A genetically engineered broad-spectrum disease resistance gene for maize, ZmLecRK1, and its application.

Abstract

This invention provides an artificially modified broad-spectrum disease resistance gene for maize. ZmLecRK1 Its characteristic is that it will ZmLecRK1 The critical amino acid N at position 341, which encodes an amino acid in the gene, was modified by a point mutation. ZmLecRK1 After modifying the N at position 341 of the amino acid sequence encoded by the gene to Q, A, E, G, H, I, K, M, R, T, V, W, or Y, the following is obtained: ZmLecRK1 Gene mutants, making ZmLecRK1 It can evade the recognition and attack of the Fusarium graminearum effector protein FgEG18, and avoid... ZmLecRK1 Protein degradation enhances maize's resistance to Fusarium stalk rot; ZmLecRK1 The nucleotide sequence of the gene is shown in SEQ ID No. 1; the encoded amino acid sequence is shown in SEQ ID No. 2. This invention relates to an artificially modified broad-spectrum disease resistance gene for maize. ZmLecRK1 The N341 site of the amino acid can enhance maize's resistance to Fusarium stalk rot and expand its range. ZmLecRK1 To improve the disease resistance spectrum and enhance the economic and ecological benefits of corn production.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, specifically relating to an artificially modified broad-spectrum disease resistance gene for maize. ZmLecRK1 And its applications. Background Technology

[0002] corn( Zea mays As a globally important food and energy crop, maize's yield and quality improvement are crucial for global food security. However, maize faces threats from various diseases during production, severely impacting its yield and quality. Fusarium stalk rot (FSR) is one of the most significant diseases affecting maize production, primarily caused by Fusarium graminearum (…). Fusarium graminearum This is caused by *Fusarium graminearum*. After infecting corn, *Fusarium graminearum* not only causes yield loss, but also produces various harmful substances to humans and animals, such as deoxynivalenol (DON), zearalenone (ZEN), and monazin (MON), seriously threatening my country's food and feed security.

[0003] ZmLecRK1 ( GRMZM2G330751 ZmLecRK1 is considered a key gene that can confer broad-spectrum disease resistance in maize. ZmLecRK1 enhances maize's resistance to diseases including Pythium spp. by regulating the maize immune response. Pythium aphanidermatum ), corn leaf blight fungus ( Bipolaris maydis ) and Rhizoctonia solani ( Rhizoctonia solani Resistance to multiple pathogens, including [list of pathogens]. However, despite [the fact that...] ZmLecRK1 While ZmLecRK1 has played an important role in controlling various maize diseases, its effect on resistance to Fusarium stalk rot is not significant, and it cannot provide effective resistance for maize.

[0004] Currently, in the field of maize disease resistance gene research, several technologies have been developed involving the discovery, identification, and improvement of disease resistance genes. For example, several genes related to disease resistance exist in maize; these genes enhance maize's resistance to various pathogens by regulating the plant's immune response. Among them... ZmLecRK1 This gene, as a broad-spectrum disease-resistance gene, has been proven to provide some resistance to a variety of diseases. However, ZmLecRK1 It showed limited effectiveness in resisting Fusarium graminearum stem rot.

[0005] The disadvantages of existing technologies are as follows:

[0006] 1.ZmLecRK1 Insufficient resistance to Maize Fusarium stalk rot

[0007] although ZmLecRK1The gene has been proven to have broad-spectrum disease resistance, but in current technologies... ZmLecRK1 Genes have limited effectiveness in controlling Fusarium wilt of cereals. Current technologies have failed to effectively address this issue. ZmLecRK1 The problem of gene dysfunction in Fusarium graminearum infection.

[0008] 2. Disease-resistant genes are unstable and easily attacked by pathogens.

[0009] Currently, many disease resistance genes obtained through transgenic or gene-editing technologies often fail to maintain stable disease resistance in practical applications. For example, ZmLecRK1 is easily degraded upon challenge with the effector protein FgEG18 of Fusarium graminearum, leading to the loss of disease resistance. Existing protective mechanisms and technical solutions cannot effectively address the interference of pathogen effector proteins, thus disease resistance traits are easily lost or unstable.

[0010] 3. The methods for protecting disease-resistant genes are limited and lack targeted solutions.

[0011] Current technologies primarily introduce disease resistance genes through traditional hybridization, transgenic technology, or methods such as CRISPR / Cas9. These methods typically focus on gene expression or genetic transmission, but lack techniques for protecting disease resistance genes against pathogenic attacks. Especially when facing specific pathogens such as Fusarium graminearum, existing methods fail to adequately consider the targeted attack of pathogen effector proteins, leading to the loss of disease resistance gene function.

[0012] 4. The diversity and complexity of disease resistance genes

[0013] In existing technologies, most disease resistance genes rely on complex polygenic inheritance mechanisms, making it difficult to guarantee their stable transmission during disease resistance breeding. The disease resistance genes required for different diseases may vary significantly, resulting in existing resistance genes being less than ideal in addressing specific disease problems and limiting the widespread application of disease-resistant varieties. Summary of the Invention

[0014] The technical problem to be solved by this invention is to provide an artificially modified broad-spectrum disease resistance gene for maize, addressing the shortcomings of the prior art. ZmLecRK1 And its applications, this artificially engineered broad-spectrum disease resistance gene in maize ZmLecRK1 Enhance maize's resistance to Fusarium stalk rot and expand its range. ZmLecRK1 To improve the disease resistance spectrum and enhance the economic and ecological benefits of corn production.

[0015] To solve the above-mentioned technical problems, the technical solution adopted in this invention is an artificially modified broad-spectrum disease resistance gene for maize. ZmLecRK1 ,Will ZmLecRK1 The critical amino acid N at position 341, which encodes an amino acid, was modified by point mutation;ZmLecRK1 The nucleotide sequence of the gene is shown in SEQ ID No. 1; ZmLecRK1 The amino acid sequence encoded by the gene is shown in SEQ ID No. 2.

[0016] Preferably, the ZmLecRK1 The amino acid critical site N at position 341 of the gene-encoded amino acid sequence was mutated to Q, A, E, G, H, I, K, M, R, T, V, W, Y. That is, based on the sequence of the wild-type ZmLecRK1 protein shown in SEQ ID No. 2, the following amino acid sites were mutated: N341Q, N341A, N341E, N341G, N341H, N341I, N341K, N341M, N341R, N341T, N341V, N341W, N341Y.

