Method for cultivating corn southern leaf blight resistant corn plant through rBm1 gene and application

Abstract

The invention provides a method for cultivating a corn southern leaf blight resistant corn plant through an rBm1 gene, which comprises the following steps: overexpressing the rBm1 gene in corn through a gene engineering method to obtain a target corn plant; the nucleotide sequence of the rBm1 gene is as shown in SEQ ID NO. 1. The invention further provides application of the method. According to the invention, the expression level of the rBm1 gene is regulated and controlled in the corn plant, so that the oriented improvement of the corn plant on the resistance of the corn southern leaf blight is realized, a new corn plant capable of stably resisting the corn southern leaf blight is further cultivated, and a technical scheme and a gene target which can be directly applied are provided for corn disease-resistant breeding.

Description

Technical Field

[0001] This invention relates to the fields of plant molecular biology and genetic engineering, and particularly to a method for... rBm1 Methods and applications of genetically bred maize plants resistant to maize leaf spot disease. Background Technology

[0002] Corn is the world's most important crop used for food, feed, and industrial raw materials, playing an irreplaceable role in ensuring food security and supporting the development of the industrial chain. As a staple food, it is a vital source of energy and nutrition for the daily diet of a large population; as feed, it is a key energy source for meat, egg, and dairy products in global livestock production; simultaneously, it is widely used in industrial fields such as bioenergy and starch processing, serving as a crucial link between agriculture and industry. With the continued expansion of the global population and the upgrading of consumption demands, the total demand for corn is showing a steady upward trend, highlighting the increasing pressure to ensure stable corn production capacity and improve yield per unit area.

[0003] However, biological stress is a core bottleneck restricting high and stable yields of maize, among which the ascomycete fungus *Sclerotium micranthum* (maize leaf spot fungus) is a major cause. Bipolaris maydis Small leaf spot disease, caused by [unspecified disease name], is one of the most devastating foliar diseases affecting maize production areas worldwide. In its early stages, the disease manifests as irregular lesions on the lower leaves, which can spread and cover the entire leaf, significantly reducing photosynthetic efficiency and impacting grain filling and yield. In severe cases, it can cause substantial yield losses and pose a significant threat to the sustainable development of the maize industry.

[0004] Breeding disease-resistant varieties is the greenest and most sustainable strategy for controlling maize leaf spot disease, and elucidating the molecular mechanisms of maize disease resistance and identifying key resistance genes are the core prerequisites for disease-resistant breeding. Over long-term evolution, plants have developed a multi-layered defense network consisting of model-triggered immunity and effector-triggered immunity. The precise regulation of this network depends on the functional expression of disease-related genes, such as genes encoding pathogen recognition receptors and disease resistance signal transduction proteins. In recent years, with the development of molecular biology techniques, researchers have identified [various pathogens] in maize. ZmH2B , ChnagG , rhm1 While there are a few genes related to resistance to corn leaf blight, the corn leaf blight pathogen exhibits rapid evolutionary characteristics, and single resistance genes are easily rendered ineffective due to pathogen mutations, making it difficult for existing resistant varieties to maintain their resistance for long.

[0005] Meanwhile, existing technologies for identifying resistance genes against maize leaf spot still have significant shortcomings: on the one hand, most discovered genes focus on specific resistance pathways, and the global regulatory network of maize's response to leaf spot fungus infection is insufficiently analyzed; on the other hand, most genes have not yet been developed into technical solutions that can be directly applied to breeding, making it difficult to quickly transform them into new resistant germplasm. Therefore, identifying new, functionally defined key genes for resistance to maize leaf spot can not only fill the theoretical gaps in the molecular mechanisms of maize disease resistance but also provide efficient candidate targets for molecular design breeding, which is of great theoretical and practical significance for breaking through existing bottlenecks in disease resistance breeding and ensuring the stability of global maize production capacity. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to provide a method through rBm1 Methods and applications of gene-based breeding of maize plants resistant to maize leaf spot disease, which involves regulating the growth of maize plants... rBm1 By improving gene expression levels, we can achieve targeted improvement of maize plant resistance to maize leaf spot disease, thereby cultivating new maize plants that are stably resistant to leaf spot disease, and providing directly applicable technical solutions and gene targets for maize disease-resistant breeding.

