Gene arf12 for regulating plant resistance to brown planthopper and application thereof

Abstract

The application provides a gene ARF12 for regulating the resistance of plants to brown planthopper and application. An insect-resistant gene ARF12 is disclosed, and the gene can be used for preparing a transgenic plant with insect-resistant effect, or used as a target for screening insect-resistant plants in the breeding field of plants in the family Poaceae.

Description

Technical Field

[0001] This invention belongs to the fields of biotechnology and botany, and more specifically, this invention relates to the gene ARF12 that regulates plant resistance to brown planthopper and its applications. Background Technology

[0002] Rice is one of the world's most important food crops, and it is susceptible to various diseases and pests throughout its life cycle, severely impacting its quality and yield. Resistance breeding is an economical, effective, and environmentally friendly method to improve crop resistance; therefore, the discovery and mechanistic analysis of endogenous resistance-related genes in rice are particularly important. Transcription factors play a crucial role in plant immunity by relaying different upstream immune signals, helping plants respond rapidly to various biotic stresses.

[0003] Transcription factors participate in the defense response of rice against brown planthoppers by regulating the expression of downstream target genes. The MYB family transcription factor MYB30 enhances rice resistance to brown planthoppers by activating the expression of OsPAL6 / 8 in the phenylpropanoid metabolic pathway, thereby increasing salicylic acid and lignin content. MYB22, through its EAR motif, forms the MYB22-TOPLESS-HDAC1 transcriptional repressor complex with TOPLESS and HDAC1, synergistically inhibiting the transcription of the downstream F3'H gene, which negatively regulates brown planthopper resistance, thus enhancing rice resistance. The GRF family transcription factor GRF8 positively regulates rice resistance to brown planthoppers; the transgenic rice line GRF8OE, overexpressing OsGRF8, exhibits a brown planthopper-resistant phenotype. OsHLH61 and OsbHLH96 participate in regulating rice resistance to brown planthoppers by modulating the expression of the PR gene. OsWRKY46 and OsWRKY72 can transcribe and activate the expression of the OsRLCK281 and LOC_Os01g67364.1 genes to regulate brown planthopper resistance. The key transcription factor encoding OsEIL1 in the ethylene signaling pathway negatively regulates rice resistance to brown planthoppers by directly regulating OsLOX9.

[0004] Rice diseases and pests have always been important factors restricting rice yield and quality. The brown planthopper (Nilaparvata lugens (Stal)) is the most widespread and damaging pest in rice production, and also a major pest of rice in my country and many Southeast Asian countries. When feeding on rice, the brown planthopper uses its piercing-sucking mouthparts to penetrate the phloem and extract sap, causing the rice plants to lose water and nutrients, eventually leading to withering and death. In the field, this manifests as a typical characteristic "hopperburn" phenomenon (Watanabe and Kitagawa, 2000). In addition to direct damage, the brown planthopper also indirectly transmits plant viruses, such as grass dwarf disease (Grass dwarf disease). Brown planthoppers, including those caused by Stunt and Ragged Stunt viruses, damage rice production. Simultaneously, brown planthopper populations exhibit rapid adaptability, evolving new biotypes within a short period to overcome existing resistance and continue to harm rice. Therefore, given the severity of brown planthopper damage and its rapid adaptability, effective control of brown planthoppers is crucial for maintaining rice production.

[0005] The main methods for controlling brown planthoppers include chemical control, biological control, and resistance breeding. Both chemical and biological control methods have inherent problems that are difficult to avoid, which highlights the advantages of resistance breeding. Integrated pest management (IPM) is an environmentally friendly and long-term control mechanism from a macro-ecological perspective, effectively controlling pests and diseases while ensuring production and protecting the environment.

[0006] In summary, there is an urgent need in this field to further explore and utilize genetic resources effectively in order to find a long-term approach to integrated pest management. Summary of the Invention

[0007] The purpose of this invention is to provide a method for regulating the rice brown planthopper resistance gene ARF12 and its application.

[0008] In a first aspect of the invention, a method for improving the insect resistance (insect resistance ability) of grass plants is provided, comprising: introducing an exogenous ARF12 encoding gene into the plant; wherein the insects include Hemiptera insects.

[0009] In one or more embodiments, the method includes: introducing the coding gene for ARF12 into a plant using an expression vector carrying the coding gene for ARF12.

[0010] In one or more embodiments, the method includes:

[0011] (1) Provide Agrobacterium carrying an expression vector, wherein the expression vector contains the gene encoding ARF12;

[0012] (2) Contact plant cells, tissues or organs with Agrobacterium in step (1) to transfer the ARF12 encoding gene into the plant.

