Application of TaALDH7 gene in regulating crop resistance to stripe rust and cultivating disease-resistant varieties

Abstract

This invention discloses TaALDH7 Application of genes in regulating crop resistance to stripe rust and breeding disease-resistant varieties. This invention screened key genes regulating wheat resistance to stripe rust through combined transcriptomic and metabolomic analysis. TaALDH7 Through transient overexpression and gene silencing technology, TaALDH7 The function of the gene in wheat resistance to stripe rust was analyzed; the experimental results showed that, compared with the control group, the gene was overexpressed in wheat. TaALDH7 The disease index and fungal biomass of the gene were significantly reduced, and the colony area and hyphal length of stripe rust fungus were significantly decreased; conversely, in silent wheat... TaALDH7 The gene significantly reduces the resistance response of wheat to stripe rust, indicating that... TaALDH7 Genes act as positive regulators in the wheat's resistance to stripe rust. This invention shows promise for applications in breeding stripe rust-resistant wheat varieties and improving wheat's resistance to stripe rust fungi.

Description

Technical Field

[0001] This invention relates to disease resistance-related genes cloned from wheat and their uses, particularly to genes cloned from wheat. TaALDH7 Genes and their role in regulating wheat resistance to stripe rust belong to TaALDH7 Applications of genes in disease resistance. Background Technology

[0002] wheat( Triticum aestivum Stripe rust (L.) is a major crop worldwide, consumed as a staple food by 35% of the global population (FAO statistics database 2024). Stripe rust is caused by a fungal pathogen. Puccinia striiformis f.sp. tritici Stripe rust (Pst) is a serious disease widely distributed in wheat-producing areas worldwide. It typically causes 10-70% yield loss, and in severe cases, up to 100%. Pst exhibits high genetic diversity, resulting in different strains with varying levels of pathogenicity. Increased virulence weakens existing plant resistance; wheat varieties usually lose their innate resistance to stripe rust after 3-5 years of field use. Artificial breeding of wheat varieties with durable and broad-spectrum resistance is considered the most effective method for disease control. Therefore, identifying new wheat stripe rust resistance genes and studying their molecular regulatory mechanisms will contribute to the breeding of resistant varieties and is of great significance for the long-term effective control of wheat stripe rust.

[0003] A series of stripe rust resistance genes play a crucial role in wheat's response to stripe rust. With the continuous advancement of wheat genome research, numerous stripe rust resistance genes have been discovered and identified; several plants within the ALDH family have been confirmed to be associated with drought stress, including *Alfalfa truncatum* (…). Medicago truncatula )middle MtALDH7A1 Drought stress significantly induced the expression of this gene. Overexpression in Arabidopsis promoted lateral root proliferation (+40.0%), enhanced water uptake efficiency, and maintained osmotic balance by increasing proline accumulation. This resulted in a 35.0% increase in the survival rate of transgenic plants under 20% PEG-simulated drought (root system). MtALDH7A1 Expression level was upregulated 3.2-fold (Huang Siyuan. Transcriptome analysis of Alfalfa Tribulus terrestris and preliminary functional study of aldehyde dehydrogenase gene MtALDH7A1 under drought stress [D]. Northwest A&F University, 2019.). ALDH21 cotton( Gossypium hirsuteUnder 15% PEG stress, the strain increased germination rate by 50.0%, plant height by 40.0%, and relative leaf water content by 25.0% by scavenging malondialdehyde (MDA) and activating SOD / CAT enzyme activity (Gereziher TM, Yanchao X, Jawad MU, et al. Multi-Omics-Based Identification and Functional Characterization of GhA06G1257 Proves Its Potential Role in Drought Stress Tolerance in Gossypium hirsutum [J]. Frontiers in Plant Science, 2021, 12746771-746771).

