Artificially modified corn broad-spectrum disease-resistant gene ZmLecRK1 and application thereof

Abstract

The invention provides an artificially modified corn broad-spectrum disease-resistant gene ZmLecRK1, which is characterized in that point mutation modification is performed on a key amino acid site N at the 341st site of amino acid coded by the ZmLecRK1 gene; after the 341st N mutation of the amino acid sequence coded by the ZmLecRK1 gene is transformed into Q, A, E, G, H, I, K, M, R, T, V, W or Y, a ZmLecRK1 gene mutant is obtained, so that the ZmLecRK1 can escape from the recognition and attack of fusarium graminearum effect protein FgEG18, the degradation of the ZmLecRK1 protein is avoided, and the resistance of corn to fusarium graminearum stem rot is improved; the nucleotide sequence of the ZmLecRK1 gene is as shown in SEQ ID No. 1; the coded amino acid sequence is as shown in SEQ ID No. 2. The N341 site of the amino acid of the corn broad-spectrum disease-resistant gene ZmLecRK1 is artificially modified, so that the resistance of corn to fusarium graminearum stem rot can be enhanced, the disease-resistant spectrum of ZmLecRK1 is expanded, and the economic benefit and ecological benefit of corn production are improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of genetic engineering, and particularly relates to a genetically engineered broad-spectrum disease-resistant gene of maize ZmLecRK1 and its application. Background Art

[0002] Maize ( Zea mays L.) is an important food, feed, and energy crop globally, and improving its yield and quality is of crucial significance for global food security. However, maize faces threats from various diseases during production, seriously affecting its yield and quality. Fusarium stalk rot (FSR) is one of the important diseases in maize production, mainly caused by Fusarium graminearum . After infecting maize,

[0003] ZmLecRK1 ( GRMZM2G330751 ) not only causes yield losses but also produces various harmful substances for humans and animals such as deoxynivalenol (DON), zearalenone (ZEN), and moniliformin (MON), seriously threatening China's food and feed safety. Pythium aphanidermatum Bipolaris maydis Rhizoctonia solani ZmLecRK1

[0004] ZmLecRK1 ZmLecRK1

[0005] Currently, in the field of maize disease-resistant gene research, many techniques have been involved in the discovery, identification, and improvement of disease-resistant genes. For example, there are multiple genes related to disease resistance in maize, and these genes improve the resistance of maize to various pathogens by regulating the plant's immune response. Among them, ZmLecRK1 gene, as a broad-spectrum disease-resistant gene, has been proven to have certain resistance to various diseases. However, ZmLecRK1 shows relatively limited effectiveness in resistance to

[0005] The disadvantages of the existing technology are as follows: 1.ZmLecRK1 Insufficient resistance of genes to Although ZmLecRK1 genes have been proven to have broad-spectrum disease resistance, in the existing technologyZmLecRK1 Genes have limited effectiveness in preventing and controlling Fusarium graminearum stalk rot. Existing technologies have not effectively solved ZmLecRK1 the problem of gene function failure in the infection of Fusarium graminearum.

[0006] 2. Poor stability of disease-resistant genes, vulnerable to pathogen attack Currently, many disease-resistant genes obtained through transgenic or gene editing technologies often cannot stably maintain disease resistance in practical applications. For example, ZmLecRK1 is prone to degradation when attacked by the effector protein FgEG18 of Fusarium graminearum, resulting in the loss of disease resistance effect. Existing protection mechanisms and technical solutions cannot effectively cope with the interference of pathogen effector proteins, so disease-resistant traits are prone to loss or instability.

[0007] 3. Single method for protecting disease-resistant genes, lack of targeted solutions Existing technologies mainly introduce disease-resistant genes through traditional hybridization breeding, transgenic technologies or CRISPR / Cas9 and other methods. These methods usually focus on gene expression or genetic transmission, but there is a lack of technology in protecting disease-resistant genes from pathogen attack. Especially when facing specific pathogens such as Fusarium graminearum, existing methods do not fully consider the targeted attack of pathogen effector proteins, resulting in the loss of disease-resistant gene function.

[0008] 4. Diversity and complexity of disease-resistant genes In existing technologies, most disease-resistant genes rely on complex polygenic inheritance mechanisms, making it difficult to ensure stable transmission during disease-resistant breeding. The disease-resistant genes required for different diseases may vary greatly, resulting in less than ideal effects of existing disease-resistant genes when solving a specific disease problem, restricting the wide application of disease-resistant varieties. Summary of the Invention

[0009] The technical problem to be solved by the present invention is to provide a manually modified broad-spectrum disease-resistant gene of maize ZmLecRK1 and its application in view of the deficiencies of the above-mentioned existing technologies. This manually modified broad-spectrum disease-resistant gene of maize ZmLecRK1 enhances the resistance of maize to Fusarium graminearum stalk rot, expands ZmLecRK1 the disease resistance spectrum, and improves the economic and ecological benefits of maize production.

[0010] To solve the above technical problem, the technical solution adopted by the present invention is a manually modified broad-spectrum disease-resistant gene of maize ZmLecRK1 , and ZmLecRK1 perform point mutation modification on the key amino acid site N at the 341st position of the amino acid encoded by the gene; the ZmLecRK1 nucleotide sequence of the gene is as shown in SEQ ID No.1; the ZmLecRK1The amino acid sequence encoded by the gene is shown in SEQ ID No. 2.

[0011] Preferably, the ZmLecRK1 amino acid at the 341st position of the amino acid sequence encoded by the gene, the key site N, is mutated to Q, A, E, G, H, I, K, M, R, T, V, W, Y. That is, on the basis of the sequence of the wild-type ZmLecRK1 protein shown in SEQ ID No. 2, the following amino acid sites are mutated: N341Q, N341A, N341E, N341G, N341H, N341I, N341K, N341M, N341R, N341T, N341V, N341W, N341Y.

[0012] In the present invention ZmLecRK1 after the N at the 341st position of the amino acid sequence encoded by the gene is mutated to Q, A, E, G, H, I, K, M, R, T, V, W or Y, ZmLecRK1 a gene mutant is obtained, enabling ZmLecRK1 to escape the recognition and attack of the Fusarium graminearum effector protein FgEG18, avoiding the degradation of the ZmLecRK1 protein. Transiently expressing the above mutant in maize leaves can improve the resistance of maize to Fusarium graminearum stalk rot.

[0013] In the present invention, the key amino acid site N341 of the Fusarium graminearum effector protein FgEG18 attacking ZmLecRK1 is located in the extracellular domain of the ZmLecRK1 protein and is obtained by glycosylation mass spectrometry analysis of the extracellular domain protein of ZmLecRK1.