[0013] In one or more embodiments, the method further includes: (3) selecting plant cells, tissues or organs into which the polynucleotide has been transferred.

[0014] In one or more embodiments, the method further includes: (4) regenerating the plant cells, tissues or organs from step (3) into a plant.

[0015] In another aspect of the invention, a transgenic plant obtained by the method described above for improving the insect resistance of grass plants is provided.

[0016] In another aspect of the invention, the use of ARF12, its encoding gene, or an expression vector carrying the encoding gene is provided for improving the insect resistance of grasses; wherein the insects include Hemiptera.

[0017] In another aspect of the invention, the use of ARF12 or its encoding gene is provided for use as an analytical target for determining the insect resistance of grasses; wherein the insects include Hemiptera.

[0018] In another aspect of the present invention, a method for identifying the insect resistance ability of grass plants is provided, comprising: detecting the expression of ARF12 in the test plant; if the expression level of ARF12 in the test plant is higher than (preferably statistically higher, such as more than 20% higher; more preferably more than 50% higher; more preferably more than 80% higher) the expression level of the protein in the insect-sensitive plant; then the plant is an insect-resistant plant.

[0019] In one or more embodiments, the method for identifying the insect resistance of grass plants involves detecting the expression of endogenous ARF12.

[0020] In one or more embodiments, the method for identifying the insect resistance of grass plants involves sequence analysis (e.g., by PCR amplification) using primers specifically targeting the ARF12 coding gene or genomic gene to determine the expression status of ARF12.

[0021] In one or more embodiments, the ARF12 is selected from the group consisting of: (a) a protein with the amino acid sequence shown in SEQ ID NO:2; (b) a protein derived from (a) having an insect-inhibiting or insect-repelling function, formed by substituting, deleting, or adding one or more (e.g., 1-20; more preferably 1-10; more preferably 1-5; more preferably 1-3) amino acid residues of the amino acid sequence shown in SEQ ID NO:2; (c) a protein derived from (a) having a sequence similarity of 70% or more (preferably 80% or more; more preferably 85% or more; more preferably 90% or more; more preferably 95% or more; more preferably 98% or more; more preferably 99% or more) to the amino acid sequence shown in SEQ ID NO:2 and having an insect-inhibiting or insect-repelling function.

[0022] In one or more embodiments, the nucleotide sequence of the gene encoding ARF12 is as shown in SEQ ID NO:1, or a degenerate sequence thereof.

[0023] In one or more embodiments, the Hemiptera includes insects of the family Delphacidae.

[0024] In one or more embodiments, the planthoppers (Delphacidae) include (but are not limited to): brown planthopper (Nilaparvata lugens), white-backed planthopper (Sogatella furcifera), or gray planthopper (Laodelphaxstriatellus).

[0025] In another aspect of the invention, a method for screening substances (potential substances) that enhance plant insect resistance is provided, comprising:

[0026] (1) Add the candidate substance to the system expressing ARF12;

[0027] (2) Detect the system and observe the expression or activity of ARF12 therein. If its expression or activity is increased (statistically significant increase, such as an increase of 10%, 20%, 40%, 60%, 80%, 90% or higher), it indicates that the candidate substance is a substance that can be used to enhance the insect resistance of plants.

[0028] In one or more embodiments, the method further includes: setting up a control group in which no candidate substance is added, thereby clearly distinguishing the difference between ARF12 expression or activity in the test group and the control group.

[0029] In one or more embodiments, the candidate substances include (but are not limited to): regulatory molecules (such as upregulators, downregulators, small molecule compound gene editing constructs, etc.) designed for ARF12 or its encoding gene or its upstream or downstream proteins or genes.

[0030] Other aspects of the invention will be apparent to those skilled in the art from the disclosure herein. Attached Figure Description

[0031] Figure 1 Changes in OsARF12 expression levels after feeding by brown planthoppers;

[0032] Error bars represent positive and negative standard errors (n=3); t-tests are used for data analysis, ** indicates p<0.01, * indicates p<0.05, and all comparisons are compared to 0h.

[0033] Figure 2 Identification of brown planthopper resistance in ARF12KO plants;

[0034] (a) Schematic diagram of the OsARF12 genome editing sites in two lines of ARF12KO plants;

[0035] (b) Identification of resistance to brown planthopper in individual plants of ARF12KO-1 and ZH11;

[0036] (c) Identification of resistance to brown planthopper in individual plants of ARF12KO-2 and ZH11;

[0037] (d) Identification of resistance to brown planthopper in small populations of ARF12KO-1 and ZH11 plants;

[0038] (e) Survival statistics of small groups of ARF12KO-1 and ZH11 against brown planthopper, with error bars representing positive and negative standard errors (n=3);

[0039] (f) Identification of resistance to brown planthopper in small populations of ARF12KO-2 and ZH11 plants;

[0040] (g) Survival statistics of small populations of ARF12KO-2 and ZH11 plants against brown planthopper, with error bars representing positive and negative standard errors (n=3).

