Rice ETD1 gene, and encoded protein thereof and use thereof

Abstract

Provided in the present invention is a hypermorphic allele ETD1 against rice blast, wherein the ETD1 gene has a nucleotide sequence as set forth in SEQ ID NO: 1, and a cDNA sequence thereof is as set forth in SEQ ID NO: 2. Further provided is an encoded protein of the gene, wherein the protein has an amino acid sequence as set forth in SEQ ID NO: 3. The gene is a hypermorphic allele of OsCNGC13, which exhibits enhanced calcium-ion transport capacity and has the function of significantly accelerating the influx of calcium ions into cells. Therefore, the normal expression or overexpression of the protein in a gene knockout plant can enhance resistance of rice, and thus the protein has a positive regulatory effect on rice blast resistance. The present invention can be used for the rapid identification of transgenic crops harboring ETD1 mutant genes, thereby improving the efficiency of molecular biological breeding.

Description

Rice ETD1 gene, its encoded protein and use TECHNICAL FIELD

[0001] The present application relates to a rice ETD1 gene and its encoded protein and use, and belongs to the field of molecular biology technology. BACKGROUND

[0002] Diseases are serious in crop production, which seriously threatens food security. Taking Hunan Province as an example, the 2023 major crop disease and pest occurrence trend forecast shows that the cumulative occurrence of rice sheath blight (44 million mu times), rice blast (3.3 million mu times), rice false smut (3 million mu times), and southern rice black-streaked dwarf (0.5 million mu times) in Hunan reaches 50.8 million mu times. Due to the problems of easy loss of major resistance genes due to changes in pathogenic bacteria, regional limitations, resistance costs, and genetic burdens in molecular breeding, the utilization rate of resistance genes in major rice production is low, and better broad-spectrum and durable disease resistance strategies are urgently needed. In addition, some diseases such as rice sheath blight, rice false smut, and southern rice black-streaked dwarf lack the cloning of major resistance genes and the breeding of resistant varieties, and their control mainly relies on pesticides. Therefore, improving the broad-spectrum resistance of crops to multiple pathogenic microorganisms while reducing pesticide use has become an inevitable demand for green prevention and control in grain production.

[0003] Taking rice blast as an example, the pathogen of rice blast belongs to the genus Gibberella in the kingdom Fungi. This fungus is a combination of sexual and asexual reproduction, with characteristics of susceptibility, high efficiency, and wide adaptability. Gibberella grows in the soil and can reproduce by absorbing water and nutrients through the plant roots. After the pathogen enters the inside of the rice plant through the wound or natural orifice (such as stomata) of the rice plant, it reproduces in large quantities and produces toxins, causing the rice leaves to develop fusiform or elliptical lesions. If not controlled in time, it will seriously affect the photosynthesis and nutrient growth of rice, eventually leading to the dwarfing, stopping of growth, and even death of rice, so rice blast is also known as "rice cancer". If chemical fungicides or biological pesticides are used to prevent and control rice blast after its outbreak, the stopped growth of rice will resume, and the yield loss will be reduced to a minimum within a controllable period.

[0004] The core of the molecular design breeding of rice against diseases including rice blast is the disease resistance gene. The immune system of rice is mainly composed of pathogen-triggered immunity (PTI) and effector-triggered immunity (ETI). The former is generally considered to confer plants with broad-spectrum and long-lasting disease resistance, and the latter can confer plants with specific resistance to specific races of pathogens. In production practice, major genes can significantly improve the resistance of crops to specific races of pathogens, but this resistance often cannot be long-lasting, and when new races of pathogens appear, the disease resistance effect of major genes disappears immediately. Therefore, the discovery of broad-spectrum disease resistance genes has more important value for rice disease resistance breeding and is also a hotspot in the field of plant disease resistance research. However, due to the limitations of identification and breeding, only a few broad-spectrum disease resistance genes in rice and their molecular mechanisms have been reported.

[0005] It is found that calcium ions are not only essential mineral elements for cells, but also essential second messengers for immune activation. Immune activation triggers calcium signals with different amplitudes and durations, which are decoded by various calcium-binding proteins to regulate downstream defense responses or even cell death. Therefore, the establishment of immune calcium signaling is a core link of plant biotic stress perception and response, and is also a frontier hotspot in plant immunity research. Calcium signaling is regulated by calcium ion channels, among which, plant cyclic nucleotide-gated channel proteins (CNGCs) are one of the main calcium ion channels. Studies have shown that plant CNGCs are involved in many physiological processes in plants, including immune response, pollen tube germination, elongation and guidance, stomatal movement, and root gravitropism, which mainly require the action of CNGC-mediated calcium ion flow in cells.

[0006] It is found that calcium ions are not only essential mineral elements for cells, but also essential second messengers for immune activation. Immune activation triggers calcium signals with different amplitudes and durations, which are decoded by various calcium-binding proteins to regulate downstream defense responses or even cell death. Therefore, the establishment of immune calcium signaling is a core link of plant biotic stress perception and response, and is also a frontier hotspot in plant immunity research. Calcium signaling is regulated by calcium ion channels, among which, plant cyclic nucleotide-gated channel proteins (CNGCs) are one of the main calcium ion channels. Studies have shown that plant CNGCs are involved in many physiological processes in plants, including immune response, pollen tube germination, elongation and guidance, stomatal movement, and root gravitropism, which mainly require the action of CNGC-mediated calcium ion flow in cells.

[0006] It is found that calcium ions are not only essential mineral elements for cells, but also essential second messengers for immune activation. Immune activation triggers calcium signals with different amplitudes and durations, which are decoded by various calcium-binding proteins to regulate downstream defense responses or even cell death. Therefore, the establishment of immune calcium signaling is a core link of plant biotic stress perception and response, and is also a frontier hotspot in plant immunity research. Calcium signaling is regulated by calcium ion channels, among which, plant cyclic nucleotide-gated channel proteins (CNGCs) are one of the main calcium ion channels. Studies have shown that plant CNGCs are involved in many physiological processes in plants, including immune response, pollen tube germination, elongation and guidance, stomatal movement, and root gravitropism, which mainly require the action of CNGC-mediated calcium ion flow in cells.

[0006] It is found that calcium ions are not only essential mineral elements for cells, but also essential second messengers for immune activation. Immune activation triggers calcium signals with different amplitudes and durations, which are decoded by various calcium-binding proteins to regulate downstream defense responses or even cell death. Therefore, the establishment of immune calcium signaling is a core link of plant biotic stress perception and response, and is also a frontier hotspot in plant immunity research. Calcium signaling is regulated by calcium ion channels, among which, plant cyclic nucleotide-gated channel proteins (CNGCs) are one of the main calcium ion channels. Studies have shown that plant CNGCs are involved in many physiological processes in plants, including immune response, pollen tube germination, elongation and guidance, stomatal movement, and root gravitropism, which mainly require the action of CNGC-mediated calcium ion flow in cells.

