Insect-resistant gene OsTIR1 and application

Abstract

The invention relates to an insect-resistant gene OsTIR1 and application thereof. Specifically, the invention relates to application of an OsTIR1 gene and / or an OsTIR1 protein as a target spot in regulation and control of insect resistance of plants.

Description

Technical Field

[0001] This invention relates to the field of molecular biology, specifically to the rice brown planthopper resistance gene OsTIR1 and its applications. Background Technology

[0002] Rice is one of the world's most important food crops, and pests and diseases have always been a major threat to rice yield and quality. The brown planthopper is the most significant pest of rice in Asia. Utilizing varietal resistance in breeding is currently the most economical, effective, and environmentally friendly method. Therefore, identifying and studying brown planthopper resistance genes from different genetic resources is fundamental and crucial for resistance breeding. To date, 17 brown planthopper resistance genes have been cloned using map-based cloning. Simultaneously, researchers have used reverse genetics to study the function of several brown planthopper resistance-related genes.

[0003] Reverse genetics has reported that rice genes involved in resistance to brown planthopper mainly include those related to plant hormone pathways, secondary metabolism, transcription factors, and miRNAs. Among these, genes related to plant hormone pathways play a key role in rice resistance to brown planthopper in the following ways: salicylic acid positively regulates rice resistance to brown planthopper. Overexpression of the salicylic acid synthesis pathway gene OsPAL8 in rice increases salicylic acid content in the plant and enhances rice resistance to brown planthopper, while silencing OsPAL8 reduces rice resistance to brown planthopper. Furthermore, exogenous application of salicylic acid to OsPAL8 RNAi plants can improve their resistance to brown planthopper (He et al., 2020). Jasmonic acid and its signal transduction pathway exhibit complex effects on rice resistance to the brown planthopper. OsAOC and Osr9-LOX1 in the jasmonic acid synthesis pathway positively regulate rice resistance to brown planthoppers, while OsLOX9 / OsHI-LOX and OsOPR3 in the same pathway have been reported to negatively regulate this resistance. Furthermore, the mutant myc2 of OsMYC2, a key transcription factor in the jasmonic acid signal transduction pathway, exhibits a brown planthopper-susceptible phenotype compared to the wild type. The OsACS2 gene in the ethylene synthesis pathway is upregulated after feeding by brown planthoppers, and silencing the OsACS2 gene reduces ethylene levels in plants, resulting in a stronger resistance phenotype. The OsEIL1 gene, a key transcription factor in the ethylene signal transduction pathway, negatively regulates rice resistance to brown planthoppers, and exogenous application of ethephon reduces rice resistance to brown planthoppers. Light inhibits ethylene biosynthesis and suppresses the expression of the ethylene signaling pathway gene OsEIL2, leading to a reduction in the constitutive protein content of OsEIL2. OsEIL2 and the ethylene pathway negatively regulate rice resistance to brown planthopper.

[0004] Auxin, one of the major hormones, plays a wide range of important roles in plant growth, development, and physiological regulation. However, its role in rice resistance to brown planthoppers remains unclear. Auxin is known to regulate the transcription of auxin-responsive genes through the TIR1 / AFB F-box protein, the Aux / IAA transcriptional repressor protein (IAA), and the auxin transcription factor ARF (Tan et al., 2007). The function of the auxin receptor-encoding gene OsTIR1 in rice resistance to brown planthoppers has not yet been investigated. Summary of the Invention

[0005] This invention has revealed that OsTIR1 plays an important role in the protection of rice against brown planthopper infestation.

[0006] This invention provides a nucleic acid construct comprising:

[0007] (1) The nucleic acid sequence encoding the OsTIR1 protein (auxin receptor), or

[0008] (2) Nucleic acid sequences that specifically interfere with the transcription and / or expression of the OsTIR1 gene.

[0009] In one or more embodiments, the OsTIR1 gene sequence is as shown in SEQ ID NO:1 or a sequence having at least 80% identity with it.

[0010] In one or more embodiments, the amino acid sequence of the OsTIR1 protein is as shown in SEQ ID NO:2 or a sequence having at least 80% identity with it.

[0011] In one or more embodiments, the OsTIR1 gene sequence number is LOC_Os05g05800 (Rice Genome Annotation Project).

[0012] In one or more embodiments, the nucleic acid construct further includes a promoter (such as a 35S promoter) operatively linked to a nucleic acid sequence.

[0013] In one or more embodiments, the nucleic acid construct from 5' to 3' has the following elements: a promoter (such as a 35S promoter), a nucleic acid sequence encoding the OsTIR1 protein, and a terminator.

[0014] This invention provides a genetically engineered host cell, which

[0015] (1) Expressing, containing, or secreting OsTIR1 protein,

[0016] (2) Contains the nucleic acid constructs described in any of the embodiments herein, or

[0017] (3) The nucleic acid sequence encoding the OsTIR1 protein.

[0018] In one or more embodiments, the host cell is not a plant cell.

[0019] The present invention also provides the use of the TIR1 gene and / or TIR1 protein as targets in regulating plant insect resistance.

[0020] In one or more embodiments, the use is to: (1) upregulate the expression or activity of the TIR1 gene and / or TIR1 protein, thereby enhancing the plant's insect resistance; or, (2) downregulate the expression or activity of the TIR1 gene and / or TIR1 protein, thereby weakening the plant's insect resistance.

[0021] In one or more embodiments, the plant is a grass (Poaceae).

[0022] In one or more embodiments, the plant is a species of rice.

[0023] In one or more embodiments, the plant is rice.

[0024] In one or more embodiments, the plant is the rice variety Oryza sativa L. subsp. Japonica, Zhonghua 11.

[0025] In one or more embodiments, the plant's insect resistance is its resistance to herbivorous insects.

[0026] In one or more embodiments, the herbivorous insect is a planthopper (Hemiptera order).

[0027] In one or more embodiments, the herbivorous insect is a brown planthopper.

[0028] In one or more embodiments, the herbivorous insect is the brown planthopper.

[0029] In one or more embodiments, the upregulation of TIR1 gene and / or TIR1 protein expression or activity in the plant includes: transferring the sequence of the TIR1 gene into the plant to obtain the transformed plant.

[0030] In one or more embodiments, downregulating the expression or activity of the TIR1 gene and / or TIR1 protein in plants includes:

[0031] (1) Specific interference with TIR1 gene transcription and / or expression

[0032] (2) Downregulate TIR1 protein activity, or

[0033] (3) Express the protein encoded by the TIR1 gene with reduced activity in plants.

[0034] In one or more embodiments, (1) the interference is interference with the transcription of the TIR1 gene or the translation of its transcript.

[0035] In one or more embodiments, (1) the interference is interference with the transcription of the TIR1 gene or the translation of its transcript.

[0036] In one or more embodiments, (1) the interference comprises inserting n bases at one or more positions in the coding sequence of the TIR1 gene, where n is a positive integer. Preferably, n is not an integer multiple of 3. In one or more embodiments, (1) the interference comprises inserting n bases at position XX of the coding sequence of the TIR1 gene, for example, inserting T or C.