[0014] The Fusarium graminearum effector protein FgEG18 in the present invention can attack ZmLecRK1, induce the degradation of the ZmLecRK1 protein, and inhibit the biological function of the wild-type ZmLecRK1 from triggering cell death in tobacco and maize.

[0015] In the present invention, the key amino acid site N341 of ZmLecRK1 is used to modify ZmLecRK1, making it have significant protein stability in the protein degradation mediated by the Fusarium graminearum effector protein FgEG18. Taking the key site modification of N341Q as an example: on the basis of the sequence of the wild-type ZmLecRK1 protein shown in SEQ ID No. 2, the mutant formed by mutating N341: ZmLecRK1 N341Q is such that the Fusarium graminearum effector protein FgEG18 cannot degrade ZmLecRK1 N341Q .

[0016] The present invention modifies ZmLecRK1 using the key amino acid site N341 of ZmLecRK1 without affecting the function of the mutant in tobacco and maize. Taking the modification of the key site N341Q as an example: based on the sequence of the wild-type ZmLecRK1 protein shown in SEQ ID No.2, the mutant formed by mutating N341: ZmLecRK1 N341Q 。ZmLecRK1 N341Q can trigger cell death in tobacco leaves and maize protoplast cells.

[0017] The present invention modifies ZmLecRK1 using the key amino acid site N341 of ZmLecRK1, enabling the mutant to evade the attack of the Fusarium graminearum effector protein FgEG18 and maintaining its function in tobacco and maize. Taking the modification of the key site N341Q as an example: based on the sequence of the wild-type ZmLecRK1 protein shown in SEQ ID No.2, the mutant formed by mutating N341: ZmLecRK1 N341Q 。ZmLecRK1 N341Q The cell death triggered in tobacco leaves and maize protoplasts cannot be inhibited by the Fusarium graminearum effector protein FgEG18.

[0018] In the present invention, when ZmLecRK1 the N at the 341st position of the amino acid sequence encoded by the gene is mutated to F, C, D, L, P, or S, that is, the following amino acid sites are mutated: N341F, N341C, N341D, N341L, N341P, N341S, and transiently expressing the above mutants in maize leaves does not affect ZmLecRK1 the biological function in maize, and is consistent with ZmLecRK1 transiently expressing the mutant of the wild type in maize leaves in terms of the resistance to Fusarium graminearum, and does not affect ZmLecRK1 the biological function in maize.

[0019] The present invention has the following advantages compared with the prior art: 1. Enhance the resistance of maize to Fusarium graminearum stalk rot By modifying the key site N341 of ZmLecRK1, the present invention enables ZmLecRK1 to evade the targeted attack of the Fusarium graminearum effector protein FgEG18, avoid the degradation of the ZmLecRK1 protein, and ensure its disease-resistant function in maize. Compared with the prior art, the present invention can significantly improve the resistance of maize to Fusarium graminearum, filling the deficiency of ZmLecRK1 broad-spectrum disease-resistant genes in maize in the prevention and control of Fusarium graminearum stalk rot.

[0020] 2. Expand the disease-resistant spectrum of ZmLecRK1 By artificially modifying the key site N341 of ZmLecRK1, the present invention enhances the ZmLecRK1 resistance of the gene to Fusarium graminearum, enabling ZmLecRK1 it to exert a more extensive disease-resistant effect, which means ZmLecRK1 it can provide resistance to more maize diseases, further expanding the ZmLecRK1 application scope of

[0021] 3. Improve the economic and ecological benefits of maize production By modifying the broad-spectrum disease-resistant gene of maize in the present invention ZmLecRK1 , enhancing the resistance of maize to Fusarium graminearum, it can reduce the impact of Fusarium stalk rot of maize caused by the infection of Fusarium graminearum on the yield and quality of maize, effectively reduce the use of chemical pesticides, and reduce environmental pollution and ecological negative impacts. In addition, stable disease resistance can improve the yield and quality of maize, and improve the economic and ecological benefits of agricultural production.

[0022] 4. Promote the innovation and development of maize disease-resistant breeding technology The present invention provides a new technical solution, breaking through the problem of limited functions of maize disease-resistant genes in the prior art and promoting the development of disease-resistant gene protection technology. This technology not only contributes to the technological progress in the field of maize breeding, but also provides experience and methods for the research and protection of disease-resistant genes of other crops.

[0023] The following further elaborates on the present invention in conjunction with the accompanying drawings and examples. Description of the Drawings

[0024] Figure 1 is a biological function diagram showing that the effector protein FgEG18 of Fusarium graminearum in Example 1 of the present invention can inhibit the cell death triggered by ZmLecRK1 in tobacco and maize.

[0025] Figure 2 is a protein degradation diagram showing that the effector protein FgEG18 of Fusarium graminearum in Example 2 of the present invention can induce the degradation of ZmLecRK1.

[0026] Figure 3 is a diagram showing the biological function influence of the effector protein FgEG18 of Fusarium graminearum on the ZmLecRK1 point mutant in tobacco in Example 3 of the present invention.

[0027] Figure 4 is that the key site N341Q mutation of ZmLecRK1 in Example 4 of the present invention can evade the targeted attack of the effector protein FgEG18 of Fusarium graminearum. Figure 4Figure (a) shows the effect of FgEG18 on the protein accumulation level of ZmLecRK1 in tobacco, (b) shows that FgEG18 inhibits the tobacco leaf cell death triggered by ZmLecRK1 in tobacco, (c) shows the effect of FgEG18 on the protein accumulation level of ZmLecRK1 in maize protoplasts, and (d) shows that FgEG18 inhibits the tobacco leaf cell death triggered by ZmLecRK1 in maize protoplasts.

