Use of zmgae1 protein and gene encoding same in regulation of fungal disease resistance of plants and stalk strength of plants

Abstract

The present invention pertains to the technical field of genetic engineering and molecular breeding of plants and particularly relates to use of the ZmGAE1 protein and the gene encoding same in the regulation of the fungal disease resistance of plants and the regulation of the stalk strength of plants. Reducing the content of the ZmGAE1 protein or a protein having at least 95% sequence identity to the ZmGAE1 protein in plants or inhibiting the expression of the nucleic acid molecule encoding the ZmGAE1 protein in plants can increase the cell wall thickness and the main component content of the cell wall, thereby increasing the physical resistance of plants to pathogenic fungi, achieving the effects of improving the fungal disease resistance of plants and improving the stalk strength of plants, improving the lodging resistance of plants, and stabilizing the yield of plants.

Description

Application of ZmGAE1 protein and its encoding gene in regulating plant resistance to fungal diseases and plant stem strength

[0001] This application claims priority to Chinese Patent Application No. CN202410496475.8, filed on April 24, 2024, entitled "Application of ZmGAE1 protein and its encoding gene in regulating plant resistance to fungal diseases and plant stem strength", the entire contents of which are incorporated herein by reference. Technical Field

[0002] This application belongs to the field of plant genetic engineering and molecular breeding technology, specifically involving the application of ZmGAE1 protein and its encoding gene in regulating plant resistance to fungal diseases and plant stem strength. Background Technology

[0003] Corn is a crop used for food, feed, and economic purposes, and is the world's largest food crop. However, corn production is frequently affected by diseases and lodging, leading to significant yield reductions. Improving corn's resistance to diseases and lodging can stabilize its yield and is of great strategic importance to ensuring my country's food security.

[0004] Ear rot, stalk rot, and leaf spot are important fungal diseases affecting the ear, stem, and leaves of maize, respectively. Ear rot causes ear decay and produces large amounts of toxins harmful to humans and animals. Stalk rot causes green wilt in maize, and in severe cases, can lead to total crop failure. Leaf spot damages leaf tissue, severely impacting photosynthetic efficiency and final yield. Lodging is one of the most serious abiotic stresses facing maize and is often a major factor in yield reduction. As the increase in maize yield per plant has gradually reached a bottleneck, increasing planting density has become the main measure to increase maize yield per unit area. However, there is a significant positive correlation between increased planting density and the occurrence of maize lodging, with the latter severely limiting the possibility of increasing planting density. Generally speaking, there are two methods to improve maize's resistance to lodging: reducing plant height and increasing stalk strength.

[0005] Molecular breeding is currently an important approach to maize genetic improvement, and cloning genes controlling target traits is a prerequisite for obtaining new varieties with ideal target traits through molecular breeding techniques. Therefore, cloning genes for resistance to maize ear rot, stalk rot, small leaf spot, and lodging can provide new genes for the breeding of stable-yielding and green maize varieties, playing an important role in the genetic improvement of stable yield and high-density planting in maize. However, there are currently no reports on the application of the ZmGAE1 gene in regulating maize fungal diseases and lodging resistance in existing technologies. Summary of the Invention

[0006] The purpose of this application is to provide the use of the ZmGAE1 protein and its encoding gene in regulating plant resistance to fungal diseases and plant stem strength, and to provide technical support for plant molecular breeding and improvement of genetic traits.

[0007] This application provides the use of ZmGAE1 protein or a protein having at least 95% sequence identity with ZmGAE1 protein in regulating plant resistance to fungal diseases and / or regulating plant stem strength, wherein the amino acid sequence of ZmGAE1 protein is shown in SEQ ID NO.1.

[0008] Preferably, the fungal disease resistance and / or stem strength of the plant are improved by reducing the content of ZmGAE1 protein or a protein with at least 95% sequence identity with ZmGAE1 protein in the plant.

[0009] This application also provides the application of ZmGAE1 protein-related biomaterials in improving plant resistance to fungal diseases and / or increasing plant stem strength, wherein the biomaterials are at least one of A1-A15:

[0010] A1: A nucleic acid molecule encoding the ZmGAE1 protein or a protein having at least 95% sequence identity with the ZmGAE1 protein, wherein the amino acid sequence of the ZmGAE1 protein is shown in SEQ ID NO.1;

[0011] A2: A nucleotide sequence for inhibiting the expression of the nucleic acid molecule described in A1; A3: An expression cassette containing the nucleotide sequence described in A2; A4: A recombinant vector containing the nucleotide sequence described in A2; A5: A recombinant vector containing the expression cassette described in A3; A6: A recombinant microorganism containing the nucleotide sequence described in A2; A7: A recombinant microorganism containing the expression cassette described in A3; A8: A recombinant microorganism containing the recombinant vector described in A4; A9: A recombinant microorganism containing the recombinant vector described in A5; A10: A transgenic plant cell line containing the nucleotide sequence described in A2; A11: A transgenic plant cell line containing the expression cassette described in A3; A12: A transgenic plant tissue containing the nucleotide sequence described in A2; A13: A transgenic plant tissue containing the expression cassette described in A3; A14: A transgenic plant organ containing the nucleotide sequence described in A2; A15: A transgenic plant organ containing the expression cassette described in A3.

[0012] Preferably, the nucleotide sequence in A2 includes the nucleotide sequence of the nucleic acid molecule in A1 that has been knocked out; the knockout is performed using a CRISPR / Cas9 system, and the PAM sequence target for knocking out the nucleic acid molecule in A1 includes PAM1 or PAM2, the nucleotide sequences of PAM1 and PAM2 being shown in SEQ ID NO.3 and SEQ ID NO.4, respectively.

[0013] Preferably, the plant includes monocotyledonous or dicotyledonous plants; the fungal disease includes one or more of ear rot, stem rot, and small spot disease.

[0014] This application also provides a biomaterial, wherein the biomaterial is at least one of B1-B15:

[0015] B1: A nucleotide sequence for inhibiting the expression of a nucleic acid molecule encoding the ZmGAE1 protein or a protein having at least 95% sequence identity with the ZmGAE1 protein, wherein the amino acid sequence of the ZmGAE1 protein is shown in SEQ ID NO.1; B2: An expression cassette containing the nucleotide sequence described in B1; B3: A recombinant vector containing the nucleotide sequence described in B1; B4: A recombinant vector containing the expression cassette described in B2;

[0016] B5: Recombinant microorganism containing the nucleotide sequence described in B1; B6: Recombinant microorganism containing the expression cassette described in B2; B7: Recombinant microorganism containing the recombinant vector described in B3; B8: Recombinant microorganism containing the recombinant vector described in B4; B9: Transgenic plant cell line containing the nucleotide sequence described in B1; B10: Transgenic plant cell line containing the expression cassette described in B2; B11: Transgenic plant tissue containing the nucleotide sequence described in B1; B12: Transgenic plant tissue containing the expression cassette described in B2; B13: Transgenic plant organ containing the nucleotide sequence described in B1; B14: Transgenic plant organ containing the expression cassette described in B2.

