Related gene for improving corn ear rot resistance and application method thereof

Abstract

The invention discloses a related gene for improving corn ear rot resistance and an application method thereof, the gene is (a1) or (a2) or (a3), and the protein is (b1) or (b2). The invention also discloses application of the ZmNFXL1 gene, the protein, or the inhibiting factor thereof, or a biological material containing the coding gene or the inhibiting factor thereof. The corn spike rot resistance gene ZmNFXL1 is found for the first time, and the corn spike rot resistance can be remarkably improved by blocking the expression of the gene through a natural mutation screening or manual intervention method. Meanwhile, according to the method disclosed by the invention, the spike rot resistant corn germplasm can be quickly identified and created, so that the influence of the spike rot on corn planting can be reduced.

Description

Technical Field

[0001] This invention relates to the fields of genetic engineering and biological control, and more specifically, to a gene related to improving resistance to corn ear rot and its application method. Background Technology

[0002] Ear rot in maize is a common fungal disease affecting maize producing regions worldwide. Various fungi can infect maize ears and kernels, causing the entire ear or part of the kernels to rot during late growth, harvest, or storage. This severely impacts maize yield and kernel quality, posing a significant threat to food, feed, and storage safety. The incidence rate of ear rot is typically 5%-10%, reaching 30%-40% in severe years, and can exceed 50% in susceptible varieties, resulting in yield losses of 30%-40%. Besides direct yield loss due to kernel rot, the pathogens in the kernels can produce various toxins, leading not only to reduced maize quality but also posing a significant threat to food and feed safety and human and animal health. Therefore, controlling the occurrence and damage caused by ear rot in maize is of paramount importance. In production, ear rot occurs in the middle and late stages of maize growth, making field control difficult and usually not specifically targeted at it. Furthermore, the pathogens causing ear rot are complex, with diverse infection pathways, and the disease's occurrence is influenced by climate, all of which make it difficult for pesticides to achieve ideal control effects. Therefore, screening and utilizing resistant varieties has become the most economical and effective measure to control maize ear rot. Due to the severity of ear rot, domestic and international research has been conducted on pathogen identification, resistant germplasm screening, resistance mechanism analysis, resistance genetics, and the discovery and localization of resistance genes. However, current research on maize ear rot-resistant varieties and resistance genes faces multiple challenges: First, resistant germplasm resources are scarce. Currently, resistant germplasm identified domestically and internationally mainly targets single physiological races, and germplasm resistant to dominant physiological races such as *Fusarium verticillioides* and *Fusarium graminearum* is extremely scarce, making it difficult to support germplasm improvement and innovation in traditional breeding. Secondly, most research on disease-resistant germplasm focuses on QTL mapping and omics analysis. The elucidation of regulatory networks for the few cloned disease-resistant genes is lagging, and there are no reports of superior haplotypes or their applications in breeding, thus failing to support molecular breeding. Thirdly, progress in research on susceptible genes is slow due to technological bottlenecks. Compared to resistance genes, susceptible genes are essential host genes for pathogen infection and disease development. Resistance mediated by susceptible gene mutations often exhibits persistence and broad-spectrum characteristics. However, susceptible gene mutations can bring various negative effects to plant growth and development, greatly limiting the application of susceptible genes in plant disease resistance breeding. Identifying usable susceptible genes has always been a key focus and challenge in plant pathology and crop disease resistance breeding. Summary of the Invention

[0003] The purpose of this invention is to provide a gene related to improving resistance to corn ear rot.

[0004] Another objective of this invention is to provide an application and method for genes related to improving resistance to corn ear rot.

[0005] The objective of this invention can be further achieved by the following technical measures:

[0006] To achieve the objective of this invention, the present invention first provides a gene related to improving resistance to ear rot in maize, wherein the nucleic acid molecule of the gene is a DNA molecule as follows (a1) or (a2) or (a3):

[0007] (a1) The nucleotide shown in SEQ ID NO: 1 or SEQ ID NO: 2;

[0008] (a2) A nucleotide encoding the amino acid shown in SEQ ID NO: 3 or SEQ ID NO: 4; or

[0009] (a3) A nucleotide that can hybridize with the complementary sequence of SEQ ID NO: 1 or SEQ ID NO: 2 under strict hybridization conditions, and the protein encoded by the nucleotide has the function of the ZmNFXL1 transcription factor.