[0007] The present invention solves the above-mentioned technical problems by adopting the following technical solutions: A way rBm1 Methods for genetically breeding maize plants resistant to maize leaf spot disease, using genetic engineering methods to... rBm1 The gene was overexpressed in maize to obtain the target maize plants; rBm1 The nucleotide sequence of the gene is shown in SEQ ID NO.1.

[0008] As one of the preferred embodiments of the present invention, the rBm1 The amino acid sequence encoded by the gene is shown in SEQ ID NO.2.

[0009] As one of the preferred embodiments of the present invention, the rBm1 The expression products of genes positively regulate the resistance of maize plants to maize leaf spot disease.

[0010] As one of the preferred embodiments of the present invention, the genetic engineering method includes the following specific steps: (1) Contains rBm1 Construction of plant expression vectors for genes The plant expression vector backbone was obtained and linearized; cDNA from maize material was used as a template for PCR amplification to obtain... rBm1 The CDS sequence of the gene is shown in SEQ ID NO.1; the linearized vector and rBm1 Recombination ligation of gene CDS sequences yields a product containing... rBm1 Plant expression vectors for genes; (2) Genetic transformation The plant expression vector constructed in step (1) was introduced into maize receptor material and cultured to obtain candidate plants; (3) Positive screening Detection step (2) among candidate plants rBm1 Gene expression levels, screening rBm1 Plants in which the gene is successfully overexpressed are the target maize plants.

[0011] As one of the preferred embodiments of the present invention, in step (1), the plant expression vector backbone is the pZZ00026 vector, and the linearization treatment is performed using the SmaI restriction endonuclease.

[0012] As one of the preferred embodiments of the present invention, in step (1), the recombination connection method is homologous recombination, the recombination reaction temperature is 37°C, and the reaction time is 2h.

[0013] As one of the preferred embodiments of the present invention, in step (2), the maize recipient material is maize inbred line KN5585, and the introduction method is Agrobacterium-mediated transformation.

[0014] As one of the preferred embodiments of the present invention, in step (3), rBm1 Gene expression level detection was performed using real-time quantitative PCR, and the screening criteria were as follows: rBm1 The relative expression level of the gene is higher than that of the wild type.

[0015] An application of the above method includes at least one of the following application scenarios: (1) Use the maize plants resistant to maize leaf spot cultivated by this method as parental materials to create new maize germplasm that combines resistance to maize leaf spot with excellent agronomic traits; (2) Use the maize plants resistant to maize leaf spot cultivated by this method to breed maize varieties or hybrids that are genetically stable and resistant to maize leaf spot; (3) Apply the maize varieties or hybrids resistant to maize leaf spot cultivated by this method to maize field planting to reduce yield loss caused by maize leaf spot.

[0016] The advantages of this invention compared to the prior art are: (1) Gene function has been fully verified and the resistance mechanism is clear. The present invention provides rBm1 Genes can directly respond to corn leaf blight pathogen ( Bipolaris maydis The experimental data showed that after inoculation with the corn leaf blight pathogen, rBm1 The expression level of [the substance] continuously increased over time, reaching a peak at 12 hours after inoculation, and then gradually decreased. This dynamic expression characteristic directly proves its deep involvement in the defense response of maize against small leaf spot fungus; simultaneously, through [other methods]... rBm1The comparative verification between overexpression materials (significantly enhanced resistance) and mutant materials (significantly weakened resistance) further clarified the regulatory role of this gene in the plant's resistance to small leaf spot disease, avoiding the technical defect of "functional prediction without empirical evidence", and providing solid theoretical and experimental support for subsequent breeding applications.

