Plant rl1 gene regulates broad-spectrum disease resistance and application

Abstract

The present application relates to plant RL1 gene regulation broad spectrum disease resistance and application. The present application provides a RL1 down-regulation agent for use in improving plant disease resistance, or for preparing a preparation for enhancing plant disease resistance. The present application also provides a method for improving traits of a plant in the family Poaceae or preparing a plant in the family Poaceae with improved traits, comprising: down-regulating the expression or activity of RL1 in the plant; the improved traits include: improving the disease resistance of the plant. The present application also provides the use of RL1 as a molecular marker for identifying plant disease resistance, or as a molecular marker for targeted screening of plants with disease resistance, and related identification methods, screening methods, etc.

Description

Technical Field

[0001] This invention belongs to the fields of botany and genetic engineering. More specifically, this invention relates to the regulation of broad-spectrum disease resistance by the RL1 gene in plants and its applications. Background Technology

[0002] Rice is an important food crop in my country, and its high and stable yields are of great strategic significance for maintaining national food security. During its natural growth process, rice is susceptible to pathogen infection, which can significantly reduce yields and even lead to complete crop failure. Therefore, the prevention and control of rice diseases is crucial for maintaining food security.

[0003] Currently, there are two main methods for controlling rice diseases: chemical control, which uses chemically synthesized fungicides to prevent pathogens, and disease-resistant breeding, which clones disease-resistant genes (such as rice blast and bacterial blight) and applies them to field production to solve the disease problem. Disease-resistant breeding is more economical, environmentally friendly, and efficient than chemical control.

[0004] Therefore, this field needs to study the disease-resistant genes in rice and use disease-resistant breeding to solve the problem of rice diseases. Summary of the Invention

[0005] The purpose of this invention is to provide the regulation of broad-spectrum disease resistance by the RL1 gene in plants and its applications.

[0006] In a first aspect, the present invention provides the use of an RL1 downregulator for improving plant disease resistance or for preparing formulations that enhance plant disease resistance.

[0007] In one or more embodiments, the RL1 downregulator includes: an agent that knocks out or silences RL1, or an agent that inhibits RL1 activity.

[0008] In one or more embodiments, the RL1 downregulator comprises: an agent that induces a loss-of-function mutation in RL1; or, an interfering molecule that specifically interferes with the expression of the gene encoding RL1.

[0009] In one or more embodiments, the reagent for performing loss-of-function mutations on RL1 includes: a mutagenic reagent for RL1, a CRISPR gene-editing reagent for RL1, a homologous recombination reagent, or a site-directed mutagenesis reagent.

[0010] A second aspect of the present invention provides a method for improving traits of grass plants or preparing trait-improved grass plants, comprising: downregulating the expression or activity of RL1 in the plant; wherein the improved trait includes: enhancing the plant's disease resistance.

[0011] In one or more embodiments, the downregulation of RL1 expression or activity in plants includes: reducing RL1 activity, and / or reducing RL1 stability, and / or downregulating RL1 expression, and / or reducing the effective duration of RL1 action, and / or inhibiting RL1 transcription and translation, and / or reducing histone H3K4me3 methylation.

[0012] In one or more embodiments, the downregulation includes: knocking out or silencing the gene encoding RL1 in a plant, inhibiting or silencing the expression of the RL1 protein, or inhibiting the activity of RL1.

[0013] In one or more embodiments, the downregulation includes: knocking out the coding gene of RL1 by gene editing with a CRISPR system through mutagenesis to inhibit or silence the expression of the RL1 protein; knocking out the coding gene of RL1 by homologous recombination; silencing RL1 by interfering molecules that specifically interfere with the expression of the coding gene of RL1; or performing a loss-of-function mutation on RL1 in plants containing RL1.

[0014] In one or more specific embodiments, inhibiting or silencing the expression of the RL1 protein by mutagenesis includes: inducing a T deletion mutation at position 1236 of the nucleic acid encoding the RL1 protein by mutagenesis, thereby inhibiting or silencing the expression of the RL1 protein.

[0015] In one or more embodiments, the RL1 protein comprises:

[0016] (a) A protein with the amino acid sequence shown in SEQ ID NO:2;

[0017] (b) A protein derived from (a) that has the function of the protein in (a) and is formed by substitution, deletion or addition of one or more amino acid residues of the amino acid sequence shown in SEQ ID NO:2.

[0018] (c) A protein whose amino acid sequence is more than 80% identical to the amino acid sequence defined in (a) and which has the function of the protein in (a); or

[0019] (d) A fragment of SEQ ID NO:2 that has the function of protein (a).

[0020] In one or more embodiments, the disease resistance is resistance to pathogenic microorganisms; preferably, the pathogenic microorganisms include bacteria; more preferably, the bacteria include bacterial blight pathogens.

[0021] In one or more embodiments, the bacterial blight pathogen races include, but are not limited to: PXO99A, PXO61, PXO71, PXO347, PXO112, ZHE173, YC19, FJ23, YN24, T1, T2, KACC10331, AH28, YC15, YC26, LYG50, YN04-5, GX4, JNXO, Oct-78 / 78-10, PXO86, LN2, JL1, LN1, and LN3.

[0022] In one or more embodiments, the improvement of plant disease resistance includes: reducing the length of plant lesions and reducing the number of plant lesions.

[0023] In one or more embodiments, the plant is a plant that expresses RL1 or its homologs.

[0024] In one or more embodiments, the plant is a cereal crop.

[0025] In one or more embodiments, the cereal crops include plants of the Poaceae family.

[0026] In one or more embodiments, the cereal crops include: rice, sorghum, corn, barley, wheat, oats, and rye.

[0027] A third aspect of the invention provides a plant cell, tissue, or organ containing an exogenous RL1 downregulator as described in any embodiment of the invention.

