Method for screening and obtaining rice ETD1 mutant

Abstract

The invention provides a method for screening and obtaining a rice ETD1 mutant. The method comprises the following steps: treating seeds by EMS mutation on the first day to the second day; on the third day, the seeds are treated with a cleaning agent to remove residual EMS, and then the seeds are washed with water and air-dried; sowing the aired seeds, raising seedlings, transplanting and cultivating, and harvesting M1-generation seeds; harvesting the M1-generation plants, and planting the M1-generation plants into an M2 group; performing seedling-stage magnaporthe oryzae identification on each M2 group to obtain resistance-enhanced scab-like plants; and carrying out multi-band selfing on the lesion-like plant to obtain the ETD1 mutant. Wherein the ETD1 gene has a nucleotide sequence as shown in SEQ ID NO: 1, and a cDNA (complementary deoxyribonucleic acid) sequence of the ETD1 gene is as shown in SEQ ID NO: 2. The gene belongs to a super-efficient allele of OsCNGC13, obtains stronger calcium ion transport capacity, and has a function of remarkably accelerating cell calcium ion influx. Therefore, the rice resistance can be improved by normal expression or overexpression of the gene in a gene knockout type plant, and the rice blast resistance can be positively regulated and controlled.

Description

Technical Field

[0001] This invention relates to a method for screening and obtaining rice ETD1 mutants, belonging to the field of biotechnology. Background Technology

[0002] Due to issues such as the susceptibility of major resistance genes to loss due to changes in pathogen races, regional limitations, resistance costs, and genetic burdens in molecular breeding, the utilization rate of resistance genes in rice varieties promoted for production is low, necessitating better broad-spectrum and long-lasting disease resistance strategies. Furthermore, for some diseases such as sheath blight, rice false smut, and southern rice black-streaked dwarf virus, the cloning of major resistance genes and the breeding of resistant varieties are lacking, and their control mainly relies on pesticides. Therefore, improving crops' broad-spectrum resistance to multiple pathogens while reducing pesticide use has become an essential requirement for green pest 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 literature on rice blast disease research indicates that although there are reports of calcium channel protein-related genes enhancing rice blast resistance, the enhancement effect is not significant. Therefore, a method is needed to screen for rice ETD1 mutants that exhibit broad-spectrum resistance to 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 and its encoded protein, 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] The third principle of this invention is to utilize the ETD1 gene for genetic improvement of crops, particularly for resistance to rice blast, thereby cultivating broad-spectrum blast-resistant rice. Specifically, ETD1 is a super-effective 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, especially for resistance to rice blast, to cultivate broad-spectrum blast-resistant rice.

[0014] Therefore, the present invention provides a method for screening and obtaining rice ETD1 mutants, the steps of which include:

[0015] (1) Treat seeds with EMS mutation on days 1-2;

[0016] (2) On the third day, treat the seeds with a cleaning agent to remove residual EMS, then rinse with water and air dry;

[0017] (3) Sow the dried seeds, raise seedlings, transplant rice seedlings and harvest M1 generation seeds;

[0018] (4) Harvest the M1 generation plants and plant them into the M2 population;

[0019] (5) Identify rice blast pathogens in seedlings for each M2 population to obtain disease-like plants with enhanced resistance;

[0020] (6) The ETD1 mutant was obtained by self-pollinating multiple disease-spotted plants;

[0021] The rice ETD1 mutant has the ETD1 gene sequence shown in SEQ ID NO:1, and / or the protein encoded by the ETD1 gene has the amino acid sequence shown in SEQ ID NO:3, wherein the gene is a 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.

[0022] In one embodiment, the cDNA sequence of the ETD1 gene is shown in SEQ ID NO:2.

[0023] In one embodiment, the encoded protein is selected from proteins having 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more of the same identity and function as proteins obtained by substitution and / or deletion and / or addition of amino acid residues of the amino acid sequence shown in SEQ ID NO:3. In a specific embodiment, the protein includes fusion proteins with the same function obtained by attaching tags to the N-terminus and / or C-terminus.

[0024] In a preferred embodiment, the gene and / or protein has the function of regulating rice blast resistance and enhancing rice's resistance to rice blast.

[0025] In any of the above embodiments, steps (1)-(3) are small-scale processing steps, which include:

[0026] (i) Soak the seeds for 24 hours on the first day, with 120 seeds for each treatment, and repeat 3 times.

[0027] (ii) On the second day, after the seeds show signs of sprouting, prepare the mutagen EMS and set up 5 concentrations. Add 120 seeds and 10 ml of the corresponding concentration of EMS solution to a 9 mm petri dish, and incubate for 16 hours at room temperature and in a shaker at 60 rpm.

[0028] (iii) On the third day, treat the seeds with 1N NaOH to remove residual EMS, then rinse with water and air dry.

[0029] In any of the above embodiments, steps (1)-(3) are large-scale processing steps, including:

[0030] (i) On the first day, soak approximately 10,000 seeds for 24 hours.

[0031] (ii) On the second day, prepare 1 liter of EMS mutagen and perform seed treatment.

[0032] (iii) On the third day, collect the seeds, treat them with 1N NaOH to remove residual EMS, then rinse them with water and air dry.

[0033] In any of the above embodiments, the cleaning agent is a 1N NaOH solution.

[0034] In any of the above embodiments, the most suitable EMS mutagenesis concentration for confirming a germination rate of 50% is approximately 0.5%, wherein a germination rate of 50% after EMS mutagenesis is the optimal mutagenesis dose, indicating that the mutagenesis basically covers the whole genome level, and the 0.5% refers to the EMS concentration.

[0035] In any of the above embodiments, the method for identifying rice blast in step (5) includes:

[0036] (2)(a) The rice blast fungus stored on filter paper at -20°C was activated in fresh plum culture medium;

[0037] (b) The activated pathogen was cultured in the dark and under light conditions for 12 hours each to obtain a large number of rice blast fungus spores;

[0038] (c) Collecting rice blast fungus spores;

[0039] (d) Observe spore activity on a hemocytometer and calculate the spore suspension concentration, and prepare the spore solution to a concentration of (10-25)×10⁴ spores / mL;

[0040] (e) Pre-treat rice seeds before germination and then sow them;

[0041] (f) After fertilizing the seedlings, inoculate and infect them with rice blast fungus at the 3-4 leaf stage, and then allow them to grow in the dark.

[0042] (g) Observe the disease incidence of seedlings 7-9 days after inoculation and compare them with positive susceptible varieties to assess the strength of disease resistance.

[0043] In one specific implementation, step (c) involves scraping rice blast fungus spores from the plum culture medium with an appropriate amount of 0.1% Tween 20 aqueous solution, filtering the mycelium with four layers of filter cloth, and collecting the rice blast fungus spores.

[0044] In another specific implementation, step (f) involves spraying rice blast fungus spore suspension into a room inoculation box using a high-pressure spray gun, spraying 15 ml of the suspension into each seedling tray, then transferring the rice seedlings to a greenhouse environment with a temperature maintained at 25°C and a humidity of 95%, and subjecting them to 24 hours of darkness to promote the infection process of the pathogen.

[0045] In another specific implementation, after the dark growth in step (f), the rice seedlings are cultured under growing conditions (6.8 × 10³ lux, 16 hours of light at 25°C, 8 hours of darkness at 25°C) while maintaining humidity.

[0046] In other specific implementation schemes, the observation process in step (g) includes: visually investigating and recording the disease incidence of various types of rice, observing the leaves, stems, and other parts of rice seedlings, and recording disease symptoms such as the size, shape, color, and distribution of lesions. Pay attention to whether there are typical symptoms of rice blast, such as spindle-shaped lesions with brown edges, grayish-white centers, and a gray mold layer.

[0047] In other specific implementations, the positive susceptible variety in step (g) is selected from the CO30 rice variety.

[0048] Technical effect

[0049] 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.

[0050] 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 ).

[0051] 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.

[0052] 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.

[0053] 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;

[0054] 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.

[0055] 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.

[0056] 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. The ETD1 gene can be created in the host by performing single base substitution editing through gene editing technology.

[0057] 9. This invention develops ETD1 gene-specific molecular marker technology, which can be applied to the genetic improvement of crops and the rapid breeding of varieties in the long term, greatly improving screening efficiency.

[0058] 10. 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 and light 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 all withered and died 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

[0059] 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).

[0060] 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), number of tillers (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).

[0061] Figure 3 shows the superalle of OsCNGC13, with A representing the distribution of SNP indexes on chromosomes in the lesion-like phenotype pool; B representing the ETD1 mutation site; C representing the protein secondary structure prediction diagram; D representing the inoculation phenotype of the ETD1 gene knockout line with IR64 non-virulent strain V86010; E representing the inoculation phenotype of IR64 virulent strain CA89; F and G representing the inoculation phenotypes of the OsCNGC13 gene knockout line and the ETD1 genetic supplementation line with rice blast fungus. The inoculation phenotypes of NIP non-virulent strain 4029-1 (F) and NIP virulent strain S5 (G) are also shown. Figure 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.

[0062] 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 ETD12+ 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).

[0063] 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.

[0064] Figure 6 ETD1 gene-specific molecular marker identification: wild-host type IR64 showed only one band of about 250bp; ETD1 showed one band of about 450bp; the F2 segregating generations showed wild-host type (250bp), ETD1 type (450bp) and heterozygous type (two bands of 250bp and 450bp). Detailed Implementation

[0065] 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.

[0066] Example 1: Chemical mutagenesis to obtain ETD1 gene mutant

[0067] 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 1AInterestingly, 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).

[0068] The specific steps for obtaining the ETD1 mutant through EMS (ethyl methanesulfonate) chemical mutagenesis of the indica rice variety IR64 are as follows:

[0069] There are no restrictions on rice varieties; theoretically, all rice varieties can be tried.

[0070] (1) The EMS mutation treatment experimental protocol includes steps for both small-scale and large-scale methods.

[0071] Small-scale methods include:

[0072] Soak the seeds for 24 hours on the first day, with 120 seeds for each treatment, and repeat 3 times.

[0073] The next day, after the seeds showed signs of sprouting, EMS mutagen was prepared, with 5 concentrations set up. 120 seeds and 10 ml of the corresponding concentration of EMS solution were added to a 9 mm petri dish and cultured for 16 hours at room temperature in a shaker at 60 rpm.

[0074] On the third day, the seeds were treated with 1N NaOH to remove residual EMS, then rinsed with water and dried.

[0075] Finally, the seeds are evaluated between the seventh and tenth day.

[0076] The treatment concentration that yielded a germination rate of 50% was confirmed to be the most suitable EMS mutagenesis concentration (generally 0.5%).

[0077] Large-scale methods include:

[0078] Soak approximately 10,000 seeds for 24 hours on the first day;

[0079] The next day, prepare 1 liter of EMS mutagen and perform seed treatment;

[0080] On the third day, collect the seeds, treat them with 1N NaOH to remove residual EMS, then rinse them with water and air dry.

[0081] Then, the dried seeds were sown in the field, and all materials that had been exposed to the mutagen were treated with 1N NaOH.

[0082] After being treated by both methods, the seeds were dried and then cultivated into seedlings. The seedlings were transplanted and the M1 generation seeds (approximately 30,000 seedlings) were harvested.

[0083] We will harvest 30,000 M1 generation plants and plant them into 30,000 M2 populations.

[0084] Each M2 population was individually identified as a seedling blast fungus, and one offspring of the M2 population showed lesion-like plants with enhanced resistance.

[0085] The ETD1 mutant was obtained by self-pollinating multiple diseased plants.

[0086] (2) Specific identification methods for rice blast:

[0087] Rice blast fungus was stored at -20°C using filter paper.

[0088] Before use, the rice blast fungus stored on filter paper at -20℃ was placed in fresh plum culture medium for activation for a total of 5 days;

[0089] The plum culture medium utilizes plums to provide the vitamins and trace elements needed by *Magnaporum oryzae*, and is more conducive to the growth and sporulation of *Magnaporum oryzae* compared to other culture media. 1L of plum culture medium contains: 3 plums, 1g yeast extract, 5g lactose, 15g agar powder, pH=5.8. Preparation process: Blend the plums using a high-speed blender, add a little pure water and boil on an induction cooker; filter the plum juice through cheesecloth; add yeast powder, lactose, and agar powder to a final volume of 1L and adjust the pH; sterilize in an autoclave, cool, and pour into petri dishes for later use.

[0090] Then, the activated rice blast fungus was transferred to fresh plum culture medium and cultured for 14 days under conditions of 12 hours in darkness and 12 hours in light, and at a temperature of 25-28℃, in order to obtain a large number of rice blast fungus spores.

[0091] When a layer of gray mycelium covers the plum culture medium, scrape the rice blast fungus spores from the plum culture medium with an appropriate amount of 0.1% Tween 20 aqueous solution, filter the mycelium with four layers of filter cloth, and collect the rice blast fungus spores.

[0092] 10 μl of spore solution was pipetted onto a hemocytometer to observe spore activity and calculate the spore suspension concentration. The spore solution with the required activity was prepared to a concentration of (10-25)×10⁴ spores / mL.

[0093] Soak rice seeds in pure water at 28℃ for 24 hours, then drain the water and transfer the treated seeds to a germination chamber set at 37℃ until the rice seeds show signs of sprouting.

[0094] Sow the seeds into seedling trays. Sow 25-30 seeds for each rice variety.

