Application of OsCMR2 gene in regulating rice resistance to rice leaf roller

Abstract

This invention relates to the application of the OsCMR2 gene in regulating rice resistance to the rice leaf roller. Specifically, this invention provides the use of the CMR2 gene or its encoded protein, or its promoter, for improving plant agronomic traits, or for preparing a composition or formulation for improving plant agronomic traits, including enhancing plant resistance to rice leaf rollers. This invention is the first to discover that increasing the expression level or activity of the CMR2 gene or its encoded protein in plants (such as rice) can improve plant agronomic traits, such as enhancing plant resistance to rice leaf rollers.

Description

Technical Field

[0001] This invention relates to the field of agronomy, specifically to the application of the OsCMR2 gene in regulating rice resistance to rice leaf roller. Background Technology

[0002] Jasmonic acid (JA) and its derivatives are an important class of plant endogenous hormones that play a central role in regulating plant growth and development and stress response, especially in mediating plant insect resistance responses.

[0003] Rice is one of the world's most important food crops, especially in my country, where its planting area is vast and consumption is high, making it a core crop for maintaining national food security and social stability. With changes in agricultural production conditions and the increasing complexity of the ecological environment, insect pests have become a major factor limiting stable and high rice yields. Among them, the rice leaf roller (Cnaphalocrocism edinalis) is a key pest that is widely distributed, causes severe damage, and is difficult to control. This insect primarily feeds on leaves as larvae and is characterized by strong concealment, rapid reproduction, and a long infestation period. Numerous studies have shown that rice leaf roller damage can lead to reduced tillering, decreased panicle number, and lower grain filling rate in rice, causing yield losses of 20%-30% in severe cases, and even exceeding 60% in some areas during outbreak years. With global climate change and adjustments in planting methods, the range of this pest continues to expand, and its damage is increasingly severe, making it one of the key biological stress factors restricting stable and high rice yields and posing a significant threat to regional food security. Although current pest control measures primarily rely on chemical pesticides, long-term dependence on insecticides has not only led to increased pesticide resistance but also exacerbated environmental pollution and food safety risks. With green agriculture and sustainable development becoming strategic directions for agriculture, developing rice's own insect resistance has become an effective way to reduce pesticide use and ensure ecological security. However, current research on the mechanisms of rice resistance to the rice leaf roller is still insufficient, and the discovery of insect-resistant germplasm resources and functional genes is significantly lacking, hindering the in-depth advancement of insect-resistant breeding work. Therefore, systematically conducting research on rice insect resistance mechanisms and discovering new resistance gene resources is of significant scientific importance and practical value for improving rice's insect resistance and promoting the development of green agriculture.

[0004] The precise elucidation of the regulatory mechanism of the jasmonic acid (JA) signaling pathway in plants, as a core defense system against herbivorous insects, has always been a focus of research in the field of plant-insect interactions.

[0005] Therefore, there is an urgent need in this field to screen and identify key regulatory factors involved in the jasmonic acid signal transduction process in rice, to provide new molecular targets for the genetic improvement of rice insect resistance, and to provide theoretical basis and technical support for the construction of an efficient and green pest and disease management system. Summary of the Invention

[0006] The purpose of this invention is to screen and identify key regulatory factors involved in the jasmonic acid signal transduction process in rice, provide new molecular targets for the genetic improvement of rice insect resistance, and provide theoretical basis and technical support for the construction of an efficient and green pest and disease management system.

[0007] The first technical solution of this invention provides the application of OsCMR2 as a target in regulating rice resistance to the rice leaf folder. It includes the following steps: improving the plant's resistance to the rice leaf folder by overexpressing the exogenous gene OsCMR2 in a plant, such as rice. Preferably, the plant is a gramineous crop selected from the group consisting of rice, wheat, maize, barley, oats, rye, sorghum, and millet, with rice being the most preferred. A direct method to test the insect resistance effect of the transgenic plant is whether the transgenic plant seedlings show increased resistance to the rice leaf folder compared to the wild type.

[0008] This invention provides a technical solution for regulating rice resistance to the rice leaf folder using the OsCMR2 gene, addressing the problems of poor resistance to the rice leaf folder and limited resistance gene resources in existing rice varieties. OsCMR2 encodes a membrane protein that plays a positive regulatory role in rice's resistance to rice leaf folder infection. Functional studies have shown that knocking out OsCMR2 significantly enhances the feeding effect of rice leaf folder larvae, leading to increased insect body weight and more severe damage in the field, indicating that this gene has an important biological function in rice insect resistance. Based on this finding, this invention further provides a molecular breeding strategy to enhance rice insect resistance by regulating the expression level of the OsCMR2 gene, including manipulating the gene using molecular marker-assisted selection, gene editing, or genetic transformation to screen for new rice materials with enhanced insect resistance. Simultaneously, this invention also constructs a recombinant expression vector containing the OsCMR2 gene, which can be used for rice genetic improvement to enhance its natural resistance to the rice leaf folder. This technical solution provides key genetic resources and operable molecular methods for the genetic improvement of rice resistance to rice leaf roller, and has significant agricultural application value. It helps to reduce the use of chemical pesticides, reduce agricultural environmental pollution, and improve the stability of rice yield, which is of great significance for ensuring food security.

[0009] A first aspect of the present invention provides the use of the CMR2 gene or its encoded protein, or an promoter thereof, for improving agronomic traits of plants, or for preparing a composition or formulation for improving agronomic traits of plants, said improved agronomic traits including enhancing plant resistance to pyralid moths.

[0010] In another preferred embodiment, the moths include the genus *Rhizoctonia solani*.

[0011] In another preferred embodiment, the moths include major pests of rice such as the rice leaf roller, rice stem borer, rice caltrop, and rice bark borer.

[0012] In another preferred embodiment, the CMR2 gene or its encoded protein is derived from grasses.

[0013] In another preferred embodiment, the CMR2 gene or the protein it encodes is derived from one or more plants selected from the group consisting of rice, wheat, corn, barley, oats, rye, sorghum, and millet, with rice being preferred.

[0014] In another preferred embodiment, the CMR2 gene includes the rice CMR2 gene (OsCMR2, accession number: XP_015620637).

[0015] In another preferred embodiment, the CMR2 gene or the protein it encodes is derived from rice or a variant thereof.

[0016] In another preferred embodiment, the CMR2 gene includes a wild-type CMR2 gene and a mutant CMR2 gene.

