Rice disease resistance defense regulation gene ECDR1 and application thereof

Abstract

The invention discloses a rice disease resistance defense regulation gene ECDR1 as well as an encoding protein and application thereof. According to the invention, a rice disease spot-like mutant ecdr1 is taken as an experimental material, a rice disease resistance defense regulation gene ECDR1 is separated through strategies such as map-based cloning and transgene complementation, the nucleotide sequence of the gene is as shown in SEQ ID NO.1, and the sequence of a protein coded by the gene is as shown in SEQ ID NO.2. According to the invention, the biological function of the ECDR1 gene is preliminarily analyzed, and a theoretical basis is provided for clarification of a molecular mechanism of plant programmed cell death and defensive reaction; particularly, the gene mutation site is introduced into a plant through gene engineering or a conventional means, the disease resistance can be remarkably enhanced, and the gene mutation site has important application value in the aspect of cultivation of disease-resistant varieties of the plant.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to the isolation and application of the coding region of ECDR1, a gene regulating disease resistance and defense in rice. Background Technology

[0002] Rice is one of the most important food crops in my country and even the world. In my country, various diseases such as rice blast, sheath blight, and bacterial leaf blight occur year-round, seriously threatening high and stable rice yields. Breeding and cultivating disease-resistant rice varieties is the most economical, effective, and environmentally friendly measure for controlling rice diseases. Discovering and utilizing broad-spectrum resistance gene resources is an ideal way to improve the disease resistance of susceptible varieties. Broad-spectrum resistance gene resources include traditional resistance genes (R genes), defense-regulator genes (DR genes), and resistance QTLs (quantitative trait loci) that can resist two or more pathogens or multiple physiological races (pathogenic types) of the same pathogen. Compared with R genes, which usually provide complete resistance, DR genes usually provide partial resistance. This type of resistance often does not show obvious race specificity and is considered a broad-spectrum and durable resistance. Compared with QTLs, which usually provide minor resistance, DR genes are relatively easy to identify and clone. Therefore, the discovery and identification of DR genes have attracted much attention from rice pathologists and geneticists.

[0003] More than 50 broad-spectrum disease resistance DR genes have been identified and cloned in rice, including Bsr-d1, Bsr-k1, Spl11, Spl33, and OsBBI1. These genes encode transcription factors, E3 ubiquitin ligases, eEF1A-like proteins, kinases, and peroxidases, respectively, and positively or negatively regulate rice resistance to rice blast, bacterial leaf blight, or bacterial leaf streak. Lesion mimic genes are important members of the DR gene family. Their mutants spontaneously produce hypersensitive response (HR)-like necrotic spots on the plant when it is not infected by pathogens, mechanically damaged, or subjected to abiotic stress. These genes are important experimental materials for studying programmed cell death (PCD) and the molecular mechanisms of disease resistance in plants. Currently, mutants of more than 30 rice lesion-like genes have been reported to significantly resist rice blast or bacterial blight. Of particular note is the lesion-like gene RBL1, encoded by cytosine diphosphate diglyceride (CDP-DAG) synthase, cloned by Sha et al. (2023) (Genome editing of a rice CDP-DAG synthase confers multipathogen resistance, Nature, 618(7967):1017-1023.). This gene regulates PCD and immunity by controlling phosphatidylinositol synthesis. Its mutant rbl1 exhibits broad-spectrum resistance to rice blast and bacterial blight. Furthermore, a new gene, RBL1, enhancing broad-spectrum disease resistance and ensuring stable yield in crops was created through gene editing. Δ12 This provides a successful example for the breeding application of defense-regulating gene resources.

[0004] Although numerous rice DR genes have been identified and cloned to date, the molecular mechanisms by which they regulate plant PCD and disease resistance defense responses remain unclear. Discovering and isolating more DR genes with breeding potential, and deeply elucidating their biological functions and roles in PCD and defense response pathways, is not only crucial for clarifying the regulatory mechanisms of plant PCD and rice disease resistance, but also provides genetic resources and theoretical support for improving rice disease resistance through molecular methods. Summary of the Invention

[0005] To further elucidate the molecular mechanisms by which human vegetative-pathogen (HR) mediates cell death and disease resistance responses in plants, the inventors isolated and identified a novel rice lesion-like mutant from anther tissue culture progeny of rice breeding materials, naming it ecdr1 (enhanced cell death and disease resistance 1). This mutant exhibits a spotted leaf phenotype and enhanced resistance to rice blast and bacterial blight. The ECDR1 gene, encoding a protein with an unknown function containing a hyccin domain, was obtained through map-based cloning and transgenic complementation. This gene is constitutively expressed in all examined tissues and organs, and its encoded protein is localized to the cell membrane. After the ecdr1 mutant produces lesion-like spots, it induces a significant upregulation of the expression of defense-related genes, significantly enhancing resistance to pathogens.

