Disease-resistant protein MSLK and application thereof

Abstract

The application belongs to the technical field of genetic engineering, and discloses an anti-disease protein MSLK and application thereof. The anti-disease protein MSLK is introduced to provide enhanced corn broad-spectrum disease resistance, and the resistance of corn stalk rot and ear rot is improved from disease to high resistance. The resistance of large and small spot diseases is improved from disease to disease resistance, and the corn yield is increased under the condition of field disease occurrence. The corn yield is not affected under the condition of no disease under pesticide disease control.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, specifically relating to a disease-resistant protein MSLK and its applications. Background Technology

[0002] corn( Zea mays Corn is the most widely planted crop in the world, with an annual global planting area exceeding 2 billion mu (approximately 133 million hectares) and a total output reaching 1 billion tons. Its edible, industrial, and feed value gives it a significant place in my country. Currently, with increasingly drastic environmental changes, corn ear rot, stalk rot, and leaf spot are prevalent and severely damaging diseases in my country's corn-producing areas. Ear rot is caused by Fusarium graminearum (…). Fusarium graminearum Fusarium moniliforme () Fusarium moniliforme Aspergillus flavus ( Aspergillus flavus Stem rot is caused by a complex infection of nearly 20 pathogens, including *Pythium*, which not only leads to grain rot and reduced yield, but also produces fungal toxins such as aflatoxin, which seriously threaten human and animal health. The main pathogen of stem rot is *Pythium*. Pythium Fusarium ( ) Fusarium ) and anthrax bacteria ( Colletotrichum spp This causes stem rot, premature aging, and lodging in the plant; while large and small leaf spot diseases spread rapidly in the later stages of corn growth, leading to leaf death and a significant reduction in photosynthetic capacity.

[0003] Maize yield and quality are threatened by diseases such as ear rot, stalk rot, and leaf spot. Ear rot, in particular, not only affects maize yield, but the virulence it causes also impacts food safety. Therefore, improving maize disease resistance and self-sufficiency is an important direction in current maize breeding.

[0004] Currently, maize disease resistance breeding mainly relies on traditional hybridization and molecular marker-assisted selection. However, disease-resistant germplasm resources are limited, and most resistance traits are inherited quantitatively, controlled by multiple genes and easily affected by environmental conditions. This results in long breeding cycles, low efficiency, and difficulty in quickly developing broad-spectrum, durable disease-resistant varieties. While chemical control can control diseases to some extent, it increases production costs and pesticide residue risks, and long-term use can induce pesticide resistance in pathogens. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention aims to provide a disease-resistant protein, MSLK, and its applications. Maize, as a representative model plant of the Gramineae family, demonstrates the disease resistance of this protein in maize, obtaining transgenic maize resistance to stalk rot, ear rot, and leaf spot. Under pesticide-controlled conditions, the resistance has no impact on yield when no disease is present. This provides valuable reference for other Gramineae plants such as rice. This invention can effectively improve the disease resistance of existing maize varieties, enhance yield stability, and improve grain quality.

[0006] This invention discloses a disease-resistant protein MSLK, comprising at least one of the following proteins: (1) The amino acid sequence is shown in SEQ ID NO.11; (2) A fusion protein obtained by attaching a protein tag to the N-terminus and / or C-terminus of the protein shown in SEQ ID NO.11, wherein the disease resistance includes one or more of stem rot, ear rot, and leaf spot.

[0007] Furthermore, the disease-resistant protein MSLK encodes a gene that includes at least one of the following genes: (1) The nucleotide sequence is shown in SEQ ID NO.12; (2) A fusion gene obtained by connecting the 3' end and / or 5' end of the gene described in (1) to the coding gene of the protein tag.

[0008] This invention discloses an MSLK protein comprising a polypeptide having the amino acid sequence of SEQ ID NO. 11, a conserved variant thereof, or a homologous polypeptide thereof. In this invention, the disease-resistant protein MSLK includes, but is not limited to, SEQ ID NO. 11. Amino acid sequences having a certain degree of homology with the amino acid sequence shown in SEQ ID NO. 11 are also included in this invention. These sequences typically exhibit greater than 78% similarity / identity with the sequences of this invention, preferably greater than 85%, more preferably greater than 90%, even more preferably greater than 95%, and may be greater than 99%. Preferred polynucleotides and proteins of this invention may also be defined according to more specific ranges of similarity and / or identity. For example, sequences exhibiting 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% similarity and / or identity with the sequences exemplified in this invention.