[0041] The t-test is used to analyze differences in data. An asterisk indicates a significant difference compared to the wild type, and ** indicates p < 0.01.

[0042] Figure 3 Identification of brown planthopper resistance in ARF12KOk plants;

[0043] (a) Schematic diagram of the OsARF12 genome editing sites in two lines of the ARF12KOk plant;

[0044] (b) Identification of resistance to brown planthopper in two lines of ARF12KOk and in individual Kasalath plants.

[0045] Figure 4 Identification of brown planthopper resistance in ARF12OE plants;

[0046] (a) Expression levels of OsARF12 in two lines of ARF12OE and ZH11 plants. Error bars represent positive and negative standard errors (n=3).

[0047] (b) Identification of resistance to brown planthopper in individual plants of ARF12OE and ZH11;

[0048] (c) Identification of resistance to brown planthopper in small populations of ARF12OE-1 and ZH11 plants;

[0049] (d) Survival statistics of small populations of ARF12OE-1 and ZH11 plants against brown planthopper, with error bars representing positive and negative standard errors (n=3);

[0050] (e) Identification of resistance to brown planthopper in small populations of ARF12OE-2 and ZH11 plants;

[0051] (f) Survival statistics of small populations of ARF12OE-2 and ZH11 plants against brown planthopper, with error bars representing positive and negative standard errors (n=3);

[0052] The t-test is used to analyze differences in data. An asterisk indicates a significant difference compared to the wild type, ** indicates p < 0.01, and * indicates p < 0.05.

[0053] Figure 5 Identification of resistance to brown planthoppers in ARF12OEn and wild-type plants. Detailed Implementation

[0054] This invention discloses an insect-resistant gene, ARF12, which exhibits resistance to harmful insects. This gene can be used to prepare transgenic plants with insect-resistant properties, or as a target for screening insect-resistant plants in the field of grass breeding.

[0055] The ARF12 gene is a member of the ARF transcription factor gene family. The inventors prepared OsARF12 knockout and overexpression materials in rice and conducted basic research, discovering that ARF12 plays an important role in the defense of gramineous plants against brown planthopper infestation. Based on this, the invention was completed.

[0056] In rice, there are 25 amino acid-releasing factor (ARF) transcription factors, which play important roles in regulating the growth and development of roots, leaves, flowers, fruits, and seeds. Several ARF transcription factors have been shown to be involved in drought and salt stress responses. Nine ARF genes—OsARF2, OsARF4, OsARF10, OsARF14, OsARF16, OsARF18, OsARF19, OsARF22, and OsARF23—have been reported to respond to drought stress. In rice, the expression of OsARF11 and OsARF15 differs under salt stress conditions, indicating their response to salt stress. Furthermore, some ARF genes have been found to be associated with cold and heat stress. OsARF4, OsARF14, OsARF18, and OsARF19 are induced by cold stress, while OsARF11, OsARF13, and OsARF16 are induced by heat stress.

[0057] Studies have found that OsARF12 and OsARF16 positively regulate rice resistance to rice black-streaked dwarf virus (RSV), while OsARF5 and OsARF11 negatively regulate it. The RSV SP8 and P8 proteins specifically interact with the CTD of OsARF17, preventing its dimerization and inhibiting its activity as a transcription factor. The RSV P2 protein interacts with OsARF17, affecting its DNA binding ability.

[0058] However, plant resistance mechanisms are diverse, involving numerous signaling pathways and a complex array of genes, with different resistance mechanisms for different types of infestation. Although ARF12 has been associated with the resistance mechanism of grasses to black-streaked dwarf virus, its role against other types of infestation (such as insects) remains unclear. Furthermore, the types of insects that infest plants are numerous and complex, and the signaling pathways and genes involved in insect resistance vary among different insects.

[0059] the term

[0060] As used herein, "plant (crop)" refers to a plant containing ARF12 or its homologs; preferably, the plant includes, but is not limited to, plants of the Poaceae family, Brassicaceae family, Solanaceae family, Euphorbiaceae family, etc. More preferably, the plant is a Poaceae plant. Even more preferably, the plant is a crop (such as a food crop or cash crop). For example, the "plant" includes, but is not limited to, rice, sorghum, corn, barley, wheat, oats, rye, etc. In embodiments of the present invention, plants of the Poaceae family are exemplified.

