Corn sheath blight disease-resistant gene GRMZM2G057329 and application thereof

Abstract

The invention relates to the field of plant genetic engineering, and discloses a corn sheath blight disease-resistant gene GRMZM2G057329 and application thereof, and the corn sheath blight disease-resistant gene GRMZM2G057329 comprises any one of the following sequences: a nucleotide sequence shown as SEQ ID No.1, a nucleotide sequence shown as SEQ ID No.2, a nucleotide sequence shown as SEQ ID No.3, a nucleotide sequence shown as SEQ ID No.4, a nucleotide sequence shown as SEQ ID No.5, a nucleotide sequence shown as SEQ ID No.6, a The nucleotide sequence is as shown in SEQ ID No.2; the amino acid sequence of the protein is shown as SEQ ID No.3; the nucleotide sequence has 90% or more of identity with the nucleotide sequence as shown in SEQ ID No. 1 or SEQ ID No. 2 and is used for coding protein with the same function as the nucleotide sequence as shown in SEQ ID No. 1 or SEQ ID No. 2. According to the present invention, the DNA fragment of the complete coding segment of the anti-disease gene GRMZM2G057329 separated and cloned from corn is adopted, and the GRMZM2G057329 gene expression quantity is increased so as to significantly enhance the sheath blight resistance of the corn genetic transformation plant;

Description

Technical Field

[0001] This invention relates to the field of plant genetic engineering technology, specifically to a maize sheath blight resistance gene. GRMZM2G057329 And its applications. Background Technology

[0002] Maize (Zea mays) is an important global food and feed crop. However, extreme weather events (such as high temperatures, torrential rains, and droughts) and the increasing frequency of common diseases seriously threaten maize yield and quality. Among the many diseases, banded leaf and sheath blight (BLSB), caused by the semi-living pathogenic fungus Rhizoctonia solani, is a common and serious disease in my country and Southeast Asia.

[0003] In plant-pathogen interaction research, plant defenses are known. R. solani The mechanism involves multiple types of disease resistance-related genes, such as those encoding chitinase, β-1,3-glucanase, osmolysin, polygalacturonase, oxalate oxidase / decarboxylase, antimicrobial peptides, and genes related to hormone signal transduction, cell wall synthesis, and transcriptional regulation. In maize, ZmFBL41 can enhance resistance to sheath blight by regulating lignin synthesis pathways. Certain natural variations of this gene can evade recognition by ZmCAD, thereby maintaining lignin accumulation and enhancing the plant's structural defense capabilities. Despite these advances, the understanding of… R. solani The pathogenic mechanisms, effector secretion, and specific pathways of the host defense network in maize remain unclear.

[0004] Existing methods for improving maize resistance to sheath blight lack single-gene targets that have been validated by knockout / overexpression, leading to blindness in hybrid breeding phenotypic screening, gene editing target selection, and gene adaptation in transgenic technology. This results in insufficient resistance to sheath blight in genetically transformed maize plants. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a maize sheath blight resistance gene. GRMZM2G057329 Its application addresses the problem of insufficient resistance to sheath blight in genetically modified maize plants during existing maize sheath blight resistance improvement processes.

[0006] To achieve the above objectives, the present invention provides the following technical solution: a maize sheath blight resistance gene. GRMZM2G057329 , including any of the following sequences: The nucleotide sequence shown in SEQ ID No. 1; The nucleotide sequence shown in SEQ ID No. 2; The amino acid sequence of the protein shown in SEQ ID No. 3; Nucleotide sequences that have ≥90% identity with the nucleotide sequences shown in SEQ ID No. 1 or SEQ ID No. 2 and encode the same functional protein.

[0007] By employing the above technical solution, disease-resistant genes were isolated and cloned from maize. GRMZM2G057329 A complete coding segment of DNA was used to improve maize's ability to defend against sheath blight. GRMZM2G057329 Increased gene expression significantly enhances the resistance of genetically transformed maize plants to sheath blight, making the transgenic maize more resistant to the disease than the recipient maize. This solves the problem of insufficient resistance to sheath blight in existing maize sheath blight resistance improvement processes.