[0095] When the rice seedlings reach the 2-leaf stage, start applying compound fertilizer, and apply compound fertilizer again 5-7 days before inoculation;

[0096] When the rice seedlings have grown to the 3-4 leaf stage, spray inoculate them with rice blast fungus. Use a high-pressure spray gun to spray inoculate in the inoculation box. Spray 15ml of rice blast fungus spore suspension into each seedling tray. Then transfer the rice seedlings to a greenhouse environment with a temperature maintained at 25℃ and humidity at 95% and implement 24 hours of darkness treatment to promote the infection process of the fungus.

[0097] After 24 hours of dark treatment, the rice seedlings were placed in growing conditions (6.8×103 lux, 16 hours of light at 25℃, 8 hours of darkness at 25℃) for cultivation, and humidity was maintained during the period.

[0098] Investigate the disease incidence in rice seedlings 7-9 days after inoculation. Use visual inspection to investigate and record the disease incidence of various rice varieties. Observe the leaves, stems, and other parts of the rice seedlings, and record the disease symptoms, such as the size, shape, color, and distribution of lesions. Pay attention to whether there are typical symptoms of rice blast, such as spindle-shaped lesions with brown edges, grayish-white centers, and a gray mold layer.

[0099] Using the susceptible rice variety CO39 as a control, the disease incidence of other rice varieties was compared with that of CO39 to assess their resistance.

[0100] 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.

[0101] 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.

[0102] Survey of agronomic traits:

[0103] 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.

[0104] 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.

[0105] 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.

[0106] Example 2: Specific implementation plan for OsCNGC13 gene knockout

[0107] 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.

[0108] The specific experimental plan is as follows:

[0109] 1. CRISPR target sites, target site adapter primer design, and gRNA acquisition

[0110] 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).

[0111] 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.

[0112] 2. Construction of intermediate vectors containing gRNA fragments

[0113] (1) Extraction of vector plasmids

[0114] The intermediate subcloning vector lentiCRISPR plasmid and the rice genetic transformation vector YLCas9-hu were constructed by alkaline cleavage to extract plasmids.

[0115] The specific steps for extracting plasmid DNA using the alkaline cleavage method are as follows:

[0116] 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;

[0117] 2) Take 2 ml of bacterial culture, centrifuge at 1000 rpm for 1 min, and collect the bacterial cells;

[0118] 3) Discard the supernatant and add 100 μl of solution I vortexed bacterial cells;

[0119] 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.

[0120] 5) Add 150 μl of solution III and gently mix.

[0121] 6) Centrifuge at 12000 rpm for 10 min;

[0122] 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.

[0123] 8) Discard the supernatant, add 600 μl of 75% ethanol, and centrifuge at 12000 rpm for 10 min;

[0124] 9) Discard the supernatant, dry the residual moisture and alcohol in a 37°C oven, and add 30-50 μl of ultrapure water.

[0125] (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.

[0126] (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.

[0127] (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.

[0128] (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.

[0129] (6) Agrobacterium-mediated genetic transformation of mature rice embryos:

[0130] 1) Callus induction: Peel mature rice seeds, treat them with 75% alcohol solution for 30 seconds, inverting them during treatment to ensure thorough disinfection, rinse 4-5 times with sterile ultrapure water, blot dry with sterile filter paper, soak in 0.15% HgCl2 for 15-20 minutes, rinse 5-10 times with sterile ultrapure water to remove surface HgCl2, and evenly place them on callus culture medium (40-50 seeds / plate). Incubate in the dark for 40-50 days to induce callus formation.

[0131] 2) Subculture: Prepare subculture medium 2-3 days in advance, sterilize it, and dry the medium; select light yellow, granular, dry, and viable callus tissue from the induced callus and transfer it into subculture medium for dark culture for 20 days; (it is best to perform infection after one subculture, and subculture a maximum of two times, otherwise the callus transformation efficiency will be very low. When subculturing for the first time, be careful to remove other tissues such as endosperm and buds attached to the callus tissue.)

[0132] 3) Pre-culture: Prepare an appropriate amount of sterile pre-culture medium in advance using a 500ml Erlenmeyer flask; before the experiment, add 300μl AS and 5ml 40% glucose to every 250ml of medium and pour into dishes, pouring the medium into 8-10 dishes per flask; from the subcultured callus, pick out the pale yellow, granular, dry, and viable callus tissue and transfer it into the pre-culture medium. Usually, inoculate about 60-80 pieces of callus the size of mung beans per dish. Larger callus can be crushed with tweezers. Incubate in the dark for 3 days.

[0133] 4) Infection and Co-culture: ① Two days before the experiment, streak the Agrobacterium strain containing the target gene onto a Petri dish containing antibiotics to activate it; ② Prepare suspension medium (100ml / fraction), co-culture medium (250ml / fraction), large Petri dishes, small Petri dishes (lined with absorbent paper and filter paper, sterilized and dried at 80℃ before use), and several 250ml sterile Erlenmeyer flasks; ③ Scrape the streaked Agrobacterium into 1 / 2N6 suspension medium (add 100μl AS + 2ml) 50% glucose), inoculate one loopful of bacteria (the amount depends on the volume of the suspension medium), incubate at 28℃ and 200 rpm for 30 minutes until slightly turbid (OD600≈0.4); ④ While shaking the bacteria, collect the pre-cultured callus tissue into a 250ml sterile Erlenmeyer flask; ⑤ Pour the Agrobacterium bacterial solution into the callus tissue and soak for 30 minutes; ⑥ Pour off the bacterial solution, first invert the Erlenmeyer flask containing the callus onto a sterile small dish of paper to pour off the bacterial solution, then spread the callus on the filter paper of a sterile large dish, cover it with another filter paper, gently press the callus with tweezers to absorb the surface bacterial solution, change the filter paper four times on each side, finally cover the callus with another filter paper, cover the large dish, and let it air dry for 3-4 hours; ⑦ When drying the callus, pour it into a co-culture dish. Add 300 μl AS + 5 ml 50% glucose to each bottle of co-culture medium and pour into 8-10 dishes per bottle; ⑧ Spread the fully dried callus evenly onto the co-culture medium with a spoon (it is best not to move it after spreading it to reduce the contact between the culture medium and the callus surface and prevent the overgrowth of Agrobacterium); seal with sealing glue; ⑨ Incubate in the dark at 19℃ for 3 days.

[0134] 5) Washing: ① Prepare sterile ultrapure water, large and small dishes (containing absorbent paper and filter paper, dried at 80℃), several 250ml Erlenmeyer flasks, and screening culture medium; ② Transfer the co-cultured callus tissue to a washing cup, pour in sterile distilled water until the callus tissue is completely submerged, cover and shake for 20-30 seconds, then discard the sterile distilled water. Repeat this 2-3 times. Add sterile ultrapure water until the callus tissue is completely submerged, cover and shake vigorously for 20-30 seconds, let stand for 5 minutes, then discard the ultrapure water. Add ultrapure water until the callus tissue is completely submerged, cover and shake vigorously for 20-30 seconds, then let stand for 10 minutes. Then observe; if the water in the washing cup is clear, it means that the Agrobacterium has been washed away; otherwise, continue washing. Finally, discard the ultrapure water, add ultrapure water containing 500mg / L Cn, and shake at 200rpm for 30 minutes. ③ Discard the sterile ultrapure water, invert the Erlenmeyer flask containing the callus onto the paper of a sterile small dish, pour off the dried bacterial solution, then spread the callus on the filter paper of a sterile large dish, cover it with another filter paper, gently press the callus with tweezers to absorb the surface bacterial solution, change the filter paper four times on each side, and finally cover the callus with another filter paper, cover the large dish, and let it air dry for 3-4 hours; ④ When drying the callus, add 400μl CN + 250μl Hn + 5ml 50% glucose to each bottle of screening medium and pour it into 8 dishes per bottle. After pouring, open the lid on the ultra-clean workbench and blow it with sterile air for 1.5-2 hours. The surface of the screening medium should not be too wet, otherwise it will not be conducive to the inhibition of Agrobacterium and the growth of resistant callus during screening; ⑤ After the callus is dried, spread the fully dried callus evenly on the co-culture medium with a spoon, and seal it with sealing glue; incubate in the dark for 20 days (S1).

[0135] 6) Screening (S2): ① Prepare screening medium. Add 300μl CN + 250μl Hn + 5ml 50% glucose to each 250ml culture medium bottle, and pour into dishes. After pouring, open the lid on a clean bench and air dry with sterile air for 1.5-2 hours. ② Select dry callus transfer dishes from S1 medium that are free from Agrobacterium contamination and place them sparsely on S2 medium (inoculate 25 to 30 callus pieces per dish). ③ Incubate in the dark for 20 days and observe whether fresh, tender yellow resistant callus has grown. If no resistant callus has grown, continue transferring to dishes for S3. Reduce the amount of CN added to the screening medium to 200μl.

[0136] 7) Differentiation: ① Prepare the differentiation medium 3-4 days in advance; ② Select small, dense, dry, pale yellow resistant callus pieces that are attached to the medium, one piece per cluster, being careful not to select those with Agrobacterium growth. Place only three pieces of resistant callus per bottle of differentiation medium, and keep them far apart. ③ Culture in light for 40 days. After the seedlings differentiate (about 5-10 cm tall), proceed to the next step. During this period, promptly remove any areas with bacterial growth.

[0137] 8) Rooting: Prepare rooting medium and 4-5 sterile empty dishes; remove the differentiated seedlings from the differentiation medium, take only one seedling from each piece of callus, cut off the excessively long leaves and roots with scissors, and inoculate them into rooting tubes, inoculating 1-2 seedlings into each tube.

[0138] 9) Hardening off: After the transformed seedlings have grown vigorously, remove the sealing film from the rooting tube, add a certain amount of tap water, and harden off for 4-7 days under light. During hardening off, small samples can be taken for positive tests. Gently remove the transformed seedlings from the rooting tube, wash off the attached culture medium on the roots, and transplant them to the field or a prepared bread box to complete the genetic transformation process.

[0139] (7) Leaf hygromycin sensitivity test: Using non-transgenic rice IR64 as a control, the transgenic T2 generation OsCNGC13 gene knockout line without transgenic elements was screened. At the three-leaf stage of rice, healthy green leaves (2cm) were cut and placed in a test solution containing 1mg / l 6-BA and 50mg / l hygromycin. Each sample was replicated 8-10 times. Using IR64 as a control, the OsCNGC13 gene knockout line without transgenic elements was selected.

[0140] Example 3: Specific implementation plan for constructing the ETD1 gene recombinant vector

[0141] 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 ligated into the pCAMBIA1305.2 backbone digested with EcoRI and PstI using T4 ligase to obtain the ETD1 transgenic vector (SEQ ID NO:6).

[0142] The specific experimental plan is as follows:

[0143] 1. Extraction of rice genomic DNA

[0144] (1) Cut a 2-3 cm long leaf into a 2 ml centrifuge tube, add steel balls, freeze with liquid nitrogen, and then crush the leaf into powder in a crusher.

[0145] (2) Centrifuge at 10000 prm for 30s, add 800 μl of 2×CTAB, and incubate in a water bath at 65℃ for 30min;

[0146] (3) Add 800 μl of chloroform, shake vigorously up and down to mix evenly, and centrifuge at 10000 rpm for 10 min;

[0147] (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.

[0148] (5) Centrifuge at 12000 rpm for 10 min, discard the supernatant, and drain the water;

[0149] (6) Add 600 μl of 75% alcohol, shake up and down, centrifuge at 12000 rpm for 10 min, discard the supernatant, and repeat twice;

[0150] (7) After opening the lid and drying the residual alcohol and water at 37°C, add 100μl ddH2O.

[0151] 2. Total RNA extraction from rice

[0152] (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.

[0153] (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;

[0154] (3) Add 200 μl of chloroform, shake vigorously for 15 seconds to form an emulsion, and let stand at 4°C for 5 minutes;

[0155] (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);

[0156] (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.

[0157] (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.

[0158] (7) Centrifuge at 12000 rpm for 10 min at 4℃ and discard the supernatant;

[0159] (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.

[0160] (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℃.

[0161] 3. Reverse transcription to obtain cDNA

[0162] (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.

[0163] (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.

[0164] (3) After aliquoting, store the product at -80℃. Avoid repeated freeze-thaw cycles on cDNA.

[0165] 4. Construction of the ETD1 gene promoter and CDS intermediate vector

[0166] (1) Obtaining the ETD1 gene promoter and some exons:

[0167] 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.

[0168] 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℃.

[0169] (2) Obtaining the terminator of the 3' fusion of the ETD1 gene with the GFP gene and the Nons gene

[0170] 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.

[0171] 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.

[0172] (3) Product purification:

[0173] 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.

[0174] 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.

[0175] 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.

[0176] 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.

[0177] 5) Discard the filtrate and place the adsorption column back into the collection tube. Centrifuge at 12,000 rpm for 2 min.

[0178] 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.

[0179] (4) TA cloning and transformation:

[0180] 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.

[0181] 2) Transformation: Refer to the above methods.

[0182] 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.

[0183] 5. Obtaining the ETD1 transgenic vector:

[0184] (1) Preparation of the pCAMBIA1305.2 support framework:

[0185] 1) EcoRI and PstI digested the vector pCAMBIA1305.2 backbone.

[0186] 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.

