ETD1, a super-effective allele for rice blast resistance

Abstract

This invention provides a super-effective allele for rice blast resistance, ETD1, which has the nucleotide sequence shown in SEQ ID NO.1 and its cDNA sequence shown in SEQ ID NO.2. This gene belongs to the OsCNGC13 super-effective allele, possessing stronger calcium ion transport capacity and significantly accelerating the influx of calcium ions into cells. Therefore, normal expression or overexpression of this gene in gene knockout plants can improve rice resistance, thus positively regulating rice blast resistance.

Description

Technical Field

[0001] This invention specifically relates to a highly effective allele for resistance to rice blast, ETD1, and belongs to the field of molecular biology technology. Background Technology

[0002] Crop diseases are severe, posing a serious threat to food security. Taking Hunan Province as an example, the 2023 forecast of major crop diseases and pests showed that rice sheath blight (44 million mu), rice blast (3.3 million mu), rice false smut (3 million mu), and southern rice black-streaked dwarf virus (0.5 million mu) had a cumulative occurrence of 50.8 million mu. Due to problems such as the susceptibility of major resistance genes to loss due to changes in pathogen races, regional limitations, resistance costs, and genetic burden in molecular breeding, the utilization rate of resistance genes in rice varieties promoted in production is low, and better broad-spectrum and long-lasting disease resistance strategies are urgently needed. In addition, for some diseases such as rice sheath blight, rice false smut, and southern rice black-streaked dwarf virus, there is a lack of cloning of major resistance genes and breeding of resistant varieties, and their control mainly relies on pesticides. Therefore, improving the broad-spectrum resistance of crops to multiple pathogens while reducing pesticide use has become an inevitable requirement for green prevention and control in grain production.

[0003] Taking rice blast as an example, the pathogen of rice blast belongs to the genus *Fusarium* of the fungus phylum. This fungus is capable of both sexual and asexual reproduction, and is characterized by susceptibility, high efficiency, and wide adaptability. *Fusarium* grows in the soil and reproduces by absorbing water and nutrients through the plant roots. The pathogen enters the rice plant through wounds or natural openings (such as stomata), multiplies rapidly, and produces toxins, causing spindle-shaped or elliptical lesions on the rice leaves. If not controlled in time, it will severely affect rice photosynthesis and vegetative growth, ultimately leading to stunted growth, cessation of growth, and even death. Therefore, rice blast is also known as "rice cancer." If timely control measures are taken after an outbreak of rice blast using chemical fungicides or biological pesticides, the rice that has stopped growing will resume growth, minimizing yield loss within a controllable period.

[0004] In molecular design breeding for rice resistance to diseases including rice blast, the most crucial aspect is the discovery of resistance genes. The rice immune system primarily consists of pathogen-associated pattern-triggered immunity (PTI) and effector-triggered immunity (ETI). The former is generally considered to confer broad-spectrum and durable resistance, while the latter confers specialized resistance to specific pathogen races. In production practice, major-effect genes can significantly enhance crop resistance to specific pathogen races, but this resistance is often short-lived; the resistance effect of major-effect genes disappears when new pathogen races emerge. Therefore, discovering broad-spectrum resistance genes is of greater value for rice disease resistance breeding and is a hot topic in plant disease resistance research. However, due to limitations in identification and breeding, only a few broad-spectrum rice resistance genes and their molecular mechanisms have been reported to date.

[0005] Research has found that calcium ions are not only an essential mineral element for cells but also a crucial second messenger for immune activation. Immune activation triggers calcium signals of varying amplitudes and durations. These signals are decoded by various calcium-binding proteins, thereby regulating downstream defense responses and even cell death. Therefore, the establishment of immune calcium signaling is a core link in plant stress perception and response, and also a cutting-edge research area in plant immunity. Calcium signaling is regulated by calcium ion channels, among which cyclic nucleotide-gated channel proteins (CNGCs) are one of the main calcium ion channels. Studies have shown that plant CNGCs participate in numerous physiological processes within plants, including immune responses, pollen tube germination, elongation and guidance, stomatal movement, and root gravitropism, which primarily require CNGC-mediated cellular calcium ion flow.

[0006] The rice genome contains 16 CNGCs (1-16), divided into four families (I-IV) and two subfamilies (IV-A and IV-B). OsCNGC13 is the first member of the OsCNGC family to be cloned in rice. Loss of function of OsCNGC13 affects the accumulation of calcium ions in the style after pollination, weakens programmed cell death in the style, leading to abnormal pollen tube pathway formation and thus causing semi-sterility in rice. Currently, no gain-of-function mutants of OsCNGC13 have been reported.

[0007] OsCNGC9 is the second member of the OsCNGCs family to be cloned in rice, and this gene plays a positive regulatory role in rice blast resistance during the seedling stage. Studies have found that OsCNGC9 is a calcium channel protein. Under pathogen-associated molecular pattern induction, the rice receptor kinase OsRLCK185 can interact with OsCNGC9, activating calcium ion influx through phosphorylation and actively regulating the expression of reactive oxygen species bursts and PTI-related genes. Therefore, OsCNGC9 directly participates in the regulation of immune calcium signaling. Under cold stress, OsCNGC9-mediated calcium ion influx can initiate downstream cold stress responses and enhance rice cold tolerance. Wang Jiachang (Map-based cloning and functional analysis of the rice immune-related gene OsCNGC9 and functional study of two rice heading-stage-related transcription factors, Doctoral dissertation, Nanjing Agricultural University, 2018) reported the discovery of a rice lesion-like mutant gene cds1 derived from OsCNGC9, showing no significant difference in leaf appearance between wild-type and mutant at the tillering stage. The mutant exhibited a distinct lesion-like phenotype on its leaves after heading and showed a significant reduction in resistance to rice blast. Further experiments revealed a 4bp deletion in the fourth exon of the mutant, leading to premature termination of translation. Both the lesion-like phenotype and decreased resistance in the mutant were attributed to the OsCNGC9 mutation. Transgenic results indicate that increasing OsCNGC9 transcription can improve broad-spectrum resistance to rice blast to some extent. However, this paper still represents basic research on OsCNGC9, merely demonstrating that the calcium ion channel OsCNGC9 participates in plant PTI immunity. Pathogens can overcome this resistance through the evolution of effector proteins. Such pathogens have already emerged in natural evolution, resulting in the presence of the OsCNGC9 gene, yet rice remains susceptible. Furthermore, this mutant significantly weakens rice resistance to rice blast, meaning this phenotype is not related to resistance. This mutant is not an acquired functional mutant and therefore lacks practical value for production applications.

[0008] Furthermore, existing research has identified numerous known lesion-like mutants (see pages 9-12 of the aforementioned paper), involving various gene regulations. However, most genes regulating the lesion-like phenotype involve metabolic-related regulatory genes, and lesion-like mutants with ion channel mutations are rarely reported. Moreover, not all lesion-like mutants are associated with resistance to rice blast or similar diseases, resulting in a lack of substantial progress in related research.

[0009] Existing research suggests that the regulatory roles of other OsCNGC family members in immune calcium signaling warrant further investigation. Although multiple CNGC genes have been identified in rice and other plants and are associated with basal immune responses, the resistance they mediate is weak and lacks practical value for industrial applications.

[0010] A review of research literature on rice blast indicates that although there are reports of calcium channel protein-related genes enhancing rice blast resistance, the enhancement effect is not significant. Therefore, a new calcium channel protein-related CNGC gene mutant is needed, which should exhibit broad-spectrum resistance against rice blast fungus, thereby improving rice's resistance to different physiological races of rice blast. Summary of the Invention

[0011] The first principle of this invention is the discovery of the ETD1 gene, a super-efficient allele mutant from CNGC13, which has the function of significantly accelerating the influx of calcium ions into cells.

[0012] The second principle of this invention is the first demonstration that the ETD1 gene mutant associated with the lesion-like phenotype not only possesses resistance to rice blast and similar diseases, but also belongs to the superalle of OsCNGC13. Since the superalle possesses a novel function, this mutant can be used to enable gene knockout plants to acquire stronger calcium ion transport capabilities, significantly accelerating the influx of calcium ions into cells and enhancing the plant's resistance to rice blast and similar diseases.

[0013] Therefore, the present invention provides a mutant ETD1 gene from CNGC13, wherein the ETD1 gene has the nucleotide sequence shown in SEQ ID NO.1, wherein the gene belongs to the super-efficient allele of OsCNGC13, which has a stronger calcium ion transport capacity and has the function of significantly accelerating the influx of calcium ions into cells.

[0014] In one embodiment, the cDNA sequence of the ETD1 gene is shown in SEQ ID NO.2.

[0015] In a preferred embodiment, the gene has the function of regulating rice blast resistance and enhancing rice's resistance to rice blast.

[0016] In any of the above embodiments, the ETD1 gene is a super-effective allele of OsCNGC13. Normal expression or overexpression in gene knockout plants can improve rice resistance, thus having a positive regulatory effect on rice blast resistance.

[0017] In any of the above embodiments, the expression of the ETD1 gene is suppressed or reduced in OsCNGC13 gene knockout or gene deletion rice, resulting in decreased rice blast resistance. In contrast, the cds1 gene, a rice lesion mutant from OsCNGC9, shows increased expression in rice, leading to decreased rice blast resistance.

