Gene ta2o3g5a-2 and encoded protein and application thereof related to resistance to wheat powdery mildew and stem base rot

Abstract

This invention belongs to the field of biotechnology and discloses genes related to resistance to both wheat powdery mildew and stem rot. Ta2O3G5A-2 Its encoded protein and applications. This invention cloned a prolyl 4-hydroxylase gene from the wheat variety Bainong Aikang 58, and named it... Ta2O3G5A-2 This invention discovers silence. Ta2O3G5A-2 After wheat was inoculated with powdery mildew fungus, its resistance to powdery mildew was significantly reduced, and overexpression of the fungus was observed. Ta2O3G5A-2 The resistance levels of wheat plants to powdery mildew and stem rot were significantly improved, indicating that... Ta2O3G5A-2 Increased expression levels in susceptible wheat plants significantly improved their resistance to powdery mildew and stem rot. Therefore, Ta2O3G5A-2 Genes can be used to breed new wheat varieties resistant to powdery mildew and stem rot.

Description

Technical Field

[0001] This invention relates to the field of biotechnology and discloses genes related to resistance to both wheat powdery mildew and stem rot. Ta2O3G5A-2 Its encoded proteins and applications. Background Technology

[0002] wheat( Triticum aestivum Wheat is one of the world's most important food crops, with one-third of the population relying on it as a staple food. Wheat is also one of my country's main food crops, and its production is directly related to ensuring national food security. Wheat powdery mildew (… Blumeria graminis f. sp. Tritici Powdery mildew, a fungal disease caused by *Fusarium graminearum*, is one of the most important diseases affecting wheat production. Due to changes in production methods, improved irrigation conditions, increased fertilizer application, and the lack of effective resistance genes in major promoted varieties, wheat powdery mildew is becoming increasingly serious in wheat-producing areas of my country, affecting approximately 100 million mu (about 6.67 million hectares) annually, becoming a major obstacle to stable and high wheat yields. Wheat stem base rot, caused by *Fusarium graminearum* and *Fusarium graminearum*, infects the base of wheat stems, leading to brown rot, easy breakage, and withered white ears, generally reducing yield by 5%-10%, with severe cases resulting in losses exceeding 50% or even total crop failure. Discovering broad-spectrum resistance genes and creating wheat germplasm resources resistant to both powdery mildew and stem base rot is of great value in disease resistance breeding.

[0003] Plant cell walls are dynamic network structures composed of polysaccharides, including components such as cellulose, hemicellulose, pectin, and lignin. Primary cell walls are rich in cellulose and its derivatives (methylated / acetylated forms), while secondary cell walls are mainly composed of cellulose (Laura et al., 2017; Kesten et al., 2017). As the first line of defense against pathogens, cell walls undergo a series of defensive changes during pathogen infection, including the synthesis of lignin polymers, accumulation of phenolic substances, callose deposition, and formation of papillae. These changes significantly enhance the mechanical strength of the cell wall. Based on this, the concept of "cell wall resistance" has been proposed, specifically referring to the comprehensive defense mechanism established by plants after pathogen infection through physical reconstruction of the cell wall, changes in chemical composition, and activation of signaling pathways (Cantu et al., 2008; Hückelhoven 2007).

[0004] α-ketoglutarate / Fe(II)-dependent dioxygenases (2-ODDs), also known as 2-OGDs, 2-OGXs, or 2OG-Fe(II)-Oxy, are a class of heme-free mononuclear iron-containing proteins widely found in animals, plants, and microorganisms (Petersen et al., 2003). The first member of this family to be identified was proline hydroxylase in collagen synthesis, a discovery that opened the door to research on 2-ODDs. Studies have shown that 2-ODD genes may be involved in plant immune responses. For example, in safflower and onion, 2-ODD genes (such as F3H, FLS, and ANS) drive flavonoid biosynthesis, and their products enhance disease resistance by inhibiting pathogen infection or neutralizing reactive oxygen species. The expression of 16 2-ODD genes in safflower is regulated by light, temperature, and hormones (such as salicylic acid), and may respond to pathogen infection through flavonoid accumulation (Ye et al., 2024). Subsequent studies have revealed their key roles in growth and development regulation, epigenetic modifications (such as DNA demethylation), fatty acid metabolism, and the synthesis of medicinal secondary metabolites (such as schisandrin) (Marcinkowski et al., 2020). As a large gene family, the Arabidopsis thaliana genome contains more than 130 2-ODD genes, which can be divided into three significantly different subfamilies—DOXA, DOXB, and DOXC—based on sequence similarity. Their catalytic diversity stems from the structural combination of a conserved double-stranded β-helical backbone and a variable substrate-binding pocket. The DOXB subfamily of genes encodes prolyl 4-hydroxylases (P4Hs), which are mainly involved in post-translational modifications of polypeptide chains and play an important role in the formation of plant cell walls and signal peptide hormones. The cell wall is the first line of defense for plants against pathogens and plays a crucial role in plant immunity. Studies have shown that 2-ODDs-like genes may be involved in plant immune responses.

[0005] The plant cell wall is a crucial structure for plant growth, development, and maintaining cell stability. Its morphogenesis and complex network structure are formed by polysaccharides linked together by proteins. The cell wall is the first line of defense against pathogens, playing a vital role in plant growth, development, and resistance to pathogen invasion. It also acts as a conduit for transmitting disease resistance signals. Pathogen invasion causes plants to release cell wall fragments, which are recognized by pattern recognition receptors on the cell membrane, triggering a kinase cascade reaction that strengthens the cell wall and enhances resistance to pathogens. Proline hydroxylases (P4Hs) catalyze the hydroxylation of proline residues in the cell wall, affecting the cross-linking and stability of cell wall components (such as extensins), thereby enhancing the plant's physical barrier against pathogen invasion. Proline hydroxylases have been reported in animals, participating in the synthesis of vertebrate collagen and hypoxia-induced transcription factors. However, reports on plant P4Hs are scarce, with only a few species reported, including Arabidopsis thaliana, Chlamydomonas, Carnation, and Tomato, and their specific functions remain unclear. Therefore, studying the function of P4Hs in plant immunity, especially in wheat, and clarifying their disease resistance mechanisms is of great significance for enriching plant disease resistance gene resources and creating disease-resistant germplasm resources. Summary of the Invention

[0006] To address the problems and shortcomings of existing technologies, the purpose of this invention is to address the issues related to resistance to both wheat powdery mildew and stem rot. Ta2O3G5A-2 Its encoded proteins and applications.