[0017] This application also provides a method for cultivating plants with enhanced resistance to fungal diseases and / or enhanced stem strength, comprising the following steps: reducing the content of ZmGAE1 protein or a protein having at least 95% sequence identity with ZmGAE1 protein in the target plant or inhibiting the expression of the ZmGAE1 protein encoding gene in the target plant, thereby obtaining a plant with enhanced resistance to fungal diseases and / or enhanced stem strength compared to the target plant; the amino acid sequence of the ZmGAE1 protein is shown in SEQ ID NO.1.

[0018] This application also provides a molecular marker InDel-884, which has an insertion polymorphism as shown in SEQ ID NO.5 at -884bp of the promoter of the ZmGAE1 gene; the nucleotide sequence of the ZmGAE1 gene is shown in SEQ ID NO.2.

[0019] This application also provides primer pairs for amplifying the molecular marker InDel-884 described in the above technical solution, characterized in that the nucleotide sequences of the upstream primer and the downstream primer of the primer pair are shown in SEQ ID NO.6 and SEQ ID NO.7, respectively.

[0020] This application also provides the application of the molecular marker InDel-884 or the primer pairs described in the above-mentioned technical solutions in plant molecular breeding.

[0021] Preferably, the plant molecular breeding includes identifying or assisting in the identification of plant fungal disease resistance and / or stem strength traits.

[0022] This application also provides a method for identifying fungal disease resistance and / or stem strength traits in plants, comprising the following steps:

[0023] The genomic DNA of the plant to be tested was amplified by PCR using the primer pairs described in the above technical solution to obtain PCR amplification products;

[0024] The PCR amplification products were detected by electrophoresis to obtain PCR amplification bands.

[0025] When the PCR amplification band is a single 371 bp band, the plant to be tested is InDel-884. + Homozygous haplotype, resistant to fungal diseases and / or resistant to lodging;

[0026] When the PCR amplification bands are a 371bp band and a 230bp band, the test plant is InDel-884. ± Heterozygous haplotype, resistant to fungal diseases and / or lodging-resistant plants;

[0027] When the PCR amplification band is a single 230 bp band, the plant to be tested is InDel-884. - Homozygous haplotype, susceptible to fungal diseases and / or prone to lodging. Beneficial effects:

[0028] This application provides the application of ZmGAE1 protein or a protein with at least 95% sequence identity to ZmGAE1 protein, and nucleic acid molecules encoding ZmGAE1 protein, in regulating plant resistance to fungal diseases and / or regulating plant stem strength. The amino acid sequence of the ZmGAE1 protein is shown in SEQ ID NO.1. By reducing the content of ZmGAE1 protein or a protein with at least 95% sequence identity to ZmGAE1 protein in plants, or by inhibiting the expression of nucleic acid molecules encoding ZmGAE1 protein in plants, cell wall thickness and the content of major cell wall components can be increased, thereby increasing the physical resistance of plants to pathogenic fungi, achieving the effects of improving plant resistance to fungal diseases and increasing plant stem strength, thereby improving the plant's lodging resistance and stabilizing plant yield. Attached Figure Description

[0029] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0030] Figure 1: Comparison of ear rot resistance between maize inbred lines BT-1 and N6 in Example 1;

[0031] Figure 2: Key insertion / deletion sites affecting ZmGAE1 gene resistance provided in Example 1;

[0032] Figure 3: Differences in ear rot resistance between the two allele inbred lines of ZmGAE1 in Example 3;

[0033] Figure 4: Comparison of ear rot-related indicators between non-transgenic and overexpression lines in Example 1;

[0034] Figure 5: Comparison of the number of grains and the area affected by ear rot in non-transgenic lines and overexpression lines in Example 1;

[0035] Figure 6: Comparison of the structure and sequence of the ZmGAE1 mutant allele and the normal allele in Example 2;

[0036] Figure 7: Comparison of indicators related to ear rot between wild-type lines and ZmGAE1 mutants in Example 2;

[0037] Figure 8: Comparison of the number of grains and the area affected by ear rot in wild-type lines and ZmGAE1 mutant in Example 2;

[0038] Figure 9: Cell wall stained sections of the rachis connection region between the wild-type line and the ZmGAE1 mutant in Example 2;

[0039] Figure 10: Comparison of stem rot resistance between wild-type lines and ZmGAE1 mutants in Example 2;

[0040] Figure 11: Cross section of stem and cell wall thickness measurement of wild-type line and ZmGAE1 mutant in Example 2;

[0041] Figure 12: Comparison of agronomic traits between wild-type lines and ZmGAE1 mutants in Example 2;

[0042] Figure 13: Gene structure changes in wild-type and ZmGAE1 knockout lines in Example 3;

[0043] Figure 14: Comparison of the disease phenotypes of wild-type strains and ZmGAE1 knockout strains in Example 3;

[0044] Figure 15: Comparison of small spot disease phenotypes between wild-type strains and ZmGAE1 knockout strains in Example 3;

[0045] Figure 16: Comparison of plant type and grain type between wild-type line and ZmGAE1 knockout line in Example 3;

[0046] Figure 17: Proportion of ZmGAE1 resistant genotypes in different germplasm resources in Example 4;

[0047] Figure 18: Agarose gel image of InDel-884 haplotype in Example 4;

[0048] Figure 19: Comparison of ear rot resistance between heterozygous and homozygous susceptible varieties of ZmGAE1 in Example 4;

[0049] Figure 20: Marker identification results of ZmGAE1 genotype in 613 hybrids in Example 4;

[0050] Figure 21: Alignment results of the publicly available ZmGAE1 gene sequence in Example 5;

[0051] Figure 22: Alignment results of the publicly disclosed sequence of ZmGAE1 protein in Example 5;

[0052] Figure 23: Map of pTF101.1-MYC plasmid vector in Example 1. Detailed Implementation

[0053] This application provides the use of ZmGAE1 protein or a protein having at least 95% sequence identity with ZmGAE1 protein in regulating plant resistance to fungal diseases and / or regulating plant stem strength, wherein the amino acid sequence of ZmGAE1 protein is shown in SEQ ID NO.1.

[0054]

[0055] This application also provides the application of biomaterials related to the ZmGAE1 protein in improving plant resistance to fungal diseases and / or increasing plant stem strength, wherein the biomaterials are at least one of A1-A15: A1: a nucleic acid molecule encoding the ZmGAE1 protein or a protein having at least 95% sequence identity with the ZmGAE1 protein, wherein the amino acid sequence of the ZmGAE1 protein is as shown in SEQ ID. As shown in NO.1; A2: Nucleotide sequence for inhibiting the expression of the nucleic acid molecule encoding the ZmGAE1 protein described in A1; A3: Expression cassette containing the nucleotide sequence described in A2; A4: Recombinant vector containing the nucleotide sequence described in A2; A5: Recombinant vector containing the expression cassette described in A3; A6: Recombinant microorganism containing the nucleotide sequence described in A2; A7: Recombinant microorganism containing the expression cassette described in A3; A8: Recombinant microorganism containing the recombinant vector described in A4; A9: Recombinant microorganism containing the recombinant vector described in A5; A10: Transgenic plant cell line containing the nucleotide sequence described in A2; A11: Transgenic plant cell line containing the expression cassette described in A3; A12: Transgenic plant tissue containing the nucleotide sequence described in A2; A13: Transgenic plant tissue containing the expression cassette described in A3; A14: Transgenic plant organ containing the nucleotide sequence described in A2; A15: Transgenic plant organ containing the expression cassette described in A3.