[0017] In another preferred embodiment, the mutants include mutants in which the function of the encoded protein remains unchanged (i.e., the function is the same as or substantially the same as the wild-type encoded protein) and mutants in which the function is enhanced.

[0018] In another preferred embodiment, the polypeptide encoded by the mutant CMR2 gene is the same as or substantially the same as the polypeptide encoded by the wild-type CMR2 gene.

[0019] In another preferred embodiment, the mutant CMR2 gene comprises polynucleotides with ≥80% (preferably ≥90%, more preferably ≥95%, even more preferably ≥98% or 99%) homology to the wild-type CMR2 gene.

[0020] In another preferred embodiment, the mutant CMR2 gene comprises a polynucleotide with 1-60 (preferably 1-30, more preferably 1-10) nucleotides truncated or added to the 5' and / or 3' ends of the wild-type CMR2 gene.

[0021] In another preferred embodiment, the amino acid sequence of the CMR2 protein is selected from the group consisting of:

[0022] (i) A polypeptide having the amino acid sequence shown in SEQ ID NO.:1;

[0023] (ii) A polypeptide derived from (i) that has the function of improving the agronomic traits of plants, formed by substituting, deleting or adding one or more (e.g., 1-10) amino acid residues of the amino acid sequence shown in SEQ ID NO.:1; or

[0024] (iii) A polypeptide having the agronomic trait function of the plant being improved, with an amino acid sequence homology of ≥80% (preferably ≥90%, more preferably ≥95% or ≥98%) to the amino acid sequence shown in SEQ ID NO.:1.

[0025] In another preferred embodiment, the nucleotide sequence of the CMR2 gene is selected from the group consisting of:

[0026] (a) A polynucleotide encoding the polypeptide shown in SEQ ID NO.:1;

[0027] (b) Polynucleotides with sequences as shown in SEQ ID NO.:2;

[0028] (c) A polynucleotide whose nucleotide sequence is ≥75% homology (preferably ≥85%, more preferably ≥90% or ≥95%) to the sequence shown in SEQ ID NO.:2;

[0029] (d) A polynucleotide with 1-60 (preferably 1-30, more preferably 1-10) nucleotides truncated or added to the 5' and / or 3' ends of the polynucleotide shown in SEQ ID NO.:2;

[0030] (e) and any of the polynucleotides complementary to the polynucleotides described in (a)-(d).

[0031] In another preferred embodiment, the promoter includes a substance that promotes the expression of the CMR2 gene or its encoded protein.

[0032] In another preferred embodiment, the promoter is selected from the group consisting of small molecule compounds, CMR2-expressing carriers, or combinations thereof.

[0033] In another preferred embodiment, the promotion of CMR2 expression or activity refers to increasing the expression or activity of the CMR2 gene or protein by ≥20%, more preferably ≥50%, and even more preferably ≥70%.

[0034] In another preferred embodiment, the composition is an agricultural composition.

[0035] In another preferred embodiment, the composition comprises (a) the CMR2 gene or its encoded protein, or an promoter thereof; and (b) an agronomically acceptable vector.

[0036] In another preferred embodiment, the dosage form of the composition or preparation is selected from the group consisting of solutions, emulsions, suspensions, powders, foams, pastes, granules, aerosols, or combinations thereof.

[0037] In another preferred embodiment, the plant includes grasses (Poaceae).

[0038] In another preferred embodiment, the plant includes rice, wheat, corn, barley, oats, rye, sorghum, and millet, with rice being the preferred variety.

[0039] In another preferred embodiment, the rice includes indica rice, japonica rice, or a combination thereof.

[0040] A second aspect of the present invention provides a composition comprising:

[0041] (a) The CMR2 gene or its encoded protein, or its promoters;

[0042] (b) An agriculturally acceptable carrier.

[0043] In another preferred embodiment, the CMR2 gene or its encoded protein is derived from grasses.

[0044] In another preferred embodiment, the CMR2 gene or the protein it encodes is derived from one or more plants selected from the group consisting of rice, wheat, corn, barley, oats, rye, sorghum, and millet, with rice being preferred.

[0045] In another preferred embodiment, the CMR2 gene includes the rice CMR2 gene (OsCMR2, accession number: XP_015620637).

[0046] In another preferred embodiment, the CMR2 gene or the protein it encodes is derived from rice or a variant thereof.

[0047] In another preferred embodiment, the composition comprises an agricultural composition.

[0048] In another preferred embodiment, the dosage form of the composition is selected from the group consisting of solutions, emulsions, suspensions, powders, foams, pastes, granules, aerosols, or combinations thereof.

[0049] In another preferred embodiment, the composition contains 0.0001-10 wt%, preferably 0.1-1 wt%, of component (a) based on the total weight of the composition.

[0050] In another preferred embodiment, the composition further includes other substances for enhancing the plant's resistance to pyralid moths.

[0051] A third aspect of the present invention provides the use of the composition described in the second aspect of the present invention for enhancing the resistance of plants to pyralid moths.

[0052] A fourth aspect of the present invention provides a method for improving the agronomic traits of plants, comprising the steps of: promoting or increasing the expression level and / or activity of the CMR2 gene or its encoded protein in the plant, thereby improving the agronomic traits of the plant; wherein the improved agronomic traits of the plant include enhancing the plant's resistance to pyralid moths.

[0053] In another preferred embodiment, the CMR2 gene or its encoded protein is derived from grasses.

[0054] In another preferred embodiment, the CMR2 gene or the protein it encodes is derived from one or more plants selected from the group consisting of rice, wheat, corn, barley, oats, rye, sorghum, and millet, with rice being preferred.

[0055] In another preferred embodiment, the CMR2 gene includes the rice CMR2 gene (OsCMR2, accession number: XP_015620637).

[0056] In another preferred embodiment, the CMR2 gene or the protein it encodes is derived from rice or a variant thereof.

[0057] In another preferred embodiment, the enhancement of plant resistance to pyralid moths includes inhibiting the growth and development of rice leaf roller larvae.

[0058] In another preferred embodiment, the method includes administering a promoter of the plant CMR2 gene or its encoded protein.

[0059] In another preferred embodiment, the promoter is selected from the group consisting of small molecule compounds, CMR2-expressing carriers, or combinations thereof.

[0060] In another preferred embodiment, the method includes the steps of:

[0061] (i) providing a plant or plant cell; and

[0062] (ii) Introducing a promoter of the CMR2 gene or its encoded protein into the plant or plant cells to obtain a transgenic plant or plant cells.