[0017] In this invention ZmLecRK1 After modifying the N at position 341 of the amino acid sequence encoded by the gene to Q, A, E, G, H, I, K, M, R, T, V, W, or Y, the following is obtained: ZmLecRK1 Gene mutants that enable ZmLecRK1 to evade recognition and attack by the Fusarium graminearum effector protein FgEG18 and avoid degradation of ZmLecRK1 protein, transiently expressing the above mutants in maize leaves can improve maize's resistance to Fusarium graminearum stem rot.

[0018] In this invention, the Fusarium graminearum effector protein FgEG18 attacks the key amino acid site N341 of ZmLecRK1, which is located in the extracellular domain of the ZmLecRK1 protein. This site was obtained through glycosylation mass spectrometry analysis of the ZmLecRK1 extracellular domain protein.

[0019] The Fusarium graminearum effector protein FgEG18 in this invention can attack ZmLecRK1, induce ZmLecRK1 protein degradation, and inhibit the biological function of wild-type ZmLecRK1 in triggering cell death in tobacco and maize.

[0020] In this invention, the key amino acid site N341 of ZmLecRK1 is used to modify ZmLecRK1, giving it significant protein stability in protein degradation mediated by the Fusarium graminearum effector protein FgEG18. Taking the N341Q key site modification as an example: Based on the sequence of the wild-type ZmLecRK1 protein as shown in SEQ ID No. 2, the mutant ZmLecRK1 is formed by mutating N341. N341Q This prevents the Fusarium graminearum effector protein FgEG18 from degrading ZmLecRK1. N341Q .

[0021] This invention utilizes the key amino acid site N341 of ZmLecRK1 to modify ZmLecRK1 without affecting the function of the mutant in tobacco and maize. Taking the N341Q key site modification as an example: Based on the sequence of the wild-type ZmLecRK1 protein as shown in SEQ ID No. 2, the mutant ZmLecRK1 is formed by mutating N341. N341Q ZmLecRK1 N341Q It can trigger cell death in tobacco leaves and corn protoplasts.

[0022] This invention utilizes the key amino acid site N341 of ZmLecRK1 to modify ZmLecRK1, enabling the mutant to evade attack by the Fusarium graminearum effector protein FgEG18 while retaining its function in tobacco and maize. Taking N341Q as an example of key site modification: Based on the sequence of the wild-type ZmLecRK1 protein as shown in SEQ ID No. 2, the mutant ZmLecRK1 is formed by mutating N341. N341Q ZmLecRK1 N341Q Cell death triggered in tobacco leaves and maize protoplasts could not be inhibited by the Fusarium graminearum effector protein FgEG18.

[0023] In this invention when ZmLecRK1 A mutation at position 341 (N) of the amino acid sequence encoded by the gene, replacing it with F, C, D, L, P, or S, results in mutations at the following amino acid sites: N341F, N341C, N341D, N341L, N341P, and N341S. Transient expression of these mutants in maize leaves does not affect... ZmLecRK1 Biological functions in maize, and ZmLecRK1 Transient expression of the mutant in maize leaves of the wild type showed consistent resistance to Fusarium graminearum and did not affect the wild type's resistance to Fusarium graminearum. ZmLecRK1 Biological functions in maize.

[0024] Compared with the prior art, the present invention has the following advantages:

[0025] 1. Enhance corn's resistance to Fusarium stalk rot.

[0026] This invention modifies the key site N341 of ZmLecRK1, enabling ZmLecRK1 to evade the targeting attack of the Fusarium graminearum effector protein FgEG18, thus preventing the degradation of the ZmLecRK1 protein and ensuring its disease resistance function in maize. Compared with existing technologies, this invention can significantly improve maize's resistance to Fusarium graminearum, filling a gap in the development of broad-spectrum disease resistance genes in maize. ZmLecRK1 The shortcomings in the prevention and control of Fusarium graminearum stem rot.

[0027] 2. Expanding the disease resistance spectrum of ZmLecRK1

[0028] By artificially modifying the key site N341 of ZmLecRK1, this invention enhances... ZmLecRK1 The gene resistance to Fusarium graminearum makes ZmLecRK1 It can exert a wider range of disease-fighting effects, which means ZmLecRK1 It can provide resistance to more corn diseases, further expanding the range of diseases covered. ZmLecRK1 Its wide range of applications makes it an important tool in maize disease-resistant breeding.

[0029] 3. Improve the economic and ecological benefits of corn production.

[0030] This invention modifies maize broad-spectrum disease resistance genes. ZmLecRK1 Enhancing maize's resistance to Fusarium graminearum can reduce the impact of Fusarium graminearum infection on maize stalk rot, thus reducing yield and quality. This can effectively decrease the use of chemical pesticides, minimizing environmental pollution and negative ecological impacts. Furthermore, stable disease resistance can improve maize yield and quality, thereby increasing the economic and ecological benefits of agricultural production.

[0031] 4. Promote the innovation and development of maize disease-resistant breeding technology.

[0032] This invention provides a novel technical solution that overcomes the problem of limited function of maize disease resistance genes in existing technologies, thus promoting the development of disease resistance gene protection technology. This technology not only contributes to technological advancements in maize breeding but also provides experience and methods for the research and protection of disease resistance genes in other crops.

[0033] The present invention will now be described in further detail with reference to the accompanying drawings and embodiments. Attached Figure Description

[0034] Figure 1 This is a diagram illustrating the biological function of the Fusarium graminearum effector protein FgEG18 in Example 1 of the present invention, which inhibits ZmLecRK1 from triggering cell death in tobacco and maize.

[0035] Figure 2 This is a protein degradation diagram of ZmLecRK1 induced by the Fusarium graminearum effector protein FgEG18 in Example 2 of the present invention.

[0036] Figure 3 This is a diagram showing the effect of the Fusarium graminearum effector protein FgEG18 on the biological function of the ZmLecRK1 point mutant in tobacco in Example 3 of the present invention.