[0028] In a fourth aspect, the present invention provides the use of the RL1 protein or its gene as a molecular marker for identifying plant disease resistance, or as a molecular marker for targeted screening of disease-resistant plants.

[0029] In one or more embodiments, a plant is considered to have high disease resistance if the test plant has low or no expression of the RL1 protein or its gene.

[0030] In one or more embodiments, the RL1 is as described in any embodiment of the present invention; and / or, the plant is as described in any embodiment of the present invention; and / or, the disease resistance is as described in any embodiment of the present invention.

[0031] A fifth aspect of the present invention provides a method for targeted selection or identification of plants, comprising: identifying the expression of RL1 in a test plant; if the test plant has low or no expression of RL1, it is a plant with high disease resistance; if the test plant has high expression of RL1, it is a plant with low disease resistance.

[0032] In one or more embodiments, the RL1 is as described in any embodiment of the present invention; and / or, the plant is as described in any embodiment of the present invention; and / or, the disease resistance is as described in any embodiment of the present invention.

[0033] A sixth aspect of the present invention provides a method for screening substances that enhance plant disease resistance, comprising:

[0034] (1) Add the candidate substance to the system expressing RL1;

[0035] (2) Detect the system and observe the expression or activity of RL1. If its expression or activity decreases, it indicates that the candidate substance is a substance that can be used to enhance plant disease resistance.

[0036] In one or more embodiments, the RL1 is as described in any embodiment of the present invention; and / or, the plant is as described in any embodiment of the present invention; and / or, the disease resistance is as described in any embodiment of the present invention.

[0037] Other aspects of the invention will be apparent to those skilled in the art from the disclosure herein. Attached Figure Description

[0038] Figure 1 Image of pUN1301-OsRL1-GFP plasmid.

[0039] Figure 2 Map of the 1300-35s-OsRL1-GFP plasmid.

[0040] Figure 3 Photos of the mature growth and development of wild-type ZJ99, mutant df68, and replacement material OsRL1-OE / df68. Scale bar = 10cm.

[0041] Figure 4 Subcellular localization map of OsRL1. The full-length CDS of OsRL1 was tandemly linked with GFP fluorescent protein and transiently expressed in tobacco via Agrobacterium-mediated infection. H2B-mCherry indicates the cell nucleus, and PIP2-mCherry indicates the cell membrane. Scale bar = 10 μm.

[0042] Figure 5 OsRL1 gene mutation enhances resistance to bacterial blight in rice. (AB) Two-month-old wild-type ZJ99, OsRL1 gene mutant df68, and supplementary material OsRL1-OE / df68 were inoculated with bacterial blight pathogen PXO99A(OD) 600=1.0) Photographs of lesions at 14 days (A) and statistical graphs of lesion length (B), and statistical graphs of wild-type ZJ99 and mutant df68 after inoculation with multiple races of white leaf blight (C). Values ​​are mean ± SD, asterisks indicate significant differences (Student's t test, p < 0.0001, scale bar = 1 cm).

[0043] Figure 6 OsRL1 can maintain H3K4me3 levels. Total protein was extracted from two-week-old wild-type ZJ99 and mutant df68; Anti-H3K4me3, Anti-H 27 Kme3 and Anti-H 27 Kme3 was used to characterize histones H3, K4me3, and H, respectively. 27 Kme3, H 27 Kme3 level. H3: Histone and Ponceau S characterize the internal reference. Detailed Implementation

[0044] Through in-depth research, the inventors discovered that the rice material df68 with a mutation in the OsRL1 gene exhibits resistance to bacterial blight in rice. Sequencing analysis revealed that OsRL1 is a T deletion mutation at position 1236 of the CDS sequence in the coding frame LOC_Os07g31450. OsRL1 negatively regulates resistance to bacterial blight in plants; downregulation of OsRL1 increases plant resistance to this bacterial disease. OsRL1 encodes a chromatin remodeling factor protein (CHR4 / MI-2-LIKE), which is located in the cell nucleus, and OsRL1 plays a crucial role in maintaining the methylation level of histone H3K4me3. Therefore, mutation or downregulation of this gene can enhance plant resistance and can be used for disease-resistant breeding.

[0045] RL1 gene / protein and plants

[0046] In this invention, RL1 can be a protein having the sequence shown in SEQ ID NO:2. This invention also includes sequence variations having the same function as RL1. These variations include (but are not limited to): deletions, insertions, and / or substitutions of several amino acids (typically 1-20, preferably 1-10, and more preferably 1-8 or 1-5), and additions or deletions of one or more amino acids (e.g., up to 20, preferably up to 10, and more preferably up to 5) at the C-terminus and / or N-terminus. For example, substitution of one amino acid. Any protein with high homology to RL1 (e.g., 70% or higher; preferably 80% or higher; more preferably 90% or higher, such as 95%, 98%, or 99% homology) and retaining the same function / activity is also included in this invention.

[0047] In this invention, RL1 also includes its homologs, that is, proteins found in species other than rice that have high homology with the sequence described above (e.g., 70% or higher; preferably 80% or higher; more preferably 90% or higher, such as 95%, 98%, or 99% homology) and function the same or similar to RL1 in this invention. This invention also includes isolated bioactive fragments of RL1 that have the same or similar functions as RL1.

[0048] The present invention also includes polynucleotides (genes) encoding the polypeptide, such as polynucleotides of the nucleotide sequence shown in SEQ ID NO:1 or degenerate sequences thereof, polypeptides encoding RL1 of SEQ ID NO:1, and homologs (homologous genes) of proteins of that sequence. Since RL1 is conserved in many species, it should be understood that, although the OsRL1 gene presented in the embodiments of the present invention is preferred, the present invention is not limited to the gene specifically listed in the embodiments.