[0006] Therefore, this application identifies a novel rice lesion mutant, ecdr1, and clones the target gene OsECDR1, which controls this trait. This gene encodes a protein containing a hyccin domain, and its mutation induces programmed cell death and significantly enhances resistance to fungal diseases (rice blast) and bacterial diseases (rice bacterial blight). This provides valuable genetic material for studying the formation mechanism of PCD and defense responses, as well as for breeding broad-spectrum and durable disease-resistant rice varieties.

[0007] In this regard, the present invention first provides a gene ECDR1 that regulates cell death and defense responses similar to allergic reactions, the nucleotide sequence of its coding region being the genome sequence shown in SEQ ID NO.1, or the CDS sequence from position 2043 to 3329 therein.

[0008] Another object of the present invention is to provide a protein encoded by the above-mentioned gene, the amino acid sequence of which is shown in SEQ ID NO.2.

[0009] A third objective of this invention is to provide a recombinant vector containing the aforementioned genes.

[0010] A fourth objective of this invention is to provide the use of the aforementioned genes, proteins, and recombinant vectors in improving the disease resistance of rice. Mutations in the aforementioned genes or proteins can regulate programmed cell death and disease defense responses in rice cells, and these genes or proteins can be used to improve the resistance of rice to rice blast and bacterial blight.

[0011] The fifth objective of this invention is to provide a method for breeding disease-resistant crop varieties using the aforementioned genes or proteins. By using rice materials carrying the ECDR1 mutant gene, and through hybridization, backcrossing, and marker-assisted selection, the mutant gene can be introduced into other rice materials, or the structure or expression of the gene can be altered through gene editing or RNAi interference, which can significantly improve the resistance of other rice materials or other crops to fungal and bacterial diseases.

[0012] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0013] This invention identifies a novel lesion-like mutant from anther tissue culture progeny of rice breeding materials and names it ecdr1. On day 22 post-sowing, sporadic reddish-brown spots first appear on the basal first leaf of the ecdr1 mutant, gradually spreading outwards. On day 25, reddish-brown spots begin to appear on the basal second leaf, and on day 28, these spots begin to appear on the third leaf, at which point the first leaf is completely dead. By the heading stage (85 days post-sowing), all leaves on the main stem of the ecdr1 mutant, except for the flag leaf, have died. The ecdr1 mutant produces excessive hydrogen peroxide (H2O2) and superoxide anions (O2). - The expression levels of some defense-related genes, such as AOS2, PBZ1, PR1a, and PR1b, were significantly upregulated, enhancing rice resistance to rice blast and bacterial blight. The gene LOC_Os11g06860, which controls the lesion-like phenotype, was isolated using a map-based cloning strategy. Functional complementation experiments confirmed that LOC_Os11g06860 is the target gene ECDR1 controlling the lesion-like phenotype. Quantitative real-time PCR analysis showed that ECDR1 is a constitutively expressed gene, expressed in all tissues and organs. The application of the mutant ecdr1 and its target mutant gene will help in breeding new rice materials and varieties with broad-spectrum and durable disease resistance.

[0014] The present invention has the following significant beneficial effects:

[0015] This invention provides a preliminary analysis of the biological function of the ECDR1 gene, offering a theoretical basis for elucidating plant PCD and disease resistance defense mechanisms. By introducing the ECDR1 mutant gene into rice or other crops using rice lesion mutants (ecdr1 and ECDR1) through conventional hybridization, marker-assisted selection, or genetic engineering, the resistance of crops to fungal diseases (such as rice blast) and bacterial diseases (such as rice bacterial blight) can be significantly enhanced. This invention has significant theoretical and applied value for further elucidating the molecular mechanisms of plant PCD and disease resistance defense responses, and for breeding broad-spectrum, durable disease-resistant varieties. Attached Figure Description

[0016] Figure 1Phenotypic identification of wild-type and mutant ecdr1. Phenotyps of wild-type and ecdr1 mutant at 20 days (A) and 85 days (B) after sowing; (C) Phenotyps of wild-type and mutant whole plants in the field at heading stage; (D) Trypan blue staining for detecting cell death; (E) DAB staining for detecting hydrogen peroxide; (F) NBT staining for detecting superoxide anion; (G) Detection of hydrogen peroxide and superoxide anion content in wild-type and mutant; (H) TUNEL assay for DNA fragmentation; (I) Detection of CAT, POD and SOD enzyme activities.