[0037] In one or more embodiments, (1) the interference includes the deletion of n bases at one or more positions in the coding sequence of the TIR1 gene, where n is a positive integer. Preferably, n is not an integer multiple of 3.

[0038] In one or more embodiments, (2) the downregulation includes in plants:

[0039] (i) Expressing specific antibodies or ligands (e.g., inhibitory antibodies) that can downregulate TIR1 protein activity, or

[0040] (ii) Import the nucleic acid sequence encoding (i) and / or a nucleic acid construct that can express (i).

[0041] In one or more embodiments, the TIR1 gene sequence is as shown in SEQ ID NO:1 or a sequence having at least 80% identity with it.

[0042] In one or more embodiments, the TIR1 gene sequence number is LOC_Os05g05800 (Rice Genome Annotation Project).

[0043] In one or more embodiments, the TIR1 protein sequence is as shown in SEQ ID NO:2 or a sequence having at least 80% identity with it.

[0044] In one or more embodiments, downregulating the expression of the TIR1 protein in the plant includes: transferring nucleic acids that specifically interfere with the transcription and / or expression of the TIR1 gene into the plant to obtain a transformed plant.

[0045] In one or more embodiments, the nucleic acid that specifically interferes with the transcription and / or expression of the TIR1 gene is selected from the group consisting of (1) dsRNA, antisense nucleic acid, small interfering RNA, microRNA, shRNA, reRNA, sgRNA that target the TIR1 gene or its transcripts for repression or silencing, or (2) constructs that can express or form (1).

[0046] This invention provides a method for regulating the insect resistance of plants, the method comprising: regulating the expression or activity of the TIR1 gene and / or TIR1 protein in the system.

[0047] In one or more embodiments, the method is to: (1) upregulate the expression or activity of the TIR1 gene and / or TIR1 protein to enhance the insect resistance of the plant; or, (2) downregulate the expression or activity of the TIR1 gene and / or TIR1 protein to weaken the insect resistance of the plant.

[0048] In one or more embodiments, the system is a cell system, a tissue system, or a plant model.

[0049] In one or more embodiments, the plant is a grass (Poaceae).

[0050] In one or more embodiments, the plant is a species of rice.

[0051] In one or more embodiments, the plant is rice.

[0052] In one or more embodiments, the plant is the rice variety Oryza sativa L. subsp. Japonica, Zhonghua 11.

[0053] In one or more embodiments, the plant's insect resistance is its resistance to herbivorous insects.

[0054] In one or more embodiments, the herbivorous insect is a planthopper (Hemiptera order).

[0055] In one or more embodiments, the herbivorous insect is a brown planthopper.

[0056] In one or more embodiments, the herbivorous insect is the brown planthopper.

[0057] This invention also provides a method for screening substances that can regulate plant insect resistance, comprising the steps of:

[0058] (1) Contact the candidate material with a system containing the TIR1 gene and / or TIR1 protein, and

[0059] (2) Detect the expression or activity of TIR1 gene and / or TIR1 protein in the detection system and compare it with the expression or activity of TIR1 gene and / or TIR1 protein in the control.

[0060] If the expression or activity of the TIR1 gene and / or TIR1 protein is lower than that of the control, the substance downregulates the expression or activity of the TIR1 gene and / or TIR1 protein, thereby weakening the plant's insect resistance; if the expression or activity of the TIR1 gene and / or TIR1 protein is higher than that of the control, the substance upregulates the expression or activity of the TIR1 gene and / or TIR1 protein, thereby enhancing the plant's insect resistance.

[0061] In one or more embodiments, the control is the same system that does not contain the substance.

[0062] In one or more embodiments, the system is a solution system, a cell system, a tissue system, or a plant model.

[0063] In one or more embodiments, the plant is a grass (Poaceae).

[0064] In one or more embodiments, the plant is a species of rice.

[0065] In one or more embodiments, the plant is rice.

[0066] In one or more embodiments, the plant is the rice variety Oryza sativa L. subsp. Japonica, Zhonghua 11.

[0067] In one or more embodiments, the plant's insect resistance is its resistance to herbivorous insects.

[0068] In one or more embodiments, the herbivorous insect is a planthopper (Hemiptera order).

[0069] In one or more embodiments, the herbivorous insect is a brown planthopper.

[0070] In one or more embodiments, the herbivorous insect is the brown planthopper.

[0071] This invention provides a method for regulating the expression of TIR1 in plants, the method comprising: regulating the feeding duration of insects on plants in the system.

[0072] In one or more embodiments, the method is to: (1) increase the feeding time of insects, thereby increasing the expression of TIR1 in the system; or, (2) decrease the feeding time of insects, thereby decreasing the expression of TIR1 in the system.

[0073] In one or more embodiments, the system is a cell system, a tissue system, or a plant model.

[0074] In one or more embodiments, the plant is a grass (Poaceae).

[0075] In one or more embodiments, the plant is a species of rice.

[0076] In one or more embodiments, the plant is rice.

[0077] In one or more embodiments, the plant is the rice variety Oryza sativa L. subsp. Japonica, Zhonghua 11.

[0078] In one or more embodiments, the insect is a planthopper (Hemiptera order).

[0079] In one or more embodiments, the insect is a brown planthopper.

[0080] In one or more embodiments, the insect is a brown planthopper.

[0081] In one or more embodiments, the feeding time is 0-72 hours. Preferably, the feeding time is 0-24 hours. More preferably, the feeding time is 0-12 hours.

[0082] This invention provides a miR393 gene and / or the use of miR393 for regulating the transcription of TIR1 in plants.

[0083] In one or more embodiments, the use is to: (1) upregulate the expression or activity of the miR393 gene and / or miR393 to promote the transcription of TIR1 in plants; or, (2) downregulate the expression or activity of the miR393 gene and / or miR393 to inhibit the transcription of TIR1 in plants.

[0084] In one or more embodiments, the upregulation of miR393 gene expression or activity in plants includes: transferring the sequence of miR393 gene into plants to obtain transformed plants.

[0085] In one or more embodiments, downregulating the expression or activity of the miR393 gene in the plant includes:

[0086] (1) Specific interference with miR393 gene transcription and / or expression.

[0087] (2) Downregulate miR393 activity, or

[0088] (3) Expression of miR393 with reduced activity in plants.

[0089] In one or more embodiments, (1) the interference is interference with the transcription of the miR393 gene or the translation of its transcripts.

[0090] In one or more embodiments, (2) the downregulation includes in plants:

[0091] (i) Expressing specific antibodies or ligands (e.g., inhibitory antibodies) that can downregulate miR393 activity, or

[0092] (ii) Import the nucleic acid sequence encoding (i) and / or a nucleic acid construct that can express (i).

[0093] In one or more embodiments, downregulating miR393 expression in plants includes: transferring nucleic acids that specifically interfere with miR393 gene transcription and / or expression into plants to obtain transformed plants.