[0056]

[0057] The term "sequence identity" as used herein refers to the degree to which two optimally aligned polynucleotide sequences or two optimally aligned polypeptide sequences are identical. Optimal sequence alignment is established by manually aligning two sequences, for example, a reference sequence and another DNA sequence, to maximize the number of nucleotide matches in sequence alignments with appropriate internal nucleotide insertions, deletions, or gaps. As used herein, the term "reference sequence" refers to the amino acid sequence shown in SEQ ID NO. 1 or the nucleic acid sequence shown in SEQ ID NO. 2 and positions 191-2407 of SEQ ID NO. 2.

[0058] The “% sequence identity” or “% identity” mentioned in this application is an identity score multiplied by 100. The “percentage of identity” of the sequence best aligned with the reference sequence is the number of nucleotide matches in the best alignment divided by the total number of nucleotides in the reference sequence, for example, the total number of nucleotides in the entire length of the reference sequence. Therefore, this application provides a DNA molecule containing a sequence that, when best aligned with the reference sequence, i.e., the amino acid sequence shown in SEQ ID NO.1 or the nucleic acid sequence shown in SEQ ID NO.2 or positions 191-2407, has at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identity with the reference sequence.

[0059] The nucleotide molecule described in this application preferably also includes variant sequences derived from the ZmGAE1 gene through mutations involving deletions, substitutions, insertions, or additions of one or more nucleotides, wherein the variant sequences retain the regulatory activity of the ZmGAE1 gene. Gene mutation refers to a sudden, heritable variation in genomic DNA molecules. At the molecular level, gene mutation refers to a change in the base pair composition or sequence of a gene. Gene mutations can occur spontaneously or induced. Artificial mutagenesis includes physical mutagenesis (such as gamma rays, X-rays, ultraviolet light, and neutron flux), chemical mutagenesis (such as alkylating agents, base analogs, and antibiotics), and biological mutagenesis (such as certain viruses and bacteria). Furthermore, recombinant DNA technology can be used to induce specific changes at designated locations in DNA molecules, thereby achieving targeted mutagenesis. Those skilled in the art can use any of these known mutagenesis methods to obtain variant sequences of the ZmGAE1 gene containing mutations involving deletions, substitutions, insertions, or additions of one or more nucleotides.

[0060] In this application, the term "strict conditions" generally refers to the conditions described by Sambrook et al., 1989, and by Haymes et al. in: Nucleic acid hybridization, A practical approach, IRO Press, Washington, DC (1985). Suitable strict conditions for DNA hybridization (e.g., 6.0 × sodium chloride / sodium citrate (SSC) at approximately 45°C, followed by a 2.0 × SSC wash at 50°C) are known to those skilled in the art or can be found in Current Protocols in Molecular Biology, John Wiley & Sons, NY, 1989, 6.3.1–6.3.6. For example, the salt concentration in the washing step can range from a low strict condition of approximately 2.0 × SSC at 50°C to a high strict condition of approximately 0.2 × SSC at 50°C. Additionally, the temperature in the washing step can be increased from a low strict condition of room temperature (approximately 22°C) to a high strict condition of approximately 65°C. Both temperature and salt concentration can be varied, or one of the temperature or salt concentration can be kept constant while the other variable changes. For example, moderately stringent conditions can be a salt concentration of approximately 2.0 × SS and a temperature of approximately 65°C, while highly stringent conditions can be a salt concentration of approximately 0.2 × SSC and a temperature of approximately 65°C.

[0061] In this application, "fungal disease resistance" refers to a quantitative trait measuring a plant's resistance to diseases induced by pathogenic fungi. Depending on the pathogenic fungus and the infected tissue, fungal disease resistance manifests differently in different plants. In maize, fungal disease resistance preferably includes one or more of ear rot resistance, stem rot resistance, and small leaf spot resistance, with ear rot resistance being more preferred. In this application, the molecular mechanism by which the ZmGAE1 protein and its encoding gene exert fungal disease resistance involves altering the composition and thickness of the plant cell wall, and this resistance mechanism is common. Therefore, those skilled in the art can reasonably expect that the ZmGAE1 gene of this application can regulate not only "fungal disease resistance" in maize but also "fungal disease resistance" in other plants.

[0062] Based on the above explanation of the molecular mechanisms, the plants described in this application preferably include monocotyledonous or dicotyledonous plants, and more preferably include maize, soybean, cotton, peanut, barley, oat, wild grass, rice, sorghum, sugarcane, tall fescue, turfgrass, wheat, alfalfa, or Arabidopsis thaliana. The fungal diseases described in this application preferably include one or more of ear rot, stem rot, and small leaf spot, more preferably ear rot, stem rot, and small leaf spot.

[0063] The nucleotide sequence described in this application for inhibiting the expression of the nucleic acid molecule encoding the ZmGAE1 protein includes the nucleotide sequence for knocking out the nucleic acid molecule. This application preferably uses a CRISPR / Cas9 system for the knockout, and the PAM sequence target for knocking out the nucleic acid molecule preferably includes PAM1 or PAM2. The nucleotide sequences of PAM1 and PAM2 are shown in SEQ ID NO.3 and SEQ ID NO.4, respectively. Specifically, the nucleotide sequences shown in SEQ ID NO.3 and SEQ ID NO.4 are 5'-CCAGCACCATGTTCCTGTGGG-3' and 5'-TACGGACTCTCCATCACCGG-3', respectively.

[0064] The expression cassette containing a nucleotide sequence that inhibits the expression of a nucleic acid molecule encoding the ZmGAE1 protein, as described in this application, preferably refers to DNA capable of reducing or inhibiting the expression of the ZmGAE1 protein in a host cell; the DNA preferably includes a promoter and / or a terminator; furthermore, the expression cassette preferably also includes an enhancer sequence.

[0065] The recombinant expression vector described in this application preferably contains a nucleic acid molecule that knocks out the ZmGAE1 gene; the initial vector of the recombinant expression vector preferably includes a Cas9 plasmid. This application does not specify the construction steps of the recombinant expression vector, and can be performed according to conventional operating procedures in the art.

[0066] The initial microorganism in the recombinant microorganisms described in this application preferably includes Agrobacterium.

[0067] The transgenic plant cell lines, transgenic plant tissues, and transgenic plant organs described in this application do not include propagation material. The transgenic plant cell lines, transgenic plant tissues, and transgenic plant organs described in this application are preferably obtained by transferring the recombinant microorganisms into the target plant. This application does not specify a particular method for transferring the recombinant microorganisms into the target plant; conventional transgenic methods in the art are acceptable.