[0017] (2) The technical solution can be directly implemented and is compatible with conventional breeding systems. around rBm1 In terms of gene application, this invention provides a complete technical process from "expression vector construction - genetic transformation - positive screening". The entire process does not require special equipment, is compatible with the existing technical conditions of most scientific research and breeding units, lowers the threshold for technology promotion, and can be quickly transformed into actual breeding results.

[0018] (3) Break through the limitations of existing resistance genes and broaden the dimensions of disease-resistant breeding. Currently discovered maize resistance genes for small leaf spot disease (such as...) ZmH2B , rhm1 Most of these methods focus on a single disease-fighting pathway and are easily rendered ineffective due to the rapid evolution of pathogens; however, this invention... rBm1 As a newly identified key functional gene, this gene not only fills the gap in the molecular regulatory network of maize resistance to small leaf spot, but its characteristic of "dynamic response of expression level to pathogen infection" can also form synergistic regulation with other stress resistance genes, providing a new target for breeding maize varieties with "broad-spectrum resistance + long-lasting resistance", effectively solving the problem of single resistance and easy degradation of existing disease-resistant varieties.

[0019] (4) It has diverse application scenarios, taking into account both scientific research and industrial value. based on rBm1 Genetically bred disease-resistant plants can be used as parental materials to create new maize germplasm with "disease resistance + excellent agronomic traits (such as high yield and lodging resistance)," or they can be directly bred to produce stable genetically resistant varieties or hybrids. In field applications, these disease-resistant varieties can significantly reduce the decline in photosynthetic efficiency and yield loss caused by leaf spot disease, and reduce the use of chemical fungicides. This not only meets the needs of green agricultural development, but also provides a stable supply of raw materials for the maize industry chain (feed, industrial processing, etc.), realizing the dual realization of scientific research value and industrial value. Attached Figure Description

[0020] Figure 1 It is after corn is infected with corn leaf blight fungus rBm1 The results of the expression level analysis; Figure 2 This is a schematic diagram of the structure of carrier pZZ00026; Figure 3 yes rBm1 In transgenic plants with positive gene overexpression, each line rBm1 Relative expression level; Figure 4 yes rbm1 Mutation sites and sequencing results of the mutants (Figure A shows the mutation sites; Figure B shows the sequencing results). Figure 5 yes rBm1 Comparative figures of leaf lesions in overexpression lines and mutant materials after inoculation with *Spodoptera exigua* (Figure A shows representative images of disease symptoms of rBm1-OE2 and rBm1-OE8 lines on day 7 after inoculation with *Spodoptera exigua*, with wild-type WT KN5585 as the control; scale bar represents 1 cm; Figure B shows a comparison of the disease index of rBm1-OE2, rBm1-OE8 lines and wild-type WT KN5585 on day 7 after infection with *Spodoptera exigua*, with at least 12 plants counted; Figure C shows the leaf lesions 7 days after inoculation with *Spodoptera exigua*). rbm1 Typical disease symptoms of the mutant are shown in Figure D, with the B73 wild-type WT as the control, scale bar 1 cm; Figure D shows the symptoms 7 days after inoculation with maize leaf spot fungus. rbm1 The disease index of the mutant was compared with that of the wild-type B73 WT, with at least 12 plants counted; Figure E shows the quantitative analysis of fungal biomass by real-time quantitative PCR: using maize as an example. GAPDH As an internal control, the relative biomass of *Sclerotium affine* in maize was assessed by measuring the 5.8S rDNA level; "*": P <0.05, "**": P < 0.01). Detailed Implementation

[0021] The embodiments of the present invention are described in detail below. These embodiments are implemented based on the technical solution of the present invention, and provide detailed implementation methods and specific operation processes. However, the scope of protection of the present invention is not limited to the following embodiments.

[0022] The culture medium formulations involved in the following examples are as follows: PDA medium: 5 g / L potato extract powder, 20 g / L glucose, 15 g / L agar, with the final pH adjusted to 5.6.

[0023] YEP solid medium (containing rifampin and kanamycin): beef extract 5g / L, yeast extract 10g / L, peptone 10g / L, agar 15g / L, pH adjusted to 7.0, rifampin and kanamycin 50mg / mL.