[0007] OsCNGC9 is the second member of the OsCNGCs family cloned in rice, which positively regulates the resistance of rice seedlings to rice blast. Studies have found that OsCNGC9 is a calcium channel protein. Under the induction of pathogen-associated molecular patterns, rice receptor-like kinase OsRLCK185 can interact with OsCNGC9, activate calcium influx by phosphorylating it, and thus positively regulate the expression of reactive oxygen species burst and PTI-related genes. Therefore, OsCNGC9 is directly involved in the regulation of immune calcium signaling. Under cold stress, OsCNGC9-mediated calcium influx can initiate downstream cold stress response to improve rice cold tolerance. Wang JC (Molecular cloning and functional analysis of rice immune-related gene OsCNGC9 and functional study of two rice heading-related transcription factors, Nanjing Agricultural University, Ph.D. thesis, 2018) reported the discovery of a rice lesion mimic mutant cds1 gene from OsCNGC9. Studies have shown that there is no obvious difference between wild type and mutant in tillering stage leaves. The mutant showed obvious lesion mimic phenotype in leaves after heading, and the resistance to rice blast was significantly reduced. Further experiments showed that a 4-bp deletion occurred in the fourth exon of the mutant, resulting in premature termination of translation. The lesion mimic phenotype and reduced resistance of the mutant are due to the mutation of OsCNGC9. Transgenic results show that increasing the transcription of OsCNGC9 can improve the broad-spectrum resistance of rice to rice blast to some extent. However, this paper is still a basic research on OsCNGC9, which only proves that the calcium channel OsCNGC9 is involved in the PTI immunity of plants, and the pathogen can use the evolution of effector proteins to attack this resistance. In natural evolution, this type of pathogen has appeared, so there is an OsCNGC9 gene, but rice is still susceptible. Secondly, this mutant significantly reduces the resistance of rice to rice blast, i.e. this phenotype and resistance are not related. The mutant is not a gain-of-function mutant, so it has no production application value.

[0008] In addition, according to existing research, there are many known lesion mimic mutants (see pages 9-12 of the above paper), involving different gene regulation. However, most of the lesion mimic phenotype regulatory genes involve metabolic-related regulatory genes, and there are few reports on ion channel-type lesion mimic mutants. Not all lesion mimic mutants are related to resistance to rice blast or similar diseases, which has not led to substantial progress in related research.

[0009] From existing research, it is worth further studying the regulatory role of other members of the OsCNGCs family in immune calcium signaling. Although multiple CNGC genes have been identified in rice and other plants and are associated with basic immune responses, the resistance mediated by them is weak and has no production application value.

[0010] From the search of rice blast related research literature, although there are reports of calcium ion channel protein related genes improving rice blast resistance, the improvement effect is not significant. Therefore, a new calcium ion channel protein related CNGC gene mutant is needed, which should have broad-spectrum resistance to Magnaporthe oryzae, so as to improve the disease resistance of rice to different rice blast physiological races. SUMMARY

[0011] The first principle of the present application is to find the coding protein of the super-effective allele mutant ETD1 gene from CNGC13 for the first time, which has the function of significantly accelerating the intracellular flow of calcium ions.

[0012] The second principle of the present application is to prove for the first time that the coding protein of the mutant ETD1 gene related to the lesion mimic phenotype not only has resistance to rice blast and similar diseases, but also belongs to the super (effective) allele of OsCNGC13. Since the super allele has new functions, the mutant can be used to make gene knockout plants have stronger calcium ion transport capacity, have the function of significantly accelerating the intracellular flow of calcium ions, and enhance the resistance of plants to rice blast and similar diseases.

[0013] The third principle of the present application is to use the coding protein of the ETD1 gene for genetic improvement of crops, especially the resistance of rice to rice blast, and to cultivate broad-spectrum rice blast resistant rice. Specifically, ETD1 is a super-effective allele of OsCNGC13, but it belongs to a recessive relationship. Under the condition of OsCNGC13, ETD1 cannot function. Therefore, the original OsCNGC13 gene of the original crop needs to be knocked out and ETD1 is introduced, which can be used for genetic improvement of crops, especially the resistance of rice to rice blast, and can cultivate broad-spectrum rice blast resistant rice.

[0014] Therefore, the present application provides a mutant ETD1 gene from CNGC13, which has a nucleotide sequence as shown in SEQ ID NO. 1, wherein the gene belongs to the super-effective allele of OsCNGC13, and has stronger calcium ion transport capacity and the function of significantly accelerating the intracellular flow of calcium ions.

[0015] In one embodiment, the cDNA sequence of the ETD1 gene is shown in SEQ ID NO. 2.

[0016] In a preferred embodiment, the gene has the function of regulating rice blast resistance and enhancing the resistance of rice to rice blast.

[0017] In any of the above embodiments, the ETD1 gene belongs to the super-effective allele of OsCNGC13, and normal expression or overexpression in a gene knockout plant can improve the resistance of rice, thus positively regulating the resistance of rice to rice blast.

[0018] In any of the above embodiments, the expression of the ETD1 gene in a gene knockout or gene deletion type of rice is inhibited or reduced, and the resistance of the rice to rice blast is reduced. In contrast, the expression of the rice lesion mimic mutant cds1 gene from OsCNGC9 in rice is increased, resulting in reduced resistance of the rice to rice blast.

[0019] In any of the above embodiments, a method for enhancing the resistance of rice to rice blast is established, comprising knocking out the OsCNGC13 gene of rice, then transgenically introducing the ETD1 gene, then culturing the transgenic rice plant, and finally obtaining a rice plant with enhanced resistance to rice blast.

[0020] In any of the above embodiments, a method for screening for an ETD1 mutant of rice is established, comprising: treating the seeds with EMS mutation on days 1-2; treating the seeds with a cleaning agent on day 3 to remove residual EMS, then rinsing with water and air-drying; sowing, seedling, transplanting and harvesting M1 generation seeds from the air-dried seeds; planting the M1 generation plants to form an M2 population; identifying the seedlings of each M2 population for rice blast to obtain lesion mimic plants with enhanced resistance; and selfing the lesion mimic plants to obtain the ETD1 mutant.

[0021] In any of the above embodiments, a method for specifically identifying the transgenic ETD1 gene of rice by molecular markers is established, characterized by identifying the ETD1 gene by the developed specific molecular markers.