[0037] Figure 4 The N341Q mutation at the ZmLecRK1 key site in Example 4 of this invention enables it to evade the targeted attack of the Fusarium graminearum effector protein FgEG18. Figure 4(a) shows the effect of FgEG18 on the protein accumulation of ZmLecRK1 in tobacco; (b) shows the effect of FgEG18 on ZmLecRK1-induced cell death in tobacco leaves; (c) shows the effect of FgEG18 on the protein accumulation of ZmLecRK1 in maize protoplasts; and (d) shows the effect of FgEG18 on ZmLecRK1-induced cell death in tobacco leaves in maize protoplasts.

[0038] Figure 5 The modification of the ZmLecRK1 key site N341 in Example 5 of this invention can enhance the resistance of maize to Fusarium graminearum. Detailed Implementation

[0039] The broad-spectrum disease resistance gene ZmLecRK1 in maize mediates resistance to Pythium stalk rot, sheath blight, and small leaf spot, but not to Fusarium stalk rot. The inventors hypothesized that an effector protein attacking ZmLecRK1 might exist in Fusarium graminearum. Since ZmLecRK1 can trigger cell death in tobacco leaves, the inventors screened for the Fusarium graminearum effector protein FgEG18, which specifically inhibits ZmLecRK1-induced cell death in tobacco leaves. In both tobacco and maize protoplast systems, FgEG18 significantly inhibited ZmLecRK1-induced cell death; furthermore, at the protein level, FgEG18 significantly reduced the accumulation of ZmLecRK1 protein. Given FgEG18's ability to hydrolyze β-1,4 glycosidic bonds, it is hypothesized that FgEG18 attacks the N-glycosylation modification site of ZmLecRK1, making the ZmLecRK1 protein more susceptible to degradation. The inventors identified 10 N-glycosylation modification sites in the extracellular structure of ZmLecRK1, including N341, using glycosylation mass spectrometry. Furthermore, this invention simulated a deglycosylation mutation (NQ) to artificially mutate N (asparagine) at position 341 to Q (glutamine), discovering a mutant ZmLecRK1. N341Q Cell death triggered in tobacco and maize protoplasts was unaffected; meanwhile, the Fusarium graminearum effector protein FgEG18 also failed to inhibit ZmLecRK1. N341Q The mutants, modified at the ZmLecRK1 key site N341, significantly enhanced maize resistance to Fusarium graminearum after transient expression in maize leaves. This indicates that the extracellular N-glycosylation modification site N341 of ZmLecRK1 is a key target of the Fusarium graminearum effector protein FgEG18. Therefore, modification of the ZmLecRK1 key site N341 can serve as a target for disease resistance gene modification, possessing significant scientific importance and application value in maize disease resistance breeding.

[0040] Based on this, the present invention provides a modification of the key site N341 of the maize broad-spectrum disease resistance gene ZmLecRK1 and its application. Specifically, the gene site modification involves mutating the following amino acid site: N341Q, based on the sequence of the wild-type ZmLecRK1 protein as shown in SEQ ID No. 2. The mutant ZmLecRK1 after key site modification... N341Q It 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, ensuring the stability of the ZmLecRK1 mutant protein. In addition, other modifications to the key site N341 of ZmLecRK1 are as follows: based on the sequence of the wild-type ZmLecRK1 protein as shown in SEQ ID No. 2, the following amino acid sites were 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. The ZmLecRK1 mutants modified in these ways, after transient expression in maize leaves, significantly improved maize resistance to Fusarium graminearum; in SEQ ID No. 2... Based on the sequence of the wild-type ZmLecRK1 protein shown in No. 2, the following amino acid sites were mutated: N341F or N341C or N341D or N341L or N341P or N341S. After transient expression of the above-mentioned modified ZmLecRK1 mutant in maize leaves, the resistance to Fusarium graminearum was consistent with that of the wild-type ZmLecRK1 in maize leaves.

[0041] Example 1

[0042] This embodiment contains Fusarium graminearum effector protein. FgEG18 Able to specifically inhibit ZmLecRK1 The biological functions of proteins, specific operational methods, and results are as follows:

[0043] Step 1, FgEG18 and ZmLecRK1 Cloning

[0044] First, total RNA was extracted from Fusarium graminearum strain FG-12 (Hao et al., 2021, Submitted GenBank assembly: GCA_019343145.1) and reverse transcribed into cDNA; total RNA was also extracted from maize inbred line B73 and reverse transcribed into cDNA.

[0045] The primer sequences are designed as shown in Table 1:

[0046] Table 1 Primers required for constructing the expression vector in Example 1

[0047]

[0048] Using cDNA from Fusarium graminearum strain FG-12 as a template, PCR amplification was performed using primers FgEG18-PF and FgEG18-PR. The PCR reaction system consisted of: 10 μL of 2×T8 High-Fidelity Master Mix (catalog number: DLP201, purchased from Beijing Qingke Biotechnology Co., Ltd.), 1 μL each of 10 μM primers (primers FgEG18-PF and FgEG18-PR) (synthesized by Beijing Qingke Biotechnology Co., Ltd.), 1 μL of template cDNA, and sterile water to a final volume of 20 μL. The PCR reaction conditions were: 98℃ pre-denaturation for 2 min; 32 cycles of 98℃ for 30 sec, 60℃ for 30 sec, and 72℃ for 30 sec; and a final extension at 72℃ for 5 min. After PCR products were subjected to 1% agarose gel electrophoresis, the target fragment was recovered using a DNA gel recovery kit (catalog number: TSP602-200, purchased from Beijing Qingke Biotechnology Co., Ltd.). The recovery procedure was performed according to the kit instructions to obtain the gene fragment. FgEG18 .

[0049] Using cDNA from the maize inbred line B73 as a template, PCR amplification was performed using primers ZmLecRK1-PF and ZmLecRK1-PR. The PCR reaction system consisted of: 10 μL of 2×T8 High-Fidelity Master Mix (Catalog No.: TSE111, purchased from Beijing Qingke Biotechnology Co., Ltd.), 1 μL each of 10 μM primers (primers ZmLecRK1-PF and ZmLecRK1-PR) (synthesized by Beijing Qingke Biotechnology Co., Ltd.), 1 μL of template cDNA, and deionized water to a final volume of 20 μL. The PCR reaction conditions were: 98℃ pre-denaturation for 2 min; 32 cycles of 98℃ for 30 sec, 60℃ for 2 min, and 72℃ for 30 sec; and a final extension at 72℃ for 5 min. After PCR products were subjected to 1% agarose gel electrophoresis, the target fragment was recovered using a DNA gel recovery kit (catalog number: TSP602-200, purchased from Beijing Qingke Biotechnology Co., Ltd.). The recovery procedure was performed according to the kit instructions to obtain the gene fragment. ZmLecRK1 The nucleotide sequence is shown in SEQ ID No. 1, encoding... ZmLecRK1 The amino acid sequence of the protein is shown in SEQ ID No. 2.