[0024] YEP liquid medium (containing rifampin and kanamycin): beef extract 5g / L, yeast extract 10g / L, peptone 10g / L, pH adjusted to 7.0, rifampin and kanamycin 50mg / mL.

[0025] MS liquid culture medium: MS powder 4.74 g / L (catalog number: M8521, Solarb, Shanghai, China), autoclaved at 115℃ for 30 minutes before use.

[0026] Screening medium (containing hygromycin): Supplement MS liquid medium with 5 mg / L hygromycin and 3 g / L agar.

[0027] Differentiation medium: MS powder 4.74 g / L (catalog number: M8521, Solarbio, Shanghai, China), sucrose 30 g / L, sorbitol 30 g / L, 6-BA 0.5 mg / L, agar 3 g / L, pH adjusted to 5.8, autoclaved at 121℃, and cooled to a temperature that can be touched by hand. Then, temetin was added to supplement the medium in a laminar flow hood to a final concentration of 200 mg / L.

[0028] Rooting medium: MS powder (catalog number: M8521, Solarbio, Shanghai, China) 2.37 g / L, sucrose 20 g / L, agar 3 g / L.

[0029] The maize inbred lines B73 and KN5585 used were provided by the National-Local Joint Engineering Laboratory for Crop Stress Resistance Breeding and Disaster Reduction, College of Life Sciences, Anhui Agricultural University, and were commercially available materials.

[0030] Used rBm1 The gene premature termination mutant material was purchased from the Maize EMS Mutant Library (EMS4-0a9cba), the official website of the EMS Mutant Library is "http: / / maizeems.qlnu.edu.cn / ", and the material ID number is "Zm00001d010399".

[0031] Unless otherwise specified, all experimental methods used are conventional. Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0032] Example 1: Analysis of rBm1 induced expression pattern: 1. Preparation of a suspension of corn leaf spot spores Corn leaf spot fungus ( Bipolaris maydis The culture was incubated on PDA medium at 25°C for 10 days to produce conidia. The culture was then rinsed with sterile water and filtered through sterile gauze to collect the conidia. The suspension concentration was adjusted using 0.1% Tween-20 solution and further adjusted to a final concentration of 10 using a hemocytometer. 5 per mL.

[0033] 2. Inoculation with corn leaf blight pathogen The prepared corn leaf spot fungus spore suspension was sprayed onto the leaves of B73 corn plants that were about three weeks old. Samples were taken at 0h, 6h, 12h, 24h, 36h and 48h to prepare for RNA extraction.

[0034] 3. Extraction of total RNA from leaves using the Trizol method Prepare 2 mL RNase EP tubes and 1 mL RNase pipette tips in advance, and pre-cool them in liquid nitrogen. Pre-cool the refrigerated centrifuge to 4°C. Pre-cool the mortar (dried in a 180°C oven for 6 hours) with liquid nitrogen. Take approximately 1 g of leaf material and grind it thoroughly and rapidly, adding liquid nitrogen during grinding until the sample powder turns slightly white. Quickly transfer the powder to the EP tube using a pipette tip (the pre-cooled pipette tip and the inside of the EP tube should not adhere to the sample powder). Add an appropriate amount of Trizol reagent for lysis, vortex, and incubate on ice in the dark for 10 min. Then add 0.25 mL of chloroform-isoamyl alcohol (24:1) to each tube, seal tightly in a fume hood, and shake rapidly for 15 seconds. Incubate on ice in the dark for 3 min. Centrifuge the mixture at 12000 g, 4°C for 5 min. Carefully transfer the supernatant to a new 1.5 mL RNase EP tube. Mix the RNA precipitate in an EP tube with an equal volume of isopropanol, incubate on ice for 10 minutes, and centrifuge again at high speed for 10 minutes. Discard the supernatant. Ideally, the RNA precipitate from young plant leaves should be white. Add 1 mL of freshly prepared 75% ethanol to wash the precipitate (be careful not to blow it away). Centrifuge at 12000g and 4℃ for 5 minutes, and discard the supernatant. Add another 1 mL of freshly prepared 75% ethanol to wash the precipitate and centrifuge again. Remove as much residual alcohol as possible and dissolve the RNA precipitate in an appropriate amount of DEPC water for later use.