[0004] Studies have shown that the aldehyde dehydrogenase (ALDH) family in plants plays a crucial role in alleviating salt stress-induced oxidative damage by scavenging aldehyde toxins (such as malondialdehyde) produced by lipid peroxidation (Baan TM, Liqaa IJ, Rana AHH, et al. Identification and characterization of aldehyde dehydrogenase (ALDH) gene superfamily in garlic and expression profiling in response to drought, salinity, and ABA. [J]. Gene, 2023, 860147215-147215.). For example, transgenic peanuts... AhALDH3H1Genetically modified soybeans maintain cell wall integrity by adjusting cell wall remodeling genes. Simultaneously, transgenic soybeans also promote the transport and metabolism of SOD, MDA, and aldehydes by activating the ABC transporter pathway, significantly reducing MDA accumulation (Yingxue C, Jing W, Siqi Z, et al. Overexpression of the aldehyde dehydrogenase AhALDH3H1 from Arachishypogaea in soybean increases saline-alkali stress tolerance.[J]. Frontiers in Plant Science, 2023, 141165384-1165384.). Wheat also belongs to the ALDH family... TaALDH7B1 The gene was strongly induced by salt stress. Silencing the gene resulted in increased MDA content, accelerated chlorophyll degradation, and a significant decrease in drought and salt tolerance in plants; conversely, overexpressing plants showed a survival rate 4–8 times higher than wild-type plants under salt stress (Jiamin C, Bo W, Guoliang L, et al.TraeALDH7B1-5A, encoding aldehyde dehydrogenase 7 inwheat,confers improved drought tolerance in Arabidopsis.[J].Planta,2015,242(1):137-51.). In common bean plants, under salt-alkali stress... PvALDH Gene expression, especially after treatment with neutral salt (NaCl) and alkaline salt (NaHCO3), increased synchronously with ALDH enzyme activity, significantly reducing oxidative stress levels (Wang X, Wu M, Yu S, et al. Comprehensive analysis of the aldehyde dehydrogenase gene family in Phaseolus vulgaris L. and their response to saline–alkali stress[J]. Frontiers in Plant Science, 2024, 151283845-1283845.). Therefore, the ALDH family has been shown to alleviate salt stress damage by scavenging aldehyde toxins in legumes (soybean, common bean, alfalfa), grasses (wheat), and malvaceaes (cotton). Its molecular mechanisms include direct catalytic detoxification, regulation of antioxidant enzyme activity, and stress signaling pathways. ALDHGene families are important targets for the genetic improvement of crop salt tolerance. To date, no [specific targets have been identified]. TaALDH7 Any reports of genes having the function of regulating resistance to stripe rust. Summary of the Invention

[0005] The main objective of this invention is to provide wheat TaALDH7 The role of genes in regulating crop resistance to stripe rust.

[0006] To achieve the above objectives, the main technical solutions adopted by the present invention include: This invention provides wheat TaALDH7 The application of genes in regulating plant resistance to stripe rust includes: by altering wheat... TaALDH7 The transcriptional or expression levels of genes in plants regulate plant resistance to stripe rust; wherein, the alteration of wheat... TaALDH7 The level of gene transcription or expression in plants can enhance wheat TaALDH7 The transcription or expression level of genes in plants can also reduce or weaken wheat. TaALDH7 The transcriptional or expression level of genes in plants; correspondingly, by altering wheat TaALDH7 Genes regulate stripe rust resistance at the transcriptional or expression level in plants or by altering wheat... TaALDH7 The regulation of stripe rust resistance by gene transcription and translation in plants should fall within the scope of protection of this invention.

[0007] As for how to increase or decrease wheat TaALDH7 The transcriptional or expression levels of genes in plants can be achieved by those skilled in the art through various conventional technical means; for example, by constructing wheat... TaALDH7 Gene overexpression vectors are used to transform plants via Agrobacterium-mediated genetic transformation to obtain wheat. TaALDH7 Overexpression of the gene can enhance the resistance of the overexpressing plant to stripe rust; alternatively, knocking out or interfering with the gene in the plant using CRISPR or VIGS methods can improve its resistance. TaALDH7 The level of gene transcription or expression affects the levels of gene transcription or expression in plants. TaALDH7 Deletion or mutation of a gene or its homologous gene can ultimately silence or reduce its effectiveness in plants. TaALDH7 The transcription or expression level of the gene or its homologous gene reduces the resistance of the resulting transgenic plants to stripe rust.