[0007] To achieve the objectives of this invention, the technical solution adopted is as follows: The first aspect of this invention provides a protein derived from the genus Triticum (tristicum). Triticum aestivum L.), named it Ta2O3G5A-2, which is a protein of the following type: A1) or A2) or A3) or A4): A1) Proteins with amino acid sequences as shown in SEQ ID NO.1; A2) A fusion protein obtained by attaching a tag to the N-terminus (i.e., amino terminus) and / or C-terminus (i.e., carboxyl terminus) of the protein shown in SEQ ID NO.1; A3) Proteins with the same function obtained by substituting and / or deleting and / or adding one or more amino acid residues of the amino acid sequence shown in SEQ ID NO.1; A4) Proteins that have 85% or more similarity to the amino acid sequence shown in SEQ ID NO.1 and have the same function.

[0008] SEQ ID NO.1 consists of 255 amino acid residues, and the specific sequence of the amino acids shown in SEQ ID NO.1 is as follows: MPPPAPDRKRRPPMEQPEAGDGAPQQRHKKSSAAKKAKKGGATGSSGGAWPAVKPKKDLLVNRLKGTQLLTVPDFLTSAEAKAFVDVAESMGFTHQGSLGPLKGEAYRDNDRISVTDPLLAQTLWES GINRVFMDINISGKVATGLNPNIRLYRYVEGQRFGRHIDESVNLGDGSRTQYTLLVYLSGKGSAKDSQALVGGETVFYDHRGGIVAEVAPVQGMALLHLHGARCMLHEARVVKKNVKYVLRSDVVFA.

[0009] To facilitate the purification of the protein in A1), a tag as shown in Table 1 can be attached to the N-terminus or C-terminus of the protein shown in SEQ ID NO.1.

[0010]

[0011] The protein in A3 above, wherein the substitution and / or deletion and / or addition of one or more amino acid residues is a substitution and / or deletion and / or addition of no more than 10 amino acid residues.

[0012] The proteins mentioned in A3 above can be synthesized artificially, or their encoding genes can be synthesized first and then expressed biologically.

[0013] The gene encoding the protein in A3) above can be obtained by deleting one or more nucleotides from the DNA sequence shown in SEQ ID NO.2, and / or by performing a missense mutation of one or more base pairs, and / or by attaching the coding sequence of the tag shown in Table 1 to its 5′ end and / or 3′ end.

[0014] A second aspect of the present invention provides a biomaterial related to the protein described in the first aspect above, wherein the biomaterial is any of the following: B1) Nucleic acid molecules encoding the proteins described in the first aspect above; B2), an expression cassette containing the nucleic acid molecule described in B1); B3) A recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2); B4) Recombinant microorganisms containing the nucleic acid molecules described in B1), or recombinant microorganisms containing the expression cassette described in B2), or recombinant microorganisms containing the recombinant vector described in B3).

[0015] Based on the above-mentioned biological material, preferably, the coding sequence of the nucleic acid molecule described in B1) is as shown in SEQ ID NO.2. The nucleotide sequence shown in SEQ ID NO.2 is specifically as follows: .

[0016] A third aspect of the present invention provides the application of the biomaterial described in the second aspect above, wherein the application is any one of the following: C1) Its application in improving plant disease resistance; C2) Its application in the preparation of products that enhance plant disease resistance; C3) Application in cultivating plants with improved disease resistance; C4) Application in the preparation of products that cultivate plants with enhanced disease resistance.

[0017] According to the above application, preferably, the disease resistance is resistance to powdery mildew and / or stem base rot.

[0018] According to the above application, preferably, the plant is a monocotyledonous plant or a dicotyledonous plant; more preferably, the dicotyledonous plant may specifically be Arabidopsis thaliana, and the monocotyledonous plant may specifically be wheat, corn, etc. Most preferably, the plant is wheat.

[0019] A fourth aspect of the present invention provides a method for cultivating transgenic plants with enhanced disease resistance, comprising the step of increasing the expression of nucleic acid molecules in a recipient plant capable of expressing the proteins described in the first aspect above, thereby obtaining transgenic plants; wherein the transgenic plants exhibit higher disease resistance than the recipient plants.

[0020] According to the above method, preferably, the disease resistance is resistance to powdery mildew and / or stem base rot.

[0021] According to the above method, preferably, the method for improving the expression of nucleic acid molecules capable of expressing the protein described in the first aspect in the recipient plant is as follows: introducing nucleic acid molecules encoding the protein described in the first aspect into the recipient plant.

[0022] According to the above method, preferably, the plant is a monocotyledonous plant or a dicotyledonous plant; more preferably, the dicotyledonous plant may specifically be Arabidopsis thaliana, and the monocotyledonous plant may specifically be wheat, corn, etc. Most preferably, the plant is wheat.

