A novel gene for regulating plant insect resistance and application thereof

Abstract

The application provides a novel gene for regulating plant insect resistance and application thereof. Based on large-scale research screening and experimental work, a novel gene MYB22 related to regulating plant insect resistance is cloned, and the loss of function of the gene MYB22 leads to a great decrease in the insect resistance of plants, which indicates that the gene MYB22 is a gene with insect resistance function. The application provides a novel way for improving the insect resistance of plants.

Description

Technical Field

[0001] This invention belongs to the fields of botany and biotechnology, and more specifically, this invention relates to a novel gene that regulates plant insect resistance and its application. Background Technology

[0002] Plants are threatened by a variety of herbivorous insects during their growth and development. As plants evolve, they develop complex defense systems to resist insect attacks.

[0003] Rice, a member of the Poaceae family, is one of the world's most important food crops, with more than half of the global population relying on it as their staple food. Pests and diseases affecting rice are significant factors influencing rice quality and yield. Among these, planthoppers, such as the brown planthopper, are monophagous pests that feed exclusively on rice and common wild rice, posing a major threat to rice food security.

[0004] In current technology, research on the molecular mechanisms of plant (such as grasses) resistance to insects (e.g., planthoppers) is still in its early stages, with efforts focused on identifying genes related to insect resistance. Although some studies have investigated the correlation between certain genes and insect-resistant traits in the breeding field, progress has not been entirely satisfactory.

[0005] Therefore, enhancing our understanding of the molecular mechanisms of insect resistance will be of great economic and practical value in helping to control insects in the field, uncover insect-resistant genes, and provide reserve resources for resistance breeding. Summary of the Invention

[0006] The purpose of this invention is to provide a novel gene for regulating plant insect resistance and its application.

[0007] In a first aspect of the invention, a method for improving plant insect resistance is provided, the method comprising upregulating the expression or activity of MYB22 in plants, including upregulating / increasing the stability or effective duration of MYB22.

[0008] In one or more embodiments, the upregulation of MYB22 expression or activity in plants includes: administering an upregulator of MYB22 to the plant to increase its expression or activity; preferably, the upregulator includes (but is not limited to): a polynucleotide or construct encoding MYB22, an upregulator that promotes the promoter-driven ability of the MYB22 gene, an upregulator that interacts with the MYB22 protein to increase its expression or activity, a downregulator of MYB22 gene-specific microRNA, a chemical upregulator of MYB22, or a combination thereof.

[0009] In one or more embodiments, the insect is a herbivorous insect.

[0010] In one or more embodiments, the herbivorous insect is a hemiptera insect.

[0011] In one or more embodiments, the hemiptera insects are planthoppers, including brown planthoppers (Nilaparvata lugens), white-backed planthoppers (Sogatella furcifera), and gray planthoppers (Lalielphaxstriatellus).

[0012] In one or more embodiments, the MYB22 is selected from the group consisting of: (a) a polypeptide with an amino acid sequence as shown in SEQ ID NO:1; (b) a MYB22 derivative having the polypeptide function of (a) or (b) formed by substituting, deleting or adding one or more (e.g., 1 to 20, 1 to 10, 1 to 5, 1 to 3 or 1 to 2) amino acid residues; and (c) a MYB22 derivative or an active fragment thereof having a sequence that is ≥70% homology to the amino acid sequence shown in SEQ ID NO:1 (e.g., homology ≥75%, ≥80%, ≥85%, ≥90%, ≥92%, ≥94%, ≥96%, ≥98% or ≥99%).

[0013] In one or more embodiments, the MYB22 includes its homologs, which are proteins or encoding genes from different species (species other than Oryza) that have sequence homology with SEQ ID NO:1 or the polynucleotide encoding it from the Oryza genus.

[0014] In one or more embodiments, the expression construct (expression vector) includes: a plant expression vector.

[0015] In one or more embodiments, the expression construct (expression vector) includes: a non-viral vector and a viral vector.

[0016] In one or more embodiments, the upregulation includes overexpression.

[0017] In one or more embodiments, the upward adjustment indicates a significant upward adjustment, such as an increase of 20%, 40%, 60%, 80%, 90%, or more.

[0018] In one or more embodiments, the plant is a plant that expresses MYB22 or its homologs.

[0019] In one or more embodiments, the plant includes: grasses.

[0020] In one or more embodiments, the grasses include (but are not limited to): rice, barley, wheat, oats, rye, corn, sorghum, and short-stalked grass.

[0021] In another aspect of the invention, an application of MYB22 or an upregulator thereof is provided for: improving plant insect resistance; preparing formulations that improve plant insect resistance; or, as a molecular marker for identifying plant insect resistance.