[0187] 2) Digest at 37℃ for 4-5 hours, then take 1 μl for electrophoresis to observe whether the digestion is complete.

[0188] 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.

[0189] (2) Obtaining the ETD1 promoter and the ETD1 CDS-GFP fragment:

[0190] 1) EcoRI and SalI digestion of the TA-promoter plasmid yielded a DNA fragment of approximately 4.1 kb.

[0191] 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.

[0192] 2) Digestion of the TA-ETD1-GFP plasmid with SalI and PstI enzymes yielded a DNA fragment of approximately 3 kb.

[0193] 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.

[0194] 3) Digest at 37℃ for 7-8 hours, then take 1 μl for electrophoresis to observe whether the digestion is complete.

[0195] 4) After the remaining enzyme digestion samples were subjected to pulsed electrophoresis, the 4.1kb and 3kb linearized ETD1 promoter and ETD1 CDS-GFP fragments were recovered by gel extraction, respectively. For specific steps, please refer to the above method.

[0196] (3) Enzyme digestion and ligation:

[0197] 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℃.

[0198] (4) Conversion: Refer to the above methods

[0199] (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.

[0200] Example 4: Verification that ETD1 is a gain-of-function mutation of the cyclic nucleotide-gated channel protein OsCNGC13

[0201] 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.

[0202] 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 after inoculation with *Magnapordica oryzae*. Figure 3F -G).

[0203] 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.

[0204] 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.

[0205] 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.

[0206] 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.

[0207] 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.

[0208] 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).

[0209] Example 6: Verification of ETD1-encoded enhanced calcium ion inward channel

[0210] 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.

[0211] 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.

[0212] 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.

[0213] Example 7: ETD1-enhanced calcium ion influx induced cell death under immune activation

[0214] 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.

[0215] Further research revealed that 12 hours after inoculation with *Magnapordica oryzae*, the intracellular calcium ion accumulation level in ETD1 mesophyll cells 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.

[0216] Example 8: Identification of ETD1-specific molecular markers

[0217] Given that the ETD1 gene differs from the OsCNGC13 gene by only one SNP, restriction enzyme markers (dCAPS) can be designed for differentiation. An ETD1 gene-specific molecular marker technology has been developed, which can be applied to crop genetic improvement and rapid variety selection. Based on this, the inventors collected leaves from transplanted IR64, ETD1, and segregating populations after their greening period and extracted genomic DNA. Specific primers C3F / C3R (SEQ ID NO:7-1 and SEQ ID NO:7-2) were designed to amplify a specific 458bp DNA fragment using the genomic DNA as a template. The PCR product was digested with the restriction endonuclease AvaII. The digested products were then differentiated by agarose gel electrophoresis (in a 2% agarose gel, the wild type showed only one band of approximately 250bp; the heterozygous type showed two bands of approximately 250bp and 450bp; and ETD1 showed one band of approximately 450bp).

[0218] 1. Extraction of rice genomic DNA

[0219] (1) Cut a 2-3 cm long leaf into a 2 ml centrifuge tube, add steel balls, freeze with liquid nitrogen, and then crush the leaf into powder in a crusher.

[0220] (2) Centrifuge at 10000 prm for 30s, add 800 μl of 2×CTAB, and incubate in a water bath at 65℃ for 30min;

[0221] (3) Add 800 μl of chloroform, shake vigorously up and down to mix evenly, and centrifuge at 10000 rpm for 10 min;

[0222] (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.

[0223] (5) Centrifuge at 12000 rpm for 10 min, discard the supernatant, and drain the water;

[0224] (6) Add 600 μl of 75% alcohol, shake up and down, centrifuge at 12000 rpm for 10 min, discard the supernatant, and repeat twice;

[0225] (7) After opening the lid and drying the residual alcohol and water at 37°C, add 100μl ddH2O.

[0226] 2. PCR reaction system preparation

[0227] (1) The 10ul PCR reaction system includes: 5ul Novizan 2×Rapid Taq Master Mix; 3ul ultrapure water; 0.5ul C3F (10uM); 0.5ul C3R (10uM); 1ul DNA.

[0228] 3. PCR reaction procedure

[0229] (1) Pre-denaturation: 95℃ for 2 min;

[0230] (2) Denaturation: 95℃ for 15s;

[0231] (3) Annealing: 58℃ for 15s;

[0232] (4) Extension: 72℃ for 30 seconds;

[0233] (5) Number of loops: 32 loops;

[0234] (6) Final extension: 72℃ for 5 min.

[0235] 4. Enzyme digestion system and reaction time

[0236] (1) 10ul enzyme digestion system: 5.7ul ultrapure water; 1ul cutsmart buffer; 0.3ul AvaII (NEB); 3ul PCR product;

[0237] (2) Enzyme digestion reaction time: 37℃ for 2 hours.

[0238] 5. Identification of enzyme digestion products by agarose gel electrophoresis

[0239] (1) Install the electrophoresis tank (Beijing Liuyi) and insert the sample comb;

[0240] (2) To prepare an agarose gel, dissolve 2g of agarose in electrophoresis buffer TAE, heat until completely dissolved, and then cool to about 60°C.

[0241] (3) Gel pouring: Pour the cooled agarose solution into the electrophoresis tank to form a gel;

[0242] (4) After the gel solidifies, add electrophoresis buffer TAE and remove the comb;

[0243] (5) Add the PCR digestion product into the sample well;

[0244] (6) Electrophoresis: Turn on the power supply, adjust the voltage to 120V / cm, and electrophore for 15 minutes;

[0245] (7) Staining and observation: Take out the gel, add it to the staining solution containing ethidium bromide, stain for 5 minutes, and then observe the DNA bands under ultraviolet light.

[0246] The results are as follows Figure 6 As shown, the molecular weights of the marker bands on the left, from top to bottom, are 2000bp, 1000bp, 750bp, 500bp, 250bp, and 100bp.

[0247] Since a 2% agarose gel can only distinguish DNA fragments of 0.1-2kb, and the 258bp and 200bp bands differ by only 58bp, they cannot be distinguished in a 2% agarose gel. Therefore, the 258bp and 200bp bands will merge into one band.

[0248] like Figure 6 As shown, the wild-type PCR product was digested into two fragments, 258 bp and 200 bp, but electrophoresis showed a single fusion band. The ETD1 PCR product could not be digested and showed a bright band of approximately 500 bp. The heterozygous PCR product, after digestion, produced three fragments: 458 bp, 258 bp, and 200 bp (the latter being a fusion band). That is, the F2 segregating generations respectively produced wild-type, ETD1 type, and heterozygous type (two bands of approximately 250 bp and 500 bp).

[0249] Therefore, it is evident that those skilled in the art can utilize this ETD1 gene-specific molecular marker technology for crop genetic improvement and rapid variety selection.

[0250] 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.

[0251] Full-length genomic sequence of ETD1 (SEQ ID NO: 1, including introns and exons):