[0028] Figure 5 It is a diagram showing that the modification of the key site N341 of ZmLecRK1 in Example 5 of the present invention can enhance the resistance of maize to Fusarium graminearum. Detailed implementation mode

[0029] The maize broad-spectrum disease-resistant gene ZmLecRK1 can mediate the resistance of maize to Pythium stalk rot, sheath blight and southern leaf blight, but does not mediate the resistance of maize to Fusarium stalk rot. The inventors speculated that there might be effector proteins in Fusarium graminearum that attack ZmLecRK1. Since ZmLecRK1 can trigger tobacco leaf cell death, the inventors screened the Fusarium graminearum effector protein FgEG18 that can specifically inhibit the tobacco leaf cell death triggered by ZmLecRK1. In the tobacco and maize protoplast systems, FgEG18 can significantly inhibit the cell death triggered by ZmLecRK1; and at the protein level, FgEG18 can significantly reduce the protein accumulation level of ZmLecRK1. Given that FgEG18 has the ability to hydrolyze β-1,4 glycosidic bonds, it is speculated that FgEG18 attacks the N-glycosylation modification site of ZmLecRK1, making the ZmLecRK1 protein prone to degradation. The inventors identified 10 extracellular structure N-glycosylation modification sites of ZmLecRK1, including N341, through glycosylation mass spectrometry detection. Further, in the present invention, by simulating deglycosylation mutation (N-Q), the N (asparagine) at position 341 was artificially mutated to Q (glutamine), and it was found that the mutant ZmLecRK1 N341Q triggered cell death in tobacco and maize protoplasts was not affected; at the same time, the Fusarium graminearum effector protein FgEG18 could also not inhibit the cell death triggered by ZmLecRK1 N341Q Moreover, after the mutants with the modification of the key site N341 of ZmLecRK1 were transiently expressed in maize leaves, they could all significantly improve the resistance of maize to Fusarium graminearum. This shows that the extracellular N-glycosylation modification site N341 of ZmLecRK1 is the key target attacked by the Fusarium graminearum effector protein FgEG18. Therefore, the modification of the key site N341 of the broad-spectrum disease-resistant gene ZmLecRK1 can be used as a target for the modification of disease-resistant genes, which has important scientific significance and application value in maize disease-resistant breeding.

[0030] Based on this, the present invention provides a modification of the key site N341 of the maize broad-spectrum disease-resistant gene ZmLecRK1 and its application. The modification of this gene site specifically is: based on the sequence of the wild-type ZmLecRK1 protein shown in SEQ ID No.2, the following amino acid sites are mutated: N341Q. The mutant ZmLecRK1 after the modification of the key site N341Q does not affect its biological function of triggering cell death in tobacco and maize; it can evade the attack of the Fusarium graminearum effector protein FgEG18 and ensure the stability of the ZmLecRK1 mutant protein. In addition, other modifications to the key site N341 of ZmLecRK1 specifically are: based on the sequence of the wild-type ZmLecRK1 protein shown in SEQ ID No.2, the following amino acid sites are mutated: N341Q or N341A or N341E or N341G or N341H or N341I or N341K or N341M or N341R or N341T or N341V or N341W or N341Y. After transient expression of the above-mentioned modified ZmLecRK1 mutants in maize leaves, the resistance of maize to Fusarium graminearum can be significantly improved; based on the sequence of the wild-type ZmLecRK1 protein shown in SEQ ID No.2, the following amino acid sites are mutated: N341F or N341C or N341D or N341L or N341P or N341S. After transient expression of the above-mentioned modified ZmLecRK1 mutants in maize leaves, the resistance to Fusarium graminearum is the same as that of the wild-type ZmLecRK1 transiently expressed in maize leaves.

[0031] Example 1

[0032] The Fusarium graminearum effector protein in this example FgEG18 can specifically inhibit ZmLecRK1 the biological function of the protein. The specific operation methods and results are as follows: Step 1, FgEG18 and ZmLecRK1 cloning of First, extract the total RNA of the Fusarium graminearum FG-12 strain (Hao et al., 2021, Submitted GenBank assembly: GCA_019343145.1) and reverse transcribe it into cDNA; extract the total RNA of the maize inbred line B73 and reverse transcribe it into cDNA.

[0033] Design the primer sequences as shown in Table 1: Table 1 Primers required for constructing the expression vector in Example 1

[0034] Using the cDNA of Fusarium graminearum strain FG-12 as a template, PCR amplification was performed with primers FgEG18-PF and FgEG18-PR. The PCR reaction system was as follows: 10 μL of 2×T8 High-Fidelity Master Mix (product number: DLP201, purchased from Beijing Tsingke Biotechnology Co., Ltd.), 1 μL each of 10 μM primers (primer FgEG18-PF and primer FgEG18-PR) (synthesized by Beijing Tsingke Biotechnology Co., Ltd.), 1 μL of template cDNA, and sterile water was added to make up to 20 μL. The PCR reaction conditions were: pre-denaturation at 98°C for 2 min; 98°C for 30 sec, 60°C for 30 sec, 72°C for 30 sec, for 32 cycles; final extension at 72°C for 5 min. After the PCR product was electrophoresed on a 1% agarose gel, the target fragment was recovered using a DNA gel recovery kit (product number: TSP602-200, purchased from Beijing Tsingke Biotechnology Co., Ltd.), and the recovery steps were carried out according to the kit instructions to obtain the gene fragment FgEG18 .

[0035] Using the cDNA of maize inbred line B73 as a template, PCR amplification was performed with primers ZmLecRK1-PF and ZmLecRK1-PR. The PCR reaction system was as follows: 10 μL of 2×T8 High-Fidelity Master Mix (product number: TSE111, purchased from Beijing Tsingke Biotechnology Co., Ltd.), 1 μL each of 10 μM primers (primer ZmLecRK1-PF and primer ZmLecRK1-PR) (synthesized by Beijing Tsingke Biotechnology Co., Ltd.), 1 μL of template cDNA, and deionized water was added to make up to 20 μL. The PCR reaction conditions were: pre-denaturation at 98°C for 2 min; 98°C for 30 sec, 60°C for 2 min, 72°C for 30 sec, for 32 cycles; final extension at 72°C for 5 min. After the PCR product was electrophoresed on a 1% agarose gel, the target fragment was recovered using a DNA gel recovery kit (product number: TSP602-200, purchased from Beijing Tsingke Biotechnology Co., Ltd.), and the recovery steps were carried out according to the kit instructions to obtain the gene fragment ZmLecRK1 , and the nucleotide sequence is shown in SEQ ID No.1, encoding a ZmLecRK1 protein with the amino acid sequence shown in SEQ ID No.2.