[0017] Figure 2 The lesion-like phenotype of mutant ECDR1 was induced by light and temperature. (A) The lesion-like phenotype of mutant ECDR1 was induced by light. 1: Phenotype of EDR1 mutant under normal growth conditions; 2: Phenotype of mutant ECDR1 after 4 days of shading; 3: Phenotype of mutant ECDR1 after removing the aluminum foil 4 days later. (B) The lesion-like phenotype of mutant ECDR1 was induced by high temperature. - No lesion-like phenotype; + Lesion-like phenotype.

[0018] Figure 3 The ecdr1 mutant exhibits premature aging. (A) The second leaf (top image) and third leaf (bottom image) of the wild type and mutant were cultured in vitro for 10 days at 30°C in the dark. (B) Expression analysis of three senescence-related genes in the wild type and mutant. (C) Relative expression levels of five chlorophyll degradation-related genes in the wild type and mutant. (D) Expression analysis of eight photosynthesis-related genes in the wild type and mutant. (E) Determination of chlorophyll a and chlorophyll b content in leaves of the wild type and mutant. Data are the mean ± standard deviation of three biological replicates (t-test, **, P < 0.01 indicates highly significant difference).

[0019] Figure 4 To analyze the expression of defense-related genes and identify disease resistance, the study included: (A) qRT-PCR analysis of defense-related genes; (B) identification of resistance to rice bacterial blight pathogen; (C) measurement of rice bacterial blight lesion length; (D) identification of rice blast pathogen resistance; and (E) determination of rice blast lesion length and calculation of pathogen biomass. Data are presented as mean ± standard deviation (A, n = 3; C, n = 30; E, n = 5) (t-test, **, P < 0.01 indicates highly significant difference).

[0020] Figure 5Map-based cloning and functional verification of the ECDR1 gene. (A) Fine mapping of the ECDR1 gene; (B) Comparison of ORF4 and ORF5 sequences in wild-type (WT) and mutant ecdr1, with ATG and TGA representing the start and stop codons, respectively; (C) Transgenic complementation of ORF4, with 4#1 and 4#2 representing two transgenic complementation lines of ORF4; Scale bar: 15cm.

[0021] Figure 6 The spectrum of the pCAMBIA1305.1 vector.

[0022] Figure 7 Predicted domains for the ECDR1 encoded protein. Detailed Implementation

[0023] The following embodiments are for further illustration of the present invention and do not limit the invention. Unless otherwise specified, the experimental methods used in the embodiments are conventional methods; the experimental materials and reagents used are all commercially available.

[0024] Example 1: Obtaining and phenotype of mutant ecdr1

[0025] The lesion-like mutant ecdr1 is a genetically stable material 15-3090, formed by multiple generations of self-pollination after anther tissue culture of the F1 generation of the cross between the japonica rice line 09-2131 (a hybrid of Chunyang and Zhonghua 11) and Kendao 2016. Its sister line 15-3089 has normal leaf development and is considered the wild type of ecdr1. On day 22 after sowing, sporadic reddish-brown spots first appear on the basal first leaf of the ecdr1 mutant, gradually spreading outwards. On day 25, reddish-brown spots begin to appear on the basal second leaf, and on day 28, these spots begin to appear on the third leaf, at which point the first leaf is completely dead. By the heading stage (85 days after sowing), all leaves on the main stem of the ecdr1 mutant, except for the flag leaf, are dead. Figure 1 (AC). After staining leaves of wild-type and mutant ecdr1 with trypan blue, it was found that ecdr1 leaves showed more and darker blue spots than wild-type leaves. Figure 1 The presence of dark brown (D) spots indicates that the mutant experienced more severe cell death. Staining with diaminobenzidine (DAB) and nitrotetrazole blue (NBT) revealed that the ecdr1 mutant produced more dark brown or dark black deposits than the wild type. Figure 1 The presence of E and F in the mutant indicates that the mutant accumulated more H2O2 and O2-. Furthermore, quantitative analysis showed that the contents of H2O2, O2-, and MDA (malondialdehyde) in the mutant were 1.1, 2.4, and 1.7 times higher than those in the wild type, respectively. Figure 1(G). Leaf cell death was detected using terminal deoxyribonucleotide transferase-mediated nick-end labeling (TUNEL). The results showed that ecdr1 mutant leaf cells exhibited strong TUNEL fluorescence signals, while wild-type cells showed very weak fluorescence signals. Figure 1 The presence of H indicates that the ecdr1 mutant leaves undergo severe DNA fragmentation caused by cell death.

