substance that can increase the activity of UGT 2B20-like protein, improve its stability, promote its expression, prolong its effective action time, or promote its gene transcription and translation can be used in the present invention as a "promoter" for the UGT 2B20-like gene. For example, vectors that increase the expression or activity of UGT 2B20-like protein.

[0114] Alternatively, to upregulate the activity of the UGT 2B20-like protein, an promoter of the UGT 2B20-like gene can be introduced into the insect. Promoters of the UGT 2B20-like gene include, but are not limited to, small molecule compounds, nucleic acid molecules, or combinations thereof. Preferably, the nucleic acid molecule is a nucleic acid construct containing the UGT 2B20-like coding sequence. The nucleic acid construct is an expression vector or an integration vector.

[0115] On the other hand, any substance that can reduce the activity of UGT 2B20-like proteins, reduce their stability, inhibit their expression, reduce their effective duration of action, or reduce their transcription and translation can be used in this invention as an inhibitor of the UGT 2B20-like gene.

[0116] In this article, inhibitors that downregulate the expression or activity of UGT 2B20-like proteins in insects include: (1) inhibitors that specifically interfere with the transcription and / or expression of UGT 2B20-like genes; (2) inhibitors that downregulate the activity of UGT 2B20-like proteins; and (3) variants of UGT 2B20-like proteins or their coding sequences that downregulate their activity. Wherein, the inhibitor in item (1) is selected from the group consisting of: (i) antisense nucleic acids, microRNAs, siRNAs, shRNAs, dsRNAs, sgRNAs, small molecule compounds or combinations thereof; and (ii) nucleic acid constructs that can express or form (i). Preferably, the inhibitor in item (1) is a nucleic acid that specifically interferes with the transcription and / or expression of UGT 2B20-like genes. Preferably, the inhibitor in item (2) is selected from the group consisting of: (i) specific antibodies or ligands of UGT 2B20-like proteins; and (ii) nucleic acid sequences encoding (i) and / or nucleic acid constructs that can express (i). In one or more embodiments, the nucleic acid that specifically interferes with the transcription and / or expression of the UGT 2B20-like gene has the sequence described in SEQ ID NO:3 or has at least 80% identity with it. Preferably, the nucleic acid is double-stranded DNA, wherein one strand has the sequence shown in SEQ ID NO:3 or has at least 80% identity with it. Alternatively, the nucleic acid is double-stranded RNA, wherein one strand has the RNA counterpart of the sequence shown in SEQ ID NO:3 or has at least 80% identity with it.

[0117] Nucleic acid

[0118] The present invention also provides polynucleotides expressing UGT 2B20-like proteins or nucleic acid constructs containing said polynucleotides.

[0119] This invention also relates to variants of the aforementioned polynucleotides, which encode fragments, analogs, and derivatives of polypeptides having the same amino acid sequence as those of this invention. These polynucleotide variants can be naturally occurring allelic variants or non-naturally occurring variants. These nucleotide variants include substitution variants, deletion variants, and insertion variants. As is known in the art, an allelic variant is a substitution of a polynucleotide, which may be a substitution, deletion, or insertion of one or more nucleotides, but does not substantially alter the function of the polypeptide it encodes. "Polynucleotide encoding a polypeptide" can include a polynucleotide encoding the polypeptide, or it can include a polynucleotide that also includes additional coding and / or non-coding sequences.

[0120] The present invention also relates to polynucleotides that hybridize with the above-described sequences and have at least 50%, preferably at least 70%, and more preferably at least 80% identity between the two sequences. The present invention particularly relates to polynucleotides that are hybridizable with the polynucleotides described herein under stringent conditions. In the present invention, “stringent conditions” means: (1) hybridization and elution at lower ionic strength and higher temperatures, such as 0.2×SSC, 0.1% SDS, 60°C; or (2) hybridization with a denaturing agent, such as 50% (v / v) formamide, 0.1% fetal bovine serum / 0.1% Ficoll, 42°C, etc.; or (3) hybridization only occurs when the identity between the two sequences is at least 90%, preferably at least 95%. Furthermore, the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide shown in SEQ ID NO:2.