[0008] Preferably, the amino acid sequence of the protein further includes a derived amino acid sequence having the same function as the sequence shown in SEQ ID No. 3, by substitution, deletion or insertion of one or more amino acids.

[0009] A molecular marker for detecting resistance to maize sheath blight, the molecular marker being used to detect maize sheath blight resistance genes. GRMZM2G057329 An 18bp insertion variant in the promoter region, wherein the 18bp insertion sequence is a disease-resistant haplotype-specific sequence.

[0010] Preferably, the molecular marker consists of a primer pair, which is a primer pair consisting of the upstream primer shown in SEQ ID No. 21 and the downstream primer shown in SEQ ID No. 22, or a primer pair consisting of the upstream primer shown in SEQ ID No. 23 and the downstream primer shown in SEQ ID No. 24.

[0011] A specific knockout GRMZM2G057329 The gRNA of the gene, the nucleotide sequence of which is shown in SEQ ID No. 4.

[0012] Based on the above application of gRNA in the preparation of transgenic maize with altered resistance to maize sheath blight.

[0013] Preferably, the application includes the following steps: The gRNA was combined with the CRISPR-Cas9 system to construct a gene knockout vector; After introducing gene knockout vectors into maize cells via Agrobacterium-mediated transformation, the cells were screened and cultured to obtain... GRMZM2G057329 Transgenic plants with missing gene function.

[0014] Based on the aforementioned maize sheath blight resistance genes GRMZM2G057329 Application in improving maize's resistance to sheath blight.

[0015] Based on the aforementioned maize sheath blight resistance genes GRMZM2G057329 Application in the breeding of maize varieties resistant to sheath blight.

[0016] An overexpression vector, the overexpression vector comprising GRMZM2G057329 The CDS sequence of the gene is used to... GRMZM2G057329 The CDS sequence of the gene was amplified, ligated into the overexpression vector CAUC2829-Ubi, and obtained by plasmid extraction after transformation.

[0017] This invention provides a disease resistance gene for maize sheath blight. GRMZM2G057329 And its applications. It has the following beneficial effects: 1. This invention utilizes disease-resistant genes isolated and cloned from maize. GRMZM2G057329 A complete coding segment of DNA was used to improve maize's ability to defend against sheath blight. GRMZM2G057329 Increased gene expression significantly enhances the resistance of genetically transformed maize plants to sheath blight, making the transgenic maize more resistant to the disease than the recipient maize. This solves the problem of insufficient resistance to sheath blight in existing maize sheath blight resistance improvement processes.

[0018] 2. This invention, through genotype-phenotype co-segregation analysis based on the F2 segregating population and association verification of 278 core maize germplasm accessions, discovered... GRMZM2G057329 The 18bp insertion variation in the gene promoter region is a key site determining maize sheath blight resistance. Plants carrying the resistant allele with this 18bp insertion have a significantly lower disease index than plants carrying the susceptible allele with the 18bp deletion. Moreover, this variation is stable in resistant haplotypes, breaking through the limitations of traditional breeding that relies on field phenotypic observation. It provides a heritable and detectable molecular criterion for screening resistant germplasm, transforming the genetic analysis of resistance traits from phenotype-genotype inference to genotype-phenotype prediction, thus improving the accuracy and reliability of resistance genetic analysis.

[0019] 3. This invention develops two pairs of specific molecular markers targeting the 18bp natural variation, enabling resistance screening to be completed in the early generations of maize breeding without waiting for field disease phenotypes, reducing screening costs, shortening the breeding cycle for maize sheath blight resistance, providing an efficient tool for large-scale resistant germplasm screening and breeding applications, and improving the problems of unstable association between markers and resistance traits and high detection costs in existing molecular marker-assisted breeding.