[0036] Homologous recombination ligation: The existing plasmids pCAMBIA1300-HA, pCAMBIA1300-Flag, pCAMBIA1300-GFP, pUC19-HA, and pUC19-Flag (Wang et al., 2023) were linearized with the restriction enzymes KpnI (product number: 1618, purchased from Baori Biotechnology (Beijing) Co., Ltd.) and Sal I (product number: 1636, purchased from Baori Biotechnology (Beijing) Co., Ltd.). The reaction system was as follows: 10 μL of 100 ng / μL vector, 2 μL of 10×QuickCut Buffer, 1 μL of restriction enzyme Kpn I, 1 μL of restriction enzyme Sal I, and deionized water was added to make up to 20 μL. The reaction system was reacted at 37°C for 1 h and then at 80°C for 20 min. After the products were electrophoresed on a 1% agarose gel, the linearized vectors were recovered using a DNA gel recovery kit (product number: TSP602-200, purchased from Beijing Tsingke Biotechnology Co., Ltd.). The recovery steps were carried out according to the kit instructions to obtain the linearized vectors pCAMBIA1300-HA, pCAMBIA1300-Flag, pCAMBIA1300-GFP, pUC19-HA, and pUC19-Flag. The linearized vector pCAMBIA1300-HA was recombined with the fragment FgEG18, and the linearized vectors pCAMBIA1300-Flag and pCAMBIA1300-GFP were respectively recombined with the fragment ZmLecRK1 using a homologous recombination kit (product number: TSV-S1, purchased from Beijing Tsingke Biotechnology Co., Ltd.) (the experimental steps were carried out according to the kit instructions), and then transformed into Escherichia coli DH5α to obtain the expression vectors pCAMBIA1300-35S::FgEG18-HA, pCAMBIA1300-35S::ZmLecRK1-Flag, and pCAMBIA1300-35S::ZmLecRK1-GFP for transient expression in plants. The linearized vector pUC19-HA was recombined with the fragment FgEG18, and pUC19-Flag was recombined with the fragment ZmLecRK1 using a homologous recombination kit and then transformed into Escherichia coli DH5α to obtain the expression vectors pUC19-35S::FgEG18-HA and pUC19-35S::ZmLecRK1-Flag for transient expression in maize protoplasts.

[0037] Using the expression vectors pCAMBIA1300-35S::FgEG18-HA and pUC19-35S::FgEG18-HA as templates respectively, with the primers FgEG18 H107A -PF and FgEG18 H107A-PR was used for PCR amplification. The PCR reaction system was as follows: 5 μL of 2×T8 High-Fidelity Master Mix, 0.5 μL each of 10 μM primers (primer FgEG18 H107A -PF and FgEG18 H107A -PR), 1 μL of template cDNA, and sterile water was added to make up to 10 μL. The PCR reaction conditions were: pre-denaturation at 98°C for 2 min; 98°C for 30 sec, 60°C for 30 sec, 72°C for 30 sec, for 20 cycles; and final extension at 72°C for 5 min. The PCR product was treated with the restriction enzyme Dpn I (product number: 1609, purchased from Takara Biotechnology (Beijing) Co., Ltd.). The enzyme digestion reaction system was: 10 μL of PCR product, 2 μL of 10×QuickCut Buffer, 1 μL of restriction enzyme Dpn I, and deionized water was added to make up to 20 μL. The reaction was carried out at 37°C for 1 h, followed by a reaction at 80°C for 20 min. Subsequently, it was transformed into Escherichia coli DH5α to obtain pCAMBIA1300-35S::FgEG18 H107A -HA and pUC19-35S::FgEG18 H107A -HA expression vectors.

[0038] Step 2, FgEG18 Regarding ZmLecRK1 the impact on biological functions (1) The effector protein of Fusarium graminearum FgEG18 was able to inhibit the cell death of ZmLecRK1 in tobacco leaves: This experiment was divided into 3 groups. The vectors pCAMBIA1300-35S::GFP-HA (Wang et al., 2023), pCAMBIA1300-35S::FgEG18 H107A -Flag (constructed in this example), pCAMBIA1300-35S::FgEG18-HA, and pCAMBIA1300-35S::ZmLecRK1-Flag (constructed in this example) were respectively transferred into Agrobacterium tumefaciens GV3101. After shaking for 18 h, the cells were resuspended with infiltration solution (10 mM MgCl2, 10 mM MES, and 150 μM acetosyringone), and the OD of each component was adjusted 600 to 0.5. According to pCAMBIA1300-35S::GFP-HA + pCAMBIA1300-35S::ZmLecRK1-Flag, pCAMBIA1300-35S::FgEG18 H107A-Flag + pCAMBIA1300-35S::ZmLecRK1-Flag, pCAMBIA1300-35S::FgEG18-HA + pCAMBIA1300-35S::ZmLecRK1-Flag were co-injected into the leaves of four-week-old Nicotiana benthamiana. The tobacco leaves were observed and photographed 48 h after injection.

[0039] The results are as Figure 1 shown in a. Co-injection of ZmLecRK1 with GFP and FgEG18 H107 both caused tobacco cell death, while co-injection with FgEG18 did not trigger cell death in tobacco leaves. This indicates that the Fusarium graminearum effector protein FgEG18 can inhibit the biological function of ZmLecRK1 in tobacco.

[0040] (2) The Fusarium graminearum effector protein FgEG18 can inhibit the cell death of ZmLecRK1 in maize protoplasts: This experiment was divided into 4 groups, with pUC19-35S::mCherry (Wang et al., 2023) and pUC19-35S::FgEG18 H107A -HA as controls, and pUC19-35S::LUC (Li et al., 2024) as the reporter gene vector. According to the combinations: pUC19-35S::LUC, pUC19-35S::LUC + pUC19-35S::mCherry + pUC19-35S::ZmLecRK1-Flag, pUC19-35S::LUC + pUC19-35S::FgEG18 H107A -HA + pUC19-35S::ZmLecRK1-Flag, pUC19-35S::LUC + pUC19-35S::FgEG18-HA + pUC19-35S::ZmLecRK1-Flag, using the PEG-mediated maize protoplast transformation system, the corresponding plasmids were transformed into maize protoplasts. After dark expression for 20 h, they were cultured under fluorescent light for 4 h. The maize protoplasts were collected by centrifugation, and after protein extraction, the fluorescence values of each experimental group were measured using an enzyme-linked immunosorbent assay (ELISA) reader, with luciferase activity as an indicator of ZmLecRK1-induced cell death in maize protoplasts.

[0041] The results are as Figure 1 shown in b. In maize protoplasts, FgEG18 can significantly inhibit the cell death triggered by ZmLecRK1, while the control mCherry and FgEG18 H107A cannot inhibit the cell death triggered by ZmLecRK1. This indicates that the Fusarium graminearum effector protein FgEG18 can inhibit the biological function of ZmLecRK1 in maize.