[0022] In one or more embodiments, the upregulator includes (but is not limited to): a polynucleotide or construct encoding MYB22, an upregulator that promotes the promoter-driven ability of the MYB22 gene, an upregulator that interacts with the MYB22 protein to increase its expression or activity, a downregulator of MYB22 gene-specific microRNA, a chemical upregulator of MYB22, or a combination thereof.

[0023] In one or more embodiments, the insect is a herbivorous insect; preferably a hemiptera insect; more preferably a planthopper, including the brown planthopper (Nilaparvata lugens), the white-backed planthopper (Sogatella furcifera), and the gray planthopper (Lalielphax striatellus).

[0024] In one or more embodiments, the plant is a plant that expresses MYB22 or its homologs; preferably, the plant includes: grasses; preferably, the grasses include (but are not limited to): rice, barley, wheat, oats, rye, corn, sorghum, and sedge.

[0025] In another aspect of the invention, a plant cell, tissue, or organ is provided, wherein the plant is a plant expressing MYB22 or a homolog thereof, and contains an exogenous upregulator of MYB22 or a homolog thereof, the upregulator comprising: a polynucleotide or construct (including an expression vector) encoding MYB22, an upregulator that promotes the driving ability of the MYB22 gene promoter, an upregulator that interacts with the MYB22 protein to increase its expression or activity, a downregulator of MYB22 gene-specific microRNA, a chemical upregulator of MYB22, or a combination thereof.

[0026] In one or more embodiments, the plant cells, tissues, or organs do not directly generate living plants or serve as plant propagation material.

[0027] In one or more embodiments, high expression or high activity refers to a statistically significant increase in expression or activity compared to the average expression or activity of similar or identical plants, such as an increase of 10%, 20%, 40%, 60%, 80%, 90%, or higher.

[0028] In another aspect of the invention, a method for selecting or identifying insect-resistant plants is provided, the method comprising: identifying the expression or activity of MYB22 in a test plant, wherein if the test plant has high MYB22 expression or activity (e.g., higher than the average value of the plant), it is an insect-resistant plant.

[0029] In another aspect of the present invention, a method for screening potential substances to enhance plant insect resistance is provided, the method comprising:

[0030] (1) A system expressing MYB22 was treated with candidate substances; and

[0031] (2) Detect the expression or activity of MYB22 in the system; if the candidate substance statistically increases (e.g., by 5%, 10%, 15%, 20%, 30% or more, preferably by 50% or more; more preferably by more than 80%) the expression or activity of MYB22, it indicates that the candidate substance is a potential substance for improving plant insect resistance.

[0032] In one or more embodiments, the screening method further includes setting up a control group to clearly distinguish the expression or activity of MYB22.

[0033] In another preferred embodiment, the candidate substances include (but are not limited to): regulatory molecules (such as upregulators, interfering molecules (e.g., those that can interfere with upstream genes that "inhibit MYB22 expression") targeting the MYB22 protein or its encoding gene, or its upstream or downstream proteins or genes), nucleic acid inhibitors, binding molecules (such as antibodies or ligands)), CRISPR constructs, small molecule compounds, etc.

[0034] Other aspects of the invention will be apparent to those skilled in the art from the disclosure herein. Attached Figure Description

[0035] Figure 1 Detection of OsMYB22 gene overexpression and gene-edited plants;

[0036] A: Schematic diagram of base mutation sites in Cas9 plants of the OsMYB22 gene;

[0037] B: Detection of OsMYB22 gene expression in OE-11 plants.

[0038] Figure 2 Changes in the expression of the OsMYB22 gene after feeding by brown planthoppers.

[0039] Figure 3 Phenotypic identification of plants overexpressing the gene against brown planthopper;

[0040] A: Identification of resistance to brown planthopper in individual OE-11 and ZH11 plants;

[0041] B: Identification of resistance to brown planthopper in OE-11 plants and ZH11 small colonies.

[0042] Figure 4 Phenotypic identification of Cas9 plants against brown planthopper;

[0043] A: Identification of resistance to brown planthopper in individual Cas9 plants;

[0044] B: Identification of resistance to brown planthopper in small populations of Cas9 plants. Detailed Implementation

[0045] Through extensive research, screening, and experimental work, the inventors have cloned a novel gene, MYB22, which is associated with regulating insect resistance in plants. The loss of MYB22's function significantly reduces plant resistance to insects, indicating that it is itself a gene with insect-resistant function. This invention provides a new approach for improving insect-resistant traits in plants.

[0046] the term

[0047] As used herein, the insects referred to are phytophagous insects; preferably including Hemiptera, Homoptera, Diptera, and Lepidoptera (such as the rice stem borer); preferably, they are planthoppers. The term "planthopper" includes, but is not limited to, the brown planthopper (BPH), white-backed planthopper (WBPH), and small brown planthopper (SBPH). The "planthopper family" is a family belonging to the order Homoptera. Planthoppers share several common characteristics: commonly known as planthoppers, all are phytophagous, many species live on grasses, and they are piercing-sucking agricultural pests. They have migratory habits and are currently the primary pests of rice in China and many Asian countries. The brown planthopper is a monophagous pest that feeds and reproduces on rice and common wild rice. The white-backed planthopper and small brown planthopper have a wide range of diets and can damage grasses such as rice, wheat, and corn.