[0026] During the heading stage, a 2cm section from the middle of the flag leaf of the main stem of ECDR1 plants that had not yet developed lesion-like spots was wrapped with aluminum foil. Four days after this shading treatment, lesion-like spots appeared on the unshaded sections, while no such spots appeared on the shaded sections. Four days after removing the aluminum foil, obvious lesion-like spots appeared on the previously shaded sections. Figure 2 (A) indicates that the formation of lesion-like spots is induced by light. When germinated wild-type and mutant plants were placed in incubators at 30℃, 25℃, and 20℃ respectively, lesion-like spots appeared on day 48 in the mutant at 20℃; on day 25 at 25℃; and on day 20 at 30℃. Figure 2 (B) indicates that the formation of ecdr1 mutant lesions is induced by light and temperature.

[0027] Example 2: Agronomic traits of wild-type and mutant strains

[0028] In Beijing, wild-type and mutant ecdr1 were planted during the regular season. An examination of agronomic traits revealed that, compared with the wild-type, the ecdr1 mutant had significantly lower plant height, number of spikelets per panicle, number of grains per panicle, thousand-grain weight, and seed setting rate, while there were no significant differences in panicle length, number of effective panicles, and heading date (Table 1).

[0029] Table 1 Comparison of agronomic traits between wild-type and ecdr1 mutants.

[0030]

[0031] The data are the mean ± standard deviation of 30 plants (t-test, **, P < 0.01 indicates extremely significant difference).

[0032] Example 3: Identification of premature aging phenotype in mutants

[0033] The second leaf from the base of both the wild-type and mutant strains after 25 days of growth, and the third leaf from the base of both strains after 28 days of growth, were selected and cultured in vitro in distilled water (30°C, darkness). After 10 days, the leaves of the ECDR1 strain turned completely yellow and even died, while the leaves of the wild-type strain remained green. Figure 3(A). qRT-PCR analysis showed that before the appearance of lesions (day 14), the expression of three aging-related genes, Osh36, Osl2, and Osl57, was not significantly different between wild-type and mutant. After the appearance of lesions (day 28), the expression of these three genes in mutants was significantly higher than that in wild-type. Figure 3 (B) indicates that the mutant has already shown obvious signs of premature aging at this time.

[0034] Premature aging is often accompanied by decreased chlorophyll content and impaired photosynthetic system. Therefore, the contents of chlorophyll a and chlorophyll b, and the expression levels of five chlorophyll degradation-related genes (SGR, NYC1, NYC3, NOL, and RCCR1) and eight photosynthesis-related genes (cab2R, CHLI, porA, PsaA, PsaB, PsbB, PsbC, rbcS, and rpoA) were examined in wild-type and mutants. The results showed that after the appearance of lesion-like spots (day 28), the expression levels of chlorophyll degradation-related genes in mutants were significantly higher than in wild-types. Figure 3 In the C group, the expression levels of photosynthesis-related genes in the mutant were significantly lower than those in the wild type. Figure 3 In the mutant (D), the contents of chlorophyll a and chlorophyll b in the leaves were significantly reduced. Figure 3 (E). The above results indicate that, along with the appearance of lesion-like spots, the leaves of ecdr1 exhibit significant premature senescence.

[0035] Example 4: Expression of defense-related genes and resistance identification

[0036] Expression of four defense-related genes (AOS2, PBZ1, PR1a, and PR1b) in wild-type and mutant ecdr1 was examined. Before the appearance of the lesion-like phenotype (day 14), there was no difference in the expression of these four genes between wild-type and mutant. However, after the appearance of the lesion-like phenotype (day 28), the expression levels in the mutant were increased by 76.5, 17.3, 60.8, and 12.6 times, respectively, compared to the wild-type. Figure 4 (A). Further inoculation with four *Bacillus oryzae* strains (P2, P6, C5, and T1) revealed that the lesion length of the mutant was significantly reduced compared to the wild type. Figure 4 (B, C). Inoculation with rice blast strain 99-26-2 significantly reduced the size of lesions and the growth of pathogens in the mutants. Figure 4 (D, E). These results indicate that the defense response is activated in the mutant ecdr1, which significantly enhances its resistance to rice bacterial blight and rice blast.