[0035] 4. cDNA Synthesis The specific steps for performing RNA reverse transcription experiments using the Takara reverse transcription kit are as follows: The concentration and quality of the extracted RNA were detected and recorded using an ultra-micro nucleic acid protein concentration analyzer.

[0036] The extracted RNA was detected by agarose gel electrophoresis. Qualified RNA should have three distinct bands, mainly 28S, 18S, 5.8S and 5S.

[0037] For RNA that passed the initial test, reverse transcription experiments were performed according to the kit instructions. For each reverse transcription reaction, 1g of RNA was accurately added based on the concentration. The sample loading system was as follows: 10μL of 2×Hifair® Ⅱ SuperMix, 1µg of Total RNA added according to the measured concentration, followed by RNase-free H2O to a final volume of 20μL. After careful mixing, the mixture was incubated at 25℃ for 5 min, then at 42℃ for 30 min, and finally inactivated at 85℃.

[0038] 5. RT-qPCR detection and analysis The cDNA obtained above was used as a template for qRT-PCR amplification. The RT-qPCR amplification system consisted of: 0.8 μL of primer rBm1-DF (10 μM), 0.8 μL of primer rBm1-DR (10 μM), 1.6 μL of cDNA template, 10 μL of SYBR Green Mix, and 6.8 μL of RNase-free Water. The nucleotide sequences of primers rBm1-DF and rBm1-DR are shown in SEQ ID NO. 3 and SEQ ID NO. 4, respectively.

[0039] The main procedures for quantitative real-time PCR are as follows: First stage: pre-denaturation at 95℃ for 2 min; Second stage: denaturation at 95℃ for 15 sec, followed by annealing and extension at 60℃ for 30 sec (data collection stage), repeated 40 times; Third stage: after the last cycle, melting curve analysis was performed in 0.5℃ increments within the temperature range of 60–95℃ to verify the reaction specificity. Amplification signals and data processing used the comparative Ct method (ΔΔCt), with a relative expression value RQ = 2(-ΔΔCt).

[0040] The RT-qPCR method was used to detect the presence of B73 maize virus in maize after inoculation with maize leaf spot. rBm1 Expression level, such as Figure 1 As shown. After inoculation with corn leaf blight pathogen, rBm1 The expression level of [the substance] continuously increased over time, reaching a peak at 12 hours after inoculation, and then gradually decreased. This dynamic expression characteristic fully demonstrates [the following]. rBm1 Genes are involved in the defense response of maize against small leaf spot fungus. rBm1 The gene nucleotide sequence is shown in SEQ ID NO.1, and the encoded amino acid sequence is shown in SEQ ID NO.2.

[0041] Example 2 rBm1 Construction of gene overexpression plants: 1. Contains rBm1 Construction of plant expression vectors for genes The plant expression vector backbone pZZ00026 (structure as shown) was obtained. Figure 2 As shown in the figure, the pZZ00026 plasmid was linearized using the SmaI restriction endonuclease. The digestion system was as follows: 17 μL pZZ00026 plasmid, 2 μL CutSmart Buffer, 1 μL SmaI, for a total volume of 20 μL. The linearized vector after digestion was detected by 2% agarose gel electrophoresis, and the vector backbone was recovered.