[0121] It should be understood that although the genes provided in the examples of this invention are derived from the cotton bollworm, gene sequences derived from other similar insects (especially those belonging to the same family or genus as the cotton bollworm) that have a certain degree of homology (e.g., 70% or more, such as 80%, 85%, 90%, 95%, or even 98% sequence identity) with the sequence of this invention (preferably, the sequence is as shown in SEQ ID NO: 1) are also included within the scope of this invention, provided that those skilled in the art can easily isolate the sequence from other insects based on the information provided in this application after reading it. Methods and tools for comparing sequence identity are also well known in the art, such as BLAST. The full-length nucleotide sequence or fragments thereof of this invention can generally be obtained by PCR amplification, recombination, or artificial synthesis. For PCR amplification, primers can be designed based on the relevant nucleotide sequences disclosed in this invention, especially the open reading frame sequences, and the relevant sequences can be amplified using commercially available DNA libraries or cDNA libraries prepared according to conventional methods known to those skilled in the art. When the sequence is long, it is often necessary to perform two or more PCR amplifications, and then splice the fragments amplified from each amplification in the correct order. Once the relevant sequence is obtained, it can be mass-produced using recombination methods. Typically, it is cloned into a vector, transformed into cells, and then the sequence is isolated from the proliferated host cells using conventional methods. Alternatively, the relevant sequence can be synthesized artificially. Currently, the DNA sequence encoding the protein of this invention (or its fragments, or its derivatives) can be obtained entirely through chemical synthesis. Furthermore, mutations can be introduced into the protein sequence of this invention through chemical synthesis.

[0122] This invention also provides nucleic acids that specifically interfere with the transcription and / or expression of UGT 2B20-like protein-coding genes and nucleic acid constructs containing said nucleic acids. Such nucleic acids are conventional techniques in the art, including but not limited to antisense nucleic acids, microRNAs, siRNAs, shRNAs, dsRNAs, and sgRNAs. Those skilled in the art, knowing the sequence of the editing gene of the target protein to be interfered with, can obtain such nucleic acids using conventional techniques and verify their function using known methods, such as PCR and Southern blotting. In an exemplary embodiment, the nucleic acid that specifically interferes with the transcription and / or expression of UGT 2B20-like protein-coding genes has the sequence shown in SEQ ID NO:3.

[0123] This invention also provides a recombinant vector comprising the polynucleotides of this invention. As a preferred embodiment, the recombinant vector contains a multiple cloning site or at least one restriction enzyme site downstream of the promoter. When it is necessary to express the target gene of this invention, the target gene is ligated into a suitable multiple cloning site or restriction enzyme site, thereby operatively linking the target gene to the promoter. As another preferred embodiment, the recombinant vector comprises (from 5' to 3' direction): a promoter (e.g., a 35S promoter), a target gene, and a terminator. If desired, the recombinant vector may further include elements selected from the group consisting of: a 3' polynucleotide signal; a non-translated nucleic acid sequence; a transport and targeting nucleic acid sequence; an resistance selection marker (dihydrofolate reductase, neomycin resistance, hygromycin resistance, and green fluorescent protein, etc.); an enhancer; or an operator. The vector can be an expression vector or an integration vector; the former is used to express genes, dsRNA, related enzymes, sgRNA, etc.; the latter is used to integrate the desired expressed nucleic acid sequence into the genome. Exemplary vectors include p1301s, pAbAi, pGADT7, p1300-GFP, pA7-GFP, pGreenII-62sk, and pGreenII-0800.

[0124] The methods used to prepare recombinant vectors are well known to those skilled in the art. Expression vectors can be bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses, or other vectors. In short, any plasmid and vector can be used as long as it can replicate and remain stable within the host.

[0125] Those skilled in the art can use well-known methods to construct expression vectors containing the genes described in this invention. These methods include in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombination technology, etc. When constructing recombinant expression vectors using the genes of this invention, any type of enhancing, constitutive, tissue-specific, or inducible promoter can be added before its transcription initiation nucleotide.

[0126] host cells

[0127] Vectors containing the gene or expression cassette of this invention can be used to transform suitable host cells to enable the host to express the protein. Host cells can be prokaryotic cells, such as *Escherichia coli*, *Streptomyces*, or *Agrobacterium*; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as plant cells. Those skilled in the art will understand how to select appropriate vectors and host cells. Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art. When the host is a prokaryote (such as *E. coli*), it can be treated with CaCl2 or electroporation. When the host is a eukaryote, the following DNA transfection methods can be used: calcium phosphate coprecipitation, conventional mechanical methods (such as microinjection, electroporation, liposome packaging, etc.).