[0063] In another preferred embodiment, the promoter refers to a substance that promotes the expression of the CMR2 gene or its encoded protein.

[0064] In another preferred embodiment, the method includes introducing an exogenous CMR2 gene or its encoded protein into a plant.

[0065] In another preferred embodiment, the method includes introducing into the plant a substance that promotes the expression of the endogenous CMR2 gene or its encoded protein.

[0066] In another preferred embodiment, the method includes promoting the expression of the endogenous CMR2 gene or its encoded protein in a plant.

[0067] In another preferred embodiment, the method includes the steps of:

[0068] (i) providing a plant or plant cell; and

[0069] (ii) The CMR2 gene sequence is introduced into the plant or plant cell to obtain a transgenic plant or plant cell.

[0070] In another preferred embodiment, the method includes the steps of:

[0071] (a) Agrobacterium providing an expression vector carrying the CMR2 gene sequence;

[0072] (b) Contacting plant cells, tissues or organs with Agrobacterium in step (a) to transfer the CMR2 gene sequence into the plant cells and integrate it into the chromosomes of the plant cells;

[0073] (c) Select plant cells, tissues, or organs that have been transfected with the CMR2 gene sequence; and

[0074] (d) Regenerate the plant cells, tissues or organs from step (c) into a plant.

[0075] In another preferred embodiment, the expression level or activity of the CMR2 gene or its encoded protein in the plant tissue or plant cell is increased by ≥20%, more preferably ≥50%, and even more preferably ≥70%.

[0076] In another preferred embodiment, "enhancement" refers to an increase in the expression or activity of the CMR2 gene or its encoded protein that meets the following conditions:

[0077] The ratio of A1 / A0 is ≥20%, preferably ≥50%, more preferably ≥70%, and most preferably 80-200%; wherein A1 is the expression or activity of (a) CMR2 gene or its encoded protein in plant tissues or plant cells; and A0 is the expression or activity of the same CMR2 gene in wild-type plant tissues or plant cells of the same species.

[0078] In another preferred embodiment, the method includes the steps of:

[0079] (a) Introducing a foreign construct into plant cells, wherein the construct contains a foreign CMR2 gene sequence, thereby obtaining plant cells in which the foreign construct has been introduced;

[0080] (b) The plant cells obtained in the previous step and introduced with the exogenous construct are then used to generate plants: and

[0081] (c) Optionally, the regenerated plants are identified to obtain plants that have activity in enhancing the plant’s resistance to pyralid moths.

[0082] In another preferred embodiment, the exogenous CMR2 gene sequence further includes a promoter and / or terminator operatively linked to the ORF sequence.

[0083] In another preferred embodiment, the promoter is selected from the group consisting of constitutive promoters, tissue-specific promoters, inducible promoters, and strong promoters, with strong promoters being preferred.

[0084] The fifth aspect of this invention provides a method for preparing genetically engineered plant tissues or plant cells, comprising the steps of:

[0085] Increase the expression level and / or activity of the CMR2 gene or its encoded protein in plant tissues or plant cells to obtain genetically engineered plant tissues or plant cells.

[0086] In another preferred embodiment, the genetic engineering includes transgenic engineering.

[0087] In another preferred embodiment, the method further includes introducing the CMR2 gene or its encoded protein or a promoter thereof into plant tissues or plant cells.

[0088] The sixth aspect of this invention provides a method for preparing plants with improved traits, comprising the steps of:

[0089] The genetically engineered plant tissues or plant cells prepared by the method described in the fifth aspect of the present invention are regenerated into plant bodies, thereby obtaining plants with improved traits.

[0090] In another preferred embodiment, the trait includes enhanced plant resistance to pyralid moths.

[0091] The seventh aspect of the present invention provides a genetically engineered plant in which the CMR2 gene or its encoded protein, or its promoter, is introduced, or the plant is prepared using the method described in the sixth aspect of the present invention.

[0092] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0093] The following figures are used to illustrate specific embodiments of the present invention and are not intended to limit the scope of the invention as defined by the claims.

[0094] Figure 1 The gene sequence of the OsCMR2 mutant region obtained using CRISPR / Cas9 is shown.

[0095] Figure 2 The image shows a violin plot of the weight statistics of rice leaf roller larvae after feeding on wild-type rice (ZH11) and OsCMR2 mutant seedlings for 6 days. Detailed Implementation

[0096] Through extensive and in-depth research, the inventors of this invention, through the study and screening of numerous plant trait loci, have for the first time discovered that increasing the expression level and / or activity of the CMR2 gene or its encoded protein in plants (such as rice) can improve agronomic traits, such as enhancing plant resistance to pests of the family Pyralidae. Specifically, this invention identified the OsCMR2 gene—a key regulatory factor involved in the jasmonic acid signal transduction process in rice—through systematic functional screening, which can be significantly induced to express in rice leaves fed on by the rice leaf folder. This invention explores the potential application of the OsCMR2 gene in regulating rice resistance to the rice leaf folder. Experiments have confirmed that the OsCMR2 gene is associated with rice resistance to the rice leaf folder; after knocking out the OsCMR2 gene using CRISPR / Cas9, the mutant rice showed significantly reduced resistance to the rice leaf folder compared to the wild type. This result indicates that the OsCMR2 gene and its encoded protein can be used to regulate rice resistance (to the rice leaf folder). Further research into the mechanism of action of OsCMR2 is expected to provide new molecular targets for the genetic improvement of rice's insect resistance, and to provide theoretical basis and technical support for building an efficient and green pest and disease management system. Based on this, the inventors completed this invention.

[0097] CMR2 gene

[0098] This invention is the first to discover that the CMR2 gene (Cnaphalocrocis medinalis resistance 2) is a functional gene in rice that is closely related to resistance to rice leaf roller.

[0099] As used herein, the terms "CMR2 gene of the present invention" and "CMR2 gene" are used interchangeably and both refer to the CMR2 gene or a variant thereof derived from plants (such as grasses, preferably rice).

[0100] In a preferred embodiment, the nucleotide sequence of the CMR2 gene of the present invention is shown in SEQ ID NO.:2. Variants of the CMR2 gene of the present invention can be obtained by inserting or deleting regulatory regions, performing random or site-directed mutations, etc.