[0037] Example 5: Map-based cloning of the ECDR1 gene

[0038] Genetic analysis of the ecdr1 mutant obtained in Example 1 showed that both the F1 generation of the ecdr1 mutant and the wild-type mutant in reciprocal crosses exhibited the wild-type phenotype, while the F2 populations showed clear segregation. In the reciprocal cross F2 population, 489 plants had the normal phenotype and 160 plants had the lesion-like phenotype, meeting a segregation ratio of 3:1. In the reciprocal cross F2 population, 410 plants had the normal phenotype and 163 plants had the lesion-like phenotype, also meeting a segregation ratio of 3:1 (Table 2). This indicates that the lesion-like phenotype of ecdr1 is regulated by a single recessive nuclear gene and is not affected by the maternal cytoplasm.

[0039] To clone the target gene ECDR1, which controls the lesion-like phenotype, DNA pools were constructed using 20 lesion-like individual plants from the F2 population of a cross between the indica rice variety CO39 and the ecdr1 mutant. Parental polymorphic markers and 167 lesion-like individual plants were also used to preliminarily locate SPL37 in an approximately 235.4 kb region between Indel markers S10 and S18 on chromosome 11. Figure 5 (A)

[0040] By developing polymorphic markers for the target region, and using 392 F2 single plants with lesion-like phenotypes, the target gene was located within a 33.2 kb region between Indel markers S20 and S25 (Table 3). This region contains 7 open reading frames (ORFs). Figure 5 Sequencing of all genes within the interval revealed a 546bp deletion at positions 127bp upstream of the start codon in ORF4 (LOC_Os11g06860) and 18bp downstream of the stop codon in ORF5 (LOC_Os11g06870). Figure 5 In the B group, the nucleotide sequences of other ORFs did not differ between the wild type and the mutant. Therefore, ORF4 and ORF5 were selected as candidate genes for controlling lesion-like genes.

[0041] Table 2. Genetic analysis of F2 populations from reciprocal crosses of wild type and mutants.

[0042]

[0043] Table 3. Map-based cloning molecular markers for the ECDR1 gene.

[0044]

[0045]

[0046] Example 6: Validation of ECDR1 gene functional complementation

[0047] Based on the Nipponbare reference sequence, specific primers for the ECDR1 gene (1305C-ECDR1F) were designed using primer design software. ATCCTCTAGAGTCGAC CCGTGCATCAGACAGCTTAA;1305C-ECDR1R: AGGATTCAATCTTAAG GAACATCGCATCCTAACGCA (underlined part is the recombinant sequence used for ligation). The full length of the candidate gene was amplified by PCR using high-fidelity KOD enzyme (TOYOBO). Sequencing confirmed that the candidate gene fragment was 4307 bp, including its own promoter region of 2042 bp, the entire coding region of 1287 bp, and the termination sequence of 978 bp (SEQ ID NO.1).