[0022] Therefore, the present application provides a mutant ETD1 gene from CNGC13, which has an amino acid sequence as shown in SEQ ID No. 3, wherein the coding gene of the protein belongs to the super-effective allele of OsCNGC13, which can make the gene knockout or gene deletion type plant have stronger calcium ion transport capacity, and has the function of significantly accelerating the calcium ion influx of cells.

[0023] In one embodiment, the coding protein is selected from a protein having 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity with the amino acid sequence shown in SEQ ID No. 3 and having the same function.

[0024] In one specific embodiment, the protein includes a fusion protein with the same function obtained by connecting a tag at the N-terminus and / or C-terminus.

[0025] In one preferred embodiment, the protein has the function of regulating rice blast resistance, enhancing the resistance of rice to rice blast.

[0026] In any of the above embodiments, the gene encoding the ETD1 protein belongs to a super-effective allele of OsCNGC13, and normal expression or overexpression in a gene knockout plant can improve the resistance of rice, thus positively regulating the resistance of rice to rice blast.

[0027] In any of the above embodiments, the gene encoding the ETD1 protein belongs to a super-effective allele of OsCNGC13, and normal expression or overexpression in a gene knockout plant can improve the resistance of rice, thus the expression of the gene encoding the ETD1 protein in the OsCNGC13 gene knockout or gene deletion rice is inhibited or the expression amount is reduced, and the resistance of rice to rice blast is reduced. In contrast, the expression amount of the rice lesion mimic mutant cds1 gene from OsCNGC9 in rice is increased, resulting in reduced resistance of rice to rice blast.

[0028] Technical effects

[0029] 1. Although there are many existing lesion mimic mutants, different genes are involved in the regulation. However, most of the lesion mimic phenotype regulation genes are related to metabolic regulation genes, and there are few reports on ion channel type lesion mimic mutants. The present application first discovers a functional gain-of-function allele of calcium ion channel, which enhances the activity of the channel on the basis of the original function of the channel, and performs directed evolution on the basic function channel. The present application provides a new solution and example for improving plant disease resistance by using immune calcium signal.

[0030] 2. The present application first obtains the mutant ETD1 protein from the OsCNGC13 gene by biological induction mutation technology. The mutation is located on the seventh exon of the LOC_Os06g10580 gene, which causes G-A variation and changes the amino acid (Figure 3B).

[0031] 3. The present application first proves that the mutant ETD1 gene encoding protein related to the lesion mimic phenotype not only has resistance to rice blast and similar diseases, but also belongs to a super-effective allele of OsCNGC13. Since the super-allele has a new function, the mutant can be used to make gene knockout plants have stronger calcium ion transport capacity, have the function of significantly accelerating the calcium ion influx of cells, and enhance the resistance of plants to rice blast and similar diseases.

[0032] 4, The application first proves that the unit point mutation of the amino acid can significantly improve the resistance of rice to rice blast.

[0033] 5, The application first proves that the ETD1 protein belongs to a mutant of a calcium ion channel protein, which can significantly accelerate the ability of calcium ion influx into cells.

[0034] 6, The application first proves that the ETD1 gene belongs to a super allele of OsCNGC13, but is in a recessive relationship, and ETD1 cannot function under the condition that OsCNGC13 exists. Therefore, the original OsCNGC13 gene of the original crop needs to be knocked out, and ETD1 is introduced, which can be used for genetic improvement of crops, especially the resistance of rice to rice blast, and a broad-spectrum rice blast-resistant rice can be cultivated.

[0035] 7, The application provides a method for cultivating a high rice blast-resistant rice variety by using the ETD1 gene and the protein encoded by the gene and related biological elements, which provides a new gene resource for breeding rice blast-resistant germplasm, and is beneficial to the breeding of rice blast-resistant varieties, thereby ensuring the safety of rice production.

[0036] 8, Since the wild type host does not contain the ETD1 gene, and the application proves that it is not necessary to improve the expression or expression amount of ETD1, but only needs to introduce the gene into a CNGC13 gene knockout or gene deletion host to obtain stronger calcium ion transport capacity, which has the function of significantly accelerating the calcium ion influx into cells, thereby improving the ability of the host to resist rice blast.

[0037] 9, The application first proves that ETD1 will cause the leaf cells of the CNGC13 gene knockout or gene deletion type to die obviously more than the wild type under the stimulation of the rice blast fungus, but this reaction mechanism is essentially beneficial to the host to resist the invasion of foreign pathogens and establish an effective immune defense mechanism. Once the invasion period of foreign pathogens is passed, and reasonable nutrition, light and other conditions are provided in the later period, the subsequent growth of the transgenic rice is basically or completely restored to normal growth, and has basically no effect on yield. In contrast, in the same field and at the same growth stage, after inoculation of the wild type strain with the rice blast fungus, it is basically all withered and died. Therefore, it is predicted that the application of ETD1 to the CNGC13 gene knockout or gene deletion host can develop transgenic crops resistant to various crop diseases, and has a wide production application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0038] Figure 1, ETD1 broad spectrum resistance to rice blast, where A is the ETD1 creation process; B is leaf phenotype after inoculation with rice blast races; C is the lower leaf cell death index after inoculation; D is the relative pathogen growth after inoculation with Magnaporthe oryzae; E is the Chitin induced ETD1 cell death phenotype; F is the cell death index of IR64 and ETD1; G is the leaf resistance phenotype of ETD1 after inoculation with different rice blast races. t-test, *** indicates extremely significant difference, p < 0.001.

[0039] Figure 2, ETD1 lesion mimic phenotype affects agronomic traits: A is the mature stage phenotype of IR64 and ETD1; B is the leaf lesion mimic phenotype of ETD1; C is the leaf mesophyll cell TUNEL staining analysis; D-I are the comparison of agronomic traits of IR64 and ETD1: plant height (D), tiller number (E), panicle length (F), seed setting rate (G), 1000-grain weight (H), heading stage (I). t-test, * indicates significant difference, p < 0.05; ** indicates extremely significant difference, p < 0.01.

[0040] Figure 3, ETD1 is a hypermorphic allele of OsCNGC13: A is the distribution of SNP index of lesion mimic phenotype pool on chromosomes; B is the ETD1 mutation site; C is the protein secondary structure prediction map; D is the phenotype of ETD1 knockout line inoculated with IR64 avirulent strain V86010; E is the phenotype of IR64 virulent strain CA89 inoculation; F-G are the phenotype of OsCNGC13 knockout line and ETD1 genetic back-complementation line inoculated with rice blast. NIP avirulent strain 4029-1 (F) and NIP virulent strain S5 (G). Figure H is the final field growth result of the genetic back-complementation line of the knockout type ETD1 (right panel) and the wild type line (left panel) after inoculation with rice blast.