[0049] It should be understood that although the RL1 gene of the present invention is preferably obtained from rice, a grass of the Poaceae family, other genes obtained from other plants that are highly homologous to the rice RL1 gene (e.g., have more than 80%, such as 85%, 90%, 95%, or even 98% sequence identity) or genes that are degenerate with the said gene 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.

[0050] Vectors containing the said coding sequence, and host cells genetically engineered using the said vector or polypeptide coding sequence, are also included in this invention. Methods well known to those skilled in the art can be used to construct suitable expression vectors.

[0051] The host cell is usually a plant cell. Transformation of plants can generally be achieved using methods such as Agrobacterium-mediated transformation or gene gun transformation, for example, the leaf disc method or rice embryo transformation; Agrobacterium-mediated transformation is preferred. Transformed plant cells, tissues, or organs can be regenerated into plants using conventional methods, thereby obtaining plants with altered traits compared to the wild type.

[0052] As used herein, "plant" refers to a plant that expresses RL1 or its homologs. The plant may include monocotyledonous or polycotyledonous plants; preferably, the plant is a cereal crop; preferably, the cereal crop includes plants of the Poaceae family, such as rice (Oryza sativa), wheat (Triticum aestivum), and corn (Zea spp.). Examples of cereal crops include: rice, sorghum, corn, barley, wheat, oats, rye, etc. It should be understood that the plants applicable to the technical solutions of this invention are not limited to those listed above, and suitable plants can be determined by identifying the presence of RL1 or its homologs.

[0053] The term "pathogen" refers to "pathogenic microorganisms," which are microorganisms that infect plants and cause disease. For example, the pathogens include, but are not limited to, bacteria; the bacteria include, but are not limited to, bacterial blight pathogens. The bacterial blight pathogen races include, but are not limited to, PXO99A, PXO61, PXO71, PXO347, PXO112, ZHE173, YC19, FJ23, YN24, T1, T2, KACC10331, AH28, YC15, YC26, LYG50, YN04-5, GX4, JNXO, Oct-78 / 78-10, PXO86, LN2, JL1, LN1, and LN3.

[0054] Application of improved plants

[0055] Based on the inventor's new discovery, the present invention provides a method for improving plant traits or preparing trait-improved grass plants, comprising: downregulating the expression or activity of RL1 in the plant; the improved trait includes: enhancing the plant's disease resistance.

[0056] Based on the inventor's new discovery, the present invention provides a use of RL1 or its downregulator for improving the traits of grass plants or preparing trait-improved grass plants; the improved traits include: enhancing the plant's disease resistance.

[0057] Therefore, the present invention provides a method for making plants, especially plants that highly express (including overexpress) RL1, exhibit enhanced disease resistance, comprising: downregulating the expression or activity of RL1.

[0058] It should be understood that, once the function of RL1 is known, various methods well known to those skilled in the art can be used to regulate the expression or activity of RL1, such as reducing or eliminating RL1 expression. Any appropriate conventional means, including reagents, temperature, and pressure conditions, can be used to implement the method.

[0059] As one implementation method, a method for downregulating RL1 expression in plants is provided, comprising:

[0060] (1) The interfering molecule that interferes with RL1 expression is transferred into plant cells, tissues, organs or seeds with reproductive capacity to obtain plant cells, tissues, organs or seeds transformed with the interfering molecule;

[0061] (2) Regenerate plants from plant cells, tissues, organs or seeds that have been infused with the interfering molecules obtained in step (1).

[0062] In some preferred embodiments, the method further includes:

[0063] (3) Screening out plant cells, tissues, or organs that have been transformed with the vector; and

[0064] (4) Regenerate plants from the plant cells, tissues or organs in step (3).

[0065] As one implementation method, the RL1 protein can be mutagenized to inhibit or silence its expression. In one or more specific embodiments, inhibiting or silencing the expression of the RL1 protein by mutagenesis includes: inducing a T deletion mutation at position 1236 of the nucleic acid encoding the RL1 protein, thereby inhibiting or silencing the expression of the RL1 protein.

[0066] As another implementation method, gene editing is performed using the CRISPR / Cas system to knock out or downregulate target genes. Suitable sgRNA target sites result in higher gene editing efficiency; therefore, suitable target sites should be designed and identified before gene editing begins. After designing specific target sites, in vitro cell activity screening is required to obtain effective target sites for subsequent experiments. Gene editing reagents can efficiently target and mutate RL1, thereby achieving appropriate and effective downregulation (e.g., downregulation of 20–30%, 30–40%, 40–50%, 50–60%, 60–70%, 80–90%, or 90–100%).

[0067] This invention also provides substances for downregulating RL1, which improve plant traits by downregulating RL1. The substances can be downregulators, nucleic acid inhibitors, antagonists, blockers, or other agents that can downregulate the expression level of RL1. The biomolecules can be at the nucleic acid level (including DNA and RNA) or at the protein level.

[0068] The RL1 downregulators refer to any substance that can reduce RL1 activity, decrease RL1 stability, downregulate RL1 expression, reduce the effective duration of RL1 action, inhibit RL1 transcription and translation, or reduce histone H3K4me3 methylation. These substances can all be used in this invention as useful for downregulating RL1. They can be compounds, small chemical molecules, or biomolecules. The biomolecules can be at the nucleic acid level (including DNA and RNA) or at the protein level. For example, the downregulators are: mutagenic agents targeting RL1, interfering RNA molecules or antisense nucleotides that specifically interfere with RL1 expression, gene editing reagents that specifically edit RL1, etc.

[0069] This invention also provides expression vectors containing the aforementioned interfering molecules, preferably plant expression vectors; more preferably expression vectors suitable for subsequent transgenic operations (such as transgenic operations using Agrobacterium). Methods well known to those skilled in the art can be used to construct expression vectors containing the promoter and / or target gene sequence described in this invention. These methods include in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombination technology, etc. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.