[0101] The present invention also includes nucleic acids having 50% or more (preferably 60%, 70%, 80%, more preferably 90%, more preferably 95%, most preferably 98%, such as 99%) homology to the preferred gene sequence of the present invention (SEQ ID NO.:2), said nucleic acids also effectively regulating traits of monocotyledonous plants such as rice. "Homology" refers to the level of similarity (i.e., sequence similarity or identity) between two or more nucleic acids according to the percentage of positions they occupy.

[0102] In this invention, the nucleotide sequence in SEQ ID NO.:2 can be modified by substitution, deletion, or addition of one or more (usually 1-90, preferably 1-60, more preferably 1-20, most preferably 1-10), and by adding several (usually up to 60, preferably up to 30, more preferably up to 10, most preferably up to 5) nucleotides to the 5' and / or 3' ends to generate a derived sequence of SEQ ID NO.:2. Due to the degeneracy of the codon, even if the homology with SEQ ID NO.:2 is low, it can still essentially encode the amino acid sequence shown in SEQ ID NO.:1.

[0103] Additionally, the meaning of "the nucleotide sequence in SEQ ID NO.:2 having been substituted, deleted, or added at least one nucleotide-derived sequence" also includes nucleotide sequences that can hybridize with the nucleotide sequence shown in SEQ ID NO.:2 under moderately stringent conditions, and preferably under highly stringent conditions. These variations include (but are not limited to): deletions, insertions, and / or substitutions of several nucleotides (typically 1-90, preferably 1-60, more preferably 1-20, and most preferably 1-10), and additions of several nucleotides (typically up to 60, preferably up to 30, more preferably up to 10, and most preferably up to 5) at the 5' and / or 3' ends.

[0104] It should be understood that although the gene provided in the examples of this invention is derived from rice, CMR2 gene sequences derived from other similar plants (especially plants belonging to the same Poaceae family as rice or other families or genera with high homology to rice) that have a certain degree of homology (conservation, such as having more than 80%, such as 85%, 90%, 95% or even 98% sequence identity) with the sequence of this invention (preferably, the sequence is as shown in SEQ ID NO.:2) are also included within the scope of this invention. As long as those skilled in the art can easily isolate the sequence from other plants based on the information provided in this application after reading this application, the methods and tools for comparing sequence identity are also well known in the art, such as BLAST.

[0105] The polynucleotides of this invention can be in DNA or RNA form. The DNA form includes DNA, genomic DNA, or artificially synthesized DNA, which can be single-stranded or double-stranded. The DNA can be a coding strand or a non-coding strand. The coding region sequence encoding the mature polypeptide can be identical to or a degenerate variant of the coding region sequence shown in SEQ ID NO.:2.

[0106] Polynucleotides encoding mature polypeptides include: coding sequences that encode only the mature polypeptide; coding sequences of the mature polypeptide and various additional coding sequences; coding sequences of the mature polypeptide (and optional additional coding sequences) and non-coding sequences.

[0107] The term "polynucleotide encoding a polypeptide" can refer to a polynucleotide that encodes the polypeptide, or it can also include polynucleotides that include additional coding and / or non-coding sequences. This invention also relates to variants of the aforementioned polynucleotides that encode fragments, analogs, and derivatives of polyglycosides or polypeptides having the same amino acid sequence as those of this invention. These polynucleotide variants can be naturally occurring allelic variants or non-naturally occurring variants. These nucleotide variants include substitution variants, deletion variants, and insertion variants. As is known in the art, an allelic variant is a substitution of a polynucleotide, which may be a substitution, deletion, or insertion of one or more nucleotides, but does not substantially alter the function of the polypeptide it encodes.

[0108] The present invention also relates to polynucleotides that hybridize with the above-described sequences and have at least 50%, preferably at least 70%, and more preferably at least 80% identity between the two sequences. The present invention particularly relates to polynucleotides that hybridize with the polynucleotides described herein under stringent conditions. In the present invention, “stringent conditions” means: (1) hybridization and elution at lower ionic strength and higher temperatures, such as 0.2×SSC, 0.1% SDS, 60°C; or (2) hybridization with a denaturing agent, such as 50% (v / v) formamide, 0.1% fetal bovine serum / 0.1% Ficoll, 42°C, etc.; or (3) hybridization only occurs when the identity between the two sequences is at least 90%, preferably at least 95%.

[0109] The full-length nucleotide sequence or fragments of the CMR2 gene of 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, especially the open reading frame sequences, and the relevant sequences can be amplified using commercially available DNA libraries or cDNA libraries prepared according to conventional methods known to those skilled in the art. When the sequence is long, two or more PCR amplifications are often required, and then the fragments amplified from each amplification are spliced ​​together in the correct order. Once the relevant sequence is obtained, it can be obtained in large quantities using recombination. Typically, it is cloned into a vector, transformed into cells, and then the relevant sequence is isolated from the proliferated host cells using conventional methods.

[0110] Furthermore, the relevant sequences can be synthesized artificially, especially when the fragment length is short. Typically, long fragments are obtained by first synthesizing multiple small fragments and then ligating them. Currently, the DNA sequence encoding the protein of the present invention (or a fragment thereof, or a derivative thereof) can be obtained entirely through chemical synthesis. This DNA sequence can then be introduced into various existing DNA molecules (or vectors) and cells known in the art. Furthermore, mutations can be introduced into the protein sequence of the present invention through chemical synthesis.

[0111] In a preferred embodiment, the nucleotide sequence (CDS sequence) of the CMR2 gene (OsCMR2) is shown below:

[0112] ATGCTCACGGCCACGCAGACTCTGGCGCCGGCAGTGCTCTCCCGGAGCCA

[0113] TGGCGCCCCTTCTTCCTTCCAGCCAGCCGCGCCGCACCGCCGCCGCCG

[0114] CCTCGAGAGTAAGCTGCACCCGCGTCGGCGCCTTGTCGGAGGTCGTCAAT

[0115] GGCGAACTCGTCGTCGGCGACCAAGAACAGACGACCGACGACCTCCTTA

[0116] CGCGGCACAAGAATGTCGTCGCCGACTACACGCTGAGCGCCACGGTGAC

[0117] GGTGAGCTTGAAGCAGGACGATTCCACTCCCCAGAAGGTGGCGGACATG

[0118] GTTAATCGAGACTGGCTTTTCCTTGATTTCTTCAGCTCGCATATAGAGGGG

[0119] ATGCACACGGAGCCTCAGCTCGCCAGGTACTCGCACATGGATGGCAAAG

[0120] GCTCCTTCATATACGAGGCCAGCTTCAGCATCCCGTCCTCGTTGGACGCC

[0121] GTCGGCGCCGTGCAGGTCGTGAACCGCTACAGCAGCGAGGTGTACATCTC

[0122] GGACATCGACGTCCACCTCTGCGGCGGCCGCCATCAGTGGACCGACATCA

[0123] CTTTCCACTGCAACTCTTGGATCGACTACAACCCCAACGACCAGCGCTTC

[0124] TTCTTCCCTCTCAAGTCGTACCTCCCGTCTCAGACGCCCAGGGGCGTGAA

[0125] GAATCTGCGCAAGGAAGAGCTCAGGGCCATCCGCGGCGATGGCCGCGGC

[0126] GAGCGCAAGGAGTGGGAGCGCATCTACGACTACGACGTCTACAACGACC

[0127] TCGGCGACCCCGACAATGACCCGGCCACTCGTCGGCCGGTGCTCGGCGGC

[0128] CGCGGGCGCCCCTACCCGCGCCGCTGCCGCACGGGCCGCCGCCGCTGCAG

[0129] GACAGACCCGTCGTCGGAGTCGCCGCCGGCCAAGGACGGCGCCGGGATC

[0130] TACGTGCCACGGGACGAGGCGTTCACGGAGCGGAAGGCCGGCGCGTTCG

[0131] CCACCAAGAAGGCGCTGTCGGCGCTGTCGGCGTTCACCACGGCGCAGAG

[0132] GGTGTCCGGCGACCGGCGGCGGGGCTTCCCGTCGCTGGCGGCCATCGACG

[0133] CGCTGTACGAGGACGGGTACAAGAACCGGCCGTCGTCGTCGCAGCAGGA

[0134] GGCGGACAACCTCGAAGGCTACTTCAGGGAGGTGCTCCAGAAGCAGGTG

[0135] AAGCTGCTGCTCAAGGGCGAGAAGGAGGAGTTCAAGGAGGAGCTACGCA

[0136] AAGTGTTCAAATTCCAAACGCCCGAGATTCACGACAAGGACAAGCTTGCA

[0137] TGGTTCAGAGACGAGGAGTTCGCGCGGCAAACGCTGGCAGGGATGAACC

[0138] CTCTCAGCATCCAACTTGTCAGGGACACGGACTTCCCTATATTCAGCAAG

[0139] CTGGACGAGGAAACCTACGGCCCAGGGGACTCCCTCATCACCAAAGAGC

[0140] TGATTGAAGAGCAGATTAATGGGGTCATGACAGCAGAGGAGGCCGTGGA

[0141] GAAGAAGAAGCTGTTCATGCTGGACTACCACCAGGTGCTCCTGCCGTTCG

[0142] TGCACGCGGTGCGCGAGCTGGACGACACCACGCTGTACGCCTCGCGGAC

[0143] GCTCTTCTTCCTGACGGAGGACGGCACGCTGAGGCCGATCGCCATCGAGC

[0144] TGACGAGGCCCAAGTCCCCCAACACGCCGCAGTGGCGCCAGGTCTTCACG

[0145] CCGGGCTCCAGCGCGCGGCGTCCTGGCTGTGGCAGCTCGCCAAAACGCA

[0146] CGTCCTCGCCCACGACACCGGCTACCACCAGCTCGTCAGCCACTGGCTGA

[0147] GGACGCACTGCTGCGTGGAGCCGTACGTGATCGCGGCGAACCGGCGGCT

[0148] GAGCCAGATGCACCCCATCTACCGACTGCTGCACCCGCACTTCCGCTTCA

[0149] CCATGGAGATCAACGCCCAAGCGCGCGGGATGCTCATCAACGCCAATGG

[0150] AATCATCGAGAGCGCCTTCGCGCCGGGGAAGCTCTGCATGGAGCTCAGCT

[0151] CGGCGGTTTACGACAAGTTTTGGAGGTTCGACATGGAGGCTCTGCCCGCC

[0152] GATCTCATCCGGAGGGGCATGGCGATCGAATGCGAGGATGGCAAGCTGG

[0153] AGCTGACGATAGAGGACTACCCGTACGCCAACGACGGCCTGCTCATCTGG

[0154] GACTCCATCAAGGAGTGGGTGTCGGATTATGTGAACCATTACTACCAGTT

[0155] GGCTTCAGACATCCACATGGACAAGGAGCTCCAGGGTTGGTGGAACGAG

[0156] GTGCGAACCAAGGGCCACCCGGACAAGGAGGAAGGGTGGCCAGAGCTGA

[0157] ACTGCCACGGGAGCCTCGTCGAGGTTCTGACCACCATCATCTGGGTCGCG

[0158] TCGGGGCACCATGCGGCGGTGAACTTTGGCCAGTACCCCTACGCCGGCTA

[0159] CTTCCCCAATCGCCCCACCATCGCCCGGCGGAACATGCCGACGGAGGGGC

[0160] AGGCGTGCAGTCACGACGGCATGCAGCCAACGTTCGTTGAGGATCCCGTC

[0161] AGGGTGCTACTAGACACGTTCCCATCGCAGTACCAGACCACCCTCGTCCT

[0162] GCCGGTGCTCAACCTGCTATCGTCACACTCGCCCGGCGAGGAGTACATGG

[0163] GCACGCATGCGGAGTCAGCGTGGATGGCGGACAGGGAGGTCAGGGCGGC

[0164] GTTCGGGAGGTTCAACGAGAGGATGATGAGCATCGCGGAGATGATCGAC

[0165] TGCCGGAACAAGGATCCGGAGCGAAAGAACCGGCAGGGCCCCGGCGTGG

[0166] TGCCGTACGTGCTGCTCAAGCCGTCCTACGTGACCCTAAGGACATGACGTCCGTGATGGAGATGGGTATCCCCAACAGCATCTCAATTTGA(SEQ ID NO.:2)

[0167] The polypeptide encoded by the CMR2 gene

[0168] As used herein, the terms "polypeptide of the present invention" and "protein encoded by the CMR2 gene" are used interchangeably and refer to a polypeptide derived from CMR2 in plants (e.g., rice), or a polypeptide of CMR2 and its variants. In a preferred embodiment, a typical amino acid sequence of the polypeptide of the present invention is shown in SEQ ID NO.:1.