[0068] This application also provides a biological material, wherein the biological material is at least one of B1-B15: B1: a nucleotide sequence for inhibiting the expression of a nucleic acid molecule encoding a ZmGAE1 protein or a protein having at least 95% sequence identity with the ZmGAE1 protein, wherein the amino acid sequence of the ZmGAE1 protein is as shown in SEQ ID. As shown in NO.1; B2: Expression cassette containing the nucleotide sequence described in B1; B3: Recombinant vector containing the nucleotide sequence described in B1; B4: Recombinant vector containing the expression cassette described in B2; B5: Recombinant microorganism containing the nucleotide sequence described in B1; B6: Recombinant microorganism containing the expression cassette described in B2; B7: Recombinant microorganism containing the recombinant vector described in B3; B8: Recombinant microorganism containing the recombinant vector described in B4; B9: Transgenic plant cell line containing the nucleotide sequence described in B1; B10: Transgenic plant cell line containing the expression cassette described in B2; B11: Transgenic plant tissue containing the nucleotide sequence described in B1; B12: Transgenic plant tissue containing the expression cassette described in B2; B13: Transgenic plant organ containing the nucleotide sequence described in B1; B14: Transgenic plant organ containing the expression cassette described in B2.

[0069] The nucleic acid molecule described in this application is preferably selected from any one of the nucleic acid sequences B1-1 to B1-3: B1-1: the nucleic acid sequence shown in SEQ ID NO.2, or its complementary sequence; B1-2: a nucleic acid sequence having at least 95% sequence identity with the nucleic acid sequence shown in positions 191-2407 of SEQ ID NO.2, or its complementary sequence; B1-3: a nucleic acid sequence that hybridizes to the nucleic acid sequence shown in SEQ ID NO.2 or its positions 191-2407 under stringent conditions, or its complementary sequence. The expression of the ZmGAE1 gene described in this application is negatively correlated with fungal disease resistance and stem strength. Therefore, by inhibiting the expression of the ZmGAE1 gene, plants with increased fungal disease resistance and stem strength can be obtained, thereby increasing yield stability.

[0070] Based on the above molecular mechanism, this application also provides a method for cultivating plants with enhanced resistance to fungal diseases and / or enhanced stem strength, comprising the following steps: reducing the content of ZmGAE1 protein or a protein having at least 95% sequence identity with ZmGAE1 protein in the target plant or inhibiting the expression of the ZmGAE1 protein encoding gene in the target plant, thereby obtaining a plant with enhanced resistance to fungal diseases and / or enhanced stem strength compared to the target plant; the amino acid sequence of the ZmGAE1 protein is shown in SEQ ID NO.1. The methods described in this application for inhibiting the expression of the ZmGAE1 protein encoding gene in the target plant or reducing the content of ZmGAE1 protein or a protein having at least 95% sequence identity with ZmGAE1 protein in the target plant are preferably including, but not limited to, transposon insertion, mutagenesis, RNA-mediated inhibition, or gene editing, further preferably gene editing, and more preferably gene knockout.

[0071] In addition, after initially locating ZmGAE1, this application developed new molecular markers that are closely linked to ZmGAE1 and corresponding primers. This is useful for screening maize fungal disease resistance and stem strength traits, and provides technical support for further fine-maize positioning of ZmGAE1 and marker-assisted selection breeding, thereby accelerating the process of high-yield maize breeding.

[0072] This application also provides a molecular marker InDel-884, which exhibits an insertion polymorphism as shown in SEQ ID NO. 5 at -884 bp of the promoter of the ZmGAE1 gene; the nucleotide sequence of the ZmGAE1 gene is shown in SEQ ID NO. 2. Specifically, the nucleotide sequence shown in SEQ ID NO. 5 of this application is: 5'-GAGGGCGTGTTCGGTTGGCTGCAAGCCGACACTGTTGCAGCTGTTTGGACTGCTGCAGCTGCAATCCATAGAGAGAAAAATACTGTAGAAGCCGCAGCCGCAGCCGGATTGCAGCCGCAGCAAGCCGCAGCGAACAAGCTG-3'. The molecular marker InDel-884 of this application is located in the chromosomal region 174809823-174810049 of the B73 V3 reference genome.

[0073] This application also provides primer pairs for amplifying the molecular marker InDel-884 described in the above technical solution. The nucleotide sequences of the upstream and downstream primers of the primer pairs are shown in SEQ ID NO. 6 and SEQ ID NO. 7, respectively. The nucleotide sequences shown in SEQ ID NO. 6 and SEQ ID NO. 7 of this application are 5'-GCAAAGTAATTTTTATAGAAACGC-3' and 5'-TTCCATGTTCAGATTGTCGAT-3', respectively.

[0074] This application also provides the application of the molecular marker InDel-884 or the primer pair described in the above technical solutions in plant molecular breeding, preferably in the identification or auxiliary identification of plant fungal disease resistance and / or stem strength traits, more preferably in the identification or auxiliary identification of plant fungal disease resistance and stem strength traits.

[0075] Specifically, this application also provides a method for identifying fungal disease resistance and / or stem strength traits in plants, comprising the following steps:

[0076] The genomic DNA of the plant to be tested is amplified by PCR using the primer pairs described in the above technical solution to obtain PCR amplification products; the PCR amplification products are then detected by electrophoresis to obtain PCR amplification bands; when the PCR amplification band is a single 371 bp band, the genotype of the plant to be tested is InDel-884. + A homozygous haplotype indicates a plant resistant to fungal diseases and / or lodging; when the PCR amplification bands are a 371 bp band and a 230 bp band, the genotype of the tested plant is InDel-884. ± A heterozygous haplotype indicates a plant resistant to fungal diseases and / or lodging; when the PCR amplification band is a single 203 bp band, the genotype of the tested plant is InDel-884. - The homozygous haplotype is a plant susceptible to fungal diseases and / or prone to lodging. The method for extracting genomic DNA described in this application is not particularly limited; any conventional genomic DNA extraction method in the art may be used. The PCR amplification system and procedure described in this application are not particularly limited; any conventional PCR amplification system and procedure in the art may be used. The electrophoretic detection described in this application preferably includes agarose gel electrophoresis.

[0077] To further illustrate this application, the technical solutions provided by this application will be described in detail below with reference to the accompanying drawings and embodiments, but they should not be construed as limiting the scope of protection of this application.

[0078] Example 1

[0079] Cloning of the maize resistance gene ZmGAE1

[0080] 1. Identification of the ZmGAE1 gene using pooled sequencing of extremely resistant and susceptible RIL populations to ear rot.