[0041] Figure 4, ETD1 encodes an enhanced calcium inward channel: A-B are the calcium accumulation in E. coli expressing ETD1 and OsCNGC13, which is dependent on extracellular calcium concentration (A) and time (B); C is the calcium accumulation in E. coli expressing ETD1 and OsCNGC13 under the treatment of calcium channel blocker Gd 2+ (0.1 mM) and activator ACC (1 mM); D is the organelle localization of ETD1-GFP and OsCNGC13-GFP in Xenopus oocytes; E is the average current-voltage result of Xenopus oocytes expressing ETD1 and OsCNGC13 under 30 mM calcium treatment. F is the average current-voltage result of Xenopus oocytes expressing ETD1 and OsCNGC13 under 30 mM barium treatment; G is the NMT measurement showing the calcium accumulation in IR64 and ETD1 after 10 mM Ca 3+ 2+ treatment.​2+ Intraflux, measured for 5 minutes under normal conditions, 10 mM Ca 2+ Measurements were taken for 6 minutes after treatment; H represents calcium-triggered Ca2+ in root cells. 2+ Quantification of flux integral; I represents calcium stimulation of cytoplasm in the living roots of IR64 and ETD1, leading to Ca2+. 2+ The temporal dynamics of the increase were assessed using the normalized ratio of cpVenus / ECFP to evaluate cytoplasmic calcium. 2+ The increase in J represents cytoplasmic Ca. 2+ Concentration curve integration; using resting cytoplasmic Ca2+ before thermal stimulation 2+ The curve integral was calculated based on concentration. A t-test was performed, with different letters indicating highly significant differences (p < 0.01).

[0042] Figure 5. Cell death induced by enhanced calcium ion influx in ETD1 under immune activation: A and B represent dynamic analysis of ROS generation in IR64 and ETD1 leaves induced by Chitin (A) and flg22 (B), respectively; C and D represent the calcium ion influx in IR64 and ETD1 mesophyll cells after Chitin (C) and flg22 (D) treatment, respectively. 2+ Comparison of influxes; E and F represent dynamic analysis of Ca in the root cytoplasm of IR64 and ETD1 roots stimulated by Chitin (E) and flg22 (F), respectively. 2+ Concentration changes; GH represented the calcium ion accumulation levels in IR64 and ETD1 mesophyll cells 12 h after inoculation with non-toxic rice blast fungus V86010 (G) and toxic rice blast fungus CA89 (H), respectively; I represented the calcium channel blocker Gd. 3+ (1mM) treatment inhibited ETD1-induced cell death under seeding conditions. Detailed Implementation

[0043] 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.

[0044] Example 1: Obtaining the ETD1 gene mutant through bio-induced mutation

[0045] Previously, an EMS chemical mutagenesis was performed on IR64 to obtain a mutant that significantly induced leaf cell death after spraying with both the non-toxic physiological race V86010 and the toxic physiological race CA89 (Fig. 1A-D). Interestingly, the fungal elicitor chitin also significantly induced cell death in the mutant leaves (Fig. 1E-F). Therefore, this mutant was named ETD1 (Elicitor triggered Cell Death 1).

[0046] Rice blast spray inoculation proved that ETD1 mutants were completely immune to different rice blast races; Rice blast strains: CA89 and V86010 were collected from the International Rice Research Institute. 17-1-1, 17-5-2, 17-6-1, 17-6-2, 17-7-1 and 19-2-1 were collected from Sichuan in 2017 and kept in our lab. ETD1 was inoculated with the above strains, and resistance evaluation was as previously described.

[0047] The differences in plant height, panicle length, seed setting rate, and tiller number were significant, and T-test was used.

[0048] Investigation of agronomic traits:

[0049] Seeding and transplanting were carried out in Changsha Chunhua base and Haitangwan base in Sanya, Hainan. At the mature stage of rice, 5 plants of IR64 and ETD1 were randomly selected, and the number of dead cells per leaf was counted.

[0050] Forty-five strong pathogenic rice blast races collected from the main rice production areas in China and five representative rice blast races from abroad were selected for spray inoculation of ETD1. Different rice blast races could trigger cell death in ETD1 leaves (part of the inoculation results are shown in Figure 1G). Therefore, we created a new rice germplasm with broad-spectrum resistance to rice blast through EMS mutagenesis.

[0051] In the field, we found that ETD1 leaves spontaneously produced cell death, and the leaves at the flowering stage were almost covered with cell death spots (Figures 2A-B), which is a typical hypersensitive-like phenotype. TUNEL experiments showed that the degree of DNA damage in ETD1 was significantly enhanced compared to the wild type (Figure 2C), which further proved that ETD1 caused significant cell death. With the strong induction of cell death, ETD1 agronomic traits changed significantly, such as dwarfing, reduced tiller number, shorter panicle length, reduced seed setting rate, reduced thousand-grain weight, and delayed growth period (Figures 2D-I). The above results indicate that ETD1 improves rice blast resistance, but the hypersensitive-like phenotype significantly affects normal rice growth.

[0052] Example 2: Specific embodiments of OsCNGC13 gene knockout

[0053] The target (SEQ ID NO. 4 and SEQ ID NO. 5) is selected on the first exon of OsCNGC13 gene, and the gRNA fragment is obtained by PCR using a synthetic target primer; the intermediate vector containing the gRNA fragment is constructed by Eco31I enzyme digestion and T4 ligase system and is verified by sequencing. Then the gRNA fragment in the intermediate vector is transferred to the rice genetic transformation vector YLCas9-hu by enzyme digestion and ligation to obtain the YLCas9-hu-CNGC13 gene knockout vector. The recombinant plasmid is transformed into Agrobacterium GV3101, and the OsCNGC13 transgenic line is obtained by Agrobacterium-mediated rice mature embryo transformation. The homozygous editing line is screened by target site sequencing in the transgenic T2 generation, and the OsCNGC13 gene knockout line without transgenic elements is obtained by detecting the leaf hygromycin sensitivity.

[0054] The specific experimental scheme is as follows:

[0055] 1. CRISPR target site, target site linker primer design and gRNA acquisition

[0056] The gRNA (SEQ ID NO. 4 and SEQ ID NO. 5) of the first exon of OsCNGC13 gene is designed through an online website (http: / / tools.genome-engineering.org),

[0057] The gRNA fragment is obtained by PCR amplification, and the PCR system is: 1 μl SEQ ID NO. 4 (100 μM), 1 μl SEQ ID NO. 5 (100 μM), 8 μl ddH2O. PCR instrument 95℃ 5min, slowly cool to room temperature 1h, 1:200 dilution duplex.