[0020] 4. This invention can transform through overexpression vector conversion. GRMZM2G057329By introducing genes into susceptible main varieties, transgenic lines highly resistant to sheath blight can be rapidly cultivated, avoiding the problems of complex genetic backgrounds and unstable segregation of target traits in traditional hybridization breeding. Furthermore, by combining 18bp variant molecular markers, resistance alleles can be precisely preserved in backcross breeding, reducing the linkage burden of non-target traits. This approach leverages the precision of genetic engineering technology while also taking into account the practicality of traditional breeding, breaking through the limitations of single technologies in resistance improvement and providing a diversified and efficient technical path for breeding resistance to sheath blight in maize. Attached Figure Description

[0021] Picture 1 Genetic modification of the disease-resistant maize materials (Z2028F, GEMS6, CIMBL156) and susceptible materials (ZHI41, D863F, SHEN137) after inoculation with *Rhizoctonia solani*. GRMZM2G057329 A graph showing the transcriptional level response detection. Picture 2 For the present invention GRMZM2G057329 Overexpression of T2 generation genetically transformed maize plants ( GRMZM2G057329 Figure 1 shows that gene expression levels are significantly increased in OE1 and OE2. Picture 3 CRISPR-Cas9 knockout for the present invention GRMZM2G057329 Schematic diagram of sequencing results of T2 generation positive homozygous mutant plants; Picture 4 For the present invention GRMZM2G057329 Schematic diagram of disease phenotype and statistical diagram of lesion length 14 days after inoculation of gene knockout and overexpression T2 generation positive homozygous plants with sheath blight. Picture 5 For the present invention GRMZM2G057329 Pr gene transcription response detection diagram 48 hours after inoculation of T2 generation positive homozygous mutant plants with sheath blight. Picture 6 For the present invention GRMZM2G057329 Resequencing analysis of disease-resistant alleles (Z2018F) and disease-susceptible alleles (D863F); Picture 7 Genotype-phenotype association analysis diagram of the F2 population (Z2018F×SHEN137 / GEMS6×D863F) of this invention; Picture 8 This diagram illustrates the molecular markers used in the present invention for the specific detection of 18bp natural variations, along with their distribution assessment and genotype-phenotype association analysis on 278 maize germplasms. Detailed Implementation

[0022] The technical solution of the present invention will now be clearly and completely described with reference to the accompanying drawings. Obviously, the described embodiments are merely some embodiments of the present invention, and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0023] Example 1: GRMZM2G057329 Gene expression pattern after inoculation with *Rhizoctonia solani* In order to detect GRMZM2G057329 The gene expression pattern induced by *Rhizoctonia solani* was investigated. qRT-PCR was used to detect gene expression after inoculation with the pathogen. Primers used were primer 9, 5′-AGCATACAGGGCCTGGACTG -3′ (sequence shown in List No. 13) and primer 10, 5′-GCAGGCAAGCTTTGGTTTGT -3′ (sequence shown in List No. 14). RNA was extracted from resistant materials Z2018F, GEMS6, and CIMBL156, and susceptible materials ZHI41, D863F, and SHEN137 at 0, 24, 48, 72, and 96 hours after inoculation with *Rhizoctonia solani*. cDNA was obtained by reverse transcription and amplified using the cDNA as a template by quantitative real-time PCR.

[0024] The quantitative real-time PCR reaction system was as follows: 10 μL of 2×Real Fast SYBR qPCR Mix (Low ROX) (Huayu Biotechnology), 0.5 μL of 10 μM upstream primer 1, 0.5 μL of 10 μM downstream primer 2, 2 μL of cDNA template, and ddH2O to a final volume of 20 μL.

[0025] The quantitative real-time PCR reaction program was as follows: pre-denaturation at 95℃ for 2 min; 95℃ for 15 sec, 60℃ for 30 sec, for 40 cycles. The relative gene expression levels were calculated using the 2^-ΔΔCt method.

[0026] After quantitative real-time PCR, the analysis results showed that the disease was present in maize varieties Z2018F, GEMS6, and CIMBL156 (resistant varieties) and ZHI41, D863F, and SHEN137 (susceptible varieties). GRMZM2G057329 Genes can be affected by Rhizoctonia solani. R. solani The induction of disease, in which disease-resistant materials and disease-susceptible materials were induced 72 hours after inoculation. GRMZM2G057329 Gene expression levels reached their peak and then declined. However, it is noteworthy that gene expression levels in disease-resistant materials... GRMZM2G057329 The peak value of the transcriptional response induced by *Rhizoctonia solani* was much higher than that of the susceptible material (see appendix). Picture 1 This indicates that the gene is involved in the resistance response of maize to sheath blight.

