Use of bx1 protein and its encoding gene to improve plant resistance

Abstract

The application discloses application of a BX1 protein and a coding gene thereof in improving plant resistance, and belongs to the field of variation or genetic engineering. The technical problem solved by the application is how to improve the insect resistance and disease resistance of corn. The BX1 protein disclosed by the application is a protein as shown in SEQ ID No. 4. BX1 After a transcription enhancer as shown in SEQ ID No. 5 is inserted into the promoter of the gene, BX1 The expression amount of the gene is significantly improved, and the resistance of the corn to fusarium graminearum, asian corn borer and beet armyworm is also significantly improved. BX1 The corn with the insect resistance and disease resistance can be bred by improving the expression amount of the gene.

Description

Technical Field

[0001] This invention belongs to the field of mutation or genetic engineering, specifically relating to the application of the BX1 protein and its encoding gene in improving plant resistance. Background Technology

[0002] corn( Zea mays Maize (L.) is an important food crop. However, in recent years, with global warming and frequent extreme weather events, maize diseases and pests have become more frequent and severe, causing serious yield losses. Stalk rot is a common and prevalent disease of maize, caused by Fusarium graminearum (Fusarium graminearum). Fusarium graminearum (This is a common pathogen that causes disease.) It usually occurs during the reproductive growth stage, especially during the tasseling and silking period of corn, causing the corn roots and stems to turn brown and rot, and also leading to lodging of the plant. Important pests of corn include the Asian corn borer ((This is a common pest that causes disease in corn). Ostrinia furnacalis ), beet armyworm ( Spodoptera exigua Fall armyworm ( Spodoptera frugiperda ), armyworms ( Mythimna separataWalker The larvae of these pests primarily feed on corn leaves, and some later-stage pests will bore into the stalks or eat the male and female ears, hindering normal growth and yield formation. Therefore, improving the pest and disease resistance of corn is essential. Summary of the Invention

[0003] The technical problem to be solved by this invention is how to improve the insect resistance and disease resistance of corn.

[0004] To solve the above-mentioned technical problems, the present invention first provides the application of protein or a substance that increases the content of said protein in improving the resistance of maize, wherein said protein is BX1 protein, and said BX1 protein is the protein shown in SEQ ID No. 4; The resistance is insect resistance and / or disease resistance, and the insect resistance is resistance to the beet armyworm ( ). Spodoptera exigua ) and / or Asian corn borer ( Ostrinia furnacalis (Guenée)) resistance; the resistance is against Fusarium graminearum ( Fusarium graminearum Resistance to ).

[0005] This invention also provides the application of biomaterials in improving maize resistance, wherein the biomaterials are any one of D1)-D6): D1) Nucleic acid molecules, expression cassettes, recombinant vectors, or recombinant microorganisms that enhance the expression level of the BX1 gene; wherein the BX1 gene encodes the BX1 protein; D2) The nucleic acid molecule encoding the BX1 protein; D3) An expression cassette containing the nucleic acid molecules described in D2); D4) A recombinant vector containing the nucleic acid molecule described in D2) or the expression cassette described in D3); D5) Recombinant microorganisms containing the nucleic acid molecule described in D2), the expression cassette described in D3), or the recombinant vector described in D4); D6) Plant cells, tissues or organs containing the nucleic acid molecule described in D2), the expression cassette described in D3), or the recombinant vector described in D4); The resistance is insect resistance and / or disease resistance, wherein the insect resistance is resistance to beet armyworm and / or Asian corn borer; and the disease resistance is resistance to Fusarium graminearum.

[0006] Specifically, the nucleic acid molecule described in D1) can be a negative regulator in the BX1 pathway. The nucleic acid molecule described in D1) can also be a DNA molecule. Further, the DNA molecule is a transcriptional enhancer. Even further, the transcriptional enhancer is the DNA molecule shown in SEQ ID No. 5.

[0007] The term "transcriptional enhancer" (also referred to herein as an enhancer) refers to DNA that enhances the transcription of the BX1 gene, which is operatively linked to it. In other words, an enhancer is DNA that has enhanced transcriptional activation relative to the natural regulatory sequence of the BX1 gene. In some embodiments of the invention, the natural regulatory sequence of the BX1 gene is the promoter of the BX1 gene. In one embodiment of the invention, the promoter of the BX1 gene is the DNA molecule shown in SEQ ID No. 2.