[0128] Those skilled in the art are well aware of how to select appropriate vectors, promoters, enhancers, and host cells. The peptides described herein can be expressed intracellularly, on the cell membrane, or secreted extracellularly. If desired, recombinant proteins can be isolated and purified using various separation methods utilizing their physical, chemical, and other properties. These methods are well known to those skilled in the art. Examples of these methods include (but are not limited to): conventional refolding treatment, treatment with protein precipitants (salting out), centrifugation, permeation, ultrafiltration, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high-performance liquid chromatography (HPLC), and various other liquid chromatography techniques and combinations thereof.

[0129] filter

[0130] This invention also provides the use of UGT 2B20-like protein or its coding sequence in screening candidate substances that can modulate insect sensitivity to pesticides. This invention also provides a method for screening candidate substances that can enhance insect sensitivity to pesticides, comprising (1) contacting the candidate substance with a system containing UGT 2B20-like protein or its coding sequence, and (2) detecting the expression or activity of the UGT 2B20-like protein and comparing it with a control group; if the expression or activity of the UGT 2B20-like protein is downregulated, then the substance is a candidate substance for enhancing insect sensitivity to pesticides. The control group may be the same system without the substance.

[0131] In this document, the systems used for screening candidate substances are conventional knowledge in the art. Exemplarily, the systems are solution systems, cell systems, tissue systems, plant systems, or animal systems, such as the insects mentioned above.

[0132] In this article, the insect is preferably a noctuid moth. In particular, the insect is a bollworm (Helicoverpa armigera), and more preferably, the insect is a cotton bollworm.

[0133] Pesticide sensitivity

[0134] In this paper, "insecticide sensitivity" refers to a physiological response characteristic of insects to insecticides. After exposure to a certain dose of insecticide, insects exhibit susceptibility to the effects of insecticides, such as stunted growth and development, behavioral changes, and death. This invention also provides a method for regulating insect sensitivity to insecticides, comprising: downregulating the expression or activity of UGT2B20-like protein in insects to increase insecticide sensitivity, or upregulating the expression or activity of UGT2B20-like protein in insects to decrease insecticide sensitivity. Preferably, the downregulation of UGT2B20-like protein expression or activity in insects includes: (1) specifically interfering with the transcription and / or expression of the UGT 2B20-like gene, (2) downregulating the activity of the UGT 2B20-like protein, or (3) expressing a variant of the UGT 2B20-like protein or its coding sequence with reduced activity in insects; the upregulation of UGT 2B20-like protein expression or activity in insects includes: transferring the coding sequence of the UGT 2B20-like protein into insects to obtain transformed insects, more preferably, (1) the interference is the transcription of the interfering gene or the translation of its transcript, and (2) the downregulation includes in insects: (i) expressing a specific antibody or ligand that can downregulate the activity of the UGT 2B20-like protein, or (ii) introducing a nucleic acid sequence encoding (i) and / or a nucleic acid construct that can express (i).

[0135] The insecticides described herein include organophosphates, carbamates, pyrethroids, organochlorines, neonicotinoids, insect growth regulators, etc., preferably including one or more of the following: imidacloprid, acetamiprid, thiamethoxam, dinotefuran, diflubenzuron, tebufenozide, tebufenozide, and tebufenozide. More preferably, the insecticides are imidacloprid and dinotezuron.

[0136] Insect control

[0137] In this article, insect “control” refers to the use of a series of methods and technologies to prevent, control or eliminate harmful insects in order to reduce their negative impact on human health, agricultural production, and the ecological environment.