[0009] In one embodiment of the invention, the polypeptide is selected from the group consisting of: (a) a polypeptide having the amino acid sequence of SEQ ID NO. 11; and (b) a polypeptide derived from (a) formed by substituting, deleting or adding one or more amino acid residues of the amino acid sequence of SEQ ID NO. 11, and having the effect of improving plant disease resistance.

[0010] On the other hand, the present invention provides a nucleotide comprising a nucleotide sequence encoding the polypeptide of the present invention; In one embodiment of the invention, the sequence of the polynucleotide is selected from one of the following groups: (a) a nucleotide sequence having SEQ ID NO. 12; (b) a nucleotide sequence having 90% or more similarity to SEQ ID NO. 12; or a polynucleotide complementary to any one of the nucleotide sequences (a)-(b).

[0011] The present invention MSLK The gene is preferably obtained from Dendrobium. Other genes from other plants that are highly homologous to the Dendrobium MSLK gene (e.g., having 50% or more, preferably 55%, 60%, 65%, 70%, 75%, 80%, more preferably 85% or even 98% sequence identity) are also within the scope of preferred consideration in this invention. Methods and tools for comparing sequence identity are also well known in the art, such as BLAST.

[0012] This invention discloses a recombinant vector containing the gene encoding the disease-resistant protein MSLK.

[0013] Furthermore, the recombinant vector contains its regulatory elements, which include a promoter and a terminator. The promoter is a maize ubiquitin promoter, the nucleotide sequence of which is shown in SEQ ID NO.3; the terminator is a terminator for carmine synthase, the nucleotide sequence of which is shown in SEQ ID NO.4.

[0014] This invention discloses a recombinant bacterium comprising the aforementioned recombinant vector.

[0015] This invention discloses an expression cassette containing the gene encoding the disease-resistant protein MSLK.

[0016] This invention discloses the application of the disease-resistant protein MSLK in improving the disease resistance of maize. The amino acid sequence of the disease-resistant protein MSLK is shown in SEQ ID NO.11. The diseases are: stalk rot, ear rot, and leaf spot.

[0017] The application is selected from one or more of the following: (1) To prepare agents with anti-disease effects; (2) Cultivate transgenic corn with or improved disease resistance characteristics.

[0018] The method for constructing transgenic maize with or improved disease resistance characteristics includes the following steps: expressing the disease resistance protein MSLK in maize to obtain transgenic maize.

[0019] This invention discloses a disease-resistant maize material, wherein the coding gene of the disease-resistant protein MSLK is inserted into the genome of the maize material.

[0020] This invention discloses the application of a protein with an amino acid sequence as shown in SEQ ID NO.2 in improving the disease resistance of maize. The application is to breed transgenic maize with or with improved disease resistance, or to prepare agents with disease resistance effects, wherein the diseases are stalk rot, ear rot and / or leaf spot.

[0021] In another preferred embodiment, the disease resistance described in this invention refers to: corn stalk rot, ear rot, large and small leaf spot, etc. Compared with the prior art, the present invention has the following beneficial effects: This invention provides a method to enhance broad-spectrum disease resistance in maize by introducing the disease-resistant protein MSLK. It improves maize resistance to stalk rot and ear rot from susceptible to highly resistant, and resistance to leaf spot from susceptible to resistant. It also increases maize yield when the disease occurs in the field, and has no impact on maize yield when the disease is not present under pesticide control conditions. Attached Figure Description

[0022] Figure 1 Disease-resistant genes MSLK Vector map of the recombinant expression vector; Figure 2 Disease resistance genes in 15 samples MSLK PCR test results; Figure 3 This diagram illustrates different disease states in the field. Different lowercase letters indicate significant differences between different treatments. p <0.05); Figure 4 For maize yield test results, different lowercase letters indicate significant differences between different treatments. p <0.05). Detailed Implementation

[0023] The technical solutions of the embodiments of this application will be further clearly and completely described below with reference to the accompanying drawings. It should be noted that the described embodiments are only a part of the embodiments of this application, and not all of the embodiments. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0024] The purpose of this invention is to provide a disease-resistant protein, MSLK, and its application, enabling transgenic maize to exhibit resistance to stalk rot, ear rot, and leaf spot, while maintaining yield even under pesticide-controlled conditions. This invention can effectively improve the disease resistance of existing maize varieties, enhance maize yield stability, and improve grain quality.