[0050] Homologous recombination ligation: Existing plasmids pCAMBIA1300-HA, pCAMBIA1300-Flag, pCAMBIA1300-GFP, pUC19-HA, and pUC19-Flag (Wang et al., 2023) were linearized using restriction endonucleases KpnI (catalog number: 1618, purchased from Baoriyi Biotechnology (Beijing) Co., Ltd.) and Sal I (catalog number: 1636, purchased from Baoriyi Biotechnology (Beijing) Co., Ltd.), respectively. The reaction system consisted of: 10 μL of 100 ng / μL vector, 2 μL of 10×QuickCutBuffer, 1 μL of restriction endonuclease KpnI, 1 μL of restriction endonuclease Sal I, and deionized water to a final volume of 20 μL. The reaction system was incubated at 37°C for 1 h, followed by incubation at 80°C for 20 min. After the products were subjected to 1% agarose gel electrophoresis, the linearized vectors were recovered using a DNA gel recovery kit (catalog number: TSP602-200, purchased from Beijing Qingke Biotechnology Co., Ltd.). The recovery procedure was performed in accordance with the kit instructions, and the linearized vectors pCAMBIA1300-HA, pCAMBIA1300-Flag, pCAMBIA1300-GFP, pUC19-HA, and pUC19-Flag were obtained. The linearized vector pCAMBIA1300-HA was recombinated with the fragment FgEG18, and the linearized vectors pCAMBIA1300-Flag and pCAMBIA1300-GFP were recombinated with the fragment ZmLecRK1 using a homologous recombination kit (product number: TSV-S1, purchased from Beijing Qingke Biotechnology Co., Ltd.) (the experimental steps were performed according to the kit instructions). The resulting vectors were transformed into Escherichia coli DH5α to obtain the expression vectors pCAMBIA1300-35S::FgEG18-HA, pCAMBIA1300-35S::ZmLecRK1-Flag, and pCAMBIA1300-35S::ZmLecRK1-GFP, which were used for transient expression in plants. The linearized vector pUC19-HA was recombinated with the fragment FgEG18, and pUC19-Flag was recombinated with the fragment ZmLecRK1 using a homologous recombination kit. The resulting vectors were transformed into Escherichia coli DH5α to obtain the expression vectors pUC19-35S::FgEG18-HA and pUC19-35S::ZmLecRK1-Flag, which were used for transient expression in maize protoplasts.

[0051] Using expression vectors pCAMBIA1300-35S::FgEG18-HA and pUC19-35S::FgEG18-HA as templates, and primer FgEG18 as the primer, respectively... H107A -PF and FgEG18 H107A-PR was used for PCR amplification. The PCR reaction system was: 5 μL of 2×T8 High-Fidelity Master Mix and 10 μM primers (primer FgEG18). H107A -PF and FgEG18 H107A 0.5 μL each of PCR product and template cDNA, 1 μL of sterile water to a final volume of 10 μL. PCR reaction conditions: 98℃ pre-denaturation for 2 min; 20 cycles of 98℃ for 30 sec, 60℃ for 30 sec, and 72℃ for 30 sec; final extension at 72℃ for 5 min. PCR products were treated with restriction endonuclease Dpn I (catalog number: 1609, purchased from Baori Biotechnology (Beijing) Co., Ltd.). The enzyme digestion reaction system was: 10 μL PCR product, 2 μL 10×QuickCut Buffer, 1 μL restriction endonuclease Dpn I, and deionized water to a final volume of 20 μL. The reaction was carried out at 37℃ for 1 h, followed by an reaction at 80℃ for 20 min. The product was then transformed into *E. coli* DH5α to obtain pCAMBIA1300-35S::FgEG18. H107A -HA and pUC19-35S::FgEG18 H107A -HA expression vector.

[0052] Step two, FgEG18 right ZmLecRK1 Impact of biological functions

[0053] (1) Fusarium graminearum effector protein FgEG18 The experiment was able to inhibit ZmLecRK1 cell death in tobacco leaves: The experiment was divided into three groups, with the vectors pCAMBIA1300-35S::GFP-HA (Wang et al., 2023), pCAMBIA1300-35S::FgEG18, and pCAMBIA1300-35S::FgEG18 being used. H107A -Flag (constructed in this example), pCAMBIA1300-35S::FgEG18-HA, and pCAMBIA1300-35S::ZmLecRK1-Flag (constructed in this example) were transformed into Agrobacterium GV3101, respectively. After shaking for 18 h, the bacterial cells were resuspended in infiltration buffer (10 mM MgCl2, 10 mM MES, and 150 μM acetylsyleugenone), and the OD of each component was adjusted. 600 Up to 0.5. Following the formula pCAMBIA1300-35S::GFP-HA + pCAMBIA1300-35S::ZmLecRK1-Flag, pCAMBIA1300-35S::FgEG18 H107AThe combination of -Flag + pCAMBIA1300-35S::ZmLecRK1-Flag and pCAMBIA1300-35S::FgEG18-HA + pCAMBIA1300-35S::ZmLecRK1-Flag was injected into four-week-old tobacco leaves. The tobacco leaves were observed and photographed 48 h after injection.

[0054] The results are as follows Figure 1 As shown in Figure a, ZmLecRK1 is associated with GFP and FgEG18. H107 Co-injection of both ZmLecRK1 and FgEG18 induced tobacco cell death, while co-injection with FgEG18 did not. This indicates that the Fusarium graminearum effector protein FgEG18 can inhibit the biological function of ZmLecRK1 in tobacco.