[0018] Technical effect

[0019] 1. Although many lesion-like mutants exist, involving different gene regulation, most lesion-like phenotypic regulatory genes involve metabolic-related regulatory genes, and lesion-like mutants with ion channel mutations are rarely reported. This invention is the first to discover a gain-of-function allele of a calcium ion channel, enhancing the channel's activity based on its original function, and performing directed evolution of the channel with basic functions. This invention provides a new solution and implementation method for enhancing plant disease resistance using immune calcium signaling.

[0020] 2. This invention utilizes bio-induced mutagenesis technology to obtain, for the first time, a mutant ETD1 gene derived from the OsCNGC13 gene. This mutation, located on the seventh exon of the LOC_Os06g10580 gene, produces a GA variation, leading to changes in amino acids. Figure 3B ).

[0021] 3. This invention is the first to demonstrate that the ETD1 gene mutant associated with the lesion-like phenotype not only possesses resistance to rice blast and similar diseases, but also belongs to the superalle of OsCNGC13. Since the superalle possesses a novel function, this mutant can be used to enable gene knockout plants to acquire stronger calcium ion transport capacity, significantly accelerating the influx of calcium ions into cells and enhancing the plant's resistance to rice blast and similar diseases.

[0022] 4. This invention is the first to demonstrate that a single point mutation of this amino acid can significantly improve the resistance of rice to rice blast.

[0023] 5. This invention is the first to demonstrate that the ETD1 protein is a mutant encoding a calcium ion channel protein, which can significantly accelerate the influx of calcium ions into cells;

[0024] 6. This invention is the first to demonstrate that the ETD1 gene is a super allele of OsCNGC13, but it is recessive. In the presence of OsCNGC13, ETD1 cannot function. Therefore, it is necessary to knock out the original OsCNGC13 gene in the crop and introduce ETD1, which can then be used for genetic improvement of the crop, particularly for improving rice resistance to rice blast, and to cultivate broad-spectrum blast-resistant rice.

[0025] 7. This invention provides a method for using the ETD1 gene and its encoded protein, as well as related biological elements, to breed rice varieties with high resistance to rice blast. It also provides new gene resources for breeding rice blast-resistant germplasm, which is beneficial for the breeding of rice blast-resistant varieties and thus ensures the safety of rice production.

[0026] 8. Since the wild-type host does not contain the ETD1 gene, and this invention proves that it is not necessary to increase the expression or expression level of ETD1, but only to introduce the gene into the CNGC13 gene knockout or gene deletion host to obtain stronger calcium ion transport capacity, which has the function of significantly accelerating the influx of calcium ions into the cell, thereby improving the host's resistance to rice blast.

[0027] 9. This invention demonstrates for the first time that ETD1, when stimulated by rice blast fungus, causes significantly greater leaf cell death in CNGC13 gene knockout or deletion genotypes than in wild-type rice. However, this response mechanism is essentially beneficial for the host to resist the invasion of foreign pathogens and establish an effective immune defense mechanism. Once the invasion period of foreign pathogens has passed, with the provision of reasonable nutrition, light, and other conditions in the later stages, and in the absence of rice blast fungus, the subsequent growth of transgenic rice basically or completely recovers to normal growth, with virtually no impact on yield. In contrast, wild-type lines in the same field and at the same growth stage almost entirely wither and die after inoculation with rice blast fungus. Therefore, this invention indicates that by reintroducing ETD1 into CNGC13 gene knockout or deletion genotype hosts, transgenic crops resistant to multiple crop diseases can be developed, with broad prospects for production applications. Attached Figure Description

[0028] Figure 1. Broad-spectrum resistance to rice blast by ETD1, where A represents the creation process of ETD1; B represents the leaf phenotype after inoculation with rice blast physiological races; C represents the cell death index of inoculated leaves; D represents the relative growth of rice blast fungus after inoculation; E represents the Chitin-induced cell death phenotype of ETD1; F represents the cell death indices of IR64 and ETD1; and G represents the leaf resistance phenotypes after inoculation with different rice blast physiological races using ETD1. t-tests were performed, with *** indicating highly significant differences (p < 0.001).

[0029] Figure 2. Effects of ETD1 disease phenotype on agronomic traits: A represents the maturity phenotypes of IR64 and ETD1; B represents leaf lesions of ETD1; C represents TUNEL staining analysis of mesophyll cells; DI represents the comparison of agronomic traits between IR64 and ETD1: plant height (D), tiller number (E), panicle length (F), seed setting rate (G), thousand-grain weight (H), and heading date (I). t-test was used; * indicates significant difference (p < 0.05); ** indicates extremely significant difference (p < 0.01).

[0030] Figure 3. ETD1 is the superalle of OsCNGC13: A shows the distribution of SNP indexes in the lesion-like phenotype pool on chromosomes; B shows the ETD1 mutation site; C shows the protein secondary structure prediction diagram; D shows the inoculation phenotype of the ETD1 gene knockout line inoculated with the IR64 virus-free strain V86010; E shows the inoculation phenotype of the IR64 virus-free strain CA89; FG shows the inoculation phenotypes of the OsCNGC13 gene knockout line and the ETD1 genetic supplementation line inoculated with rice blast fungus. The inoculation phenotypes of the NIP virus-free strain 4029-1 (F) and the NIP virus-free strain S5 (G) are also shown. H shows the final field growth results of the gene knockout line genetically supplemented with ETD1 (right) and the wild-type line (left) after inoculation with rice blast fungus.

[0031] Figure 4. ETD1 encodes an enhanced calcium inward channel: AB represents the intracellular calcium channel in *E. coli* expressing ETD1 and OsCNGC13. 2+ Accumulation is dependent on extracellular calcium ion concentration (A) and time (B); C is the calcium channel blocker Gd. 3+ Calcium accumulation in *E. coli* expressing ETD1 and OsCNGC13 was observed under treatment with 0.1 mM calcium and activator ACC (1 mM); D shows organelle localization of ETD1-GFP and OsCNGC13-GFP in *Xenopus laevis* oocytes; E shows the average current-voltage results of patch-clamp recordings in *Xenopus laevis* oocytes expressing ETD1 and OsCNGC13 under 30 mM calcium treatment; F shows the average current-voltage results of patch-clamp recordings in *Xenopus laevis* oocytes expressing ETD1 and OsCNGC13 under 30 mM barium treatment; G shows NMT measurements indicating the effect of 10 mM calcium... 2+ After treatment, extracellular Ca of IR64 and ETD1 2+ Intraflux, measured for 5 minutes under normal conditions, 10 mM Ca 2+ Measurements were taken for 6 minutes after treatment; H represents calcium-triggered Ca2+ in root cells. 2+ Quantification of flux integral; I represents calcium stimulation of cytoplasm in the living roots of IR64 and ETD1, leading to Ca2+. 2+ The temporal dynamics of the increase were assessed using the normalized ratio of cpVenus / ECFP to evaluate cytoplasmic calcium. 2+ The increase in J represents cytoplasmic Ca. 2+ Concentration curve integration; using resting cytoplasmic Ca2+ before thermal stimulation 2+ The curve integral was calculated based on concentration. A t-test was performed, with different letters indicating highly significant differences (p < 0.01).

[0032] Figure 5. Cell death induced by enhanced calcium ion influx in ETD1 under immune activation: A and B represent dynamic analysis of ROS generation in IR64 and ETD1 leaves induced by Chitin (A) and flg22 (B), respectively; C and D represent the calcium ion influx in IR64 and ETD1 mesophyll cells after Chitin (C) and flg22 (D) treatment, respectively. 2+ Comparison of influxes; E and F represent dynamic analysis of Ca in the root cytoplasm of IR64 and ETD1 roots stimulated by Chitin (E) and flg22 (F), respectively. 2+ Concentration changes; GH represented the calcium ion accumulation levels in IR64 and ETD1 mesophyll cells 12 h after inoculation with non-toxic rice blast fungus V86010 (G) and toxic rice blast fungus CA89 (H), respectively; I represented the calcium channel blocker Gd. 3+ (1mM) treatment inhibited ETD1-induced cell death under seeding conditions. Detailed Implementation

[0033] The present invention will now be further described with reference to embodiments, but it is not limited to any one of these embodiments or similar examples.

[0034] Example 1: Obtaining the ETD1 gene mutant through bio-induced mutation

[0035] A mutant strain was obtained by EMS chemical mutagenesis of IR64. This mutant strain significantly induced leaf cell death after spray inoculation with both the non-toxic physiological race V86010 and the toxic physiological race CA89. Figure 1A Interestingly, the fungal elicitor Chitin also significantly induced cell death in mutant leaves (-D). Figure 1E -F). Therefore, this mutant was named ETD1 (Elicitor triggered Cell Death 1).

[0036] Rice blast spraying inoculation demonstrated that the ETD1 mutant exhibited complete immunity to different rice blast physiological races. The rice blast strains CA89 and V86010 were from rice blast physiological races collected by the International Rice Research Institute. Races 17-1-1, 17-5-2, 17-6-1, 17-6-2, 17-7-1, and 19-2-1 were from rice blast physiological races collected and preserved in Sichuan Province in 2017 by our laboratory. Resistance evaluation using these strains after inoculation with ETD1 was as described above.

[0037] Its agronomic traits, such as plant height, ear length, seed setting rate, and number of tillers, were all assessed. Significance was determined using the T-test.