[0025] The disease-resistant protein MSLK is derived from Dendrobium officinale (… Dendrobium officinale Kimura&Migo Disease-resistant proteins.

[0026] Example 1: Construction, preservation, and detection of recombinant expression vectors Nanjing Genscript Biotechnology Co., Ltd. synthesized a disease resistance gene encoding the disease resistance protein MSLK by attaching an Nco I restriction site to its 5' end and an EcoR I restriction site to its 3' end. MSLK The nucleotide sequence is shown in SEQ ID NO.1 or SEQ ID NO.12.

[0027] The disease resistance genes shown in SEQ ID NO.1 and SEQ ID NO.12 were synthesized. MSLK The nucleotide sequences were ligated into the cloning vector pEASY-T5 (TransGen Biotech, Beijing, Cat. No.: CT501-01), following the instructions for use of the pEASY-T5 vector. Different recombinant cloning vectors, LP-T1 and LP-T2, were obtained. These recombinant cloning vectors were then transformed into *E. coli* T1 competent cells (TransGen Biotech, Beijing, Cat. No.: CD501) using a heat shock method. The heat shock conditions were as follows: (1) 50 μL of Escherichia coli T1 competent cells and 10 μL of plasmid DNA (recombinant cloning vector LP-T) were incubated in a water bath at 42℃ for 30 s and then incubated in a water bath at 37℃ for 45 min (shaking at 200 rpm). The mixture was then spread on LB agar plates containing ampicillin (100 mg / L) (tryptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L, agar 15 g / L, pH adjusted to 7.5 with NaOH) and grown overnight.

[0028] (2) Pick white colonies and incubate them overnight at 37°C in LB liquid medium (tryptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L, ampicillin 100 mg / L, pH adjusted to 7.5 with NaOH).

[0029] (3) Alkaline extraction of plasmids: Centrifuge the bacterial culture at 12,000 rpm for 1 min, discard the supernatant, and suspend the precipitated bacterial cells in 100 μL of ice-cold solution I (25 mM Tris-HCl, 10 mM EDTA (ethylenediaminetetraacetic acid), 50 mM glucose, pH 8.0); add 150 μL of freshly prepared solution II (0.2 M NaOH, 1% SDS (sodium dodecyl sulfate)), invert the tube 4 times to mix, and place on ice for 3-5 min; add 150 μL of ice-cold solution III (4 M potassium acetate, 2 M acetic acid), mix thoroughly immediately, and place on ice for 5-10 min; centrifuge at 4℃ and 12,000 rpm for 5 min, add 2 volumes of anhydrous ethanol to the supernatant, mix well, and place at room temperature for 5 min; centrifuge at 4℃ and 12,000 rpm for 5 min. After 30 min, discard the supernatant, wash the precipitate with 70% ethanol and air dry; add 30 μL of TE (10mM Tris-HCl, 1mM EDTA, pH 8.0) containing RNase (20 μg / mL) to dissolve the precipitate; digest the RNA in a water bath at 37℃ for 30 min; store at -20℃ for later use.

[0030] After the extracted plasmids were identified by Nco I and EcoRI digestion, positive clones were sequenced for verification. The results showed that the nucleotide sequence inserted into the recombinant cloning vector LP-T1 was the disease resistance gene shown in SEQ ID NO.1 of the sequence listing. MSLK The nucleotide sequence inserted into the recombinant cloning vector LP-T2 is the disease resistance gene shown in SEQ ID NO.12 of the sequence listing. MSLK The nucleotide sequence of the disease-resistant gene. MSLK The nucleotide sequence was correctly inserted.

[0031] The recombinant cloning vectors LP-T1 and LP-T2 and the expression vector LP-BB1 (vector backbone: pCAMBIA3301 (available from the CAMBIA organization)) were digested with restriction endonucleases Nco I and EcoRI, respectively, to extract the disease resistance gene. MSLK Nucleotide sequence fragments were inserted between the Nco I and EcoRI sites of the expression vector LP-BB1 to construct the recombinant expression vector LP-PTMSLK, into which the disease resistance gene was inserted. MSLK The vector map of the recombinant expression vector is as follows Figure 1 As shown.