[0027] Example 2: Cloning GRMZM2G057329 Gene According to the database Maize GDB GRMZM2G057329 Primers were designed for gene sequencing. Primer 1, 5′-ATGGCGTCCGGCGGAGCGGC-3′ has the sequence shown in List No. 5, and primer 2, 5′-CATTTTCACCACACTCACTTA-3′ has the sequence shown in List No. 6. These primers were used to obtain... GRMZM2G057329 Gene. RNA was extracted from maize B73 and cDNA was obtained through reverse transcription. The cDNA was then used as a template for PCR amplification.

[0028] The PCR reaction system consisted of: 25 μL of 2×T8 High-Fidelity Master Mix (Qingke Biotechnology), 2 μL of 10 μM upstream primer, 2 μL of 10 μM downstream primer, 1 μL of cDNA template, and ddH2O to a final volume of 50 μL.

[0029] The PCR reaction program was as follows: 98℃ pre-denaturation for 2 min; 98℃ for 30 sec, 60℃ for 30 sec, 72℃ for 1 min, 35 cycles; 72℃ final extension for 5 min.

[0030] After PCR, the amplified fragments were recovered and purified using a DNA recovery kit from Qingke Biotechnology Co., Ltd. The purified DNA fragments were then ligated into the vector CAUC2829-Ubi (provided by the Crop Functional Genomics and Molecular Breeding Center of China Agricultural University) and transformed. E. coli DH5α competent cells were used, positive clones were selected and plasmids were extracted, and sequencing was performed by Qingke Biotechnology, yielding a 606 bp plasmid. GRMZM2G057329 The fragment has the DNA sequence listed as No. 2.

[0031] Example 3: GRMZM2G057329 Functional verification of genes The purified cDNA fragment was ligated into the overexpression vector CAUC2829 via homologous recombination and then transformed by heat shock. E. coli Colony PCR and agarose gel electrophoresis were performed on the recombinant colonies after DH5α competent cells were cultured. The recombinant colonies contained bands of the same size as the target fragment. After expanding the identified recombinant colonies, plasmids were extracted, and sequencing of the plasmids was performed. The sequences were compared with the original sequences, and the inserted fragment was found to be full-length cDNA without any base mutations or deletions, proving that... GRMZM2G057329 Overexpression vector CAUC2829-Ubi :: GRMZM2G057329 Successfully built.

[0032] Genetic transformation of the overexpression vector was carried out at the Crop Functional Genomics and Molecular Breeding Center of China Agricultural University on the platform. GRMZM2G057329 Overexpression vectors were introduced into maize variety ND101, resulting in genetically transformed maize plants. This invention yielded two independent T2 generation positive homozygous transformant lines. Using primers 9, 5′-AGCATACAGGGCCTGGACTG-3′ (sequence shown in List No. 13) and 10, 5′-GCAGGCAAGCTTTGGTTTGT-3′ (sequence shown in List No. 14), the positive lines were detected. GRMZM2G057329 Genes in -OE1, OE2) GRMZM2G057329 Expression levels were significantly increased (see appendix) Picture 2 ).

[0033] against GRMZM2G057329 A gRNA was designed to specifically target bases 180-198 downstream of the start codon ATG in the gene coding region. The gRNA sequence is shown in List No. 4. GRMZM2G057329 Gene information and specific gRNA sequences were uploaded to the Crop Functional Genomics and Molecular Breeding Center of China Agricultural University. This platform was used for the construction of CRISPR / Cas9 knockout vectors and the genetic transformation of these vectors. The knockout vector was introduced into the maize variety ND101, obtaining one T2 generation homozygous mutant line. Using primers 1, 5′-ATGGCGTCCGGCGGAGCGGC-3′ (sequence shown in List No. 5) and 2, 5′-CATTTCACCACACTCACTTA-3′ (sequence shown in List No. 6), gene sequence amplification and sequencing were performed. The sequencing results showed that in the homozygous mutant plants… GRMZM2G057329 The A base is deleted at position 182 downstream of the ATG start codon (see appendix). Picture 3 ).