[0061] As used herein, "insect" refers to any insect that can be killed or suppressed by the ARF12 gene of this invention or by the killing method based on this invention. Preferably, it refers to insects of the order Hemiptera, more preferably insects of the family Planthopper, and especially brown planthoppers.

[0062] As used herein, "insect-sensitive plants" refers to plants that have no or low resistance to insects and are easily infected. In methods for identifying plants with resistance to a particular insect, insect-sensitive plants are generally selected from plants of the same genus as the plant to be identified, or wild-type plants of the same species. When it is necessary to identify whether a plant to be tested has an anti-inhibitory effect, the general or average expression level of ARF12 protein in insect-sensitive plants of the same genus, type, or species as the plant to be tested is used as the "threshold" for judgment. If the expression level is higher than this "threshold," the plant to be tested can be considered to have the ability to inhibit insects.

[0063] As used in this article, "exogenous" or "heterogeneous" refers to the relationship between two or more nucleic acid or protein sequences from different sources. For example, if the combination of a promoter and a target gene sequence is not naturally occurring, then the promoter is exogenous to the target gene. A particular sequence is "exogenous" to the cell or organism into which it is inserted.

[0064] ARF12 and its encoding gene

[0065] The ARF12 protein of the present invention can be a recombinant protein, a natural protein, or a synthetic protein, preferably a recombinant protein. The protein of the present invention can be a naturally purified product, a chemically synthesized product, or produced from a prokaryotic or eukaryotic host (e.g., plant, bacteria, yeast, insect cells) using recombinant technology.

[0066] This invention also includes fragments, derivatives, and analogs of the ARF12 protein (peptide). As used herein, the terms “fragment,” “derivative,” and “analyte” refer to proteins that substantially retain the same biological function or activity as the ARF12 protein of this invention. The protein fragments, derivatives, or analogs of this invention may be (i) proteins with one or more conserved or non-conserved amino acid residues (preferably conserved amino acid residues) substituted, and such substituted amino acid residues may or may not be encoded by the genetic code; or (ii) proteins having substituent groups in one or more amino acid residues; or (iii) proteins formed by fusing an additional amino acid sequence to this protein sequence (such as a leader sequence or secretory sequence, or a sequence used to purify this protein, or a proteomic 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.

[0067] Any bioactive fragment of the ARF12 protein can be used in this invention. Here, a bioactive fragment of the ARF12 protein means a protein that retains all or part of the function of the full-length ARF12 protein. Typically, the bioactive fragment retains at least 50% of the activity of the full-length ARF12 protein. Under more preferred conditions, the bioactive fragment can retain 60%, 70%, 80%, 90%, 95%, 99%, or 100% of the activity of the full-length ARF12 protein.

[0068] In this invention, the term "ARF12 protein" refers to the protein of the sequence SEQ ID NO:2 or a variant thereof with the same function. These variants 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 the addition or deletion 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. For example, in the art, substitution with amino acids of similar or comparable properties generally does not alter the function of the protein. Similarly, adding or removing one or more amino acids at the C-terminus and / or N-terminus generally does not alter the function of the protein. The term also includes active fragments and active derivatives of the ARF12 protein.

[0069] Protein variants include: homologous sequences, conserved variants, allelic variants, naturally occurring mutants, induced mutants, and proteins encoded by DNA that hybridizes with ARF12 protein DNA under high or low stringency conditions. This invention also provides other proteins, such as fusion proteins comprising ARF12 protein or fragments thereof. In addition to nearly full-length proteins, this invention also includes soluble fragments of the ARF12 protein. Typically, this fragment has at least about 20 consecutive amino acids of the ARF12 protein sequence, typically at least about 30 consecutive amino acids, preferably at least about 50 consecutive amino acids, more preferably at least about 80 consecutive amino acids, and most preferably at least about 100 consecutive amino acids.

[0070] This invention also provides analogs of the ARF12 protein. These analogs may differ from the natural ARF12 protein in amino acid sequence, in form of modification that does not affect the sequence, or both. These proteins include natural or induced genetic variants. It should be understood that the proteins of this invention are not limited to the representative proteins exemplified above.

[0071] In this invention, "ARF12 protein conserved variant protein" refers to a protein in which up to 20, more preferably up to 10, more preferably up to 5, and most preferably up to 3 amino acids are replaced by amino acids with similar or related properties compared to the amino acid sequence of SEQ ID NO:2.