[0042] Example 2 The effector protein of Fusarium graminearum can induce the protein degradation of ZmLecRK1. The specific operation methods and results are as follows: The following expression vectors were used in the experiment: pCAMBIA1300-35S::GFP-HA (Wang et al., 2023), pCAMBIA1300-35S::ZmLecRK1-Flag (prepared in Example 1), pCAMBIA1300-35S::FgEG18-HA (prepared in Example 1), and pCAMBIA1300-35S::FgEG18 H107A -HA (prepared in Example 1). The above vectors were respectively transferred into Agrobacterium tumefaciens GV3101. After shaking culture for 18 h, the bacterial cells were resuspended with infiltration solution (10 mM MgCl2, 10 mM MES, and 150 μM acetosyringone), and the OD of each component was adjusted 600 to 0.5. According to the combinations of pCAMBIA1300-35S::ZmLecRK1-Flag + pCAMBIA1300-35S::GFP-HA and pCAMBIA1300-35S::ZmLecRK1-Flag + pCAMBIA1300-35S::FgEG18-HA, the leaves of four-week-old Nicotiana benthamiana were injected. After 48 h of injection, samples were taken to extract proteins, and the protein accumulation of ZmLecRK1 in each group of samples was analyzed by western-blot.

[0043] The results are as Figure 2 shown. FgEG18 can significantly reduce the protein accumulation of ZmLecRK1, while the mutant FgEG18 with lost enzyme activity H107A does not affect the protein accumulation of ZmLecRK1. This indicates that the effector protein FgEG18 of Fusarium graminearum can affect the protein accumulation of ZmLecRK1.

[0044] Example 3 Analysis of the key extracellular glycosylation sites of the maize broad-spectrum disease resistance gene ZmLecRK1. The specific operation methods and results are as follows: Step 1: Cloning and protein enrichment of ZmLecRK1 ECD+TM of ZmLecRK1 (1) Construction of the expression vector of ZmLecRK1 ECD+TM of ZmLecRK1 Primers were designed as shown in Table 2: Table 2 Primers required for the construction of the expression vector of ZmLecRK1 ECD+TM of ZmLecRK1

[0045] Using the expression vector pCAMBIA1300-35S::ZmLecRK1-Flag constructed in Example 1 as a template, with primers ZmLecRK1 ECD+TM -PF and primer ZmLecRK1 ECD+TM -PR for PCR amplification. The reaction system is: 2×T8 High-Fidelity Master Mix 10 μL, 10 μM primers (primer ZmLecRK1 ECD+TM -PF and primer ZmLecRK1 ECD+TM -PR) 1 μL each, template 1 μL, and sterile water added to make up to 20 μL. The PCR reaction conditions are: pre-denaturation at 98°C for 2 min; 98°C for 30 sec, 60°C for 30 sec, 72°C for 1 min, for 32 cycles; final extension at 72°C for 5 min. After the PCR product is electrophoresed on a 1% agarose gel, the target fragment is recovered using a DNA gel recovery kit (product number: TSP602-200, purchased from Beijing Tsingke Biotechnology Co., Ltd.). The recovery steps are carried out according to the kit instructions to obtain the gene fragment ZmLecRK1 ECD+TM .

[0046] Homologous recombination ligation: The linearized vector pCAMBIA1300-Flag and the fragment ZmLecRK1 ECD+TM are recombined using a homologous recombination kit (product number: TSV-S1, purchased from Beijing Tsingke Biotechnology Co., Ltd.) (the experimental steps are carried out according to the kit instructions), and transformed into Escherichia coli DH5α to obtain the pCAMBIA1300-35S::ZmLecRK1 ECD+TM -Flag expression vector.

[0047] (2) Protein expression and enrichment of ZmLecRK1 ECD+TM Transfer the above expression vector pCAMBIA1300-35S::ZmLecRK1 ECD+TM -Flag into Agrobacterium tumefaciens GV3101. After shaking for 18 h, resuspend the cells with an infiltration solution (10 mM MgCl2, 10 mM MES, and 150 μM acetosyringone), adjust the OD 600 to 0.5, inject the leaves of four-week-old Nicotiana benthamiana, and take samples 48 h after injection to obtain tobacco leaves expressing the ZmLecRK1 ECD+TM protein.

[0048] ​Quickly grind the above tobacco leaves into powder in liquid nitrogen, add protein extraction buffer (25 mM Tris-HCl pH7.5, 1 mM EDTA, 150 mM NaCl, 10% glycerol, 2% PVP, 0.5% Triton-X100, 1 mM DTT, protease inhibitor), let it stand on ice for 30 min, centrifuge at 4°C, 8000×g for 10 min, take the supernatant, add 5 μL of anti-Flag magnetic beads (product number: HY-K0207, MedChemexpress Biotechnology Company, USA), incubate at 4°C for 2 h to enrich ZmLecRK1 ECD+TM protein. After the incubation and enrichment are completed, wash the magnetic beads 3 - 5 times with washing buffer (25 mM Tris-HCl pH7.5, 1 mM EDTA, 150 mM NaCl, 0.5% Triton-X100, 1 mM DTT), then wash the magnetic beads 10 times with PBS (pH7.5), and collect the magnetic beads for standby.

[0049] Step 2: Glycosylation mass spectrometry analysis of extracellular glycosylation modification sites of ZmLecRK1 Perform glycosylation mass spectrometry analysis on the magnetic beads enriched with ZmLecRK1 ECD+TM protein to obtain information such as extracellular domain glycosylation modification sites of ZmLecRK1.

[0050] The results are shown in Table 3. Through glycosylation mass spectrometry analysis, a total of 10 N-glycosylation modification sites including N34, N85, N121, N142, N215, N227, N322, N341, N380, and N435 were identified in the extracellular domain of ZmLecRK1. The naming method of the sites is as follows: Taking N341 as an example, it means that ZmLecRK1 the 341st amino acid encoded by the gene is N (asparagine).

[0051] Table 3 Glycosylation mass spectrometry analysis of extracellular glycosylation modification sites of ZmLecRK1

[0052]

[0053] Note: The letters in parentheses represent the amino acids at both ends of the peptide segment.

[0054] Step 3: ZmLecRK1 Analysis of key glycosylation sites in the extracellular domain (1) ZmLecRK1Construction of mutant vectors for mimicking deglycosylation modification of extracellular domain N-glycosylation sites. Primers were designed as shown in Table 4. The naming method of point mutation vectors is exemplified by mutating the 341st amino acid encoded by the ZmLecRK1 gene from N (asparagine) to Q (glutamine). The point mutation vector is named ZmLecRK1 N34Q mutant.