[0070] This invention also provides genetically engineered host cells containing gene-editing molecules (such as CRISPR / Cas editing reagents), interference molecules, or silencing sequences, or containing vectors containing interference molecules or silencing sequences. The host cells are typically plant cells. Transformed plants can generally be achieved using methods such as Agrobacterium-mediated transformation or gene gun transformation, for example, leaf disc transformation or embryo transformation.

[0071] Plant-directed or targeted screening of regulatory molecules

[0072] Based on the inventors' new findings, this invention also relates to using RL1 as a tracking marker for the progeny of gene-transformed plants. Furthermore, this invention relates to using RL1 as a molecular marker to determine the disease resistance of plants at an early stage by detecting the expression or activity of RL1 in plants.

[0073] Therefore, the present invention provides a method for specifically identifying the disease resistance of plants, comprising: identifying RL1 in the plant to be tested; if the RL1 in the test plant is expressed at low levels or not expressed at all, then the plant is a disease-resistant plant.

[0074] 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.

[0075] Identifying plant disease resistance early in the planting process or even before planting greatly facilitates plant breeding.

[0076] After understanding the function and molecular mechanism of RL1, targeted screening of plants can be conducted. This new discovery can also be used to screen for potential substances that can target and regulate plant disease resistance by modulating RL1.

[0077] The present invention provides a method for screening potential substances to improve plant disease resistance, the method comprising: (1) treating an expression system expressing RL1 with a candidate substance; and (2) detecting the expression or activity of RL1 in the system; if the candidate substance statistically reduces the expression or activity of RL1, it indicates that the candidate substance is a potential substance to improve plant disease resistance.

[0078] The method of screening substances that act on a protein or gene or a specific region thereof as a target can 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 will understand how to select an appropriate screening method.

[0079] Various techniques can be used to identify gene transcription or expression in a system. These techniques include, but are not limited to, oligonucleotide hybridization (e.g., probes), polymerase chain reaction (PCR), and polyacrylamide gel electrophoresis. Protein expression or localization can be detected using various techniques, such as, but not limited to, immunoprecipitation, immunofluorescence, and Western blotting.

[0080] Through large-scale screening, a class of potential substances that specifically act on RL1 and regulate plant disease resistance can be obtained.

[0081] 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 or as recommended by the manufacturer.

[0082] Experimental Materials and Methods

[0083] 1. Experimental materials

[0084] 1.1 Rice (Oryza sativa) variety: ZJ99 (Zhejiang Japonica 99)

[0085] 1.2 Construction of mutants and transgenic plants

[0086] Df68 and OsRL1-GFP / df68. These rice materials are grown twice a year: summer at Songjiang Farm in Shanghai and winter at Lingshui Base in Hainan.

[0087] 1.3 Strains and Vectors

[0088] Escherichia coli strain: DH5α (preserved in our laboratory); Agrobacterium tumefaciens strain: EHA105 (Weidi, Shanghai), GV3101 (Weidi, Shanghai); Xanthomonas oryzae pv.oryzae: PXO99A, PXO61, PXO71, PXO347, PXO112, ZHE173, YC19, FJ23, YN24, T1, T2, KACC10331, AH28, YC15, YC26, LYG50, YN04-5, GX4, JNXO, Oct-78 / 78-10, PXO86, LN2, JL1, LN1, LN3.

[0089] pUN1301-UBI-GFP (rice overexpression vector); pCambia1300-35S-YFP (for transient expression in protoplasts and tobacco).

[0090] 1.4 Commonly Used Culture Medium Formulations

[0091] LB medium (1L): NaCl 10g, yeast extract 5g, Tryptone 10g, pH 7.0, and 15g agar powder should be added for solids.

[0092] PSA medium (1L): Peptone 10g, sucrose 10g, sodium glutamate 1g, pH 7.0, and 15g agar powder should be added for solids.

[0093] 1 / 2MS rice / Arabidopsis thaliana germination medium (1L): Murashige & Skoog Basal Salt Mixture (PhytoTech) 2.165g, pH 5.8. For solids, add 4.2g Phytagel (rice) or 6g agar powder (Arabidopsis thaliana).

[0094] AB liquid culture medium (1L): KH2PO4 3g, NaH2PO4 1g, NH4Cl 1g, MgSO4·7H2O 300mg, KCl 150mg, CaCl2 10mg; FeSO4·7H2O 2.5mg, Glucose 5g.

[0095] NBD medium (1L): NB Basal Medium (PhytoTech) 4.1g, sucrose 30g, 1ml 2,4-D solution (1000×), pH 5.8, and 4.5g Phytagel should be added for solids.

[0096] MS rice differentiation medium (1L): Murashige & Skoog Basal Medium with Vitamins (PhytoTech) 4.43g, sucrose 30g, 6-BA 3mg / L, NAA 0.5mg / L, pH 5.8, and 4.5g Phytagel should be added for solids.

[0097] 1 / 2MS rice rooting medium (1L): Murashige & Skoog Basal Medium with Vitamins (PhytoTech) 2.215g, sucrose 20g, pH 5.8, and 4.2g Phytagel should be added for solids.

[0098] 2. Construction of OsRL1 overexpression vector

[0099] 2.1 Extraction of RNA from rice leaves

[0100] RNA was extracted from rice using TRIzol (Thermofisher, 15596018). All procedures were performed using RNAnase-Free pipette tips.

[0101] Take 0.2g of the sample after it has been crushed by liquid nitrogen, add it to 1ml of RNA extraction solution TRIzol, and shake vigorously for 2min to mix.

[0102] Place at room temperature for 6 minutes, then at 4°C, 12000 rpm for 10 minutes.

[0103] Pipette 200 μl of supernatant into a new tube, add 200 μl of chloroform, and shake vigorously for 5 min; let stand at room temperature for 5 min to separate the layers, then incubate at 4 °C, 12000 rpm for 10 min.