[0094] In one or more embodiments, the nucleic acid that specifically interferes with the transcription and / or expression of the miR393 gene is selected from the group consisting of (1) dsRNA, antisense nucleic acid, small interfering RNA, microRNA, shRNA, reRNA, sgRNA that target the miR393 gene or its transcripts for inhibition or silencing, or (2) constructs that can express or form (1).

[0095] In one or more embodiments, the system is a cell system, a tissue system, or a plant model.

[0096] In one or more embodiments, the plant is a grass (Poaceae).

[0097] In one or more embodiments, the plant is a species of rice.

[0098] In one or more embodiments, the plant is rice.

[0099] In one or more embodiments, the plant is the rice variety Oryza sativa L. subsp. Japonica, Zhonghua 11.

[0100] The present invention also provides a method for regulating TIR1 gene transcription, the method comprising: regulating the expression or activity of miR393 gene or miR393 in the system.

[0101] In one or more embodiments, the method is to: (1) upregulate the expression or activity of the miR393 gene or miR393 in the system, thereby promoting the transcription of TIR1 in the system; or (2) downregulate the expression or activity of the miR393 gene or miR393 in the system, thereby inhibiting the transcription of TIR1 in the system.

[0102] In one or more embodiments, the system is a cell system, a tissue system, or a plant model.

[0103] In one or more embodiments, the plant is a grass (Poaceae).

[0104] In one or more embodiments, the plant is a species of rice.

[0105] In one or more embodiments, the plant is rice.

[0106] In one or more embodiments, the plant is the rice variety Oryza sativa L. subsp. Japonica, Zhonghua 11.

[0107] This invention also provides a method for screening substances that can regulate the transcription of the TIR1 gene in a plant system, comprising the following steps:

[0108] (1) Contact the candidate material with a system containing the miR393 gene, and

[0109] (2) Detect the expression or activity of TIR1 protein in the system and compare it with the expression or activity of TIR1 protein in the control.

[0110] If TIR1 protein expression or activity is lower than the control, the substance downregulates TIR1 transcription; if TIR1 protein expression or activity is higher than the control, the substance upregulates TIR1 transcription.

[0111] In one or more embodiments, the control is the same system that does not contain the substance.

[0112] In one or more embodiments, the system is a solution system, a cell system, a tissue system, or a plant model.

[0113] In one or more embodiments, the plant is a grass (Poaceae).

[0114] In one or more embodiments, the plant is a species of rice.

[0115] In one or more embodiments, the plant is rice.

[0116] In one or more embodiments, the plant is the rice variety Oryza sativa L. subsp. Japonica, Zhonghua 11.

[0117] This invention provides a method for obtaining transgenic plants, comprising the steps of:

[0118] (1) Provide Agrobacterium carrying the nucleic acid constructs described in any of the embodiments herein,

[0119] (2) Contact the plant cells, tissues or organs with Agrobacterium in step (1) to transfer the nucleic acid construct into the plant tissues or organs.

[0120] (3) Select plant tissues, organs, or seeds into which a nucleic acid sequence has been transferred, wherein the nucleic acid sequence is: (a) a nucleic acid sequence encoding the OsTIR1 protein, or (b) a nucleic acid sequence that specifically interferes with the transcription and / or expression of the OsTIR1 gene; and

[0121] (4) Regenerate plants from the plant tissues, organs or seeds in step (3).

[0122] In one or more embodiments, the genetically modified plant is insect-resistant rice. Attached Figure Description

[0123] Figure 1 The expression level of OsTIR1 in brown planthoppers after feeding is shown. Error bars represent positive and negative standard errors (n=3). t-tests were used for data analysis; ** indicates p<0.01, and all comparisons are compared to 0h.

[0124] Figure 2 The expression levels of OsTIR1 and OsmiR393 in overexpressed and edited materials are shown. Error bars represent positive and negative standard errors (n=3). A t-test was used to analyze data differences; an asterisk indicates a significant difference compared to the wild type, and ** indicates p<0.01.

[0125] Figure 3 The study investigated the OsTIR1 editing and overexpression in plants. (a) shows a schematic diagram of OsTIR1 genome editing sites in two TIR1KO lines; (b) shows the OsTIR1 expression levels in TIR1OE and NIP plants. Error bars represent positive and negative standard errors (n=3). A t-test was used to analyze data differences; an asterisk indicates a significant difference compared to the wild type, and ** indicates p<0.01.

[0126] Figure 4 Resistance to brown planthopper was assessed in TIR1KO plants. The results are as follows: (a) Individual resistance to brown planthopper was assessed in TIR1KO and NIP plants; (b) Small population resistance was assessed in TIR1KO-1 and NIP plants; (c) Survival rate statistics were presented for small population resistance in TIR1KO-1 and NIP plants, with error bars representing positive and negative standard errors (n=3); (d) Small population resistance was assessed in TIR1KO-2 and NIP plants; (e) Survival rate statistics were presented for small population resistance in TIR1KO-2 and NIP plants, with error bars representing positive and negative standard errors (n=3). A t-test was used to analyze data differences; an asterisk indicates a significant difference compared to the wild type, and ** indicates p<0.01.

[0127] Figure 5 To identify the resistance of TIR1OE plants to brown planthoppers. (a) shows the resistance of individual TIR1OE-1 and NIP plants to brown planthoppers; (b) shows the resistance of small populations of TIR1OE-1 and NIP plants to brown planthoppers; (c) shows the survival rate statistics of small populations of TIR1OE-1 and NIP plants to brown planthoppers, with error bars representing positive and negative standard errors (n=3); (d) shows the resistance of individual TIR1OE-2 plants to brown planthoppers.

[0128] (e) shows the resistance of TIR1OE-2 plants to brown planthopper in a small population; (f) shows the survival rate statistics of TIR1OE-2 and NIP plants against brown planthopper in a small population. The error bars represent the positive and negative standard errors (n=3). The t-test was used to analyze the differences in data. An asterisk indicates a significant difference compared with the wild type, and ** indicates p<0.01.

[0129] Figure 6 The spectrum of the p1301-35SNos vector. Detailed Implementation

[0130] In this invention, OsTIR1 gene editing and overexpression materials were constructed to verify its function, and it was found that OsTIR1 plays an important role in the process of rice resisting brown planthopper damage.

[0131] As used herein, "plant" refers to a plant carrying the OsTIR1 gene or a sequence having at least 80% identity with it. The plant may include monocotyledonous or polycotyledonous plants; preferably, it includes grasses, such as rice (Oryza sativa). It should be understood that the plants suitable for the technical solutions of this invention are not limited to those listed above, and suitable plants can be determined by identifying the presence of the OsTIR1 gene or a sequence having at least 80% identity with it. Transformed plants can generally be transformed using methods such as Agrobacterium-mediated transformation or gene gun transformation, for example, the leaf disc method; the Agrobacterium-mediated transformation is preferred. Transformed plant cells, tissues, or organs can be regenerated into plants using conventional methods to obtain plants with altered traits relative to the wild type.