[0055] (2) The Fusarium graminearum effector protein FgEG18 can inhibit ZmLecRK1 cell death in maize protoplasts: This experiment was divided into 4 groups, pUC19-35S::mCherry (Wang et al., 2023) and pUC19-35S::FgEG18. H107A -HA served as the control, and pUC19-35S::LUC (Li et al., 2024) was used as the reporter gene vector. The vectors were selected in the following combinations: pUC19-35S::LUC, pUC19-35S::LUC + pUC19-35S::mCherry + pUC19-35S::ZmLecRK1-Flag, and pUC19-35S::LUC + pUC19-35S::FgEG18. H107A The plasmids pUC19-35S::ZmLecRK1-Flag and pUC19-35S::LUC+pUC19-35S::FgEG18-HA+pUC19-35S::ZmLecRK1-Flag were transformed into maize protoplasts using a PEG-mediated maize protoplast transformation system. After 20 h of expression in the dark, the protoplasts were cultured under fluorescent light for 4 h. The protoplasts were collected by centrifugation, and proteins were extracted. The fluorescence values ​​of each experimental group were measured using a microplate reader, with luciferase activity serving as an indicator of ZmLecRK1-induced cell death in maize protoplasts.

[0056] The results are as follows Figure 1 As shown in Figure b, in maize protoplasts, FgEG18 significantly inhibited ZmLecRK1-triggered cell death, while control mCherry and FgEG18... H107A It could not inhibit ZmLecRK1-triggered cell death. This indicates that the Fusarium graminearum effector protein FgEG18 can inhibit the biological function of ZmLecRK1 in maize.

[0057] Example 2

[0058] Fusarium graminearum effector proteins can induce the degradation of ZmLecRK1 protein. The specific operation method and results are as follows:

[0059] 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) was used to transform the above-mentioned vector into Agrobacterium GV3101. After shaking culture for 18 h, the bacterial cells were resuspended in infiltration buffer (10 mM MgCl2, 10 mM MES and 150 μM acetylsylcholine) and the OD of each component was adjusted. 600 Up to 0.5. Four-week-old tobacco leaves were injected with a combination of pCAMBIA1300-35S::ZmLecRK1-Flag + pCAMBIA1300-35S::GFP-HA and pCAMBIA1300-35S::ZmLecRK1-Flag + pCAMBIA1300-35S::FgEG18-HA. Samples were taken 48 h after injection to extract protein, and the protein accumulation of ZmLecRK1 in each group of samples was analyzed by Western blot.

[0060] The results are as follows Figure 2 As shown, FgEG18 can significantly reduce the protein accumulation of ZmLecRK1, while the mutant FgEG18, which loses enzyme activity, is significantly reduced. H107A It does not affect the protein accumulation of ZmLecRK1. This indicates that the Fusarium graminearum effector protein FgEG18 can affect the protein accumulation of ZmLecRK1.

[0061] Example 3

[0062] Analysis of key extracellular glycosylation sites in the maize broad-spectrum disease resistance gene ZmLecRK1: The specific procedures and results are as follows:

[0063] Step 1: ZmLecRK1 ECD+TM Cloning and protein enrichment

[0064] (1) ZmLecRK1 ECD+TM Construction of expression vectors

[0065] The primers are designed as shown in Table 2:

[0066] Table 2 ZmLecRK1 ECD+TM Primers required for constructing expression vectors

[0067]

[0068] Using the expression vector pCAMBIA1300-35S::ZmLecRK1-Flag constructed in Example 1 as a template, and primer ZmLecRK1 ECD+TM -PF and primer ZmLecRK1 ECD+TM -PR was used for PCR amplification, and the reaction system was: 10 μL of 2×T8 High-Fidelity Master Mix and 10 μM of primers (primer ZmLecRK1). ECD+TM -PF and primer ZmLecRK1 ECD+TM 1 μL each of -PR and template, 1 μL, and sterile water were added to a final volume of 20 μL. PCR reaction conditions were: 98℃ pre-denaturation for 2 min; 32 cycles of 98℃ for 30 sec, 60℃ for 30 sec, and 72℃ for 1 min; final extension at 72℃ for 5 min. After PCR products were subjected to 1% agarose gel electrophoresis, the target fragment was recovered using a DNA gel recovery kit (catalog number: TSP602-200, purchased from Beijing Qingke Biotechnology Co., Ltd.). The recovery procedure was performed according to the kit instructions, yielding the gene fragment ZmLecRK1. ECD+TM .

[0069] Homologous recombination ligation: linearized vector pCAMBIA1300-Flag and fragment ZmLecRK1 ECD+TM Recombination was performed using a homologous recombination kit (product number: TSV-S1, purchased from Beijing Qingke Biotechnology Co., Ltd.) (the experimental procedure was performed according to the kit instructions), transforming *E. coli* DH5α to obtain pCAMBIA1300-35S::ZmLecRK1. ECD+TM -Flag expression carrier.

[0070] (2) ZmLecRK1 ECD+TM Protein expression and enrichment

[0071] The above expression vector pCAMBIA1300-35S::ZmLecRK1 was used. ECD+TM - The cells were transferred into Agrobacterium GV3101 via Flag and shaken for 18 h. The cells were then resuspended in infiltration buffer (10 mM MgCl2, 10 mM MES, and 150 μM acetylsylcholine) and the OD was adjusted. 600 0.5 mg / L was injected into four-week-old tobacco leaves. Samples were taken 48 h after injection to obtain samples expressing ZmLecRK1. ECD+TMProtein-rich tobacco leaves.

[0072] The tobacco leaves were rapidly ground into powder in liquid nitrogen. Protein extraction solution (25 mM Tris-HCl pH 7.5, 1 mM EDTA, 150 mM NaCl, 10% glycerol, 2% PVP, 0.5% Triton-X100, 1 mM DTT, and protease inhibitor) was added. The mixture was incubated on ice for 30 min, then centrifuged at 8000×g for 10 min at 4°C. The supernatant was discarded, and 5 μL of anti-Flag magnetic beads (Catalog No.: HY-K0207, MedChemexpress Biotechnology, USA) was added. The mixture was incubated at 4°C for 2 h to enrich ZmLecRK1. ECD+TM Protein. After incubation and enrichment, the magnetic beads were washed 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), and then washed 10 times with PBS (pH7.5). The magnetic beads were then collected for later use.

[0073] Step 2: Glycosylation mass spectrometry analysis of ZmLecRK1 extracellular glycosylation modification sites

[0074] The above-mentioned ZmLecRK1 enrichment ECD+TM Glycosylation mass spectrometry analysis of the protein was performed using magnetic beads to obtain information such as the glycosylation modification sites of the ZmLecRK1 extracellular domain.