[0038] Survey of agronomic traits:

[0039] Rice seedlings were sown and transplanted at the Changsha Chunhua base and the Sanya Haitang Bay base in Hainan. At the rice maturity stage, five seedlings from each of IR64 and ETD1 were randomly selected for statistical analysis.

[0040] Forty-five highly pathogenic rice blast fungus physiological races collected from major rice-producing areas in China and five representative rice blast fungus physiological races from abroad were selected for spray inoculation identification of ETD1. Different rice blast physiological races could all trigger leaf cell death in ETD1 (partial inoculation results are shown in the figure). Figure 1G Therefore, we created a new rice germplasm with broad-spectrum resistance to rice blast through EMS mutagenesis.

[0041] During field planting, we observed spontaneous cell death in the leaves of ETD1, with the leaves almost entirely covered with spots of cell death during the flowering period. Figure 2A -B), this is a typical lesion-like phenotype. TUNEL assays showed that the degree of DNA damage in ETD1 was significantly enhanced compared to the wild type. Figure 2C This further demonstrates that ETD1 induces significant cell death. Along with the strong induction of cell death, ETD1 agronomic traits were significantly altered, such as shorter plant height, reduced tiller number, shorter ear length, decreased seed setting rate, reduced thousand-grain weight, and delayed growth period. Figure 2D -I). In summary, while ETD1 improved rice blast resistance, the lesion-like phenotype significantly affected normal rice growth.

[0042] Example 2: Specific implementation plan for OsCNGC13 gene knockout

[0043] Targets (SEQ ID NO.4 and SEQ ID NO.5) were selected on the first exon of the OsCNGC13 gene. Using synthesized target primers, a gRNA fragment was obtained by PCR. An intermediate vector containing the gRNA fragment was constructed using Eco31I digestion and T4 ligase, and sequenced for verification. The gRNA fragment from the intermediate vector was then transferred to the rice genetic transformation vector YLCas9-hu to obtain the YLCas9-hu-CNGC13 gene knockout vector. The recombinant plasmid was transformed into Agrobacterium tumefaciens GV3101, and OsCNGC13 transgenic lines were obtained through Agrobacterium-mediated transformation of mature rice embryos. Homozygous edited lines were screened by target site sequencing in the T2 generation of transgenic cells, and OsCNGC13 gene knockout lines without transgenic elements were obtained by leaf hygromycin sensitivity testing.

[0044] The specific experimental plan is as follows:

[0045] 1. CRISPR target sites, target site adapter primer design, and gRNA acquisition

[0046] The gRNAs (SEQ ID NO. 4 and SEQ ID NO. 5) of the first exon of the OsCNGC13 gene were designed using an online website (http: / / tools.genome-engineering.org).

[0047] The gRNA fragment was obtained by PCR amplification. The PCR system consisted of 1 μl SEQ ID NO.4 (100 μM), 1 μl SEQ ID NO.5 (100 μM), and 8 μl ddH2O. The PCR instrument was heated at 95℃ for 5 min, then slowly cooled to room temperature for 1 h, and the duplex was diluted 1:200.

[0048] 2. Construction of intermediate vectors containing gRNA fragments

[0049] (1) Extraction of vector plasmids

[0050] The intermediate subcloning vector lentiCRISPR plasmid and the rice genetic transformation vector YLCas9-hu were constructed by alkaline cleavage to extract plasmids.

[0051] The specific steps for extracting plasmid DNA using the alkaline cleavage method are as follows:

[0052] 1) Remove the glycerol bacteria from the -80℃ ultra-low temperature freezer and put them into 4-6 ml of LB medium containing antibiotics, and incubate overnight at 37℃ and 200 rpm in a shaker;

[0053] 2) Take 2 ml of bacterial culture, centrifuge at 1000 rpm for 1 min, and collect the bacterial cells;

[0054] 3) Discard the supernatant and add 100 μl of solution I vortexed bacterial cells;

[0055] 4) Add 200 μl of solution II, mix gently from top to bottom, and let stand for 2 minutes. This operation should be completed within 5 minutes.

[0056] 5) Add 150 μl of solution III and gently mix.

[0057] 6) Centrifuge at 12000 rpm for 10 min;

[0058] 7) Transfer 440 μl of supernatant to a new 1.5 ml centrifuge tube, add 880 μl of anhydrous ethanol, mix well, and centrifuge at 12000 rpm for 10 min.

[0059] 8) Discard the supernatant, add 600 μl of 75% ethanol, and centrifuge at 12000 rpm for 10 min;

[0060] 9) Discard the supernatant, dry the residual moisture and alcohol in a 37°C oven, and add 30-50 μl of ultrapure water.

[0061] (2) 20 μl plasmid digestion system: 2 μg lentiCRISPR plasmid, 1 μl Eco31I (10 U / μl), 2 μl 10×rcutsmart buffer, add ultrapure water to 20 μl. Digest for 6-8 hours.

[0062] (3) 10 μl enzyme digestion and ligation system: 1 μl T4 DNA ligase, 2 μl 5× DNA ligase buffer, 1 μl digested lentiCRISPR plasmid, 6 μl duplex (diluted 200 times). Ligate at room temperature for 4-6 hours.

[0063] (4) Transformation: Take out from -80℃, quickly insert 100μl of DH5α competent cells into ice, wait for the bacterial block to thaw, add the ligation product and gently mix by tapping the bottom of the EP tube (avoid pipetting), and let stand on ice for 25min; then heat shock in a 42℃ water bath for 45s, quickly put back on ice and let stand for 2min, add 700μl of sterile LB medium without antibiotics to the centrifuge tube, mix well, and revive at 37℃, 200rpm for 60min; then centrifuge at 5000rpm for 1min to collect the bacterial cells, keep 100μl of supernatant, gently pipette to resuspend the bacterial block and spread it on LB medium containing Kana antibiotic; finally, invert the plate and incubate overnight in a 37℃ incubator.

[0064] (5) Agrobacterium transformation: Remove GV3101 competent Agrobacterium cells from the -80℃ freezer and quickly insert them into an ice box to thaw them; add DNA sample and mix gently, then incubate sequentially on an ice box for 5 min, in liquid nitrogen for 5 min (or in a dry ice ethanol bath at -80℃ and -80℃), in a 37℃ water bath for 5 min, and in an ice bath for 5 min; add 900 μl of antibiotic-free YEB liquid medium, mix well, and incubate at 28℃ with shaking for 2-3 h. 4. Centrifuge at 6000 rpm for 1 min to collect the bacterial cells, and resuspend approximately 100 μl of the supernatant by gently pipetting and spreading the bacterial block onto a YEB plate containing the appropriate antibiotic, then invert and incubate at 28℃ for 2-3 days.

[0065] (6) Agrobacterium-mediated genetic transformation of mature rice embryos:

[0066] 1) Induction of callus: After the mature rice seeds are peeled and thoroughly disinfected, they are soaked in 0.15% HgCl2 and then placed in callus culture medium for dark culture to induce the production of callus tissue.

[0067] 2) Subculture: From the induced callus, dilute callus tissue is transferred to subculture medium for dark culture;

[0068] 3) Pre-culture: Add subcultured callus to a sterile pre-culture medium containing 300 μl of 40% glucose and culture in the dark;

[0069] 4) Infection and co-culture: Pour the activated Agrobacterium bacterial solution into the callus tissue, soak it, and when drying the callus, add AS + 50% glucose to the co-culture medium in advance and pour it into a dish. Then, spread the fully dried callus evenly on the co-culture medium with a spoon and carry out dark culture.

[0070] 5) Wash the Agrobacterium with water and then continue the dark culture;

[0071] 6) Screening: Prepare a screening medium pre-added with CN, Hn and 50% glucose. Place the callus without Agrobacterium contamination in the screening medium and incubate in the dark.

[0072] 7) Differentiation: Place small pieces of resistant callus on differentiation medium and culture under light until seedlings differentiate.

[0073] 8) Rooting: Transfer the seedlings to a rooting medium;

[0074] 9) Hardening off seedlings: After the transformed seedlings have grown vigorously, remove the sterile film sealing the seedlings, add tap water, and carry out light culture to harden off the seedlings until the transformed seedlings are transferred to the field to complete the genetic transformation process.

[0075] 10) Leaf hygromycin sensitivity test: Using non-transgenic rice IR64 as a control, the OsCNGC13 gene knockout line without transgenic elements in the transgenic T2 generation was screened.

[0076] Example 3: Specific implementation plan for constructing the ETD1 gene recombinant vector

[0077] ETD1 seedling leaves were collected, and genomic DNA and RNA were extracted. Specific primers were designed to amplify a 4.1 kb DNA fragment containing the ETD1 gene promoter and some exons using the genomic DNA as a template. A TA-promoter clone was constructed and sequenced for verification. RNA was reverse-engineered into cDNA, and primers were designed to amplify the complete CDS DNA fragment of the ETD1 gene using the cDNA as a template. An approximately 3 kb DNA fragment was obtained by fusion of the GFP gene and the Nons gene terminator at the 3' end of the ETD1 gene using overlapping PCR. A TA-ETD1-GFP clone was constructed and sequenced for verification. The approximately 4.1 kb DNA fragment was recovered by digesting the TA-promoter plasmid with EcoRI and SalI, and the approximately 3 kb DNA fragment was recovered by digesting the TA-ETD1-GFP plasmid with SalI and PstI. The fragment was then introduced into the pCAMBIA1305.2 backbone digested with EcoRI and PstI using T4 ligase to obtain the ETD1 transgenic vector.