[0104] Carefully transfer the upper aqueous phase to another centrifuge tube, add an equal volume of isopropanol, invert to mix, and let stand at room temperature for 10 min; then centrifuge at 4°C, 12000 rpm for 10 min, and discard the supernatant.

[0105] Add 1 ml of 75% ethanol to the centrifuge tube, wash the precipitate, 4°C, 7500 rpm, 5 min, and carefully discard the supernatant; 4°C, 12000 rpm, 1 min, and use a pipette tip to completely aspirate the remaining liquid.

[0106] Open the centrifuge tube cap and let it stand at room temperature for 5-10 minutes to dry the RNA. Add 25 μl of DEPC-treated water to dissolve the RNA and let it stand at room temperature for 10 minutes to fully dissolve the RNA.

[0107] RNA integrity and content were detected by electrophoresis. Simultaneously, 1 μl was used to determine RNA concentration and purity. RNA was stored at -80℃.

[0108] 2.2 Reverse transcription of RNA from rice leaves

[0109] mRNA reverse transcription was performed using the Invitrogen Reverse Transcription Kit. For detailed operating procedures, please refer to the Materials and Methods (SuperScript III first-strand synthesis system) section of the Invitrogen Reverse Transcription Kit. RNA was reverse transcribed into cDNA for gene cloning.

[0110] The specific steps are as follows:

[0111] Preparation of reaction system

[0112]

[0113] After mixing, incubate at 65°C for 5 minutes to denature; then immediately place on ice.

[0114] Preparation of cDNA synthesis reaction solution

[0115]

[0116]

[0117] Add 10 μl of cDNA synthesis reaction solution to the RNA / Oligo(dT) mixture from the previous step and mix gently.

[0118] Incubate the reaction at 50°C for 50 min, then terminate the reaction at 85°C for 5 min, and place on ice.

[0119] Add 1 μl RNase H, react at 37℃ for 30 min, and store at -20℃.

[0120] 2.3 Cloning of the OsRL1 gene in rice

[0121] PCR of the target gene was performed using reverse transcribed cDNA as a template. The gene was cloned by PCR using pUN1301-OsRL1-GFP-F and pUN1301-OsRL1-GFP-R. The following reaction system was prepared:

[0122]

[0123] Reaction steps: 95℃, 3 min; 95℃, 30 s; 58℃, 30 s; 68℃, 1 min; 32 cycles; 68℃, 5 min.

[0124] The primers are as follows:

[0125] pUN1301-OsRL1-GFP-F (SEQ ID NO:3):

[0126] GGTCGACTCTAGAGGATCcATGATGAAGGAGCGGAGC

[0127] pUN1301-OsRL1-GFP-R(SEQ ID NO:4):

[0128] gcccttgctcacggtaccATGCTCATCTGTGATCATCTCGT

[0129] 1300-OsRL1-GFP-F (SEQ ID NO:5):

[0130] cagggtacccggggatccATGATGAAGGAGCGGAGC

[0131] 1300-OsRL1-GFP-R(SEQ ID NO:6):

[0132] gctcaccatggtactagtATGCTCATCTGTGATCATCTCGT

[0133] 2.4 Linearization of the target vector by enzyme digestion

[0134] The pUN1301-GFP empty vector was digested with enzymes to prepare the enzyme digestion reaction system:

[0135]

[0136]

[0137] The reaction was carried out at 37℃ for 1 hour; incubation at 70℃ for 10 minutes resulted in inactivation. The completed PCR reaction system and enzyme digestion system were subjected to 1% agarose gel electrophoresis.

[0138] 2.5 DNA Agarose Recovery and Purification

[0139] DNA recovery and purification should be performed according to the recommended procedures for the Hligene Agarose Gel DNA Recovery Kit (Hlingene, NG202S):

[0140] PCR products were separated by 1% agarose gel electrophoresis. The target amplification band was cut out and placed in a 2ml centrifuge tube. 300μl of sol solution was added to each 0.1g gel.

[0141] Place at 65℃ for 10 minutes, mixing every 2-3 minutes.

[0142] After mixing, add the solution to the adsorption column, centrifuge at 12000 rpm for 30 s, and discard the waste liquid;

[0143] Add 700 μl of wash buffer WB, centrifuge at 12000 rpm for 1 min, and discard the waste liquid. Add another 600 μl of wash buffer WB, centrifuge at 12000 rpm for 1 min, and discard the waste liquid.

[0144] Centrifuge the empty column at 12000 rpm for 3 min, transfer the adsorption column to a new centrifuge tube, open the cap and let it stand for 2 min to evaporate the alcohol;

[0145] Add 30 μl of elution buffer EB preheated to 65 °C to the adsorption column, incubate at room temperature for 2 min, and centrifuge at 12000 rpm for 2 min.

[0146] 2.6 Target Fragment and Vector Recombination

[0147] Utilizing the principle of homologous recombination, using according to The reaction was performed using the IIOne Step Cloning Kit (Vazyme, Cat#C112-02), and the reaction system is as follows;

[0148]

[0149] Incubate at 37°C for 30 minutes to complete the recombination reaction.

[0150] 2.7 Escherichia coli transformation

[0151] The transformation steps using competent cells produced by strain DH5α (Shanghai Weidi) are as follows: Thaw competent E. coli cells stored at -80℃ on ice. Add the above recombinant product, mix gently, and incubate on ice for 30 min; heat shock at 42℃ for 60 s, then incubate on ice for 5 min; add 1 ml of liquid LB medium, and incubate at 37℃ for 1 h; plate onto selection plates containing Kans and incubate overnight. Pick single colonies for colony PCR identification, or after liquid culture for 16 h, extract plasmid DNA for restriction enzyme digestion or sequencing identification.