[0032] in, KanR: Kanamycin gene; Ori: pUCorigin represents the replication region sequence of plasmid pUC, which can guide the double-stranded DNA replication process; RB: right boundary; pZmUbi1: from the maize ubiquitin gene promoter (SEQ ID NO.3). MSLK Disease-resistant genes MSLK Nucleotide sequence (SEQ ID NO.1 or SEQ ID NO.12); Nos: terminator of carmine synthase (SEQ ID NO.4); PAT: phosphinic acid acetyltransferase, used for transformation screening and conferring glufosinate resistance in transgenic plants (SEQ ID NO.5); 35S: terminator from cauliflower mosaic virus (CaMV) (SEQ ID NO.6); LB: left border.

[0033] Example 2: Plant transformation and detection, detection of transgenic maize plants 2.1 Plant Transformation and Detection Transformation was performed using the conventional Agrobacterium infection method. The aseptically cultured transgenic maize embryos were co-cultured with the Agrobacterium in Example 1 to transfer the T-DNA from the constructed recombinant expression vector LP-PTMSLK into the maize chromosome set, thereby generating transgenic maize events.

[0034] (1) Infection For Agrobacterium-mediated maize transformation, briefly, immature embryos are isolated from maize and contacted with an Agrobacterium suspension, in which Agrobacterium can transmit disease resistance genes. MSLK nucleic acid sequence and PAT The nucleic acid sequence of the gene is delivered to at least one cell of one of the immature embryos, in which the immature embryo is specifically immersed in Agrobacterium suspension (OD660 = 0.4-0.6, infection medium (MS salt 4.3 g / L, MS vitamins, casein 300 mg / L, sucrose 68.5 g / L, glucose 36 g / L, acetylsuccinone (AS) 40 mg / L, 2,4-dichlorophenoxyacetic acid (2,4-D) 1 mg / L, pH 5.3)) to initiate inoculation.

[0035] (2) Co-cultivation The embryos were co-cultured with Agrobacterium for a period of time (3 days). Specifically, after the infection step, the embryos were cultured on solid medium (MS salt 4.3 g / L, MS vitamins, casein 300 mg / L, sucrose 20 g / L, glucose 10 g / L, acetylsylgenone (AS) 100 mg / L, 2,4-dichlorophenoxyacetic acid (2,4-D) 1 mg / L, agar 8 g / L, pH 5.8).

[0036] (3) Recovery Following this co-culture phase, a selective "recovery" step may be performed. In the "recovery" step, the recovery medium (MS salt 4.3 g / L, MS vitamins, casein 300 mg / L, sucrose 30 g / L, 2,4-dichlorophenoxyacetic acid (2,4-D) 1 mg / L, plant gel 3 g / L, pH 5.8) contains at least one known antibiotic that inhibits the growth of Agrobacterium (such as cephalosporin), without the addition of selectants for plant transformants.

[0037] (4) Choose Specifically, the embryos were cultured on a solid medium containing antibiotics but without a selector to eliminate Agrobacterium and provide a recovery period for infected cells. Next, the inoculated embryos were cultured on a medium containing a selector (N-(phosphonocarboxymethyl)glycine) and the growing transformed callus was selected. Specifically, the embryos were cultured on a selection solid medium containing a selector (MS salt 4.3 g / L, MS vitamins, casein 300 mg / L, sucrose 30 g / L, N-(phosphonocarboxymethyl)glycine 0.25 mol / L, 2,4-dichlorophenoxyacetic acid (2,4-D) 1 mg / L, plant gel 3 g / L, pH 5.8), resulting in selective growth of transformed cells.

[0038] (5) Regeneration Then, the callus tissue regenerates into a plant. Specifically, callus tissue grown on a medium containing a selector is cultured on solid media (MS differentiation medium and MS rooting medium) to regenerate a plant.