[0078] The specific experimental plan is as follows:

[0079] 1. Extraction of rice genomic DNA

[0080] (1) Cut a 2-3cm long leaf into a 2ml centrifuge tube, add steel balls, freeze with liquid nitrogen, and then crush the leaf into powder in a crusher;

[0081] (2) Centrifuge at 10000 prm for 30s, add 800 μl of 2×CTAB, and incubate in a water bath at 65℃ for 30min;

[0082] (3) Add 800 μl of chloroform, shake vigorously up and down to mix evenly, and centrifuge at 10000 rpm for 10 min;

[0083] (4) Pipette 600 μl of supernatant into a 1.5 ml centrifuge tube, add the same volume of isopropanol, and incubate at -20 °C for 2 h.

[0084] (5) Centrifuge at 12000 rpm for 10 min, discard the supernatant, and drain the water;

[0085] (6) Add 600 μl of 75% alcohol, shake up and down, centrifuge at 12000 rpm for 10 min, discard the supernatant, and repeat twice;

[0086] (7) After opening the lid and drying the residual alcohol and water at 37°C, add 100μl ddH2O.

[0087] 2. Total RNA extraction from rice

[0088] (1) Quickly freeze fresh leaves with liquid nitrogen, and then quickly place them in a mortar that has been sterilized and pre-frozen with liquid nitrogen for grinding. During this process, liquid nitrogen is continuously added to keep the sample at a low temperature until the sample is ground into powder.

[0089] (2) Transfer the powdered sample to a 2ml RNA-free centrifuge tube using a sterilized spatula, add 1ml RNAisolater and vortex, let stand at room temperature for 5min to completely separate the nucleoprotein complex;

[0090] (3) Add 200 μl of chloroform, shake vigorously for 15 seconds to form an emulsion, and let stand at 4°C for 5 minutes;

[0091] (4) After centrifuging at 12,000 rpm for 15 min at 4℃, carefully remove the sample and place it on ice. At this time, the sample is divided into three layers: a colorless aqueous phase (upper layer), a white middle layer, and a red organic layer (lower layer);

[0092] (5) Transfer the upper aqueous phase to a new enzyme-free sterile 1.5ml centrifuge tube, add the same volume of isopropanol pre-cooled at 4℃, mix gently and place on ice for 10min.

[0093] (6) Centrifuge at 12000rpm for 10min at 4℃. At this time, a white precipitate can be seen at the bottom of the tube. Carefully discard the supernatant, wash off the protein with 1ml of 75% alcohol (prepared with RNase-free ddH2O), and carefully blow the white precipitate with a pipette tip to make it float. Let it stand at room temperature for 3-5min.

[0094] (7) Centrifuge at 12000 rpm for 10 min at 4℃ and discard the supernatant;

[0095] (8) Open the cap of the centrifuge tube in a pre-sterilized ultraviolet laminar flow hood and let the precipitate air dry for 5 minutes. Be careful not to over-dry it to avoid making the extracted RNA difficult to dissolve.

[0096] (9) Dissolve the RNA with ddH2O (RNase-free), take a small amount of RNA for detection, and store the rest of the sample in an ultra-low temperature freezer at -80℃.

[0097] 3. Reverse transcription to obtain cDNA

[0098] (1) Genomic DNA removal system (16 μl): 10 pg-100 ng or Poly A+ RNA, 1 pg-1 μg Total RNA, 1 μl Random hexamers (50 ng / μl), 1 μl Oligo(dT)23VN (50 μM), 4 μl 4×g DNA wiperMix, and up RNase-free ddH2O to 16 μl. After gentle mixing, place in a PCR instrument at 42℃ for 2 min.

[0099] (2) First-strand cDNA synthesis reaction system (20 μl): 16 μl genomic DNA removal mixture, 2 μl HiScript II Enzyme Mix, 2 μl 10×RT Mix. Mix gently until homogeneous. Reaction conditions: 50℃ for 15 min, 85℃ for 2 min.

[0100] (3) After aliquoting, store the product at -80℃. Avoid repeated freeze-thaw cycles on cDNA.

[0101] 4. Construction of the ETD1 gene promoter and CDS intermediate vector

[0102] (1) Obtaining the ETD1 gene promoter and some exons:

[0103] PCR system (50 μl): 10 μl 5×SF Buffer (with 10 mM MgSO4), 1 μl dNTP Mix (10 mM each), 1 μl rice genomic DNA, 1.5 μl upstream primer (10 μM), 1.5 μl downstream primer (10 μM), 1 μl Phanta Super-Fidelity DNA Polymerase, 34 μl ddH2O.

[0104] PCR amplification was performed in a PCR instrument. The PCR conditions were as follows: 95℃ pre-denaturation for 3 min, followed by 95℃ denaturation for 15 s, 55℃ annealing for 15 s, 72℃ extension for 30 s / kb, for 32 cycles, then 72℃ double extension for 5 min, and finally incubation at 16℃.

[0105] (2) Obtaining the terminator of the 3' fusion of the ETD1 gene with the GFP gene and the Nons gene

[0106] Overlapping PCR System: The first round of PCR amplification was performed using primers to amplify the complete CDS of the ETD1 gene, the 3' fusion of the ETD1 gene with the GFP gene, and the Nons gene terminator. The products were recovered by column chromatography to obtain intermediate fragments A' (complete ETD1 gene CDS) and B' (GFP gene and Nons gene terminator). The PCR amplification system and conditions were the same as the previous step. The second round of PCR used intermediate fragments A' and B' as templates. The concentrations of both fragments were measured, and they were added in equimolar amounts, ensuring the total amount did not exceed 100 ng. Primers A1 and B2 were used only; all other components remained unchanged. The PCR system and conditions were identical.

[0107] The PCR products were subjected to nucleic acid gel electrophoresis to verify whether the length of the amplified gene fragment was correct. If correct, the gel was excised, recovered, and transformed for sequencing.

[0108] (3) Product purification:

[0109] 1) After DNA electrophoresis, quickly cut the gel containing the target DNA fragment under UV light, removing as much excess gel as possible. Weigh the gel (excluding the weight of the empty tube); 100 mg of gel is equivalent to 100 μl, which is considered one gel volume. Add an equal volume of Buffer GDP. Incubate in a 50–55°C water bath, inverting twice during the bath to accelerate gel dissolution.

[0110] 2) Briefly centrifuge to collect droplets on the tube wall. Place the FastPure DNA Mini Columns-G adsorption column in a 2ml Collection Tube, transfer ≤700μl of sol to the adsorption column, and centrifuge at 12,000 rpm for 30-60 seconds. If the sol volume is >700μl, return the adsorption column to the collection tube, transfer the remaining sol to the adsorption column, and centrifuge at 12,000 rpm for 30-60 seconds.

[0111] 3) Discard the filtrate and place the adsorption column in the collection tube. Add 300 μl of Buffer GDP to the adsorption column. Let stand for 1 min. Centrifuge at 12,000 rpm for 30-60 s.

[0112] 4) Discard the filtrate and place the adsorption column in the collection tube. Add 700 μl of Buffer GW (with anhydrous ethanol added) to the adsorption column and mix by inverting 2-3 times. Centrifuge at 12,000 rpm for 30-60 seconds. Repeat twice.

[0113] 5) Discard the filtrate and place the adsorption column back into the collection tube. Centrifuge at 12,000 rpm for 2 min.

[0114] 6) Place the adsorption column in a 1.5 ml sterile centrifuge tube, add 20-30 μl of Elution Buffer to the center of the adsorption column, and incubate for 2 min. Centrifuge at 12,000 rpm for 1 min. Discard the adsorption column and store the DNA at -20℃ for later use.

[0115] (4) TA cloning and transformation:

[0116] 1) TA cloning reaction system (5 μl): 1 μl 5×TA / Blunt-Zero Cloning Mix, 200 ng DNA fragment (2-5 kb in size), and finally add ddH2O to bring the total system volume to 5 μl. The PCR instrument was set at 25℃ for 5 min.

[0117] 2) Transformation: Refer to the above methods.

[0118] 3) Selection and verification of candidate intermediate clones: 24 single colonies of TA-promoter clone and TA-ETD1-GFP clone were selected respectively. They were digested with EcoRI and SalI, SalI and PstI respectively, and electrophoresed with 1% agarose gel. The correct TA-promoter clone and TA-ETD1-GFP clone were selected and sent to the sequencing company for sequencing verification.

[0119] 5. Obtaining the ETD1 transgenic vector:

[0120] (1) Preparation of the pCAMBIA1305.2 support framework:

[0121] 1) EcoRI and PstI digested the vector pCAMBIA1305.2 backbone.

[0122] 50 μl enzyme digestion system: 10 μl pCAMBIA1305.2 vector, 1 μl EcoRI (10 U / μl), 1 μl PstI (10 U / μl), 5 μl 10×rcutsmart buffer, 33 μl ddH2O.

[0123] 2) Digest at 37℃ for 4-5 hours, then take 1 μl for electrophoresis to observe whether the digestion is complete.

[0124] 3) After the remaining enzyme digestion samples are processed by pulsed electrophoresis, the 12kb linearized pCAMBIA1305.2 vector is recovered by gel excision. For specific steps, please refer to the method described above.