[0152] 2.8 Transformation of Agrobacterium EHA105

[0153] Transformation was performed using strain EHA105 (Shanghai Weidi). The culture was thawed on ice, and 1-2 μg of plasmid was added and gently mixed. The mixture was then incubated on ice for 5 min, flash-frozen in liquid nitrogen for 5 min, bathed in a 37°C water bath for 5 min, and then incubated on ice for 5 min. 800 μl of liquid YEP medium was added, and the culture was incubated at 28°C for 2 h. The culture was then plated onto selection plates containing the appropriate antibiotic and incubated upside down at 28°C for 2-3 days. Single colonies were picked, plasmids were extracted for identification, and the strain was stored at -80°C.

[0154] 3. Transformation of callus tissue in mature rice embryos

[0155] 3.1 Select df68 rice seeds

[0156] After shelling, soak in 75% ethanol for 60 seconds, rinse once with sterile water, then soak in 30% (v / v) NaClO3 and shake at 180 rpm for 30 minutes at room temperature. Rinse 5-6 times with sterile water until no obvious yellowing is observed. Blot dry the surface moisture with sterile filter paper, and sow the seeds on NBD medium to induce callus. After culturing at 26℃ for 14 days, the obtained callus tissue can be used for transgenic culture or subcultured on NBD medium, with subculture every 14 days.

[0157] 3.2 Preparation of Agrobacterium-mediated transformation culture

[0158] Agrobacterium strains stored at -80℃ were activated on plates. Single colonies were picked and placed in YEP liquid medium containing the appropriate antibiotic for secondary activation, and cultured overnight at 28℃ with shaking. 2 ml of the overnight culture was transferred to 20 ml of AB (20 mg / L Lfif + 50 mg / L Kan + 100 mg / L AS) liquid medium and incubated at 28℃ until OD (open-circuit retrieval). 600 = Approximately 0.5 (about 3 hours)

[0159] 3.3 Co-culture of rice callus and bacterial solution

[0160] OD of bacterial solution 600Centrifuge at 5000 rpm for 10 min and collect the bacterial cells; precipitate the bacterial cells by suspending them in AAM containing 100 mg / L AS until the bacterial solution OD600 = 0.4-0.6; soak the bacterial solution with rice callus for 20 min while shaking continuously; then, blot dry the bacterial solution and co-culture the callus tissue in the dark on sterile filter paper on NBD medium for 2-3 days.

[0161] 3.4 Screening for transformed callus

[0162] Blot the callus tissue dry with sterile filter paper and transfer it to a selection medium containing hygromycin to screen for resistant callus. Culture for approximately one month, changing the medium about every two weeks. Selection medium: First selection: NBD (0.4% Phytagel) + 100 mg / L Timentin + 30 mg / L Hyg; Second selection: NBD (0.4% Phytagel) + 100 mg / L Timentin + 40 mg / L Hyg; Third selection: NBD (0.4% Phytagel) + 100 mg / L Timentin + 50 mg / L Hyg.

[0163] 3.5 Differentiation

[0164] Selected rice callus was transferred to rice differentiation medium and cultured under light. The medium was changed approximately every 14 days (3-4 times) until the callus differentiated into seedlings.

[0165] 3.6 Rooting

[0166] Transfer the seedlings from the differentiation medium to the rooting medium to grow roots. After about 2-3 weeks, once the seedlings have grown roots, remove them, wash off the agar medium, and culture them in water for 5-7 days before transplanting them into the soil.

[0167] 4. Extraction of rice DNA

[0168] Take rice leaves (about 1-2 cm) and place them in a sampling tube. Add steel balls and 400 μl of TPS solution, and crush the leaves using a ball mill (frequency 60 Hz, time 30 s, 2 times). Incubate in a 65℃ water bath for 30 min.

[0169] Centrifuge at 3700 rpm for 10 min.

[0170] Take 120 μl of supernatant into a new PCR plate, add an equal volume of isopropanol, mix by pipetting, and precipitate at -20℃ for 30 min.

[0171] Centrifuge at 3700 rpm for 10 min.

[0172] Discard the supernatant, add 200 μl of 70% ethanol to the precipitate and wash the precipitate, then centrifuge at 3700 rpm for 10 min.

[0173] Discard the supernatant and allow the precipitate to dry at room temperature. After about 10 minutes, add 30 μl of deionized water to dissolve the precipitate.

[0174] 5. Extraction of total protein from rice and Western blot

[0175] 5.1 Total Protein Extraction

[0176] After the rice sample was crushed with liquid nitrogen, 100 μL of 5% SDS was added to the powder, vortexed to mix, and then centrifuged at 12,000 rpm for 10 min in a boiling water bath for 5 min. The supernatant was then transferred to a new centrifuge tube with 2×SDS loading buffer, boiled in a water bath for 5 min, and stored at -20°C.

[0177] 2×SDS loading buffer (50mM Tris-Cl, pH 8.0; 20% (v / v) glycerol; 4.6% SDS; 0.02% bromophenol blue; add 0.2M DTT before use).

[0178] 5.2 Western blot hybridization

[0179] 5.2.1 Preparation of PAGE gel

[0180] Refer to the instructions for the PAGE gel preparation kit (Promoton) and select an appropriate separating gel (12.5%, 10%, and 7.5%) based on the size of the target protein. Prepare the PAGE gel according to the instructions. Alternatively, use a continuous gradient precast gel (Tanon).

[0181] 5.2.2 Protein Electrophoresis: Use a Bio-Rad or Tanon protein electrophoresis system, adding an appropriate amount of electrophoresis buffer (3.03 g / L Tris base, 14.4 g / L Glycine, 1 g / L SDS) or pre-mixed gel electrophoresis solution. Load approximately 20 μl of protein extraction solution into each well, run at a constant voltage of 50 V for 30 min, until the protein loading buffer is compressed into a straight line, then adjust the voltage to 120 V and electrophoresis for 1-2 h. Stop electrophoresis according to the position of the target band.