[0138] The present invention also provides a method for controlling insects, comprising the steps of: (a) downregulating the expression or activity of a UGT 2B20-like protein in insects, and (b) exposing the insects to an insecticide. Steps (a) and (b) may be performed simultaneously or in any order. The present invention also provides the use of reagents and insecticides for downregulating the expression or activity of UGT 2B20-like proteins in controlling insects. In one or more embodiments, the downregulation comprises in the insect: (1) specifically interfering with the transcription and / or expression of a UGT 2B20-like gene, (2) downregulating the activity of a UGT 2B20-like protein, and / or (3) expressing a variant of the UGT 2B20-like protein with reduced activity or its coding sequence. In one or more embodiments, (1) the interference is interfering with the transcription of a gene or the translation of its transcript. In one or more embodiments, (2) the downregulation comprises in the insect: (i) expressing a specific antibody or ligand capable of downregulating protein activity, or (ii) introducing a nucleic acid sequence encoding (i) and / or a nucleic acid construct capable of expressing (i). For example, in order to downregulate the expression or activity of UGT 2B20-like protein, the inhibitors described herein that specifically interfere with the transcription and / or expression of UGT 2B20-like gene or downregulate the activity of UGT 2B20-like protein can be introduced into insects, so that the insects do not express or reduce the expression of UGT 2B20-like gene.

[0139] In one or more embodiments, the method includes the step of contacting an insect with an inhibitor described herein that downregulates the expression or activity of a UGT 2B20-like protein in the insect. The contact may include ingestion via the insect's mouthparts, injection, or transgenic introduction.

[0140] Those skilled in the art are familiar with conventional methods for introducing nucleic acids into insects, including but not limited to: gene gun method, electroporation method, PEG-mediated method, viral vector method, nanomaterial-mediated method, and microinjection method. Those skilled in the art can choose an appropriate transformation method based on actual needs and the specific insect species. Transformation results can be verified by combining screening markers (such as antibiotic resistance, fluorescent proteins) and molecular detection (such as PCR).

[0141] In some embodiments, the method for controlling insects described herein includes the steps of: (a) applying a composition containing an inhibitor as described in any embodiment herein, and (b) contacting the insect with the insecticide. The plant is intended to control the insect. The composition is selected from solids, liquids, powders, suspensions, and emulsions; preferably, the composition is an aqueous solution. In one or more embodiments, the inhibitor is a nucleic acid that specifically interferes with the transcription and / or expression of a protein-encoding gene, for example, a nucleic acid having the sequence shown in SEQ ID NO:3 or having at least 80% identity with it. Preferably, the nucleic acid is double-stranded DNA, wherein one strand has the sequence shown in SEQ ID NO:3 or its RNA counterpart. Alternatively, the nucleic acid is double-stranded RNA, wherein one strand has the RNA counterpart of the sequence shown in SEQ ID NO:3 or a nucleic acid having at least 80% identity with it.

[0142] In this document, "application" includes any method that enables an insecticidal composition or its active ingredient to enter an insect, be applied to the insect's body surface, or expose the insect to the environment of the composition. Such methods are conventional techniques in the art. For example, application involves cultivating plants using the composition, such as watering plants with the composition. Alternatively, application involves introducing the inhibitor into a plant (e.g., plant leaves), such as introducing nucleic acids transcribed and / or expressed from the gene encoding the specific interfering protein into the plant. The insect dies or loses its activity after feeding on the plant, thus achieving the control.

[0143] Those skilled in the art are familiar with conventional methods for introducing nucleic acids into plants, including but not limited to: Agrobacterium-mediated transformation, gene gun transformation, electroporation, PEG-mediated transformation, viral vector transformation, nanomaterial-mediated transformation, pollen tube pathway transformation, and microinjection. Those skilled in the art can select appropriate transformation methods based on actual needs and the specific plant species. Transformation results can be verified by combining screening markers (such as antibiotic resistance, fluorescent proteins) and molecular detection (such as PCR).

[0144] In some embodiments, the method for controlling insects described herein includes the step of applying a composition containing an inhibitor as described in any embodiment herein to a plant. The plant is intended to control the insect. The composition is selected from solids, liquids, powders, suspensions, and emulsions. In one or more embodiments, the inhibitor is a nucleic acid that specifically interferes with the transcription and / or expression of a protein-encoding gene, for example, a nucleic acid having the sequence shown in SEQ ID NO:3 or having at least 80% identity with it. Preferably, the nucleic acid is double-stranded DNA, wherein one strand has the sequence shown in SEQ ID NO:3 or its RNA counterpart. Alternatively, the nucleic acid is double-stranded RNA, wherein one strand has the RNA counterpart of the sequence shown in SEQ ID NO:3 or a nucleic acid having at least 80% identity with it.