[0034] Two homozygous overexpressing lines and one homozygous mutant line were inoculated with *Cephalotaxus fortunei* for identification. The results showed that inoculation with *Cephalotaxus fortunei*... R. solani Knockout 14 days later GRMZM2G057329 Compared with wild-type maize plants, the maize plants in this study showed significantly increased symptoms of sheath blight, with the average lesion length increasing by 3.7235 cm (results are shown in Table 1, and see Appendix). Picture 4 Overexpression GRMZM2G057329 Compared with the wild type, the maize plants in the control group showed significantly reduced symptoms of sheath blight, with the average lesion length shortened by 4.0062 cm and 4.4876 cm, representing a reduction of approximately 20.02% (results are shown in Table 1 and see Appendix). Picture 4 ),illustrate GRMZM2G057329 It participated in the resistance response of maize to sheath blight.

[0035] Example 4: Knockout GRMZM2G057329 PR gene detection in T2 generation homozygous mutant plants Knockout GRMZM2G057329 The expression level of the PR gene was detected in T2 generation homozygous mutant plants 48 hours after inoculation with *Rhizoctonia solani*. Primers 3, 5′-TGTGCATGCACCTATATGTACT-3′ (sequence shown in List No. 7) and 4, 5′-TTCGTGAGACATGACGATACAT-3′ (sequence shown in List No. 8) were used to detect the gene expression. ZmPR4 (Zm00001d048949) The gene was detected using primers 5, 5′-AGATCACTAAAGCCAAGGAGTC -3′ (sequence shown in List No. 9) and 6, 5′-CATGGTCTAGTTGTAGGCTTCC -3′ (sequence shown in List No. 10). ZmPR10 (Zm00001d028816) The 18S reference gene in maize was detected using primers 7, 5′-CCATCCCTCCGTAGTTAGCTTCT -3′ (sequence shown in List No. 11) and 8, 5′-CCTGTCGGCCAAGGCTATATAC -3′ (sequence shown in List No. 12). Extraction and knockout were performed. GRMZM2G057329 RNA was collected from T2 generation homozygous mutant plants and background material ND101 48 hours after inoculation with Rhizoctonia solani. cDNA was obtained by reverse transcription and amplified by real-time quantitative PCR using cDNA as a template.

[0036] The quantitative real-time PCR reaction system was as follows: 10 μL of 2×Real Fast SYBR qPCR Mix (Low ROX) (Huayu Biotechnology), 0.5 μL of 10 μM upstream primer 1, 0.5 μL of 10 μM downstream primer 2, 2 μL of cDNA template, and ddH2O to a final volume of 20 μL.

[0037] The quantitative real-time PCR reaction program was as follows: pre-denaturation at 95℃ for 2 min; 95℃ for 15 sec, 60℃ for 30 sec, for 40 cycles. The relative gene expression levels were calculated using the 2^-ΔΔCt method.

[0038] After the quantitative real-time PCR was completed, data analysis revealed that, compared with the background material ND101, the knockout... GRMZM2G057329 In T2 generation homozygous mutant plants, the PR gene was significantly reduced 48 hours after inoculation with *Rhizoctonia solani* (see appendix). Picture 5 This indicates that after inoculation with *Rhizoctonia solani*, knockout was performed. GRMZM2G057329 In T2 generation homozygous mutant plants, immune defense pathways associated with resistance to sheath blight were suppressed, thereby reducing resistance to maize sheath blight.

[0039] Example 6: [Regarding...] GRMZM2G057329 Resequencing analysis of disease-resistant and disease-susceptible haplotypes of the gene. Using primers 11, 5′-CATCATACAAGAGGCTGCAG -3′ (sequence shown in List No. 15) and 12, 5′-ACCCGATGGTCACGTCGAA -3′ (sequence shown in List No. 16), this gene was analyzed. GRMZM2G057329 Resequencing of disease-resistant and disease-susceptible haplotypes revealed that... GRMZM2G057329 The promoter regions of the disease-resistant allele lines (represented by the disease-resistant material Z2018F, sequence shown in List No. 17) and the disease-susceptible allele lines (represented by the disease-susceptible material D863F, sequence shown in List No. 18) generally exhibit an 18bp insertion (see Appendix). Picture 6 ),Right now GRMZM2G057329 Ecotype materials with an 18bp insertion in the gene promoter region exhibit resistance to sheath blight.