[0058] 2. Construction of intermediate vector containing gRNA fragment

[0059] (1) Extraction of vector plasmid

[0060] The intermediate subcloned vector lentiCRISPR plasmid and the rice genetic transformation vector YLCas9-hu are extracted by alkaline lysis method.

[0061] The specific steps of alkaline lysis method for extracting plasmid DNA are as follows:

[0062] 1) Take the glycerol bacteria from the -80℃ ultra-low temperature freezer to 4-6ml LB medium containing antibiotics, and incubate overnight at 37℃ 200rpm in a shaking bed;

[0063] 2) Take 2ml of bacterial solution, centrifuge at 1000rpm for 1min, and collect the bacterial body;

[0064] 3) Discard the supernatant, add 100 μl solution I, vortex the bacteria;

[0065] 4) Add 200 μl solution II, mix gently, stand for 2 min, and perform this operation within 5 min;

[0066] 5) Add 150 μl solution III, mix gently;

[0067] 6) Centrifuge at 12000 rpm for 10 min;

[0068] 7) Take 440 μl supernatant into a new 1.5 ml centrifuge tube, add 880 μl absolute ethanol, mix, and centrifuge at 12000 rpm for 10 min;

[0069] 8) Discard the supernatant, add 600 μl 75% ethanol, and centrifuge at 12000 rpm for 10 min;

[0070] 9) Discard the supernatant, dry the residual water and alcohol in a 37°C oven, and add 30-50 μl ultrapure water.

[0071] (2) 20 μl plasmid digestion system: 2 μg lentiCRISPR plasmid, 1 μl Eco31I (10 U / μl), 2 μl 10x rcutsmart buffer, and ultrapure water to 20 μl. Digest for 6-8 hours.

[0072] (3) 10 μl digestion and ligation system: 1 μl T4 DNA ligase, 2 μl 5x DNA ligase buffer, 1 μl digested lentiCRISPR plasmid, and 6 μl duplex (diluted 200 times). Ligate at room temperature for 4-6 hours.

[0073] (4) Transformation: Take out 100 μl DH5α competent cells from -80°C, quickly insert into ice, and add the ligation product after the bacterial mass is melted. Gently mix the bottom of the EP tube with hand (avoid using gun suction), stand in ice for 25 min; then heat shock at 42°C for 45 s, quickly put back on ice and stand for 2 min, add 700 μl sterile LB medium without antibiotics to the centrifuge tube, mix, and recover at 37°C, 200 rpm for 60 min; then centrifuge at 5000 rpm for 1 min to collect the bacteria, take 100 μl supernatant, resuspend the bacterial mass by gently blowing, and spread on LB medium containing kana antibiotic; finally, place the plate upside down in a 37°C incubator and culture overnight.

[0074] (5) Agrobacterium transformation: GV3101 Agrobacterium competent cells were taken out from the -80°C refrigerator, quickly inserted into an ice box, and dissolved; DNA sample was added and mixed gently, and then sequentially placed on the ice box for 5 min, liquid nitrogen for 5 min (or dry ice ethanol bath box at -80°C and -80°C freezing), 37°C water bath for 5 min, and ice bath for 5 min; 900 μl of YEB liquid medium without antibiotics was added, mixed uniformly, and cultured at 28°C for 2-3 h. The bacteria were collected by centrifugation at 4.6000 rpm for 1 min, and about 100 μl of supernatant was taken to resuspend the bacterial block by blowing gently and then plated on YEB plates containing the corresponding antibiotics, and placed in a 28°C incubator for 2-3 days.

[0075] (6) Agrobacterium-mediated genetic transformation of mature rice embryos:

[0076] 1) Inducing callus: After the mature rice seeds were sterilized and soaked with 0.15% HgCl2, they were placed in callus culture medium for dark culture to induce the generation of callus;

[0077] 2) Subculture: The light callus was selected from the induced callus and transferred to subculture medium for dark culture;

[0078] 3) Pre-culture: The subculture callus was added to sterile pre-culture medium containing 300 μl of 40% glucose, and dark culture was performed;

[0079] 4) Infection and co-culture: The activated Agrobacterium liquid was poured into the callus, soaked, and then the callus was dried, and AS+50% glucose was added to the co-culture medium and poured into the dish, and then the fully dried callus was evenly spread on the co-culture medium with a spoon for dark culture;

[0080] 5) Washing the Agrobacterium with water, and then continuing dark culture;

[0081] 6) Screening: The screening medium was prepared by pre-adding CN, Hn, and 50% glucose, and the callus without Agrobacterium contamination was placed in the screening medium for dark culture;

[0082] 7) Differentiation: The resistant callus was placed in the differentiation medium for light culture until seedlings were differentiated;

[0083] 8) Rooting: The seedlings were transferred to the rooting medium;

[0084] 9) Hardening: After the transformed seedlings grew vigorously, the sterile film was removed, tap water was added, and light culture was performed for hardening until the transformed seedlings were transferred to the field, and the genetic transformation process was completed;

[0085] 10) Leaf hygromycin sensitivity detection: The untransgenic rice IR64 was used as control, and the transgenic T2 generation OsCNGC13 gene knockout line without transgenic elements was screened out.

[0086] Example 3: Specific embodiments of construction of ETD1 gene recombinant vector

[0087] The ETD1 leaves at the seedling stage were collected, and the genomic DNA and RNA were extracted. Specific primers were designed to amplify the ETD1 gene promoter and part of the exon about 4.1 kb size DNA fragment using the genomic DNA as a template, to construct a TA-promoter clone and verify by sequencing; the RNA was reversed into cDNA, and the primers were designed to amplify the DNA fragment of the complete CDS of the ETD1 gene using the cDNA as a template, to obtain a DNA fragment of about 3 kb size by fusing the 3' end of the ETD1 gene with the GFP gene and the Nons gene terminator through overlapping PCR, to construct a TA-ETD1-GFP clone and verify by sequencing; the TA-promoter plasmid was recovered by EcoRI and SalI enzyme digestion about 4.1 kb size DNA fragment, the TA-ETD1-GFP plasmid was recovered by SalI and PstI enzyme digestion about 3 kb size DNA fragment, and the fragment was introduced into the pCAMBIA1305.2 skeleton digested by EcoRI and PstI using T4 ligase to obtain the ETD1 transgenic vector.