[0145] Compositions and kits

[0146] This invention also provides a composition or kit for insect control, comprising an agent and an insecticide for downregulating the expression or activity of UGT 2B20-like protein as described herein. Preferably, the agent for downregulating the expression or activity of UGT 2B20-like protein is an inhibitor of the expression or activity of UGT 2B20-like protein. The insect control composition of this invention comprises a nucleic acid that specifically interferes with the transcription and / or expression of the gene encoding the UGT 2B20-like protein, said nucleic acid comprising the sequence described in SEQ ID NO:3 or having at least 80% identity with it. Preferably, the nucleic acid is double-stranded DNA, wherein one strand has the sequence shown in SEQ ID NO:3 or its RNA counterpart. Alternatively, the nucleic acid is double-stranded RNA, wherein one strand has the RNA counterpart of the sequence shown in SEQ ID NO:3 or a nucleic acid having at least 80% identity with it.

[0147] The insect control compositions or kits described herein also contain other components besides the active ingredient in insecticides known in the art, such as at least one component of a carrier, surfactant, cationic lipid, organosilicon, organosilicon surfactant, polynucleotide herbicides, non-polynucleotide herbicides, non-polynucleotide insecticides, safeners, and insect growth regulators.

[0148] The insect control composition described herein can be in any physical form, and the insect control composition is selected from at least one of the following forms: solid, liquid, powder, suspension, emulsion, spray, encapsulant, microbeads, carrier microparticles, film, matrix, seed treatment agent, soil irrigation agent, implantable formulation, and furrow irrigation formulation.

[0149] Other aspects of the invention will be apparent to those skilled in the art from the disclosure herein. The invention will be further described below by way of specific embodiments. It should be understood that these embodiments are merely illustrative and not intended to limit the scope of the invention. Unless otherwise stated, the methods and reagents used in the embodiments are conventional methods and reagents in the art.

[0150] Example

[0151] I. Materials and Methods

[0152] 1. Insect rearing:

[0153] The cotton bollworms used in this study were purchased from Jiyuan Baiyun Industrial Co., Ltd. and raised in a greenhouse at 26±1℃, relative humidity of 75%, and a day-night photoperiod of 14:10h.

[0154] 2. RNA extraction

[0155] Total RNA extraction and use Reagent (Invitrogen) and follow the instructions: 1) Add 50-100 mg of well-ground corn borer sample to 1 mL of TRIzol, mix well, and let stand at room temperature for 5 min. 2) Add 200 μL of chloroform, vortex to mix well, and let stand at room temperature for 3 min. 3) Centrifuge at 12,000 rpm (4℃) for 15 min, transfer the upper aqueous phase to another new centrifuge tube, add 500 μL of pre-chilled isopropanol, vortex to mix well, and let stand at room temperature for 10 min. 4) Centrifuge at 12,000 rpm (4℃) for 15 min, and carefully aspirate the supernatant. 5) Wash with 500 μL of pre-chilled 75% ethanol, and gently vortex for 10 sec. 6) Centrifuge at 12,000 rpm (4℃) for 2 min, carefully aspirate the supernatant, and dry at room temperature for 5 min. Dissolve in an appropriate amount of DEPC sterile water to obtain the total RNA sample. The absorbance was measured using a spectrophotometer, and the total RNA mass was determined by 1% agarose gel electrophoresis. The sample was then stored at -80℃ for later use.

[0156] 3. dsRNA synthesis

[0157] Use the kit The RNAi Kit (Ambion) was used to synthesize dsRNA, and the experimental procedures were performed according to the instructions. A T7 promoter sequence was added to the 5' end of the primers used for amplification to facilitate subsequent dsRNA synthesis. pPigbac A3 EGFP was used as the template for synthesizing the control group dsEGFP, which served as a negative control in subsequent experiments. The primers for dsRNA synthesis are shown in SEQ ID NO:6-7. During synthesis, template DNA and single-stranded RNA were removed using DNase and RNase, respectively.

[0158] 4. Detection of gene expression levels (qRT-PCR)

[0159] use Total RNA was extracted using the Invitrogen reagent, following strict adherence to the instruction manual. 1 μg of total RNA was extracted using the ReverTra reagent kit. The qPCR RT Master Mix with gDNA Remover (TOYOBO) was used for the synthesis of the first strand of cDNA. The kit used for the RT-qPCR reaction was... Premix Ex Taq TMII (Takara), primers are shown in SEQ ID NO:6-7. Each gene sample was tested in triplicate, and the expression level was normalized using 18S rRNA expression levels. Data analysis is described in section 2. -ΔΔCT Method (Livak & Schmittgen, 2001). Values ​​were obtained by calculating the mean and standard error. To eliminate individual differences, each experimental group consisted of a sample pool of two surviving larvae after treatment, and each experimental group was subjected to three biological replicates.