[0252] ATGTCTGGCCAAGAGAGAGATGATGTTCCAATGCTAGAGCTACAAAGATTTCCTACTAGAAGTGTATCGATGTGTATACCTGTCA

[0253] GGGATGATATATATGAAGACTCAATCATATCCCACAGTGGTCCTATTTTCACCCCAGCACCAACTCAGTATACATCCGTGGCTATTC

[0254] CATCAGGAAACAGAGACATGCTCGATAAGCTGCCTCGTCCAAAGGTTAAGAGCAAACCACATGTTGTTACGCCGGAAGAAGTT

[0255] GGAATAAGTAACTGGCCTTATGACCAGCATGTTCCGAAGAACAAACACCTGATGATGTACTCTGAGCCTCTGGGACTGTGTGAT

[0256] AATCCTGATTGTGTCGACTGTCCTCGTGCTTGTAAAAACAAAAGGCATTTCCAGAGAAGTTTAGCTCCGTTTGATAACAAGGTGA

[0257] TATTCTCAGTCATGAACACCACTGACTTGCCACCTCCCTTTAAACACATTTCCTTTTGAGAAAAGCATAAGCATATTGATTCATGT

[0258] GGTGGCTTGGATTGGAGCTCTATATTAAGAGGTCTTCAAAACTATTTGGTTGCCTTGTTATAGTTGTCAAATTTTAGATGTTTATA

[0259] GATCAACTGACTGACTCTTGATTCATTCTGTGCAACCTTTAAGGAAACAACTGTTTCACACATATTGTATTTTCATGCTTTAGCTGT

[0260] GGATTATTTGAGTTATCTAAGATCTGTTTGGTTTGACAAATTAAACCATCCTATGATTAGTCAATTATAGGGTCATCCCATGGTTTT

[0261] GGAGGCATGATCCCGTTCCTCATTTTCCTACCAAGGATATGTTTTAAGGGATGAAGTGGTTACGTACCACTTTACTCTATTCATTG

[0262] AACCAAGCAAGTAGCTAGCCAGGTTTGAGCTAGAATCCAATGGTTGATTGACCAACATATTCCTCAAACTAAAAGTCCCATTAAA

[0263] CATTCTCAACTCGCCCCTTTCATAAATGTTAAAGCACAAAACTGAAGCAATGTAAAGAAATTTACATTGTACAAATCCCCTAGAAT

[0264] TTATTAAATTCAAGTGGTGTTTTTATTTTTGAACTTTGCAAAGCTGCAAATTTTACAAATGACTTTAAATCATCATAATTGTCGTTG

[0265] TTGTTGTTATTGTCACTGTCTGTTGTTGACTTTTTGCAATTTCTTGGAGTATTTGTTACTGAATTTATTCATGTCTCCGTGGCTCT

[0266] GTGCATCTCTCTACTGATGAATGTGCTATTGATATATCTCGTTTATTATTGTAGTTTCACAACATTCTTTATGGCTACGGTGATCGAT

[0267] GGAAGAAGAAAGCTGGGCATTACCTTTCATACATTCCAATTATGAAGCCACATGATAAGGCTGTTCATCGGTGGAACCAGTTTTT

[0268] TGTGATATCATGCTTGCTAGCCATATTCAATGACCCTCTGTTTTTCTTCCTATTGTCAGTAGATAAGGTAAGGCTACAAGTTTGACA

[0269] ACTGTTATATGAAAGAATGGACTGAGGCCATCTCTTTACATGTTCATTTCTTTCCTCAATATCAAATGTTGCAGATTTACATCCACA

[0270] ATGGCTCTAACACAAATGAGCCCACTACTATTAGTCTGTGTGAAGTGTCATTGTCAAGCCACTAGTACAGTTTGAACATGTAGGC

[0271] TAACGCTATAAAAAAATCAACCATAACAGTTCATCTCAATTGTTTCTATTCTTGCAACTATTTATATAAAATTATATTACAGAGAAT

[0272] GGATCCAATTTAACCACAGTACATATATGTATTTCTGTATATTTGTTTTTTTGTGCAGGATTATAAGTGTATAGTGTTCAACTGG

[0273] AATTTCGCGATAGCATTAGCTGTTGGAAGAAGTGTGACTGACGCCATATATTTTTTACACATGCTTCTTCAGGTGATATTAACAT

[0274] GTAATTTATTGTATTTATACTTTTCCTTCTCCATTAAATGAATATGCAGATCCAATGTATTCTTAAGTGAGAGATTGGTTTATTCTTT

[0275] TAATTTCCTATGATGGGCCAAATACACCTTTTGGATTTGCACAGGTTTTGGGTGCATGTACATTATGGCATGGCTTGTTGGACAA

[0276] AAAACTGCACATGCCCTTAGTTTTAAAGAAATCTCTTAAAAGTTAGCATGCAAGGCTATTATATTTTTTCCATATGTTGTTTACCAT

[0277] GAAGTAGTAATTTTTGCAGCAGAGGTTTTCCTTCTGTTAAATATGGAAATCTACTTCCATGTTTAGTTTCTTCTTTTTCATGATAGT

[0278] ACTTCTCTATTTTATGAACTATGTGGCATTTTGGTTATTAAGATTCCAAGAATGTATTTATATTACAAATCCAAGAAAAAGCACCAT

[0279] GCTCATACTGTCATACATTAAGTGTTCCCCGCCAAAAAAAAATGCTCATACATTTGAATAACTTTTGCAGTTCAGGCTAGCATATGT

[0280] AGCACCGGAGTCGAGAGTAGTGGGAACTGGAGATTTGGTTGATGAGCCTATGAAAATTGCTATGCGCTATCTTCGTGGGTTTTT

[0281] TGTACTCGACTTATTTGTTGTTCTTCCACTCCCTCAGGTATTGTATGCTTTGTTCACTTTGACATATGTTTGCCATATTAGAAAGCG

[0282] GAGTAGGTTTGAGTTATGTGACCGATGGGTTTGCAGCAATGTTCTTTTTTCCATTCTTCCTTCTTCTTGTCAGCACCCAGCACTCTT

[0283] CATTTCTTCGAAAACCCATTAGTTTTGCTTGTTCGTACTGGTCCTGGTTAATATATTACAGGTGGTTGGCCTGATTTGGCACAGGT

[0284] GGTTTGCTGTTCAAATGAGCAATAACCAGGCTGATTTGGCCTGTGTTAAAGATGAACACTTTGCTGTTTGGCATTGCTCATTTGAA

[0285] GCACTAAGAGGCTTCTGACACTGTAGTGTGAGAGACTCAAGAACTTTATGCAATTAACAGTGTAGAATATAAGGTAGTTTTCCTA

[0286] CATATCTTCAATCAATATATTGAATGATTGTTCCTAACTAGTGTCAAAACAAATCTATTGGAGCTATATATTTTTGATCTGGTCTAAA

[0287] GCTAGAAAAGTTACAGACTTCTGCAGGCTAATTGCTTACTACATTATCTGAAAGAGACTGTCCTGTTTTTCAGAAATTACTTTTGT

[0288] CATATATGTATATCTTTGATGATTCCATTCCTCCTTGATTCAGGTGATGATATTGCTAGTTATCCCTAAATATGTGGGATTATCAAGTG

[0289] CAAACTATGCAAAAAACTTGTTGCGTGCTACAGTTCTTCTTCAGTATGTACCACGTATTATAAGATTTGTGCCACTGCTTGGTGGT

[0290] CAATCTACAAATGGATTCATTTTTGAGTCAGCATGGTCTACTTTTGTGATCAATCTTCTAATGTTTGTTTTGGCTGGGCATGTCGTT

[0291] GGTTCATGTTGGTACCTCTTCGGACTACAAGTAAGTCTCTTCTTTAATGACAATTCCATATGGTATATAGCTATGGGCGCAATTAAT

[0292] CAGATACTTCCCAACCACCCAAGCCTGTTCAGATTGTATTCAGAAAGGCAGGTCTGTAATTCCCCAGATTTGGAACCATATATTTT

[0293] GTTGCCTATCAGTAGGTTCTGTTATCTTTTTCAAGATTTTTTTTCTTTTTCCAATTATTTGGAGTTGTCATTAGCACGTTGGACATA

[0294] AAGTTGATTTATCAATGTTACATTTCTGCTATGATCTGGGCTAACAGGTACTGGATTACAGTATAAACTGACCATGCAACACCAAA

[0295] GGTTGGGGACTCAGGGTGTTACAATATCATACCTGTTTTCACTCAACATTCAACTAAAAATGTTCACCTGGGATATATTCTCATTT

[0296] GCATTTTTTTTAGATATGGAAGCTTTATTTAGACTCAGTTAATTACATTCTCATTTGCATGATTGTCCACTTTACTGACTTGTTTTC

[0297] TTGTTTTATTCGATGATTGAAAAAGCTGTAGTCAGAATATTGTTGAAGCCTCAACTAAGTATAAAAAATATATCTCATTTTCATTCA

[0298] TGTTTTGTTGCAGAGGGTTAATCAATGTCTACGGGATTCTTGTGCTGCATCAAACATATCAAAAGCGTTGTGTAATAATTGTACAG

[0299] ATTGTGGAATTACTGAATAAATAGGACCAATTGGTTGAACAACTCAGACTTAACTGGCTGTTTTGATACTAAAAGCGGTAATTT

[0300] CCCTTATGGCATCTACCAACAGGCAGTGTTGCTAACCACAGAACCTGGACTTAAGCGTTATATATATTCGCTCTTTTGGGGGTTTC

[0301] AGGTACCTTAATTTCTTCTTATAAGATGAATTTGTTGTCTTTTATCCATATGGTTAGGAGTGGGTCATTGTATGGGTATCCATTATCC

[0302] ACTCTTAATTCAACTAAAACAACTAAATATGTTTTTTTCTGTCGCGTTACATTTTTAACCAGTAGTATCCAGCTGTATATGGAAAGG

[0303] AGGAAAGGTTCCAACCTCCTTCCTAAATATCTACTCCCTTTTTTACCCAAAGCAATGGGCCACTGTTGTACTTCTGGTCCAGCTTT

[0304] TTATCTTTTATATGCCTTTACTGACCTTACTATCTTTTTTATTTTCTTCAATTATCTGATGCAGCAAATTAGTACATTAGCTGGCAATT

[0305] TGATCCCAAGTTACTTTGTATGGGAAGTAATATTCACTATGGCTATTATTGGACTGGGGTTGTTGCTTTTCGCATTACTCATAGAGT

[0306] CCATGCAGAATTTTCTCCAAGCTCTTGGAAAAAGGTGTGCAGTTGTACCACAAGTTTATCCTATATGAAGTGTACTGATGAGATG

[0307] CCAATGGTTTAGTATATATACATGTAAATGATGTTACACATATGCACAGAATTGAAGAGAACACAAATAGCAACAACAATCAGTT

[0308] TGAATAATAGAAACTGTCGGCCACTCTGGTAGTGCCTTTTCCTAATGAAGTCTGATAACTAATGTTCCAAAGGGTATATGTTTTTT

[0309] TTTTGTTTTTTTTGCAGGAGACTAGAGATGCAATTAAGAAGGCGTGATGTTGAACAGTGGATGAGCCATAGGCGGCTGCCAGA

[0310] AGACTGAGAAGGTCTTATATCTTCCAAATCAAATAACTGAGATATGAGGAAACTGACCTTTTTTTTTAATTTTCTTTCCTTTCACA

[0311] TTATTAAAGAAGTAAACATCATATTACCTGAAAAGGATATAAATGTTCTCCTAGATCTGTTTACCTAGTGAAATGTCCGGTTTCAGG

[0312] CCTTCAGTGGGGTAAATGAACTTCAATCCAATTTAAAATGTGACTAAGAATCGAAATGGAGAAAACTGCTACCATCATGTTGTTT

[0313] TAGTAACAATTTCGAACAAGGGGGAATGGATGAGTATTAGAGATAAATGCTGGAATTATATGCATACATATCTTGATAATGTAAAA

[0314] TGGCTAAATTTCAAGCAGAAGATATAAATGGTTAATAAATCTGACTTTTATTCATGAGTGAAGATAGTACCATTTTTCTTCGTGT

[0315] TAGTACTAGTAAAATGTTTTGTTATCAACAAGCTGTAGTAAGTAGTAACACATCAGTAACAAACCCCAAATTTCTATGCTGCAAGG

[0316] TGAGAATGCCTAAAGCTTTGTGGGTGTGCAGGAGGGTTTAGATCTGCGAGAGGTTCAGCTGGGTAGCTACTAGAGGAGTGAA

[0317] TGAAGAGAGCTTTTGAGCAATTTGCCAGAAGATATTCAAAGGGGCATACGCCGCCATTTCTTTGGGTTCCTTAAGAAGGTTTG

[0318] TTGATTGTGTCTTTGCAACTGTTTTTTCCTCTCCATCTTCAATTAGTTGACACTTGACACTGGGTAGTGCATATTTTAATTATTAAT

[0319] GCAAGTTTTCCTGATAGCCTGGACATATCCCCGTTCGCAATTGCTTCTTGTCTTTCAGATAATTATTCATGTAGTAACAACTTTACT

[0320] AGGTACCGGTATGAGTTCCTGTTACTGGTACTGGTTTTTTTTTTGAATGAATACTGGTAGTGGTTTTACTCAGTAGATCCTTACA

[0321] CAGGAAAGGACTGAAAACAAACTTCCTGTAGACCAAGTTAATCCAGGTGGTACAGGTGTAAAATTTCGGTTCTGTGCGGATGT

[0322] CTTCTTTTGAATTTTGCTTTATGAAAGGAAACAGCAAGTTTTGCGATGTGTATATGCTTAATGATTTCCTCTGTTTTGCCTTCCTTT

[0323] TTCTTGCGAGAACTCTGTTTTTCTTTTAAGAATAATGATATGCGACTCTGTTTTTCTTTTAAGAATAATGATACGCCACCACCAATC

[0324] CATTACAGCCCCTGCACTATATAGTACTGGGTGGCTGCGTACTTGTTATTCAGAATCAGAATAAGTTCTGTTGCCTATATCATTGGG

[0325] GTTGCTGTGCCATCATTTAGTTGATCTCATGATGCCAATGCACTGTGCTGGCATAGTTCCAAGTCCAGTTGCACCGAATATGTTTT

[0326] ATGTTCAACTACTTTGTCATTTTTGCATATCATGAAAAATTTCTATGCAATTATTTTTTGGAACTTGAGGTGACTTTAACTTGCACT

[0327] TTCCTTCTTTCACCATCTATTTTAGGTCCGGCTGTTTAACCTGATGGACAATGCAACCTGGGATGCAATTTGTGACAAGCTAAGG

[0328] CAGAACTTGTATATTACAGGAAGTGATATTCTTTATCAGGGTGGTCCTGTTGAAAAGATGGTTTTTATAGTCAGGGGTAGATTGG

[0329] AAGCATCAGTGCAGATGGAAATAAGTCTCCCTTGCAAGAAGGAGAGTTTGTGGTGAGGAACTCCTTTCCTGGTACTTGGAG

[0330] CAATCTTCAGTGAACCGAGGTCTGCAAATGTTATGCTTTCTTGACTTCTCGTGCCTTCTGATCTTTCCCTTTATTATGTCATCTAAA

[0331] ATGCTACCATCCTACCGTATCAGATGGTGGGAAGATCAAGTTGCATGGCATGCGTTTGGTCGCCATACGTACTGTCAGATGTTTA

[0332] ACAAATGTTGAAGCTTTTGTACTGCGAGCACGTGATCTGGAAGAAGTGACTTCACAATTTTCAAGATTCTTGCGCAATCCACTTG

[0333] TGCTAGGTACAATCAGGTAAGAAAATACCCGAGCAAATTACACCTTTATTTTATGAATCACAAAACAATATAGTTAATTACTAGTAT

[0334] TAGGTATTTAGGTTGAGGAAAACCCAAATTAATCTAAGCAATAAATCAAATCGAAGATTTAGGGCCTATTTGGCACAGCTCCACC

[0335] TCCACCTCCACCCCTCCTGGAGTTGGAGCTCAGCCAAACAGTTTCAGCTCCACCAAAACTGGGAGTGGAGTTGGGTGGAGTTA

[0336] TCTCACAAAATGTACTAGAGTTGTAGAGCTGGGTTTAGGCAGCTCCACAACTCCACTCTAGCCTCAACTCCTAGAGCAATATTTA

[0337] GGAGTTGGAGCTGTACCAAACAGACCCTTACTCCTACATAATTTCAAGTTCTATTATCGATGTGTGACAGGAAATGTGCGAAAAG

[0338] TGTATTTATTTCTCCCAAGTATATTTGTCTGTTTTCCAGCAAATAACCTTTCAAAATATTTATCCATTGAGGAGACTAAGACGTACCA

[0339] CCTCTGTAGAATTTTATAGGGTGTATTTTAATTGATAAAACTCTTAAGGTCACACTTTGACAGTATATTCCTGATATCTATAAAACTA

[0340] ACATTTTGTGAAAGTATTTGAAATATAATATACTTGTACTACTACTTCCATCACAAAATGAGTTCATTTTTCACCCACTTTATCAAAT

[0341] CCCAGTAATTGTTTTTCACTTTATCTACTTTCAATGCATTTGTCCCCTACTTTTACAAATTCCAATGCAATGATTGCTTAAAAATGA

[0342] ACTTATTTTGGGACAAACGGGAGGGGGCAAAAAAATATCTTATTTTGGGATAGAGGAAGTATAATTTTATACAGTAAATATACTTAT

[0343] AATTTGAATAATCATTAGTCAAAGTTCACAAAGTGACTTCTTTTTCAAATCAAAACACGTGGAGTGTGTAGTTATTTTAGACTTTT

[0344] TTATTTTTTGCCCTAACTAGTTATTTTAGACTTTTTTATTTTTTGCCCTAACTAGATTCTTTGAAAGACATAATTAGGTTATATTATGG

[0345] CGGTGTCGTCATGTTTCCTTGTTAACTTTTTCTTAATTTTTTTTTTTTACTTCATAGATATAGGCACATGGTGGGATATAAACATATAAG

[0346] GTGAAACAGAATGAAGTAGTTTATTAATGAAAATGTTTTGAGAGCTCTTATTTGTGCATGATTTATGTGATGGATGAGCATTGCTC

[0347] TCTCCATTTAAAAATATAGGGCATATAAGCTTTGACACGGTCTTCAACATAGAACTTTGACTATTAGTTCTTTTGCAAATATTACCA

[0348] ACGACTATGAAATGAATATCATACAAAGGTATTTTCAAATATGAATTTAATGATATCACATACGTAACACACAAATACACAGCATAA

[0349] AGACACTCCTCAGATGCGCAGTCTATTGTATCACATAGGAATTTTGAATTTTTCAATTAGTGTAGCTGGAGCCTGGAGGATATCAT

[0350] ATTCCCATGGCCCTAACTTACTCTGTTCACTGATGCTTTCGTGAACATGAAAAACAGGTATGAATCACCTTACTGGAAGAACCTTG

[0351] CAGCAAATCGCATCCAAGTCGCATGGAGGTATCGGAAAAGGCGACTGAAGAGAGCTGAGATGCAAAGGTTGCAATAG

[0352] Full-length cDNA sequence of ETD1 gene (SEQ ID NO:2):