[0169] This invention relates to a CMR2 polypeptide and its variants for improving plant agronomic traits. In a preferred embodiment of the invention, the amino acid sequence of the polypeptide is shown in SEQ ID NO.:1. The polypeptide of the present invention can effectively improve the traits of plants (such as rice), for example, by enhancing the plant's resistance to moths, such as rice leaf rollers.

[0170] The present invention also includes polypeptides or proteins having the same or similar functions and having 50% or more (preferably 60%, 70%, 80%, more preferably 90%, more preferably 95%, most preferably 98%, such as 99%) homology to the sequence shown in SEQ ID NO.:1 of the present invention.

[0171] The "same or similar functions" mainly refer to: "regulating the traits of plants or crops (such as rice), such as enhancing the plant's resistance to moths, such as rice leaf rollers."

[0172] The polypeptides of the present invention can be recombinant polypeptides, natural polypeptides, or synthetic polypeptides. The polypeptides of the present invention can be naturally purified products, chemically synthesized products, or produced from prokaryotic or eukaryotic hosts (e.g., bacteria, yeast, higher plants, insects, and mammalian cells) using recombinant technology. Depending on the host used in the recombinant production protocol, the polypeptides of the present invention can be glycosylated or non-glycosylated. The polypeptides of the present invention may or may not include an initial methionine residue.

[0173] This invention also includes plant-derived CMR2 protein fragments and analogs having the protein activity of the invention. As used herein, the terms “fragment” and “analyte” refer to polypeptides that substantially retain the same biological function or activity as the native CMR2 protein of the invention.

[0174] The polypeptide fragments, derivatives, or analogs of the present invention may be: (i) polypeptides in which one or more conserved or non-conserved amino acid residues (preferably conserved amino acid residues) are substituted, and such substituted amino acid residues may or may not be encoded by the genetic code; or (ii) polypeptides having substituent groups in one or more amino acid residues; or (iii) polypeptides formed by fusing a mature polypeptide with another compound (e.g., a compound that extends the half-life of the polypeptide, such as polyethylene glycol); or (iv) polypeptides formed by fusing an additional amino acid sequence to this polypeptide sequence (e.g., a leader sequence or secretion sequence or a sequence used to purify this polypeptide or a proteogen sequence, or a fusion protein). These fragments, derivatives, and analogs, as defined herein, are within the scope well known to those skilled in the art.

[0175] In this invention, the polypeptide variant is a derived sequence obtained by substituting, deleting, or adding at least one amino acid to the amino acid sequence shown in SEQ ID NO.:1, and by adding one or more amino acids (usually 1-60, preferably 1-30, more preferably 1-20, most preferably 1-10) at the C-terminus and / or N-terminus. For example, in the protein, substitution with amino acids of similar or comparable properties generally does not change the protein's function, and adding one or more amino acids to the C-terminus and / or N-terminus generally does not change the protein's function either. These conserved variations are preferably generated by substitutions according to Table I.

[0176] Table I

[0177]

[0178]

[0179] This invention also includes analogs of the claimed protein. These analogs may differ from the natural SEQ ID NO.:1 by differences in amino acid sequence, by differences in modifications that do not affect the sequence, or by both. These protein analogs include natural or induced genetic variants. Induced variants can be obtained by various techniques, such as random mutagenesis through radiation or exposure to a mutagen, or by site-directed mutagenesis or other known biochemical techniques. Analogs also include those having residues different from the natural L-amino acid (e.g., D-amino acids), and those having non-naturally occurring or synthetic amino acids (e.g., β, γ-amino acids). It should be understood that the proteins of this invention are not limited to the representative proteins exemplified above.

[0180] Modifications (which typically do not alter the primary structure) include chemically derived forms of proteins, such as acetate or carboxylation, either in vivo or in vitro. Modifications also include glycosylation, such as those occurring during protein synthesis and processing. This modification can be accomplished by exposing the protein to glycosylating enzymes, such as mammalian glycosylation or deglycosylation enzymes. Modifications also include sequences containing phosphorylated amino acid residues, such as phosphotyrosine, phosphotyserine, and phosphotythreonine.

[0181] In a preferred embodiment, the amino acid sequence of CMR2 (OsCMR2) is as follows:

[0182] MLTATQTLAPAVLSRSHGAPSSFSSQPRRTAAAASRVSCTRVGALSEVVNGELVVGDQEQ

[0183] TTDDLLTRHKNVVADYTLSATVTVSLKQDDSTPQKVADMVNRDWLFLDFFSSHIEGMHTE

[0184] PQLARYSHMDGKGSFIYEASFSIPSSLDAVGAVQVVNRYSSEVYISDIDVHLCGGRHQWT

[0185] DITFHCNSWIDYNPNDQRFFFPLKSYLPSQTPRGVKNLRKEELRAIRGDGRGERKEWERI

[0186] YDYDVYNDLGDPDNDPATRRPVLGGRGRPYPRRCRTGRRRCRTDPSSESPPAKDGAGIYV

[0187] PRDEAFTERKAGAFATKKALSALSAFTTAQRVSGDRRRGFPSLAAIDALYEDGYKNRPSS

[0188] SQQEADNLEGYFREVLQKQVKLLLKGEKEEFKEELRKVFKFQTPEIHDKDKLAWFRDEEF

[0189] ARQTLAGMNPLSIQLVRDTDFPIFSKLDEETYGPGDSLITKELIEEQINGVMTAEEAVEK

[0190] KKLFMLDYHDVLLPFVHAVRELDDTTLYASRTLFFLTEDGTLRPIAIELTRPKSPNTPQW

[0191] RQVFTPGSSVAASWLWQLAKTHVLAHDTGYHQLVSHWLRTHCCVEPYVIAANRRLSQMHP

[0192] IYRLLHPHFRFTMEINAQARGMLINANGIIESAFAPGKLCMELSSAVYDKFWRFDMEALP

[0193] ADLIRRGMAIECEDGKLELTIEDYPYANDGLLIWDSIKEWVSDYVNHYYQLASDIHMDKE

[0194] LQGWWNEVRTKGHPDKEEGWPELNCHGSLVEVLTTIIWVASGHHAAVNFGQYPYAGYFPN

[0195] RPTIARRNMPTEGQACSHDGMQPTFVEDPVRVLLDTFPSQYQTTLVLPVLNLLSSHSPGE

[0196] EYMGTHAESAWMADREVRAAFGRFNERMMSIAEMIDCRNKDPERKNRQGPGVVPYVLLKP

[0197] SYGDPKDMTSVMEMGIPNSISI(SEQ ID NO.1)

[0198] Expression vector

[0199] The present invention also relates to vectors containing the polynucleotides of the present invention, host cells generated by genetic engineering using the vectors of the present invention or the coding sequences of mutant proteins of the present invention, and methods for generating the polypeptides of the present invention via recombinant technology.