[0008] In this application, the term "operable link" refers to an operable link between segments of a nucleic acid that are functionally related to each other. In some embodiments, the operably linked promoter and enhancer of the BX1 gene result in transcription of the open reading frame of the BX1 gene.

[0009] The term "vector" refers to any construct that can introduce heterologous DNA into corn cells.

[0010] The term "construct" refers to any recombinant DNA molecule. Recombinant DNA molecules can be plasmids, granules, viruses, or bacteriophages.

[0011] In one embodiment of the present invention, the enhancer is the DNA molecule shown in SEQ ID No. 5.

[0012] In one embodiment of the present invention, D1) the recombinant vector is a GRAND editing vector, wherein the GRAND editing vector is formed by combining two elements from the p5175-GZ vector. Bsa The recombinant vector was obtained by replacing the sequence between the I restriction sites with the DNA fragment shown in SEQ ID No. 6.

[0013] D2) The nucleic acid molecule can be DNA, such as cDNA of the BX1 gene, genomic DNA, or recombinant DNA; the nucleic acid molecule can also be RNA, such as mRNA or hnRNA.

[0014] Specifically, the BX1 gene is any one of the following: b11) The coding sequence is a DNA molecule of SEQ ID No. 3; b12) The nucleotide sequence is a DNA molecule of SEQ ID No. 3 or SEQ ID No. 1.

[0015] The expression cassette (BX1 gene expression cassette) containing a nucleic acid molecule encoding the BX1 protein, as described in D3, refers to a single-stranded or double-stranded nucleic acid molecule capable of expressing the BX1 protein in a host cell. This DNA includes all regulatory sequences necessary for the expression of the BX1 protein in the nucleic acid molecule. The regulatory sequences, under compatible conditions, guide the expression of the aforementioned BX1 protein in a suitable host cell. The regulatory sequences include, but are not limited to, a leader sequence, a polyadenylated sequence, a propeptide sequence, a promoter, an enhancer, a signal sequence, and a transcription terminator. At a minimum, the regulatory sequences must include a promoter and termination signals for transcription and translation. To introduce specific restriction enzyme sites into the vector for linking the regulatory sequences to the coding region of the nucleic acid sequence encoding the protein, a regulator-linked regulatory sequence may be provided. The regulatory sequence may be a suitable promoter sequence, i.e., a nucleic acid sequence that can be recognized by the host cell expressing the nucleic acid sequence. The promoter sequence contains transcriptional regulatory sequences that mediate protein expression. The promoter can be any nucleic acid sequence that is transcriptionally active in the selected host cell, including mutated, truncated, and heterozygous promoters, and can be derived from genes encoding extracellular or intracellular proteins that are homologous or heterologous to those of the host cell. The regulatory sequence can also be a suitable transcription termination sequence, i.e., a sequence that can be recognized by the host cell and thus terminate transcription. The termination sequence is operatively attached to the 3' end of the nucleic acid sequence encoding the protein. Any terminator that can function in the selected host cell can be used in this invention. The regulatory sequence can also be a suitable leader sequence, i.e., an untranslated region of mRNA that is crucial for translation in the host cell. The leader sequence is operatively attached to the 5' end of the nucleic acid sequence encoding the protein. Any leader sequence that can function in the selected host cell can be used in this invention. The regulatory sequence can also be a signal peptide coding region, which encodes an amino acid sequence linked to the amino terminus of a protein that guides the encoded protein into the cellular secretion pathway. Signal peptide coding regions that can guide the expressed protein into the secretion pathway of the host cell can be used in this invention. Adding a regulatory sequence that can regulate protein expression according to the growth status of the host cell may also be necessary. Examples of regulatory systems are those that respond to chemical or physical stimuli (including in the presence of regulatory compounds), thereby turning gene expression on or off. Other examples of regulatory sequences are those that enable gene amplification. In these cases, the nucleic acid sequence encoding the protein should be operatively linked to the regulatory sequence.

[0016] Recombinant vectors containing the BX1 gene expression cassette can be constructed using existing expression vectors.