[0081] A Reactive Inbred Line (RIL) population was constructed using the resistant inbred line BT-1 and the susceptible inbred line N6 (the RIL population has been published in the following reference: Wu Y, Zhou Z, Dong C, et al. Linkage mapping and genome-wide association study reveals conservative QTL and candidate genes for Fusarium rot resistance in maize[J]. BMC Genomics, 2020, 21(1):357.). The resistance of BT-1 and N6 to ear rot is shown in Figure 1 (the left figure represents BT-1, and the right figure represents N6). Through joint analysis of ear rot inoculation identification data from four environments (summer sowing in Zhengzhou in 2007, summer sowing in Zhengzhou in 2008, summer sowing in Wenxian County in 2015, and summer sowing in Xuchang in 2016), 25 highly resistant lines and 25 highly susceptible lines were identified. Whole-genome resequencing was performed on the mixed DNA pools of highly resistant and highly susceptible strains, revealing 4,317,573 single nucleotide polymorphisms (SNPs) and 445,777 insertion / deletion (InDels) variants. At a 95% significance level, the ΔSNP-index value showed an extreme peak on chromosome 4, located between 172.9 and 174.9 Mb. Within this region, two important SNPs fell precisely into the promoter region of the GRMZM2G042179 gene. This gene encodes UDP-D-glucuronide 4-epiisomerase 1 (GAE1), which is involved in galacturonic acid synthesis in the cell wall; therefore, this gene was named ZmGAE1.

[0082] 2. Identification of key functional variant sites in the ZmGAE1 gene

[0083] The ZmGAE1 gene regions on BT-1 and N6 were amplified and sequenced, including a 1218 bp promoter region and a 1323 bp open reading frame (ORF) (SEQ ID NO. 8). Sequence analysis revealed that the ORF regions were identical between the two materials, while a 141 bp insertion / deletion site (InDel-884) was found 884 bp upstream of the start codon (see Figure 2). Subsequently, InDel-884 was validated in four randomly selected susceptible inbred lines and four resistant inbred lines. InDel-884 may be the molecular basis for ZmGAE1's influence on ear rot resistance.

[0084] This application developed a marker containing InDel-884. The upstream and downstream primer pairs used to amplify this marker are 5'-GCAAAGTAATTTTTATAGAAACGC-3' (SEQ ID NO.6) and 5'-TTCCATGTTCAGATTGTCGAT-3' (SEQ ID NO.7), respectively. This marker was used to detect RIL populations. The specific detection steps were as follows: PCR amplification of 155 RIL strains was performed using the above primer pairs. The product length was detected by electrophoresis, and different strains were divided into InDel strains with bands consistent (length) with the resistant parent. + Type, and InDel consistent with the infected parent (short). - type.

[0085] Using a RIL population of 155 strains, combined with phenotypic data of ear rot from four environments (summer sowing in Zhengzhou in 2007, 2008, Wenxian in 2015, and Xuchang in 2016), a significant correlation was found between Indel-884 and ear rot resistance, which can explain 16.6%–29.6% of the variation in ear rot resistance phenotype.

[0086] Meanwhile, this application analyzed the InDel-884 promoter fragment corresponding to ZmGAE1 in 164 maize inbred lines. Genome-wide association analysis (GWAS) using a mixed linear model (MLM) incorporating population structure revealed a significant correlation between InDel-884 and resistance to front rot (FER) (P = 6.59 × 10⁻⁶). -5 This explains 9.7% of the phenotypic variation in ear rot resistance. Based on the haplotype of InDel-884, the 164 inbred lines were divided into InDel... + and InDel - Two haplotype groups. (Similar to InDel) - Compared to the group, InDel + The group showed significantly improved resistance to ear rot (see Figure 3).

[0087] 3. Verify the disease susceptibility function of ZmGAE1 using overexpression transgenic lines.

[0088] Two independent transgenic lines, OE1 and OE2, overexpressing ZmGAE1 were obtained in the context of maize inbred line B104, with a significant increase in the transcription level of ZmGAE1 in them.

[0089] The transgenic lines overexpressing ZmGAE1 were obtained as follows:

[0090] The coding region of the target gene was ligated to the pTF101.1-MYC plasmid vector (a gift from Professor Li Tao of the College of Life Sciences, Henan Agricultural University) using homologous recombination, resulting in a recombinant expression vector driven by the Ubi promoter. The upstream and downstream sequences of the primer pair used to amplify the target gene were 5'-cgactctagaggatccATGCGGGTGCTGGAGGAGGA-3' (SEQ ID NO. 9) and 5'-cagatcctccactagtATGTCGCGAGTTCTTGG-3' (SEQ ID NO. 10), where lowercase letters represent homologous arms of the vector sequence and uppercase letters represent matching sequences with the target gene sequence. The obtained recombinant expression vector was transformed into the maize inbred line B104 using Agrobacterium-mediated transformation, resulting in transgenic lines OE1 and OE2 overexpressing ZmGAE1.

[0091] Two transgenic maize lines exhibited more severe ear rot symptoms than non-transgenic (NT1) plants across multiple indicators. The abundance of pathogens in the kernels increased by 3.14–7.21 times, the severity of ear rot increased by 1.16–2.33 times, the fumonisin content increased by 3.73–5.79 times, and the number of diseased kernels and the area of ​​ear rot were significantly increased (see Figures 4 and 5). These results demonstrate that ZmGAE1 is a susceptibility gene for ear rot in maize.

[0092] Example 2

[0093] Enhancing the overall resistance of maize using the zmgae1 mutant

[0094] 1. The zmgae1 mutant enhances maize's resistance to ear rot.

[0095] The EMS-induced ZmGAE1 mutant allele from the inbred line B73 background was obtained from the MEMD database (http: / / www.elabcaas.cn / memd / , mutantID:EMS3-04bde5). This allele produced a stop codon mutation in the ORF of zmgae1, causing premature termination of its protein translation (see Figure 6). To ensure sufficient material, the mutant was self-crossed for two generations. Subsequently, based on the genotype of zmgae1, three homozygous mutant lines (zmgae1-1, zmgae1-2, and zmgae1-3) and one homozygous non-mutant line were isolated as wild-type (WT) controls for identification of ear rot resistance.

[0096] To accurately identify its resistance, five criteria were used for multi-environment identification (summer sowing in Hebi in 2021 and 2022), including the number of diseased grains per ear, ear rot area, ear rot grade, fumonisin content, and relative biomass of pathogens.

[0097] The results showed that, compared with the wild type, the mutant line exhibited stronger resistance to ear rot and toxin accumulation in all five indicators. Pathogen abundance was reduced by 64%–76%, ear rot severity was reduced by 1.45–2.21, fumonisin content was reduced by 53%–89%, and the number of diseased grains and the area of ​​ear rot were significantly reduced (see Figures 7 and 8).

[0098] 2. The zmgae1 mutant increases cell wall thickness and the content of major cell wall components.

[0099] Histological sections were prepared from the upper (UPC) and lower (DPC) regions of maize seeds and ears from the three homozygous mutant lines and the wild-type control in step 1. Cell wall components were then stained and observed. The mutant seeds showed significantly higher cell wall density in these two regions compared to the wild type (see Figure 9). Further analysis of the enzyme activity of UDP-D-gluconate-4-epiisomerase and the contents of galacturonic acid, pectin, hemicellulose, and cellulose in the whole seed revealed significantly decreased enzyme activity in all mutant lines, while the contents of galacturonic acid, pectin, hemicellulose, and cellulose all showed an increasing trend. Therefore, the zmgae1 mutant can enhance the physical resistance of maize to ear rot pathogens by increasing the content and thickness of maize cell wall components.