[0072] When the ARF12 gene described in this invention is expressed in plants, the plants will have an antimicrobial effect against harmful insects (such as rice planthoppers). Furthermore, in plants that have expressed a large amount of this gene, the survival rate of harmful insects can be significantly reduced, or they may migrate or die. Therefore, based on this target gene, efforts can be made to induce its expression in plants at large quantities or on a timed basis, thereby effectively resisting the damage caused by harmful insects.

[0073] The present invention also provides a polynucleotide encoding the ARF12 gene of the present invention or a conserved variant thereof.

[0074] The polynucleotides of this invention can be in DNA or RNA form. DNA form includes cDNA, genomic DNA, or artificially synthesized DNA. The DNA 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 ARF12 protein can be identical to or a degenerate variant of the coding region sequence shown in SEQ ID NO:1.

[0075] The term "polynucleotide encoding a protein" can include a polynucleotide that encodes the protein, or it can include a polynucleotide that also includes additional coding and / or non-coding sequences.

[0076] This invention also relates to variants of the aforementioned polynucleotides that encode proteins or protein fragments, analogs, and derivatives 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 protein it encodes.

[0077] 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. Particularly, polynucleotides that hybridize with the polynucleotides described in this invention under stringent conditions.

[0078] It should be understood that although the ARF12 gene of the present invention is preferably obtained from rice, other genes or proteins (homologous genes or homologous proteins) obtained from other plants that are highly homologous to the ARF12 gene or protein (e.g., having more than 80%, such as 85%, 90%, 95%, or even 98% sequence identity) are also within the scope of the present invention. Methods and tools for comparing sequence identity are also well known in the art, such as BLAST.

[0079] The full-length nucleotide sequence or fragment thereof encoding the ARF12 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 cDNA libraries or cDNA libraries prepared according to conventional methods known to those skilled in the art as templates. When the sequence is long, it is often necessary to perform two or more PCR amplifications, and then splice the fragments amplified from each amplification in the correct order.

[0080] Currently, the DNA sequence encoding the protein of this 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 this invention through chemical synthesis.

[0081] In this invention, the ARF12 gene can be inserted into a recombinant expression vector. "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 within the host. The ARF12 DNA sequence can be efficiently ligated to an appropriate promoter in the expression vector to guide mRNA synthesis. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.

[0082] 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.

[0083] This invention also provides a method for improving plants, which includes increasing the expression of ARF12 in plants. The improved plants include those with enhanced insect resistance. Knowing the function of ARF12, various methods well-known to those skilled in the art can be used to increase its expression. For example, an expression unit carrying the ARF12 gene (such as an expression vector or virus) can be delivered to a target site via methods known to those skilled in the art, thereby enabling the expression of active ARF12.

[0084] application

[0085] The inventors have discovered that the ARF12 gene plays an important role in plants, and that introducing it into plants for recombinant expression will lead to the plants' insecticidal effects.

[0086] Based on the above new findings, the ARF12 protein or its encoding gene of the present invention has multiple uses, including but not limited to: for suppressing insects; for preparing insect-resistant plants (transgenic plants); and for use as a molecular marker to determine the ability of plants to resist insects.

[0087] Therefore, based on this target gene, a variety of transgenic vectors can be designed, such as transgenic plants that express large amounts, express at specific times, or induce expression, thereby improving the plant's insect resistance without affecting yield.

[0088] The present invention also relates to a method for improving the insect resistance of plants, the method comprising transferring a polynucleotide encoding the ARF12 protein of the present invention or a conserved variant thereof into a plant, thereby giving the plant a superior insect resistance.

[0089] As a preferred embodiment of the present invention, the method for transferring a polynucleotide encoding the ARF12 protein of the present invention or its conserved variant into a plant (method for preparing transgenic plants) is as follows:

[0090] (1) Provide Agrobacterium carrying an expression vector, wherein the expression vector contains the DNA coding sequence of the ARF12 protein;

[0091] (2) Contact plant cells, tissues or organs with Agrobacterium in step (1) so that the ARF12 protein DNA coding sequence is transferred into the plant cells and integrated into the chromosomes of the plant cells;

[0092] (3) Select plant cells or tissues into which the ARF12 protein DNA coding sequence has been transferred; and

[0093] (4) Regenerate the plant cells or tissues from step (3) into plants.

[0094] This method can be implemented using any appropriate conventional means, including reagents, temperature, pressure conditions, etc.

[0095] Transgenic plants and their hybrid offspring obtained through the methods described above for improving plant insect resistance are also included in this invention.

[0096] This invention addresses the challenge of controlling harmful insects such as rice planthoppers by identifying an inherent antibiotic resistance gene in plants. Through transgenic technology or marker-assisted selection breeding, this gene can be transferred into plants, achieving broad-spectrum resistance to insects. The gene in this invention possesses antibiotic activity; therefore, plants overexpressing this gene can effectively resist the damage caused by harmful insects. The method is convenient, rapid, accurate, and pollution-free.