[0010] Furthermore, the present invention also provides a protein related to improving resistance to maize ear rot, as follows (b1) or (b2):

[0011] (b1) A protein consisting of the amino acid sequence shown in SEQ ID NO: 3 or SEQ ID NO: 4 in the sequence listing;

[0012] (b2) The fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of (b1).

[0013] Those skilled in the art can easily mutate the ZmNFXL1 gene provided by this invention using known methods, such as directed evolution and point mutation. Artificially modified nucleic acids that have 90% or higher identity with the ZmNFXL1 gene provided by this invention, as long as they encode the ZmNFXL1 protein and have the function of the ZmNFXL1 protein, are all derived from and equivalent to the nucleotide sequence of this invention.

[0014] The term "identity" used here refers to sequence similarity to a natural nucleic acid sequence. Identity can be evaluated visually or using computer software. Using computer software, the identity between two or more sequences can be expressed as a percentage (%), which can be used to evaluate the identity between related sequences.

[0015] Furthermore, the present invention provides the application of the ZmNFXL1 nucleic acid molecule, the protein, or its inhibitor, or biological materials containing its encoding gene or inhibitor, as follows (c1) or (c2) or (c3):

[0016] (c1) Regulates resistance to corn ear rot;

[0017] (c2) Improve resistance to corn ear rot;

[0018] (c3) Select and breed maize with high resistance to ear rot.

[0019] One application method involves inhibiting the expression of the ZmNFXL1 protein or its encoding gene to improve maize's resistance to ear rot.

[0020] Specific application methods include inhibiting ZmNFXL1 protein expression through transcriptional regulation, post-transcriptional regulation, translational regulation, and post-translational regulation; and inhibiting the expression of the ZmNFXL1 encoding gene through gene knockout, gene silencing, and gene expression vector optimization.

[0021] In a preferred embodiment, inhibiting ZmNFXL1 protein expression includes blocking transcription factor binding by binding to specific regions of DNA; inhibiting gene promoter activity by epigenetic modifications (such as methylation); targeting and degrading mRNA by miRNA or antisense oligonucleotides (ASO); binding to mRNA and recruiting degrading enzymes (such as nucleases) to shorten its half-life; blocking translation initiation or elongation using specific inhibitors (such as antibiotics or small molecule compounds); interfering with the function or stability of the target protein by binding to the target protein with antibodies or competitive molecules; and targeted degradation of the target protein via the ubiquitin-proteasome system (UPS) or lysosomal pathway.

[0022] In a preferred embodiment, inhibiting the expression of the ZmNFXL1 encoding gene includes precisely editing the target gene using a CRISPR-Cas9 or TALEN system to achieve gene knockout by introducing frameshift mutations or deletion sequences; designing specific siRNA or shRNA using RNA interference (RNAi) technology to degrade the target mRNA; avoiding vector construction errors (such as reading frame errors) and optimizing codon usage frequency to reduce translation efficiency; and designing mRNA secondary structure interference sequences to prevent ribosome binding or translation elongation.

[0023] Furthermore, the present invention provides a method for creating maize germplasm resistant to ear rot, wherein the method alters the performance of gene ZmNFXL1 or inhibits gene ZmNFXL1 expression through natural mutation screening or artificial intervention.

[0024] In a preferred embodiment, the artificial intervention method includes gene editing technology and viral vector introduction.

[0025] Furthermore, the present invention provides a method for identifying whether maize is resistant to ear rot, the method comprising the following steps:

[0026] (1) The ZmNFXL1 gene was amplified by PCR using specific primers.

[0027] (2) Compare whether the amplification products have the function of the ZmNFXL1 gene.