[0017] The vector may be a plasmid, granule, bacteriophage, or viral vector.

[0018] D6) The plant referred to is a plant cell, plant tissue, plant organ and / or whole plant.

[0019] "Plant cell" is the biological cell of a plant, which is taken from the plant or derived from a culture obtained by culturing cells taken from the plant.

[0020] The plants described in this application can be viable, non-viable, renewable, and / or non-renewable. The plants described in this application include propagules or propagation material. "Propagules or propagation material" can include any plant part that can grow into a whole plant. "Plant part" can refer to any organ or tissue of a plant, such as meristematic tissue, bud organs / structures (e.g., leaves, stems, or nodes), roots, flowers or floral organs / structures (e.g., flowers, bracts, sepals, petals, stamens, carpels, anthers, and ovules), seeds (e.g., embryo, endosperm, and seed coat), fruits (e.g., mature ovaries), propagules or other plant tissues (e.g., vascular tissue, dermal tissue, ground tissue, etc.) or any part thereof.

[0021] The present invention also provides a method for breeding maize with enhanced resistance, the method comprising increasing the expression level of the gene encoding the BX1 protein in the maize to be improved, thereby obtaining the target maize with enhanced resistance; The resistance is insect resistance and / or disease resistance, wherein the insect resistance is resistance to beet armyworm and / or Asian corn borer; and the disease resistance is resistance to Fusarium graminearum.

[0022] The target corn exhibits higher insect resistance and / or disease resistance than the corn to be improved.

[0023] The target maize includes at least one of its seeds, hybrid F1 and its offspring.

[0024] In some embodiments of the present invention, the maize to be improved contains the BX1 protein or the gene encoding the BX1 protein (BX1 gene).

[0025] In one embodiment of the present invention, the corn to be improved is sweet waxy corn inbred line N355.

[0026] The enhancement of the expression level of the gene encoding the BX1 protein in the maize to be improved is achieved by: inserting the enhancer into the natural promoter of the BX1 gene, modifying the promoter region of the BX1 gene, and / or driving the transcription of the BX1 gene with a heterologous strong promoter. In some embodiments, the heterologous strong promoter is a DNA molecule that has a stronger ability to drive the transcription of the BX1 gene than the natural promoter of the BX1 gene.

[0027] In one embodiment of the present invention, the natural promoter of the BX1 gene is the DNA molecule shown in SEQ ID No. 2.

[0028] Specifically, the expression level of the gene encoding the BX1 protein in the maize to be improved is achieved by inserting an enhancer shown in SEQ ID No. 5 of the maize genome into the promoter of the BX1 gene in the maize to be improved.

[0029] In one embodiment of the invention, the enhancer shown in SEQ ID No. 5 replaces the DNA fragment shown in the reverse complementary sequence at positions 881-898 of SEQ ID No. 2.

[0030] The present invention also provides a stress-resistant maize, wherein the stress-resistant maize is obtained by replacing the DNA fragment with the nucleotide sequence of SEQ ID No. 11 in the promoter of the gene encoding the BX1 protein of the maize to be improved with a DNA fragment with the nucleotide sequence shown in SEQ ID No. 12. The stress-resistant maize has higher insect resistance and / or disease resistance than the maize to be improved. The insect resistance is resistance to beet armyworm and / or Asian corn borer; the disease resistance is resistance to Fusarium graminearum.

[0031] The stress-resistant maize is obtained by inserting the enhancer shown in SEQ ID No. 5 into the promoter of the BX1 gene of the maize to be improved.

[0032] Furthermore, the DNA fragment shown in the reverse complementary sequence at positions 881-898 of SEQ ID No. 2 in the enhancer maize genome of SEQ ID No. 5 is a replacement for the DNA fragment shown in the sequence.

[0033] The resistance in this invention refers to comparisons made under comparable conditions. "Comparable conditions" are the same or similar environmental conditions and agronomic practices used to make meaningful comparisons between two or more maize genotypes, such that neither the environmental conditions nor the agronomic practices significantly promote or explain any differences observed between the two or more maize genotypes. Environmental conditions include, for example, light, temperature, water, humidity, soil, and nutrients (e.g., nitrogen and phosphorus).