[0075] The results are shown in Table 3. Glycosylation mass spectrometry analysis identified 10 N-glycosylation modification sites in the extracellular domain of ZmLecRK1, including N34, N85, N121, N142, N215, N227, N322, N341, N380, and N435. The nomenclature of these sites is as follows: taking N341 as an example, it refers to… ZmLecRK1 The 341st amino acid in the gene encoding is N (asparagine).

[0076] Table 3. Glycosylation Mass Spectrometry Analysis of Extracellular Glycosylation Modification Sites in ZmLecRK1

[0077]

[0078]

[0079] Note: The letters in parentheses represent the amino acids at both ends of the peptide.

[0080] Step 3: ZmLecRK1 Analysis of key glycosylation sites in extracellular domains

[0081] (1)ZmLecRK1 The extracellular domain N-glycosylation site was used to simulate the deglycosylation modification of the mutant vector. Primers were designed as shown in Table 4. The naming convention for the point mutant vector was as follows: taking the mutation of N (asparagine) to Q (glutamine) at amino acid position 341 of the ZmLecRK1 gene as an example, the point mutant vector was named ZmLecRK1. N34Q Mutant.

[0082] Table 4 ZmLecRK1 Primers required for constructing extracellular glycosylation modification sites to mimic deglycosylation modification mutant vectors

[0083]

[0084] ZmLecRK1 N34Q Taking the construction of a point mutation vector as an example: using the expression vector pCAMBIA1300-35S::ZmLecRK1-Flag constructed in Example 1 as a template, and primer ZmLecRK1 N34Q -PF and primer ZmLecRK1 N34Q -PR was used for PCR amplification. The PCR reaction system was: 5 μL of 2×T8 High-Fidelity Master Mix and 10 μM primers (primer ZmLecRK1). N34Q -PF and primer ZmLecRK1 N34Q 0.5 μL each of PCR product and template, 1 μL of sterile water to a final volume of 10 μL. PCR reaction conditions: 98℃ pre-denaturation for 2 min; 20 cycles of 98℃ for 30 sec, 60℃ for 30 sec, and 72℃ for 30 sec; final extension at 72℃ for 5 min. PCR products were treated with restriction endonuclease Dpn I (catalog number: 1609, purchased from Baori Biotechnology (Beijing) Co., Ltd.). The enzyme digestion reaction system was: 10 μL PCR product, 2 μL 10×QuickCut Buffer, 1 μL restriction endonuclease Dpn I, and deionized water to a final volume of 20 μL. The reaction was carried out at 37℃ for 1 h, followed by an reaction at 80℃ for 20 min. The product was then transformed into *E. coli* DH5α to obtain pCAMBIA1300-35S::ZmLecRK1. N34Q -Flag point mutation expression vector.

[0085] Using the above method and primer pairs, the remaining point mutation expression vectors were constructed, yielding: pCAMBIA1300-35S::ZmLecRK1 N85Q -Flag、

[0086] pCAMBIA1300-35S::ZmLecRK1 N121Q -Flag、

[0087] pCAMBIA1300-35S::ZmLecRK1 N142Q -Flag、

[0088] pCAMBIA1300-35S::ZmLecRK1 N215Q -Flag、

[0089] pCAMBIA1300-35S::ZmLecRK1 N227Q -Flag、

[0090] pCAMBIA1300-35S::ZmLecRK1 N322Q -Flag、

[0091] pCAMBIA1300-35S::ZmLecRK1 N341Q -Flag、

[0092] pCAMBIA1300-35S::ZmLecRK1 N380Q -Flag、

[0093] pCAMBIA1300-35S::ZmLecRK1 N435Q -Flag point mutation expression vector for transient expression in plants.

[0094] (2) Effect of FgEG18 on the function of ZmLecRK1 mutant

[0095] The ZmLecRK1 point mutation expression vector used for transient plant expression was transformed into Agrobacterium GV3101, and tobacco leaves were injected according to the method in step two of Example 1. Agrobacterium transformed with the above point mutation vector was co-injected with Agrobacterium transformed with the pCAMBIA1300-35s::FgEG18-HA expression vector, with co-injection of Agrobacterium transformed with the pCAMBIA1300-35S::GFP-HA expression vector serving as a control. The tobacco leaves were observed and photographed 48 h after Agrobacterium injection.

[0096] The results are as follows Figure 3 As shown, ZmLecRK1 N34Q ZmLecRK1 N121Q ZmLecRK1 N215Q ZmLecRK1 N227Q ZmLecRK1 N322Q ZmLecRK1 N341Q and ZmLecRK1 N380Q The mutant can still trigger cell death in tobacco leaves, but ZmLecRK1 N85QZmLecRK1 N142Q and ZmLecRK1 N435Q The mutant could not trigger cell death in tobacco leaves. Furthermore, the Fusarium graminearum effector protein FgEG18 could not inhibit ZmLecRK1. N341Q The mutant triggered cell death in tobacco leaves, but not in ZmLecRK1. N34Q ZmLecRK1 N121Q ZmLecRK1 N215Q ZmLecRK1 N227Q ZmLecRK1 N322Q and ZmLecRK1 N380Q The mutant-induced tobacco leaf cell death was still significantly inhibited. This indicates that the N341 glycosylation modification site of ZmLecRK1 is an important target of FgEG18.

[0097] Example 4

[0098] Fusarium graminearum effector protein FgEG18 against ZmLecRK1 N341Q The impact of protein accumulation on biological function was analyzed, and the specific procedures and results are as follows:

[0099] Step 1: Fusarium graminearum effector protein FgEG18 on ZmLecRK1 N341Q Effect of protein accumulation

[0100] Using the expression vector pUC19-35S::ZmLecRK1-Flag constructed in Example 1 as a template, and primer ZmLecRK1 N341Q -PF and primer ZmLecRK1 N341Q -PR was used for PCR amplification. The PCR reaction system was: 5 μL of 2×T8 High-Fidelity Master Mix and 10 μM primers (primer ZmLecRK1). N341Q -PF and primer ZmLecRK1 N341Q0.5 μL each of PCR product and template, 1 μL of sterile water to a final volume of 10 μL. PCR reaction conditions: 98℃ pre-denaturation for 2 min; 20 cycles of 98℃ for 30 sec, 60℃ for 30 sec, and 72℃ for 30 sec; final extension at 72℃ for 5 min. PCR products were treated with restriction endonuclease Dpn I (catalog number: 1609, purchased from Baori Biotechnology (Beijing) Co., Ltd.). The enzyme digestion reaction system was: 10 μL PCR product, 2 μL 10×QuickCut Buffer, 1 μL restriction endonuclease Dpn I, and deionized water to a final volume of 20 μL. The reaction was carried out at 37℃ for 1 h, followed by an reaction at 80℃ for 20 min. The product was then transformed into *E. coli* DH5α to obtain pUC19-35S::ZmLecRK1. N341Q -Flag point mutation expression vector for transient expression in maize protoplasts.