[0125] (2) Obtaining the ETD1 promoter and the ETD1 CDS-GFP fragment:

[0126] 1) EcoRI and SalI digestion of the TA-promoter plasmid yielded a DNA fragment of approximately 4.1 kb.

[0127] 50 μl enzyme digestion system: 20 μl TA-promoter vector, 1 μl EcoRI (10 U / μl), 1 μl SalI (10 U / μl), 5 μl 10×rcutsmart buffer, 23 μl ddH2O.

[0128] 2) Digestion of the TA-ETD1-GFP plasmid with SalI and PstI enzymes yielded a DNA fragment of approximately 3 kb.

[0129] 50 μl enzyme digestion system: 20 μl TA-promoter vector, 1 μl PstI (10 U / μl), 1 μl SalI (10 U / μl), 5 μl 10×rcutsmart buffer, 23 μl ddH2O.

[0130] 3) Digest at 37℃ for 7-8 hours, then take 1 μl for electrophoresis to observe whether the digestion is complete.

[0131] 4) After the remaining enzyme digestion samples are processed by pulse electrophoresis, the 4.1kb and 3kb linearized ETD1 promoter and ETD1 CDS-GFP fragments are recovered by gel extraction, respectively. For specific steps, please refer to the above method.

[0132] (3) Enzyme digestion and ligation:

[0133] 10 μl restriction enzyme ligation system: 1 μl T4 DNA ligase, 2 μl 5× DNA ligase buffer, 1 μl pCAMBIA1305.2 linearized backbone, 2 μl ETD1 promoter digested and recovered fragment, 1.5 μl ETD1 CDS-GFP digested and recovered fragment, 2.5 μl ddH2O. Ligate overnight at 16℃.

[0134] (4) Transformation: Refer to the above methods

[0135] (5) Selection and verification of candidate clones: 24 single clones were selected, digested with EcoRI and PstI, and electrophoresed with 1% agarose. Single clones with the correct digestion size were selected and sent to a sequencing company for sequencing verification.

[0136] Example 4: Verification that ETD1 is a gain-of-function mutation of the cyclic nucleotide-gated channel protein OsCNGC13

[0137] In the F2 population constructed by crossing ETD1 with CO39, the ratio of normal plants to lesion-like plants was approximately 3:1, indicating that ETD1 is a recessive gene. To further locate ETD1, 50 normal plants and 50 lesion-like plants from the F2 population were selected for BSA sequencing. Analysis showed that a missense mutation located on rice chromosome 6 was highly linked to the lesion-like phenotype. Figure 3A The mutation, located on the seventh exon of the LOC_Os06g10580 gene, resulted in a GA variant causing an amino acid alteration. Figure 3B Previous studies have shown that LOC_Os06g10580 encodes the cyclic nucleotide-gated protein OsCNGC13, and the amino acid alteration is located in a conserved region of the 6th transmembrane domain of this protein. Figure 3C Specifically, the glycine at position 483 is mutated into glutamic acid.

[0138] Knockout of the ETD1 gene did not induce cell death after inoculation with rice blast fungus. Figure 3D -E). Under NIP background conditions, knocking out OsCNGC13 did not result in cell death phenotype observed in OsCNGC13 knockout lines inoculated with *Magnapordica oryzae*. Figure 3F -G).

[0139] A genetic complement plasmid (PGETD11-GFP) including a wild-type 4kb promoter, the complete ETD1 CDS sequence, and a GFP sequence was constructed and complemented. Inoculation with *Blastoma oryzae* showed that when ETD1 was successfully introduced into the OsCNGC13 gene knockout line, the transgenic line exhibited significant cell death. Figure 3F-G), and also exhibits a lesion-like phenotype during growth and development. The above experiments indicate that ETD1 is a gain-of-function mutant gene of OsCNGC13.

[0140] Example 5: Verification that ETD1 is beneficial in improving the early and mid-stage resistance of gene knockout rice to rice blast fungus and reducing damage at maturity.

[0141] It should be noted that ETD1 is a superalle of OsCNGC13, but in a recessive relationship. ETD1 cannot function in the presence of OsCNGC13. Therefore, the original gene needs to be knocked out and replaced with ETD1 to function.

[0142] Therefore, for OsCNGC13 knockout rice, although ETD1, under the stimulation of rice blast fungus, leads to a significantly higher degree of leaf cell death than the wild type and results in significantly inferior agronomic traits (see Figure 2), this response mechanism is actually beneficial to the host in resisting the invasion of foreign pathogens and establishing an effective immune defense mechanism. As long as the rice blast fungus invasion does not occur during the rice grain-filling and ripening stage, once the invasion period has passed, with the provision of reasonable nutrition, light, and other conditions in the later stages, in the absence of rice blast fungus, the subsequent growth of transgenic rice basically or completely recovers to normal growth, and has virtually no impact on yield.

[0143] like Figure 3H As shown, for Figure 3F In late-stage field experiments of the -G gene knockout transgenic lines, transgenic lines in early to mid-stage rice (seedling stage, transplanting stage, tillering stage, or heading and flowering stage) gradually recovered their original growth without showing obvious wilting (see...). Figure 3H (See right figure) In subsequent experiments, this strain eventually reached maturity.

[0144] In contrast, wild-type rice lines in the same field and at the same growth stage almost all withered and died after inoculation with rice blast fungus, indicating that the wild-type lines would face total crop failure (see...). Figure 3H (Left image).

[0145] Example 6: Verification of ETD1-encoded enhanced calcium ion inward channel

[0146] OsCNGC13 encodes a cyclic nucleotide-gated calcium channel protein involved in rice fertility regulation. To analyze the ion channel function of ETD1, we first expressed ETD1 in *E. coli* to examine the dependence of intracellular calcium accumulation on extracellular calcium concentration and time. The results showed that ETD1 increases intracellular calcium accumulation in a time-dependent manner relative to extracellular calcium concentration. Furthermore, compared to OsCNGC13, ETD1 exhibits stronger calcium transport capacity. Figure 4A -B). To further prove whether ETD1 has Ca 2+ Selective use of 100 μM exogenous Gd 3+ (Ca 2+ Channel blockers can significantly inhibit intracellular calcium. 2+ The accumulation of calcium ions, and the use of 1mMACC (calcium channel activator) can significantly increase intracellular calcium levels. 2+ accumulation ( Figure 4C This indicates that ETD1 has calcium ion selective permeability.

[0147] Patch-clamp experiments showed that Xenopus laevis expressing ETD1 responded to 30 mM Ca2+. 2+ A large inward current was observed in the bath fluid, but only a small inward current was observed in Xenopus oocytes expressing OsCNGC13. Figure 4D -E). Some plant plasma membranes Ca 2+ The channel has Ba 2+ To further analyze the permeability, we analyzed the effects of ETD1 and OsCNGC13 on Ba. 2+ The permeability. We used the same concentration of Ba... 2+ Replace the Ca in the bath liquid 2+ Significant inward currents were observed in Xenopus laevis oocytes expressing ETD1, while only minute inward currents were observed in Xenopus laevis oocytes expressing OsCNGC13. This result is consistent with Ca... 2+ Consistent observations were made in the bath solution. Figure 4F Electrophysiological experiments have shown that ETD1 has a significantly enhanced calcium ion transport capacity compared to OsCNGC13.

[0148] Next, non-destructive microelectrometry (NMT) was used to detect the rice roots. Under normal circumstances, there was no difference in the NMT signals of IR64 and ETD1. Figure 4F ). After adding 10mM Ca 2+ Subsequently, significant extracellular Ca2+ was detected in IR64 and ETD1. 2+ The influx of NMT signal was higher than that mediated by ETD1 than that IR64. Figure 4G -H). We also constructed transgenic lines for the NES-YC3.6 calcium imaging system in IR64 and ETD1 backgrounds, under 10 mM Ca 2+ Treatment of ETD1 relative to IR64 root cell cytoplasmic Ca 2+ The concentration increased significantly. These results indicate that the ETD1-encoded calcium ion channel directs calcium ions inward. 2+ Transportation capacity has been significantly enhanced.

[0149] Example 7: ETD1-enhanced calcium ion influx induced cell death under immune activation

[0150] Reactive oxygen species (ROS) bursts are an important immune event. We used PAMP to induce immune activation and observe ROS bursts. After treatment with Chitin and flg22, the ROS production level in ETD1 leaves was higher than that in IR64. Figure 5A -B). Ca 2+ Influx precedes and is necessary for ROS burst. NMT experiments showed that, under Chitin or flg22 stimulation, ETD1 mesophyll cells produced robust and rapid Ca2+ production compared to IR64. 2+ internal flow ( Figure 5C ). Calcium imaging analysis based on YC3.6 revealed that, upon stimulation by Chitin or flg22, ETD1 relative to IR64 cytoplasmic calcium... 2+ Significantly increased ( Figure 5D The above results indicate that ETD1 enhances calcium ion influx and ROS burst under immune activation.

[0151] Further research revealed that 12 hours after inoculation with *Magnapordica oryzae*, the intracellular calcium ion accumulation level in ETD1 mesophyll cells was significantly increased (10 G-H). Intracellular calcium ion accumulation can lead to cell death; to further verify this, we used Gd... 3+ Inhibition of ETD1 channel activity. Results indicate that Gd... 3+ It can significantly inhibit cell death induced by ETD1 immune activation. Figure 5I The above results indicate that immune-triggered ETD1-enhanced calcium ion influx leads to cell death.