[0034] In some embodiments, the drought resistance is compared to maize without the enhancer. In one embodiment, the maize without the enhancer is the sweet waxy maize inbred line N355.

[0035] This invention targets corn by editing GRAND. BX1 A 26bp transcriptional enhancer sequence was inserted into the promoter region of a gene, resulting in homozygous edited maize. BX1Significantly increased gene expression levels led to significantly improved resistance to Fusarium graminearum, Asian corn borer, and beet armyworm. Plant height was significantly reduced, ear size and ear weight were significantly increased, and there was no negative impact on yield. This indicates that increasing gene expression levels can significantly improve resistance to Fusarium graminearum, Asian corn borer, and beet armyworm. BX1 Gene expression levels were used to cultivate maize with insect-resistant and disease-resistant traits. Attached Figure Description

[0036] Figure 1 . Schematic diagram of the GRAND editing media.

[0037] Figure 2 STE-26 strain BX1 Gene expression levels. Note: t-test used. ****: P <0.0001. STE-26 indicates the STE-26 strain.

[0038] Figure 3 Stalk disease incidence after field inoculation with Fusarium graminearum. Note: The blank control is WT maize plants injected with the same volume of H2O; STE-26 represents the STE-26 strain. The figure shows the injected stem node (the fourth stem node of the maize plant closest to the ground). Scale bar = 5 cm.

[0039] Figure 4 Body weight change in beet armyworm. Note: (a) Body weight change curve; (b) Body weight growth rate. STE-26 indicates the STE-26 strain. Using t-test, *: P <0.05.

[0040] Figure 5 Day 11: Beet armyworm feeding on leaves. STE-26 indicates the STE-26 strain. Note: Scale bar = 1 cm.

[0041] Figure 6 Body weight change in Asian corn borer. Note: (a) Body weight change curve; (b) Body weight growth rate. STE-26 represents the STE-26 strain. Using t-test, ***: P <0.001.

[0042] Figure 7 Day 11: Corn borer leaf-eating situation. STE-26 indicates the STE-26 strain. Note: Scale bar = 1 cm.

[0043] Figure 8 Results of maize agronomic traits. Note: (a) ear height; (b) number of rows per ear; (c) number of kernels per row; (d) number of kernels per ear; (e) 100-kernel weight; (f) kernel weight per ear; (g) kernel length; (h) kernel width; (i) tip barrenness length. STE-26 indicates the STE-26 line. t-test was used, ns: no significant difference. Detailed Implementation

[0044] In this document, unless otherwise defined herein, terms should be understood according to their common usage by those skilled in the art. Examples of resources describing many of the molecular biology-related terms used herein can be found in the following references: Alberts et al., Molecular Biology of The Cell, 5th ed., Garland Science Publishing, Inc.: New York, 2007; Rieger et al., Glossary of Genetics: Classical and Molecular, 5th ed., Springer-Verlag: New York, 1991; King et al., A Dictionary of Genetics, 6th ed., Oxford University Press: New York, 2002; and Lewin, GenesIX, Oxford University Press: New York, 2007.

[0045] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0046] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials, reagents, instruments, etc., used in the following examples are commercially available.

[0047] In the quantitative experiments described below, at least three replicate experiments were performed.

[0048] The waxy maize inbred line N355 in the following examples is "the waxy maize cultivars N355" disclosed in the following literature. This biological material is available to the public from the applicant and is used solely for replicating the relevant experiments of this invention; it may not be used for any other purpose. (Wang, Y.,) et al . (2021). Creation of aromaticmaize by CRISPR / Cas. J. Integr. Plant Biol. 63: 1664–1670.

[0049] Fusarium graminearum in the following examples ( Fusarium graminearum ): Shandong Shunfeng Biotechnology Co., Ltd.

[0050] Example 1: Improving corn BX1 Gene expression levels can enhance the insect resistance and disease resistance of maize. This embodiment found that improving corn BX1 Gene expression levels can enhance insect resistance and disease resistance in maize. In the sweet waxy maize inbred line N355, BX1 The genome sequence of the gene is SEQ ID No. 1 (exons are positions 669-974, 1112-1421, 1516-1592, 1672-1749, 1881-2018, and 2117-2251 of SEQ ID No. 1), driving... BX1 The sequence of the promoter for gene expression is SEQ ID No. 2. BX1 The CDS sequence of the gene is SEQ ID No. 3, which encodes the BX1 protein shown in SEQ ID No. 4.