[0023] Compared with the prior art, the positive and beneficial effects achieved by the present invention are as follows: This invention is the first to identify and isolate the prolyl 4-hydroxylase gene from the wheat variety Bainong Aikang 58. Ta2O3G5A-2 and its encoded protein Ta2O3G5A-2, and found Ta2O3G5A-2 The gene and its encoded protein Ta2O3G5A-2 are associated with resistance to powdery mildew and stem rot in plants (such as wheat); gene silencing can inhibit wheat... Ta2O3G5A-2 Gene expression can reduce the resistance of plants (such as wheat) to powdery mildew; through overexpression... Ta2O3G5A-2 The gene significantly enhances the resistance of transgenic lines to powdery mildew and stem rot. Therefore, Ta2O3G5A-2 Genes that positively regulate resistance to powdery mildew and stem rot in plants (such as wheat) are overexpressed in plants (such as wheat). Ta2O3G5A-2 Genes can enhance the resistance of plants (such as wheat) to powdery mildew and stem rot. Ta2O3G5A-2Genes can be used in genetic engineering breeding. Introducing them into wheat varieties susceptible to powdery mildew will improve the wheat's resistance to powdery mildew and stem rot. Attached Figure Description

[0024] Figure 1 Different time points after inoculating Bainong 207 wheat with powdery mildew Ta2O3G5A-2 Gene expression analysis; the horizontal axis represents different time points after infection of Bainong 207 with a mixed strain of wheat powdery mildew (h: hours postinoculation); the vertical axis represents the inoculated leaves. Ta2O3G5A-2 Relative gene expression levels; Figure 2 To utilize VIGS to silence wheat Ta2O3G5A-2 Phenotypic results of wheat leaves inoculated with powdery mildew after gene therapy and the presence of [unclear text - possibly related to wheat leaves] Ta2O3G5A-2 Gene expression level detection results; the left image shows silent wheat. Ta2O3G5A-2 Phenotypic results of gene production; right figure shows silent wheat. Ta2O3G5A-2 In post-gene wheat Ta2O3G5A-2 Gene expression level detection results; γ represents wheat plants infected with BSMV: γ empty vector control; 1, 2, and 3 all represent wheat plants infected with BSMV: Ta2O3G5A-2; Figure 3 A schematic diagram of the spectral structure of the overexpression vector pBI220:Ta2O3G5A-2; Figure 4 GUS staining results for some T0 generation regenerated plants; Figure 5 for Ta2O3G5A-2 Identification results of T2 generation positive plants of Transgenic Kenong 199; among which Figure 5 A is Ta2O3G5A-2 A diagram showing the positive identification results of genes at the DNA level. Ta2O3G5A -2-T2-OE1 indicates the T2-OE1 strain, Ta2O3G5A-2-T2-OE2 indicates the T2-OE2 strain, Negative indicates a transgenic negative plant, Marker indicates a DNA Marker, positive indicates that the transgenic vector plasmid is used as a DNA template, and Water indicates that no DNA template is added to the PCR reaction system. Figure 5 B is Ta2O3G5A-2 The relative expression levels of genes in transgenic positive and transgenic negative plants, where CK represents the transgenic negative line, OE1 represents the T3-OE1 line, and OE2 represents the T3-OE2 line; Figure 5 C is Ta2O3G5A-2 Phenotypic diagrams of resistance identification in transgenic positive plants after inoculation with powdery mildew. Ta2O3G5A-2-T3-OE1 indicates resistance identification of the T3-OE1 line; Ta2O3G5A-2-T3-OE2 indicates resistance identification of the T2-OE3 line; Negative indicates resistance identification of transgenic negative plants; KN199 indicates that the album uses the recipient material Ke Nong 199; Figure 6 for Ta2O3G5A-2 Photographs of seedling resistance identification for stem base rot and results of disease index significance analysis of gene overexpression lines; the top image shows the seedling resistance identification for stem base rot; the bottom image shows the results of disease index significance analysis; the negative control is a transgenic negative plant. Ta2O3G5A-2 -T3- OE1 indicates the T3- OE1 strain. Ta2O3G5A-2 -T3- OE2 represents the T3- OE2 strain, OE1 represents the T3- OE1 strain, and OE2 represents the T3- OE2 strain. Detailed Implementation

[0025] The following examples are provided to better understand the present invention, but do not limit the invention. Unless otherwise specified, the experimental methods in the following examples are conventional methods. Unless otherwise specified, the test reagents used in the following examples were purchased from conventional biochemical reagent stores. All quantitative experiments in the following examples were performed in triplicate, and the results were averaged.

[0026] Example 1: Ta2O3G5A-2 Cloning of genes 1. Obtaining cDNA Seeds of the common wheat variety Bainong Aikang 58 were soaked in water at room temperature for 24 hours. After the seeds absorbed the water and the liquid was drained, the seeds were kept moist at room temperature for 24 hours. Once the seeds showed signs of sprouting, they were planted in pots. The seedlings were grown in a light incubator at 18℃ for 10 hours, 22℃ for 14 hours, and 70% humidity. At the two-leaf stage, the seedlings were infected with fresh powdery mildew spores. Leaves from three infected wheat seedlings were flash-frozen in liquid nitrogen after 24 hours of infection. The leaf materials were ground, and total RNA (Vazyme, catalog number: RC411-01) was extracted from both materials. The obtained RNA was reverse transcribed using primers with polyT to obtain cDNA.

[0027] 2. PCR amplification Using the cDNA obtained in step 1 as a template, primers P1 and P2, designed based on the TraesCS5A02G522300 transcript sequence, were used for PCR amplification with a high-fidelity enzyme to obtain the PCR amplification product. The primer sequences are as follows: Primer P1: 5'-AACCCGATGCTATGCCACC-3' (SEQ ID NO.3); Primer P2: 5'-TTCAGCTAAGCAAACACGA-3' (SEQ ID NO.4).

[0028] The PCR amplification reaction conditions were as follows: 95℃ for 5 min; then 95℃ for 15 s, 56℃ for 15 s, 72℃ for 1 min, for 32 cycles; and finally 72℃ for 5 min.

[0029] 3. Electrophoresis and sequencing The PCR amplification product obtained in step 2 above was ligated into the TA / Blunt-Zero Cloning Kit vector (Vazyme, catalog number: C601-01) for sequencing, and the full length of the target gene was obtained. The gene was named... Ta2O3G5A- 2 , Ta2O3G5A-2 The ORF sequence of the gene is shown in SEQ ID NO.2, and the amino acid sequence it encodes is shown in SEQ ID NO.1.