[0039] The selected resistant callus tissues were transferred to MS differentiation medium (MS salt 4.3 g / L, MS vitamins, casein 300 mg / L, sucrose 30 g / L, 6-benzyladenine 2 mg / L, N-(phosphonocarboxymethyl)glycine 0.125 mol / L, plant gel 3 g / L, pH=5.8) and cultured at 25℃ for differentiation. The differentiated seedlings were transferred to MS rooting medium (MS salt 2.15 g / L, MS vitamins, casein 300 mg / L, sucrose 30 g / L, indole-3-acetic acid 1 mg / L, agar 8 g / L, pH=5.8) and cultured at 25℃ until approximately 10 cm tall, then transferred to a greenhouse for further cultivation until fruit set. In the greenhouse, the seedlings were cultured at 28℃ for 16 hours daily, followed by 8 hours at 20℃.

[0040] 2.2 Detection of transgenic maize plants (1) The disease resistance gene was verified by conventional PCR using 2×EasyTaq PCRSuperMix (Cat. No: AS111-11) from Beijing TransGen Biotech Co., Ltd. MSLK The corn plants are OE1 and OE2.

[0041] The primers for OE1 PCR are: F1: 5'-ATGGCCTCTCCCCCA-3' (SEQ ID NO. 7); R1: 5'-TCAAGGATACAGCTCCTTCATCTT-3' (SEQ ID NO. 8).

[0042] PCR detection fragment size: 513 bp.

[0043] The primers for OE2 PCR are: F2: 5'-ATGGCATCATCCCCTTCTT-3' (SEQ ID NO.9); R2: 5'-TCAAGGATAGATGTCCTTCATG-3' (SEQ ID NO. 10).

[0044] PCR detection fragment size: 513 bp.

[0045] The PCR reaction conditions were: 30 cycles, each cycle being 95℃ for 30 min; 58℃ for 30 min; 72℃ for 40 min.

[0046] (2) Verify the introduction of the disease resistance gene using qRT-PCR MSLK corn plants Take the disease-resistant gene separately MSLK Approximately 100 mg of leaves from maize plants with nucleotide sequences (SEQ ID NO. 1, SEQ ID NO. 12) were used as samples. Genomic DNA was extracted using the EasyPure Plant Genomic DNA Kit (containing RNase A) (Cat. No: EE111-01) from TransGen Biotech Co., Ltd., Beijing. Disease resistance genes were detected using TransStart Green real-time PCR. MSLK The copy number was determined. Wild-type maize plants were used as a control, and the results were analyzed using the same method. The experiment was repeated in triplicate, and the average value was taken.

[0047] (3) Detection of disease resistance genes MSLK The specific method for determining gene copy number is as follows: (a) Removing the disease-resistant gene MSLK 100 mg of maize plant nucleotide sequence and 100 mg of leaves from wild-type maize plants were ground into homogenates in a mortar with liquid nitrogen, and three replicates were taken for each sample. (b) Use the EasyPure Plant Genomic DNA Kit (containing RNase A) from Beijing TransGen Biotech Co., Ltd. to extract genomic DNA from the above samples. Refer to the product instructions for specific methods. (c) The concentration of genomic DNA in the above samples was determined using a NanoDrop 2000 (Thermo Scientific); (d) Adjust the genomic DNA concentration of the above samples to the same concentration value, wherein the concentration value ranges from 80 to 100 ng / μL; (e) The copy number of the samples was identified using TransStart Green real-time PCR. Samples with known copy numbers were used as standards, and wild-type maize plant samples were used as controls. Each sample was tested in triplicate, and the average value was taken. The PCR reaction system is shown in Table 1, and the specific reaction conditions are shown in Table 2. The primer and probe sequences for real-time PCR are as follows: The following primers are used to detect disease resistance genes. MSLK Nucleotide sequence: Primer 1 (qF1): 5'-TTCGGCTGCGTTCTCAAGAT-3' (SEQ ID NO.13); Primer 2 (qR1): 5'-CGCCCATATCACTTTGCTGC-3' (SEQ ID NO.14).

[0048] Primer 3 (qF2): 5'- GAGCTTGGCTCAAGGACCAT-3' (SEQ ID NO.15); Primer 4 (qR2): 5'-AACACAGCCAAAAGCACGTC-3' (SEQ ID NO.16).

[0049] Table 1. Quantitative Real-Time PCR System

[0050] Table 2. Reaction conditions for quantitative real-time PCR

[0051] The data was analyzed using SDS2.3 software (Applied Biosystems).