[0028] Advantages and beneficial effects of the present invention:

[0029] This invention is the first to discover the maize ear rot resistance gene ZmNFXL1. Blocking the expression of this gene through natural mutation screening or artificial intervention can significantly improve maize resistance to ear rot. Furthermore, the method of this invention can rapidly identify and create maize germplasm resistant to ear rot, thereby reducing the impact of this disease on maize cultivation. Attached Figure Description

[0030] Figure 1 Expression analysis of ZmNFXL1 in resistant and susceptible self-crossed lines.

[0031] Figure 2 Schematic diagram of ZmNFXL1 mutant detection.

[0032] Figure 3 Phenotypic identification of ear rot in ZmNFXL1 mutants: A: Ears of B73 and its mutants (zmnfxl1-1 and zmnfxl1-3) before and after inoculation with *Fusarium verticillatum* and *Fusarium graminearum*. B: Phenotypic analysis of B73 and its mutants (zmnfxl1-1 and zmnfxl1-3) after inoculation with *Fusarium verticillatum*, *Fusarium graminearum*, and after natural disease.

[0033] Figure 4 Phenotypic identification of agronomic traits of the ZmNFXL1 mutant: A and B: Schematic diagrams of the mutants (zmnfxl1-1 and zmnfxl1-3) and their respective isolated wild-type plants. CG: Field agronomic trait survey and analysis of tasseling date, pollen shedding date, silking date, plant height, and ear height of zmnfxl1-1 and isolated wild-type plants. HL: Field agronomic trait survey and analysis of tasseling date, pollen shedding date, silking date, plant height, and ear height of zmnfxl1-3 and isolated wild-type plants.

[0034] Figure 5Phenotypic identification of ear rot in ZmNFXL1 gene-edited materials: A: Schematic diagram of ZmNFXL1 gene editing detection results. B and C: Field phenotypic identification and analysis of two gene-edited lines and wild-type B73-329 after inoculation with Fusarium verticillatum and Fusarium graminearum. D and E: Schematic diagram of ear phenotypes of two gene-edited lines and wild-type B73-329 after inoculation with Fusarium verticillatum and Fusarium graminearum.

[0035] Figure 6 Phenotypic identification of agronomic traits in ZmNFXL1 gene-edited materials: A: Schematic diagram of field plants of the two gene-edited types and wild-type B73-329. BF: Field agronomic trait survey and analysis of tasseling period, pollen shedding period, silking period, plant height, and ear height of the two gene-edited types and wild-type B73-329. Detailed Implementation

[0036] The following embodiments are used to illustrate the present invention, but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art.

[0037] Unless otherwise specified, all examples were performed under standard experimental conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (Sambrook J & Russell DW, 2001), or as recommended by the manufacturer.

[0038] Example 1: ZmNFXL1 expression induced by pathogens

[0039] Fifteen days after pollination, the resistant inbred line Qi 319 and the susceptible inbred line B104 were inoculated with Fusarium oxysporum using the filament channel method. Transcriptome analysis of maize kernels was performed at 0h, 12h, and 72h. The results showed that ZmNFXL1 expression was significantly induced in the susceptible inbred line B104, and was higher than that in the resistant inbred line Qi 319. Figure 1 This suggests that ZmNFXL1 may act as a susceptible gene involved in regulating resistance to maize ear rot.

[0040] Example 2: ZmNFXL1 gene CDS and protein sequence

[0041] Based on the gene and sequence information of the B73 reference genome, the nucleotide sequence of ZmNFXL1 in Qi319 was obtained by amplification using the primers in Table 1. Alignment analysis with the coding sequence of the ZmNFXL1 gene in the B73 reference genome showed a nucleic acid sequence homology of 99.58% and a protein homology of 99.27%. The CDS and protein sequences of ZmNFXL1 in Qi319 are shown in SEQ ID NO: 1 and SEQ ID NO: 3. The CDS and protein sequences of ZmNFXL1 in B73 are shown in SEQ ID NO: 2 and SEQ ID NO: 4.