[0048] As used herein, "plant" includes plants that express MYB22 or contain MYB22 and the signaling pathways it participates in. According to knowledge in the art, plants expressing MYB22 inherently possess the mechanisms of action claimed by this invention and can achieve the technical effects claimed by this invention. In some preferred embodiments, the plant is a crop, preferably a cereal crop, and more preferably, the cereal crop is a crop with grains (ears). The "cereal crop" can be a grass family plant. Preferably, the grass family plants include: plants of the genus *Oryza* such as rice, plants of the genus *Wheat* such as wheat, plants of the genus *Maize* such as corn, etc. More specifically, examples include rice, barley, wheat, oats, rye, corn, sorghum, and *Brachys edulis*.

[0049] Regarding "control plants," selecting appropriate control plants is a routine part of experimental design. These can include corresponding wild-type plants or transgenic plants without the target gene. Control plants are generally the same plant species or even varieties of the same species or class as the plant being evaluated. Control plants can also be individuals from transgenic plants that have lost their transgenic components due to segregation. As used in this article, control plants refer not only to whole plants but also to plant parts, including seeds and seed portions.

[0050] As used herein, the terms “enhancement,” “improvement,” or “enhancement” are interchangeable and, in their application, should mean an increase of at least 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10%, preferably at least 15% or 20%, more preferably 25% or 30%, compared to the control plant as defined herein.

[0051] The MYB22 gene and the protein it encodes

[0052] The MYB22 gene was screened and obtained by the inventors, who then endowed it with a new function. The function of the MYB22 gene had not been previously studied; through in-depth research, the inventors discovered that it plays an important role in regulating plant insect resistance.

[0053] In this invention, unless otherwise specified, the MYB22 protein includes its homologs (homologous proteins). MYB22 is a polypeptide (protein) having the amino acid sequence shown in SEQ ID NO:1. This invention also includes sequence variations having the same insect-resistant function as the MYB22 protein.

[0054] The variations include (but are not limited to): deletions, insertions, and / or substitutions of several amino acids (typically 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10, and even more preferably 1-8 or 1-5); and additions or deletions of one or more amino acids (typically up to 20, preferably up to 10, and more preferably up to 5) at the C-terminus and / or N-terminus. Any protein with high homology to the MYB22 protein (e.g., 70% or higher homology to the polypeptide sequence shown in SEQ ID NO:1; preferably 80% or higher; more preferably 90% or higher, such as 95%, 98%, or 99% homology) and having the same insect-resistant function as the MYB22 protein is also included in this invention.

[0055] In this invention, the term "MYB22 protein" also includes its homologs. It should be understood that while this invention preferably studies MYB22 proteins derived from specific species, other polypeptides or genes derived from other species, particularly grasses, that are highly homologous to the MYB22 protein (e.g., having more than 70%, more particularly 80%, 85%, 90%, 95%, or even more than 98% sequence identity) are also within the scope of this invention.

[0056] In this invention, polypeptides derived from species other than rice that have high homology with the sequence shown in SEQ ID NO:1, or that play the same or similar insect-resistant role in the same or similar signaling pathways, are also included.

[0057] The present invention also provides isolated proteins, which are fragments of the MYB22 protein or formed by adding other proteins or tags at both ends.

[0058] This invention also relates to a polynucleotide sequence encoding the MYB22 protein of this invention or a sequence variant thereof. The polynucleotide may be in DNA or RNA form. DNA form includes cDNA, genomic DNA, or synthetically produced DNA. The DNA may be single-stranded or double-stranded. The DNA may be a coding strand or a non-coding strand. The coding region sequence encoding the mature polypeptide may be identical to or a degenerate variant of the coding region sequence shown in SEQ ID NO:1. As used herein, "degenerate variant" refers to a nucleic acid sequence encoding a polypeptide having the sequence of SEQ ID NO:1, but differing from the genomic sequence shown in SEQ ID NO:3 or the coding region sequence shown in SEQ ID NO:1. This invention also relates to variants (variants) of the aforementioned polynucleotide that encode polypeptides or fragments, analogs, and derivatives of polypeptides having the same amino acid sequence as those of this invention.

[0059] The present invention also relates to a vector containing the aforementioned polynucleotide, and a host cell genetically engineered using the aforementioned vector or polypeptide to encode nucleic acids.

[0060] In this invention, the polynucleotide sequence encoding the polypeptide of this invention can be inserted into a recombinant expression vector. The term "recombinant expression vector" refers to bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses, or other vectors well known in the art. In short, any plasmid and vector can be used as long as it can replicate and remain stable in the host. An important characteristic of an expression vector is that it typically contains an origin of replication, a promoter, a marker gene, and translation control elements. Preferably, the expression vector may also selectively contain resistance elements, selection elements, or reporter gene elements, such as Bar or GUS.