[0055] Table 4 ZmLecRK1 Primers required for constructing mutant vectors for mimicking deglycosylation modification of extracellular glycosylation sites

[0056] Taking the construction of the point mutation vector of ZmLecRK1 N34Q as an example: Using the expression vector pCAMBIA1300-35S::ZmLecRK1-Flag constructed in Example 1 as a template, and using primers ZmLecRK1 N34Q -PF and primer ZmLecRK1 N34Q -PR for PCR amplification. The PCR reaction system is: 5 μL of 2×T8 High-Fidelity Master Mix, 0.5 μL of each 10 μM primer (primer ZmLecRK1 N34Q -PF and primer ZmLecRK1 N34Q -PR), 1 μL of template, and sterile water is added to make up to 10 μL. The PCR reaction conditions are: pre-denaturation at 98°C for 2 min; 98°C for 30 sec, 60°C for 30 sec, 72°C for 30 sec, for 20 cycles; termination and extension at 72°C for 5 min. The PCR product is treated with the restriction enzyme Dpn I (product number: 1609, purchased from Takara Biotechnology (Beijing) Co., Ltd.). The enzyme digestion reaction system is: 10 μL of PCR product, 2 μL of 10×QuickCut Buffer, 1 μL of restriction enzyme Dpn I, and deionized water is added to make up to 20 μL. React at 37°C for 1 h, and then react at 80°C for 20 min. Subsequently, transform Escherichia coli DH5α to obtain the point mutation expression vector of pCAMBIA1300-35S::ZmLecRK1 N34Q -Flag.

[0057] Using the above method, the remaining point mutation expression vectors were constructed using the above primer pairs, and the following were obtained respectively: pCAMBIA1300-35S::ZmLecRK1 N85Q -Flag, pCAMBIA1300-35S::ZmLecRK1 N121Q -Flag, pCAMBIA1300-35S::ZmLecRK1N142Q -Flag, pCAMBIA1300 - 35S::ZmLecRK1 N215Q -Flag, pCAMBIA1300 - 35S::ZmLecRK1 N227Q -Flag, pCAMBIA1300 - 35S::ZmLecRK1 N322Q -Flag, pCAMBIA1300 - 35S::ZmLecRK1 N341Q -Flag, pCAMBIA1300 - 35S::ZmLecRK1 N380Q -Flag, pCAMBIA1300 - 35S::ZmLecRK1 N435Q Point - mutant expression vectors of - Flag, for transient expression in plants.

[0058] (2)Effect of FgEG18 on the function of ZmLecRK1 mutants The above - mentioned ZmLecRK1 point - mutant expression vectors for transient expression in plants were respectively transformed into Agrobacterium tumefaciens GV3101, and tobacco leaf injection was carried out according to the method in Step 2 of Example 1. The Agrobacterium tumefaciens transformed with the above - mentioned point - mutant vectors was co - injected with the Agrobacterium tumefaciens transformed with the pCAMBIA1300 - 35s::FgEG18 - HA expression vector, and the Agrobacterium tumefaciens transformed with the pCAMBIA1300 - 35S::GFP - HA expression vector was used as a control. Tobacco leaves were observed and photographed 48 h after Agrobacterium injection.

[0059] The results are as Figure 3 shown, ZmLecRK1 N34Q 、ZmLecRK1 N121Q 、ZmLecRK1 N215Q 、ZmLecRK1 N227Q 、ZmLecRK1 N322Q 、ZmLecRK1 N341Q and ZmLecRK1 N380Q mutants can still trigger cell death in tobacco leaves, but ZmLecRK1 N85Q 、ZmLecRK1 N142Q and ZmLecRK1 N435Q mutants cannot trigger cell death in tobacco leaves. In addition, the Fusarium graminearum effector protein FgEG18 cannot inhibit the cell death in tobacco leaves triggered by the ZmLecRK1 N341Q mutant, but for ZmLecRK1 N34Q, ZmLecRK1 N121Q , ZmLecRK1 N215Q , ZmLecRK1 N227Q , ZmLecRK1 N322Q , and ZmLecRK1 N380Q The mutants still significantly inhibited the tobacco leaf cell death triggered by ZmLecRK1. This indicates that the N341 glycosylation modification site of ZmLecRK1 is an important target for FgEG18 attack.

[0060] Example 4 Analysis of the effects of the Fusarium graminearum effector protein FgEG18 on the protein accumulation and biological function of ZmLecRK1. The specific operation methods and results are as follows: N341Q Step 1: Effects of the Fusarium graminearum effector protein FgEG18 on the protein accumulation of ZmLecRK1 N341Q Using the expression vector pUC19-35S::ZmLecRK1-Flag constructed in Example 1 as a template, and primers ZmLecRK1 N341Q -PF and primer ZmLecRK1 N341Q -PR for PCR amplification. The PCR reaction system is: 5 μL of 2×T8 High-Fidelity Master Mix, 0.5 μL of 10 μM primers (primer ZmLecRK1 N341Q -PF and primer ZmLecRK1 N341Q -PR), 1 μL of template, and sterile water to make up to 10 μL. The PCR reaction conditions are: pre-denaturation at 98°C for 2 min; 98°C for 30 sec, 60°C for 30 sec, 72°C for 30 sec, for 20 cycles; and final extension at 72°C for 5 min. The PCR product is treated with the restriction enzyme Dpn I (product number: 1609, purchased from Takara Biotechnology (Beijing) Co., Ltd.). The enzyme digestion reaction system is: 10 μL of PCR product, 2 μL of 10×QuickCut Buffer, 1 μL of restriction enzyme Dpn I, and deionized water to make up to 20 μL. React at 37°C for 1 h, and then react at 80°C for 20 min. Subsequently, transform Escherichia coli DH5α to obtain the point mutation expression vector of pUC19-35S::ZmLecRK1 N341Q N341Q -Flag for transient expression in maize protoplasts.

[0061] (1) The following expression vectors were used in the experiment: pCAMBIA1300-35S::GFP-HA (Wang et al., 2023), pCAMBIA1300-35S::ZmLecRK1-Flag (prepared in Example 1), pCAMBIA1300-35S::FgEG18-HA (prepared in Example 1), and pCAMBIA1300-35S::ZmLecRK1 N341Q -Flag (prepared in Example 3). After the above vectors were separately transferred into Agrobacterium tumefaciens GV3101 and cultured with shaking for 18 h, the bacterial cells were resuspended with an infiltration solution (10 mM MgCl2, 10 mM MES, and 150 μM acetosyringone), and the OD 600 of each component was adjusted to 0.5. The leaves of four-week-old Nicotiana benthamiana were injected with the combinations of pCAMBIA1300-35S::ZmLecRK1-Flag + pCAMBIA1300-35S::GFP-HA, pCAMBIA1300-35S::ZmLecRK1-Flag + pCAMBIA1300-35S::FgEG18-HA, pCAMBIA1300-35S::ZmLecRK1 N341Q -Flag + pCAMBIA1300-35S::GFP-HA, pCAMBIA1300-35S::ZmLecRK1 N341Q -Flag + pCAMBIA1300-35S::FgEG18-HA. Samples were taken 48 h after injection to extract proteins, and the protein accumulation levels of ZmLecRK1 or ZmLecRK1 N341Q in each group of samples were analyzed by western-blot.