[0353] ATGTCTGGCCAAGAGAGAGATGATGTTCCAATGCTAGAGCTACAAAGATTTCCTACTAGAAGTGTATCGATGTGTATACCTGTCA

[0354] GGGATGATATATATGAAGACTCAATCATATCCCACAGTGGTCCTATTTTCACCCCAGCACCAACTCAGTATACATCCGTGGCTATTC

[0355] CATCAGGAAACAGAGACATGCTCGATAAGCTGCCTCGTCCAAAGGTTAAGAGCAAACCACATGTTGTTACGCCGGAAGAAGTT

[0356] GGAATAAGTAACTGGCCTTATGACCAGCATGTTCCGAAGAACAAACACCTGATGATGTACTCTGAGCCTCTGGGACTGTTGTAT

[0357] AATCCTGATTGTGTCGACTGTCCTCGTGCTTGTAAACAAAAGGCATTTCCAGAGAAGTTTAGCTCCGTTTGATAACAAGTTTC

[0358] ACAACATTCTTTATGGCTACGGTGATCGATGGAAGAAGAAAGCTGGGCATTACCTTTCATACATTCCAATTATGAAGCCACATGAT

[0359] AAGGCTGTTCATCGGTGGAACCAGTTTTTTGTGATATCATGCTTGCTAGCCATATTCAATGACCCTCTGTTTTTCTTCCTATTGTCA

[0360] GTAGATAAGGATTATAAGTGTATAGTGTTCAACTGGAATTTCGCGATAGCATTAGCTGTTGGAAGAAGTGTGACTGACGCCATAT

[0361] ATTTTTTACACATGCTTCTTCAGTTCAGGCTAGCATATGTAGCACCGGAGTCGAGAGTAGTGGGAACTGGAGATTTGGTTTGATG

[0362] AGCCTATGAAAATTGCTATGCGCTATCTTCGTGGGTTTTTTGTACTCGACTTATTTGTTGTTCTTCCACTCCCTCAGGTGATGATAT

[0363] TGCTAGTTATCCCTAAAATATGTGGGATTATCAAGTGCAAACTATGCAAAAAACTTGTTGCGTGCTACAGTTCTTCTTCAGTATGTAC

[0364] CACGTATTATAAGATTTGTGCCACTGCTTGGTGGTCAATCTACAAATGGATTCATTTTTGAGTCAGCATGGTCTACTTTTGTGATC

[0365] AATCTTCTAATGTTTGTTTTGGCTGGGCATGTCGTTGGTTCATGTTGTACCTCTTCGGACTACAAAGGTTAATCAATGTCTACG

[0366] GGATTCTTGTGCTGCATCAAACATATCAAAAGCGTTGTGTAATAATTGTACAGATTGTGGAATTACTGGAATAAATAGGACCAATT

[0367] GGTTGAACAACTCAGACTTAACTGGCTGTTTTGATACTAAAAGCGGTAATTTCCCTTATGGCATCTACCAACAGGCAGTGTTGCT

[0368] AACCACAGAACCTGGACTTAAGCGTTATATATATTCGCTCTTTTGGGGGTTTCAGCAAATTAGTACATTAGCTGGCAATTTGATCC

[0369] CAAGTTACTTTGTATGGGAAGTAATATTCACTATGGCTATTATTGGACTGGGGTTGTTGCTTTTCGCATTACTCATAGAGTCCATGC

[0370] AGAATTTTCTCCAAGCTCTTGGAAAAAGGAGACTAGAGATGCAATTAAGAAGGCGTGATGTTGAACAGTGGATGAGCCATAGG

[0371] CGGCTGCCAGAAGATCTGAGAAGGAGGGTTAGATCTGCCGAGAGGTTCAGCTGGGTAGCTACTAGAGGAGTGAATGAAGAA

[0372] GAGCTTTTGAGCAATTTGCCAGAAGATATTCAAAGGGGCATACGCCGCCATTTCTTTGGGTTCCTTAAGAAGGTCCGGCTGTTT

[0373] AACCTGATGGACAATGCAACCTGGGATGCAATTTGTGACAAGCTAAGGCAGAACTTGTATATTACAGGAAGTGATATTCTTTATC

[0374] AGGGTGGTCCTGTTGAAAAGATGGTTTTTATAGTCAGGGGTAGATTGGAAAGCATCAGTGCAGATGGAAATAAGTCTCCCTTGC

[0375] AAGAAGGAGATGTTTGTGGTGAGGAACTCCTTTCCTGGTACTTGGAGCAATCTTCAGTGAACCGAGATGGTGGGAAGATCAAG

[0376] TTGCATGGCATGCGTTTGGTCGCCATACGTACTGTCAGATGTTTAACAAATGTTGAAGCTTTTGTACTGCGAGCACGTGATCTGG

[0377] AAGAAGTGACTTCACAATTTTCAAGATTCTTGCGCAATCCACTTGTGCTAGGTACAATCAGGTATGAATCACCTTACTGGAAGAA

[0378] CCTTGCAGCAAATCGCATCCAAGTCGCATGGAGGTATCGGAAAAGGCGACTGAAGAGAGCTGAGATGCAAAGGTTGCAATAGETD1 gene-encoded amino acid sequence (SEQ ID NO:3):

[0379] MSGQERDDVPMLELQRFPTRSVSMCIPVRDDIYEDSIISHSGPIFTPAPTQYTSVAIPSGNRDMLDKLPRPKVKSKPHVVTPEEVGIS

[0380] NWPYDQHVPKNKHLMMYSEPLGLCDNPDCVDCPRACKNKRHFQRSLAPFDNKFHNILYGYGDRWKKKAGHYLSYIPIMKPHDK

[0381] AVHRWNQFFVISCLLAIFNDPLFFFLLSVDKDYKCIVFNWNFAIALAVGRSVTDAIYFLHMLLQFRLAYVAPESRVVGTGDLVDEPM

[0382] KIAMRYLRGFFVLDLFVVLPLPQVMILLVIPKYVGLSSANYAKNLLRATVLLQYVPRIIRFVPLLGGQSTNGFIFESAWSTFVINLLMFV

[0383] LAGHVVGSCWYLFGLQRVNQCLRDSCAASNISKALCNNCTDCGITGINRTNWLNNSDLTGCFDTKSGNFPYGIYQQAVLLTTEPGL

[0384] KRYIYSLFWGFQQISTLAGNLIPSYFVWEVIFTMAIIGLGLLLFALLIESMQNFLQALGKRRLEMQLRRRDVEQWMSHRRLPEDLRR

[0385] RVRSAERFSWVATRGVNEEELLSNLPEDIQRGIRRHFFGFLKKVRLFNLMDNATWDAICDKLRQNLYITGSDILYQGGPVEKMVFIV

[0386] RGRLESISADGNKSPLQEGDVCGEELLSWYLEQSSVNRDGGKIKLHGMRLVAIRTVRCLTNVEAFVLRARDLEEVTSQFSRFLRNPLVLGTIRYESPYWKNLAANRIQVAWRYRKRRLKRAEMQRLQ*

[0387] OsCNGC13 gene editing target 1 (SEQ ID NO:4):

[0388] agcatgtctctgtttcctgatgg OsCNGC13 gene editing target 2 (SEQ ID NO:5):

[0389] gttgttacgccggaagaagttgg Base sequence of ETD1 genetic transformation vector (SEQ ID NO:6):