[0100] 3. The zmgae1 mutant enhances maize's resistance to stalk rot and seed rot.

[0101] Resistance to maize stalk rot was assessed in all zmgae1 mutant lines in three environments (summer-sown maize in Hebi in 2021, summer-sown maize in Zhengzhou in 2022, and summer-sown maize in Hebi in 2022). Fifteen days after pollination, maize stalks were inoculated with *Fusarium verticillatum* spores and *Fusarium graminearum* using a toothpick method. Twenty-five days after inoculation, the stalks were longitudinally sectioned, and the number of rotten nodes (the ratio of rotten node length to node length) was quantified. The zmgae1 mutation was found to enhance maize resistance to stalk rot (see Figure 10). For example, in the inoculation test in the 2021 Hebi mutant line, the number of *Fusarium graminearum*-infected stalk nodes was reduced by 0.41–0.57 compared to the wild type. Furthermore, mature seeds of all mutant lines showed higher resistance to *Fusarium verticillatum* compared to the wild type. These results indicate that the zmgae1 mutant can induce broad-spectrum resistance in maize.

[0102] 4. The zmgae1 mutant enhances the stalk strength of maize.

[0103] Compared with the wild type, the stem strength of the mutant lines was significantly increased. The stem bending strength of the mutant lines increased by 22-28%, and the stem penetration resistance increased by 25-30%. The vascular tissue of the mutant and wild types was observed using scanning electron microscopy, and it was found that the thickness of the vascular bundle sheath cell wall was significantly increased in the mutant lines (see Figure 11).

[0104] 5. Effects of the zmgae1 mutant on conventional agronomic traits

[0105] Agronomic trait surveys of the mutant line showed no significant changes in key plant morphology traits (plant height, ear height, number of nodes, internode distance, and tassel length) and key ear traits (number of rows per ear, weight per few hundred grains per row). However, the grain bulk density increased by 10-11% compared to the wild type, and the number of tassel branches increased slightly (see Figure 12). The results indicate that the ZmGAE1 mutant has no negative impact on major agronomic traits.

[0106] Example 3

[0107] Gene editing to knock out ZmGAE1 enhances the overall resistance of maize.

[0108] 1. ZmGAE1 knockout lines show enhanced resistance to ear rot.

[0109] A knockout line (KO1) was generated using CRISPR / Cas9 in the background of inbred line B104, and sequencing confirmed that it had a 691bp deletion of ZmGAE1 (see Figure 13).

[0110] Gene editing was performed on two PAM sequence targets of the gene to obtain mutations that delete partial gene segments: PAM1: 5'-CCAGCACCATGTTCCTGTGGG-3' (SEQ ID NO.3) and PAM2: 5'-TACGGACTCTCCATCACCGG-3' (SEQ ID NO.4).

[0111] Targets were screened using the online target screening software "CRISPR-PLANT (https: / / www.genome.arizona.edu / crispr / CRISPRsearch.html)," with the best targets containing restriction enzyme sites (Cas9 cleavage site -- 3bp away from PAM / NGG -- located within the restriction enzyme site). Off-target effects were assessed using the online software "Cas-OFFinder" (http: / / www.rgenome.net / cas-offinder / ). Primers ZmGAE1-MT1T2-F / R were designed with reference to (Xing, Hui-Li, et al. "A CRISPR / Cas9 toolkit for multiplex genome editing in plants." BMC plant biology 14(2014):1-12.), F: 5'-AATAATGGTCTCAGGCGACCAGCACCATGTTCCTGT-3' (SEQ ID NO.11), R: 5'-ATTATTGGTCTCTAAACGTGATGGAGAGTCCGTAGA-3' (SEQ ID NO.11). NO.12); ZmGAE1-MT1T2-F0 / R0, F0: 5'-GACCAGCACCATGTTCCTGTGTTTTAGAGCTAGAAATAGC-3' (SEQ ID NO.13) R0: 5'-GTGATGGAGAGTCCGTAGACGCTTCTTGGTGCC-3' (SEQ ID NO.14).

[0112] PCR amplification was performed using four primers with pCBC-MT1T2 plasmid DNA diluted 100-fold (Xing, Hui-Li, et al. "A CRISPR / Cas9 toolkit for multiplex genome editing in plants." BMC plant biology 14(2014):1-12.) diluted 100-fold as a template. Primers ZmGAE1-MT1T2-F / R were used at their normal concentrations, while primers ZmGAE1-MT1T2-F0 / R0 were diluted 20-fold before use. After PCR amplification, all products were subjected to agarose gel electrophoresis and then recovered by gel extraction. Subsequently, enzyme digestion and ELISA were performed, and the enzyme digestion and ELISA products were transformed into DH5α Escherichia coli and plated.

[0113] Positive colonies were detected by colony PCR using primers OsU3-FD3: 5'-GACAGGCGTCTTCTACTGGTGCTAC-3' (SEQ ID NO.15) and TaU3-RD: 5'-CTCACAAATTATCAGCACGCTAGTC-3' (SEQ ID NO.16). Positive colonies were selected, cultured, and plasmids were extracted and sequenced. The correctly sequenced plasmids were transformed into Agrobacterium GV3101, and colony PCR was performed again to detect positive colonies, followed by culture.

[0114] The positive bacteria were transferred into the inbred line B104 using the Agrobacterium-mediated transformation method to obtain the knockout line KO1.

[0115] Compared with the non-transgenic line (NT2), the resistance phenotype to ear rot was significantly enhanced in all five indicators (number of diseased grains per ear, ear rot area, ear rot grade, fumonisin content, and relative biomass of pathogen), similar to the mutant line (see Figure 14). Pathogen abundance decreased by 46%–54%, ear rot grade decreased by 0.82–1.77, fumonisin content decreased significantly by 71%, and both the number of diseased grains and the ear rot area decreased.

[0116] 2. ZmGAE1 knockout lines exhibit enhanced resistance to stem rot and leaf spot.

[0117] Resistance to maize stalk rot was assessed in all ZmGAE1 knockout lines in two environments (2022 Zhengzhou and 2023 Zhengzhou summer sowing). Fifteen days after pollination, maize stalks were inoculated with *Fusarium verticillatum* spores and *Fusarium graminearum* using a toothpick method, respectively. Twenty-five days after inoculation, the stalks were longitudinally sectioned, and the number of rotten nodes (the ratio of rotten node length to node length) was quantified. Similar to the mutant, knocking out ZmGAE1 enhanced maize resistance to stalk rot. Furthermore, under natural field infection conditions, the knockout lines showed increased resistance to maize small leaf spot (SLB) compared to non-transgenic lines (see Figure 15). These results indicate that the ZmGAE1 mutant can induce broad-spectrum disease resistance in maize.

[0118] 4. Effects of ZmGAE1 gene editing knockout on conventional agronomic traits

[0119] A two-year agronomic trait survey of the knockout lines showed that, compared with the non-transgenic lines, the knockout lines had slightly increased plant height, ear height, number of stem nodes, and internode distance. For example, the plant height in Zhengzhou increased by 4.4 cm in 2022 (see Figure 16). However, other major agronomic traits (number of male branches, length of male ear, number of rows of ears, number of grains per row, 100-grain weight, etc.) had no significant agronomical negative impact.