[0132] The term "herbivorous insect" or "insect" as used herein refers to insects of the family Planthopperidae in the order Hemiptera. Preferably, the herbivorous insect or insect is an insect of the genus *Brown planthopper*. More preferably, the herbivorous insect or insect is *Brown planthopper*. The term "insect resistance" refers to the ability of a plant to resist pest infestation. In this invention, insect resistance refers to the ability to resist infestation by *Brown planthopper*. *Brown planthopper* is a significant pest in rice production, causing various harms to rice, such as affecting rice growth, spreading rice diseases, and reducing rice yield.

[0133] This invention provides a nucleic acid construct comprising: (1) a nucleic acid sequence encoding the OsTIR1 protein (auxin receptor), or (2) a nucleic acid sequence specifically interfering with the transcription and / or expression of the OsTIR1 gene. The nucleic acid construct may be a vector. Preferably, 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. Alternatively, 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 (for expressing genes, dsRNA, related enzymes, sgRNA, etc.) or an integration vector (for integrating the desired nucleic acid sequence into the genome). Exemplary vectors include p1301s, pAbAi, pGADT7, p1300-GFP, pA7-GFP, pGreenII-62sk, and pGreenII-0800. Any plasmid and vector can be used as long as it can replicate and remain stable in the host. Methods for preparing recombinant vectors are well known to those skilled in the art.

[0134] Those skilled in the art can construct expression vectors containing the genes described in this invention using well-known methods. 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. In this invention, the nucleic acid construct has the following elements from 5' to 3': a promoter (such as a 35S promoter), a nucleic acid sequence encoding the OsTIR1 protein, and a terminator.

[0135] The full-length nucleotide sequences or fragments thereof involved in this invention can generally be obtained by PCR amplification, recombination, or artificial synthesis. For PCR amplification, primers can be designed based on the nucleotide sequences disclosed in this invention, and commercially available DNA libraries or cDNA libraries prepared using conventional methods known to those skilled in the art can be used as templates to amplify the relevant sequences. Typically, these sequences are cloned into a vector, transformed into cells, and then isolated from the proliferated host cells using conventional methods. Alternatively, the relevant sequences can be synthesized artificially.

[0136] The polynucleotides described herein can be in DNA or RNA form. DNA form includes cDNA, genomic DNA, or synthetically produced DNA. DNA can be single-stranded or double-stranded. DNA can be coding or non-coding. The OsTIR1 gene sequence number encoding the OsTIR1 protein in this article is LOC_Os05g05800 (Rice Genome Annotation Project).

[0137] In this invention, the nucleic acid sequence encoding the OsTIR1 protein can be the sequence shown in SEQ ID NO:1. This invention also includes sequence variations that have the same function as encoding the OsTIR1 protein. These variations include (but are not limited to): deletions, insertions, and / or substitutions of several bases (typically 1-20, preferably 1-10, and more preferably 1-8 or 1-5), and additions or deletions of one or more bases (e.g., up to 20, preferably up to 10, and more preferably up to 5) at the C-terminus and / or N-terminus. Any other gene that is highly homologous to the OsTIR1 gene described in this invention (e.g., has more than 80%, such as 85%, 90%, 95%, or even 98% sequence identity) or that is degenerate with the described gene is also within the scope of this invention. These other genes are, for example, TIR1 genes from other plants, plants of the same family, plants of the same genus, or plants of the same species. Methods and tools for comparing sequence similarities are also well known in the field, such as BLAST.

[0138] In this document, the amino acid sequence of the OsTIR1 protein may have the sequence shown in SEQ ID NO:2. Any other TIR1 protein that is highly homologous to the OsTIR1 protein described in this invention (e.g., having more than 80%, such as 85%, 90%, 95%, or even 98% sequence identity) is also within the scope of this invention. Such other TIR1 proteins are, for example, TIR1 proteins from other plants, plants of the same family, plants of the same genus, or plants of the same species.

[0139] This invention also provides genetically engineered host cells containing the nucleic acid constructs described herein. In this invention, the genes, expression cassettes, or vectors can be used to transform suitable host cells to enable the host to express proteins. 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 (e.g., *E. coli*), treatment with CaCl2 or electroporation can be used. When the host is a eukaryote, DNA transfection methods such as calcium phosphate co-precipitation, conventional mechanical methods (e.g., microinjection, electroporation, liposome packaging, etc.) can be used. Transformation of plants can also be performed using methods such as Agrobacterium-mediated transformation or gene gun transformation, for example, spraying, leaf disc transformation, embryo transformation, flower bud soaking, etc. Transformed plant cells, tissues, or organs can be regenerated into plants using conventional methods to obtain transgenic plants. When the polynucleotides are expressed in higher eukaryotic cells, the insertion of enhancer sequences into the vector will enhance transcription. Enhancers are cis-acting factors of DNA, typically consisting of approximately 10 to 300 base pairs, that act on promoters to enhance gene transcription. Those skilled in the art will understand how to select appropriate vectors, promoters, enhancers, and host cells. In this invention, the host cell expresses, contains, or secretes the OsTIR1 protein, or contains the nucleic acid constructs described in any of the embodiments herein.

[0140] This article also provides the use of the OsTIR1 gene as a target in regulating plant insect resistance. The term "regulation" as used herein includes both "upregulation" and "downregulation." In one or more embodiments, the uses described herein include: upregulating the expression or activity of the OsTIR1 gene and / or OsTIR1 protein, thereby enhancing plant resistance to brown planthoppers; or downregulating the expression or activity of the OsTIR1 gene and / or OsTIR1 protein, thereby weakening plant resistance to brown planthoppers. OsTIR expression is responsive to brown planthopper feeding.

[0141] The present invention also provides a method for regulating the insect resistance of plants, the method comprising regulating the expression or activity of the OsTIR1 gene and / or OsTIR1 protein in a plant system. Specifically, the method comprises: (1) upregulating the expression or activity of the OsTIR1 gene and / or OsTIR1 protein, thereby enhancing the plant's resistance to brown planthoppers; or, (2) downregulating the expression or activity of the OsTIR1 gene and / or OsTIR1 protein, thereby weakening the plant's resistance to brown planthoppers.

[0142] The present invention also provides a method for regulating the expression of OsTIR1, the method being: (1) upregulating the feeding time of brown planthoppers, thereby upregulating the expression of OsTIR1 in the system; or, (2) downregulating the feeding time of brown planthoppers, thereby downregulating the expression of OsTIR1 in the system. In one or more embodiments, the feeding time is 0-72 hours, preferably 0-24 hours, more preferably 0-12 hours.

[0143] The present invention also provides a method for regulating OsTIR1 transcription, the method comprising: regulating the expression or activity of the OsmiR393 gene or OsmiR393 in the system. Specifically, the method comprises: (1) upregulating the expression or activity of the OsmiR393 gene or OsmiR393 in the system, thereby promoting the transcription of OsTIR1 in the system; or (2) downregulating the expression or activity of the OsmiR393 gene or OsmiR393 in the system, thereby inhibiting the transcription of OsTIR1 in the system.