[0030] Example 2: Ta2O3G5A-2 Gene expression analysis in Bainong 207 1. Experimental Method: Seeds of the common wheat variety Bainong 207 were soaked in water at room temperature for 24 hours. After the seeds absorbed water and the liquid was drained, the seeds were kept moist at room temperature for 24 hours. Once the seeds showed signs of sprouting, they were planted in pots. The seeds were grown in incubators at 18℃ for 10 hours and 22℃ for 14 hours with 70% humidity. At the two-leaf stage, the seedlings were infected with fresh powdery mildew spores. Leaves from wheat seedlings at 0h, 24h, 48h, and 72h of infection were flash-frozen in liquid nitrogen, with leaves from three plants collected at each time point. The leaf materials were ground, and total RNA was extracted. Reverse transcription was performed using the HiScript III 1st Strand cDNA Synthesis Kit (+gDNA wiper) (Vazyme, catalog number: R312-01) to obtain cDNA. This cDNA was then used as a template for amplification using primers P3 and P4. Ta2O3G5A-2 The Tubulin fragment was amplified using TaTubulin internal control primers, and then analyzed by real-time quantitative PCR. Ta2O3G5A-2 Gene expression status. The specific nucleotide sequences of primers P3 and P4 are as follows: Primer P3: 5'-CAGCGCCACAAGAAATCCTC-3' (SEQ ID NO.5); Primer P4: 5'-TCAGCGGAAGTGAGGAAGTC-3' (SEQ ID NO. 6).

[0031] The PCR procedure was as follows: PCR reactions were amplified and fluorescence detected using a real-time quantitative PCR instrument (Roche, Germany). A 20 μL PCR reaction mixture contained 10 μL of 2×Taq Pro Universal SYBR qPCR Master Mix, 0.5 μM primers P3 and P4, and 2 μL of reverse-transcribed cDNA template. The amplification conditions were: 95℃ for 5 minutes, followed by 95℃ for 15 seconds and 60℃ for 20 seconds, for a total of 40 cycles. After the reaction, melting curves were measured. Gene expression levels were analyzed using SigmaPlot 14 software.

[0032] 2. Experimental Results: The detection results of real-time quantitative PCR are as follows Figure 1 As shown. By Figure 1 It can be seen that in wheat farmer 207, Ta2O3G5A-2 The gene expression was significantly upregulated after induction by powdery mildew at 48 h, reaching a peak at 72 h. This indicates that... Ta2O3G5A-2 Gene expression can respond to powdery mildew induction; when powdery mildew infection occurs... Ta2O3G5A-2 Gene expression levels are increased.

[0033] Example 3: Silence Ta2O3G5A-2 Obtaining genetically modified wheat and its resistance to powdery mildew 1. Obtaining the gene silencing fragment Ta2O3G5A-2 (VIGS): With Ta2O3G5A-2 Using the TA / Blunt-Zero Cloning vector (Vazyme, catalog number: C601-01) containing the ORF sequence of the gene as a template, PCR amplification was performed using primers P5 and P6 (P5 and P6 contain SmaI restriction sites and a 15 bp complementary sequence to the vector), yielding a 261 bp fragment of the target gene. Ta2O3G5A-2 The gene silencing fragment, denoted as Ta2O3G5A-2(VIGS) (the specific nucleotide sequence is shown in SEQ ID NO.7), was used to construct the VIGS silencing vector.

[0034] SEQ ID NO.7 is as follows: GAAGGTCAGCGCTTCGGCAGGCATATCGATGAGAGCGTCAACCTTGGGGACGGTTCTAGGACGCAGTATACATTGCTGGTATACCTTTCTGGCAAGGGAAGCGCGAAAGATTCGCAGGCTCTTGTTGGAGG GGAGACTGTCTTTTATGATCACCGAGGAGGAATCGTTGCTGAGGTTGCTCCCGTGCAAGGAATGGCTCTACTCCATCTACATGGCGCCAGGTGCATGCTGCATGAAGCTCGCGTCGTGAAAAAGAACGTC.

[0035] The nucleotide sequences of primers P5 and P6 are as follows: P5: 5'-TAGCTGATTAATTAACCCGGGGAAGGTCAGCGCTTCGGCA-3' (SEQ ID NO. 8); P6: 5'-TAGCTGAGCGGCCGCCCCGGGGACGTTCTTTTTCACGACG-3' (SEQ ID NO. 9).

[0036] The PCR amplification reaction system was as follows: 1 μL template (20 ng / μL), 2 μL upstream primer P5, 2 μL downstream primer P6, 25 μL PCR mix, and water added to 50 μL.

[0037] The PCR reaction conditions were as follows: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 10 s, 55℃ annealing for 15 s, 72℃ extension for 20 s, 30 cycles, 72℃ final extension for 5 min, and storage at 4℃. The PCR amplification product was recovered after detecting the amplified bands by 1.5% agarose gel electrophoresis, yielding Ta2O3G5A-2(VIGS).

[0038] 2. Construction of BSMV recombinant viral vector: Following standard molecular biology procedures, the material obtained in step 1 above... Ta2O3G5A-2 The silent gene fragment Ta2O3G5A-2 (VIGS) was inserted in reverse between the SmaⅠ restriction sites of the BSMV-VIGS viral vector γ via homologous recombination, while keeping the other sequences of the BSMV-VIGS viral vector γ unchanged, to obtain the recombinant vector γ-Ta2O3G5A-2.

[0039] 3. BSMV-VIGS Carrier System: The BSMV-VIGS viral vector system consists of α, β, and γ vectors.

[0040] The BSMV-VIGS viral vectors α and β, along with the recombinant vector γ-Ta2O3G5A-2, together constitute a silencing vector. Ta2O3G5A-2 The viral silencing vector system for genes, BSMV: Ta2O3G5A-2.

[0041] The BSMV-VIGS viral vector α, β and vector γ-PDS together constitute a silencing agent. TaPDS The viral silencing vector system BSMV:γ-PDS, which is derived from Scofield Laboratory (Scofield et al. 2005), contains a 185bp conserved fragment of the barley phytoene dehydrogenase gene (PDS), and can be used as a positive control for gene silencing. After gene silencing, the plant leaves show whitening, which can be used to visually detect whether the silencing of the VIGS system is effective.