[0051] I. Construction of Recombinant Vectors Using GRAND editing methods BX1 The transcriptional enhancer STE-26 (STE: Short Transcriptional Enhancer) was inserted upstream of the gene. This clarified the N355 maize gene. BX1 Gene promoter sequences were analyzed to identify the regulatory elements present within them. BX1 STE-26 was inserted 139 bp upstream of the transcription start site, avoiding important regulatory elements to prevent fragmentation and disruption after insertion. The inserted transcription enhancer STE-26 is 26 bp in length and its sequence is SEQ ID No. 5.

[0052] To construct the GRAND editing vector, the target was first designed and synthesized. BX1 DNA fragments of epigRNAs that are gene promoters and inserted into transcription enhancers (named Spacer1-scaffold-RTT1-PBS-linker-evopreQ1-HDV-linker-tRNA (Met)-Spacer2-scaffold-RTT2-PBS2-linker-evopreQ1, sequence SEQ ID No. 6).

[0053] In SEQ ID No. 6, positions 1-20 are the DNA sequence of Spacer 1, positions 21-106 are the DNA sequence of scaffold, positions 107-124 are the DNA sequence of RTT1, positions 125-137 are the DNA sequence of PBS, positions 138-145 are the DNA sequence of linker, positions 146-183 are the DNA sequence of evopreQ1, positions 1-183 are the DNA sequence of epigRNA1; positions 184-251 are the DNA sequence of HDV, and positions 252-259 are the DNA sequence of linker (…). The DNA sequence of the linker is as follows: positions 260-331 are the DNA sequence of tRNA (Met); positions 332-351 are the DNA sequence of Spacer2; positions 352-437 are the DNA sequence of scaffold; positions 438-455 are the DNA sequence of RTT2; positions 456-468 are the DNA sequence of PBS2; positions 469-476 are the DNA sequence of the linker; positions 477-514 are the DNA sequence of evopreQ1; and positions 332-514 are the DNA sequence of epigRNA2.

[0054] The ApR and scaffold sequences of the maize guided editing (PE3) vector p5175 were deleted, and the resulting vector was named p5175-GZ. p5175-GZ is a double-stranded plasmid with a length of 27848 bp. The first 15000 positions of one strand are the first 15000 positions of SEQ ID No. 7, and the first 15001-27848 positions are the first 12848 positions of SEQ ID No. 8.

[0055] Bits 391-1384 of SEQ ID No. 7 represent the promoter. pU6-Com Positions 1392-1462 show the DNA sequence of tRNA (Gly), and positions 1464-1469 and 1478-1483 show... Bsa The recognition sequence for I, positions 1493-1560, is the DNA sequence of HDV.

[0056] Using the p5175-GZ vector as the basic framework, the two vectors in the p5175-GZ vector were separated using the Golden Gate method. Bsa The sequence between the I restriction sites was replaced with DNA fragments of synthesized epigRNAs. The resulting recombinant vector with the correct sequence was designated as the GRAND editing vector. This GRAND editing vector contains a promoter... pU6-ComThe transcription of epigRNA is driven by the ZmUBI promoter, which drives the expression of the nCas9 (H840A) recombinant gene bpNLS-nCas9(H840A)-XTEN-NC-NLS-LINKER-M-MLV-bpNLS protein. Figure 1 ).

[0057] The sequence of the nCas9(H840A) recombinant gene is SEQ ID No. 9, encoding the nCas9(H840A) recombinant protein shown in SEQ ID No. 10.

[0058] In SEQ ID No. 9, positions 1-117 are the DNA sequence of bpNLS, positions 118-4218 are the DNA sequence of nCas9 (H840A), positions 4219-4335 are the DNA sequence of XTEN, positions 4336-4503 are the DNA sequence of NC, positions 4504-4524 are the DNA sequence of NLS, positions 4525-4632 are the DNA sequence of LINKER, positions 4633-6174 are the DNA sequence of M-MLV, and positions 6175-6306 are the DNA sequence of bpNLS.