[0062] (2) The following expression vectors were used in the experiment: pUC19-35S::mCherry-HA (Wang et al., 2023), pUC19-35S::ZmLecRK1-Flag (prepared in Example 1), pUC19-35S::ZmLecRK1 N341Q -Flag, and pUC19-35S::FgEG18-HA (prepared in Example 1). The above expression vectors were respectively combined as pUC19-35S::ZmLecRK1-Flag + pUC19-35S::mCherry-HA, pUC19-35S::ZmLecRK1-Flag + pUC19-35S::FgEG18-HA, pUC19-35S::ZmLecRK1 N341Q-Flag + pUC19-35S::mCherry-HA and pUC19-35S::ZmLecRK1 N341Q -Flag + pUC19-35S::FgEG18-HA combination, using the PEG-mediated maize protoplast transformation system, co-transferred the expression vectors into maize protoplasts. After 20 h of dark expression, proteins were extracted, and the protein accumulation levels of ZmLecRK1 or ZmLecRK1 N341Q in each group of samples were analyzed by western-blot.

[0063] The results are shown in Figure 4 a and c below. In tobacco, FgEG18 can significantly reduce the protein accumulation level of ZmLecRK1, but basically does not affect the protein accumulation level of ZmLecRK1 N341Q ; in maize protoplasts, FgEG18 also hardly affects the protein accumulation level of ZmLecRK1 N341Q . This indicates that ZmLecRK1 N341Q can avoid protein degradation under the attack of FgEG18.

[0064] Step 2: Effects of FgEG18 on the biological function of ZmLecRK1 N341Q (1) The following expression vectors were used in the experiment: pCAMBIA1300-35S::GFP-HA (Wang et al., 2023), pCAMBIA1300-35S::ZmLecRK1-Flag (prepared in Example 1), pCAMBIA1300-35S::FgEG18-HA (prepared in Example 1), and pCAMBIA1300-35S::ZmLecRK1 N341Q -Flag (prepared in Example 3). The above vectors were separately transferred into Agrobacterium tumefaciens GV3101. After shaking culture for 18 h, the bacterial cells were resuspended with infiltration solution (10 mM MgCl2, 10 mM MES, and 150 μM acetosyringone), and the OD 600 of each component was adjusted N341Q to 0.5. According to pCAMBIA1300-35S::ZmLecRK1-Flag + pCAMBIA1300-35S::GFP-HA, pCAMBIA1300-35S::ZmLecRK1-Flag + pCAMBIA1300-35S::FgEG18-HA, pCAMBIA1300-35S::ZmLecRK1 N341Q ​-Inject a combination of Flag + pCAMBIA1300-35S::FgEG18-HA into the leaves of four-week-old Nicotiana benthamiana, and observe and photograph the tobacco leaves 48 h after injection.

[0065] (2) The following expression vectors were used in the experiment: pUC19-35S::LUC (Li et al., 2024), pUC19-35S::mCherry-HA (Wang et al., 2023), pUC19-35S::ZmLecRK1-Flag (prepared in Example 1), pUC19-35S::ZmLecRK1 N341Q -Flag and pUC19-35S::FgEG18-HA (prepared in Example 1). Respectively, the above expression vectors were arranged as pUC19-35S::LUC, pUC19-35S::LUC + pUC19-35S::ZmLecRK1-Flag + pUC19-35S::mCherry-HA, pUC19-35S::LUC + pUC19-35S::ZmLecRK1-Flag + pUC19-35S::FgEG18-HA, pUC19-35S::LUC + pUC19-35S::ZmLecRK1 N341Q -Flag + pUC19-35S::mCherry-HA and pUC19-35S::LUC + pUC19-35S::ZmLecRK1 N341Q -Flag + pUC19-35S::FgEG18-HA. Using the PEG-mediated maize protoplast transformation system, the expression vectors were co-transformed into maize protoplasts. After 20 h of dark expression, they were cultured under a fluorescent lamp for 4 h. The maize protoplasts were collected by centrifugation, and after protein extraction, the fluorescence values of each experimental group were measured using a microplate reader, with luciferase activity as an indicator of ZmLecRK1-induced cell death in maize protoplasts.

[0066] The results are as Figure 4 shown in b and d below. In tobacco, FgEG18 can significantly inhibit ZmLecRK1-triggered cell death in tobacco leaves, but cannot inhibit ZmLecRK1 N341Q -triggered cell death in tobacco leaves; in maize protoplasts, ZmLecRK1 N341Q can cause cell death to the same extent as ZmLecRK1, but FgEG18 cannot inhibit ZmLecRK1 N341Q -induced cell death in maize protoplasts. This indicates that ZmLecRK1 N341QIt can evade the targeted attack of FgEG18 and continuously exert immune functions in tobacco and maize.

[0067] Example 5 Transient expression of ZmLecRK1 in maize leaves N341Q Analysis of resistance to Fusarium graminearum. The specific operation methods and results are as follows: Step 1: Agrobacterium-mediated transient expression in maize leaves (1) Construction of the expression vector with point mutation at the key site N341 of ZmLecRK1 Design primers as shown in Table 5: Table 5 Primers required for the construction of the expression vector with point mutation at the key site N341 of ZmLecRK1

[0068]