[0042] 4. BSMV in vitro transcription: (1) Linearization of the carrier The BSMV-VIGS viral vector α chain, γ chain, recombinant vector γ-Ta2O3G5A-2, and vector γ-PDS were digested with MluⅠ, and the BSMV-VIGS viral vector β chain was digested with SpeⅠ to obtain linearized vectors.

[0043] (2) Using the linearized vectors obtained in step (1) as templates, in vitro transcription was performed to obtain RNA vectors α, β, γ, γ-Ta2O3G5A-2, and γ-PDS. The in vitro transcription reaction was performed according to mMESSAGEmMACHINE T7. in vitro The transcription kit (Invitrogen, catalog number: AM1344) was used according to the manufacturer's instructions. The transcription reaction system and conditions were as follows: total reaction volume 10.0 μL, including 3 μL linearized vector, 1 μL 10X Reaction Buffer, 5 μL 2 x NTP / CAP, and 1 μL Enzyme Mix. The reaction was carried out in a PCR instrument at 37℃ for 2 h. The transcription product was then stored at -80℃ for later use.

[0044] 5. Wheat plant cultivation and BSMV inoculation Select plump seeds of Bainong Aikang 58, place them in petri dishes to absorb water for one day, then drain the water and rinse the dishes clean. Keep the seeds moist for one day. Once the seeds show white sprouts, select wheat seeds of uniform growth and sow them in pots (seeds are arranged neatly with a spacing of about 2.5 cm between seedlings to facilitate subsequent virus inoculation). Place the sown wheat in a light incubator for growth at a temperature of 12℃ / 10℃ and a light-to-dark cycle of 14 hours of light / 10 hours of darkness. After a period of cultivation until the wheat has grown to two leaves and one bud, and the second leaf is identical to the first leaf, select uniformly growing Bainong Aikang 58 wheat and inoculate them using a friction inoculation method. Ta2O3G5A-2 The recombinant viral vector solution was applied to the second leaf of wheat. The inoculated wheat was then placed in a 23°C incubator and cultured in darkness for 24 hours. The plants were then transferred to a 23°C, 14-hour light / 10-hour dark environment to obtain BSMV:Ta2O3G5A-2 infected plants (i.e., silent plants). Ta2O3G5A-2 (Wheat plants with genetically modified genes).

[0045] Meanwhile, for control purposes, some plants were inoculated with BSMV:γ-PDS virus vector solution using the same method to obtain BSMV:γ-PDS infected control plants; another group of plants were inoculated with BSMV:γ virus vector solution using the same method to obtain BSMV:γ empty vector control plants.

[0046] The above BSMV: Ta2O3G5A-2 The recombinant viral vector solution is prepared by mixing in vitro transcription products in the following order: 10 μL vector α, 10 μL vector β, and 10 μL vector γ. Ta2O3G5A-2 The solution was prepared by mixing with 225 μL of FES Buffer (0.1 M glycine, 0.06 MK2HP04 buffer containing 1% sodium pyrophosphate, 1% macaloid, 1% celite; pH to 8.5-9.0 with phosphoric acid).

[0047] The above BSMV:γ-PDS viral vector solution is a solution obtained by mixing the in vitro transcription product in the following proportions: 10 μL vector α, 10 μL vector β, 10 μL vector γ-PDS and 225 μL FES Buffer.

[0048] The above BSMV:γ virus vector solution was prepared by mixing the in vitro transcription product in the following proportions: 10 μL vector α, 10 μL vector β, 10 μL vector γ and 225 μL FES Buffer.

[0049] 6. Silence Ta2O3G5A-2 wheat identification In this experiment, 14 days after inoculation with the viral vector, the fourth leaf of the positive control wheat inoculated with BSMV:γ-PDS viral vector solution showed a PDS albino phenotype, indicating that the gene had been silenced in wheat leaves at this stage and corresponding leaf age. This demonstrates that the BSMV-VIGS system can be successfully applied to gene silencing function studies in the wheat variety Bainong Aikang 58.

[0050] To further verify the silence of the BSMV-VIGS system Ta2O3G5A-2 The effect of the gene was investigated, and its expression level was detected using quantitative real-time PCR. The specific detection method is as follows: 14 days after inoculation with the virus, the fourth leaf of barley stripe mosaic virus showing obvious symptoms was cut from plants infected with BSMV: Ta2O3G5A-2 and control plants infected with BSMV: γ empty vector. Total RNA was extracted, reverse transcribed, and detected by quantitative real-time PCR. Ta2O3G5A-2 The relative expression levels of the genes (the specific primers, internal reference genes, reaction system and amplification reaction conditions for real-time quantitative PCR detection are the same as in Example 2).

[0051] Ta2O3G5A-2 The results of the detection of relative gene expression levels are as follows: Figure 2 As shown in the middle right figure. (By...) Figure 2 It can be seen that, compared with the control plants infected with BSMV:γ, the BSMV:γ-infected empty vector plants... Ta2O3G5A-2 In the plant Ta2O3G5A-2 The extremely significant decrease in the relative expression level of the gene indicates that the silencing sequence selected in this experiment is effective.

[0052] 7. Silence Ta2O3G5A-2 Analysis of powdery mildew resistance in wheat Effective silence Ta2O3G5A-2 BSMV gene infection: Ta2O3G5A-2 Plants infected with BSMV: Leaf segments of the 4th leaf from the empty control plant were placed on 6-BA medium and inoculated with fresh powdery mildew spores. They were then cultured for 7 days at 22℃ / 18℃ under 14h light / 8h dark conditions. Phenotypic results were observed after 7 days. Figure 2 As shown in the middle left figure.

[0053] Depend on Figure 2 It was observed that a large number of spore masses were produced on the leaf surface of plants infected with BSMV: Ta2O3G5A-2, while only a small number of spore masses were produced on the leaf surface of plants infected with BSMV: γ (empty control). This indicates that the disease incidence in the BSMV: γ (empty control) plants was significantly lower than that in the BSMV: γ (empty control) plants 7 days after inoculation with powdery mildew. Ta2O3G5A-2 Plants. This result indicates that the common wheat variety, Bainong Aikang 58, was silenced. Ta2O3G5A-2The gene weakened the resistance of Bainong Dwarf Anti-58 to powdery mildew.