[0097] Furthermore, this invention relates to using ARF12 or its encoding gene as a tracking marker for the progeny of gene-transformed plants. This invention also relates to using ARF12 or its encoding gene as a molecular marker to identify plant insect resistance by detecting the expression of ARF12 in plants. When evaluating test plants, the expression level or mRNA level of ARF12 can be measured to determine whether the expression or mRNA level in the test plant is higher than the average level for this type of plant; if it is significantly higher, it has higher insect resistance.

[0098] Therefore, the present invention provides a method for specifically identifying the insect resistance of plants, comprising: identifying the expression of ARF12 in the plant to be tested; if the plant has high expression of ARF12, then it is an insect-resistant plant.

[0099] Those skilled in the art can employ any of the well-known or developing techniques to perform nucleic acid sequence analysis or protein analysis, 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, immunohistochemistry, etc.

[0100] The ability to identify plant insect resistance early in the planting process greatly facilitates plant breeding.

[0101] Having learned about the function and molecular mechanism of ARF12, targeted screening of plants can be conducted based on this understanding. This new discovery can also be used to screen for potential substances that can target and regulate plant insect resistance by modulating ARF12.

[0102] The present invention provides a method for screening potential substances to improve plant insect resistance, the method comprising: (1) treating an expression system expressing ARF12 with a candidate substance; and (2) detecting the expression or activity of ARF12 in the system; if the candidate substance statistically improves the expression or activity of ARF12, it indicates that the candidate substance is a potential substance to improve plant insect resistance.

[0103] 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.

[0104] Through large-scale screening, a class of potential substances that specifically regulate ARF12 or its upstream and downstream pathway-related genes can be obtained.

[0105] The invention will be better understood from the following examples. However, those skilled in the art will understand that the specific methods and results are merely for illustrating the invention and not for limiting it. Experimental methods in the following examples 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, Science Press, or according to the manufacturer's recommendations.

[0106] Materials and methods

[0107] plant materials

[0108] The rice material used in this embodiment of the invention is the japonica rice variety (Oryza sativa L. subsp. Japonica) Zhonghua 11.

[0109] The tested rice varieties were grown in fields in Shanghai during the summer using standard rice field planting and management methods; in the winter, they were grown in the greenhouse of the Center for Excellence in Molecular Plant Sciences, Chinese Academy of Sciences, Shanghai. The temperature was 29℃±1℃, the humidity was 50-70%±5%, and the light duration was 7:00-19:00.

[0110] Methods for identifying resistance of single rice plants to brown planthopper

[0111] 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 mesh covering the top.

[0112] During identification, approximately 15 third-instar nymphs were placed in each enclosure. The number of nymphs was counted again the following day. Under normal growth and management conditions, the survival status of the seedlings was observed after 5-8 days.

[0113] Methods for identifying resistance to brown planthopper in small rice colonies

[0114] After the rice seeds germinated, they were planted in blue plastic boxes with 8 seedlings per row, for a total of 5 rows. The control group and the experimental group were planted symmetrically.

[0115] When the rice plants reach the two-leaf-one-heart stage, inoculate them with 10 brown planthoppers per plant. After inoculation, observe the growth of the rice plants daily, take photos and collect data, and set up 3 control groups.

[0116] Protein and coding gene sequences

[0117] OSARF12 gene (SEQ ID NO:1):

[0118]

[0119] OsARF12 protein (SEQ ID NO: 2):

[0120] mssssaasigppqpppppappeeekkclnselwhacagplvclptvgtrvvyfpqghseqvaastnkeveghipnypnlpaqlicqlhdvtmhadvetdevyaqmtlqplnpqeqndaylpaemgimskqptnyfcktltasdtsthggfsvprraaervfppldftqqppaqeliardihdiewkfrhifrgqpkrhllttgwsvfvsakrlvagdsvlfiwneknqlllgirrasrpqtvmpssvlssdsmhigllaaaahaaatnsrftifynpraspsefviplskyikavfhtrisvgmrfrmlfeteessvrrymgtitevsdadpvrwpssywrsvkvgwdestagerpprvslweieplttfpmypslfplrvkhpwysgvaslhddsnalmwlrgvageggfqslnfqspgigswgqqrlhpsllssdhdqyqavvaaaaasqsggylkqqflhlqqpmqspqehcnlnpllqqqilqqasqqqiinpdaqniqtmlspsaiqqqlqqlqqmqqvqndqkqkiqpdqsyqvptsavlpsptslpshlrekfgfsdpnansssfitssssdnmldssflqgsskavdlsrfnqpvaseqqqqqqqawkqkfmgsqsvsfggsvlhnsptskdgsvenkigrdvqnqslfspqvdsssllynmvpnltsnvsdgnlstipsgstylqnamygclddssgllqntgendpatrtfvkvyksgsvgrslditrfsnyaelreelgqmfgikgqlddpdrsgwqlvfvdrendvlllgddpwesfvnsvwyikilspedvhkmgkqgndpryls