[0059] II. Agrobacterium-mediated genetic transformation and genotype identification of maize Using sweet waxy maize inbred line N355 as the recipient plant, the GRAND editing vector was introduced into Agrobacterium tumefaciens EHA105. The embryos were then infected using Agrobacterium-mediated maize genetic transformation to obtain callus tissue, which was then further cultured to obtain T0 generation transgenic plants.

[0060] To identify the genotype of T0 generation transgenic plants, genomic DNA was extracted from leaves. Two rounds of PCR were used to amplify the target sites, followed by next-generation sequencing (NGS) analysis. Editing efficiency was determined using a 10% threshold in the Hi-TOM assay.

[0061] Sequencing results showed that the editing efficiency of precise insertion of the transcriptional enhancer STE-26 was 13.04% (3 / 23), while the efficiency of non-targeted editing insertions and deletions was 17.39% (4 / 23) (Table 1). The target edited plants that underwent precise insertion of STE-26 (plant numbers #1, #10, and #21) were all heterozygous (Table 2), and further self-pollination is needed to isolate homozygous plants.

[0062] Table 1. Editing efficiency of GRAND editing in T0 generation transgenic plants

[0063] Table 2. Genotypes of T0 generation target edited plants

[0064] Note: The bold nucleotides indicate inserted nucleotides.

[0065] III. Detection of Maize by Real-time Fluorescent Quantitative PCR (qPCR) BX1 Gene Expression Level To identify changes in the expression level of maize genes after insertion of the transcriptional enhancer, BX1 RNA was extracted from leaves of homozygous T3 generation of target edited plant #1 containing STE-26 (designated as STE-26 line, which is homozygous maize comprising the DNA fragment shown by the reverse complementary sequence at positions 881-898 of SEQ ID No.2 replaced by the enhancer shown in SEQ ID No.5) and reverse transcribed into cDNA. Primers were designed according to the CDS sequence of the maize BX1 gene, with a length of 18-24 nt, a GC content of 40%-60%, a Tm value of 58-62°C, and an amplification product length of 100-200 bp. The qPCR reaction was performed using Hieff UNICON® Universal Blue qPCR SYBR Green Master Mix (Yeasen Biotech), the relative gene expression level was calculated according to the resulting ct value, and a differential bar graph was drawn. As shown below: Primers Used BX1 Gene primers are: q-BX1-F: 5′-CCCGAGCACGTAAAGCAGAT-3′ (SEQ ID No.14); q-BX1-R: 5′-CTTCATGCCCCTGGCATACT-3′ (SEQ ID No.15).

[0066] The internal reference gene is GAPDH gene, with the following primers: q-GAPDH-F: 5′-AGCAGGTCGAGCATCTTCG-3′ (SEQ ID No.16); q-GAPDH-R: 5′-CTGTAGCCCCACTCGTTGTC-3′ (SEQ ID No.17).

[0067] The qPCR results showed that in the edited maize line inserted with STE-26 BX1 the gene expression level was 7.7 times that of wild-type maize N355 (WT) ( Figure 2 ), indicating that this transcriptional enhancer effectively up-regulated BX1 the expression level of the gene.

[0068] IV. Identification of Maize Disease Resistance by Field Inoculation Maize to be tested: STE-26 line, wild-type maize N355 (WT).

[0069] Fusarium graminearum ( Fusarium graminearum The bacterial strain was inoculated onto PDA medium and cultured at 28°C for approximately 5 days. Mycelial discs (approximately 0.5 cm in diameter) were pressed out from the medium and transferred into mung bean soup medium using a sterile syringe needle, 20 discs per 100 mL culture bottle. The culture bottles were then placed in a shaker at 28°C and 200 rpm to induce sporulation. After 4 days of culture, the bacterial suspension was filtered through double-layered sterile gauze, and the spores were collected by centrifugation. The spores were resuspended in sterile water to prepare a spore suspension, and the concentration was adjusted to 10. 6 / mL. Inoculation was performed during the flowering stage of the corn to be tested. A cone was inserted at a 45° angle downwards into the middle of the fourth stem node closest to the ground, ensuring the cone reached the middle of the stem without penetrating it. 1.5 mL of spore suspension was injected into the wound using a syringe. Simultaneously, an equal volume of sterile water was injected into wild-type corn as a blank control. 25 plants were inoculated for each line. The field was watered once before inoculation, followed by normal fertilizer and water management. No pesticides were applied before or after inoculation. Twelve days later, each plant was identified. The injected stem node was cut off with a sickle, and the stem was longitudinally sectioned along the wound. The percentage of lesions on the cross-section was counted for each plant, photographed, and the disease severity grade of each plant was recorded. The grading criteria are shown in Table 3. The disease severity grade of each line was calculated to evaluate the disease resistance and susceptibility level.