[0144] Any substance that can enhance the activity, stability, expression, duration of action, or transcription and translation of the OsTIR1 protein can be used in this invention as a "promoter" of the OsTIR1 gene to regulate plant insect resistance. For example, vectors that enhance OsTIR1 expression or activity.

[0145] Alternatively, to upregulate the activity of the OsTIR1 protein, an OsTIR1 protein promoter can be introduced into the plant. OsTIR1 protein promoters 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 OsTIR1 protein coding sequence. The nucleic acid construct is an expression vector or an integration vector.

[0146] On the other hand, any substance that can reduce the activity, stability, expression, duration of action, transcription, and translation of the OsTIR1 protein can be used in this invention as an inhibitor of the OsTIR1 protein. This inhibitor can be used to regulate plant insect resistance.

[0147] For example, to downregulate the expression or activity of the OsTIR1 protein, an inhibitory molecule that specifically interferes with the transcription and / or expression of the OsTIR1 protein, or downregulates its activity, can be introduced into cells or plants, causing the cells or plants to not express or reduce the expression of the OsTIR1 gene. The inhibitory molecule targets the OsTIR1 gene or its transcript or expressed protein. Therefore, the inhibitory molecule can use the sequence shown in SEQ ID NO:1 as its target. The inhibitory molecule can be a small molecule compound known to inhibit the activity of the OsTIR1 protein, an antibody or ligand of the OsTIR1 protein or its binding fragment, or an antisense nucleic acid, microRNA, siRNA, shRNA, dsRNA, or sgRNA that interferes with the expression of the OsTIR1 gene.

[0148] Furthermore, to downregulate OsTIR1 gene expression or activity, a gene knockout vector can be introduced into the cell. Therefore, the inhibitor can be a reagent that knocks out or reduces the OsTIR1 gene using a technology selected from ZFN, TALEN, and CRISPR, such as sgRNA. ZFN, TALEN, and CRISPR / Cas9 technologies suitable for use in this invention are well known in the art. Each technology achieves target gene knockout through the combined action of a DNA recognition domain and a nuclease. In these embodiments, the inhibitor further comprises a Cas enzyme (e.g., Cas9), its coding sequence, and / or a nucleic acid construct expressing the Cas enzyme.

[0149] The present invention also provides a use of the OsmiR393 gene and / or OsmiR393 for regulating the transcription of OsTIR1 in plants. In one or more embodiments, the use is to: (1) upregulate the expression or activity of the OsmiR393 gene and / or OsmiR393, thereby promoting the transcription of OsTIR1 in plants; or, (2) downregulate the expression or activity of the OsmiR393 gene and / or OsmiR393, thereby inhibiting the transcription of OsTIR1 in plants. In one or more embodiments, the upregulation of the expression or activity of the OsmiR393 gene in plants includes: transferring the sequence of the OsmiR393 gene into a plant to obtain a transformed plant. In one or more embodiments, downregulating the expression or activity of the OsmiR393 gene in plants includes: (1) specifically interfering with the transcription and / or expression of the OsmiR393 gene, (2) downregulating the activity of OsmiR393, or (3) expressing a protein encoded by the OsmiR393 gene with reduced activity in the plant. (1) The interference is interfering with the transcription of the OsmiR393 gene or the translation of its transcripts; (2) The downregulation includes in the plant: (i) expressing a specific antibody or ligand (e.g., an inhibitory antibody) capable of downregulating the activity of OsmiR393, or (ii) introducing a nucleic acid sequence encoding (i) and / or a nucleic acid construct capable of expressing (i). Downregulating the expression of OsmiR393 in plants includes: transferring a nucleic acid that specifically interferes with the transcription and / or expression of the OsmiR393 gene into the plant to obtain a transformed plant. Nucleic acids that specifically interfere with the transcription and / or expression of the OsmiR393 gene are selected from the following group: (a) dsRNA, antisense nucleic acid, small interfering RNA, microRNA, shRNA, reRNA, sgRNA that target the OsmiR393 gene or its transcripts for repression or silencing, or (b) constructs that can express or form (a).

[0150] The present invention also provides a method for screening substances that can regulate plant insect resistance, comprising the steps of: (1) contacting a candidate substance with a system containing the OsTIR1 gene and / or OsTIR1 protein, and (2) detecting the expression or activity of the OsTIR1 gene and / or OsTIR1 protein in the system and comparing it with the expression or activity of the OsTIR1 gene and / or OsTIR1 protein in the control; if the expression or activity of the OsTIR1 gene and / or OsTIR1 protein is lower than that in the control, the substance downregulates the expression or activity of the OsTIR1 gene and / or OsTIR1 protein, thereby weakening the plant's insect resistance; if the expression or activity of the OsTIR1 gene and / or OsTIR1 protein is higher than that in the control, the substance upregulates the expression or activity of the OsTIR1 gene and / or OsTIR1 protein, thereby enhancing the plant's insect resistance.

[0151] The present invention also provides a method for screening substances that can regulate OsTIR1 transcription, comprising the steps of: (1) contacting a candidate substance with a system containing the OsmiR393 gene, and (2) detecting the expression or activity of OsTIR1 protein in the system and comparing it with the expression or activity of OsTIR1 protein in a control. If the expression or activity of OsTIR1 protein is lower than that in the control, the substance downregulates the transcription of OsTIR1; if the expression or activity of OsTIR1 protein is higher than that in the control, the substance upregulates the transcription of OsTIR1.

[0152] In one or more embodiments, the control is an identical system without the substance. In one or more embodiments, the system is a solution system, a cell system, a tissue system, or a plant model.

[0153] The candidate substances can be selected from: peptides, polymeric peptides, peptide-like substances, non-peptide compounds, carbohydrates, lipids, antibodies or antibody fragments, ligands, small organic molecules, small inorganic molecules, and nucleic acid sequences, etc. Those skilled in the art will understand how to select an appropriate screening method based on the type of substance to be screened.

[0154] The plant described herein can be any plant, preferably a grass, such as a plant of the genus Oryza, preferably rice, and more preferably the japonica rice variety (Oryza sativa L. subsp. Japonica) Zhonghua 11.

[0155] Example

[0156] Experimental Materials and Methods

[0157] 1. Materials: The rice used in this study was the japonica rice variety (Oryza sativa L. subsp. Japonica) Zhonghua 11. The tested rice was planted in Shanghai fields during the summer using standard rice field planting and management methods; and in a greenhouse at the Shanghai Academy of Agricultural Sciences during the winter. Temperature was 29℃±1℃, humidity was 50-70%±5%, and light duration was 7:00-19:00.

[0158] 2. Construction of OsTIR1 gene supervector: The OsTIR1 CDS sequence was amplified in rice cDNA (primer sequence: TIR1OE-F:acgggggactctagaggatccatggggcgcggcggctcgcgc).