[0054] Example 4: Overexpression Ta2O3G5A-2 Obtaining genetically modified wheat plants 1. Ta2O3G5A-2 Overexpression vector PBI220: Ta2O3G5A-2 Construction With Ta2O3G5A-2 Using the TA / Blunt-Zero Cloning vector (Vazyme, catalog number: C601-01) containing the gene's ORF sequence as a template, primers P7 and P8 (primer P7 containing a BamHI restriction site and primer P8 containing a StuI restriction site) were used as amplification primers for PCR amplification. The PCR amplified fragments were recovered, and the amplified products were inserted into the vector pBI220, which had been double-digested with BamHI and StuI, using homologous recombination. Ta2O3G5A-2 It is placed at the multiple cloning site downstream of the 35S promoter. This allows the target gene to be... Ta2O3G5A-2 The vector was cloned downstream of the strong promoter 35S to obtain the overexpression vector pBI220:Ta2O3G5A-2 (its structural schematic diagram is shown below). Figure 3 (As shown in the image). Sequencing verification confirmed that the vector was successfully constructed.

[0055] The nucleotide sequences of primers P7 and P8 are as follows: Primer P7: 5'-GGAGAGAACACGGG GGATCC ATGCCACCACCGGCGCCGG-3' (SEQ ID NO.10, the underlined part is the BamHI recognition base sequence) Primer P8: 5'-AACGTCGTATGGGTA AGGCCT CTAAGCAAACACGACGTCT-3' (SEQ ID NO.11, the underlined part is the Stu I recognition base sequence).

[0056] 2. Overexpression Ta2O3G5A-2 wheat plant acquisition The overexpression vector pBI220:Ta2O3G5A-2 constructed in step 1 above and the pAHC-25 vector containing the Bar and GUS genes (Jefferson RA, Kavanagh TA, Bevan MW. GUS fusions: beta-glucuronidase as a sensitive and versatile gene fusion marker in higherplants. EMBO J.1987,6:3901-3907.) were co-transformed into the callus tissue of wheat seedlings susceptible to powdery mildew to obtain overexpression. Ta2O3G5A-2 Wheat plants.

[0057] The specific experimental steps are as follows: (1) Select about 2,000 pieces of KENON 199 embryonic callus tissue that have been precultured for 7 days and pretreat them on hypertonic medium (MS + ABA 0.5 mg / L + hydrolyzed casein 500 mg / L + 2,4-D 2 mg / L + glucose 30 g / L + 0.4 mol / L mannitol, pH 5.8) for 4-5 hours; (2) Carrying the target gene Ta2O3G5A-2 The overexpression vector pBI220:Ta2O3G5A-2 and the bar-labeled pAHC-25 vector were co-transformed into KNON 199 callus tissue by gene gun bombardment. After bombardment, the tissue was cultured on hypertonic medium for 16 hours. (3) The callus tissue was transferred to recovery medium (1 / 2 MS + hydrolyzed casein 500 mg / L + 2,4-D 2 mg / L + sucrose 30 g / L, pH 5.8) and cultured in the dark for 2 weeks; (4) The callus tissue treated in step (3) was transferred to a selection medium containing herbicide (1 / 2 MS + ABA 0.5 mg / L + hydrolyzed casein 500 mg / L + 2,4-D 1 mg / L + sucrose 30 g / L + 4 mg / L Bialaphos, pH 5.8) and cultured for 2 weeks. (5) The herbicide-resistant callus obtained in step (4) was transferred to differentiation medium (1 / 2MS + L-glutamine 1 mmol / L + hydrolyzed casein 200 mg / L + KT 1 mg / L + IAA 0.5 mg / L + sucrose 30 g / L + agar 0.8%, pH 5.8) for differentiation. When the differentiated shoots grew to 2-4 cm, they were transferred to rooting medium (1 / 2MS + KT 1 mg / L + sucrose 30 g / L + agar 0.8%, pH 5.8).

[0058] (6) When the regenerated seedlings are about 8cm long and the root system is relatively strong, the tubes can be opened for hardening off for 1-2 days. Finally, wash away the culture medium residue carried by the roots and transplant them into pots to obtain T0 generation regenerated plants.

[0059] Since the co-transformation vector pAHC-25 contains a GUS reporter gene, GUS staining of the leaves of regenerated wheat plants can be performed (GUS staining is a well-known procedure in this field) for identification. Wheat plants whose leaves stain blue are preliminarily identified as positive transgenic wheat plants. Leaf tips were cut from T0 generation regenerated plants and subjected to GUS staining. The GUS staining results of some T0 generation regenerated plants are shown below. Figure 4 As shown. By Figure 4 It can be seen that the leaves of some T0 generation regenerated plants were stained blue, indicating that T0 generation Ta2O3G5A-2 transgenic wheat positive plants (referred to as T0 generation positive plants) were successfully obtained.

[0060] Example 5: Overexpression Ta2O3G5A-2 Identification of powdery mildew resistance in genetically modified wheat plants Seeds from the T0 generation positive plants obtained in Example 4 were harvested and planted in pots to obtain the T1 generation lines. The expression levels of the target gene in the T1 generation lines were analyzed by DNA molecular identification and qRT-PCR (the specific primers, internal reference genes, reaction system, and amplification conditions for qRT-PCR were the same as in Example 2) to identify positive T1 generation Ta2O3G5A-2 transgenic wheat lines (referred to as T1 generation positive lines). Seeds from the T1 generation positive lines were harvested. The seeds of the T1 generation positive lines were planted in pots to obtain the T2 generation lines, which were then identified by DNA molecular identification and qRT-PCR. The expression level of the target gene was analyzed to identify the T2 generation lines. T2 generation Ta2O3G5A-2 transgenic wheat positive lines (referred to as T2 generation positive lines) were screened and seeds of T2 generation positive lines were harvested. Seeds of two T2 generation independent positive lines were selected and planted in pots to obtain T3 generation positive lines (T3-OE1 line and T3-OE2 line, respectively).