[0152] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the above-disclosed technical content to create equivalent embodiments without departing from the scope of the present invention. Any simple modifications, equivalent changes, and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

[0153] The full-length genome sequence of ETD1 (SEQ ID NO:1, containing introns and exons):

[0154] ATGTCTGGCCAAGAGAGAGATGATGTTCCAATGCTAGAGCTACAAAGATTTCCTACTAGAAGTGTATCGA

[0155] TGTGTATACCTGTCAGGGATGATATATATGAAGACTCAATCATATCCCACAGTGGTCCTATTTTCACCCCA

[0156] GCACCAACTCAGTATACATCCGTGGCTATTCCATCAGGAAACAGAGACATGCTCGATAAGCTGCCTCGTCC

[0157] AAAGGTTAAGAGCAAACCACATGTTGTTACGCCGGAAGAAGTTGGAATAAGTAACTGGCCTTATGACCAG

[0158] CATGTTCCGAAGAACAAACACCTGATGATGTACTCTGAGCCTCTGGGACTGTGTGATAATCCTGATTGTGT

[0159] CGACTGTCCTCGTGCTTGTAAAAACAAAAGGCATTTCCAGAGAAGTTTAGCTCCGTTTGATAACAAGGTGA

[0160] TATTCTCAGTCATGAACACCACTGACTTGCCACCTCCCTTTAAACACATTTCCTTTTGAGAAAAGCATAAGC

[0161] ATATTGATTCATGTGGTGGCTTGGATTGGAGCTCTATATTAAGAGGTCTTCAAAACTATTTGGTTGCCTTGT

[0162] TATAGTTGTCAAATTTTAGATGTTTATAGATCAACTGACTGACTCTTGATTCATTCTGTGCAACCTTTAAGG

[0163] AAACAACTGTTTCACACATATTGTATTTTCATGCTTTAGCTGTGGATTATTTGAGTTATCTAAGATCTGTTTG

[0164] GTTTGACAAATTAAACCATCCTATGATTAGTCAATTATAGGGTCATCCCATGGTTTTGGAGGCATGATCCC

[0165] GTTCCTCATTTTCCTACCAAGGATATGTTTTAAGGGATGAAGTGGTTACGTACCACTTTACTCTATTCATTG

[0166] AACCAAGCAAGTAGCTAGCCAGGTTTGAGCTAGAATCCAATGGTTGATTGACCAACATATTCCTCAAACTA

[0167] AAAGTCCCATTAAACATTCTCAACTCGCCCCTTTCATAAATGTTAAAGCACAAAACTGAAGCAATGTAAAG

[0168] AAATTTACATTGTACAAATCCCCTAGAATTTATTAAATTCAAGTGGTGTTTTTATTTTTGAACTTTGCAAAGC

[0169] TGCAAATTTTACAAATGACTTTAAATCATCATAATTGTCGTTGTTGTTGTTATTGTCACTGTCTGTTGTTGAC

[0170] TTTTTGCAATTTCTTGGAGTATTTGTTACTGGAATTTATTCATGTCTCCGTGGCTCTGTGCATCTCTCTACTG

[0171] ATGAATGTGCTATTGATATATCTCGTTTATTATTGTAGTTTCACAACATTCTTTATGGCTACGGTGATCGAT

[0172] GGAAGAAGAAAGCTGGGCATTACCTTTCATACATTCCAATTATGAAGCCACATGATAAGGCTGTTCATCG

[0173] GTGGAACCAGTTTTTGTGATATCATGCTTGCTAGCCATATTCAATGACCCTCTGTTTTTCTTCCTATTGTCA

[0174] GTAGATAAGGTAAGGCTACAAGTTTGACAACTGTTATATGAAAGAATGGACTGAGGCCATCTCTTTACAT

[0175] GTTCATTTCTTTCCTCAATATCAAATGTTGCAGATTTACATCCACAATGGCTCTAACACAAATGAGCCCACT

[0176] ACTATTAGTCTGTGTGAAGTGTCATTGTCAAGCCACTAGTACAGTTTGAACATGTAGGCTAACGCTATAAA

[0177] AAAATCAACCATAACAGTTCATCTCAATTGTTTTCTATTCTTGCAACTATTTATATATAAATTATATTACAGA

[0178] GAATGGATCCAATTTAACCACAGTACATATATATGTATTTCTGTATATTTGTTTTTTTGGTGGTGCAGGATTATA

[0179] AGTGTATAGTGTTCAACTGGAATTTCGCGATAGCATTAGCTGTTGGAAGAAGTGTGACTGACGCCATATA

[0180] TTTTTTACACATGCTTCTTCAGGTGATATATTAACATGTAATTTATTGTATTTATACTTTTCCTTCTCCATTAA

[0181] ATGAATATGCAGATCCAATGTATTCTTAAGTGAGATGATTGGTTTATTCTTTTAATTTCCTATGATGGGCCA

[0182] AATACACCTTTTGGATTTGCACAGGTTTTGGGTGCATGTACATTATGGCATGGCTTGTTGGACAAAAAACT

[0183] GCACATGCCCTTAGTTTTAAAGAAATCTCTTAAAAGTTAGCATGCAAGGCTATTATATTTTTTCCATATGTT

[0184] GTTTACCATGAAGTAGTAATTTTTGCAGCAGAGGTTTTCCTTCTGTTAAATATGGAAATCTACTTCCATGTT

[0185] TAGTTTCTTCTTTTTCATGATAGTACTTCTCTATTTTATGAACTATGTGGCATTTTGGTTATATTAAGATTCCA

[0186] AGATGTATTTATATTACAAATCCAAGAAAAAGCACCATGCTCATACTGTCATACATTAAGTGTTCCCCGCCA

[0187] AAAAAAAATGCTCATACATTTGAATAACTTTTGCAGTTCAGGCTAGCATATGTAGCACCGGAGTCGAGAG

[0188] TAGTGGGAACTGGAGATTTGGTTGATGAGCCTATGAAAATTGCTATGCGCTATCTTCGTGGGTTTTTTGTA

[0189] CTCGACTTATTTGTTGTTCTTCCACTCCCTCAGGTATTGTATGCTTTGTTCACTTTGACATATGTTTGCCATAT

[0190] TAGAAAGCGGAGTAGGTTTGAGTTATGTGACCGATGGGTTTGCAGCAATGTTCTTTTTTCCATTCTTCCTTC

[0191] TTCTGTCAGCACCCAGCACTCTTCATTTCTTCGAAAACCCATTAGTTTTGCTTGTTCGTACTGGTCCTGGTTA

[0192] ATATATTACAGGTGGTTGGCCTGATTTGGCACAGGTGGTTTGCTGTTCAAATGAGCAATAACCAGGCTGA

[0193] TTTGGCCTGGTTAAAGATGAACACTTTGCTGTTTGGCATTGCTCATTTGAAGCACTAAGAGGCTTCTGACA

[0194] CTGTAGTGTGAGAGACTCAAGAACTTTATGCAATTAACAGTGTAGAATATAAGGTAGTTTTCCTACATATC

[0195] TTCAATCAATTATTGAATGATTGTTCCTAACTAGTGTCAAAACAAATCTATTGGAGCTATATATTTTTGAT

[0196] CTGGTCTAAAGCTAGAAAAGTTACAGACTTCTGCAGGCTAATTGCTTACTACATTATCTGAAAGAGACTGT

[0197] CCTGTTTTTCAGAAATTACTTTTGTCATATATGTATATCTTTGATGATTCCATTCCTCCTTGATTCAGGTGAT

[0198] GATATTGCTAGTTATCCCTAAATATGTGGGATTATCAAGTGCAAACTATGCAAAAAACTGTTGCGTGCTA

[0199] CAGTTCTTCTTCAGTATGTACCACGTATTATAAGATTTGTGCCACTGCTTGGTGGTCAATCTACAAATGGAT

[0200] TCATTTTTGAGTCAGCATGGTCTACTTTTGTGATCAATCTTCTAATGTTTGTTTTGGCTGGGCATGTCGTTG

[0201] GTTCATGTTGGTACCTCTCGGACTACAAGTAAGTCTCTTCTTTAATGACAATTCCATATGGTATATAGCTA

[0202] TGGGCGCAATTAATCAGATACTTCCCAACCACCCAAGCCGTGTTCAGATTGTATTCAGAAAGGCAGGTCTGT

[0203] AATCCCCAGATTTGGAACCATATATTTTGTTGCCTATCAGTAGGTTCTGTTATCTTTTTCAAGATTTTTTTT

[0204] CTTTTTCCAATTATTTGGAGTTGTCATTAGCACGTTGGACATAAAGTTGATTTATCAATGTTACATTTCTGCT

[0205] ATGATCTGGGCTAACAGGTACTGGATTACAGTATAAACTGACCATGCAACACCAAAGGTTGGGGACTCAG

[0206] GGTGTTACAATATCATACCTGTTTTCACTCAACATTCAACTAAAAATGTTCACCTGGGATATATTCTCATTT