[0008] Those skilled in the art can construct wheat overexpression gene vectors using conventional methods. TaALDH7 A plant vector for overexpressing the gene or its homologous gene; or a wheat gene overexpression vector constructed using conventional gene editing techniques or gene knockout vector construction methods in the art. TaALDH7The editing vectors of genes or their homologous genes are methods well-known to those skilled in the art; for example, the wheat... TaALDH7 A gene or its homologous gene is operatively linked to an expression regulatory element to obtain an overexpression plant vector that can express the gene in plants; the overexpression plant vector contains a promoter, wheat TaALDH7 The vector contains the CDS sequence and terminator of a gene or its homologous gene; the promoter can be a constitutive promoter, an inducible promoter, or a tissue or organ-specific promoter, and the terminator sequence can be derived from the Ti-plasmid of *Agrobacterium tumefaciens*, such as the termination regions of octopine synthase and carmine synthase. The vector may also contain selective marker genes for selecting transformed cells or tissues. These marker genes include genes encoding antibiotic resistance and genes conferring herbicide resistance. Furthermore, the marker genes may also include phenotypic markers, such as β-galactosidase and fluorescent proteins.

[0009] In this invention, any plant transformation method can be used to transform the overexpression plant vector or gene editing vector constructed in this invention into the tissues or cells of the target plant to obtain the transformant. The transformant can then be regenerated by plant tissue culture to obtain the complete plant and its clone or its offspring. The transformation methods include Agrobacterium-mediated genetic transformation, protoplast transformation, plant virus vector, microinjection, electroporation, etc.

[0010] As a preferred embodiment of the present invention, the present invention provides a method for improving plant resistance to stripe rust, comprising: constructing wheat TaALDH7 The overexpression vector of the gene or its homologous gene; the wheat TaALDH7 Overexpression of the gene or its homologous gene in plants results in overexpressed plants with increased resistance to stripe rust.

[0011] As another preferred embodiment of the present invention, the present invention provides a method for breeding stripe rust-resistant plant varieties, comprising: constructing wheat... TaALDH7 overexpression vectors of genes or their homologs; wheat TaALDH7 The gene or its homologous gene is overexpressed in plants; overexpressing plants with enhanced resistance to stripe rust are screened from the obtained positive overexpression plants.

[0012] The plants described in this invention include, but are not limited to, monocotyledonous or dicotyledonous plants, with wheat being the most preferred plant.

[0013] The wheat described in this invention TaALDH7 The nucleotide sequence of the gene is selected from any one of (a)-(e): (a) The polynucleotide sequence shown in SEQ ID No. 1; (b) A polynucleotide that can hybridize with the complementary sequence of SEQ ID No. 1 under stringent hybridization conditions; (c) A polynucleotide that has at least 90% or more homology with the polynucleotide shown in SEQ ID No. 1; (d) A mutant of the polynucleotide shown in SEQ ID No. 1 by deletion, substitution or insertion of one or more bases, and the mutant still has the function or activity of regulating stripe rust resistance; (e) A nucleotide sequence encoding the amino acid sequence shown in SEQ ID No. 2.

[0014] In addition, those skilled in the art can optimize the nucleotides shown in SEQ ID No. 1 to enhance their expression efficiency in plants; for example, the preferred codons of the target plant can be used to optimize the synthesis of polynucleotides to enhance their expression efficiency in the target plant.

[0015] Chimeric genes or expression cassettes obtained by chimerizing or connecting the gene shown in SEQ ID No. 1 of this invention with other genes are all within the scope of protection of this invention; recombinant expression vectors containing the chimeric gene or expression cassette are also within the scope of protection of this invention.