[0061] The specific procedures for DNA molecular identification were as follows: Genomic DNA was extracted from wheat lines, and PCR amplification was performed using promoter primer P9 (AGTGGAAAAGGAAGGTGGCT) and gene primer P10 (TGTAAGCCTCCCCCTTGAGT). The PCR amplification products were then analyzed by 1% agarose gel electrophoresis to detect whether the target band could be amplified for positive identification of transgenic lines. The PCR reaction system consisted of: 100 ng genomic DNA template, 0.4 μl each of 10 μM P9 and P10; 5 μl 2×mix; and 3.2 μl ddH2O. The PCR reaction conditions were: 95℃ pre-denaturation for 5 min; 26 cycles of 95℃ for 15 s, 59℃ for 45 s, and 72℃ for 50 s; and a 72℃ extension for 10 min.

[0062] Among them, the DNA molecular identification results of the T3 generation positive lines (T3-OE1 line and T3-OE2 line) are as follows: Figure 5 As shown in a. From Figure 5 As can be seen from a, the target band, which is consistent with the plasmid positive control, can be amplified in transgenic positive lines, while the target band cannot be amplified in negative lines.

[0063] T3 generation positive lines (T3-OE1 and T3-OE2 lines) were detected by qRT-PCR. Ta2O3G5A-2 The results of the detection of relative gene expression levels are as follows: Figure 5 As shown in b. From Figure 5 b indicates that the expression level of the Ta2O3G5A-2 gene in the T3-OE1 and T3-OE2 lines was significantly higher than that in the transgenic negative plants.

[0064] Leaf segments of the first leaf from the T3-OE1 and T3-OE2 lines were cut at the two-leaf stage and placed on 6-BA medium. Transgenic negative plants were used as negative controls. Fresh powdery mildew spores were inoculated and cultured for 7 days at 22℃ / 18℃ under 14h light / 8h dark conditions. Phenotypic results were observed 7 days after inoculation with powdery mildew spores. Figure 5 As shown in c.

[0065] Depend on Figure 5 As shown in c, the transgenic negative-positive plants exhibited high susceptibility to powdery mildew, with leaves covered in powdery mildew spore masses; while the T3-OE1 and T3-OE2 strains showed only a small number of powdery mildew spores on their leaves. This indicates that, compared to the transgenic negative-positive plants, the T3 generation positive strains (T3-OE1 and T3-OE2 strains) showed significantly improved powdery mildew resistance.

[0066] Example 6: Overexpression Ta2O3G5A-2 Identification of resistance to stem base rot in genetically modified wheat plants Currently, wheat stem rot seriously harms wheat growth and development, and has a huge impact on wheat yield. Therefore, this study focuses on the resistance of the T3 generation positive lines (T3-OE1 and T3-OE2 lines) obtained in Example 5 to stem rot. At the same time, the transgenic negative plants and Cunmai 218 were used as controls. The wheat line Cunmai 218 was donated by Henan Agricultural University (the specific literature of Cunmai 218 is as follows: Li Qiaoyun, Guo Zhenfeng, Yin Zhao, et al. Identification of resistance to stem rot in seedlings and detection of resistance sites in 194 wheat varieties (lines) [J]. Journal of Triticeae Crops, 2025, 45(3):329-336.).

[0067] 1. Straw base rot strain tested: Fusarium pseudograss strain WZ-8A.

[0068] 2. Culture media: PDA medium, millet medium.

[0069] Preparation of PDA medium: Take 200 g of peeled potato, cut into chunks, add 1 L of distilled water, and boil until soft. Filter and collect the filtrate, add 20 g of sucrose, stir thoroughly to dissolve, and bring the volume to 1 L with distilled water. Add agar powder at a ratio of 20 g / L, dispense into containers, autoclave at 121℃ for 20 minutes, cool, and pour into plates for later use. This medium provides various nutrients required for microbial growth through potato extract, with sucrose as the carbon source and agar as the solidifying agent.

[0070] Preparation of millet culture medium: Boil an appropriate amount of distilled water, add millet and cook for 2 minutes. Quickly drain and rinse with cold water to remove surface starch. Spread the treated millet evenly on clean newspaper to air dry. After the moisture evaporates, dispense into sterile Erlenmeyer flasks. Seal and autoclave at 121℃ for 20 minutes. After cooling, it is ready for use. This preparation process retains nutrients through short-time cooking, ensures the particles are loose by rinsing with cold water, and prevents clumping by air drying, ultimately obtaining a solid substrate culture medium suitable for microbial culture.

[0071] 3. Experimental methods: (1) Reproduction of pathogenic bacteria: First, *Fusarium graminearum* strain WZ-8A was inoculated onto a PDA plate and incubated in the dark at 25°C for 3 days to obtain a pure culture. Then, mycelial pellets were obtained using a perforator and inoculated into sterilized millet medium. The culture was continued under the same conditions for 5-7 days, with periodic shaking of the conical flask to promote uniform mycelial growth. Once the mycelium completely covered the surface of the medium, the pellets were removed and dried to obtain inoculum with stable inoculum content.

[0072] (2) Indoor seedling resistance identification 1) Wheat variety preparation: First, the seeds (T3 generation) of the T2 generation transgenic positive line obtained in Example 5 were disinfected with a 1% sodium hypochlorite solution for 5 minutes. After rinsing three times with sterile water, they were placed at 4 ℃ for 24 hours for low-temperature induction. Then, the seeds were placed in petri dishes lined with sterile, moistened filter paper, with the ventral groove facing down, and cultured in the dark at 25 ℃ for 48 hours to obtain pre-germinated seeds. Throughout the treatment process, the filter paper should be kept moist to promote uniform seed germination.

[0073] 2) Inoculation method: Take 180 g of sterilized potting soil (the potting soil was taken from the original dike experimental field of Henan University of Science and Technology, and sterilized in an autoclave at 121℃ for 30 min after sieving) and fill it into 7 cm × 7 cm flower pots. Sow 9 pre-germinated seeds evenly in each pot. For inoculation treatment, use 0.3 g of millet culture containing bacteria, which is evenly placed around the base of the wheat stems. Each treatment group has 3 biological replicates to eliminate experimental error.