[0048] SEQ ID No: 1

[0049] CCGTGCATCAGACAGCTTAAAGATCAATCTACATGCATGCAGAGCAACCATGCCAAAGGCATCACCTTTCCTTCCTCC

[0050] TCCTCCTCCTCCTCCTGATCACCATCTACATTGCCCATGGCGCCGCCGGTGACGCCATCGGCGGCGACCCATGGCAGGAG

[0051] CCCGAGGTGGCCCAGCAGCCAGCCGTCGTCCTCGCCGGCGAGTGGCAGCTCCTGCACCAGAACACCGGCGTCTCGGCGAT

[0052] GCACATGCAGCTGCTCCCCGGCGACTACGTCCTCATGTTCGACCGCACCGACTCCGGCCCCTCCAACATCTCCCTCGACG

[0053] CGCTCTCGCCGTGCGCCGCCGCGGCGACCACCGCCCTCGCCGCCGGCGGCGGCGGCGCCGTTGACTGCACCGCGCACTCG

[0054] GTGCTGCTCGACCTCCGGTCGAACGCCCTGCGGCCGTACCCGCTCGCCACCAACCCATGGTGTTCGTCCGCGGCGCTGCT

[0055] CCCCAATGGCACGCTGCTGCAGACCGGTGGGTTCTCCAATGGCGACCGCATCGCGCGCCTTCTCGCCGTCGACGGGGT

[0056] GGGTCGATCTCCCGTCGTTCTTGGCCGTCCGGCGGTGGTACGCCACCGACATCCTCCTCGCCGACGGCCGCGTGCTCATC

[0057] CTCGGCGGCCGGCGGCAGTTCAACTTCGAGTTCTTCCCCCACGACGACGCGCCGGCGCCGCAGCCGACCTTGTTCCCGTT

[0058] CTTGGAGGAGACGACCGACATGGACGCCGAGGACAACCTCTACCCGTTCCTCCACCTCCTCCCCGACGCCACCGTCTTCG

[0059] TCTTCGCCAACGACCGCGCCGTCGTGTTCGACCCCTACAACCGCGCCCCGCTCCGCCGCCTCCCCGCCATCCCCGGCGGC

[0060] GTGCCGAGGAACTACCCGTCGTCGGGATCCTCCGTCCTCCTCCCGCTCCGGCCGGACTCGCCGTCGCACGCCGAGGTGCT

[0061] CGTCTGCGGCGGCGCGCCGCGCGGCGCGTACCGCCTCGCCCTCCGCAACGGCACGTTCGCCCCGGCCGACCGGACCTGCG

[0062] GCCGGATCGCGCCGACCGACGCGAACCCGGTGTGGGCGATGGAGGAGATGCCGCTGCCGCGCGCCATGGGCGACATGGTG

[0063] CTGCTCCCCACCGGCGACGTGCTCATCGTCAACGGCGCGGCGGCCGGCACGGCGGGGTGGGAGCTCGGGCGCGAGCCGGT

[0064] CACCTACCCGGTGCTGTACAAGCCGGACATGCAGCTCGGCGCGCGGTTCGAGGTGCTCGCCGCGTCAACCATCCCGAGGA

[0065] TGTACCACTCGTCGGCGACGCTGGACACGCTCGGCCGCGTGCTCGTCGGCGGCAGCAACCCGCACGTCGGCTACGTGTTC

[0066] GACAACGTGACGTACCCGACAGAGCTCAGCCTGGAGGCGTTCCTACCACCATACTTCGACGCACGGCTCGACGGCGTGCG

[0067] GCCGCGGCTGGTGGCGGCGCCGTCGGAGGTCGGGTACGGGGAGGCGGCGGCGGTGAGGTTCGAGGTGCCCGGCGGCGCGG

[0068] TCTCCGGTGGCCCCGAGGAGGTGCGCGTGGCGGCCGTGGCGCCGGCGTTCGCGACGCACTCCTTCGGGATGAACCAGAGG

[0069] GTGGTGTCCCTGGCCGTGGGCACCGTCGCGCAGCTGGCCGCCGGGCTGTACGAGGCGCAGGTGGCCGCGCCGCCGTCGCC

[0070] GTCGGTGGCGCCGCCGGGGTACTACCTCTGGTTCGTGCTCCACGCCGGCGTGCCCAGCACCGCGGCATGGGTGCGCATGC

[0071] GGCCGCTCGGTGCAGCGACGTGATGACGACGACGACGTCGACGTGTGAACCAGTCAACTGCCACGTCTCACTATTTTTCT

[0072] CTCTCAAAAAAAATGAAAAGAAAAAGAAATGGTCAACTACCACCTACTAGTAGTACTAGTAATAACACCTCGGGCTTGTG

[0073] GTTTATTTACAAAAGAGCCCATGGAGAAGTAGGCGAGACGCAGAAAGGTCCCTCGATTCGAGAGCTGTGGAGAGAGATCG

[0074] CCCAAATCTCATCTCGCTTTCCTCTCTCTCTCGCCGCCGGCGATGCCTCCGTCCCCGTCCGCCGCCGGGGCGGCCTCGCC

[0075] GTCGAACTCGTCGGCGGCGTCCGCATCCGACCCCACCCCGTCGTGGTGGGAGTCGGTCTCGCAGGCGAGGTCCCGCATCC

[0076] TGGCGCTGTCGTCCATCCTCCCGGCGCCGGCCGATTCCGACGTCGCGGCGCTCGCCGACTCCGACCGCCCGGCGCGCGCG

[0077] CTGCTCCGGTCGTCCGCCGCGTACGTCGCGCTGTCCGCGGCGCTCCGGTCGGGGGGCGGCGCCGACGACCCGGCCTGCCA