[0390] CATGGCTACTACTAAGCATTTGGCTCTTGCCATCCTTGTCCTCCTTAGCATTGGTATGACCACCAGTGCAAGAACCCTCCTAGATC

[0391] TGAGGGTAAATTTCTAGTTTTTCTCCTTCATTTTCTTGGTTAGGACCCTTTTCTCTTTTTATTTTTTTGAGCTTTGATCTTTCTTTAA

[0392] ACTGATCTATTTTTTAATTGATTGGTTATGGTGTAAATATTACATAGCTTTAACTGATAATCTGATTACTTTATTTCGTGTGTCTATGA

[0393] TGATGATGATAGTTACAGAACCGACGAACTAGTCTGTACCCGATCAACACCGAGACCCGTGGCGTCTTCGACCTCAATGGCGTC

[0394] TGGAACTTCAAGCTGGACTACGGGAAAGGACTGGAAGAGAAGTGGTACGAAAGCAAGCTGACCGACACTATTAGTATGGCCG

[0395] TCCCAAGCAGTTACAATGACATTGGCGTGACCAAGGAAATCCGCAACCATATCGGATATGTCTGGTACGAACGTGAGTTCACGG

[0396] TGCCGGCCTATCTGAAGGATCAGCGTATCGTGCTCCGCTTCGGCTCTGCAACTCACAAAGCAATTGTCTATGTCAATGGTGAGCT

[0397] GGTCGTGGAGCACAAGGGCGGATTCCTGCCATTCGAAGCGGAAATCAACAACTCGCTGCGTGATGGCATGAATCGCGTCACCG

[0398] TCGCCGTGGACAACATCCTCGACGATAGCACCCTCCCGGTGGGGCTGTACAGCGAGCGCCACGAAGAGGGCCTCGGAAAAGT

[0399] CATTCGTAACAAGCCGAACTTCGACTTCTTCAACTATGCAGGCCTGCACCGTCCGGTGAAAATCTACACGACCCCGTTTACGTAC

[0400] GTCGAGGACATCTCGGTTGTGACCGACTTCAATGGCCCAACCGGGACTGTGACCTATACGGTGGACTTTCAAGGCAAAGCCGA

[0401] GACCGTGAAAGTGTCGGTCGTGGATGAGGAAGGCAAAGTGGTCGCAAGCACCGAGGGCCTGAGCGGTAACGTGGAGATTCC

[0402] GAATGTCATCCTCTGGGAACCACTGAACACGTATCTCTACCAGATCAAAGTGGAACTGGTGAACGACGGACTGACCATCGATGT

[0403] CTATGAAGAGCCGTTCGGCGTGCGGACCGTGGAAGTCAACGACGGCAAGTTCCTCATCAACAACAAACCGTTCTACTTCAAGG

[0404] GCTTTGGCAAACATGAGGACACTCCTATCAACGGCCGTGGCTTTAACGAAGCGAGCAATGTGATGGATTTCAATATCCTCAAAT

[0405] GGATCGGCGCCAACAGCTTCCGGACCGCACACTATCCGTACTCTGAAGAGTTGATGCGTCTTGCGGATCGCGAGGGTCTGGTC

[0406] GTGATCGACGAGACTCCGGCAGTTGGCGTGCACCTCAACTTCATGGCCACCACGGGACTCGGCGAAGGCAGCGAGCGCGTCA

[0407] GTACCTGGGAGAAGATTCGGACGTTTGAGCACCATCAAGACGTTCTCCGTGAACTGGTGTCTCGTGACAAGAACCATCCAAGC

[0408] GTCGTGATGTGGAGCATCGCCAACGAGGCGGCGACTGAGGAAGAGGGCGCGTACGAGTACTTCAAGCCGTTGGTGGAGCTG

[0409] ACCAAGGAACTCGACCCACAGAAGCGTCCGGTCACGATCGTGCTGTTTGTGATGGCTACCCCGGAGACGGACAAAGTCGCCG

[0410] AACTGATTGACGTCATCGCGCTCAATCGCTATAACGGATGGTACTTCGATGGCGGTGATCTCGAAGCGGCCAAAGTCCATCTCCG

[0411] CCAGGAATTTCACGCGTGGAACAAGCGTTGCCCAGGAAAGCCGATCATGATCACTGAGTACGGCGCAGACACCGTTGCGGGC

[0412] TTTCACGACATTGATCCAGTGATGTTCACCGAGGAATATCAAGTCGAGTACTACCAGGCGAACCACGTCGTGTTCGATGAGTTT

[0413] GAGAACTTCGTGGGTGAGCAAGCGTGGAACTTCGCGGACTTCGCGACCTCTCAGGGCGTGATGCGCGTCCAAGGAAACAAG

[0414] AAGGGCGTGTTCACTCGTGACCGCAAGCCGAAGCTCGCCGCGCACGTCTTTCGCGAGCGCTGGACCAACATTCCAGATTTCGG

[0415] CTACAAGAACGCTAGCCATCACCATCACCATCACGTGTGAATTGGTGACCAGCTCGAATTTCCCCGATCGTTCAAACATTTGGCA

[0416] ATAAAGTTTCTTAAGATTGAATCCTGTTGCCGGTCTTGCGATGATTATCATATAATTTCTGTTGAATTACGTTAAGCATGTAATAATT

[0417] AACATGTAATGCATGACGTTATTTATGAGATGGGTTTTTATGATTAGAGTCCCGCAATTATACATTTAATACGCGATAGAAAACAAA

[0418] ATATAGCGCGCAAACTAGGATAAATTATCGCGCGCGGTGTCATCTATGTTACTAGATCGGGAATTAAACTATCAGTGTTTGACAGG

[0419] ATATATTGGCGGGTAAACCTAAGAGAAAAGAGCGTTTATTAGAATAACGGATATTTAAAAGGGCGTGAAAAGGTTTATCCGTTCG

[0420] TCCATTTGTATGTGCATGCCAACCACAGGGTTCCCCTCGGGATCAAAGTACTTTGATCCAACCCCTCCGCTGCTATAGTGCAGTC

[0421] GGCTTCTGACGTTCAGTGCAGCCGTCTTCTGAAAACGACATGTCGCACAAGTCCTAAGTTACGCGACAGGCTGCCGCCCTGCC

[0422] CTTTTCCTGGCGTTTTCTTGTCGCGTGTTTTAGTCGCATAAAGTAGAATACTTGCGACTAGAACCGGAGACATTACGCCATGAAC

[0423] AAGAGCGCCGCCGCTGGCCTGCTGGGCTATGCCCGCGTCAGCACCGACGACCAGGACTTGACCAACCAACGGGCCGAACTGC

[0424] ACGCGGCCGGCTGCACCAAGCTGTTTTCCGAGAAGATCACCGGCACCAGGCGCGACCGCCCGGAGCTGGCCAGGATGCTTG

[0425] ACCACCTACGCCCTGGCGACGTTGTGACAGTGACCAGGCTAGACCGCCTGGCCCGCAGCACCCGCGACCTACTGGACATTGCC

[0426] GAGCGCATCCAGGAGGCCGGCGCGGGCCTGCGTAGCCTGGCAGAGCCGTGGGCCGACACCACCACGCCGGCCGGCCGCATG

[0427] GTGTTGACCGTGTTCGCCGGCATTGCCGAGTTCGAGCGTTCCCTAATCATCGACCGCACCCGGAGCGGGCGCGAGGCCGCCAA

[0428] GGCCCGAGGCGTGAAGTTTGGCCCCCGCCCTACCCTCACCCCGGCACAGATCGCGCACGCCCGCGAGCTGATCGACCAGGAA

[0429] GGCCGCACCGTGAAAGAGGCGGCTGCACTGCTTGGCGTGCATCGCTCGACCCTGTACCGCGCACTTGAGCGCAGCGAGGAAG

[0430] TGACGCCCACCGAGGCCAGGCGGCGCGGTGCCTTCCGTGAGGACGCATTGACCGAGGCCGACGCCCTGGCGGCCGCCGAGA

[0431] ATGAACGCCAAGAGGAACAAGCATGAAACCGCACCAGGACGGCCAGGACGAACCGTTTTTCATTACCGAAGAGATCGAGGCG

[0432] GAGATGATCGCGGCCGGGTACGTGTTCGAGCCGCCCGCGCACGTCTCAACCGTGCGGCTGCATGAAATCCTGGCCGGTTTGTC

[0433] TGATGCCAAGCTGGCGGCCTGGCCGGCCAGCTTGGCCGCTGAAGAAACCGAGCGCCGCCGTCTAAAAAGGTGATGTGTATTT

[0434] GAGTAAAACAGCTTGCGTCATGCGGTCGCTGCGTATATGATGCGATGAGTAAATAAACAAATACGCAAGGGGAACGCATGAAG

[0435] GTTATCGCTGTACTTAACCAGAAAGGCGGGTCAGGCAAGACGACCATCGCAACCCATCTAGCCCGCGCCCTGCAACTCGCCGG

[0436] GGCCGATGTTCTGTTAGTCGATTCCGATCCCCAGGGCAGTGCCCGCGATTGGGCGGCCGTGCGGGAAGATCAACCGCTAACCG

[0437] TTGTCGGCATCGACCGCCCGACGATTGACCGCGACGTGAAGGCCATCGGCCGGCGCGACTTCGTAGTGATCGACGGAGCGCC

[0438] CCAGGCGGCGGACTTGGCTGTGTCCGCGATCAAGGCAGCCGACTTCGTGCTGATTCCGGTGCAGCCAAGCCCTTACGACATAT

[0439] GGGCCACCGCCGACCTGGTGGAGCTGGTTAAGCAGCGCATTGAGGTCACGGATGGAAGGCTACAAGCGGCCTTTGTCGTGTC

[0440] GCGGGCGATCAAAGGCACGCGCATCGGCGGTGAGGTTGCCGAGGCGCTGGCCGGGTACGAGCTGCCCATTCTTGAGTCCCGT

[0441] ATCACGCAGCGCGTGAGCTACCCAGGCACTGCCGCCGCCGGCACAACCGTTCTTGAATCAGAACCCGAGGGCGACGCTGCCC

[0442] GCGAGGTCCAGGCGCTGGCCGCTGAAATTAAATCAAAACTCATTTGAGTTAATGAGGTAAAGAGAAAATGAGCAAAAGCACAA

[0443] ACACGCTAAGTGCCGGCCGTCCGAGCGCACGCAGCAGCAAGGCTGCAACGTTGGCCAGCCTGGCAGACACGCCAGCCATGA

[0444] AGCGGGTCAACTTTCAGTTGCCGGCGGAGGATCACACCAAGCTGAAGATGTACGCGGTACGCCAAGGCAAGACCATTACCGA

[0445] GCTGCTATCTGAATACATCGCGCAGCTACCAGAGTAAATGAGCAAATGAATAAATGAGTAGATGAATTTTAGCGGCTAAAGGAG

[0446] GCGGCATGGAAAATCAAGAACAACCAGGCACCGACGCCGTGGAATGCCCCATGTGTGGAGGAACGGGCGGTTGGCCAGGCG

[0447] TAAGCGGCTGGGTTGTCTGCCGGCCCTGCAATGGCACTGGAACCCCCAAGCCCGAGGAATCGGCGTGACGGTCGCAAACCAT

[0448] CCGGCCCGGTACAAATCGGCGCGGCGCTGGGTGATGACCTGGTGGAGAAGTTGAAGGCCGCGCAGGCCGCCCAGCGGCAAC

[0449] GCATCGAGGCAGAAGCACGCCCCGGTGAATCGTGGCAAGCGGCCGCTGATCGAATCCGCAAAGAATCCCGGCAACCGCCGGC

[0450] AGCCGGTGCGCCGTCGATTAGGAAGCCGCCCAAGGGCGACGAGCAACCAGATTTTTTCGTTCCGATGCTCTATGACGTGGGCA

[0451] CCCGCGATAGTCGCAGCATCATGGACGTGGCCGTTTTCCGTCTGTCGAAGCGTGACCGACGAGCTGGCGAGGTGATCCGCTAC

[0452] GAGCTTCCAGACGGGCACGTAGAGGTTTCCGCAGGGCCGGCCGGCATGGCCAGTGTGTGGGATTACGACCTGGTACTGATGG

[0453] CGGTTTCCCATCTAACCGAATCCATGAACCGATACCGGGAAGGGAAGGGAGACAAGCCCGGCCGCGTGTTCCGTCCACACGTT

[0454] GCGGACGTACTCAAGTTCTGCCGGCGAGCCGATGGCGGAAAGCAGAAAGACGACCTGGTAGAAACCTGCATTCGGTTAAACA

[0455] CCACGCACGTTGCCATGCAGCGTACGAAGAAGGCCAAGAACGGCCGCCTGGTGACGGTATCCGAGGGTGAAGCCTTGATTAG

[0456] CCGCTACAAGATCGTAAAGAGCGAAACCGGGCGGCCGGAGTACATCGAGATCGAGCTAGCTGATTGGATGTACCGCGAGATCA

[0457] CAGAAGGCAAGAACCCGGACGTGCTGACGGTTCACCCCGATTACTTTTTGATCGATCCCGGCATCGGCCGTTTTCTCTACCGCC

[0458] TGGCACGCCGCGCCGCAGGCAAGGCAGAAGCCAGATGGTTGTTCAAGACGATCTACGAACGCAGTGGCAGCGCCGGAGAGT

[0459] TCAAGAAGTTCTGTTTCACCGTGCGCAAGCTGATCGGGTCAAATGACCTGCCGGAGTACGATTTGAAGGAGGAGGCGGGGCA

[0460] GGCTGGCCCGATCCTAGTCATGCGCTACCGCAACCTGATCGAGGGCGAAGCATCCGCCGGTTCCTAATGTACGGAGCAGATGCT

[0461] AGGGCAAATTGCCCTAGCAGGGGAAAAAGGTCGAAAAGGTCTCTTTCCTGTGGATAGCACGTACATTGGGAACCCAAAGCCGT

[0462] ACATTGGGAACCGGAACCCGTACATTGGGAACCCAAAGCCGTACATTGGGAACCGGTCACACATGTAAGTGACTGATATAAAA

[0463] GAGAAAAAAGGCGATTTTTCCGCCTAAAACTCTTTAAAACTTATTAAAACTCTTAAAACCCGCCTGGCCTGTGCATAACTGTCTG

[0464] GCCAGCGCACAGCCGAAGAGCTGCAAAAAGCGCCTACCCTTCGGTCGCTGCGCTCCCTACGCCCCGCCGCTTCGCGTCGGCCT

[0465] ATCGCGGCCGCTGGCCGCTCAAAAATGGCTGGCCTACGGCCAGGCAATCTACCAGGGCGCGGACAAGCCGCGCCGTCGCCAC

[0466] TCGACCGCCGGCGCCCACATCAAGGCACCCTGCCTCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCC

[0467] GGAGACGGTCACAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTG

[0468] TCGGGGCGCAGCCATGACCCAGTCACGTAGCGATAGCGGAGTGTATACTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTG

[0469] AGAGTGCACCATATGCGGTGTGAAATACCGCACAGATGCGTAAGGAGAAAATACCGCATCAGGCGCTCTTCCGCTTCCTCGCTC

[0470] ACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCA

[0471] GGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTT

[0472] TTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAG

[0473] ATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTC

[0474] CCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGT

[0475] GTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTAT

[0476] CGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCC

[0477] TAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTC

[0478] TTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTC

[0479] AAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGCATTCTA

[0480] GGTACTAAAACAATTCATCCAGTAAAATATAATATTTTATTTTCTCCCAATCAGGCTTGATCCCCAGTAAGTCAAAAAATAGCTCGA

[0481] CATACTGTTCTTCCCCGATATCCTCCCTGATCGACCGGACGCAGAAGGCAATGTCATACCACTTGTCCGCCCTGCCGCTTCTCCCA

[0482] AGATCAATAAAGCCACTTACTTTGCCATCTTTCACAAAGATGTTGCTGTCTCCCAGGTCGCCGTGGGAAAAGACAAGTTCCTCTT

[0483] CGGGCTTTTCCGTCTTTAAAAAATCATACAGCTCGCGCGGATCTTTAAATGGAGTGTCTTCTTCCCAGTTTTCGCAATCCACATCG

[0484] GCCAGATCGTTATTCAGTAAGTAATCCAATTCGGCTAAGCGGCTGTCTAAGCTATTCGTATAGGGACAATCCGATATGTCGATGGA

[0485] GTGAAAGAGCCTGATGCACTCCGCATACAGCTCGATAATCTTTTCAGGGCTTTGTTCATCTTCATACTCTTCCGAGCAAAGGACG

[0486] CCATCGGCCTCACTCATGAGCAGATTGCTCCAGCCATCATGCCGTTCAAAGTGCAGGACCTTTGGAACAGGCAGCTTTCCTTCC

[0487] AGCCATAGCATCATGTCCTTTTCCCGTTCCACATCATAGGTGGTCCCTTTATACCGGCTGTCCGTCATTTTTAAATATAGGTTTTCA

[0488] TTTTCTCCCACCAGCTTATATACCTTAGCAGGAGACATTCCTTCCGTATCTTTTACGCAGCGGTATTTTTCGATCAGTTTTTTCAAT

[0489] TCCGGTGATATTCTCATTTTAGCCATTTATTATTTCCTTCCTCTTTTCTACAGTATTTAAAGATACCCCAAGAAGCTAATTATAACAA

[0490] GACGAACTCCAATTCACTGTTCCTTGCATTCTAAAACCTTAAATACCAGAAAACAGCTTTTTCAAAGTTGTTTTCAAAGTTGGCG