[0069] Taking ZmLecRK1 N341A as an example for the construction of the point mutation expression vector: Using the expression vector pCAMBIA1300-35S::ZmLecRK1-GFP constructed in Example 1 as a template, and using the above primers ZmLecRK1 N341A -PF and primer ZmLecRK1 N341A -PR for PCR amplification. The PCR reaction system is: 2×T8 High-Fidelity Master Mix 5 μL, 10 μM primers (primer ZmLecRK1 N341A -PF and primer ZmLecRK1 N341A -PR) 0.5 μL each, template 1 μL, and sterile water to make up to 10 μL. The PCR reaction conditions are: pre-denaturation at 98°C for 2 min; 98°C for 30 sec, 60°C for 30 sec, 72°C for 30 sec, for 20 cycles; termination and extension at 72°C for 5 min. The PCR product is treated with the restriction endonuclease Dpn I (product number: 1609, purchased from Takara Biotechnology (Beijing) Co., Ltd.). The enzyme digestion reaction system is: PCR product 10 μL, 10×QuickCut Buffer 2 μL, restriction endonuclease Dpn I 1 μL, and deionized water to make up to 20 μL. React at 37°C for 1 h, and then react at 80°C for 20 min. Subsequently, transform Escherichia coli DH5α to obtain the point mutation expression vector of pCAMBIA1300-35S::ZmLecRK1 N341A -GFP. Using the above method, use the above primer pairs to construct the remaining point mutation expression vectors respectively to obtain pCAMBIA1300-35S::ZmLecRK1 N341F -GFP, pCAMBIA1300-35S::ZmLecRK1 N341C -GFP, pCAMBIA1300-35S::ZmLecRK1 N341D -GFP, pCAMBIA1300-35S::ZmLecRK1 N341E -GFP, pCAMBIA1300-35S::ZmLecRK1 N341G -GFP, pCAMBIA1300-35S::ZmLecRK1 N341H -GFP, pCAMBIA1300-35S::ZmLecRK1 N341L -GFP, pCAMBIA1300-35S::ZmLecRK1 N341I -GFP, pCAMBIA1300-35S::ZmLecRK1 N341K -GFP, pCAMBIA1300-35S::ZmLecRK1 N341M -GFP, pCAMBIA1300-35S::ZmLecRK1 N341P -GFP, pCAMBIA1300-35S::ZmLecRK1 N341R -GFP, pCAMBIA1300-35S::ZmLecRK1 N341S -GFP, pCAMBIA1300-35S::ZmLecRK1 N341T -GFP, pCAMBIA1300-35S::ZmLecRK1 N341V -GFP, pCAMBIA1300-35S::ZmLecRK1 N341W -GFP, pCAMBIA1300-35S::ZmLecRK1 N341Y Point mutation expression vector of -GFP.

[0070] (2)Agrobacterium-mediated transient expression in maize leaves pCAMBIA1300-35S::GFP (Wang et al., 2023), pCAMBIA1300-35S::ZmLecRK1-GFP (prepared in Example 1), and the above point mutation expression vectors were separately transferred into Agrobacterium tumefaciens GV3101. According to previous reports on Agrobacterium-mediated transient gene expression in barley leaves (Xu et al., 2023), the inventors made slight adjustments to make it suitable for transient expression in maize leaves. Specifically as follows: After the Agrobacterium was cultured with shaking for 18 h, the Agrobacterium cells were washed twice with sterile water and then resuspended in an infiltration solution (10 mM MgCl2, 10 mM MES, and 300 μM acetosyringone), and the bacterial liquid concentration was adjusted to OD 600 = 0.5. The first leaf of maize seedlings at the V2 stage was used for injection. Before injection, the back of the leaf was gently scratched with a sterile needle to create a wound for convenient infiltration of Agrobacterium. After injection, it was cultured at 25°C in the dark with 98% humidity for 24 h; then it was cultured at 25°C under weak light conditions for 72 h, and the leaves were cut and set aside.

[0071] Step 2: Resistance analysis of the artificially modified mutant of the key site N341 of ZmLecRK1 to Fusarium graminearum The Fusarium graminearum FG-12 strain was cultured in a PDA plate for 3 d, and a 2-mm-diameter fungal plug was punched out along the edge of the colony. The maize leaves after transient expression of the above ZmLecRK1 wild type and point mutants were placed with the back facing up in a tray lined with moist filter paper, and the Fusarium graminearum fungal plug was inoculated onto the maize leaves with the mycelium facing downwards, and attention should be paid to making the inoculation point coincide with the Agrobacterium injection point. After moisturizing treatment, it was cultured in the dark at 25°C for 24 h, then photographed and recorded, and the lesion area was statistically analyzed.

[0072] The results are as Figure 5As shown, the point mutation types at the key site N341 of ZmLecRK1 are N341Q, N341A, N341E, N341G, N341H, N341I, N341K, N341M, N341R, N341T, N341V, N341W, and N341Y. The disease severity and lesion area of the transiently expressed maize leaves with these 13 groups of mutations were significantly reduced compared to the control group (GFP) and the wild-type ZmLecRK1 (WT) after inoculation with Fusarium graminearum FG-12. However, the disease severity and lesion area of the transiently expressed maize leaves with the point mutation types at the key site N341 of ZmLecRK1 being N341F, N341C, N341D, N341L, N341P, and N341S were the same as those of the wild-type ZmLecRK1 after inoculation with Fusarium graminearum FG-12. This indicates that artificial modification of the key site N341 of ZmLecRK1 does not affect the biological function of ZmLecRK1 in maize. Furthermore, 13 groups of mutation types including N341Q can significantly improve the resistance of ZmLecRK1 to Fusarium graminearum in maize. Therefore, the resistance of maize to Fusarium graminearum can be improved through the above modification, making it possible for the broad-spectrum disease-resistant gene ZmLecRK1 in maize to have resistance to Fusarium stalk rot, which has important significance and application value in maize disease-resistant breeding.

[0073] As described above, the above are only the preferred embodiments of the present invention and do not impose any limitations on the present invention. Any simple modifications, changes, and equivalent variations made to the above embodiments based on the technical essence of the invention still fall within the protection scope of the technical solution of the present invention.

Claims

1. An artificially modified broad-spectrum disease-resistant gene of corn ZmLecRK1 , characterized in that Mutate the key amino acid site N at the 341st position of the amino acid encoded by the ZmLecRK1 gene by point mutation; the ZmLecRK1 nucleotide sequence of the gene is shown in SEQ ID No.1; the ZmLecRK1 amino acid sequence encoded by the gene is shown in SEQ ID No.

2.

2. An artificially modified broad-spectrum disease-resistant gene of corn according to claim 1 ZmLecRK1 , characterized in that The ZmLecRK1 amino acid at the 341st position in the amino acid sequence encoded by the gene, which is the key site N, is mutated into Q, A, E, G, H, I, K, M, R, T, V, W, Y.

3. Use of an artificially modified broad-spectrum disease-resistant gene of maize as described in claim 2 ZmLecRK1 , characterized in that The ZmLecRK1 341st N of the amino acid sequence encoded by the gene is mutated to Q, A, E, G, H, I, K, M, R, T, V, W or Y, resulting in ZmLecRK1 a gene mutant, enabling ZmLecRK1 to evade the recognition and attack of the effector protein FgEG18 of Fusarium graminearum, avoid ZmLecRK1 the degradation of the protein, and improve the resistance of maize to Fusarium graminearum stalk rot.

Images