[0200] Using conventional recombinant DNA technology, the polynucleotide sequence of this invention can be used to express or produce the protein or its variants described herein. Generally, the following steps are involved:

[0201] (1) Transform or transduce suitable host cells with a polynucleotide encoding the protein of the present invention or a variant thereof, or with a recombinant expression vector containing the polynucleotide;

[0202] (2) Host cells cultured in a suitable culture medium;

[0203] (3) Isolate and purify proteins from culture media or cells.

[0204] This invention also provides a recombinant vector comprising the gene of this invention. As a preferred embodiment, the recombinant vector contains a multiple cloning site or at least one restriction enzyme site downstream of the promoter. When it is necessary to express the target gene of this invention, the target gene is ligated into a suitable multiple cloning site or restriction enzyme site, thereby operatively linking the target gene to the promoter. As another preferred embodiment, the recombinant vector comprises (from 5' to 3' direction): a promoter, a target gene, and a terminator. If desired, the recombinant vector may further comprise 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 fluorescent proteins, etc.); an enhancer; or an operator.

[0205] In this invention, a polynucleotide sequence encoding a protein 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 such as adenoviruses, retroviruses, or other vectors well-known in the art. Any plasmid and vector can be used as long as it can replicate and remain stable within the host. An important characteristic of expression vectors is that they typically contain an origin of replication, a promoter, a marker gene, and translational control elements.

[0206] Methods well known to those skilled in the art can be used to construct expression vectors containing the protein-coding DNA sequence of the present invention and suitable transcription / translation control signals. 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 the present invention, any type of enhancing, constitutive, tissue-specific, or inducible promoter can be added before its transcription initiation nucleotide.

[0207] The DNA sequence can be efficiently ligated to an appropriate promoter in the expression vector to direct mRNA synthesis. Representative examples of these promoters include: the *E. coli* lac or trp promoter; the *λ* phage PL promoter; eukaryotic promoters including the CMV immediate early promoter, the HSV thymidine kinase promoter, early and late SV40 promoters, retroviral LTRs, and other known promoters that control gene expression in prokaryotic or eukaryotic cells or their viruses. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.

[0208] Vectors including the genes, expression cassettes, or other components of this invention can be used to transform suitable host cells to enable the host to express proteins. Host cells can be prokaryotic cells, such as *Escherichia coli*, *Streptomyces*, or *Agrobacterium*; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as plant cells. Those skilled in the art will understand how to select appropriate vectors and host cells. Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art. When the host is a prokaryote (such as *E. coli*), competent cells capable of uptake DNA can be harvested after the exponential growth phase and treated with CaCl2, the steps of which are well known in the art. Another method is to use MgCl2. If desired, transformation can also be performed using electroporation. When the host is a eukaryote, DNA transfection methods such as calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, and liposome packaging can be used.

[0209] Transformed plants can also be achieved using methods such as Agrobacterium-mediated transformation or gene gun transformation, including leaf disc transformation, embryo transformation, and flower bud soaking. Transformed plant cells, tissues, or organs can be regenerated into new plants using conventional methods, thus obtaining transgenic plants.

[0210] In addition, the expression vector preferably contains one or more selective marker genes to provide phenotypic traits for selecting host cells for transformation, such as dihydrofolate reductase, neomycin resistance, and green fluorescent protein (GFP) for eukaryotic cell culture, or tetracycline or ampicillin resistance for Escherichia coli.

[0211] Vectors containing the appropriate DNA sequence and appropriate promoter or control sequence can be used to transform appropriate host cells so that they can express proteins.

[0212] The host cell can be a prokaryotic cell, such as a bacterial cell; a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell. Representative examples include: Escherichia coli, Streptomyces; Salmonella typhimurium bacterial cells; fungal cells such as yeast; and plant cells (such as rice cells).

[0213] When the polynucleotides of this invention are expressed in higher eukaryotic cells, the insertion of an enhancer sequence into the vector will enhance transcription. Enhancers are cis-acting factors of DNA, typically approximately 10 to 300 base pairs, that act on the promoter to enhance gene transcription. Examples include the SV40 enhancer (100 to 270 base pairs) located late on the replication origin side, the polyoma enhancer located late on the replication origin side, and adenovirus enhancers.

[0214] Those skilled in the art are well aware of how to select appropriate vectors, promoters, enhancers, and host cells.

[0215] The obtained transformants can be cultured using conventional methods to express the polypeptide encoded by the gene of this invention. Depending on the host cells used, the culture medium can be selected from various conventional media. Culture is carried out under conditions suitable for host cell growth. Once the host cells have grown to an appropriate cell density, the selected promoter is induced using a suitable method (such as temperature adjustment or chemical induction), and the cells are cultured for a further period.

[0216] The proteins described in this invention can be expressed intracellularly, on the cell membrane, or secreted extracellularly. If desired, recombinant proteins can be separated and purified using various separation methods based on their physical, chemical, and other properties. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional refolding treatment, treatment with protein precipitants (salting out), centrifugation, permeation, ultrafiltration, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high-performance liquid chromatography (HPLC), and various other liquid chromatography techniques, as well as combinations of these methods.

[0217] Improve plant traits

[0218] In this invention, a method for improving plant traits is also provided, specifically, promoting or increasing the expression of the CMR2 gene or its encoded protein, thereby improving plant traits, including enhanced plant resistance to pyralid moths.

[0219] The main advantages of this invention include:

[0220] (1) This invention is the first to discover that increasing the expression level or activity of the CMR2 gene or its encoded protein in the plant (such as rice) can improve the agronomic traits of the plant, such as enhancing the plant's resistance to moths.

[0221] (2) This invention explores the potential application of the OsCMR2 gene in regulating rice resistance to the rice leaf folder. Experiments confirmed that the OsCMR2 gene is associated with rice resistance to the rice leaf folder. After knocking out the OsCMR2 gene using CRISPR / Cas9, the mutant rice showed significantly reduced resistance to the rice leaf folder compared to the wild type. This result indicates that the OsCMR2 gene and its encoded protein can be used to regulate rice resistance to the rice leaf folder. Further research into the mechanism of action of OsCMR2 is expected to provide new molecular targets for the genetic improvement of rice insect resistance, and to provide theoretical basis and technical support for building an efficient and green pest management system.