[0182] 5.2.3 Transfer: First, activate the PVDF membrane: immerse in methanol for 10 seconds, wash in deionized water for 5 minutes, then immerse in transfer buffer (3.03 g / L Tris base, 14.4 g / L Glycine, 200 ml / L methanol) for at least 10 minutes. After protein electrophoresis, remove the PAGE gel and immerse it in transfer buffer for at least 10 minutes. Choose either semi-dry or wet transfer as needed. Semi-dry transfer: Follow the Bio-Rad semi-dry transfer unit method, from bottom to top: thick filter paper - membrane - gel - thick filter paper, wetting each layer with transfer buffer and removing air bubbles. Transfer at a constant voltage of 15V for 30-60 minutes. Wet transfer: Assemble correctly in the order of sponge - filter paper - PVDF membrane - gel - filter paper - sponge, with black as the negative electrode and white as the positive electrode. Transfer in an ice-water bath at a constant current of 180 mA for 1.5-3 hours.

[0183] 5.2.4 Blocking: After the transfer is complete, place the membrane in blocking buffer (5% skim milk powder dissolved in TBST) and incubate with shaking for 2 hours. TBST (20mM Tris-HCl, pH 7.5; 150mM NaCl; 0.05% (v / v) Tween 20).

[0184] 5.2.5 Primary antibody incubation: Dilute the corresponding antibody with TBST or Solution I (Toyobo, primary antibody enhancer) containing 1% skim milk powder at a certain ratio. After rinsing the blocked membrane in TBST for 2 min, place the membrane in a sealing bag, add the primary antibody, and incubate at low speed for 1 h or overnight at 4°C.

[0185] 5.2.6 Primary antibody rinsing: Rinse the membrane in TBST 5 times, 10 min each time.

[0186] 5.2.7 Secondary antibody incubation: Dilute the corresponding secondary antibody with TBST or Solution II (Toyobo, secondary antibody enhancer) containing 1% skim milk powder at a certain ratio, put the membrane in a sealed bag, add the secondary antibody, and incubate at 37°C with low-speed shaking for 1 hour.

[0187] 5.2.8 Secondary antibody rinsing: Rinse the membrane in TBST 5 times, 10 min each time.

[0188] 5.2.9 Development: Mix ECL Plus solution A and solution B in a 1:1 ratio (Tanon). Approximately 400 μl of developer is needed for each membrane. Place the membrane in a sealed bag, add the developer, remove any air bubbles, and scan with a CCD camera.

[0189] 6. White leaf blight inoculation

[0190] Strawberries of bacterial blight, including strains PXO99A, PXO61, PXO71, PXO347, PXO112, ZHE173, YC19, FJ23, YN24, T1, T2, KACC10331, AH28, YC15, YC26, LYG50, YN04-5, GX4, JNXO, Oct-78 / 78-10, PXO86, LN2, JL1, LN1, or LN3, stored at -80℃, were streaked onto PSA plates and incubated at 28℃ for 2-3 days. After single colonies emerged, a single colony was picked and transferred to PSA medium and shaken for 24 hours. 100 μl of the bacterial suspension was then plated and incubated at 28℃ for 2-3 days. The medium was washed with sterile water to collect the bacterial cells. The inoculum concentration for rice was determined by OD0.05. 600 =0.5-1.0. Rice plants that have grown for about 8 weeks can be used for inoculation. Inoculate with PXO99A, PXO61, PXO71, PXO347, PXO112, ZHE173, YC19, FJ23, YN24, T1, T2, KACC10331, AH28, YC15, YC26, LYG50, YN04-5, GX4, JNXO, Oct-78 / 78-10, PXO86, LN2, JL1, LN1, or LN3. Dip scissors in the bacterial solution and cut the tip of the rice leaf 1-2 cm diagonally downwards. Re-dip the scissors in the bacterial solution after every 3-5 leaves. Cut 3-4 leaves from each rice plant. Measure the length of the leaf lesions about 2 weeks after inoculation.

[0191] 7. Sequence

[0192] OsRL1 coding region sequence (SEQ ID NO:1)

[0193]

[0194] OsRL1 protein sequence (SEQ ID NO:2)

[0195]

[0196] Example 1: The OsRL1 gene and its relationship with phenotype

[0197] To uncover potential disease-resistant genes, the laboratory used EMS mutagenesis to screen for these genes in rice. In the early field trials, a mutant, df68, was discovered. Compared to the wild type, this mutant exhibited significantly reduced plant height and tillering, shorter and curled leaves with a white, powdery waxy coating. The inventors named this gene OsRL1 (RollingLeaf). Further sequencing analysis revealed that OsRL1 is a mutation of the rice leaf-rolling phenotype gene LOC_Os07g31450, with a T deletion mutation at position 1236 of its coding frame (CDS) sequence, resulting in an incomplete protein.

[0198] To verify that the mutant phenotype was indeed caused by a mutation in OsRL1, the inventors constructed genetic material of 35S::OsRL1 / df68. The results are as follows... Figure 3 As shown, reintroducing the OsRL1 gene into the mutant resulted in the reintroduction of both the reduced plant height phenotype and the growth and development phenotype. This indicates that OsRL1 is a gene that affects plant height and growth and development phenotypes in rice.

[0199] Example 2: Subcellular localization of the OsRL1 gene

[0200] Using the protein sequence of OsRL1 to predict its potential function, gene annotation revealed that OsRL1 is a potential chromatin remodeling factor (CHR4 / MI-2-LIKE). Previous reports indicate that chromatin remodeling factors typically function in the cell nucleus.

[0201] To verify the localization of the OsRL1 protein, the inventors used the full-length CDS of OsRL1 and GFP fluorescent protein tandemly to transiently express it in tobacco using Agrobacterium-mediated infection.