[0042] Table 1. Primer sequences for ZmNFXL1 amplification

[0043]

[0044] Example 3: Phenotypic identification of the ZmNFXL1 mutant with ear rot

[0045] To further validate the phenotype of ZmNFXL1, two allelic mutants with a B73 background were purchased from the Institute of Modern Agricultural Technology, Qilu Normal University (http: / / maizeems.qlnu.edu.cn / ), and named zmnfxl1-1 and zmnfxl1-3, respectively. Sequence analysis (Table 2) revealed that the two mutations were CAG to TAG and TGG to TGA, respectively, both of which led to premature termination of protein translation. Figure 2 ).

[0046] In 2024, field phenotypic identification was conducted at the Changping Experimental Base of the Institute of Crop Science, Chinese Academy of Agricultural Sciences. The silk pathway method was used, and artificial inoculation was performed 7 days after maize pollination. The physiological races were *Fusarium verticillatum* and *Fusarium graminearum*. Natural disease identification was also conducted simultaneously. The results showed that compared to the control B73, the ZmNFXL1 mutant exhibited increased resistance to both *Fusarium verticillatum* and *Fusarium graminearum*. The average disease index of artificially inoculated *Fusarium verticillatum* B73 was 80.11%, while the average disease indices of the mutants (zmnfxl1-1 and zmnfxl1-3) were 44.88% and 63.21%, respectively. Figure 3 The average disease index of artificially inoculated Fusarium graminearum B73 was 62.22%, while the average disease index of mutants (zmnfxl1-1 and zmnfxl1-3) was 41.14% and 50.35%, respectively. Figure 3 Under natural disease conditions, the average disease index of the control B73 was 85.55%, while the average disease index of the mutants (zmnfxl1-1 and zmnfxl1-3) was 36.74% and 48.34%, respectively. Figure 3 ).

[0047] Table 2 Primer sequences for ZmNFXL1 mutant detection

[0048]

[0049] Example 4: Phenotypic identification of agronomic traits in the ZmNFXL1 mutant

[0050] To further verify whether the ZmNFXL1 mutation affects the phenotype of agronomic traits, wild-type mutants (controls) were isolated from the two mutants and named CK-zmnfxl1-1 and CK-zmnfxl1-3, respectively. Phenotypic identification of agronomic traits was then performed. The results showed that there were no significant differences between CK-zmnfxl1-1 and zmnfxl1-1, and between CK-zmnfxl1-3 and zmnfxl1-3, in agronomic traits such as tasseling date, pollen shedding date, silking date, plant height, and ear height. Figure 4 In summary, the ZmNFXL1 mutation enhances maize's resistance to Fusarium verticillatum and Fusarium graminearum without altering agronomic traits.

[0051] Example 5: Identification of ear rot phenotype in ZmNFXL1 gene-edited materials

[0052] To further validate the phenotype of ZmNFXL1, two allelic mutants, named KO-1 and KO-2, were obtained through gene editing using B73-329 (a susceptible inbred line) as the genetic background. Vector construction and genetic transformation were performed by Wimi Biotechnology Co., Ltd. (http: / / www.wimibio.com / ). Sequence analysis (Table 3) revealed that gene-edited line 1 was of the +1A type, and gene-edited line 2 was of the -175bp type. Both editing types led to premature termination of protein translation. Figure 5 A).

[0053] Two gene-edited strains and the wild-type B73-329 were artificially inoculated 7 days after maize pollination using the filamentous pathway method. The physiological races were identified as *Fusarium verticillatum* and *Fusarium graminearum*. The results showed that compared to the control B73-329, ZmNFXL1 simultaneously improved resistance to both *Fusarium verticillatum* and *Fusarium graminearum*. The average disease index of artificially inoculated *Fusarium verticillatum* B73-329 was 80.98%, while the average disease indices of the ZmNFXL1 gene-edited lines (KO-1 and KO-2) were 63.97% and 68.04%, respectively. Figure 5 The average disease index of artificially inoculated Fusarium graminearum B73-329 was 61.65%, while the average disease index of the ZmNFXL1 gene-edited lines (KO-1 and KO-2) was 49.15% and 47.31%, respectively. Figure 5 ).

[0054] Table 3 Primer sequences for detecting ZmNFXL1 gene-edited materials.