[0491] TATAACATAGTATCGACGGAGCCGATTTTGAAACCGCGGTGATCACAGGCAGCAACGCTCTGTCATCGTTACAATCAACATGCTA

[0492] CCCTCCGCGAGATCATCCGTGTTTCAAACCCGGCAGCTTAGTTGCCGTTCTTCCGAATAGCATCGGTAACATGAGCAAAGTCTGC

[0493] CGCCTTACAACGGCTCTCCCGCTGACGCCGTCCCGGACTGATGGGCTGCCTGTATCGAGTGGTGATTTTGTGCCGAGCTGCCGG

[0494] TCGGGGAGCTGTTGGCTGGCTGGTGGCAGGATATATTGTGGTGTAAACAAATTGACGCTTAGACAACTTAATAACACATTGCGG

[0495] ACGTTTTTAATGTACTGAATTAACGCCGAATTAATTCGGGGGATCTGGATTTTAGTACTGGATTTTGGTTTTAGGAATTAGAAATT

[0496] TTATTGATAGAAGTATTTTACAAATACAAATACATACTAAGGGTTTCTTATATGCTCAACACATGAGCGAAACCCTATAGGAACCCT

[0497] AATTCCCTTATCTGGGAACTACTCACACATTATTATGGAGAAACTCGAGCTTGTCGATCGACAGATCCGGTCGGCATCTACTCTAT

[0498] TTCTTTGCCCTCGGACGAGTGCTGGGGCGTCGGTTTCCACTATCGGCGAGTACTTCTACACAGCCATCGGTCCAGACGGCCGC

[0499] GCTTCTGCGGGCGATTTGTGTACGCCCGACAGTCCCGGCTCCGGATCGGACGATTGCGTCGCATCGACCCTGCGCCCAAGCTG

[0500] CATCATCGAAATTGCCGTCAACCAAGCTCTGATAGAGTTGGTCAAGACCAATGCGGAGCATATACGCCCGGAGTCGTGGCGATC

[0501] CTGCAAGCTCCGGATGCCTCCGCTCGAAGTAGCGCGTCTGCTGCTCCATACAAGCCAACCACGGCCTCCAGAAGAAGATGTTG

[0502] GCGACCTCGTATTGGGAATCCCCGAACATCGCCTCGCTCCAGTCAATGACCGCTGTTATGCGGCCATTGTCCGTCAGGACATTGT

[0503] TGGAGCCGAAATCCGCGTGCACGAGGTGCCGGACTTCGGGGCAGTCCTCGGCCCAAAGCATCAGCTCATCGAGAGCCTGCGC

[0504] GACGGACGCACTGACGGTGTCGTCCATCACAGTTTGCCAGTGATACACATGGGGATCAGCAATCGCGCATATGAAATCACGCCA

[0505] TGTAGTGTATTGACCGATTCCTTGCGGTCCGAATGGGCCGAACCCGCTCGTCTGGCTAAGATCGGCCGCAGCGATCGCATCCAT

[0506] AGCCTCCGCGACCGGTTGTAGAACAGCGGGCAGTTCGGTTTCAGGCAGGTCTTGCAACGTGACACCCTGTGCACGGCGGGA

[0507] GATGCAATAGGTCAGGCTCTCGCTAAACTCCCCAATGTCAAGCACTTCCGGAATCGGGAGCGCGGCCGATGCAAAGTGCCGAT

[0508] AAACATAACGATCTTTGTAGAAACCATCGGCGCAGCTATTTACCCGCAGGACATATCCACGCCCTCCTACATCGAAGCTGAAAGC

[0509] ACGAGATTCTTCGCCCTCCGAGAGCTGCATCAGGTCGGAGACGCTGTCGAACTTTTCGATCAGAAACTTCTCGACAGACGTCG

[0510] CGGTGAGTTCAGGCTTTTTCATATCTCATTGCCCCCCGGGATCTGCGAAAGCTCGAGAGAGATAGATTTGTAGAGAGAGACTGG

[0511] TGATTTCAGCGTGTCCTCTCCAAATGAAATGAACTTCCTTATATAGAGGAAGGTCTTGCGAAGGATAGTGGGATTGTGCGTCATC

[0512] CCTTACGTCAGTGGAGATATCACATCAATCCACTTGCTTTGAAGACGTGGTTGGAACGTCTTCTTTTTCCACGATGCTCCTCGTG

[0513] GGTGGGGGTCCATCTTTGGGACCACTGTCGGCAGAGGCATCTTGAACGATAGCCTTTCCTTTATCGCAATGATGGCATTTGTAG

[0514] GTGCCACCTTCCTTTTCTACTGTCCTTTTGATGAAGTGACAGATAGCTGGGCAATGGAATCCGAGGAGGTTTCCCGATATTACCC

[0515] TTTGTTGAAAAGTCTCAATAGCCCTTTGGTCTTCTGAGACTGTATCTTTGATATTCTTGGAGTAGACGAGAGTGTCGTGCTCCAC

[0516] CATGTTATCACATCAATCCACTTGCTTTGAAGACGTGGTTGGAACGTCTTCTTTTTCCACGATGCTCCTCGTGGGTGGGGGTCCA

[0517] TCTTTGGGACCACTGTCGGCAGAGGCATCTTGAACGATAGCCTTTCCTTTATCGCAATGATGGCATTTGTAGGTGCCACCTTCCT

[0518] TTTCTACTGTCCTTTTGATGAAGTGACAGATAGCTGGGCAATGGAATCCGAGGAGGTTTCCCGATATTACCCTTTGTTGAAAAGT

[0519] CTCAATAGCCCTTTGGTCTTCTGAGACTGTATCTTTGATATTCTTGGAGTAGACGAGAGTGTCGTGCTCCACCATGTTGGCAAGC

[0520] TGCTCTAGCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGA

[0521] AAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTC

[0522] GTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCTATGACCATGATTACGAATTCCTTTCACCCATCAC

[0523] CCTTTGTCACATCACATGTCCTTTACTACTTCCTTAATTCTTGCCAAAAAGGGTATAATCGCTTATATTCACGGACTGAGGGAGTG

[0524] CTTACTAGGCTATGTTCTTCCCCTCCTTCTCCAACTCCACTCTCATTTTTCATGCGCACATAAACAGTGTGTTTTTTCAAATAAATTA

[0525] TACAAAAGTTATTTTAGAAAAAAAATCATATTAATCCATTTTTCATATTCTTTTAGCTAATACTTAATTAGTCATACATTAATAGTTCAT

[0526] CACGTTTTCCATGCGGGGAGAAACTCTTACCTTCCCAAAGCAGCCTTAGAGCAGGTACAATAGCAAGCTATAAACATATTTTTAAA

[0527] GAGATAAAGGAAGAGAGGAAGAGCAACGGGATACAGATCTATAGCCAGCTGTAGCACGGGCTCCAAGACATAATGTTTGTAT

[0528] GACAGGTAGGATCAATTATTAATAGTATAATAAGCATCTATTGTATGATTAGCTATTACATTGACTATAGATGATTTGAAGTTAGTAC

[0529] TTGGATATACTATTAAACTTGCTCTTAGAGCAAGTACAATAGCAGGCTATAAGCCAGCTATAAACATATTTAAAAGAGATAAAGAA

[0530] ATAGAGAGAAGAGCAGCGGGCTACAGATTTGTAGCCACCTGCAGCATGAACTGTCGAACTCCAAGACATAATGTGTGTATGATA

[0531] GGTAGGACTAGGTATTAATAGTATAGTAAGCAATTATTATAAATCGGCTATTACGTTAGTTATAGATGATTTGGAGCTAGTAGTG

[0532] TGCTATACTATTAATATTAAACGGCCCATTAGGCCGTGCACCTAGGGACGCCCGGGGTGCGGGGGCGGAGCCCCCGCGAAAC

[0533] GGATCGTCATCCATTTTAGGGTTCCACCATATATATACCTCTTGTAAGCCGCCGTGCGGCGTTGTAACCTCACCCCAGATATGGTG

[0534] AAGATATTTTACTGCTAGCGCCTGTGGTTTTTTCTCCTCTGTTTTGGGAGGGTTTTCCACGTTAAATCTCGTGTCTTCTGTGATTG

[0535] ATCTATTGTTCTTTGTCGTTTGTTCGCTTATCGTTTCCTAACACTGCGGGCTAATAGCCCATGGCTAAAGCCAACTATTAGTACAAT

[0536] AGATTGTCTATTGACTGACTGAATCTCATAATCAAATAATTAATTATGACATGCACATATCAATGTATATGATCAAGAGTGAATTGTA

[0537] TCATTGTAGCTATTTGTGTTGTGAGATGAACTTTGTTCACCAAACACCAGGATCATATCACAGATCATACCAACCAATTTCTCTGTA

[0538] GTACTAAAATTACAAAGCACTAGTGTTTGTTGGCAAGAAGAGCTCGTGAGAAAGGAGTGGATTTCTTTGGCTGATTTTCGTCAA

[0539] ACGATGTGGCATCAGAGTTTTTCACGGCACAGTTTTCGCTCACGATCGCATGGATGGAAGACGAAGCACGCGGAAGGTGATGA

[0540] GATCGCGCACGAACGGCGGAGATGGATTTGCACACCGCTGGGGGAAGGGGATCGCGCGCGGTGGGCGAAAGGGGATCGCG

[0541] CGCCGCGGGGTAGGCAGTTCTCACGGGATCGTCTGAGGACGTGAGGGCCCCACAAACAGCGTTTTTAGCACTAGGGTCGGCT

[0542] GCACACGACCAATACTGCGTGGAGAAAAGTGCTAACGCCGGGCGACAATTTAGTCGTCCGCTTTATTCTCTCTCATCCACATAG

[0543] GAAATTGATGATGTGGCATTATTTTAGAGCCCGCTGAGTCAAATTATTGTACCTGCTCTTACAATGCATCCCCTGTTCCAACTTCCA

[0544] AGTGGATCGCATACTTCTGTGAATGACGGTGCGACGAACACAATGGCACAGGCGAGCGCCAGCAAGGATGGGTTACACTGAC

[0545] ATGTGGGCCCGGACAACTCCGTGACAATGGGACGTCGCGTTGGACGACTCGCCGCTCGGGGTCACCCGCGAGCCGCAACTAG

[0546] TCCACCGCCCTCTCTCTCGCTTTCTTCTCAAGTCCCAACTCGCGCGAAGCGGAGAGCGAGAGGCGAGAGCGAAGTGAAGCTTC

[0547] TTCGTGGCGCGCGCGCCTCGAGTTCGTCTCTTCCCTCCCGCGGCCCAGGCAGCGCCGGCCGGCGGCGCTCTCACCCTCCTCGT

[0548] TTCGTGGCGCCGCGGCGGATCAACGCGGTGATGCATTTTTTATTTTTTCCTGATTTTTTGGGGAAAGTTTCGCGCCGAACGAGT

[0549] TGAGGGTTCTTCTAACTAGGGGAGCCGAGAGGAGGAGAGAGCTTTTATTTTTTCTACTATATTTGTTGGGGTTTGATTCTGATGT

[0550] TTTTTTTTTGTATGGATGTTCTAGCCAGTTAGTGTACGGTGACTCCGTGAGTGGAGAGTGGAGAAGGGTTTAGCACTCAAACTT

[0551] TTTTTTTTCGAACTTTTTTGTGCATTTATCCAGGGAGATGAGACGCGATGCATTTGGCTTGTTTTTTTTCCGGATGAATTAGTTGC

[0552] TTGTTCCTCGCTCCCGAATGAGTTCTCGACCATTTTTTATTTTTTATTTTTTTTGCCTTGTACTAGATTCGCTGTTCGGAGCTTCAG

[0553] AAAATGCAACCATGGTGATAGTGGGTGGTTGGTTAGCGTATGAACATCGTGGAGCATTTTTTCTCTAATAATATATTTTTTTCAGG

[0554] AGGTGATGTTTAATCAGCCTCTTCTGATGGATTTCCTAGCGCCTACAGTTTCTAGCCATTTGTAGATTTTTTATATCGATTCTAATGC

[0555] GCAGTCAAACGCATGGGCGAATTTTCACTCGCTTCCCTATGCGGTGCTCTGAATTTTCAGTTTCCAGTTGCTGCTTGCTGCGCTA

[0556] TCAAACTAACACCTTGTGTTCTGCGCAGCTTCTCAGCACTTGCTGTCAAGTTTGCCGGCAATTGGTTGTGGTAAATATTAGTCAA

[0557] GTCTGAAGTCTCCATCTCTCGGAGATAATTATCTTTTAATAAATAGCCCTGTATCCATTCATTTTTGTGGTTCTAATTGCAGCCGTCG

[0558] CCCTTGTCCTTCTGAATTGGTTATGTGAGCTGGGGATGTGAGTGACAAGGCTTCGTCCTGGATTGGTGGTTCGCTAGTTGCTGC

[0559] AGTTCTTGTTTTTTTTGGGGAATACTGGTTGCTGCAGTTCTTGCATATCCTGATAGGTGTTTGTGCCTCTTTTTGCCTGTTTCGCA

[0560] TGAATATTTGTTTGATTAGTGATTAGTGTCGTTGGTATGGTTTGAGTTACCGCGTGTGTGCTTTTAGCTACGGATTCAGAAAGTAT

[0561] TTTTGATATAAGTACTGTTTGAGATGGAGTGCCTTTTGGCTTTTAACAGATTTACCTGCTGAAGTATGACCGATTTTTTCATGTGTA

[0562] TGTCGTTATTATGCTTTATGTAGACAGTTTTCAGTCTCTCCTAACGTTCTTTTTAAGATAAAATGACATGTGTGTACTCTGTGTTTTTACAC

[0563] CTTTCTACATTTTGCTTTAGTTTATTATGGTTATCCCTTTCTTGGGTCTTAATGTAGATTCAAGCAAATTGGATAACCAATCTTACA

[0564] TATTACTCTATTTTACCAAGAATGCTAGAGCATGAATGGTGTCCCTTGTATTTAACAACTTTTCCTGAAGAAGCTTTATAAAAATCT

[0565] GATTTGCTGTTGTTGAGTCGACTGAAATAGAGCAAGAATTTGCTTGTAGGAATGCTTATCAGAGTAATCTTTTAAATTTAAGCG

[0566] CATACCTTCTGTATACAAGAATATTATACTTTATATGCAAGTAACAGAGATCATTTTACTTATCTACTTCAGCACTTAATGCAATGTCA

[0567] TCTAATGCAGTGGATGTTGGGTTCCGTGTAAGGAAATGTCTGGCCAAGAGAGAGATGATGTTCCAATGCTAGAGCCTACAAAGAT

[0568] TTCCTACTAGAAGTGTATCGATGTGTATACCTGTCAGGGATGATATATATGAAGACTCAATCATATCCCACAGTGGTCCTATTTTCA

[0569] CCCCAGCACCAACTCAGTATACATCCGTGGCTATTCCATCAGGAAACAGAGACATGCTCGATAAGCTGCCTCGTCCAAAGGTTAA

[0570] GAGCAAACCACATGTTGTTACGCCGGAAGAAGTTGGAATAAGTAACTGGCCTTATGACCAGCATGTTCCGAAGAACAAACACC

[0571] TGATGATGTACTCTGAGCCTCTGGGACTGTGTGATAATCCTGATTGTGTCGACTGTCCTCGTGCTTGTAAAAACAAAAGGCATTT

[0572] CCAGAGAAGTTTAGCTCCGTTTGATAACAAGTTTCACAACATTCTTTATGGCTACGGTGATCGATGGAAGAAGAAAGCTGGGCA

[0573] TTACCTTTCATACATTCCAATTATGAAGCCACATGATAAGGCTGTTCATCGGTGGAACCAGTTTTTTGTGATATCATGCTTGCTAGC

[0574] CATATTCAATGACCCTCTGTTTTTCTTCCTATTGTCAGTAGATAAGGATTATAAGTGTATAGTGTTCAACTGGAATTTCGCGATAGC

[0575] ATTAGCTGTTGGAAGAAGTGTGACTGACGCCATATATTTTTTACACATGCTTCTTCAGTTCAGGCTAGCATATGTAGCACCGGAG

[0576] TCGAGAGTAGTGGGAACTGGAGATTTGGTTGATGAGCCTATGAAAATTGCTATGCGCTATCTTCGTGGGTTTTTTGTACTCGACT

[0577] TATTTGTTGTTCTTCCACTCCCTCAGGTGATGATATTGCTAGTTATCCCTAAATATGTGGGATTATCAAGTGCAAACTATGCAAAAA

[0578] ACTTGTTGCGTGCTACAGTTCTTCTTCAGTATGTACCACGTATTATAAGATTTGTGCCACTGCTTGGTGGTCAATCTACAAATGGA

[0579] TTCATTTTTGAGTCAGCATGGTCTACTTTTGTGATCAATCTTCTAATGTTTGTTTTGGCTGGGCATGTCGTTGGTTCATGTTGGTAC

[0580] CTCTTCGGACTACAAAGGGTTAATCAATGTCTACGGGATTCTTGTGCTGCATCAAACATATCAAAAGCGTTGTGTAATAATTGTAC

[0581] AGATTGTGGAATTACTGGAATAAATAGGACCAATTGGTTGAACAACTCAGACTTAACTGGCTGTTTTGATACTAAAAGCGGTAAT

[0582] TTCCCTTATGGCATCTACCAACAGGCAGTGTTGCTAACCACAGAACCTGGACTTAAGCGTTATATATATTCGCTCTTTTGGGGGTT

[0583] TCAGCAAATTAGTACATTAGCTGGCAATTTGATCCCAAGTTACTTTGTATGGGAAGTAATATTCACTATGGCTATTATTGGACTGG

[0584] GGTTGTTGCTTTTCGCATTACTCATAGAGTCCATGCAGAATTTTCTCCAAGCTCTTGGAAAAAGGAGACTAGAGATGCAATTAAG

[0585] AAGGCGTGATGTTGAACAGTGGATGAGCCATAGGCGGCTGCCAGAAGATCTGAGAAGGAGGGTTAGATCTGCCGAGAGGTTC

[0586] AGCTGGGTAGCTACTAGAGGAGTGAATGAAGAAGAGCTTTTGAGCAATTTGCCAGAAGATATTCAAAGGGGCATACGCCGCCA

[0587] TTTCTTTGGGTTCCTTAAGAAGGTCCGGCTGTTTAACCTGATGGACAATGCAACCTGGGATGCAATTTGTGACAAGCTAAGGCA