[0121] Example 1. Expression of OsARF12 in response to brown planthopper feeding

[0122] Analysis showed that the OsARF12 (auxin response factor) gene encodes an auxin transcription factor.

[0123] To investigate whether OsARF12 plays a role in rice resistance to brown planthopper, wild-type rice materials were selected at different time points (1, 4, 8, 12, 24, and 48 h) after feeding by brown planthopper, and the changes in OsARF12 expression were detected.

[0124] Test results as follows Figure 1 As shown, OsARF12 expression was upregulated after feeding by brown planthoppers, indicating that OsARF12 responds to feeding by brown planthoppers.

[0125] Example 2. Knocking out OsARF12 reduces rice resistance to brown planthopper.

[0126] To investigate the function of OsARF12 in regulating rice resistance to brown planthopper, OsARF12 knockout (KO) lines were prepared and their genome-level analysis was performed.

[0127] 1. Gene editing using ZH11 as a background

[0128] OsARF12 gene-edited materials: OsARF12KO was prepared using CRISPR / Cas9 technology against ZH11 as the background. Several independent OsARF12KO rice lines were obtained, including ARF12KO-1 and ARF12KO-2, in which different codons in the OsARF12 coding sequence were mutated.

[0129] Sequence analysis showed that ARF12KO-1 had a 5-base deletion in its coding sequence, while ARF12KO-2 had a 1-base T insertion in its coding sequence. Figure 2 a).

[0130] Individual resistance tests for brown planthoppers were performed on these two lines. After being fed by the same number of brown planthoppers, ARF12KO-1 and ARF12KO-2 withered and died earlier than the ZH11 wild-type plants. Figure 2 b, Figure 2 c).

[0131] Simultaneously, small-group resistance to brown planthoppers was assessed, and similarly, ARF12KO plants died earlier than wild-type ZH11. Figure 2 d, Figure 2 f).

[0132] Seedling survival rate statistics show that the survival rate of ARF12KO plants is significantly lower than that of wild-type plants. Figure 2 d, Figure 2 e, Figure 2 f, Figure 2 g).

[0133] The above results indicate that knocking out OsARF12 in plants significantly reduces the resistance of rice to brown planthoppers.

[0134] 2. Gene editing using Kasalath as a background

[0135] OsARF12 gene-edited material with Kasalath background: OsARF12KO was prepared using CRISPR / Cas9 technology. Several independent OsARF12KO rice lines were obtained. To distinguish them from edited plants with ZH11 background, the lines used for subsequent validation were named ARF12KOk-1 and ARF12KOk-2, respectively.

[0136] Genome-level analysis of ARF12KOk revealed that the ARF12KOk-1 strain had an A base inserted into its coding sequence, while the ARF12KOk-2 strain had a C base inserted into its coding sequence. Figure 3 a).

[0137] Individual resistance to brown planthoppers was assessed in two lines, ARF12KOk-1 and ARF12KOk-2. The results showed that, compared to the wild-type Kasalath plants, ARF12KOk-1 and ARF12KOk-2 plants withered and died earlier. Figure 3 b).

[0138] The results of the above studies on rice with different genetic backgrounds show that, in both the ZH11 and Kasalath genetic backgrounds, knocking out OsARF12 reduces the resistance of rice to brown planthopper.

[0139] Therefore, it is shown that OsARF12 plays a key role in rice resistance to brown planthopper.

[0140] Example 3. OsARF12 overexpression enhances rice resistance to brown planthopper.

[0141] 1. Gene editing using ZH11 as a background

[0142] Using ZH11 as a background, the coding sequence of OsARF12 was inserted after the 35S promoter of pCambia2300:35S to prepare the recombinant vector pCambia2300-OsARF12. The constructed recombinant vector was transformed into ZH11 using Agrobacterium tumefaciens to obtain transgenic lines. The overexpression materials were designated ARF12OE-1 and ARF12OE-2. Figure 4 a).

[0143] One-month-old plants with uniform growth were selected for individual plant insect resistance assessment. After the same number of brown planthoppers fed on the plants for seven days, the insect resistance was analyzed.