[0159] The vector TIR1OE-R (gggaaattcgagctcggtaccctacacaatctggacgcaggc) was constructed into the p1301-35S-Nos vector via homologous recombination. Subsequently, it was transformed into the wild-type rice variety Nipponbare using Agrobacterium-mediated transformation to obtain OsTIR1 overexpression materials TIR1OE-1 and TIR1OE-2.

[0160] 3. The OsTIR1 gene was edited using CRISPR / Cas9 technology. First, an OsTIR1 gene-specific sgRNA was designed: AGATGATCGCCGCGTCCTTC. Specific primers were synthesized based on this sgRNA sequence, and the sgRNA expression cassette was constructed using overlapping PCR. The sgRNA expression cassette was cloned into the pYLCRISPR / Cas9 system final vector Ubi-H, and then transformed into the wild-type rice variety NIP using Agrobacterium-mediated genetic transformation. Finally, several homozygous mutant plants were obtained through molecular biological analysis.

[0161] TIR1-gRT1:AGATGATCGCCGCGTCCTTCgttttagagctagaaat

[0162] TIR1-OsU6aT1:GAAGGACGCGGCGATCATCTCggcagccaagccagca

[0163] 4. Rice genetic transformation was carried out according to the method in the literature (Hiei et al., 1994).

[0164] 5. Method for Identifying Rice Plant Resistance to Brown Planthoppers: After rice seeds have been germinated, individual plants are sown in small plastic pots. Under normal growth and management, after approximately one month, when the seedlings are about to enter the tillering stage, resistance to the planthopper is assessed. Prepare a 40cm high, approximately 8cm diameter permeable plastic cover, leaving a 6*10cm ventilation opening on one side, and cover it with a mesh screen. Cover the seedlings with the cover, and the top with the mesh screen. During the assessment, inoculate each pot with approximately 15 third-instar nymphs. Count the nymphs the following day. Under normal growth and management conditions, observe the survival status of the seedlings after 5-8 days.

[0165] 6. Method for identifying resistance to brown planthoppers in small rice populations: After rice seeds germinate, they are planted in blue plastic boxes with 10 plants per row, for a total of 5 rows. The control group and experimental group are planted symmetrically. When the rice reaches the two-leaf-one-heart stage, 10 brown planthoppers are inoculated per plant. After inoculation, the growth of the rice plants is observed daily, and photos are taken for statistical analysis. Three controls are set up.

[0166] Example 1: OsTIR1 response to brown planthopper feeding

[0167] The OsTIR1 (auxin transport inhibitor response) gene encodes an auxin receptor, with the sequence number LOC_Os05g05800 (Rice Genome Annotation Project). We analyzed the expression level of OsTIR1 induced by brown planthoppers to detect whether it responded to feeding. The results showed that the expression of OsTIR1 generally increased with the duration of feeding by brown planthoppers, indicating a response to feeding.

[0168] Example 2: OsTIR1 is regulated transcriptionally by OsmiR393

[0169] The OsTIR1 gene encodes the auxin receptor. It is also a target gene of OsmiR393 (https: / / www.mirbase.org / ). Rice contains two genes encoding OsmiR393: OsmiR393a and OsmiR393b. We previously constructed a double knockout transgenic material, 393DKO, of OsmiR393a and OsmiR393b, and further constructed OsmiR393a overexpression material 393aOE and OsmiR393b overexpression material 393bOE. To further demonstrate this, we examined the transcriptional regulation of OsmiR393 on OsTIR1. We measured the expression levels of the OsTIR1 gene in 393DKO, 393aOE, 393bOE, and wild-type plants. It was found that it was significantly upregulated in 393DKO plants, while it was significantly downregulated in 393aOE and 393bO plants. Figure 2 This demonstrates that OsmiR393 regulates the transcription of the OsTIR1 gene.

[0170] Example 3, Construction of OsTIR1-related genetic materials

[0171] Based on the OsTIR1 response to brown planthopper feeding, we inferred that it may play a role in the rice's defense response against brown planthoppers. To investigate the function of OsTIR1 in regulating brown planthoppers in rice, it was edited using Crisper-CAS9 technology to obtain the TIR1KO edited material, and an overexpression genetic material TIR1OE (using the 35S promoter) was constructed. Genomic analysis of the gene-edited materials showed that TIR1KO-1 had a T base inserted into its coding sequence, and TIR1KO-2 had a C base inserted into its coding sequence. Figure 3 a). The expression of OsTIR1 in overexpressing transgenic plants and wild-type plants was detected by qRT-PCR. The results showed upregulated expression ( Figure 3 b).

[0172] Example 4: Knocking out OsTIR1 reduces rice resistance to brown planthopper.

[0173] Individual insect resistance was assessed in two strains of TIR1KO, TIR1KO-1 and TIR1KO-2. After being fed on by the same number of brown planthoppers, TIR1KO-1 and TIR1KO-2 withered and died earlier than wild-type NIP plants. Figure 4 a). Simultaneously, small-group insect resistance assessments were conducted; similarly, TIR1KO plants died earlier than the wild type. Figure 4 b, d), seedling survival rate statistics showed that the survival rate of TIR1KO plants was significantly lower than that of wild-type plants (b, d). Figure 4 (c, e). The above results indicate that knocking out OsTIR1 reduces rice resistance to brown planthopper.

[0174] Example 5: Overexpression of OsTIR1 enhances rice resistance to brown planthopper.

[0175] Single-plant insect resistance was assessed in two strains of TIR1OE, TIR1OE-1 and TIR1OE-2. After the same number of brown planthoppers fed on the plants, while the wild-type NIP plants withered and died, the TIR1OE-1 and TIR1OE-2 plants still grew well. Figure 5 a, d). Simultaneously, small-group insect resistance assessments were conducted; similarly, NIP plants died earlier than TIR1OE plants. Figure 5 b, e), seedling survival rate statistics showed that the survival rate of TIR1OE plants was significantly higher than that of wild-type plants ( Figure 5 (c, f). The above results indicate that overexpression of OsTIR1 enhances rice resistance to brown planthopper. In summary, we have demonstrated that OsTIR1 positively regulates rice resistance to brown planthopper.

[0176] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments are given in this invention, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.