[0588] GAACTTGTATATTACAGGAAGTGATATTCTTTATCAGGGTGGTCCTGTTGAAAAGATGGTTTTTATAGTCAGGGGTAGATTGGAA

[0589] AGCATCAGTGCAGATGGAAATAAGTCTCCCTTGCAAGAAGGAGATGTTTGTGGTGAGGAACTCCTTTCCTGGTACTTGGAGCA

[0590] ATCTTCAGTGAACCGAGATGGTGGGAAGATCAAGTTGCATGGCATGCGTTTGGTCGCCATACGTACTGTCAGATGTTTAACAAA

[0591] TGTTGAAGCTTTTGTACTGCGAGCACGTGATCTGGAAGAAGTGACTTCACAATTTTCAAGATTCTTGCGCAATCCACTTGTGCTA

[0592] GGTACAATCAGGTATGAATCACCTTACTGGAAGAACCTTGCAGCAAATCGCATCCAAGTCGCATGGAGGTATCGGAAAAGGCG

[0593] ACTGAAGAGAGCTGAGATGCAAAGGTTGCAAATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGT

[0594] CGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGAC

[0595] CCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTT

[0596] CAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCT

[0597] TCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAA

[0598] GGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGG

[0599] CCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCA

[0600] CTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCA

[0601] AAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCT

[0602] GTACAAGTAACGGCCGCTAAGTGAGTAGATCGTTCAAACATTTGGCAATAAAGTTTCTTAAGATTGAATCCTGTTGCCGGTCTTG

[0603] CGATGATTATCATATAATTTCTGTTGAATTACGTTAAGCATGTAATAATTAACATGTAATGCATGACGTTATTTATGAGATGGGTTTTT

[0604] ATGATTAGAGTCCCGCAATTATACATTTAATACGCGATAGAAAACAAAATATAGCGCGCAAACTAGGATAAATTATCGCGCGCGGT

[0605] GTCATCTATGTTACTAGATCGGgCTGCAGGCATGCAAGCTTGGCACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCT

[0606] GGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCT

[0607] TCCCAACAGTTGCGCAGCCTGAATGGCGAATGCTAGAGCAGCTTGAGCTTGGATCAGATTGTCGTTTCCCGCCTTCAGTTTAGC

[0608] TTCATGGAGTCAAAGATTCAAATAGAGGACCTAACAGAACTCGCCGTAAAGACTGGCGAACAGTTCATACAGAGTCTCTTACGA

[0609] CTCAATGACAAGAAGAAAATCTTCGTCAACATGGTGGAGCACGACACACTTGTCTACTCCAAAAATATCAAAGATACAGTCTCA

[0610] GAAGACCAAAGGGCAATTGAGACTTTTCAACAAAGGGTAATATCCGGAAACCTCCTCGGATTCCATTGCCCAGCTATCTGTCAC

[0611] TTTATTGTGAAGATAGTGGAAAAGGAAGGTGGCTCCTACAAATGCCATCATTGCGATAAAGGAAAGGCCATCGTTGAAGATGCC

[0612] TCTGCCGACAGTGGTCCCAAAGATGGACCCCCACCCACGAGGAGCATCGTGGAAAAAGAAGACGTTCCAACCACGTCTTCAA

[0613] AGCAAGTGGATTGATGTGATATCTCCACTGACGTAAGGGATGACGCACAATCCCACTATCCTTCGCAAGACCCTTCCTCTATATAA

[0614] GGAAGTTCATTTCATTTGGAGAGAACACGGGGGACTCTTGAC ETD1 gene-specific molecular marker amplification primer

[0615] SEQ ID NO:7-1: AGAGTGCCGCACAGTTTCTA

[0616] SEQ ID NO:7-2: TACCCAAAGCAATGGGCCAC

[0617] Base information of the amplified fragment of the ETD1 gene-specific molecular marker (SEQ ID NO:8):

[0618] GAAAGGTTCCAACCTCCTTCCTAAATATCTACTCCCTTTTTTACCCAAAGCAATGGGCCACTGTTGTACTTCTGGTCCAGCTTTTT

[0619] ATCTTTTATATGCCTTTACTGACCTTACTATCTTTTTTATTTTCTTCAATTATCTGATGCAGCAAATTAGTACATTAGCTGGCAATTTG

[0620] ATCCCAAGTTACTTTGTATGGGAAGTAATATTCACTATGGCTATTATTGGACTGGGGTTGTTGCTTTTCGCATTACTCATAGAGTCC

[0621] ATGCAGAATTTTCTCCAAGCTCTTGGAAAAAGGTGTGCAGTTGTACCACAAGTTTATCCTATATGAAGTGTACTGATGAGATGCC

[0622] AATGGGTTTAGTATATATACATGTAAATGATGTTACACATATGCACAGAATTGAAGAGAACACAAATAGCAACAACAATCAGTTTG

[0623] AATAATAGAAACTGTGCGGCACTCT.

Claims

1. A method for screening to obtain rice ETD1 mutants, characterized by the following steps: include: (1) Treat seeds with EMS mutation on days 1-2; (2) On the third day, treat the seeds with a cleaning agent to remove residual EMS, then rinse with water and air dry; (3) Sow the dried seeds, raise seedlings, transplant rice seedlings and harvest M1 generation seeds; (4) Harvest the M1 generation plants and plant them into the M2 population; (5) Identify rice blast pathogens in seedlings for each M2 population to obtain disease-like plants with enhanced resistance; (6) The rice ETD1 mutant was obtained by self-pollinating multiple diseased plants; The rice ETD1 mutant has the ETD1 gene sequence shown in SEQ ID NO:1, and / or the protein encoded by the ETD1 gene has the amino acid sequence shown in SEQ ID NO:3, wherein the gene is a 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.

2. The method for screening and obtaining rice ETD1 mutants according to claim 1, characterized in that... The cDNA sequence of the ETD1 gene is shown in SEQ ID NO:2, and / or the encoded protein is selected from proteins that have 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity and the same function as proteins obtained by substitution and / or deletion and / or addition of amino acid residues in the amino acid sequence shown in SEQ ID NO:

3.

3. The method for screening and obtaining rice ETD1 mutants according to claim 2, characterized in that... The gene and / or protein described herein have the function of regulating rice blast resistance and enhancing rice resistance to rice blast, and / or the protein includes a fusion protein with the same function obtained by attaching a tag to the N-terminus and / or C-terminus.

4. A method for screening to obtain rice ETD1 mutants according to claim 1, 2, or 3, characterized in that... Steps (1)-(3) are small-scale processing steps, which include: (i) Soak the seeds for 24 hours on the first day, with 120 seeds for each treatment, and repeat 3 times. (ii) On the second day, after the seeds show signs of sprouting, prepare the mutagen EMS and set up 5 concentrations. Add 120 seeds and 10 ml of the corresponding concentration of EMS solution to a 9 mm petri dish, and incubate for 16 hours at room temperature and in a shaker at 60 rpm. (iii) On the third day, treat the seeds with 1N NaOH to remove residual EMS, then rinse with water and air dry.

5. A method for screening to obtain rice ETD1 mutants according to claim 1, 2, or 3, wherein the method is characterized in that... Steps (1)-(3) are large-scale processing steps, including: (i) On the first day, soak approximately 10,000 seeds for 24 hours. (ii) On the second day, prepare 1 liter of EMS mutagen and perform seed treatment. (iii) On the third day, collect the seeds, treat them with 1N NaOH to remove residual EMS, then rinse them with water and air dry.

6. A method for screening to obtain rice ETD1 mutants according to claim 4 or 5, characterized in that... The cleaning agent is a 1N NaOH solution, and / or the most suitable EMS mutagenesis concentration for confirming a germination rate of 50% is approximately 0.5%, wherein a germination rate of 50% after EMS mutagenesis is the optimal mutagenesis dose, indicating that the mutagenesis basically covers the whole genome level, and the 0.5% refers to the EMS concentration.

7. The method for screening and obtaining rice ETD1 mutants according to claim 6, characterized in that... The method for identifying rice blast in step (5) includes: (a) The rice blast fungus stored on filter paper at -20°C was activated by placing it in fresh plum culture medium; (b) The activated pathogen was cultured in the dark and under light conditions for 12 hours each to obtain a large number of rice blast fungus spores; (c) Collecting rice blast fungus spores; (d) Observe spore activity on a hemocytometer and calculate the spore suspension concentration, and prepare the spore solution to a concentration of (10-25)×10⁴ spores / mL; (e) Pre-treat rice seeds before germination and then sow them; (f) After fertilizing the seedlings, inoculate and infect them with rice blast fungus at the 3-4 leaf stage, and then allow them to grow in the dark. (g) Observe the disease incidence of seedlings 7-9 days after inoculation and compare them with positive susceptible varieties to assess the strength of disease resistance.

8. The method for screening and obtaining rice ETD1 mutants according to claim 7, characterized in that... In step (c), the rice blast fungus spores are scraped from the plum culture medium with an appropriate amount of 0.1% Tween 20 aqueous solution, the mycelium is filtered through four layers of filter cloth, and the rice blast fungus spores are collected.

9. The method for screening and obtaining rice ETD1 mutants according to claim 7, characterized in that... In step (f), a high-pressure spray gun is used to spray inoculate rice seedlings in a room inoculation box, spraying 15 ml of rice blast fungus spore suspension into each seedling tray. The rice seedlings are then transferred to a greenhouse environment with a temperature maintained at 25°C and a humidity of 95%, and subjected to 24 hours of darkness treatment to promote the infection process of the pathogen; and / or, after the dark growth in step (f), the rice seedlings are cultured under growth conditions (6.8 × 10³ lux, 16 hours of light at 25°C, 8 hours of darkness at 25°C) while maintaining humidity.

10. The method for screening and obtaining rice ETD1 mutants according to claim 7, characterized in that... The observation process in step (g) includes: visually investigating and recording the disease incidence of various rice varieties, observing the leaves, stems and other parts of rice seedlings, and recording the disease symptoms, such as the size, shape, color and distribution of lesions; and / or, the positive susceptible varieties in step (g) are selected from the CO30 rice variety.

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