[0052] Disease-resistant genes MSLK The PCR test results are as follows Figure 2 As shown, the results indicate that the disease resistance gene... MSLKThe nucleotide sequence has been integrated into the chromosome set of the tested maize plants, and the disease-resistant gene has been transferred. MSLK The maize plants with the specified nucleotide sequences were all transgenic maize plants containing a single copy.

[0053] WT represents wild-type plants, PC represents plasmid control, NC represents water control, 1-7 represent transgenic positive plants of SEQ ID NO.1, expressing the disease-resistant protein MSLK with the amino acid sequence shown in SEQ ID NO.2; 8-15 represent transgenic positive plants of SEQ ID NO.12, expressing the amino acid sequence shown in SEQ ID NO.11.

[0054] Example 3 Disease Resistance Test 1) Field occurrence of ear rot During the waxy ripening stage, the incidence of natural disease in the field was observed. Diseased grains were dull, not plump, brittle, and hollow inside, often filled with interwoven mycelia. The bracts of the diseased parts of the ears were often penetrated by dense mycelia, sticking together and adhering to the ears, making them difficult to peel off – these were signs of disease. Results were as follows... Figure 3 As shown in Table 3, compared with wild-type maize plants, the overexpressing plants OE1 (overexpressing SEQ ID NO.1 gene line 1) and OE2 (overexpressing SEQ ID NO.12 gene line 2) showed significantly improved resistance to ear rot, with the resistance level increasing from susceptible to highly resistant. The incidence of ear rot in OE2 was slightly lower than that in OE1, but there was no significant difference.

[0055] 2) Stem rot disease occurs in the field. During the waxy ripening and large trumpet stage, the incidence of natural disease in the field was recorded. The presence of irregular water-soaked lesions on the middle stem and leaf sheaths, the degree of plant rot, plant wilting, leaf death from top to bottom, and the presence of irregularly spreading brown spots at the base of the stem internodes, as well as the degree of internal hollowing, were used to determine the extent of stem rot infection. The incidence rate in the field was recorded during the waxy ripening stage. The results are as follows: Figure 3 As shown in Table 3, compared with wild-type maize plants, the overexpression plants OE1 and OE2 showed significantly improved resistance to stem rot. The stem rot resistance level of OE1 improved from susceptible to resistant, and the stem rot resistance level of OE2 improved from susceptible to highly resistant. The incidence of stem rot in OE2 was significantly lower than that in OE1, and the stem rot resistance level was one level higher than that of OE1; the stem rot resistance of OE2 was higher than that of OE1.

[0056] 3) Field occurrence of large and small leaf spot diseases At the waxy maturity stage, the area of ​​diseased leaves above the ear in the field was counted. The disease status of the plant was judged based on the spindle-shaped yellowish-brown lesions on the leaves. The results are as follows: Figure 3As shown in Table 3, compared with wild-type maize plants, the overexpression plants OE1 and OE2 showed significantly improved resistance to both large and small leaf spot diseases. The resistance level of OE1 to large and small leaf spot diseases improved from susceptible to moderately resistant, while the resistance level of OE2 to large and small leaf spot diseases improved from susceptible to resistant. The incidence of large and small leaf spot diseases in OE2 was significantly lower than that in OE1, and the resistance level of large and small leaf spot diseases was one level higher than that in OE1; the resistance of OE2 to large and small leaf spot diseases was higher than that in OE1.

[0057] Table 3 Disease resistance of transgenic MSLK maize plants

[0058] Disease resistance evaluation: Disease resistance levels for ear rot and stem rot: High resistance means incidence rate of 0.0-5.0%; Resistance means incidence rate of 5.1-10.0%; Moderate resistance means incidence rate of 10.1-30.0%; Susceptible means incidence rate of 30.1-40.0%; Highly susceptible means incidence rate of 40.1% or higher.

[0059] Resistance levels for large and small leaf spot diseases: High resistance refers to an affected area of ​​0.0-5.0%; Resistance refers to an affected area of ​​5.1-10.0%; Moderate resistance refers to an affected area of ​​10.1-30.0%; Susceptible refers to an affected area of ​​30.1-40.0%; Highly susceptible refers to an affected area of ​​more than 40.1%.