[0042] Using pre-designed specific primers and maize B73 cDNA as a template, amplification was performed under specific PCR conditions. rBm1After agarose gel electrophoresis, a band of approximately 1497 bp was excised and the PCR product was recovered using a DNA gel recovery kit to obtain the PCR product ( rBm1 The CDS sequence of the gene is shown in SEQ ID NO.1. The PCR amplification system is as follows: 1 μL of specific primer F, 1 μL of specific primer R, 2 μL of B73 maize inbred line cDNA template, 8.5 μL of ddH2O, 12.5 μL of Primer STARMax, and a total volume of 25 μL. The nucleotide sequences of specific primer F and specific primer R are shown in SEQ ID NO.5 and SEQ ID NO.6, respectively. The PCR amplification program is as follows: 98℃ pre-denaturation for 10 min, 98℃ denaturation for 10 s, 60℃ extension for 1 min 30 s, 35 cycles; 72℃ extension for 10 min. Using homologous recombination method rBm1 The CDS sequence was ligated into a linearized vector, and the ligation reaction was carried out at 37°C for 2 hours. After completion, the mixture was placed on ice for 5 minutes to obtain the ligation product. The ligation product was transformed into *E. coli* for amplification and propagation, and the vector plasmid was obtained using a plasmid extraction kit and named [name missing]. rBm1 -pZZ00026. The recombination ligation reaction system was as follows: 5 μL of 2×Hieff Clone® Enzyme Premix, 2 μL of the recovered product of SmaI digestion of the pZZ00026 vector, 3 μL of the PCR recovered product, and a total volume of 10 μL.

[0043] 2. Genetic transformation The constructed vector was introduced into Agrobacterium strain EHA105 and plated on YEP solid medium containing 50 mg / L rifampicin and 50 mg / L kanamycin to screen for positive clones. Single colonies were picked and inoculated into YEP liquid medium containing rifampicin and kanamycin, and cultured at 28°C with shaking at 200 rpm until OD500. 600 The value reaches 0.6~0.8.

[0044] Collect bacterial cells, resuspend them in MS liquid medium, and adjust OD. 600 The concentration was adjusted to 0.5-0.6 as the inoculum concentration. Callus tissue from vigorous maize inbred line KN5585 was selected and soaked in the inoculum concentration for 20-30 minutes in a clean bench, gently shaking it during the process to ensure full contact.

[0045] After infection, the callus tissue was blotted dry with sterile filter paper and cultured at room temperature in the dark for 3 days. Then, it was transferred to a selection medium containing 5 mg / L hygromycin for selection of resistant callus tissue. Subculture was performed every 2 weeks for 4 weeks.

[0046] The obtained resistant callus was transferred to differentiation medium and cultured for 3 weeks at 25°C under 16h light / 8h dark conditions to induce green seedling differentiation.

[0047] When the seedlings grow to 3-4 cm in height, transfer them to a rooting medium to promote root development. After rooting, the complete plants are hardened off in a greenhouse for one week before being transplanted into nutrient soil and cultured until the T0 generation plants mature.

[0048] 3. Identification of positive maize plants Total RNA was extracted from transgenic maize leaves according to the method in Example 1. 1 μg of total RNA was used to obtain cDNA of the corresponding material using the Vazyme R323-01 reverse transcription kit for ordinary reverse mRNA transcription.

[0049] The obtained cDNA was used as a template for qRT PCR amplification; the system and procedure for the RT qPCR amplification were as described in Example 1; the maize Actin gene and 18S RNA were used as internal controls, and the primers for the internal control gene were Actin-F, Actin-R, 18S-F, and 18S-R, with sequences as shown in SEQ ID NO. 7~10, respectively.

[0050] The positive transgenic plants obtained were detected using RT-qPCR. rBm1 The relative expression levels, the results are as follows Figure 3 As shown, two lines with high expression levels and stable inheritance were finally obtained, named rBm1-OE2 and rBm1-OE8, respectively.

[0051] Example 3 rBm1 Identification of gene premature termination mutant materials: rBm1 The gene premature termination mutant material was obtained from the maize EMS mutant library, material ID number "Zm00001d010399", hereinafter referred to as "mutant". rbm1 ".