[0061] When the aforementioned polynucleotide is expressed in higher eukaryotic cells, the insertion of an enhancer sequence into the vector will enhance transcription. An enhancer is a cis-acting factor of DNA, typically consisting of approximately 10 to 300 base pairs, that acts on the promoter to enhance gene transcription.

[0062] Transforming host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art. Plant transformation can be performed using methods such as Agrobacterium-mediated transformation or gene gun transformation, including spraying, leaf disc transformation, and rice embryo transformation.

[0063] Plant insect resistance modification

[0064] This invention, through extensive systematic research and large-scale screening, identified the MYB22 gene, which regulates plant insect resistance. By editing this gene using CRISPR / Cas9 technology, materials with altered insect resistance were obtained.

[0065] In a specific embodiment, the inventors targeted and deleted the MYB22 gene, and then observed the changes in traits. The results showed that plants with the MYB22 gene deleted had significantly reduced insect resistance compared to wild-type plants. Therefore, the MYB22 gene itself is a positive regulatory gene that promotes plant insect resistance.

[0066] Based on the inventors' new discovery, this invention provides a use for the MYB22 protein or its regulatory molecule to enhance plant insect resistance. Knowing the function of MYB22, various methods well-known to those skilled in the art can be used to overexpress MYB22, thereby improving plant insect resistance. For example, expression units carrying the MYB22 gene (such as expression vectors or viruses) can be delivered to the target site via methods known to those skilled in the art, thereby expressing active MYB22.

[0067] As a preferred method, a method for preparing transgenic plants is provided, comprising: (1) transferring exogenous MYB22 encoding nucleic acid into plant tissues, organs or seeds to obtain plant tissues, organs or seeds converted into MYB22 encoding polynucleotides; and (2) regenerating plant plants from the plant tissues, organs or seeds converted into exogenous encoding nucleic acid obtained in step (1).

[0068] Other methods for increasing the expression of the MYB22 gene or its homologs are well known in the art. For example, the expression of the MYB22 gene or its homologs can be enhanced by using a strong promoter. Alternatively, the expression of MYB22 can be enhanced by enhancers (such as the first intron of the rice waxy gene, the first intron of the Actin gene, etc.). Suitable strong promoters include, but are not limited to, the 35S promoter, the Ubi promoter of rice and maize, etc.

[0069] In this invention, the upregulators of the MYB22 polypeptide or its encoding gene include promoters, agonists, and activators. The terms "upregulation" and "promotion" include "upregulation" and "promotion" of polypeptide activity or polypeptide expression. Any substance that can increase the activity of MYB22, improve the stability of MYB22, upregulate MYB22 expression, or increase the effective duration of MYB22 can be used in this invention as a useful substance for upregulating MYB22. These substances can be compounds, small chemical molecules, or biomolecules. The biomolecules can be at the nucleic acid level (including DNA and RNA) or at the protein level. The MYB22 or its upregulators are particularly suitable for application to a class of plants whose MYB22 expression is below the average of that class of plants or whose MYB22 is not expressed; thereby, the application of the MYB22 protein or its upregulators can revert this class of plants to a wild-type phenotype or a better phenotype.

[0070] As a preferred embodiment, a method for upregulating the expression of MYB22 protein in plants is provided, the method comprising: transferring the MYB22 protein or an expression construct or vector of the protein encoded by it into plants.

[0071] Preferably, a method for preparing transgenic plants is provided, comprising:

[0072] (1) Transforming the exogenous nucleic acid encoding the polypeptide of the present invention into a plant organ or tissue to obtain a plant tissue or organ transformed with the nucleic acid encoding the polypeptide; and

[0073] (2) Regenerate plant plants from plant tissues or organs that have been converted into the nucleic acid encoding the polypeptide of the present invention obtained in step (1).

[0074] As a preferred example, the method includes the steps of: (s1) providing Agrobacterium carrying an expression vector containing nucleic acid encoding the MYB22 protein of the present invention; (s2) contacting a plant tissue or organ with the Agrobacterium in step (s1) to transfer and integrate the nucleic acid encoding the polypeptide into the chromosome of a plant cell; (s3) selecting plant tissues or organs that have been transferred with the nucleic acid encoding the MYB22 protein; and (s4) regenerating the plant tissues or organs in step (s3) into plants.

[0075] The present invention also includes plants obtained using any of the foregoing methods, said plants comprising: transgenic plants into which the coding nucleic acid of the polypeptide has been transferred.