[0088] The specific experimental scheme is as follows:

[0089] 1. Rice genomic DNA extraction

[0090] (1) Cut 2-3 cm length leaves into 2 ml centrifuge tubes, add steel balls, freeze in liquid nitrogen, and crush the leaves into powder in a crusher;

[0091] (2) Centrifuge at 10000 rpm for 30 s, add 800 μl 2x CTAB, and incubate at 65°C for 30 min;

[0092] (3) Add 800 μl chloroform, mix well by shaking up and down, and centrifuge at 10000 rpm for 10 min;

[0093] (4) Take 600 μl supernatant into 1.5 ml centrifuge tubes, add the same volume of isopropanol, and place at -20°C for 2 h;

[0094] (5) Centrifuge at 12000 rpm for 10 min, discard the supernatant, and dry the water;

[0095] (6) Add 600 μl 75% alcohol, shake up and down, centrifuge at 12000 rpm for 10 min, discard the supernatant, and repeat twice;

[0096] (7) Open the lid, dry the residual alcohol and water at 37°C, then add 100 μl ddH2O.

[0097] 2. Total RNA extraction from rice

[0098] (1) Freeze the fresh leaves with liquid nitrogen, then put them into a mortar which has been sterilized and pre-frozen with liquid nitrogen. Add liquid nitrogen during the grinding process to keep the sample in a low temperature state until it is ground into powder;

[0099] (2) Transfer the powder into a 2 ml RNA-free centrifuge tube with a sterilized spatula, add 1 ml RNA isolater and vortex, then stand at room temperature for 5 min to separate the nucleoprotein complex completely;

[0100] (3) Add 200 μl chloroform and shake vigorously for 15 s to form an emulsion, then stand at 4°C for 5 min;

[0101] (4) After centrifugation at 12000 rpm for 15 min at 4°C, carefully take out the sample and place it on ice. At this time, the sample is divided into three layers: a colorless water phase (upper layer), a white middle layer, and a red organic layer (lower layer);

[0102] (5) Transfer the upper water phase into a new enzyme-free and sterile 1.5 ml centrifuge tube, add the same volume of 4°C pre-cooled isopropanol, mix gently and uniformly, and place it on ice for 10 min;

[0103] (6) Centrifuge at 12000 rpm for 10 min at 4°C. At this time, a white precipitate can be seen at the bottom of the tube. Carefully discard the supernatant, wash the precipitate with 1 ml of 75% alcohol (RNase-free ddH2O), and gently blow it up with a syringe to make the white precipitate float. Stand at room temperature for 3-5 min;

[0104] (7) Centrifuge at 12000 rpm for 10 min at 4°C, discard the supernatant;

[0105] (8) Open the lid of the centrifuge tube in the ultraviolet sterilized clean bench, and let the precipitate dry naturally for 5 min. Note that it should not be dried too much to avoid difficulty in dissolving the extracted RNA;

[0106] (9) Dissolve the RNA with ddH2O (RNase-free), take a small amount of RNA for detection, and store the rest of the sample in a -80°C ultra-low temperature freezer.

[0107] 3. Reverse transcription to obtain cDNA

[0108] (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 4x gDNA wiper Mix, up RNase-free ddH2O to 16 μl. After gentle mixing, place in PCR machine at 42 °C for 2 min.

[0109] (2) First strand cDNA synthesis reaction system (20 μl): 16 μl genomic DNA removal mixed solution, 2 μl HiScript II Enzyme Mix, 2 μl 10x RT Mix. After gentle mixing, the reaction conditions are: 50 °C for 15 min, 85 °C for 2 min.

[0110] (3) The product is aliquoted and stored at -80 °C. Avoid repeated freezing and thawing of cDNA.

[0111] 4. Construction of ETD1 gene promoter and CDS intermediate vector

[0112] (1) Obtaining ETD1 gene promoter and partial exon:

[0113] PCR system (50 μl): 10 μl 5x 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.

[0114] PCR reaction amplification is carried out in a PCR machine. The PCR conditions are: 95 °C pre-denaturation for 3 min, then 95 °C denaturation for 15 s, 55 °C annealing for 15 s, 72 °C extension for 30 s / kb, 32 cycles, then 72 °C re-extension for 5 min, and finally 16 °C incubation.

[0115] (2) Obtaining ETD1 gene 3' end fusion GFP gene and Nons gene terminator

[0116] Overlapping PCR system: the first round of PCR amplification was performed with primer pair ETD1 gene complete CDS, ETD1 gene 3' end fusion GFP gene and Nons gene terminator, the product was column recovered to obtain A'(ETD1 gene complete CDS) and B'(GFP gene and Nons gene terminator) intermediate fragments, the PCR amplification system and conditions were referred to the previous step. The second round of PCR was performed with intermediate fragments A' and B' as templates, the two were added in equal molar amounts after concentration, and the total amount did not exceed 100 ng. Primer only A1 and B2, other components were unchanged. The PCR system and conditions were the same.

[0117] The PCR product was subjected to nucleic acid gel electrophoresis to verify whether the length of the amplified gene fragment was correct, if correct, the gel was cut and recovered for transformation and sequencing.

[0118] (3) Product purification:

[0119] 1) After DNA electrophoresis, the gel containing the target DNA fragment was quickly cut under the ultraviolet lamp, and the excess gel was removed as much as possible. The weight of the gel was weighed (the weight of the empty tube was removed), 100 mg of gel was equivalent to 100 μl of volume, which was used as a gel volume. An equal volume of Buffer GDP was added. 50-55°C water bath, invert mix twice during water bath to accelerate gel dissolution.

[0120] 2) Centrifuge briefly to collect the droplets on the wall of the tube. Place FastPure DNA Mini Columns-G adsorption columns in Collection Tubes 2ml collection tubes, transfer ≤700 μl of gel solution to the adsorption column, centrifuge at 12,000 rpm for 30-60 s. If the gel volume is >700 μl, place the adsorption column back into the collection tube, transfer the remaining gel solution to the adsorption column, centrifuge at 12,000 rpm for 30-60 s.

[0121] 3) Discard the filtrate, place the adsorption column in the collection tube. Add 300 μl of Buffer GDP to the adsorption column. Stand for 1 min. Centrifuge at 12,000 rpm for 30-60 s.

[0122] 4) Discard the filtrate, place the adsorption column in the collection tube. Add 700 μl of Buffer GW (with anhydrous ethanol) to the adsorption column, invert mix 2-3 times. Centrifuge at 12,000 rpm for 30-60 s. Repeat twice.

[0123] 5) Discard the filtrate, place the adsorption column back into the collection tube. Centrifuge at 12,000 rpm for 2 min.

[0124] 6) Place the column in a 1.5 ml sterile centrifuge tube, add 20-30 μl Elution Buffer to the center of the column, and let stand for 2 min. Centrifuge at 12,000 rpm for 1 min. Discard the column, and store the DNA at -20°C until use.