[0120] Example 4

[0121] Identification of maize ear rot resistance genotypes using ZmGAE1 functional insertion / deletion molecular markers

[0122] 1. Analysis of the ZmGAE1 gene in maize germplasm resources using molecular markers.

[0123] The ZmGAE1 promoter fragments from 77 representative inbred lines, 19 *Tetrandrus davidii* lines, and 21 tropical maize species from the GWAS population were resequencing. These lines were then divided into two groups based on the promoter insertion / deletion marker "InDel-884". The results showed that "InDel-884" was absent in *Tetrandrus davidii*. + The proportion of the disease-resistant genotype in tropical endemic varieties was 5%, in Tangsipingtou inbred lines it was 8%, in Ruide inbred lines it was 10%, in Lancaster inbred lines it was 20%, and in P-group inbred lines it was as high as 44% (see Figure 17). This indicates that the ZmGAE1 gene can be selected with the help of molecular markers, and the resistance to ear rot in temperate inbred lines can be improved using tropical resistance sources such as the P-group.

[0124] 2. Identification of resistance genes for ear rot in mainstream maize varieties

[0125] The “InDel-884” marker and ear rot resistance of 47 major maize hybrids from the Huang-Huai-Hai region of China were identified using the following methods:

[0126] The genome of the maize to be tested was amplified by PCR using the primer pairs described in SEQ ID NO.6 and SEQ ID NO.7 in Example 1. The amplification products were detected by agarose gel electrophoresis, and the band lengths were divided into homozygous disease-resistant genotype (371bp), homozygous disease-susceptible genotype (230bp), and heterozygous genotype (370bp and 230bp), as shown in Figure 18.

[0127] The results showed that none of these mainstream varieties had a homozygous disease-resistant genotype of ZmGAE1, while the heterozygous genotype varieties had a 0.92-degree lower incidence of ear rot than the homozygous susceptible genotype varieties (see Figure 19).

[0128] Further analysis of 613 maize hybrids from China using this marker revealed that only 6.53% of the varieties carried the homozygous disease-resistant ZmGAE1 genotype (see Figure 20). Therefore, the use of the ZmGAE1 gene to improve the ear rot resistance of maize hybrids has broad applicability.

[0129] Example 5

[0130] The ORF sequence of the ZmGAE1 gene on the maize reference genome B73 was compared with other publicly available ZmGAE1 gene sequences. The comparison results are shown in Figure 21.

[0131] The ZmGAE1 protein on the maize reference genome B73 was compared with other publicly available ZmGAE1 protein sequences, and the comparison results are shown in Figure 22.

[0132] From Figures 21 and 22, it can be concluded that, excluding the mutant zmgae1 with the background of inbred line B73 and the knockout transgenic line KO1 with the background of inbred line B104 (KO in Figures 21 and 22), the sequences of ZmGAE1 on 33 sequenced maize inbred lines can be found in the MaizeGDB database (https: / / maizegdb.org / ), as shown in Table 1 (Gene model number in the MaizeGDB database, DNA and protein sequences are in the same number):

[0133] Table 1. Gene model numbers of inbred lines in the MaizeGDB database.

[0134]

[0135] The ORF sequence of the knockout transgenic line KO1 with a B104 background is: 5'-ATGCGGGTGCTGGAGGAGGACCTCTTCCCCTCCACCCCGGGCAAGGTGAAGATCGAGCGGGCGGGCGCCATGAACCGGCAGCTCCACCGCTGCTTCGCGTCCACCAGCACCATGTTCCCACCGGCCTCCGCTTCTTCACCGTGTACGGGCCCTGGGGCCGCCCCGACATGGCCTACTTCTCCTTCACCCGCAACATCCTTCAGGGGAAGCCCATCACGGTTTACCGCGGCAAGGACCACGTGGACCTGGCCCGCGACTTCACCTACATCGACGATATTGTCAAGGGCTGCCTCGCCTCCCTGGAAACGGCCGGCAAGAGCACCGGCACCGGCGGCAAGAAGCGCGGGCCGGCGCCCTACAGGATCTTCAACCTCGGCAACACCTCTCCCGTTACGGTGCCCAACCTGGTGTCCATCCTGGAGAAGCACCTCCGCGTCAAGGCCAAGAAGAACGTGGTCGAGATGCCCGGCAACGGCGACGTGCCCTTCACGCACGCCAACATCTCCCTAG-3' (SEQ ID NO.19); The amino acid sequence is: MRVLEEDLFPSTPGKVKIERAGAMNRQLHRCFASTSTMFPPASASSPCTGPGAAPTWPTSPSPATSFRGSPSRFTAARTTWTWPATSPTSTILSRAASPPWKRPARAPAPAARSAGRRPTGSSTSATPLPLRCPTWCPSWRSTSASRPRRTWSRCPATATCPSRTPTSP (SEQ ID NO.20).

[0136] Compared to the B73 and B104 inbred lines used in the experiment (both with identical sequences), the other inbred lines showed the lowest ORF sequence similarity at 99.62%. The similarity between the two types of protein sequences was 95.04%. Compared to the reference group, the mutant sequence had a protein sequence length shortened to 91%, while the knockout line had a shortened protein sequence length of 8%. This indicates that the known homology of maize GAE1 proteins in nature is above 95%, and a decrease in GAE1 protein homology to 91% can produce a mutant phenotype. Furthermore, this demonstrates that proteins with at least 95% sequence identity with ZmGAE1 protein can regulate plant resistance to fungal diseases and regulate plant stem strength.

[0137] Although the above embodiments have provided a detailed description of this application, they are only some embodiments of this application, not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of this application.

Claims

1. The application of ZmGAE1 protein or a protein having at least 95% sequence identity with ZmGAE1 protein in regulating plant resistance to fungal diseases and / or regulating plant stem strength, wherein the amino acid sequence of said ZmGAE1 protein is shown in SEQ ID NO.

1.

2. The application according to claim 1, characterized in that, The application is to improve the plant's resistance to fungal diseases and / or increase the plant's stem strength by reducing the content of ZmGAE1 protein or a protein with at least 95% sequence identity with ZmGAE1 protein in the plant.

3. The application according to claim 1 or 2, characterized in that, The nucleotide sequence of the gene encoding the ZmGAE1 protein is shown in SEQ ID NO.

2.