[0160] Example 1: Analysis of the expression pattern of UGT 2B20-like in cotton bollworm

[0161] To clarify the expression pattern of the UGT 2B20-like gene in *Helicoverpa armigera*, its transcriptional levels in different tissues (head, midgut, body, and epidermis) and different developmental stages (eggs, 1st-6th-instar larvae, and pupa) were investigated. The results showed that UGT 2B20-like was expressed to varying degrees in all tested tissues. The highest expression level was observed in the whole-body sample, with the expression levels decreasing sequentially in the epidermis, head, and midgut. Figure 1 A). UGT 2B20-like was highly expressed in fifth and sixth instar larvae, and relatively low in the other four larval stages. Figure 1 B).

[0162] Example 2: Downregulation of the UGT 2B20-like gene significantly increased the sensitivity of cotton bollworm to insecticides. To further verify the relationship between UGT 2B20-like and insecticide sensitivity, we used RNA interference (RNAi) to downregulate the expression of this gene by injecting cotton bollworms with dsUGT 2B20-like, and evaluated its effect on the toxicity of imidacloprid and diflubenzuron. Compared with the control, after RNAi, the expression level of dsUGT 2B20-like was significantly inhibited by more than 75% ( Figure 2 A). We also found that injecting dsRNA alone did not affect the survival rate of cotton bollworms ( Figure 2 B). Following injection of dsUGT 2B20-like, cotton bollworms were treated with imidacloprid and diflubenzuron to evaluate changes in their insecticide sensitivity. Results showed that downregulation of the UGT 2B20-like gene increased larval sensitivity to imidacloprid and diflubenzuron, decreased pest survival, and increased pest mortality by more than 10%. Figure 2 CD).

[0163] Example 3: UGT 2B20-like substances participate in the antioxidant defense response of insects.

[0164] To determine whether UGT 2B20-like possesses antioxidant activity and thus participates in insect detoxification responses, we examined H2O2 concentration, antioxidant capacity, and MDA concentration in cotton bollworms after UGT 2B20-like gene downregulation. Compared to the control, CAT, SOD, and POD activities in larvae were significantly reduced after UGT 2B20-like silencing. Figure 3 AC), while the concentrations of H2O2 and MDA increase ( Figure 3 These results indicate that UGT 2B20-like plays an important role in the antioxidant defense of cotton bollworm.

[0165] Example 4: Downregulation of the UGT 2B20-like gene affects the antioxidant capacity of insects.

[0166] To further characterize the effect of UGT 2B20-like gene downregulation on insect antioxidant capacity, we examined the expression levels of doux, Nox, and other antioxidant genes SOD, CAT, GSTD, POD, Trx, Grx, Grx3, and Grx5. The results showed that although H2O2 levels increased, doux and Nox expression were not significantly upregulated. SOD and Grx3 were slightly induced, suggesting that these genes may have compensatory functions. Furthermore, after UGT 2B20-like gene downregulation, the transcriptional levels of CAT, GSTD, Trx, Grx5, and POD were all lower than in the control group. Figure 4 This further explains why the insect's antioxidant capacity decreased after the UGT 2B20-like gene was downregulated, but the MDA and H2O2 content increased.

[0167] Partial sequence

[0168] Full-length sequence of SEQ ID NO:1UGT 2B20-like gene

[0169] atgccaccattgtgggatctgcagcgaggactcggcgctgcacgcgatctccaaaagctgatggatgaagctaaaaacggtgtgatttacttcagcatgggttctatagtacaaagcgatggaatgtcggaagaaatgaagagatctttactggacatgttcagcaaatatgaacaaacagccatttggaagtttgagagtgaactgaccgatgtacctaaaaatgttcatttagtcaaatgggcaccacaacccagtattttggctcatccaaacttaaaactcttcatcacacatggtggtcagctttccacctcagaagcgatccactacggagtgcctctagtaggtctacctgtgatggctgatcagcattacaacatgatatccgtggaggcaaagggattcgggattaaggtgaccttggctgaagatatggttcctgaactggatgctgctggcaggaagatactgacagataaaacgtacacaaacagagccaaagagctatcggccctattccacgatcgcgaaatgcctccaggagttgcgcttacgcattgggtggaattcgtggtgtga