[0040] Meanwhile, the results in Example 1 revealed that the disease-resistant material Z2018F contains genes... GRMZM2G057329 The peak value of the response induced by *Rhizoctonia solani* infection was much higher than that of the transcriptional expression in the susceptible material D863F, i.e. GRMZM2G057329 Ecotype materials with an 18bp insertion in the gene promoter region showed a significantly increased transcriptional response level after infection by pathogens.

[0041] Example 7: Genotype-phenotype association analysis of the F2 population Phenotypic gene association analysis was performed using the F2 population (Z2018F×SHEN137 / GEMS6×D863F). Gene amplification and sequencing analysis were performed using primers 11, 5′-CATCATACAAGAGGCTGCAG -3′ (sequence shown in List No. 15) and 12, 5′-ACCCGATGGTCACGTCGAA -3′ (sequence shown in List No. 16). The results showed that GEMS6 contains the same genes as the representative disease-resistant ecotype Z2018F. GRMZM2G057329 Promoter and coding sequence (the sequence of which is shown in List No. 19). SHEN137 contains the same sequence as the representative symptomatic ecotype D863F. GRMZM2G057329 Promoter and coding sequence (the sequences are shown in List No. 20). Molecular markers that can distinguish the 18 bp variation in the susceptibility-resistance allele were designed for the F2 population (see Appendix). Picture 7(B, E) The primers used, 13, 5′-CCCTTTACCATGTATGTGCTT-3′ (sequence shown in List No. 21) and 14, 5′-GAATCAAGTAAGCTTACCCCA-3′ (sequence shown in List No. 22), were used to identify the genotype of each plant in both populations and to inoculate them in the field with the sheath blight pathogen. The severity of disease was classified into six levels (0, 1, 3, 5, 7, 9) based on the disease incidence in each population, with higher levels indicating more severe disease (see Appendix). Picture 7 (A, D). Based on the disease severity classification, each plant in each population was scored, and the disease incidence in each population was statistically analyzed. It was found that plants with an 18bp insertion in both populations showed significantly higher resistance to sheath blight than plants with an 18bp deletion (see appendix). Picture 7 C and F), i.e., genotype-phenotype co-segregation. These results indicate that... GRMZM2G057329 An 18 bp natural variation in the gene is the reason for the differences in resistance to corn sheath blight.

[0042] Example 8: Developing a set of molecular markers specifically for detecting 18bp natural variation to evaluate maize germplasm and perform genotype-phenotype association analysis. Furthermore, a molecular marker was developed to effectively distinguish the 18bp natural variation between maize sheath blight-resistant and susceptible lines. The primers used were primer 15, 5′-GATAACCTAGGGTTTGGGG-3′ (sequence shown in List No. 23) and primer 16, 5′-CCACCCGAAGCGCAAGAG-3′ (sequence shown in List No. 24). This effectively distinguished between resistance and susceptibility alleles (see Appendix). Picture 8 A). Screening of 278 maize germplasm accessions revealed that 57 accessions carried an 18bp inserted disease-resistance allele, 102 accessions did not carry an 18bp inserted disease-susceptibility allele, and 119 accessions were heterozygous (see appendix). Picture 8 B). Genotype-phenotype association analysis showed that susceptible alleles without the 18bp insertion exhibited higher resistance to *Rhizoctonia solani* compared to resistant alleles carrying the 18bp insertion (see Appendix). Picture 8 C), which further demonstrates that the 18 bp insertion conferred resistance to maize sheath blight.

[0043] This application provides a gene for resistance to corn sheath blight. GRMZM2G057329 Functional verification and application, utilizing strong promoter-driven transgenic technology to... GRMZM2G057329 When the gene overexpression vector was introduced into maize plant ND101, it was found that... GRMZM2G057329Genetically transformed maize with significantly increased gene expression levels showed markedly enhanced resistance to sheath blight; specific knockout of genes using CRISPR-Cas9 technology... GRMZM2G057329 Gene analysis revealed that homozygous mutant plants exhibited significantly reduced resistance to sheath blight, and the expression of the PR gene was significantly decreased, demonstrating... GRMZM2G057329 Genes play an important role in resistance to sheath blight.