[0202] The results are as follows Figure 4 As shown, OsRL1-GFP and the nuclear marker H2B histone are well co-localized, but not with the cell membrane marker PIP2. This indicates that OSRL1 functions in the cell nucleus.

[0203] Example 3: OsRL1 negatively regulates rice bacterial blight resistance

[0204] To verify the relationship between OsRL1 and plant disease resistance, the inventors sowed wild-type ZJ99, mutant df68, and 35S::OsRL1-GFP rice seeds, and conducted resistance assessments for bacterial blight race (99A) two months after transplanting. Disease resistance assessments were conducted two weeks after inoculation.

[0205] The results are as follows Figure 5 As shown, the mutant exhibits stronger resistance to bacterial blight than the wild type. Reintroducing OsRL1 into the mutant significantly reduces the plant's resistance. Therefore, OsRL1 negatively regulates resistance to bacterial blight in rice, and downregulating OsRL1 can improve rice's resistance to the bacterial disease bacterial blight.

[0206] Example 4: OsRL1 can maintain the methylation level of histone H3K4me3.

[0207] Previous reports indicated that chromatin remodeling factors function by regulating histone methylation. To investigate whether OsRL1 functions through some form of histone methylation, the inventors extracted total protein from wild-type and mutant strains, using H3K4me3 and H3K... 27 ME3 and H3K 36 Histone-specific antibodies against me3 were used to detect different types of methylation levels. Results were as follows: Figure 6 As shown, the level of H3K4me3 was significantly reduced in the mutant, suggesting that OsRL1 functions by regulating the level of histone H3K4me3.

[0208] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this patent should be determined by the appended claims. Furthermore, all documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference.

Claims

1. The use of an RL1 downregulator for improving plant disease resistance or for preparing formulations that enhance plant disease resistance.

2. The use as described in claim 1, characterized in that, The RL1 downregulators include: reagents that knock out or silence RL1, and reagents that inhibit RL1 activity; Preferably, the RL1 downregulator comprises: a reagent that induces a loss-of-function mutation in RL1; or, an interfering molecule that specifically interferes with the expression of the gene encoding RL1; More preferably, the reagent for performing loss-of-function mutations on RL1 includes: mutagenic reagents targeting RL1, CRISPR gene editing reagents targeting RL1, homologous recombination reagents, or site-directed mutagenesis reagents.

3. A method for improving traits of grasses or preparing grasses with improved traits, comprising: Downregulate the expression or activity of RL1 in plants; the improved traits include: enhancing plant disease resistance.

4. The method as described in claim 3, characterized in that, The downregulation of RL1 expression or activity in plants includes: reducing RL1 activity, and / or reducing RL1 stability, and / or downregulating RL1 expression, and / or reducing the effective duration of RL1 action, and / or inhibiting RL1 transcription and translation, and / or reducing histone H3K4me3 methylation. Preferably, the downregulation includes: knocking out or silencing the gene encoding RL1 in plants, inhibiting or silencing the expression of RL1 protein, or inhibiting the activity of RL1; More preferably, the downregulation includes: knocking out the coding gene of RL1 by gene editing with a CRISPR system through mutagenesis to inhibit or silence the expression of the RL1 protein; knocking out the coding gene of RL1 by homologous recombination; silencing RL1 by interfering molecules that specifically interfere with the expression of the coding gene of RL1; or performing loss-of-function mutations on RL1 in plants containing RL1.

5. As described in any one of claims 1-4, characterized in that, The RL1 protein includes: (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 the protein in (a) and is formed by substitution, deletion or addition of one or more amino acid residues of the amino acid sequence shown in SEQ ID NO:

2. (c) A protein whose amino acid sequence is more than 80% identical to the amino acid sequence defined in (a) and which has the function of the protein in (a); or (d) A fragment of SEQ ID NO:2 that has the function of protein (a).

6. As described in any one of claims 1-5, characterized in that, The disease resistance refers to resistance to pathogenic microorganisms; preferably, the pathogenic microorganisms include bacteria; more preferably, the bacteria include bacterial blight pathogens; and / or, The improvement of plant disease resistance includes: reducing the length of plant lesions and reducing the number of plant lesions.

7. As described in any one of claims 1-6, characterized in that, The plant mentioned is a plant that expresses RL1 or its homologs; Preferably, the plant is a cereal crop; More preferably, the cereal crops include plants of the Poaceae family; More preferably, the cereal crops include: rice, sorghum, corn, barley, wheat, oats, and rye.

8. A plant cell, tissue, or organ containing an exogenous RL1 downregulator as described in claim 1 or 2.

9. The use of an RL1 protein or its gene as a molecular marker for identifying plant disease resistance, or as a molecular marker for targeted screening of disease-resistant plants; Preferably, the plant being tested has high disease resistance if its RL1 protein or its gene is expressed at low levels or not at all. More preferably, the RL1 is as described in claim 5; and / or, the plant is as described in claim 7; and / or, the disease resistance is as described in claim 6.

10. A method for targeted selection or identification of plants, comprising: Identify the expression of RL1 in the test plant; if the test plant has low or no RL1 expression, it is a plant with high disease resistance; if the test plant has high RL1 expression, it is a plant with low disease resistance. Preferably, the RL1 is as described in claim 5; and / or, the plant is as described in claim 7; and / or, the disease resistance is as described in claim 6.

11. A method for screening substances that enhance plant disease resistance, comprising: (1) Add the candidate substance to the system expressing RL1; (2) Detect the system and observe the expression or activity of RL1. If its expression or activity decreases, it indicates that the candidate substance is a substance that can be used to enhance plant disease resistance. Preferably, the RL1 is as described in claim 5; and / or, the plant is as described in claim 7; and / or, the disease resistance is as described in claim 6.

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