[0144] The results showed that when the wild-type ZH11 plants withered and died, the ARF12OE-1 and ARF12OE-2 plants still grew well. Figure 4 b).

[0145] Simultaneously, small-group insect resistance assessments were conducted, and similarly, wild-type plants died earlier than ARF12OE plants (ARF12OE-1 and ARF12OE-2). Figure 4 c, Figure 4 e).

[0146] Seedling survival rate statistics show that the survival rate of ARF12OE plants is significantly higher than that of wild-type plants. Figure 4 d, Figure 4 f).

[0147] The results above demonstrate that overexpression of OsARF12 in plants significantly enhances the resistance of rice to brown planthopper.

[0148] 2. Gene editing using Nipponbare as a background

[0149] OsARF12 overexpression materials with Nipponbare (NIP) as the background were prepared using the same method as the previously mentioned ZH11 background lines, except that the background rice variety was replaced with NIP. To distinguish them from the ZH11 background plants, the obtained transgenic lines were named ARF12OEn-1 and ARF12OEn-2, respectively.

[0150] Individual resistance to brown planthoppers was assessed in two lines, ARF12OEn-1 and ARF12OEn-2. The results showed that while wild-type NIP plants withered and died, ARF12OEn-1 and ARF12OEn-2 plants continued to grow well. Figure 5 ).

[0151] The results above indicate that overexpression of OsARF12 in plants enhances rice resistance to brown planthoppers under both ZH11 and NIP backgrounds.

[0152] The results repeatedly confirmed by gene editing and overexpression indicate that OsARF12 is a gene that positively regulates rice resistance to brown planthopper and has application value in rice breeding optimization.

[0153] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are 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 modifications and improvements 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.

Claims

1. A method for improving the insect resistance of grasses, comprising: The exogenous ARF12 coding gene is introduced into plants; wherein the insects include Hemiptera.

2. The method as described in claim 1, characterized in that, The method includes: introducing the coding gene for ARF12 into a plant using an expression vector carrying the coding gene for ARF12.

3. The method as described in claim 2, characterized in that, The method includes: (1) Provide Agrobacterium carrying an expression vector, wherein the expression vector contains the gene encoding ARF12; (2) Contact plant cells, tissues or organs with Agrobacterium in step (1) to transfer the ARF12 encoding gene into the plant.

4. The use of ARF12, its encoding gene, or expression vectors carrying this encoding gene, to improve the insect resistance of grasses; among which, The insects mentioned include Hemiptera.

5. The use of ARF12 or its encoding gene as an analytical target for assessing the insect resistance of grasses; among which, The insects mentioned include Hemiptera.

6. A method for identifying the insect resistance of grasses, comprising: The expression of ARF12 in the test plant was detected; if the expression level of ARF12 in the test plant was higher than that in the insect-resistant plant, then the plant was an insect-resistant plant.

7. The method for identifying the insect resistance of grasses as described in claim 6, characterized in that, Sequence analysis was performed using primers specifically targeting the ARF12 coding gene or genomic genes to determine the expression status of ARF12.

8. The method as described in any one of claims 1 to 3 and 6 to 7, or the use as described in any one of claims 4 to 5, characterized in that, The ARF12 mentioned above is selected from the following group: (a) A protein with the amino acid sequence shown in SEQ ID NO:2; (b) A protein derived from (a) that has the function of inhibiting or repelling insects, formed by substituting, deleting or adding one or more amino acid residues of the amino acid sequence shown in SEQ ID NO:

2. (c) A protein derived from (a) that has more than 70% sequence similarity to the amino acid sequence shown in SEQ ID NO:2 and has the function of inhibiting or repelling insects.

9. The method as described in any one of claims 1 to 3 and 6 to 7, or the use as described in any one of claims 4 to 5, characterized in that, The nucleotide sequence of the gene encoding ARF12 is shown in SEQ ID NO:1, or is a degenerate sequence thereof.

10. The method as described in any one of claims 1 to 3 and 6 to 7, or the use as described in any one of claims 4 to 5, characterized in that, The Hemiptera insects mentioned include those of the family Delphacidae. Preferably, the planthoppers (Delphacidae) include: brown planthopper (Nilaparvatalugens), white-backed planthopper (Sogatella furcifera), or gray planthopper (Laodelphax striatellus).

11. A method for screening substances that enhance plant resistance to insects, comprising: (1) Add the candidate substance to the system expressing ARF12; (2) Detect the system and observe the expression or activity of ARF12. If its expression or activity is increased, it indicates that the candidate substance can be used to enhance the insect resistance of plants.

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