[0078] CTGGCTCTACGATACGCTGCTGTCCCCGGACCCCGACCTCCGCCTCGCCGCGCTCGCGTTCCTCCCGCTCCTCTCGTCGC

[0079] TCTACCTCCTCCGCCTGCCGCCCGCGCTCCCCTCCTCGCTCTCCGGCTTCGAGGCCGTCCTCCTCGCCGTCTACTCCTCC

[0080] GAGGCCAAGAATCGCCAGGGCAAGCCCGTCCTTGTCCAGGTACCCGACCTCTCCGTCCCTTCCCTCTACCACACGCCGCT

[0081] GTCCAGCCCCAGCTCGAAGTCCCCTCGCCGGCCACAGCCGCCGCCGATCCCCCCACCCGCGGGGAATGTTGTGGTCGGGG

[0082] TGCTATCGCCGCCGCTTGAGCCACAGGCGGCGGTGAAGTCGACCAAGCGCGCGGGGATTATCGGAGTTGCCTTCGAGGCG

[0083] TACTATGCCAAGATTTCGCAGATGCCGCCTGCATCGAAGGTGGATGCTTGCAACGCCGTGGCGGCGTGGGCGGGGCAGTA

[0084] CTGCAAGTGCCGATTTGAGCTTGATGAGAAGGAGTTGGAAGAGGAGGAGGCTGATTCATTGGGGTCCGTTTCGCCATTGT

[0085] CATCGGAGGCCGAGAACGGGAAGGCCTTGGAGGAGGAAATGGCAAAGATGCGCGTCAACGGAGACACCAATGGTCGGAAT

[0086] TGTGGCGAGAGAGAGGGGAGGGTGCCACTTCCATGGGAGCTTCTACAGCCAGTGATGAGAGTTCTTGGTCACTGCCTGTT

[0087] AGCTCCGCTGAATCCGACGGAGGTGCGGGATACCGCCGCAGAAGCTGTGCGGGTTGTCTATGCCCGCGCGTGTCATGAGC

[0088] TTGTGCCACAGGCTATCTTGGCGTCCCGTAGCTTGATCGAGCTCGACAAAAGCGCACGCAAGGCTGCCAAGGAGGCAGCT

[0089] GCAGCAGCTTCTGGGGCAATCGTGTCAGTAGGCACAGCTGGTAGCACCGCGTCGAGCTCTAGACCCAGCTCCAAGCCAAA

[0090] CACACCAGGCAAGCAGCGGAAGCCTGATGTACTGCTCCTGTCGAAATGATTCTGATTGCATTACATCTTGTAAGATTTTC

[0091] TGCAGTGGTGATAAAAGAGTTCATTTTTCTCATGAGTTTGTCACTGGAATTTCATTGAAGTCTACTAAACCTTGCAATGC

[0092] GTAAAGGTCGACTCTTGTGGCTTGGAAAAGCATTGGTTTAGTATGTAGCCAGTTCTGTATTGTAAGTTATCTTGTCTTAT

[0093] GTTATTGTTTTTGCATATAGCTGATGAATTAATATATTTCACATTTATGTGTGCGGTTGATGAAAAGAACATGGTGTAGT

[0094] GTGCTATCTACTACTAGTAATGGATTTTAGCAGGCGATTATGTATTGACTTGTTTTATGCATTTCTTACCGAACATATAT

[0095] TAATTTGATGTTCCTTACTACAACTACTATCTTATGGAATTCTCAGCAGCATAAATTGCAATGATTTGTGTCTTGATGTG

[0096] TCCCTTGGTTTTGAAAGAGTCCATTAATTGATATTTCATATAGTCACATACCTAAGTTGAGTTTATTTAACCAAATATG

[0097] GTTAGAATGAAAAGCGATTAAAGAGTCACTTGTGATCGTCCCTGTCATTGTTTGTCTTAATGTTCTACCGAAAAAGGGGA

[0098] AAGAAAGGAAATGAATAGAAAGAAGCAATATTGACAATGGAAAGCATCAAAGACCTACTAATTGCCTGTTTGCTTTGAGC

[0099] CTTTGATCATATGAACTGCAATAAGTTTATTTCTTTACTATAATAACAGCGGCTATTTATTTGGTTTTTGCTGTATGTAT

[0100] GGAGTTCATTAGTAGTCGGGCTAGTGCTAAACTGCTAATGCATTCCAGGCGCAGGGCATTGCTGTTGATCTGTTCTCTCT

[0101] CACTAGGTGATATTGCTGTAGAGTTGTAGAAAGGGTTTACCCCCTTCAATATCTCTCTGTTTTTAAGAGGAGTTCCCAGT

[0102] TAGTAATTTGAGATGTGCAAACGATGTTACTGATTCTTAGACTTAAGCTCAAGTAATACAAGTGAAATGCGTTAGGATGCGATGTTC.

[0103] The sequence consists of the promoter sequence (positions 1-2042), the 5' UTR sequence (positions 1992-2042), the CDS sequence (positions 2043-3329), the 3' UTR sequence (positions 3330-3739), and the terminator sequence (positions 3740-4307). The encoded amino acid sequence is SEQ ID NO: 2.