[0076] Plant breeding screening or targeted screening of regulatory molecules

[0077] Based on the inventors' new findings, this invention provides a molecular marker suitable for identifying plant insect resistance, namely the MYB22 gene. This invention also relates to specific molecular markers designed for the MYB22 gene, and identification strategies. This allows for early determination of plant insect resistance.

[0078] Therefore, the present invention provides a method for specifically identifying the insect resistance of plants, comprising: identifying the expression of MYB22 in the plant to be tested; if the plant to be tested has high expression of MYB22, then it is a plant with insect resistance.

[0079] Therefore, the present invention provides a method for targeted selection or identification of plants with regulated agronomic traits, comprising: identifying the expression or activity of MYB22 protein in a test plant; if the expression or activity of MYB22 protein in the test plant is higher than the average expression or activity of MYB22 protein in the same type of plant (control plant), then it is a plant with increased insect resistance; or, if the expression or activity of MYB22 protein in the test plant is lower than the average expression or activity of MYB22 protein in the same type of plant (control plant), then it is a plant with decreased insect resistance.

[0080] Based on the novel findings of this invention, those skilled in the art can employ any of the various techniques known in the art or under development to analyze nucleic acid sequences, and these techniques are all included in this invention. The methods described include, but are not limited to: sequencing, PCR amplification, probe methods, hybridization, restriction enzyme digestion analysis, allele polymorphism analysis (such as melting curve analysis) for nucleic acid sequence identification, etc.

[0081] The identification method of this invention only requires PCR reaction and / or agarose gel electrophoresis, and by judging the length of the corresponding PCR product, the phenotype of the sample can be accurately and quickly determined. It is low-cost, suitable for large-scale identification, and requires very little sample. If needed, those skilled in the art can design primers for identifying the molecular markers.

[0082] Methods for obtaining DNA from the sample to be tested are well-known to those skilled in the art, such as the traditional phenol / chloroform / isoamyl alcohol method, or commercially available DNA extraction kits. Polymerase chain reaction (PCR) is also well-known to those skilled in the art; its basic principle is the in vitro enzymatic synthesis of specific DNA fragments. The method of this invention can be performed using conventional PCR techniques.

[0083] This invention has promising applications in molecular design breeding and crop variety improvement using genetic engineering technology.

[0084] After understanding the function of the MYB22 gene, it can be used as a molecular marker for targeted plant screening. This new discovery can also be used to screen for substances or potential substances that can be targeted to regulate insect resistance by modulating this mechanism.

[0085] The present invention provides a method for screening substances (potential substances) that promote the improvement of plant traits, wherein the trait improvement includes: enhanced insect resistance; the method includes: (1) adding the candidate substance to a system expressing MYB22 protein; (2) detecting the system and observing the expression or activity of MYB22 protein therein, and if its expression or activity is increased, it indicates that the candidate substance is a substance that promotes and enhances the insect resistance of plants.

[0086] The present invention provides a method for screening substances (potential substances) that promote the improvement of plant traits, wherein the trait improvement includes: enhanced insect resistance; the method includes: (1) adding the candidate substance to a system expressing MYB22 protein; (2) detecting the system and observing the expression or activity of MYB22 protein therein, and if its expression or activity is reduced, it indicates that the candidate substance is a substance that reduces the insect resistance of plants.

[0087] Methods for screening substances that act on proteins or genes or specific regions thereof as targets are well known to those skilled in the art, and these methods can all be used in this invention. 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. Depending on the type of substance to be screened, those skilled in the art understand how to select an appropriate screening method.

[0088] The detection of protein-protein interactions and their strength can be achieved using a variety of techniques well-known to those skilled in the art, such as GST-Pull Down, bimolecular fluorescence complementation assays, yeast two-hybrid systems, or immunoprecipitation techniques.

[0089] Through large-scale screening, a class of substances that specifically act on the MYB22 protein or its encoding gene and have a regulatory effect on improving insect resistance traits can be obtained.

[0090] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments that do not specify specific conditions are generally performed according to conventional conditions such as those described in J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd Edition, Science Press, or according to the manufacturer's recommendations.

[0091] Materials and methods

[0092] 1. Plant materials

[0093] The rice material used in this invention is the japonica rice variety (Oryza sativa L. subsp. Japonica) Zhonghua 11.

[0094] The tested rice materials were planted in fields in Shanghai during the summer using standard rice field planting and management methods; in the winter, they were planted in the artificial climate chamber of the Center for Excellence in Molecular Plant Sciences, Chinese Academy of Sciences. The temperature was 29℃±1℃, the humidity was 50-70%±5%, and the light duration was 7:00-19:00.

[0095] 2. Construction of OsMYB22 overexpression vector

[0096] The OsMYB22 gene fragment was amplified from the total cDNA of Zhonghua 11 using primers PUN1301-OsMYB22F and PUN1301-OsMYB22R in Table 1, and then constructed into the PUN1301-3×Flag vector using homologous recombination.