[0177] Partial sequence of this article:

[0178] Full-length cDNA sequence of SEQ ID NO.1 OsTIR1 gene

[0179] ATGGGGCGCGGCGGCTCGCGCGCGGCGTGCGCCGCCGCGGCGCCGCCGTGGCACTCGCTCCCGGACGAGGTCTGGGAGC

[0180] ACGCCTTCTCCTTCCTCCCCGCCGCCGCGGACAGGGGCGCCGCGGCGGGGGCGTGCAGCTCGTGGCTCCGCGCCGAGCGC

[0181] CGGTCGCGCCGCCGCCTCGCCGTCGCCAACTGCTACGCCGCCGCGCCGCGGGACGCCGTCGAGCGGTTCCCGTCCGTGCG

[0182] CGCCGCCGAGGTCAAGGGCAAGCCCCACTTCGCCGACTTCGGCCTCGTCCCCCCCGCCTGGGGCGCCGCCGCGGCGCCGT

[0183] GGATCGCCGCCGCCGCCGACGGGTGGCCGCTGCTCGAGGAGCTCAGCTTCAAGCGCATGGTCGTCACCGACGAGTGCCTC

[0184] GAGATGATCGCCGCGTCCTTCAGGAACTTCCAGGTGCTCCGCCTCGTCTCCTGCGACGGCTTCAGCACCGCGGGCCTCGC

[0185] CGCCATTGCTGCCGGTTGCAGACACCTAAGAGAACTTGACCTGCAAGAGAACGAGATTGAGGATTGTTCTATTCATTGGC

[0186] TCAGCCTCTTCCCGGAATCGTTCACTTCTCTAGTAACTCTAAACTTTTCATGCTTAGAGGGGGAGGTCAATATCACTGTAC

[0187] TTGAACGGTTAGTGACCAGATGTCACAACCTGAAGACTCTTAAGCTCAACAATGCTATCCCCCTTGACAAGCTTGCTAGC

[0188] CTCCTTCATAAGGCTCCTCAGCTAGTTGAACTCGGAACTGGCAAATTCTCTGCTGATTACCATTCCGATCTGTTTGCAAAG

[0189] CTGGAGGCGGCGTTTGGAGGTTGTAAAAGCTTGAGAAGGCTTTCTGGGGCTTGGGATGCTGTTCCAGATTATCTGCCAGC

[0190] ATTCTATTGTGTATGTGAAGGCCTCACATCACTTAATCTGAGTTATGCTACTGTGCGAGGTCCTGAGCTCATCAAATTCAT

[0191] TAGTAGATGCAGAAATTTGCAACAATTATGGGTGATGGACCTCATTGAGGATCATGGTTTAGCTGTTGTGGCATCATCTTG

[0192] CAATAAACTTCAAGAGTTGCGGGTCTTCCCTTCTGACCCTTTTGGTGCAGGATTCTTGACTGAAAGAGGTCTTGTTGATGT

[0193] CTCTGCAAGTTGTCCAATGTTGGAGTCAGTGCTCTACTTCTGCAGACGGATGACAAATGAGGCACTTATTACCATTGCAA

[0194] AGAACCGTCCCAACTTCACTTGCTTCCGCCTATGCATCCTTGAGCCACACACTCCAGACTACATCACACGGGAGCCTCTTG

[0195] ATGCAGGTTTCAGCGCCATTGTGGAGTCATGCAGGGGCCTTAGGCGTCTCTCTATCTCAGGCCTTCTCACAGATCTTGTGT

[0196] TTAAATCCATTGGGGCACATGCTGATCGTCTTGAGATGCTTTCAATCGCCTTCGCTGGGAACAGCGACTTGGGCCTGCATT

[0197] ACATCCTCTCAGGCTGCAAGAGCCTGAAGAAACTGGAGATCAGGGACTGCCCATTTGGTGATAAGCCATTGCTGGCGAAC

[0198] GCAGCAAAGCTGGAGACAATGCGATCCCTTTGGATGTCGTCGTGCTTGTTGACCCTGGGCGCATGCCGACAGCTTGCACG

[0199] CAAGATGCCCCGCCTTAGTGTGGAGATCATGAACGATCCTGGAAGGTCATGCCCCTTGGATTCGCTTCCGGATGAAACAC

[0200] CTGTTGAGAAACTGTACGTCTACCGGACGATCGCAGGTCCAAGGTGTAGAATGAAAATTCACAGCCTTTTTCAGGACATG

[0201] GAAATGCATGGACTCTAG

[0202] SEQ ID NO.2 OsTIR1 protein sequence:

[0203] MGRGGSRAACAAAAPPWHSLPDEVWEHAFSFLPAAADRGAAAGACSSWLRAERRSRRRLAVANCYAAAPRDAVERFPSVR

[0204] AAEVKGKPHFADFGLVPPAWGAAAAPWIAAAADGWPLLEELSFKRMVVTDECLEMIAASFRNFQVLRLVSCDGFSTAGLAAI

[0205] AAGCRHLRELDLQENEIEDCSIHWLSLFPESFTSLVTLNFSCLEGEVNITVLERLVTRCHNLKTLKLNNAIPLDKLASLLHKAPQL

[0206] VELGTGKFSADYHSDLFAKLEAAFGGCKSLRRLSGAWDAVPDYLPAFYCVCEGLTSLNLSYATVRGPELIKFISRCRNLQQLW

[0207] VMDLIEDHGLAVVASSCNKLQELRVFPSDPFGAGFLTERGLVDVSASCPMLESVLYFCRRMTNEALITIAKNRPNFTCFRLCILE

[0208] PHTPDYITREPLDAGFSAIVESCRGLRRLSISGLLTDLVFKSIGAHADRLEMLSIAFAGNSDLGLHYILSGCKSLKKLEIRDCPFGD

[0209] KPLLANAAKLETMRSLWMSSCLLTLGACRQLARKMPRLSVEIMNDPGRSCPLDSLPDETPVEKLYVYRTIAGPRCRMKIHSLFQDMEMHGL*

[0210] SEQ ID NO.3 TIR1OE-F

[0211] Acgggggactctagaggatccatggggcgcggcggctcgcgc

[0212] SEQ ID NO.4 TIR1OE-R

[0213] Gggaattcgagctcggtaccctacacaatctggacgcaggc

[0214] SEQ ID NO.5 TIR1-gRT1

[0215] AGATGATCGCCGCGTCCTTCgttttagagctagaaat

[0216] SEQ ID NO.6 TIR1-OsU6aT1

[0217] GAAGGACGCGGCGATCATCTCggcagccaagccagca.

Claims

1. A nucleic acid construct, comprising (1) The nucleic acid sequence encoding the TIR1 protein, or (2) Nucleic acid sequences that specifically interfere with the transcription and / or expression of the TIR1 gene. Preferably, the coding sequence of the TIR1 protein is as shown in SEQ ID NO:1 or a sequence having at least 80% identity with it, and the amino acid sequence of the TIR1 protein is as shown in SEQ ID NO:2 or a sequence having at least 80% identity with it. Preferably, the nucleic acid construct further includes a promoter operatively linked to the nucleic acid sequence.

2. A genetically engineered host cell, whose (1) Expressing, containing, or secreting TIR1 protein, (2) Contains the nucleic acid construct as described in claim 1, or (3) The nucleic acid sequence encoding the TIR1 protein, The host cell is not a plant cell. Preferably, the amino acid sequence of the TIR1 protein is as shown in SEQ ID NO:1 or a sequence having at least 80% identity with it.