[0052] 1. Mutant rbm1 Leaf genomic DNA extraction Extraction of mutants using the CTAB method rbm1 The genomic DNA of the material was analyzed using the following steps: Take a 2cm leaf and place it in a 2mL centrifuge tube. Add a small steel ball, freeze quickly in liquid nitrogen, and then place in a grinder. Shake at 45Hz for 2 minutes. After thorough shaking, add 800µL of CTAB extraction buffer, vortex to mix, and incubate in a 65℃ water bath for 1 hour, turning several times during incubation. Add an equal volume of chloroform-isoamyl alcohol (24:1) to a fume hood and vortex to mix. Centrifuge at 12000rpm for 10 minutes, carefully aspirate about 700µL of the supernatant, transfer to a 1.5mL centrifuge tube, add an equal volume of isopropanol to precipitate the nucleic acid, and let stand for 2 minutes. Then centrifuge at 12000rpm for 10 minutes, discard the supernatant, add 500µL of 70% alcohol and gently rinse twice. Finally, discard the alcohol, air dry in a fume hood, add 100µL of deionized water, and store at -20℃.

[0053] 2. Mutants rbm1 Mutation site detection With mutants rbm1 The plant's genomic DNA was used as a template for PCR amplification. The reaction system was as follows: total volume 25 µL, including 12.5 µL of Primer STAR Max premix (2×), 1 µL each of primers EMS-F and EMS-R, 2 µL of mutant genomic DNA, and 8.5 µL of sterile water added at the end. The sequences of primers EMS-F and EMS-R are shown in SEQ ID NO. 11 and 12, respectively.

[0054] After adding the sample, centrifuge at low speed to mix, and then perform the reaction in a PCR instrument. The reaction program is as follows: pre-denaturation at 98 ℃ for 10 min, followed by denaturation at 98 ℃ for 10 s, annealing at 60 ℃ for 5 s, extension at 72 ℃ for 1 min, run for 35 cycles, and finally extend at 72 ℃ for 10 min. Store the PCR product at 4 ℃.

[0055] After PCR, the product size was detected by agarose gel electrophoresis. The band of about 1500 bp was recovered and sent to Sangon Biotech for Sanger sequencing and compared with SEQ ID NO.1.

[0056] Mutation sites and sequence comparison results are shown in Figure 4 This indicates that in the mutant, rBm1 The C base at position 1384 of the gene was replaced by the T base (compared to the wild type), and the final nucleotide sequence is shown in SEQ ID NO.13, and the encoded amino acid sequence is shown in SEQ ID NO.14.

[0057] Example 4 Bm1 Validation of disease resistance phenotypes in transgenic and mutant materials: I. Experimental Materials rBm1Overexpression transgenic maize lines: Two high-expression and stable genetic lines (rBm1-OE2 and rBm1-OE8) obtained in Example 2 were used, with the background being the maize inbred line KN5585; rBm1 Mutant material: obtained from the maize EMS mutant library rBm1 Loss-of-function mutant (named rbm1 Its mutation type is rBm1 The point mutation at base 1384 of the gene is C→T, with the background being maize inbred line B73; Wild-type control materials: Non-transgenic maize inbred lines KN5585 and B73, which are in the same background as the above-mentioned transgenic lines and mutant materials, were used for phenotypic comparison analysis.

[0058] All maize materials were grown in an artificial climate chamber under the following conditions: 28℃ / 22℃ (day / night), 12h / 12h (light / dark), and 70% relative humidity. Disease resistance phenotypes were identified when the maize reached the seven-leaf stage.

[0059] II. Experimental Methods Use corn leaf blight fungus ( Bipolaris maydis ) Spore suspension (1×10 5 Spraying inoculation was performed on maize plants at the seven-leaf stage using spores / mL. Leaf samples were collected on day 3 post-inoculation for DNA extraction and subsequent quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis of fungal DNA content. The 5.8S rDNA level of maize leaf spot pathogen was correlated with maize leaf spot causal agent levels using qRT-PCR. GAPDH Expression levels were standardized to quantify fungal biomass, and disease severity was assessed on day 7 post-inoculation.