[0044] Phenotypic-genotypic association analysis of the F2 population and 278 maize accessions showed that... GRMZM2G057329 An 18bp natural variation in the promoter region of a gene can determine the resistance phenotype of maize to sheath blight. Of particular note is the clear correspondence between the resistance allele (18bp insertion) and the susceptibility allele (18bp deletion), providing a quantifiable molecular marker selection standard for the creation of resistant germplasm. It also offers an innovative technological paradigm for molecular design breeding of crop disease resistance. The discovery of disease resistance genes... GRMZM2G057329 A natural variation site of 18 bp in the promoter region is associated with resistance to maize sheath blight.

[0045] By analyzing key resistance genes for maize sheath blight, the resistance of maize to the pathogen has been effectively enhanced. The application of this transgenic technology system not only alleviates the threat of the disease to grain yield and nutritional quality, but also significantly reduces the amount of chemical pesticides used by establishing an endogenous disease resistance mechanism, thereby reducing the risk of pesticide residue pollution and promoting the sustainable operation of farmland ecosystems. From a production practice perspective, the durable resistance acquired by crops results in the stable expression of excellent genetic traits, ensuring yield, safeguarding food security, and improving the economic and ecological benefits of maize crop production.

[0046] Table 1: GRMZM2G057329 Statistics on knockout and overexpression homologs and the length of maize sheath blight lesions in field-planted maize.

[0047]

[0048]

[0049] SEQ ID No.1 2279 bp GRMZM2G057329 cDNA sequence SEQ ID No.2 606 bp GRMZM2G057329 CDS sequence ATGGCGTCCGGCGGAGCGGCCATCTCGGCGGGGCCTACACCGCCGTCGGCGACGGCGTCGTCGGTTGAGTGGCACCAGCGGCCGCCGAACCCGAAGAACCCCGTGGTGTTCTTCGACGTGACCATCGGGTCCATCGCCGCCGGCCGCATCAAGATGGAGCTCTTCGCCGACATCGCCCCCAAGACCGCGGAGAACTTCCGGCAGTTCTGCACTGGGGAGCATAGAAAAAATGCTTTGCCACAAGGTTATAAGGGCTGCCAATTCCATAGAGTGATCAAAGATTTCATGATTCAGGGTGGTGACTTCTTGAAGAATGATGGTACTGGATGCACATCAATCTACGGTACCAAATTTGATGATGAAAATTTTATAGCAAAGCATACAGGGCCTGGACTGCTCTCGATGGCAAACAGTGGAATTAACTCTAATGGATCTCAGTTCTTTATAACATGTGCAAAGTGTGACTGGCTTGACAACAAACATGTGGTATTTGGGAGAGTGCTTGGAGACGGTTTGCTCGTCGTGAGGAAGATTGAAAATGTTGCAACTGGAGCGAATAACAAACCAAAGCTTGCCTGCGTTATAAGTGAGTGTGGTGAAATGTAG SEQ ID No.3 201 aa GRMZM2G057329 Protein sequence MASGGAAISAGPTPPSATASSVEWHQRPPNPKNPVVFFDVTIGSIAAGRIKMELFADIAPKTAENFRQFCTGEHRKNALPQGYKGCQFHRVIKDFMIQGGDFLKNDGTGCTSIYGTKFDDENFIAKHTGPGLLSMANSGINSNGSQFFITCAKCDWLDNKHVVFGRVLGDGLLVVRKIENVATGANNKPKLACVISECGEM SEQ ID No. 4 19 bp DNA Artificial sequence GAAGTTCTCCGCGGTCTTG SEQ ID No. 5 20 bp DNA Artificial sequence ATGGCGTCCGGCGGAGCGGC SEQ ID No. 6 20 bp DNA Artificial sequence CATTTCACCACACTCACTTA SEQ ID No.7 22 bp DNA Artificial sequence TGTGCATGCACCTATATGTACT SEQ ID No. 8 22 bp DNA Artificial sequence TTCGTGAGACATGACGATACAT SEQ ID No. 9 22 bp DNA Artificial sequence AGATCACTAAAGCCAAGGAGTC SEQ ID No. 10 22 bp DNA Artificial sequence CATGGTCTAGTTGTAGGCTTCC SEQ ID No. 11 23 bp DNA Artificial sequence CCATCCCTCCGTAGTTAGCTTCT SEQ ID No. 12 22 bp DNA Artificial sequence CCTGTCGGCCAAGGCTATATAC SEQ ID No. 13 20 bp DNA Artificial sequence AGCATACAGGGCCTGGACTG SEQ ID No. 14 20 bp DNA Artificial sequence GCAGGCAAGCTTTGGTTTGT SEQ ID No. 15 20 bp DNA Artificial sequence CATCATACAAGAGGCTGCAG SEQ ID No. 16 19 bp DNA Artificial sequence ACCCGATGGTCACGTCGAA SEQ ID No. 17 5089 bp GRMZM2G057329 Z2018F genomic sequence SEQ ID No. 18 4979 bp GRMZM2G057329 D863F genomic sequence SEQ ID No. 19 5089 bp GRMZM2G057329 GEMS6 genomic sequence SEQ ID No.20 4979 bp GRMZM2G057329 SHEN137 genomic sequence SEQ ID No. 21 21 bp DNA Artificial sequence CCCTTTACCATGTATGTGCTT SEQ ID No. 22 21 bp DNA Artificial sequence GAATCAAGTAAGCTTACCCCA SEQ ID No. 23 19 bp DNA Artificial sequence GATAACCTAGGGTTTGGGG SEQ ID No. 24 18 bp DNA Artificial sequence CCACCCGAAGCGCAAGAG Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. Maize sheath blight resistance gene GRMZM2G057329 Its characteristics are, Includes any of the following sequences: The nucleotide sequence shown in SEQ ID No. 1; The nucleotide sequence shown in SEQ ID No. 2; The amino acid sequence of the protein shown in SEQ ID No. 3; Nucleotide sequences that have ≥90% identity with the nucleotide sequences shown in SEQ ID No. 1 or SEQ ID No. 2 and encode the same functional protein.