[0060] 4) Analysis of resistance differences among different individual plants of the same transgenic line To investigate the consistency of disease resistance phenotypes among different progeny plants from the same transformation event, three independent positive progeny plants (OE1-1, OE1-2, OE1-3; OE2-1, OE2-2, OE2-3) were selected from each of the OE1 and OE2 transgenic lines. The incidence rates of panicle rot and stem rot, as well as the affected area of ​​large and small leaf spot, were statistically analyzed under identical field conditions. The results showed that different plants from the same line all exhibited resistance phenotypes, but there were certain differences in disease resistance indicators, resulting in inconsistencies. Specific results are shown in Table 4.

[0061] Table 4. Comparison of disease incidence rates (%) among different individual plants of the same transgenic line.

[0062] The results showed that within the OE1 line, the incidence of ear rot fluctuated between 3.2% and 7.6% among different individual plants, while the incidence of stem rot ranged from 6.5% to 12.4%, with disease resistance levels ranging from "moderately resistant" to "highly resistant." Within the OE2 line, the incidence of ear rot fluctuated between 2.1% and 6.8% among different individual plants, while the incidence of stem rot ranged from 2.5% to 8.3%, with disease resistance levels ranging from "resistant" to "highly resistant." Inconsistencies in disease resistance phenotypes existed among different progeny plants of the same transgenic line, which may be related to factors such as T-DNA insertion site position effects, differences in insertion copy number, and segregation between homozygous and heterozygous states. Therefore, in practical breeding and application, it is necessary to conduct individual plant selection and phenotypic identification of transgenic progeny plants to screen for stable and superior disease resistance traits for variety improvement.

[0063] Production test results are as follows Figure 4 As shown, the disease-resistant MSLK transgenic plants showed increased yield compared to the control. The test samples included all plants. The yield before disease was achieved under greenhouse conditions with pesticide control, while the yield after disease was achieved for all individual plants naturally infected in the field.

[0064] Obviously, those skilled in the art can make various modifications and variations to this application without departing from the spirit and scope of this application. Therefore, if such modifications and variations fall within the scope of the claims of this application and their equivalents, this application also intends to include such modifications and variations.

Claims

1. A disease-resistant protein MSLK, characterized in that, The amino acid sequence of the disease-resistant protein is shown in SEQ ID NO.

11.

2. The disease-resistant protein MSLK according to claim 1, characterized in that, The nucleotide sequence of its encoding gene is shown in SEQ ID NO.

12.

3. A recombinant vector, characterized in that, It contains the gene encoding the disease-resistant protein MSLK as described in claim 1.

4. A recombinant bacterium, characterized in that, It includes the recombinant vector as described in claim 3.

5. An expression box, characterized in that, It contains the gene encoding the disease-resistant protein MSLK as described in claim 1.

6. The application of the disease-resistant protein MSLK according to claim 1 in the disease resistance characteristics of maize, wherein overexpression of the disease-resistant protein MSLK enhances the disease resistance characteristics of maize, characterized in that... The disease is one or more of stem rot, ear rot, or leaf spot.

7. The application according to claim 6, characterized in that, The application includes at least one of the following: 1) Prepare agents with disease resistance; the disease resistance refers to resistance to stem rot, ear rot, or leaf spot; 2) Develop transgenic corn with or improved resistance to diseases; the diseases mentioned are stalk rot, ear rot or leaf spot.

8. The application according to claim 7, characterized in that, The method for cultivating transgenic maize with or with enhanced disease resistance characteristics includes the following steps: expressing the disease-resistant protein MSLK as described in claim 1 in maize to obtain transgenic maize; wherein the disease is stalk rot, ear rot, or leaf spot.

9. A disease-resistant corn material, characterized in that, Insert the gene encoding the disease-resistant protein MSLK as described in claim 1 into the genome of the material, wherein the disease includes one or more of stem rot, ear rot, and leaf spot.

10. The application of a gene encoding a protein with the amino acid sequence shown in SEQ ID NO.2 in improving maize disease resistance, characterized in that... The application is as follows: overexpressing the gene in maize to breed transgenic maize with or with improved disease resistance, or preparing agents with disease resistance effects, wherein the disease is stalk rot, ear rot, or leaf spot.

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