[0016] This invention screened key genes regulating wheat resistance to stripe rust through combined transcriptomic and metabolomic analysis. TaALDH7 Through transient overexpression and gene silencing technology, TaALDH7 Preliminary investigation into the function of the gene in wheat resistance to stripe rust revealed that, compared to the control group, overexpression of the gene in wheat... TaALDH7 The disease index and fungal biomass of the gene were significantly reduced, and the colony area and hyphal length of stripe rust fungus were significantly decreased; wheat TaALDH7 Gene silencing significantly reduces the resistance response of wheat to stripe rust; the results indicate that... TaALDH7 Genes, as positive regulators, participate in the wheat's resistance to stripe rust and have promising applications in improving wheat's resistance to stripe rust or breeding stripe rust-resistant wheat varieties.

[0017] Definition of the terms of the invention Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. While any methods, apparatus, and materials similar to or equivalent to those described herein may be used in the practice or testing of this invention, preferred methods, apparatus, and materials are now described.

[0018] The terms "polynucleotide" or "nucleotide" refer to deoxyribonucleotides, deoxyribonucleosides, ribonucleosides, or ribonucleotides and their polymers, either in single-stranded or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogs of natural nucleotides, which have binding properties similar to a reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise specifically limited, the term also refers to oligonucleotide analogs, including PNAs (peptide nucleic acids), DNA analogs (phosphate thioesters, phosphoramidites, etc.) used in antisense techniques. Unless otherwise specified, a specific nucleic acid sequence implicitly encompasses variants of its conserved modifications (including, but not limited to, degenerate codon substitutions) and complementary sequences, as well as explicitly specified sequences. Specifically, degenerate codon substitution can be achieved by generating a sequence in which the 3rd position of one or more selected (or all) codons is substituted with a mixed base and / or deoxyinosine residue.

[0019] The term "homology" refers to the level of similarity or percentage identity between polynucleotide sequences in terms of percentage nucleotide positional similarity (i.e., sequence similarity or identity). As used here, homology also refers to the concept of similar functional properties between different polynucleotide molecules; for example, promoters with similar functions may have homologous cis elements. Polynucleotide molecules are homologous when they specifically hybridize under certain conditions to form a double-stranded molecule. Under these conditions (called stringent hybridization conditions), one polynucleotide molecule can be used as a probe or primer to identify another polynucleotide molecule sharing homology.

[0020] In this invention, "rigorous hybridization conditions" refers to conditions of low ionic strength and high temperature known in the field. Typically, under rigorous conditions, the detectability of a probe hybridizing with its target sequence is significantly higher than that with other sequences (e.g., at least twice the background level). Rigorous hybridization conditions are sequence-dependent and will vary under different environmental conditions; longer sequences hybridize specifically at higher temperatures. Target sequences that are 100% complementary to the probe can be identified by controlling the rigor of hybridization or washing conditions. Detailed guidance on nucleic acid hybridization can be found in the relevant literature (Tijssen, Techniques in biochemistry and molecular biology hybridization with nucleic probes, "Overview of principles of hybridization and the strategy of nucleic acid assays. 1993"). More specifically, the rigorous conditions are typically chosen to be below the melting point (T0) of the specific sequence at a specified ionic strength pH. m Approximately 5-10℃.m The temperature at which 50% of the probe complementary to the target sequence hybridizes to the target sequence under equilibrium conditions (at specified ionic strength, pH, and nucleic acid concentration) (because the target sequence is present in excess, therefore at T...). m (Under equilibrium conditions, 50% of the probe is occupied). Strict conditions may include: a salt concentration of less than approximately 1.0 M sodium ions at pH 7.0 to 8.3, typically approximately 0.01 to 1.0 M sodium ions (or other salts), and a temperature of at least approximately 30 °C for short probes (including, but not limited to, 10 to 50 nucleotides) and at least approximately 60 °C for long probes (including, but not limited to, greater than 50 nucleotides). Strict conditions can also be achieved by adding a destabilizing agent such as formamide. For selective or specific hybridization, the positive signal may be at least twice the background hybridization, and, where appropriate, 10 times the background hybridization. Exemplary strict hybridization conditions may be as follows: 50% formamide, 5×SSC and 1% SDS, incubated at 42 °C; or 5×SSC, 1% SDS, incubated at 65 °C, washed in 0.2×SSC and washed in 0.1% SDS at 65 °C. The washing can be performed for 5, 15, 30, 60, 120 minutes or longer.