2. The maize sheath blight resistance gene according to claim 1 GRMZM2G057329 Its features are: The amino acid sequence of the protein also includes a derived amino acid sequence having the same function as the sequence shown in SEQ ID No. 3, by substitution, deletion or insertion of one or more amino acids.

3. A molecular marker for detecting resistance to maize sheath blight, characterized in that, The molecular markers were used to detect maize sheath blight resistance genes. GRMZM2G057329 An 18bp insertion variant in the promoter region, wherein the 18bp insertion sequence is a disease-resistant haplotype-specific sequence.

4. The molecular marker for detecting maize sheath blight resistance according to claim 3, characterized in that: The molecular marker consists of primer pairs, which are primer pairs consisting of the upstream primer shown in SEQ ID No. 21 and the downstream primer shown in SEQ ID No. 22, or primer pairs consisting of the upstream primer shown in SEQ ID No. 23 and the downstream primer shown in SEQ ID No.

24.

5. A specific knockout GRMZM2G057329 The gRNA of a gene is characterized by, The nucleotide sequence of the gRNA is shown in SEQ ID No.

4.

6. The application of the gRNA according to claim 5 in the preparation of transgenic maize with altered resistance to maize sheath blight.

7. The application according to claim 6, characterized in that: The application includes the following steps: The gRNA was combined with the CRISPR-Cas9 system to construct a gene knockout vector; After introducing gene knockout vectors into maize cells via Agrobacterium-mediated transformation, the cells were screened and cultured to obtain... GRMZM2G057329 Transgenic plants with missing gene function.

8. The maize sheath blight resistance gene according to claim 1 GRMZM2G057329 Application in improving maize's resistance to sheath blight.

9. The maize sheath blight resistance gene according to claim 1 GRMZM2G057329 Application in the breeding of maize varieties resistant to sheath blight.

10. An overexpression vector, characterized in that: The overexpression vector contains GRMZM2G057329 The CDS sequence of the gene is used to... GRMZM2G057329 The CDS sequence of the gene was amplified, ligated into the overexpression vector CAUC2829-Ubi, and obtained by plasmid extraction after transformation.

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