[0101] (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-mentioned vectors were respectively transferred into Agrobacterium GV3101, and after shaking culture for 18 h, the bacterial cells were resuspended in infiltration buffer (10 mM MgCl2, 10 mM MES and 150 μM acetylsylcholine), and the OD of each component was adjusted. 600 to 0.5. Follow 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 The combination of -Flag + pCAMBIA1300-35S::FgEG18-HA was injected into four-week-old tobacco leaves. Samples were collected 48 h after injection for protein extraction, and ZmLecRK1 or ZmLecRK1 in each group of samples was analyzed by Western blot. N341Q The amount of protein accumulation.

[0102] (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), and pUC19-35S::ZmLecRK1 N341Q -Flag and pUC19-35S::FgEG18-HA (prepared in Example 1). The above expression vectors were respectively prepared according to the following orders: pUC19-35S::ZmLecRK1-Flag + pUC19-35S::mCherry-HA, pUC19-35S::ZmLecRK1-Flag + pUC19-35S::FgEG18-HA, pUC19-35S::ZmLecRK1-Flag + pUC19-35S::FgEG18-HA, pUC19-35S::ZmLecRK1-Flag + pUC19-35S::FgEG18-HA, and pUC19-35S::ZmLecRK1-Flag + pUC19-35S::FgEG18-HA. N341Q -Flag + pUC19-35S::mCherry-HA and pUC19-35S::ZmLecRK1 N341Q The combination of -Flag + pUC19-35S::FgEG18-HA was used to co-transfer the expression vector into maize protoplasts using a PEG-mediated maize protoplast transformation system. After 20 h of expression in the dark, the protein was extracted, and the levels of ZmLecRK1 or ZmLecRK1 in each group of samples were analyzed by Western blot. N341Q The amount of protein accumulation.

[0103] The results are as follows Figure 4 As shown in Figures a and c, FgEG18 significantly reduces the protein accumulation of ZmLecRK1 in tobacco, but has little effect on ZmLecRK1 protein levels. N341Q The amount of protein accumulation; in maize protoplasts, FgEG18 also has almost no effect on ZmLecRK1. N341Q The amount of protein accumulation. This indicates that ZmLecRK1 N341Q It can prevent protein degradation under FgEG18 attack.

[0104] Step 2: FgEG18 against ZmLecRK1 N341Q Impact of biological functions

[0105] (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-mentioned vectors were respectively transferred into Agrobacterium GV3101, and after shaking culture for 18 h, the bacterial cells were resuspended in infiltration buffer (10 mM MgCl2, 10 mM MES and 150 μM acetylsylcholine), and the OD of each component was adjusted. 600 to 0.5. Follow 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 The combination of -Flag + pCAMBIA1300-35S::FgEG18-HA was injected into four-week-old Tobacco Benedictine leaves, and the leaves were observed and photographed 48 hours after injection.

[0106] (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), and pUC19-35S::ZmLecRK1 N341Q -Flag and pUC19-35S::FgEG18-HA (prepared in Example 1), the above expression vectors were respectively prepared according to the following formulations: pUC19-35S::LUC, pUC19-35S::LUC+ pUC19-35S::ZmLecRK1-Flag + pUC19-35S::mCherry-HA, pUC19-35S::LUC+ pUC19-35S::ZmLecRK1-Flag + pUC19-35S::FgEG18 ...ZmLecRK1-Flag + pUC19-35S::FgEG18-HA, pUC19-35S: N341Q -Flag + pUC19-35S::mCherry-HA and pUC19-35S::LUC + pUC19-35S::ZmLecRK1 N341QThe combination of -Flag + pUC19-35S::FgEG18-HA was used in a PEG-mediated maize protoplast transformation system to co-transfer the expression vector into maize protoplasts. After 20 h of expression in the dark, the protoplasts were cultured under fluorescent light for 4 h. The protoplasts were collected by centrifugation, and the protein was extracted. The fluorescence values ​​of each experimental group were measured using a microplate reader, with luciferase activity serving as an indicator of ZmLecRK1-induced cell death in maize protoplasts.

[0107] The results are as follows Figure 4 As shown in Figures b and d, in tobacco, FgEG18 significantly inhibited ZmLecRK1-triggered cell death in tobacco leaves, but did not inhibit ZmLecRK1. N341Q Triggered cell death in tobacco leaves; ZmLecRK1 in maize protoplasts N341Q It can induce the same degree of cell death as ZmLecRK1, but FgEG18 cannot inhibit ZmLecRK1. N341Q This causes protoplast death in maize. This indicates that ZmLecRK1... N341Q It can evade the targeted attack of FgEG18 and continue to exert its immune function in tobacco and corn.

[0108] Example 5

[0109] Transient expression of ZmLecRK1 in maize leaves N341Q The specific procedures and results of the resistance analysis against Fusarium graminearum are as follows:

[0110] Step 1: Agrobacterium-mediated transient expression in maize leaves

[0111] (1) Construction of the expression vector for the N341 point mutation at the key site of ZmLecRK1

[0112] The primers are designed as shown in Table 5:

[0113] Table 5 Primers required for constructing the ZmLecRK1 key site N341 point mutation expression vector.