[0097] Table 1. Primer sequences used for constructing vectors related to OsMYB22 transgenic material

[0098]

[0099] 3. Construction of the OsMYB22-Cas9 vector

[0100] Using the primers OsMYB22-sgRNAF and OsMYB22-sgRNAR in Table 1, double-stranded DNA is first formed; then, the double-stranded fragment is constructed into the pOs-sgRNA vector; then, it is transferred into the pH-Ubi-Cas9 vector, and the resulting recombinant vector is called the OsMYB22-Cas9 vector.

[0101] 4. Genetic transformation of rice

[0102] The method was mainly based on the reference Hiei Y, Ohta S, Komari T, Kumashiro T: Efficient transformation of rice (Oryza sativa L.) mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA. The Plant journal: for cell and molecular biology 1994, 6(2): 271-282.

[0103] 5. Single-plant resistance identification method for brown planthopper

[0104] After germination, rice seeds are sown individually in small plastic pots. With normal growth and management, the seedlings will enter the tillering stage after about one month, at which point insect resistance will be assessed. A permeable plastic cover, 40cm high and about 8cm in diameter, is prepared beforehand, with a 6*10cm ventilation opening on one side, and covered with mesh. The seedlings are then covered with the cover, with the top covered with mesh.

[0105] During identification, approximately 15 third-instar nymphs were placed in each enclosure. The count was repeated the next day. Under normal growth and management conditions, the survival rate of the seedlings was observed after 5-8 days.

[0106] 6. Small-group identification method for resistance to brown planthoppers

[0107] After the rice seeds were germinated, the experimental group and the control group were symmetrically sown in rectangular plastic boxes. After about 20 days of normal growth and management, 8 second- to third-instar brown planthopper nymphs were inoculated into each rice plant. The survival of the rice was then observed and photographed every day.

[0108] Example 1: OsMYB22 overexpression and the acquisition of gene-edited plants

[0109] The accession number for the OsMYB22 gene is LOC_Os01g65370 (http: / / rice.plantbiology.msu.edu / ). The amino acid sequence and cDNA sequence of OsMYB22 are as follows:

[0110] The amino acid sequence of OsMYB22 (SEQ ID NO:1):

[0111]

[0112] Note: Cas9-1 has a frameshift mutation at amino acid R (48th amino acid) due to base deletion; Cas9-2 has a frameshift mutation at amino acid C (49th amino acid) due to base insertion.

[0113] The cDNA sequence of OsMYB22 (SEQ ID NO:2):

[0114]

[0115]

[0116] Note: The underlined locations indicate the sgRNA target sites. Cas9-1 is a G deletion at 143bp (bold bold), and Cas9-2 is a T insertion after 143bp, resulting in a frameshift.

[0117] The inventors cloned the CDS fragment from the total cDNA of the natural rice variety Zhonghua11 (ZH11) and ligated it into the overexpression vector PUN1301-3×Flag.

[0118] Meanwhile, the OsMYB22 gene was edited using CRISPR-Cas9 technology. First, a specific sg-RNA for the OsMYB22 gene was designed, and specific primers were designed based on the sequence of this sgRNA (Table 1). After annealing, the RNA was formed into a double strand and ligated into a CRISPR (UBI, OsU6) vector. The RNA was then transformed into the wild-type rice variety Zhonghua 11 using Agrobacterium-mediated genetic transformation. Subsequently, molecular biology techniques were used to screen for overexpressing transgenic plants and homozygous mutant plants.

[0119] After molecular detection and analysis, a series of mutant lines were obtained from the transgenic plants. The inventors selected two homozygous edited mutant lines, Cas9-1 and Cas9-2, and one overexpression line, OE-11, as examples.

[0120] The sequence information of the OsMYB22 gene edited in Cas9-1 and Cas9-2 is as follows: Figure 1 As shown in Figure A, compared to the wild type, these two lines exhibited deletions of the "G" and insertions of the "T" at the sgRNA site of the OsMYB22 gene, respectively. Figure 1 A).

[0121] OE-11 was detected using real-time quantitative PCR technology.

[0122] The results showed that, compared to wild-type ZH11, the expression level of OsMYB22 in OE-11 was upregulated by approximately 14-fold. Figure 1 B).

[0123] Example 2: OsMYB22 response to brown planthopper feeding

[0124] To investigate whether the OsMYB22 gene may play a role in rice resistance to brown planthopper, the inventors examined the changes in the expression level of this gene at 0, 4, 8, 12, 24, 48, and 72 hours after flowering during brown planthopper feeding.

[0125] The results showed that the expression of the OsMYB22 gene was significantly upregulated after feeding by brown planthoppers. Figure 2 ).

[0126] The above results suggest that OsMYB22 is involved in regulating rice resistance to brown planthopper.

[0127] Example 3: Plants overexpressing the OsMYB22 gene exhibited a resistance phenotype to brown planthoppers.