[0207] GCATTTTTTTTAGATAATGGAAGCTTTATTTAGACTCAGTTAATTACATTCTCATTTGCATGATTGTCCACTT

[0208] TACTGACTTGTTTTCTTGTTTTATTCGATGATTGAAAAAGCTGTAGTCAGAATATTGTTGAAGCTACCAAGT

[0209] ATATATAAAAAATATATCTCATTTTCATTCATGTTTTGTTGCAGAGGGTTAATCAATGTCTACGGGATTCTT

[0210] GTGCTGCATCAAACATATCAAAAGCGTTGTGTAATAATTGTACAGATTGTGGAATTACTGGAATAAATAG

[0211] GACCAATTGGTTGAACAACTCAGACTTAACTGGCTGTTTTGATACTAAAAGCGGTAATTTCCCTTATGGCA

[0212] TCTACCAACAGGCAGTGTTGCTAACCACAGAACCTGGACTTAAGCGTTATATATATTCGCTCTTTTGGGGG

[0213] TTTCAGGTACCTTAATTTCTTCTTATAAGATGAATTTGTTGTCTTTTATCCATATGGTTAGGAGTGGGTCATT

[0214] GTATGGGTATCCATTATCCACTCTTAATTCAACTAAAACAACTAAATATGTTTTTTTCTGTCGCGTTACATTT

[0215] TTAACCAGTAGTATCCAGCTTGTATATGGAAAGGAGGAAAGGTTCCAACCTCCTTCCTAAATATCTACTCCC

[0216] TTTTTTACCCAAAGCAATGGGCCACTGTTGTACTTCTGGTCCAGCTTTTATCTTTTATATGCCTTTTACTGAC

[0217] CTTACTATCTTTTTTTATTTCTTCAATTATCTGATGCAGCAAATTAGTACATTAGCTGGCAATTTGATCCCAA

[0218] GTTACTTTGTATGGGAAGTAATATTCACTATGGCTATTATTGGACTGGGGTTGTTGCTTTTCGCATTACTCA

[0219] TAGAGTCCATGCAGAATTTTCTCCAAGCTCTTGGAAAAAGGTGTGCAGTTGTACCACAAGTTTATCCTATA

[0220] TGAAGTGTACTGATGAGATGCCAATGGGTTTAGTATATATACATGTAAATGATGTTACACATATGCACAGA

[0221] ATTGAAGAGAACACAAATAGCAACAACAATCAGTTTGAATAATAGAAACTGTGCGGCACTCTGGTAGTGC

[0222] CTTTTCCTAATGAAGTCTGATAACTAATGTTCCAAAGGGTATATGTTTTTTTTTTGTTTTTTTTGCAGGAGAC

[0223] TAGAGATGCAATTAAGAAGGCGTGATGTTGAACAGTGGATGACCATAGGCGGCTGCCAGAAGATCTGA

[0224] GAAGGTCTTTATATCTTCCAAATCAAATAACTGAGATATGAGGAAACTGACCTTTTTTTTTAATTTTCTTTCCT

[0225] TTCHELPTTAAGTAAACATTATTACCTGAAAAGGATAATATGTTCTCCTAGATCTGTTTACCT

[0226] AGTGAATGTCCGGTTTCAGGCCTTCAGTGGGGTAAATGAACTTCAATCCAATTTAAAATGTGACTAAGA

[0227] ATCGAATGGAGAAACTGCTACCATCATGTTGTTTGTAACAATTTCGAACAAGGGGATGGATGAG

[0228] TATTAGAGATAATGCTGGAATTAATGCATACATATCTTGAATATGTAAAATGGCTAATATTTCAAGCAG

[0229] AAGATATAAATGGTTAATAAATCTGACTTTTTCATTCAGTGAAGATAGTACCATTTTTCTTCGTGTTAGT

[0230] ACTAGTAAAATGTTTTGTTATCAWAGCTGTAGTAGTAGTAACACATTAACAACCCCAAATTTCT

[0231] ATGCTGCAAGGTGAGAATGCCTAAAGCTTTGTGGGTGTGCAGGAGGGTTAGATCTGCCGAGGTTCAG

[0232] CTGGGTAGCTACTAGAGGAGTGAATGAAGAAGAGCTTTTGAGCAATTTGCCAGAAGATTCAAAGGGG

[0233] CATACGCCGCCATTTCTTTGGGTTCCTTAAAGGTTTGTTGATTGTGTCTTTGCAACTGTTTTTTCCTCTCC

[0234] ATCTTCAATTAGTTGACACTTGACACTGGGTAGGTGCATATTTTTATTAATTAATGCAAGTTTTCCTGATAGC

[0235] CTGGACATATCCCCGTTCGCAATTGCTTCTTGTCTTTCAGATAATTATTCATGTAGTAACAACTTTACTAGGT

[0236] ACCGGTATGAGTTCCTGTTACTGGTACTGGTTTTTTTTTTTGAATGAATACTGGTAGTGGTTTTACTCAGTA

[0237] GATCCTTACACAGGAAAGGACTGAACAAACTTCCTGTAGACCAAGTTAATCCAGGTGGTACAGGTGTA

[0238] AAATTTCGGTTCTGTGCGGATGTCTTCTTTTGAATTTTGCTTTATGAAAGGAAACAGCAAGTTTTGCGATGT

[0239] GTATATGCTTAATGATTTCCTCTGTTTTGCCTTCCTTTTTCTTGCGAGAACTCTGTTTTTCTTTTAAGAATAAT

[0240] GATATGCGACTCTGTTTTTCTTTTAAGAATAATGATACGCCACCACCAATCCATTACAGCCCCTGCACTATA

[0241] TAGTACTGGGTGGCTGCGTACTTGTTATCAGAATCAGAATAAGTTCTGTTGCCTATATCATTGGGGTTGCT

[0242] GTGCCATCATTTAGTTGATCTCATGATGCCAATGCACTGTGCTGGCATAGTTCCAAGTCCAGTTGCACCGA

[0243] ATATGTTTATGTTCAACTACTTTGTCATTTTTGCATATCATGAAAAATTTCTATGCAATTATTTTTTGGAACT

[0244] TGAGGTGACTTTAACTTGCACTTTCCTTCTTTCACCATCTATTTTTAGTCCGGCTGTTTAACCTGATGGACA

[0245] ATGCAACCTGGGATGCAATTTGTGACAAGCTAAGGCAGAACTTGTATATTACAGGAAGTGATATTCTTTAT

[0246] CAGGGTGGTCCTGTTGAAAAGATGGTTTTTATAGTCAGGGGTAGATTGGAAAGCATCAGTGCAGATGGA

[0247] AATAAGTCTCCCTTGCAAGAAGGAGATGTTTGTGGTGAGGAACTCCTTTCCTGGTACTTGGAGCAATCTTC

[0248] AGTGAACCGAGGTCTGCAAATGTTATGCTTTCTTGACTTCTCGTGCCTTCTGATCTTTCCCTTTATTATGTCA

[0249] TCTAAAATGCTACCATCCTACCGTATCAGATGGTGGGAAGATCAAGTTGCATGGCATGCGTTTGGTCGCC

[0250] ATACGTACTGTCAGATGTTTAACAAATGTTGAAGCTTTTGTACTGCGAGCACGTGATCTGGAAGAAGTGA

[0251] CTTCACAATTTTCAAGATTCTTGCGCAATCCACTTGTGCTAGGTACAATCAGGTAAGAAAATACCCGAGCA

[0252] AATTACACCTTTATTTTATGAATCACAAAACAATATAGTTAATTACTAGTATTAGGTATTTAGGTTGAGGAA

[0253] AACCCAAATTAATCTAAGCAATAAATCAAATCGAAGATTTAGGGCCTATTTGGCACAGCTCCACCTCCACC

[0254] TCCACCCCTCCTGGAGTTGGAGCTCAGCCAAACAGTTTCAGCTCCACCAAAACTGGGAGTGGAGTTGGGT

[0255] GGGTTATCTCACAAAATGTACTAGAGTTGTAGAGCTGGGTTTAGGCAGCTCCACAACTCCACTCTAGCCT

[0256] CAACTCCTAGAGCAATATTTAGGAGTTGGAGCTGTACCAAACAGACCCTTACTCCTACATAATTTCAAGTT

[0257] CTATTATCGATGTGTGACAGGAAATGTGCGAAAAGTGTATTTATTTCTCCCAAGTATATTTGTCTGTTTTCC

[0258] AGCAAATAACCTTTCAAAATATTTATCCATTGAGGAGACTAAGACGTACCACCTCTGTAGAATTTTATAGG

[0259] GTGTATTTTAATTGATAAAACTCTTAAGGTCACACTTTGACAGTATATTCCTGATATCTATAAAACTAACAT

[0260] TTTGTGAAAGTATTTGAAATATAATATACTTGTACTACTACTTCCATCACAAAATGAGTTCATTTTTCACCCA

[0261] CTTTATCAAATCCCATGTAATTGTTTTTCACTTTATCTACTTTCAATGCATTTGTCCCCTACTTTTACAAATTC

[0262] CAATGCAATGATTGCTTAAAATGAACTTATTTTGGGACAAACGGGAGGGGGCAAAAAATATCTTATTTT

[0263] GGGATAGAGGAAGTATAATTTTATACAGTAAATATACTTATAATTTGAATAATCATTAGTCAAAGTTCACA