[0114]

[0115]

[0116] ZmLecRK1 N341A Taking the construction of a point mutation expression vector as an example: using the expression vector pCAMBIA1300-35S::ZmLecRK1-GFP constructed in Example 1 as a template, and using the above primer ZmLecRK1... N341A -PF and primer ZmLecRK1 N341A-PR was used for PCR amplification. The PCR reaction system was: 5 μL of 2×T8 High-Fidelity Master Mix and 10 μM primers (primer ZmLecRK1). N341A -PF and primer ZmLecRK1 N341A 0.5 μL each of PCR product and template, 1 μL of sterile water to a final volume of 10 μL. PCR reaction conditions: 98℃ pre-denaturation for 2 min; 20 cycles of 98℃ for 30 sec, 60℃ for 30 sec, and 72℃ for 30 sec; final extension at 72℃ for 5 min. PCR products were treated with restriction endonuclease Dpn I (catalog number: 1609, purchased from Baori Biotechnology (Beijing) Co., Ltd.). The enzyme digestion reaction system was: 10 μL PCR product, 2 μL 10×QuickCut Buffer, 1 μL restriction endonuclease Dpn I, and deionized water to a final volume of 20 μL. The reaction was carried out at 37℃ for 1 h, followed by an reaction at 80℃ for 20 min. The product was then transformed into *E. coli* DH5α to obtain pCAMBIA1300-35S::ZmLecRK1. N341A -GFP point mutation expression vector. Using the above method and primer pairs, the remaining point mutation expression vectors were constructed to obtain pCAMBIA1300-35S::ZmLecRK1. N341F -GFP、

[0117] pCAMBIA1300-35S::ZmLecRK1 N341C -GFP、

[0118] pCAMBIA1300-35S::ZmLecRK1 N341D -GFP、

[0119] pCAMBIA1300-35S::ZmLecRK1 N341E -GFP、

[0120] pCAMBIA1300-35S::ZmLecRK1 N341G -GFP、

[0121] pCAMBIA1300-35S::ZmLecRK1 N341H -GFP、

[0122] pCAMBIA1300-35S::ZmLecRK1 N341L -GFP、

[0123] pCAMBIA1300-35S::ZmLecRK1 N341I -GFP、

[0124] pCAMBIA1300-35S::ZmLecRK1 N341K -GFP、

[0125] pCAMBIA1300-35S::ZmLecRK1 N341M -GFP、

[0126] pCAMBIA1300-35S::ZmLecRK1 N341P -GFP、

[0127] pCAMBIA1300-35S::ZmLecRK1 N341R -GFP、

[0128] pCAMBIA1300-35S::ZmLecRK1 N341S -GFP、

[0129] pCAMBIA1300-35S::ZmLecRK1 N341T -GFP、

[0130] pCAMBIA1300-35S::ZmLecRK1 N341V -GFP、

[0131] pCAMBIA1300-35S::ZmLecRK1 N341W -GFP、

[0132] pCAMBIA1300-35S::ZmLecRK1 N341Y -GFP point mutation expression vector.

[0133] (2) Agrobacterium-mediated transient expression in maize leaves

[0134] pCAMBIA1300-35S::GFP (Wang et al., 2023), pCAMBIA1300-35S::ZmLecRK1-GFP (prepared in Example 1), and the above-mentioned point mutation expression vector were transformed into Agrobacterium GV3101, respectively. Based on previous reports of 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, after Agrobacterium culture for 18 h, the Agrobacterium cells were washed twice with sterile water and then resuspended in infusion solution (10 mM MgCl2, 10 mM MES, and 300 μM acetylsyleugenone), and the bacterial concentration was adjusted to OD. 600=0.5, the first leaf of V2 stage maize seedlings was used for injection. Before injection, the underside of the leaf was gently scratched with a sterile needle to create a wound to facilitate Agrobacterium infiltration. After injection, the seedlings were incubated in the dark at 25°C and 98% humidity for 24 h; then incubated at 25°C under low light conditions for 72 h, and the leaves were cut off for later use.

[0135] Step 2: Resistance analysis of the artificially modified mutant of ZmLecRK1 key site N341 to Fusarium graminearum

[0136] Fusarium graminearum strain FG-12 was cultured in PDA plates for 3 days. Mycelial cakes with a diameter of 2 mm were punched along the edge of the colony. Maize leaves, after transient expression of the ZmLecRK1 wild-type and point mutant strains, were placed, back side up, on a tray lined with moist filter paper. The Fusarium graminearum mycelial cakes were inoculated onto the maize leaves, mycelium side down, ensuring the inoculation point coincided with the Agrobacterium injection point. After moistening and incubation in the dark at 25°C for 24 hours, photographs were taken, and the lesion area was recorded.

[0137] The results are as follows Figure 5 As shown, the transient expression of 13 mutations at the ZmLecRK1 key site N341, namely N341Q, N341A, N341E, N341G, N341H, N341I, N341K, N341M, N341R, N341T, N341V, N341W, and N341Y, in maize leaves after inoculation with Fusarium graminearum FG-12 significantly reduced the disease severity and lesion area compared to the control group (GFP) and the ZmLecRK1 wild type (WT). However, the transient expression of the ZmLecRK1 key site N341 point mutations, namely N341F, N341C, N341D, N341L, N341P, and N341S, in maize leaves after inoculation with Fusarium graminearum FG-12 showed the same disease severity and lesion area as the ZmLecRK1 wild type. This indicates that artificially modifying the key site N341 of ZmLecRK1 does not affect the biological function of ZmLecRK1 in maize. Furthermore, 13 mutant types, including N341Q, significantly enhance the resistance of ZmLecRK1 to Fusarium graminearum in maize. Therefore, the above modification can improve maize's resistance to Fusarium graminearum, making it possible for the broad-spectrum disease-resistant gene ZmLecRK1 to possess resistance to Fusarium stalk rot, which has important significance and application value in maize disease resistance breeding.

[0138] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention in any way. Any simple modifications, alterations, and equivalent changes made to the above embodiments based on the inventive essence shall still fall within the protection scope of the present invention.

Claims

1. An artificially modified broad-spectrum disease resistance gene for maize ZmLecRK1 Its characteristics are, The gene encoding ZmLecRK1 The mutant protein is the wild-type protein shown in SEQ ID No.

2. ZmLecRK1 Based on the protein sequence, the N-point mutation at the critical amino acid site of position 341 is modified to Q, A, E, G, H, I, K, M, R, T, V, W, or Y.

2. The application of the artificially modified maize broad-spectrum disease resistance gene ZmLecRK1 as described in claim 1, characterized in that, By modifying the N at position 341 of the amino acid sequence encoded by the wild-type ZmLecRK1 gene to Q, A, E, G, H, I, K, M, R, T, V, W, or Y, a ZmLecRK1 gene mutant is obtained, which improves the resistance of maize to Fusarium graminearum stem rot caused by Fusarium graminearum.

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