[0170] SEQ ID NO: 2 UGT 2B20-like protein sequence

[0171] MPPLWDLQRGLGAARDLQKLMDEAKNGVIYFSMGSIVQSDGMSEEMKRSLLDMFSKYEQTAIWKFESELTDVPKNVHLVKWAPQPSILAHPNLKLFITHGGQLSTSEAIHYGVPLVGLPVMADQHYNMISVEAKGFGIKVTLAEDMVPELDAAGRKILTDKTYTNRAKELSALFHDREMPPGVALTHWVEFVV

[0172] SEQ ID NO: 3 Synthetic dsRNA sequence

[0173] cacatggtggtcagctttccacctcagaagcgatccactacggagtgcctctagtaggtctacctgtgatggctgatcagcattacaacatgatatccgtggaggcaaagggattcgggattaaggtgaccttggctgaagatatggttcctgaactggatgctgctggcaggaagatactgacagataaaacgtacacaaacagagccaaagagctatcggccctattccacga

[0174] SEQ ID NO:4UGT2B20-like F primer

[0175] atgccaccattgtgggatc

[0176] SEQ ID NO:5UGT2B20-like R primer

[0177] tcacaccacgaattccacc

[0178] SEQ ID NO:6dsUGT2B20-like F primer

[0179] taatacgactcactatagggcacatggtggtcagctttcc

[0180] SEQ ID NO:7dsUGT2B20-like R primer

[0181] taatacgactcactatagggtcgtggaatagggccgatag。

Claims

1. The use of UGT 2B20-like proteins or their coding sequences in screening candidate substances that can regulate insect sensitivity to pesticides.

2. A method for screening candidate substances that can enhance insect sensitivity to pesticides, comprising: (1) Contact the candidate material with a system containing a UGT 2B20-like protein or its coding sequence, and (2) Detect the expression or activity of UGT 2B20-like protein and compare it with the control group. If the expression or activity of UGT 2B20-like protein is downregulated, the substance is a candidate substance for enhancing the sensitivity of insects to pesticides.

3. A nucleic acid construct, comprising (1) The nucleic acid sequence encoding the UGT 2B20-like protein, or (2) Nucleic acid sequences that specifically interfere with the transcription and / or expression of UGT 2B20-like genes. Preferably, the amino acid sequence of the UGT 2B20-like protein is as shown in SEQ ID NO:2 or a sequence having at least 80% identity with it.

4. A genetically engineered host cell, whose (1) Expression, inclusion, or secretion of UGT 2B20-like protein, or (2) Contains a nucleic acid sequence encoding a UGT 2B20-like protein, or (3) It includes the nucleic acid construct according to claim 3.

5. A method for regulating the sensitivity of insects to insecticides, the method comprising: Downregulating the expression or activity of UGT 2B20-like proteins in insects can increase their sensitivity to pesticides, while upregulating their expression or activity can decrease their sensitivity to pesticides. Preferably, the downregulation of UGT 2B20-like protein expression or activity in insects includes: (1) specifically interfering with the transcription and / or expression of the UGT2B20-like gene, (2) downregulating the activity of the UGT 2B20-like protein, or (3) expressing a variant of the UGT 2B20-like protein or its coding sequence with reduced activity in insects; the upregulation of UGT 2B20-like protein expression or activity in insects includes: transferring the coding sequence of the UGT 2B20-like protein into insects to obtain transformed insects. More preferably, (1) the interference is interference with gene transcription or the translation of its transcripts. (2) The downregulation includes, in insects: (i) expressing a specific antibody or ligand that can downregulate the activity of UGT 2B20-like protein, or (ii) introducing a nucleic acid sequence encoding (i) and / or a nucleic acid construct that can express (i).