[0104] MPPSPSAAGAASPSNSSAASASDPTPSWWESVSQARSRILALSSILPAPADSDVAALADSDRPARALLRSSAAYVALSAALRSGGGADDPACHWLYDTLLSPDPDLRLAALAFLPLLSSLYLLRLPPALPSSLSGFEAVLLAVYSSEAKNRQGKPVLVQVPDLSVPSLYHTPLSSPSSKSPRRPQPPPIPPPAGNVVVGVLSPPLEPQAAVKST KRAGIIGVAFEAYYAKISQMPPASKVDACNAVAAWAGQYCKCRFELDEKELEEEEADSLGSVSPLSSEAENGKALEEEMAKMRVNGDTNGNRNCGEREGRVPLPWELL QPVMRVLGHCLLAPLNPTEVRDTAAEAVRVVYARACHELVPQAILASRSLIELDKSARKAAKEAAAAASGAIVSVGTAGSTASSSRPSSKPNTPGKQRKPDVLLLLSK.

[0105] Leveraging Clonetech The purified PCR product was ligated into the pCAMBIA1305.1 vector, which had been digested and purified by EcoRI and NcoI, using the PCR Cloning system (TaKaRa) to obtain the pCAMBIA1305.1-ORF4 / ORF5 genome complementation vector (verified by sequencing). A map of the pCAMBIA1305.1 vector is shown in [link to image]. Figure 6The pCAMBIA1305.1-ORF4 / ORF5 complementary vector was introduced into Agrobacterium strain EHA105 (American Junction Biotechnology Co., Ltd.) to obtain recombinant Agrobacterium. The mutant ecdr1 callus was then infected using Agrobacterium-mediated transformation. Results showed that all positive plants transfected with ORF4 reverted to the wild-type phenotype, i.e., the normal phenotype without lesion-like spots. However, all positive plants transfected with ORF5 still exhibited a lesion-like phenotype. Figure 5 (C). The above results indicate that ORF4(LOC_Os11g06860) is the target gene ECDR1 that controls the lesion phenotype of mutants.

[0106] The ECDR1 gene encodes a protein containing 428 amino acids (SEQ ID NO.2) with a molecular weight of 44.73 kDa and a pI of 7.345. SMART software predicted ( http: / / smart.embl-heidelberg.de This protein contains a hyccin domain. Figure 7 The biological functions of this type of protein have not yet been reported in rice.

Claims

1. A gene regulating disease resistance in rice. ECDR1 Its characteristics are, Its encoded amino acid sequence is shown in SEQ ID NO.

2.

2. The defense regulation gene of rice according to claim 1. ECDR1 characterized in that The nucleotide sequence is the genome sequence shown in SEQ ID NO.1, or the CDS sequence from position 2043 to 3329 therein.

3. Contains the regulatory gene for regulating rice disease resistance as described in claim 1 or 2. ECDR1 Plant expression vectors or host cell lines.

4. The host cell line as described in claim 3, characterized in that: The host cell line is Escherichia coli, Agrobacterium, or plant cells.

5. The regulatory gene for regulating rice disease resistance as described in claim 1 or 2 ECDR1 Application in the breeding of disease-resistant plant varieties.

6. The application as described in claim 5, characterized in that: The disease-resistant plant variety is a rice variety. Disease resistance refers to resistance to fungal or bacterial diseases, preferably resistance to rice blast or rice bacterial blight.

7. The application as described in claim 5, characterized in that: The cultivation method involves identification through artificial inoculation or natural disease occurrence in the field. OsECDR1 Resistance of gene mutants to pathogens.

8. The application as described in claim 6, characterized in that: The artificial inoculation refers to inoculating rice blast fungus by spraying or detaching leaves during the seedling stage, or by injection during the tillering stage, or by leaf cutting during the tillering and heading stages; the natural disease occurrence in the field refers to the natural spread of pathogens in the field that induces rice bacterial blight or rice blast.

9. A plant genome editing vector comprising the one described in claim 9.

10. A method that includes applications ECDR1 The method for breeding disease-resistant rice varieties using mutant genes is characterized by, Its use of carrying ECDR1 The mutant gene in rice materials can be introduced into other rice materials through hybridization, backcrossing, and molecular marker-assisted selection, or the structure or expression of the gene can be altered through gene editing or RNAi interference, in order to improve the rice's resistance to fungal and / or bacterial diseases.

Images