[0070] Table 3. Disease severity classification of maize inoculated with Fusarium graminearum

[0071] Disease severity of each strain = (M1 + M2 + … + M) R ) / R, where R is the total number of replicates for the strain; M1, M2...M R These represent the disease severity of the 1st, 2nd...Rth repeating plants, respectively.

[0072] Compared with wild-type maize, the lesions on the stems of the STE-26 line were significantly reduced. Figure 3 Based on this assessment, the disease grade of the STE-26 strain was also significantly reduced (Table 4), decreasing from grade 5.67 for wild-type maize to grade 4.52. (Note: This likely refers to a separate point in the original text.) BX1 Maize with increased gene expression levels exhibits enhanced resistance to Fusarium graminearum.

[0073] Table 4. Disease severity of plants after field inoculation with Fusarium graminearum

[0074] Note: Data is presented as mean ± standard deviation. **: P <0.01.

[0075] V. Indoor Insect Inoculation Test to Determine Corn's Insect Resistance The maize to be tested was the STE-26 line, wild-type maize N355 (WT).

[0076] beet armyworm ( Spodoptera exigua ) or Asian corn borer ( Ostrinia furnacalis (Guenée) Insect eggs were placed in a culture box and cultured at 26℃ with 16 h light / 8 h dark to obtain newly hatched larvae. Young heart leaves of the test corn (four to five leaves and one heart stage) grown in a greenhouse were taken, cut into appropriate sizes, and placed in a 12-well culture plate. One newly hatched larva was inoculated into each well using a paintbrush. Each strain had one 12-well plate, inoculated with 12 larvae, and cultured at 26℃ with 16 h light / 8 h dark. The day of inoculation was recorded as day 0 (0d). Three days after inoculation, the leaves were photographed to show signs of larval damage, the larvae's weight was measured, and fresh leaves were replaced. Fresh leaves were then replaced every other day, and the larvae's weight was recorded for 11 days. The larvae's growth curve was statistically analyzed, and the weight growth rate was calculated. The weight on day 3 after inoculation was taken as the initial weight (W0), and the weight on day 11 (t=11) was taken as the final weight (W). t ), calculate the weight gain rate, weight gain rate = (W t -W0) / W0. During the experiment, it is necessary to keep edible leaves in the culture plate. Therefore, the frequency of replacing fresh leaves can be adjusted according to the grazing situation after 8-11 days.

[0077] Feeding trials involving beet armyworm showed that beet armyworms fed STE-26 strain leaves experienced significantly slower weight gain compared to those fed wild-type corn. Figure 4 In the middle (a) group, the weight gain rate on day 11 (d) was significantly reduced, with a decrease of 26.81%. Figure 4 (b) indicates that insect growth was inhibited. Observing the leaf erosion, both leaves of the wild-type corn were eaten, while in the STE-26 strain, most leaves had only one obvious insect hole or neither leaf had obvious insect holes. Figure 5 ).

[0078] Feeding trials involving Asian corn borers showed that the weight change curves of corn borers fed with STE-26 strain leaves were significantly slower than those of wild-type corn. Figure 6 In the middle (a) group, the weight gain rate on day 11 (d) decreased significantly, with a decrease of 39.36%. Figure 6 (b)). Regarding leaf erosion, wild-type corn leaves typically show insect holes the size of match heads, while the STE-26 strain leaves show more pinhole-like insect holes. Figure 7 These results illustrate BX1 Increased gene expression levels can enhance corn's resistance to insects.

[0079] VI. Field Agronomic Traits Survey The maize to be tested was the STE-26 line, wild-type maize N355 (WT).