[0264] AAGTGACTTCTTTTTCAAATCAAAACACGTGGAGTGTGTAGTTATTTTAGACTTTTTTATTTTTTGCCCTAAC

[0265] TAGTTATTTTAGACTTTTTTATTTTTTGCCCTAACTAGATTCTTTGAAAGACATAATTAGGTTATATTATGGC

[0266] GGTGTCGTCATGTTTCCTTGTTAACTTTTTCTTAATTTTTTTTTTACTTCATAGATATAGGCACATGGTGGGA

[0267] TATAAACATATAAGGTGAAACAGATGAAGTAGTTTATTAATGAAAATGTTTTGAGAGCTGCTTATTTGTGC

[0268] ATGATTTATGTGATGGATGAGCATTGCTCTCTCCATTTAAAAATATAGGGCATATAAGCTTTGACACGGTC

[0269] TTCAACATAGAACTTTGACTATTAGTTCTTTTGCAAATATTACCAACGACTATGAAATGAATATCATACAAA

[0270] GGTATTTTCAAATATGAATTTAATGATATCACATACGTAACACACAAATACACAGCATAAAGACACTCCTC

[0271] AGATGCGCAGTCTATTGTATCACATAGGAATTTTGAATTTTTCAATTAGTGTAGCTGGAGCCTGGAGGATA

[0272] TCATATTCCCATGGCCCTAACTTACTCTGTTCACTGATGCTTTCGTGAACATGAAAAACAGGTATGAATCAC

[0273] CTTACTGGAAGAACCTTGCAGCAAATCGCATCCAAGTCGCATGGAGGTATCGGAAAAGGCGACTGAAGA

[0274] GAGCTGAGATGCAAAGGTTGCAATAG

[0275] Full - length cDNA sequence of ETD1 gene (SEQ ID NO:2):

[0276] ATGTCTGGCCAAGAGAGAGATGATGTTCCAATGCTAGAGCTACAAAGATTTCCTACTAGAAGTGTATCGA

[0277] TGTGTATACCTGTCAGGGATGATATATATGAAGACTCAATCATATCCCACAGTGGTCCTATTTTCACCCCA

[0278] GCACCAACTCAGTATACATCCGTGGCTATTCCATCAGGAAACAGAGACATGCTCGATAAGCTGCCTCGTCC

[0279] AAAGGTTAAGAGCAAACCACATGTTGTTACGCCGGAAGAAGTTGGAATAAGTAACTGGCCTTATGACCAG

[0280] CATGTTCCGAAGAACAAACACCTGATGATGTACTCTGAGCCTCTGGGACTGTGTGATAATCCTGATTGTGT

[0281] CGACTGTCCTCGTGCTTGTAAAAACAAAAGGCATTTCCAGAGAAGTTTAGCTCCGTTTGATAACAAGTTTC

[0282] ACAACATTCTTTATGGCTACGGTGATCGATGGAAGAAGAAAGCTGGGCATTACCTTTCATACATTCCAATT

[0283] ATGAAGCCACATGATAAGGCTGTTCATCGGTGGAACCAGTTTTTTGTGATATCATGCTTGCTAGCCATATT

[0284] CAATGACCCTCTGTTTTTCTTCCTATTGTCAGTAGATAAGGATTATAAGTGTATAGTGTTCAACTGGAATTT

[0285] CGCGATAGCATTAGCTGTTGGAAGAAGTGTGACTGACGCCATATATTTTTTACACATGCTTCTTCAGTTCA

[0286] GGCTAGCATATGTAGCACCGGAGTCGAGAGTAGTGGGAACTGGAGATTTGGTTGATGAGCCTATGAAAA

[0287] TTGCTATGCGCTATCTTCGTGGGTTTTTTGTACTCGACTTATTTGTTGTTCTTCCACTCCCTCAGGTGATGAT

[0288] ATTGCTAGTTATCCCTAAATATGTGGGATTATCAAGTGCAAACTATGCAAAAAACTTGTTGCGTGCTACAG

[0289] TTCTTCTTCAGTATGTACCACGTATTATAAGATTTGTGCCACTGCTTGGTGGTCAATCTACAAATGGATTCA

[0290] TTTTTGAGTCAGCATGGTCTACTTTTGTGATCAATCTTCTAATGTTTGTTTTGGCTGGGCATGTCGTTGGTT

[0291] CATGTTGGTACCTCTTCGGACTACAAAGGGTTAATCAATGTCTACGGGATTCTTGTGCTGCATCAAACATA

[0292] TCAAAAGCGTTGTGTAATAATTGTACAGATTGTGGAATTACTGGAATAAATAGGACCAATTGGTTGAACA

[0293] ACTCAGACTTAACTGGCTGTTTTGATACTAAAAGCGGTAATTTCCCTTATGGCATCTACCAACAGGCAGTG

[0294] TTGCTAACCACAGAACCTGGACTTAAGCGTTATATATATTCGCTCTTTTGGGGGTTTCAGCAAATTAGTAC

[0295] ATTAGCTGGCAATTTGATCCCAAGTTACTTTGTATGGGAAGTAATATTCACTATGGCTATTATTGGACTGG

[0296] GGTTGTTGCTTTTCGCATTACTCATAGAGTCCATGCAGAATTTTCTCCAAGCTCTTGGAAAAGGAGACTA

[0297] GAGATGCAATTAAGAAGGCGTGATGTTGAACAGTGGATGACCATAGGCGGCTGCCAGAAGATCTGAGA

[0298] AGGAGGGTTAGATCTGCCGAGAGGTTCAGCTGGTAGCTACTAGAGGAGTGAATGAAGAGCTTTTG

[0299] AGCAATTTGCCAGAAGATATTCAAAAGGGGCATACGCCGCCATTTCTTTGGGTTCCTTAAGAAGGTCCGGC

[0300] TGTTTAACCTGATGGACAATGCAACCTGGGATGCAATTTGTGACAAGCTAAGGCAGAACTTGTATATTACA

[0301] GGAAGTGATATTCTTTTATCAGGGTGGTCCTGTTGAAAAGATGGTTTTTATAGTCAGGGGTAGATTGGAAA

[0302] GCATCAGTGCAGATGGAAATAAGTCTCCCTTGCAAGAAGGAGATGTTTGTGGTGAGGAACTCCTTTCCTG

[0303] GTACTTGGAGCAATCTTCAGTGAACCGAGATGGTGGGAAAGATCAAGTTGCATGGCATGCGTTTGGTCGCC

[0304] ATACGTACTGTCAGATGTTTAACAAATGTTGAAGCTTTGTACTGCGAGCACGTGATCTGGAAGAAGTGA

[0305] CTTCACAATTTTCAAGATTCTTGCGCAATCCACTTGTGCTAGGTACAATCAGGTATGAATCACCTTACTGGA

[0306] AGAACCTTGCAGCAAATCGCATCCAAGTCGCATGGAGGTATCGGAAAAGGCGACTGAAGAGAGCTGAGA

[0307] TGCAAAGGTTGCAATAG

[0308] Amino acid sequence encoded by the ETD1 gene (SEQ ID NO:3):

[0309] MSGQERDDVPMLELQRFPTRSVSMCIPVRDDIYEDSIISHSGPIFTPAPTQYTSVAIPSG

[0310] NRDMLDKLPRPKVKSKPHVVTPEEVGISNWPYDQHVPKNKHLMMYSEPLGLCDNPDCVDC

[0311] PRACKNKRHFQRSLAPFDNKFHNILYGYGDRWKKKAGHYLSYIPIMKPHDKAVHRWNQFF

[0312] VISCLLAIFNDPLFFFLLSVDKDYKCIVFNWNFAIALAVGRSVTDAIYFLHMLLQFRLAY

[0313] VAPESRVVGTGDLVDEPMKIAMRYLRGFFVLDLFVVLPLPQVMILLVIPKYVGLSSANYA

[0314] KNLLRATVLLQYVPRIIRFVPLLGGQSTNGFIFESAWSTFVINLLMFVLAGHVVGSCWYL

[0315] FGLQRVNQCLRDSCAASNISKALCNNCTDCGITGINRTNWLNNSDLTGCFDTKSGNFPYG

[0316] IYQQAVLLTTEPGLKRYIYSLFWGFQQISTLAGNLIPSYFVWEVIFTMAIIGLGLLLFAL

[0317] LIESMQNFLQALGKRRLEMQLRRRDVEQWMSHRRLPEDLRRRVRSAERFSWVATRGVNEE

[0318] ELLSNLPEDIQRGIRRHFFGFLKKVRLFNLMDNATWDAICDKLRQNLYITGSDILYQGGP

[0319] VEKMVFIVRGRLESISADGNKSPLQEGDVCGEELLSWYLEQSSVNRDGGKIKLHGMRLVA

[0320] IRTVRCLTNVEAFVLRARDLEEVTSQFSRFLRNPLVLGTIRYESPYWKNLAANRIQVAWRYRKRRLKRAEMQRLQ*

[0321] OsCNGC13 Gene Editing Target 1 (SEQ ID NO:4)

[0322] agcatgtctctgtttcctgatgg OsCNGC13 Gene Editing Target 2 (SEQ ID NO:5)

[0323] gttgttacgccggaagaagttgg。

Claims

1. An ETD1 gene, the nucleotide sequence of which is shown in SEQ ID NO.

1.

2. The use of the ETD1 gene as described in claim 1 for enhancing rice resistance to rice blast.

Images