[0060] See results Figure 5 Compared to the wild-type control KN5585, rBm1 Overexpressing transgenic lines (rBm1-OE2 and rBm1-OE8) showed significantly enhanced resistance to maize leaf spot fungus infection, with a disease index significantly lower than the control on day 7 post-infection. Figure 5 A, Figure 5 B); compared to the B73 wild-type control, rBm1 Gene mutant lines ( rbm1 ) exhibited more severe disease symptoms and a higher disease index ( Figure 5 C, Figure 5 D); and the quantitative results of fungal biomass were consistent with the observation results. rBm1 Gene mutants ( rbm1 The colonization of maize leaf spot pathogens increased significantly in ( ) Figure 5 E), indicating rBm1 Functional loss weakens corn's resistance to the pathogen.

[0061] In conclusion, rBm1 The gene is a key functional gene that positively regulates maize's resistance to small leaf spot disease; its overexpression can effectively enhance maize's disease resistance. This invention provides... rBm1 The gene mining, vector construction, genetic transformation, and phenotypic validation technology system not only provides new theoretical basis for elucidating the molecular mechanism of maize resistance to small leaf spot, but can also be directly applied to molecular breeding of maize for disease resistance: through targeted regulation. rBm1 Gene expression can efficiently create new maize germplasm resistant to small leaf spot and breed new disease-resistant varieties, providing feasible gene resources and technical solutions for solving the yield loss problem caused by small leaf spot in maize. It has important scientific research value and application prospects for ensuring the sustainable development of the maize industry.

[0062] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method of breeding corn plants resistant to Goss' Wilt by rBm1 a method of breeding corn plants resistant to Goss' Wilt, characterized in that, Through genetic engineering methods rBm1 The gene was overexpressed in maize to obtain the target maize plants; rBm1 The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

2. The method of claim 1, wherein, The rBm1 The amino acid sequence encoded by the gene is shown in SEQ ID NO.

2.

3. The method of claim 1, wherein, The rBm1 The expression products of the gene positively regulate the resistance of maize plants to maize leaf spot disease.

4. The method according to any one of claims 1 to 3, characterized in that, The genetic engineering method comprises the following specific steps: (1) Plants containing rBm1 Construction of plant expression vectors for genes Taking a plant expression vector skeleton, linearizing the same; taking the cDNA of the corn material as a template, obtaining by PCR amplification rBm1 the CDS sequence of the gene, as shown in SEQ ID NO. 1; recombining and connecting the linearized vector with rBm1 the CDS sequence of the gene, to obtain a plant expression vector containing rBm1 the gene; (2) Genetic transformation The plant expression vector constructed in step (1) is introduced into a corn receptor material, and candidate plants are obtained through culture. (3) Positive screening detecting the expression level of the gene in the candidate plants in step (2) rBm1 screening rBm1 the plants in which the gene is successfully overexpressed are the target corn plants.

5. The method of claim 4, wherein, In the step (1), the plant expression vector skeleton is pZZ00026 vector, and linear treatment adopts Smal restriction endonuclease.

6. The method of claim 4, wherein, In the step (1), the recombination connection mode is homologous recombination, the recombination reaction temperature is 37℃, and the reaction time is 2h.

7. The method of claim 4, wherein, In the step (2), the corn receptor material is corn inbred line KN5585, and the introduction mode is Agrobacterium-mediated transformation method.

8. The method of claim 4, wherein, In the step (3), rBm1 The gene expression level is detected by real-time quantitative PCR method, and the screening standard is rBm1 The relative expression of the gene is higher than that of the wild type.

9. Use of a method according to any one of claims 1 to 8, characterized in that The method comprises at least one of the following application scenarios: (1) The corn plant resistant to corn small spot disease cultivated by the method is used as parent material to create new corn germplasm with resistance to corn small spot disease and excellent agronomic traits; (2) The corn plant resistant to corn small spot disease cultivated by the method is used to select a corn variety or hybrid resistant to corn small spot disease with stable heredity; (3) The corn variety or hybrid resistant to corn small spot disease cultivated by the method is applied to corn field planting to reduce yield loss caused by corn small spot disease.

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