6. A method for controlling insects, comprising the steps of: (a) Downregulation of UGT 2B20-like protein expression or activity in insects, and (b) Exposing insects to pesticides. Preferably, (a) the downregulation includes, in insects: (1) specifically interfering with the transcription and / or expression of the UGT 2B20-like gene, (2) downregulating the activity of the UGT 2B20-like protein, and / or (3) expressing a variant of the UGT 2B20-like protein with reduced activity or its coding sequence. More preferably, (1) the interference is to interfere with gene transcription or the translation of its transcripts; (2) the downregulation includes in insects: (i) expressing a specific antibody or ligand capable of downregulating protein activity, or (ii) introducing a nucleic acid sequence encoding (i) and / or a nucleic acid construct capable of expressing (i).

7. The use of substances that regulate the expression or activity of UGT 2B20-like proteins in insects in modulating insect sensitivity to pesticides, among which... The substance is a promoter of UGT2B20-like protein expression or activity, and the regulation is to upregulate the expression or activity of UGT2B20-like protein in insects, thereby reducing the sensitivity of insects to insecticides. The substance is an inhibitor of UGT2B20-like protein expression or activity, and the regulation involves downregulating the expression or activity of UGT2B20-like proteins in insects, thereby increasing the insects' sensitivity to insecticides. Preferably, the promoter is a UGT 2B20-like protein or its coding sequence; the inhibitor is selected from: (1) inhibitors that specifically interfere with the transcription and / or expression of the UGT 2B20-like gene, (2) inhibitors that downregulate the activity of the UGT 2B20-like protein, and (3) UGT 2B20-like protein variants with downregulated activity or their coding sequences. More preferably, the inhibitor of item (1) is selected from the group consisting of: (i) antisense nucleic acids, microRNA, siRNA, shRNA, dsRNA, sgRNA, small molecule compounds or combinations thereof, and (ii) nucleic acid constructs that can express or form (i), and the inhibitor of item (2) is selected from the group consisting of: (i) specific antibodies or ligands of UGT 2B20-like proteins, and (ii) nucleic acid sequences encoding (i) and / or nucleic acid constructs that can express (i).

8. A method for regulating the antioxidant capacity of insects, the method comprising: Downregulating the expression or activity of UGT 2B20-like proteins in insects reduces their antioxidant capacity. Alternatively, it can upregulate the expression or activity of UGT 2B20-like proteins in insects, thereby enhancing their antioxidant capacity.

9. The use of reagents and insecticides that downregulate the expression or activity of UGT 2B20-like proteins in insect control. Preferably, the reagent for downregulating the expression or activity of UGT 2B20-like protein is an inhibitor of the expression or activity of UGT 2B20-like protein, wherein the inhibitor is selected from: (1) inhibitors that specifically interfere with the transcription and / or expression of UGT 2B20-like genes, (2) inhibitors that downregulate the activity of UGT 2B20-like proteins, and (3) UGT 2B20-like protein variants or their coding sequences that downregulate activity. More preferably, the inhibitor of item (1) is selected from the group consisting of: (i) antisense nucleic acids, microRNA, siRNA, shRNA, dsRNA, sgRNA, small molecule compounds or combinations thereof, and (ii) nucleic acid constructs that can express or form (i); the inhibitor of item (2) is selected from the group consisting of: (i) specific antibodies or ligands of UGT 2B20-like proteins, and (ii) nucleic acid sequences encoding (i) and / or nucleic acid constructs that can express (i).

10. A composition or kit for controlling insects, comprising an agent that downregulates the expression or activity of UGT 2B20-like protein and an insecticide. Preferably, the reagent for downregulating the expression or activity of UGT 2B20-like protein is an inhibitor of the expression or activity of UGT 2B20-like protein, wherein the inhibitor is selected from: (1) inhibitors that specifically interfere with the transcription and / or expression of UGT 2B20-like genes, (2) inhibitors that downregulate the activity of UGT 2B20-like proteins, and (3) UGT 2B20-like protein variants or their coding sequences that downregulate activity. More preferably, the inhibitor of item (1) is selected from the group consisting of: (i) antisense nucleic acids, microRNA, siRNA, shRNA, dsRNA, sgRNA, small molecule compounds or combinations thereof, and (ii) nucleic acid constructs that can express or form (i); the inhibitor of item (2) is selected from the group consisting of: (i) specific antibodies or ligands of UGT 2B20-like proteins, and (ii) nucleic acid sequences encoding (i) and / or nucleic acid constructs that can express (i).

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