4. The application of ZmGAE1 protein-related biomaterials in improving plant resistance to fungal diseases and / or increasing plant stem strength, wherein the biomaterials are at least one of A1-A15: A1: A nucleic acid molecule encoding the ZmGAE1 protein or a protein having at least 95% sequence identity with the ZmGAE1 protein, wherein the amino acid sequence of the ZmGAE1 protein is shown in SEQ ID NO.1: A2: A nucleotide sequence used to inhibit the expression of the nucleic acid molecule described in A1; A3: An expression cassette containing the nucleotide sequence described in A2; A4: A recombinant vector containing the nucleotide sequence described in A2; A5: A recombinant vector containing the expression cassette described in A3; A6: Recombinant microorganisms containing the nucleotide sequence described in A2; A7: Recombinant microorganisms containing the expression cassette described in A3; A8: Recombinant microorganisms containing the recombinant vector described in A4; A9: Recombinant microorganisms containing the recombinant vector described in A5; A10: A transgenic plant cell line containing the nucleotide sequence described in A2; A11: Transgenic plant cell lines containing the expression cassette described in A3; A12: Transgenic plant tissue containing the nucleotide sequence described in A2; A13: Transgenic plant tissue containing the expression cassette described in A3; A14: A transgenic plant organ containing the nucleotide sequence described in A2; A15: Transgenic plant organ containing the expression cassette described in A3.

5. The application according to claim 4, characterized in that, The nucleic acid molecule described in A1 is selected from any one of the nucleic acid sequences from A1-1 to A1-3: A1-1: A nucleic acid sequence as shown in SEQ ID NO.2, or a complementary sequence thereof; A1-2: A sequence that has at least 95% sequence identity with the nucleic acid sequence shown in positions 191-2407 of SEQ ID NO.2, or a sequence that is complementary to it; A1-3: A nucleic acid sequence that hybridizes under stringent conditions with the nucleic acid sequence shown in SEQ ID NO.2 or the nucleic acid sequence shown in positions 191-2407 of SEQ ID NO.2, or a sequence complementary to it.

6. The application according to claim 4, characterized in that, The nucleotide sequence described in A2 includes the nucleotide sequence of the nucleic acid molecule described in A1 that has been knocked out.

7. The application according to claim 6, characterized in that, The knockout is performed using a CRISPR / Cas9 system. The target PAM sequence for knocking out the nucleic acid molecule A1 includes PAM1 or PAM2, and the nucleotide sequences of PAM1 and PAM2 are shown in SEQ ID NO.3 and SEQ ID NO.4, respectively.

8. The application according to any one of claims 1 to 7, characterized in that, The plants include monocotyledonous plants or dicotyledonous plants.

9. The application according to claim 8, characterized in that, The plants mentioned include corn, soybeans, cotton, peanuts, barley, oats, wild grass, rice, sorghum, sugarcane, tall fescue, turfgrass, wheat, alfalfa, or Arabidopsis thaliana.

10. The application according to any one of claims 1 to 7, characterized in that, The fungal diseases include one or more of ear rot, stem rot, and small spot disease.

11. A biomaterial, characterized in that, The biomaterial is at least one of B1-B15: B1: A nucleotide sequence for inhibiting the expression of a nucleic acid molecule encoding a ZmGAE1 protein or a protein having at least 95% sequence identity with the ZmGAE1 protein, wherein the amino acid sequence of the ZmGAE1 protein is shown in SEQ ID NO.1; B2: An expression cassette containing the nucleotide sequence described in B1; B3: A recombinant vector containing the nucleotide sequence described in B1; B4: A recombinant vector containing the expression cassette described in B2; B5: Recombinant microorganisms containing the nucleotide sequence described in B1; B6: Recombinant microorganisms containing the expression cassette described in B2; B7: Recombinant microorganisms containing the recombinant vector described in B3; B8: Recombinant microorganisms containing the recombinant vector described in B4; B9: A transgenic plant cell line containing the nucleotide sequence described in B1; B10: Transgenic plant cell lines containing the expression cassette described in B2; B11: Transgenic plant tissue containing the nucleotide sequence described in B1; B12: Transgenic plant tissue containing the expression cassette described in B2; B13: A transgenic plant organ containing the nucleotide sequence described in B1; B14: Transgenic plant organ containing the expression cassette described in B2.

12. The biomaterial according to claim 11, characterized in that, The nucleic acid molecule described in B1 is selected from any one of the nucleic acid sequences from B1-1 to B1-3: B1-1: A nucleic acid sequence as shown in SEQ ID NO.2, or a complementary sequence thereof; B1-2: A sequence that has at least 95% sequence identity with the nucleic acid sequence shown in positions 191-2407 of SEQ ID NO.2, or a sequence that is complementary to it; B1-3: A nucleic acid sequence that hybridizes under stringent conditions with the nucleic acid sequence shown in SEQ ID NO.2 or the nucleic acid sequence shown in positions 191-2407 of SEQ ID NO.2, or a sequence complementary to it.

13. The biomaterial according to claim 12, characterized in that, The initial microorganisms in the recombinant microorganisms include Agrobacterium.

14. A method for cultivating plants with enhanced resistance to fungal diseases and / or increased stem strength, characterized in that, The method includes the following steps: reducing the content of ZmGAE1 protein or a protein with at least 95% sequence identity with ZmGAE1 protein in the target plant, or inhibiting the expression of the ZmGAE1 protein encoding gene or a protein with at least 95% sequence identity with ZmGAE1 protein in the target plant, to obtain a plant with enhanced resistance to fungal diseases and / or stem strength compared to the target plant; the amino acid sequence of the ZmGAE1 protein is shown in SEQ ID NO.

1.

15. The method according to claim 14, characterized in that, The methods for reducing the content of ZmGAE1 protein or proteins with at least 95% sequence identity with ZmGAE1 protein in the target plant or inhibiting the expression of the ZmGAE1 protein encoding gene in the target plant include transposon insertion, mutagenesis, RNA-mediated inhibition, or gene editing.

16. A molecular marker InDel-884, characterized in that, The molecular marker InDel-884 exhibits an insertion polymorphism as shown in SEQ ID NO.5 at -884 bp of the promoter of the ZmGAE1 gene; the nucleotide sequence of the ZmGAE1 gene is shown in SEQ ID NO.

2.

17. A primer pair for amplifying the molecular marker InDel-884 of claim 16, characterized in that, The nucleotide sequences of the upstream and downstream primers of the primer pair are shown in SEQ ID NO.6 and SEQ ID NO.7, respectively.

18. The application of the molecular marker InDel-884 of claim 16 or the primer pair of claim 17 in plant molecular breeding.

19. The application according to claim 18, characterized in that, The plant molecular breeding includes the identification or auxiliary identification of plant fungal disease resistance and / or stem strength traits.

20. A method for identifying fungal disease resistance and / or stem strength traits in plants, characterized in that, Includes the following steps: The genomic DNA of the plant to be tested was amplified by PCR using the primers described in claim 17 to obtain PCR amplification products; The PCR amplification products were detected by electrophoresis to obtain PCR amplification bands. When the PCR amplification band is a single 371 bp band, the plant to be tested is InDel-884. + Homozygous haplotype, resistant to fungal diseases and / or resistant to lodging; When the PCR amplification bands consist of one 371bp band and one 230bp band, the plant to be tested is InDel-884. ± Heterozygous haplotype, resistant to fungal diseases and / or lodging-resistant plants; When the PCR amplification band is a single 230 bp band, the plant to be tested is InDel-884. - Homozygous haplotype, susceptible to fungal diseases and / or non-lodging resistant plants.

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