[0222] (3) This invention aims to solve the technical problems of scarce resistance gene resources and unclear resistance mechanisms in existing rice varieties resistant to rice leaf folder. In the prior art, the number of insect-resistant rice varieties is limited, and there is a lack of effective resistance genes, leading farmers to rely on chemical pesticides to combat rice leaf folder, increasing agricultural environmental pollution and production costs. Through this study, we have for the first time identified the key regulatory role of the OsCMR2 gene and its encoded protein in the process of rice resistance to rice leaf folder, filling a research gap in this field. Experiments have confirmed that knockout of the OsCMR2 gene leads to a significant increase in the body weight of rice leaf folder, further demonstrating its positive regulatory role in insect resistance. Compared with the prior art, this invention not only provides new insect-resistant gene resources but also provides clear targets for the genetic improvement of insect-resistant rice varieties, with significant economic benefits and environmental protection value. It can effectively reduce dependence on chemical pesticides and promote the development of sustainable agriculture.

[0223] (4) The advantage of this invention is that it is possible to pre-assess the potential resistance of candidate plant varieties to rice leaf rollers in the laboratory alone. Since the entire life cycle of crops such as rice is usually one year or six months, it is necessary to examine their biological traits and phenotypes through field planting under normal circumstances, which would inevitably take up a lot of time and consume huge land and labor costs. In contrast, the gene identification scheme can be completed in the laboratory. Gene sequencing can be carried out in a short period of time, such as a few weeks of seedling stage, or even just seed sequencing. The efficiency of the examination is greatly improved, which will inevitably save a lot of time, space and labor costs, and has significant economic significance.

[0224] 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, unless otherwise specified, are generally performed under conventional conditions as described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise specified, all materials and reagents used in the embodiments are commercially available products.

[0225] Example 1: Identification of OsCMR2 gene knockout mutants

[0226] The CMR2 mutant was purchased from Baige Gene. Upon receiving the seeds, we identified them as homozygous for subsequent experiments.

[0227] To investigate the effect of OsCMR2 gene knockout on rice resistance to rice leaf folder, we used the coding region sequence of the rice gene OsCMR2 (SEQ ID NO.2) as the sgRNA target and constructed a gene editing vector according to the instructions of the CRISPR / Cas vector construction kit (Baige Gene, catalog number: BG101389E10). The sgRNA was GGACCGACGACCTCCTTACG (SEQ ID NO.3).

[0228] Subsequently, TPS method was used to extract DNA from rice leaves for gene identification (referencing a simplified method for extracting genomic DNA from rice leaves, Jin Su-kui, Guo Qian-qian, Liu Qiao-quan, Gao Ji-ping, 2024). OsCMR2_seq_F and OsCMR2_seq_R were used as amplification primers (Table 1). Phanta high-fidelity enzyme (Novizan, catalog number P525) was used to amplify the target gene (SEQ ID NO. 2) fragment of transgenic rice (ZH11 background, obtained from the Center for Excellence in Molecular Plant Sciences, Chinese Academy of Sciences), followed by bioengineering sequencing. The transgenic rice obtained through the above methods was identified at the genome level as the rice OsCMR2 gene knockout mutant OsCMR2 (…). Figure 1 ).

[0229] Table 1. Primers used in the examples

[0230]

[0231]

[0232] Example 2: Determination of resistance to rice seedling feeding by rice leaf roller larvae

[0233] To investigate the effect of OsCMR2 gene knockout on insect resistance in rice, we planted wild-type (ZH11) and OsCMR2 mutant rice seedlings in a greenhouse (14h / 10h light / dark, 28℃, 65% humidity). Each group was planted in six small square pots (11cm*11cm), with 20 rice seedlings in each pot. When the seedlings reached 12 days old, we selected 30 early second instar rice leaf roller larvae of uniform growth (collected from the Songjiang experimental field in Shanghai, and reared indoors). The larvae were evenly inoculated onto the surface of rice seedling leaves and placed in a well-ventilated and well-lit rearing cage at 25℃, with 16 hours of light followed by 8 hours of darkness. On day 6, each larva was individually weighed and its weight recorded. The weight data were analyzed and plotted using GraphPad Prism 8. The results showed that, compared with wild-type rice (ZH11), the body weight of rice leaf folder larvae after feeding on the mutant OsCMR2 was significantly increased, indicating that the mutant OsCMR2 had significantly reduced resistance to rice leaf folder. Figure 2 ).

[0234] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. The use of a CMR2 gene or its encoded protein, or a promoter thereof, characterized in that, Used to improve the agronomic traits of plants, or to prepare a composition or formulation for improving the agronomic traits of plants, including enhancing the plant's resistance to pyralid moths.

2. The use as described in claim 1, characterized in that, The insects of the family Pyralidae include the genus *Rhizoctonia solani*.

3. The use as described in claim 1, characterized in that, The term "pyral moths" refers to major pests of rice, including the rice leaf roller, rice stem borer, rice caltrop, and rice bark borer.

4. The use as described in claim 1, characterized in that, The CMR2 gene or its encoded protein is derived from one or more plants selected from the group consisting of rice, wheat, corn, barley, oats, rye, sorghum, and millet, with rice being the preferred variety.

5. The use as described in claim 1, characterized in that, The CMR2 gene includes the rice CMR2 gene (OsCMR2, accession number: XP_015620637).

6. A composition, characterized in that, include: (a) The CMR2 gene or its encoded protein, or its promoters; (b) An agriculturally acceptable carrier.

7. Use of the composition according to claim 6, characterized in that, Used to enhance plant resistance to pyralid moths.

8. A method for improving the agronomic traits of a plant, comprising the steps of: promoting or increasing the expression level and / or activity of the CMR2 gene or its encoded protein in the plant, thereby improving the agronomic traits of the plant; wherein the improved agronomic traits of the plant include enhancing the plant's resistance to pyralid moths.

9. A method for preparing genetically engineered plant tissues or plant cells, characterized in that, Including the following steps: Increase the expression level and / or activity of the CMR2 gene or its encoded protein in plant tissues or plant cells to obtain genetically engineered plant tissues or plant cells.

10. A method for preparing plants with improved traits, characterized in that, Including the following steps: The genetically engineered plant tissues or plant cells prepared by the method described in claim 9 are regenerated into a plant body, thereby obtaining a plant with improved traits.

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