[0125] (4) TA cloning and transformation:

[0126] 1) TA cloning reaction system (5 μl): 1 μl 5x TA / Blunt-Zero Cloning Mix, 200 ng DNA fragment (2-5 kb in size), and finally add ddH2O to make the system reach 5 μl. PCR instrument temperature control 25°C, reaction 5 min.

[0127] 2) Transformation: refer to the above method.

[0128] 3) Selection and verification of candidate intermediate clones: 24 single colonies of TA-promoter clone and TA-ETD1-GFP clone are selected respectively, and are fully digested with EcoRI and Sail, Sail and PstI, and electrophoresed on 1% agarose gel. The correct TA-promoter clone and TA-ETD1-GFP clone are selected and sent to a sequencing company for sequencing verification.

[0129] 5, Obtaining of ETD1 transgenic vector:

[0130] (1) Preparation of vector pCAMBIA1305.2 skeleton:

[0131] 1) EcoRI and PstI digestion of vector pCAMBIA1305.2 skeleton

[0132] 50 μl of enzyme digestion system: pCAMBIA1305.2 vector 10 μl, EcoRI (10 U / μl) 1 μl, PstI (10 U / μl) 1 μl, 10x rcutsmart buffer 5 μl, ddH2O 33 μl.

[0133] 2) 37°C enzyme digestion for 4-5 h, take 1 μl for electrophoresis to observe whether the enzyme digestion is complete.

[0134] 3) After the remaining enzyme digestion sample is completed by pulse electrophoresis, the 12 kb linearized pCAMBIA1305.2 vector is recovered by gel cutting, and the specific method steps are referred to the above method.

[0135] (2) Obtaining of ETD1 promoter and ETD1 CDS-GFP fragment:

[0136] 1) EcoRI and Sail enzyme digestion of TA-promoter plasmid to recover about 4.1 kb size DNA fragment

[0137] 50 μl enzyme digestion system: TA-promoter vector 20 μl, EcoRI (10 U / μl) 1 μl, Sail (10 U / μl) 1 μl, 10 x rcutsmart buffer 5 μl, ddH2O 23 μl.

[0138] 2) Sail and Pstl enzyme digestion of TA-ETD1-GFP plasmid to recover about 3 kb size DNA fragment

[0139] 50 μl enzyme digestion system: TA-promoter vector 20 μl, Pstl (10 U / μl) 1 μl, Sail (10 U / μl) 1 μl, 10 x rcutsmart buffer 5 μl, ddH2O 23 μl.

[0140] 3) 37 °C enzyme digestion for 7-8 h, take 1 μl for electrophoresis to observe whether the enzyme digestion is complete.

[0141] 4) After the remaining enzyme digestion sample is completed by pulse electrophoresis, cut the gel to recover the 4.1 kb and 3 kb size linearized ETD1 promoter and ETD1 CDS-GFP fragments, respectively. The specific method steps are referred to the above method.

[0142] (3) Enzyme digestion and ligation:

[0143] 10 μl enzyme digestion and ligation system: 1 μl T4 DNA ligase, 2 μl 5 x DNA ligase buffer, 1 μl pCAMBIA1305.2 linearized skeleton, 2 μl ETD1 promoter enzyme digestion recovery fragment, 1.5 μl ETD1 CDS-GFP enzyme digestion recovery fragment, 2.5 μl ddH2O. 16 °C ligation overnight.

[0144] (4) Transformation: refer to the above method

[0145] (5) Selection and verification of candidate clones: 24 single clone colonies are selected, fully digested with EcoRI and Pstl, and electrophoresed on 1% agarose gel. The correct size of the enzyme digestion single colony is selected and sent to a sequencing company for sequencing verification.

[0146] Example 4: Verification that ETD1 is a functional gain-of-function mutation of cyclic nucleotide-gated channel protein OsCNGC13

[0147] The ratio of normal plants to plants with lesion mimic phenotype in the F2 population hybridized with ETD1 is about 3:1, indicating that ETD1 is a recessive gene. In order to further locate ETD1, 50 normal plants and 50 plants with lesion mimic phenotype in the F2 population were selected for BSA sequencing. Analysis shows that a SNP located on chromosome 6 of rice produces a missense mutation and is highly linked to the lesion mimic phenotype (Figure 3A). The mutation is located in the seventh exon of the LOC_Os06g10580 gene, which causes a G-A variation and changes the amino acid (Figure 3B). Previous studies have shown that LOC_Os06g10580 encodes the cyclic nucleotide-gated protein OsCNGC13, and the amino acid change is located in a conserved region of the sixth transmembrane domain of the protein (Figure 3C), specifically the mutation of glycine at position 483 to glutamic acid.

[0148] Knocking out the ETD1 gene, the ETD1 gene knockout line does not induce cell death after Magnaporthe oryzae inoculation (Figures 3D-E). Knocking out OsCNGC13 in the NIP background, no cell death phenotype was observed in the OsCNGC13 gene knockout line after Magnaporthe oryzae inoculation (Figures 3F-G).

[0149] A genetic back complementation plasmid (PGETD11-GFP) including a wild-type 4kb promoter, the complete CDS sequence of ETD1, and a GFP sequence was constructed and back complemented. Magnaporthe oryzae inoculation identification showed that when ETD1 was successfully introduced into the OsCNGC13 gene knockout line, the transgenic line showed obvious cell death (Figures 3F-G), and also showed a lesion mimic phenotype at the growth and development stage. The above experiments show that ETD1 is a gain-of-function mutation gene of OsCNGC13.

[0150] Example 5: Verification of ETD1 to improve early and middle stage resistance of gene knockout type rice to Magnaporthe oryzae and reduce damage at the mature stage

[0151] It should be noted that ETD1 is a superallele of OsCNGC13, but is recessive, and ETD1 cannot function under the presence of OsCNGC13. Therefore, the original gene knockout needs to be introduced into ETD1 to function.

[0152] 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.

[0153] As shown in Figure 3H, in the later stage of field experiments on the gene knockout transgenic lines of Figure 3F-G, the transgenic lines of rice in the early and mid-stages (seedling stage, transplanting stage, clearing stage, tillering stage or heading and flowering stage) gradually recovered their original growth through later growth and did not show obvious wilting phenomenon (see the right figure of Figure 3H). In subsequent experiments, the lines eventually entered the maturity stage smoothly.

[0154] In contrast, wild-type lines in the same field and at the same growth stage withered and died almost entirely after being inoculated with rice blast fungus, and it is foreseeable that the wild-type lines will face a complete crop failure (see Figure 3H, left).