[0128] To clarify the role of the OsMYB22 gene in rice resistance to brown planthopper, the inventors conducted brown planthopper resistance assessments on OE-11 plants using two methods: single-plant resistance assessment and small-group resistance assessment.

[0129] A representative figure of the phenotypic identification results of overexpressing plants against brown planthoppers is shown in Figure 1. Figure 3 As shown in AB, where A represents the resistance of individual OE-11 and ZH11 plants to brown planthoppers, and B represents the resistance of small populations of OE-11 and ZH11 plants to brown planthoppers.

[0130] The results of both methods showed that overexpression of the OsMYB22 gene could significantly improve the resistance of rice to brown planthopper.

[0131] Example 4: Plants with edited OsMYB22 gene exhibited a phenotype susceptible to brown planthoppers.

[0132] To further clarify the function of the OsMYB22 gene in rice resistance to brown planthopper, the inventors further identified the resistance of Cas9-1 and Cas9-2 plants with OsMYB22 gene editing (OsMYB22 deletion expression) to brown planthopper.

[0133] Experimental results showed that the OsMYB22 gene-edited plants Cas9-1 and Cas9-2 exhibited significantly lower resistance to brown planthoppers than the wild-type plants. Figure 4AB).

[0134] This result further indicates that the OsMYB22 gene plays a positive regulatory role in regulating rice resistance to brown planthopper.

[0135] Example 5: Screening Method

[0136] Cells: OsMYB22 is expressed in rice leaf cells.

[0137] Test group: Candidate substances were introduced into the cell culture system expressing OsMYB22;

[0138] Control group: No candidate substance was introduced into the cell culture system expressing OsMYB22.

[0139] The expression or activity of OsMYB22 in the test group and the control group were detected and compared. If the expression or activity of OsMYB22 in the test group was statistically higher (e.g., 20% or more higher) than that in the control group, it indicates that the candidate substance is a potential substance that can improve plant disease resistance.

[0140] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims. Furthermore, all documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. sequence list <110> Center for Excellence in Molecular Plant Sciences, Chinese Academy of Sciences <120> A novel gene regulating plant insect resistance and its application. <130> 217656 <160> 6 <170> SIPOSequenceListing 1.0 <210> 1 <211> 287 <212> PRT <213> Oryza sativa L. <400> 1 Met Gly Arg Ser Pro Cys Cys Glu Lys Ala His Thr Asn Lys Gly Ala 1 5 10 15 Trp Thr Lys Glu Glu Asp Gln Arg Leu Ile Ala Tyr Ile Lys Ala His 20 25 30 Gly Glu Gly Cys Trp Arg Ser Leu Pro Lys Ala Ala Gly Leu Leu Arg 35 40 45 Cys Gly Lys Ser Cys Arg Leu Arg Trp Met Asn Tyr Leu Arg Pro Asp 50 55 60 Leu Lys Arg Gly Asn Phe Thr Asp Asp Asp Asp Glu Leu Ile Ile Lys 65 70 75 80 Leu His Ala Leu Leu Gly Asn Lys Trp Ser Leu Ile Ala Gly Gln Leu 85 90 95 Pro Gly Arg Thr Asp Asn Glu Ile Lys Asn Tyr Trp Asn Thr His Ile 100 105 110 Lys Arg Lys Leu Leu Ser Arg Gly Ile Asp Pro Gln Thr His Arg Pro 115 120 125 Val Ser Ala Gly Ser Ser Ala Ala Ala Ala Ser Gly Leu Thr Thr Thr 130 135 140 Ala Ser Thr Ala Ala Phe Pro Ser Leu Ala Pro Ala Pro Pro Pro Gln 145 150 155 160 Gln His Arg Leu His Asn Pro Val His Ala Ala Ala Pro Ser Asn Ala 165 170 175 Ser Phe Ala Arg Ser Ala Ala Ser Pro Pro Ser Glu Asp Gly His Ser 180 185 190 Ser Ser Gly Gly Ser Ser Asp Ala Pro Arg Cys Pro Asp Leu Asn Leu 195 200 205 Asp Leu Asp Leu Asp Leu Ser Met Ser Leu Pro Ser Ser Pro Pro Lys 210 215 220 Thr Pro Ala Ala Ala Ser Ser Thr Thr Ala Ser Arg His His His His 225 230 235 240 Gln Gln Gln Lys Thr Ile Cys Leu Cys Tyr His Leu Gly Val Arg Asn 245 250 255 Gly Asp Val Cys Ser Cys Lys Ala Ala Ala Pro Ser Pro Ala Gly Pro 260 265 270 Arg Ala Phe Arg Phe Leu Arg Pro Leu Glu Glu Gly Gln Tyr Ile 275 280 285 <210> 2 <211> 864 <212> DNA <213> Oryza sativa L. <400> 2 atggggaggt cgccatgctg cgagaaggcg cacacgaaca agggggcgtg gacgaaggag 60 gaggaccagc ggctgatcgc ctacatcaag gcgcacggcg agggttgctg gcggtcgctg 120 cccaaggcgg cggggctcct ccgctgcggc aagagctgcc gcctccgctg gatgaactac 180 ctccgccccg acctcaagcg cggcaacttc accgacgacg acgacgagct catcatcaag 240 ctccacgccc ttctcggcaa caagtggtcg ttgattgcgg ggcagctgcc ggggaggacg 300 gacaacgaga tcaagaacta ctggaacacg cacatcaagc gcaagctcct gagccggggc 360 atcgacccgc agacgcaccg gccggtcagc gccgggagca gcgccgccgc ggcgagcggg 420 ctgaccacga cggccagcac cgccgccttt ccgtcccttg cgccggcgcc gccgccgcag 480 cagcacaggc tacacaaccc ggtgcacgcc gcggcgccga gcaatgcgag cttcgccagg 540 tccgcggcgt ccccgccgtc ggaggacggc cacagcagca gcggcggcag ctcggacgcg 600 ccgcggtgcc ccgacctcaa cctcgacctc gacctcgacc tgtccatgag cctgccgagc 660 tcgccgccca agacgccggc cgccgcgtcg tccacgaccg cgtcgcgcca ccatcaccac 720 cagcagcaga agaccatctg cctctgctac cacctcggcg tccgcaacgg cgacgtctgc 780 agctgcaagg cggccgcgcc atcgccggcc ggcccacgcg cgttccggtt tctcaggcca 840 ctggaggagg gccagtacat atag 864 <210> 3 <211> 48 <212> DNA <213> Artificial Sequence <400> 3 caggtcgact ctagaggatc catggggagg tcgccatgct gcgagaag 48 <210> 4 <211> 46 <212> DNA <213> Artificial Sequence <400> 4 atggtctttg tagtcggtac ctatgtactg gccctcctcc agtggc 46 <210> 5 <211> 24 <212> DNA <213> Artificial Sequence <400> 5 ggcagccagg gagccgagca gcag 24 <210> 6 <211> 24 <212> DNA <213> Artificial Sequence <400> 6 aaacctgctg ctcggctccc tggc 24