[0055]

[0056] Example 6: Phenotypic Identification of Agronomic Traits in ZmNFXL1 Gene-Edited Materials

[0057] To determine whether ZmNFXL1 gene editing affects agronomic traits, field evaluation results showed that the ZmNFXL1 gene-edited lines (KO-1 and KO-2) did not differ significantly from the wild-type B73-329 in agronomic traits such as tasseling date, pollen shedding date, silking date, plant height, and ear height. Figure 6 In summary, ZmNFXL1 gene editing improves maize's resistance to Fusarium verticillatum and Fusarium graminearum without altering agronomic traits.

[0058] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A gene related to enhancing resistance to ear rot in maize, wherein the nucleic acid molecule of the gene is a DNA molecule of the following type (a1), (a2), or (a3): (a1) The nucleotide shown in SEQ ID NO: 1 or SEQ ID NO: 2; (a2) A nucleotide encoding the amino acid shown in SEQ ID NO: 3 or SEQ ID NO: 4; or (a3) A nucleotide that can hybridize with the complementary sequence of SEQ ID NO: 1 or SEQ ID NO: 2 under strict hybridization conditions, and the protein encoded by the nucleotide has the function of the ZmNFXL1 transcription factor.

2. A protein associated with improved resistance to maize ear rot, as shown in (b1) or (b2): (b1) A protein consisting of the amino acid sequence shown in SEQ ID NO: 3 or SEQ ID NO: 4 in the sequence listing; (b2) The fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of (b1).

3. The application of the ZmNFXL1 nucleic acid molecule of claim 1, the protein of claim 2, or its inhibitor, or biological material containing its encoding gene or inhibitor, is as follows (c1) or (c2) or (c3): (c1) Regulates resistance to corn ear rot; (c2) Improve resistance to corn ear rot; (c3) Select and breed maize with high resistance to ear rot.

4. The application according to claim 3, characterized in that, Inhibiting the expression of the ZmNFXL1 protein or its encoding gene can enhance maize's resistance to ear rot.

5. The application according to claim 4, characterized in that, Suppressing ZmNFXL1 protein expression includes transcriptional regulation, post-transcriptional regulation, translational regulation, and post-translational regulation; suppressing the expression of the ZmNFXL1 encoding gene includes gene knockout, gene silencing, and gene expression vector optimization.

6. The application according to claim 5, characterized in that, Inhibition of ZmNFXL1 protein expression includes blocking transcription factor binding by binding to specific regions of DNA; inhibiting gene promoter activity through epigenetic modifications (such as methylation); targeting and degrading mRNA through miRNA or antisense oligonucleotides (ASO); and binding to mRNA and recruiting degrading enzymes (such as nucleases) to shorten its half-life. Using specific inhibitors (such as antibiotics or small molecule compounds) to block translation initiation or elongation; interfering with the function or stability of the target protein by binding to the target protein with antibodies or competitive molecules; and the ubiquitin-proteasome system (UPS) or lysosomal pathway to directionally degrade the target protein.

7. The application according to claim 5, characterized in that, Inhibiting ZmNFXL1 gene expression includes precise editing of the target gene using CRISPR-Cas9 or TALEN systems, gene knockout by introducing frameshift mutations or deletion sequences; designing specific siRNAs or shRNAs using RNA interference (RNAi) technology to degrade the target mRNA; avoiding vector construction errors (such as reading frame errors); and optimizing codon usage frequency to reduce translation efficiency. Design mRNA secondary structure interference sequences to prevent ribosome binding or translational elongation.

8. A method for creating maize germplasm resistant to ear rot, characterized in that, The method described above alters the performance of the ZmNFXL1 gene or suppresses its expression through natural mutation screening or artificial intervention.

9. The method as described in claim 8, characterized in that, The artificial intervention methods include gene editing technology and viral vector introduction.

10. A method for identifying whether maize is resistant to ear rot, characterized in that, The method includes the following steps: (1) The ZmNFXL1 gene was amplified by PCR using specific primers. (2) Compare whether the amplification products have the function of the ZmNFXL1 gene.

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