[0155] Example 6: Verification of ETD1-encoded enhanced calcium ion inward channel

[0156] 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 demonstrate whether ETD1 possesses calcium transport capabilities... 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+ The accumulation of [something] (Figure 4C) indicates that ETD1 has calcium ion selective permeability.

[0157] Patch-clamp experiments showed that Xenopus laevis expressing ETD1 responded to 30 mM Ca2+. 2+There was a large inward current in the bath solution, but only a small inward current was observed in the Xenopus oocytes expressing OsCNGC13 (Fig. 4D-E). Some plant plasma membrane Ca 2+ channels have Ba 2+ permeability, we further analyzed the Ba 2+ permeability of ETD1 and OsCNGC13. We replaced Ca 2+ in the bath solution with the same concentration of Ba 2+ , and observed a clear inward current in the Xenopus oocytes expressing ETD1 but only a small inward current in the Xenopus oocytes expressing OsCNGC13, which was consistent with the observation in the Ca 2+ bath solution (Fig. 4F). The electrophysiological experiment demonstrated that ETD1 has a significantly enhanced Ca2+transport capacity relative to OsCNGC13.

[0158] We then detected the rice roots using non-invasive micro-test (NMT) technology. Under normal conditions, there was no difference in the NMT signals of IR64 and ETD1 (Fig. 4F). After the addition of 10 mM Ca 2+ , we detected a significant extracellular Ca 2+ influx NMT signal in IR64 and ETD1, and the Ca2+influx mediated by ETD1 was significantly higher than that of IR64 (Fig. 4G-H). We also constructed NES-YC3.6 calcium imaging system transgenic lines in the background of IR64 and ETD1, and the cytoplasmic Ca 2+ concentration in the root cells of ETD1 was significantly higher than that of IR64 under 10 mM Ca 2+ treatment. The above results indicate that the Ca2+inward transport capacity of the calcium channel encoded by ETD1 is significantly enhanced. 2+

[0159] Example 7: ETD1 enhances Ca2+influx to cause cell death under immune activation

[0160] ROS burst is an important immune event, and we used PAMP-induced immune activation to observe ROS burst. The ROS production level of ETD1 leaves was higher than that of IR64 after chitin and flg22 treatment (Fig. 5A-B). Ca 2+ influx preceded and was necessary for ROS burst. NMT experiments found that under chitin or flg22 stimulation, ETD1 mesophyll cells produced a strong and rapid Ca 2+ influx compared to IR64 (Fig. 5C). YC3.6-based calcium imaging analysis found that under chitin or flg22 stimulation, the cytoplasmic Ca 2+ concentration of ETD1 was significantly higher than that of IR64 (Fig. 5D). The above results indicate that ETD1 enhances Ca2+influx and ROS burst under immune activation.​

[0161] Further studies found that the level of calcium ion accumulation in ETD1 mesophyll cells increased significantly 12h after Magnaporthe grisea inoculation (10G-H). Intracellular calcium ion accumulation can cause cell death, in order to further verify this we used Gd 3+ to inhibit ETD1 channel activity. The results showed that Gd 3+ significantly inhibited cell death caused by ETD1 immune activation (Fig. 5I). The above results show that ETD1 enhances calcium ion influx to cause cell death under immune triggering.

[0162] The above is only the preferred embodiment of the present application, not any form of the present application is limited, although the present application has been disclosed as above, however, not to limit the present application, any skilled in the art, without departing from the scope of the present application, when the above disclosed technical content can be made some changes or modifications for equivalent embodiments, but as long as not beyond the scope of the present application, according to the technical essence of the present application, the above examples of any simple modification, equivalent changes and modifications, still belong to the scope of the present application.

[0163] ETD1 full-length genomic sequence (SEQ ID NO: 1, including intron and exon):

[0164] [According to Rule 26 correction 09.06.2025] ETD1 gene full-length cDNA sequence (SEQ ID NO: 2):

[0165] [According to Rule 26 correction 09.06.2025] ETD1 gene encoded amino acid sequence (SEQ ID NO: 3):

[0166] OsCNGC13 gene editing target 1 (SEQ ID NO: 4)

[0167] [According to Rule 26 correction 09.06.2025] OsCNGC13 gene editing target 2 (SEQ ID NO: 5)

Claims

A mutant ETD1 gene from CNGC13, wherein the ETD1 gene has a nucleotide sequence as shown in SEQ ID NO. 1, and wherein the gene is a super-effective allele of OsCNGC13, and wherein the gene has a stronger calcium ion transport capacity and a function of significantly accelerating the calcium ion influx of cells. The ETD1 gene of claim 1, wherein the cDNA sequence of the gene is shown in SEQ ID NO.

2. The ETD1 gene of claim 1 or 2, wherein the gene has a function of regulating the rice blast resistance of rice and enhancing the resistance of rice to rice blast. The ETD1 gene of claim 3, wherein the gene is a super-effective allele of OsCNGC13, and wherein the normal expression or overexpression of the gene in a gene knockout plant can improve the resistance of rice, and thus the gene has a positive regulation function on the rice blast resistance of rice. The ETD1 gene of claim 3, wherein the expression of the gene in a gene knockout or gene deletion type of rice is inhibited or reduced in expression amount, and the rice blast resistance of the rice is reduced. The ETD1 gene of claim 3, wherein the gene is used in a method for screening a rice ETD1 mutant, a method for establishing the rice blast resistance of rice, or a method for specific molecular marker identification of a transgenic rice ETD1 gene. A coding protein of a mutant ETD1 gene from CNGC13, wherein the coding protein has an amino acid sequence as shown in SEQ ID NO. 3, and wherein the coding gene of the protein is a super-effective allele of OsCNGC13, and wherein the gene knockout or gene deletion type of plant can obtain a stronger calcium ion transport capacity and a function of significantly accelerating the calcium ion influx of cells. The ETD1 protein of claim 7, wherein the coding protein is selected from a protein having 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity with the amino acid sequence shown in SEQ ID No. 3 and having the same function through substitution and / or deletion and / or addition of amino acid residues. The ETD1 protein of claim 8, wherein the protein includes a fusion protein having the same function through connection of a tag at the N-terminus and / or C-terminus. The ETD1 protein of claim 9, wherein the coding gene of the protein is a super-effective allele of OsCNGC13, and wherein the normal expression or overexpression of the gene in a gene knockout plant can improve the resistance of rice, and thus the gene has a positive regulation function on the rice blast resistance of rice, or the expression of the coding gene of the ETD1 protein in a gene knockout or gene deletion type of rice is inhibited or reduced in expression amount, and the rice blast resistance of the rice is reduced.

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