Claims

1. A method for improving plant insect resistance, characterized in that, This includes overexpression of MYB22 in plants; the plant is rice; the insect is the brown planthopper (Brown planthopper, family Planthopperidae). Nilaparvata lugens ); The MYB22 is a polypeptide with the amino acid sequence shown in SEQ ID NO:

1.

2. The method as described in claim 1, characterized in that, The overexpression of MYB22 in plants includes: encoding... MYB22 Polynucleotides or constructs are introduced into plants.

3. An application of MYB22 or an upregulator thereof, for: Improve plant insect resistance; Prepare formulations to enhance plant insect resistance; or, As a molecular marker for identifying plant insect resistance; The plant is rice; the insect is the brown planthopper (Brown planthopper, family Planthopperidae). Nilaparvata lugens ); The MYB22 is a polypeptide with the amino acid sequence shown in SEQ ID NO: 1; The MYB22 upregulator is coded MYB22 Polynucleotides or constructs.

4. A method for selecting or identifying insect-resistant plants, the method comprising: The expression or activity of MYB22 in the test plant was identified. If the expression or activity of MYB22 in the test plant was higher than the average expression or activity of MYB22 protein in this type of plant, then it was a plant with increased insect resistance; the plant was rice; the insect was the brown planthopper (Brassica napus), a planthopper (family Planthopperidae). Nilaparvata lugens ); The MYB22 is a polypeptide with the amino acid sequence shown in SEQ ID NO:

1.

5. A method for screening potential substances to enhance plant insect resistance, the method comprising: (1) A system expressing MYB22 was treated with a candidate substance; and (2) Detect the expression or activity of MYB22 in the system; if the candidate substance statistically increases the expression or activity of MYB22, it indicates that the candidate substance is a potential substance for improving plant insect resistance; The plant is rice; the insect is the brown planthopper (Brown planthopper, family Planthopperidae). Nilaparvata lugens ); The MYB22 is a polypeptide with the amino acid sequence shown in SEQ ID NO:

1.

6. The method for screening potential substances to enhance plant insect resistance as described in claim 5, characterized in that, The candidate substances include: regulatory molecules, agonists, nucleic acid inhibitors, binding molecules, CRISPR constructs, and small molecule compounds targeting the MYB22 protein or its encoding gene, or its upstream or downstream proteins or genes.

7. The method for screening potential substances to enhance plant insect resistance as described in claim 5, characterized in that, The screening method also includes setting up a control group to clearly distinguish the expression or activity of MYB22.

8. The method for screening potential substances to enhance plant insect resistance as described in claim 5, characterized in that, The system described is a cell culture system.

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