[0080] A randomized block design was used, with three replicate plots for each maize variety. Ridges were prepared in the field, 1.15 m wide, with two rows per ridge. The standard plot size was 5 m² with four rows, plant spacing of 24 cm, and 20 plants per row, with single or double seeding depending on seed quantity. Four protective rows were placed around the perimeter of the maize. Before sowing, 25 kg of 15-15-15 Stanley compound fertilizer and 20 kg of microbial fertilizer were applied per mu (approximately 0.067 hectares). One week after pollination, 10 plants from the middle two rows of each plot were surveyed, and ear height was measured and recorded. Forty-five days after pollination, the middle two rows of ears were harvested from each plot, and yield-related agronomical traits such as number of rows per ear, number of kernels per row, number of kernels per ear, 100-kernel weight, ear weight, kernel length, kernel width, and tip barrenness length were measured, along with yield per mu. Kernel moisture content was measured, and yield calculations were based on a standard moisture content of 14%.

[0081] The results showed that homozygous T3 generation edited maize did not differ significantly from wild-type maize in traits such as ear height, number of rows per ear, number of kernels per row, number of kernels per ear, 100-kernel weight, kernel weight per ear, tip barrenness length, and kernel size (including kernel length and kernel width). Figure 8 In terms of yield, there was no significant difference between homozygous T3 generation edited maize and wild-type maize, indicating that the improved resistance did not have a negative impact on yield (Table 5).

[0082] Table 5. T3 generation maize yield

[0083] Note: Data is presented as mean ± standard deviation.

[0084] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.

Claims

1. The application of protein in improving maize resistance, characterized by: The protein is BX1 protein, which is the protein shown in SEQ ID No. 4; The resistance mentioned refers to resistance to the Asian corn borer.

2. The application of biomaterials in improving maize resistance, characterized by: The biomaterial is any one of D1)-D6): D1) Nucleic acid molecules, expression cassettes, recombinant vectors, or recombinant microorganisms that enhance the expression level of the BX1 gene; wherein the BX1 gene encodes the BX1 protein as described in claim 1; D2) The nucleic acid molecule encoding the BX1 protein; D3) An expression cassette containing the nucleic acid molecules described in D2); D4) A recombinant vector containing the nucleic acid molecule described in D2) or the expression cassette described in D3); D5) Recombinant microorganisms containing the nucleic acid molecule described in D2), the expression cassette described in D3), or the recombinant vector described in D4); D6) A plant containing the nucleic acid molecule described in D2), the expression cassette described in D3), or the recombinant vector described in D4); The resistance mentioned refers to resistance to the Asian corn borer.

3. The application according to claim 2, characterized in that: The nucleic acid molecule mentioned in D1) is a DNA molecule.

4. The application according to claim 3, characterized in that: The DNA molecule is an enhancer, and the enhancer is the DNA molecule shown in SEQ ID No.

5.

5. The application according to any one of claims 2, 3 or 4, characterized in that: The BX1 gene is any one of the following: b11) The coding sequence is a DNA molecule of SEQ ID No. 3; b12) The nucleotide sequence is a DNA molecule of SEQ ID No. 3 or SEQ ID No.

1.

6. A method for breeding maize with enhanced resistance, characterized in that: The method includes increasing the expression level of the gene encoding the BX1 protein of claim 1 in the maize to be improved, thereby obtaining maize with improved resistance; The resistance mentioned refers to resistance to the Asian corn borer.

7. The method according to claim 6, characterized in that: The enhancement of the expression level of the gene encoding the BX1 protein of claim 1 in the maize to be improved is achieved by inserting the enhancer shown in SEQ ID No. 5 into the promoter of the BX1 gene in the maize to be improved.

8. Stress-resistant corn, characterized by: The stress-resistant corn is obtained by replacing the DNA fragment with the nucleotide sequence SEQ ID No. 11 in the promoter of the gene encoding the BX1 protein of the corn to be improved in claim 1 with a DNA fragment with the nucleotide sequence shown in SEQ ID No.

12. The insect resistance of the stress-resistant corn is higher than that of the corn to be improved, and the insect resistance is resistance to the Asian corn borer.

9. The stress-resistant maize according to claim 8, characterized in that: The stress-resistant corn is prepared according to the method described in claim 7.

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