3. The use of the TIR1 gene and / or TIR1 protein as targets in regulating plant insect resistance, wherein the regulation is as follows: (1) Upregulating the expression or activity of the TIR1 gene and / or TIR1 protein, thereby enhancing the plant's insect resistance; or, (2) Downregulating the expression or activity of the TIR1 gene and / or TIR1 protein, thereby weakening the plant's insect resistance. Preferably, The upregulation of TIR1 gene and / or TIR1 protein expression or activity in plants includes: The sequence of the TIR1 gene was transferred into plants to obtain transformed plants. The downregulation of TIR1 gene and / or TIR1 protein expression or activity in plants includes: (a) specifically interfering with TIR1 gene transcription and / or expression, (b) downregulating TIR1 protein activity, or (c) expressing a protein encoded by the TIR1 gene with reduced activity in plants. More preferably, The interference described in (a) refers to interference with the transcription of the TIR1 gene or the translation of its transcripts. The downregulation described in (b) includes (i) expressing a specific antibody or ligand (e.g., an inhibitory antibody) capable of downregulating TIR1 protein activity, or (ii) introducing a nucleic acid sequence encoding (i) and / or a nucleic acid construct capable of expressing (i). Preferably, the plant is a grass (Poaceae); more preferably, the plant is an oregano (Oryza). Preferably, the insect resistance is the resistance to herbivorous insects. Preferably, the herbivorous insect is a planthopper belonging to the family Planthopperidae in the order Hemiptera; more preferably, the herbivorous insect is a brown planthopper.

4. A method for regulating plant insect resistance, the method comprising: (1) Upregulate the expression or activity of TIR1 gene and / or TIR1 protein, thereby weakening the plant’s insect resistance; or, (2) Downregulating the expression or activity of the TIR1 gene and / or TIR1 protein can enhance the plant's insect resistance. Preferably, The upregulation of TIR1 gene and / or TIR1 protein expression or activity in plants includes: transferring the coding sequence of the IAA10 protein into plants to obtain transformed plants. The downregulation of TIR1 gene and / or TIR1 protein expression or activity in plants includes: (a) specifically interfering with TIR1 gene transcription and / or expression, (b) downregulating TIR1 protein activity, or (c) expressing TIR1 protein with reduced activity in plants. More preferably, The interference described in (a) refers to interference with the transcription of the TIR1 gene or the translation of its transcripts. The downregulation described in (b) includes, in plants: (i) expressing a specific antibody or ligand (e.g., an inhibitory antibody) that can downregulate the activity of the TIR1 protein, or (ii) introducing a nucleic acid sequence encoding (i) and / or a nucleic acid construct that can express (i).

5. A method for screening substances that regulate plant insect resistance, comprising the steps of: (1) Contact the candidate material with a system containing the TIR1 protein or its coding sequence, and (2) Detect the expression or activity of TIR1 protein in the system and compare it with the expression or activity of TIR1 protein in the control. If TIR1 protein expression or activity is lower than the control, the substance downregulates TIR1 protein expression or activity, thereby weakening the plant's insect resistance; if TIR1 protein expression or activity is higher than the control, the substance upregulates TIR1 protein expression or activity, thereby enhancing the plant's insect resistance. Preferably, The system can be a solution system, a cell system, a tissue system, or a plant model. The plant is a grass (Poaceae); preferably, the plant is a rice (Oryza) species. The insect resistance mentioned refers to the resistance to herbivorous insects, and / or The herbivorous insect is a planthopper in the family Hemiptera; more preferably, the herbivorous insect is a brown planthopper.

6. A method for regulating the expression of TIR1 in plants, wherein the method comprises: regulating the feeding duration of insects on plants in the system, Preferably, the method is as follows: (1) increasing the feeding time of insects, thereby increasing the expression of TIR1 in the system; or, (2) decreasing the feeding time of insects, thereby decreasing the expression of TIR1 in the system. Preferably, the feeding time is 0-72 hours; more preferably, the feeding time is 0-24 hours.

7. A method for regulating TIR1 transcription, the method comprising: The expression or activity of miR393 or its coding sequence in the regulatory system. Preferably, the method is as follows: (1) Upregulate the expression or activity of miR393 or its coding sequence in the system, thereby promoting the transcription of TIR1 in the system; or (2) Downregulate the expression or activity of miR393 or its coding sequence in the system, thereby inhibiting the transcription of TIR1 in the system. Preferably, The system is a cell system, tissue system, or plant model. The plant is a grass family plant; preferably, the plant is a rice genus plant.

8. An use of miR393 or its coding sequence for regulating the transcription of TIR1 in plants. Preferably, the purpose is to (1) upregulate the expression or activity of miR393 or its coding sequence, thereby promoting the transcription of TIR1 in plants; or, (2) downregulate the expression or activity of miR393 or its coding sequence, thereby inhibiting the transcription of TIR1 in plants. Preferably, The upregulation of miR393 or its coding sequence expression or activity in plants includes: The sequence of the miR393 gene was transferred into plants to obtain transformed plants. The downregulation of miR393 or its coding sequence expression or activity in plants includes: (a) specifically interfering with miR393 gene transcription and / or expression, (b) downregulating miR393 activity, or (c) expressing the product encoded by a miR393 gene with reduced activity in plants. More preferably, The interference mentioned in (a) refers to interference with the transcription of the miR393 gene or the translation of its transcripts. The downregulation described in (b) includes (i) expressing a specific antibody or ligand (e.g., an inhibitory antibody) capable of downregulating miR393 activity, or (ii) introducing a nucleic acid sequence encoding (i) and / or a nucleic acid construct capable of expressing (i). Preferably, the plant is a grass (Poaceae); more preferably, the plant is an oregano (Oryza).

9. A method for screening substances that can regulate TIR1 transcription in plant systems, comprising the following steps: (1) Contact the candidate material with a system containing miR393 or its coding sequence and TIR1 or its coding sequence or gene, and (2) Detect the expression or activity of TIR1 protein in the system and compare it with the expression or activity of TIR1 protein in the control. If TIR1 protein expression or activity is lower than the control, the substance downregulates TIR1 transcription; if TIR1 protein expression or activity is higher than the control, the substance upregulates TIR1 transcription. Preferably, the control is an identical system that does not contain the substance. Preferably, the system is a solution system, a cell system, a tissue system, or a plant model.

10. A method for obtaining transgenic plants, comprising the steps of: (1) Provides Agrobacterium carrying the nucleic acid construct of claim 1, (2) Contacting plant cells, tissues, or organs with Agrobacterium in step (1) to transfer the nucleic acid construct into plant tissues or organs. (3) Select plant tissues, organs, or seeds into which a nucleic acid sequence has been transferred, wherein the nucleic acid sequence is: (a) a nucleic acid sequence encoding the TIR1 protein, or (b) a nucleic acid sequence that specifically interferes with the transcription and / or expression of the TIR1 gene; and (4) Regenerate plants from the plant tissues, organs, or seeds obtained in step (3). Preferably, the genetically modified plant is insect-resistant rice.

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