[0074] 3) Cultivation method: The plants were incubated in a 25 ℃ constant temperature incubator using a 14-hour light / 10-hour dark photoperiod, with soil moisture maintained through bottom irrigation. During cultivation, the substrate was kept moderately moist, avoiding drought, compaction, or waterlogging. Thirty days after inoculation, the disease incidence in wheat plants from each treatment group was systematically investigated to assess the severity of the disease.

[0075] 4) Methods for investigating seedling diseases: The severity of disease was investigated at the base of potted wheat seedlings, with each plant assessed individually. The investigation was conducted 30 days after wheat sowing. A nine-level classification method was used to categorize wheat stem base rot disease severity. Grade 0: The plant is not diseased; Grade 1: The stem in the ground is obviously brown or the first leaf sheath shows mild symptoms; Grade 3: The first leaf sheath is noticeably brown, but the leaf sheath itself has not turned black; Level 5: The first leaf sheath turns black or the second leaf sheath turns brown; Level 7: The third leaf sheath shows signs of browning, or the plant is slow to grow or is close to death due to the disease; Level 9: The plant died due to disease.

[0076] The resistance levels of various wheat varieties to wheat stem base rot were evaluated according to the wheat seedling stage stem base rot resistance evaluation criteria. The wheat seedling stage stem base rot resistance evaluation criteria are as follows: the average disease indexes for immune (I), highly resistant (HR), moderately resistant (MR), moderately susceptible (HS), and highly susceptible (HS) varieties are 0, 0–25, 25–35, 35–45, and >45, respectively. The formula for calculating the disease index (DI) is as follows:

[0077] The disease grading results of T3 generation positive lines (T3-OE1 and T3-OE2 lines) 30 days after inoculation with *Fusarium graminearum* during the seedling stage were as follows: Transgenic negative plants: 0 plants at level 0; 4 plants at level 1; 9 plants at level 3; 11 plants at level 5; 2 plants at level 7; 0 plants at level 9, with a disease index of 42.73, indicating moderate susceptibility to stem base rot. The resistant control, Cunmai 218, had 0 plants at level 0; 6 plants at level 1; 20 plants at level 3; 0 plants at level 5; 0 plants at level 7; 0 plants at level 9, with a disease index of 28.21, indicating moderate resistance to stem base rot. The T3-OE1 line had 1 plant at level 0; 6 plants at level 1; 12 plants at level 3; 5 plants at level 5; 0 plants at level 7; 0 plants at level 9, with a disease index of 29.78, indicating moderate resistance to stem base rot. The T3-OE2 strain has 5 plants at disease severity level 0; 6 plants at disease severity level 1; 11 plants at disease severity level 3; 5 plants at disease severity level 5; 0 plants at disease severity level 7; and 0 plants at disease severity level 9. The disease index is 26.34, classifying it as moderately resistant to stem base rot (e.g., ...). Figure 6 (As shown).

[0078] The above identification results indicate that: Ta2O3G5A-2 Increased expression of the gene in wheat plants susceptible to powdery mildew or stem rot can significantly improve the powdery mildew resistance of susceptible wheat plants to both diseases. Therefore, this gene can be used to breed wheat plants that are resistant to both powdery mildew and stem rot using genetic engineering techniques.

[0079] Finally, it should be noted that the above embodiments are merely preferred embodiments of the present invention and are not intended to limit the present invention in any other way. Any person skilled in the art may use the above technical content as inspiration to make changes or modifications. These are equivalent embodiments with variations. However, any simple modifications, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention without departing from the technical concept of the present invention still fall within the protection scope of the claims of the present invention.

Claims

1. A protein, which is a protein of the following type: A1), A2), A3), or A4. A1) Proteins with amino acid sequences as shown in SEQ ID NO.1; A2) A fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of the protein shown in SEQ ID NO.1; A3) Proteins with the same function obtained by substituting and / or deleting and / or adding one or more amino acid residues of the amino acid sequence shown in SEQ ID NO.1; A4) Proteins that have 85% or more similarity to the amino acid sequence shown in SEQ ID NO.1 and have the same function.

2. The biomaterial related to the protein of claim 1 is any one of the following: B1) A nucleic acid molecule encoding the protein of claim 1; B2), an expression cassette containing the nucleic acid molecule described in B1); B3) A recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2); B4) Recombinant microorganisms containing the nucleic acid molecules described in B1), or recombinant microorganisms containing the expression cassette described in B2), or recombinant microorganisms containing the recombinant vector described in B3).

3. The biomaterial according to claim 2, characterized in that, B1) The coding sequence of the nucleic acid molecule is shown in SEQ ID NO.

2.

4. Any of the following applications of the protein of claim 1 or the biomaterial of claim 2 or 3: C1) Its application in improving plant disease resistance; C2) Its application in the preparation of products that enhance plant disease resistance; C3) Application in cultivating plants with improved disease resistance; C4) Application in the preparation of products that cultivate plants with enhanced disease resistance.

5. The application according to claim 4, characterized in that, The disease resistance refers to resistance to powdery mildew and / or stem base rot.

6. The use according to claim 4 or 5, characterized in that, The plant is a monocotyledonous plant or a dicotyledonous plant.

7. A method for cultivating transgenic plants with enhanced disease resistance, comprising the step of increasing the expression of a nucleic acid molecule capable of expressing the protein of claim 1 in a recipient plant to obtain a transgenic plant; wherein the transgenic plant exhibits higher powdery mildew resistance than the recipient plant.

8. The method according to claim 7, characterized in that, The method for improving the expression of nucleic acid molecules that can express the protein of claim 1 in recipient plants is as follows: introducing nucleic acid molecules encoding the protein of claim 1 into recipient plants.

9. The method according to claim 7 or 8, characterized in that, The disease resistance refers to resistance to powdery mildew and / or stem base rot.

10. The method according to claim 9, characterized in that, The plant is a monocotyledonous plant or a dicotyledonous plant.

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