[0021] In this invention, "multiple" generally means 2-8, preferably 2-4; "replacement" means replacing one or more amino acid residues with different amino acid residues; "deletion" means a reduction in the number of amino acid residues, that is, the absence of one or more amino acid residues; "insertion" means a change in the amino acid residue sequence, which, relative to the natural molecule, results in the addition of one or more amino acid residues.

[0022] The term "promoter" refers to a polynucleotide molecule that, in its native state, is located upstream or 5' of the translation start codon in the reading frame (or protein-coding region) and participates in the recognition and binding of RNA polymerase II and other proteins (trans-acting transcription factors) to initiate transcription.

[0023] The term "operably linked" refers to the linkage of a first polynucleotide molecule (e.g., a promoter) to a second transcribed polynucleotide molecule (e.g., a target gene), wherein the polynucleotide molecules are arranged such that the first polynucleotide molecule influences the function of the second polynucleotide molecule. Preferably, the two polynucleotide molecules are portions of a single, consecutive polynucleotide molecule, and more preferably, they are adjacent. For example, if a promoter regulates or mediates the transcription of a target gene within the cell, then the promoter is operably linked to the target gene.

[0024] The term "overexpression vector" refers to one or more DNA vectors used to achieve plant transformation; these vectors are often referred to as binary vectors in the art. Binary vectors, along with vectors containing helper plasmids, are commonly used for Agrobacterium-mediated transformation. Binary vectors typically include: the cis-acting sequence required for T-DNA transfer, a selection marker engineered for expression in plant cells, and a heterologous DNA sequence to be transcribed.

[0025] The term "conversion" refers to the method of introducing a heterologous DNA sequence into a host cell or organism.

[0026] The term "expression" refers to the transcription and / or translation of endogenous genes or transgenes in plant cells.

[0027] The terms "recombinant host cell line" or "host cell" refer to a cell containing the polynucleotides of the present invention, regardless of the method used for insertion to produce the recombinant host cell, such as direct uptake, transduction, pairing, or other methods known in the art. The exogenous polynucleotides may remain as, for example, non-integrating vectors of plasmids or may be integrated into the host genome. The host cell may be a prokaryotic or eukaryotic cell, and may also be a monocotyledonous or dicotyledonous plant cell. Attached Figure Description

[0028] Figure 1 is TaALDH7 Gene expression pattern identification and tissue expression analysis; A: Detection using qRT-PCR TaALDH7 Results of gene expression levels during the pathogen infection stage; B: TaALDH7 Results of qRT-PCR analysis of genes in wheat root, stem, and leaf tissues.

[0029] Figure 2 shows the subcellular localization results of TaALDH7 in tobacco leaves.

[0030] Figure 3 for TaALDH7 Results of transient gene silencing validation; Note: A: Albinism phenotype and phenotype of CYR34 inoculation 14 days; B: Silencing fragment amplification gel; C: Disease index 14 days after CYR34 inoculation; D: Fungal biomass detection 14 days after inoculation; E: Silencing efficiency detection before inoculation; F: Silencing efficiency detection after inoculation.

[0031] Figure 4 for TaALDH7 Results of DAB staining and reactive oxygen species (ROS) content detection in leaves after gene silencing; Note: A: H2O2 accumulation in wheat leaves at 24 hpi, 48 hpi, and 120 hpi after inoculation; B: H2O2 area measurement at 24 hpi after inoculation; C: H2O2 area measurement at 48 hpi after inoculation; D: H2O2 area measurement at 120 hpi after inoculation.

[0032] Figure 5 for TaALDH7 Results of WGA staining of leaves and detection of hyphal length and area after gene silencing; Note: A: Growth of stripe rust fungus in wheat leaves at 24 hpi, 48 hpi and 120 hpi after inoculation (SV: substomatal vesicles; HMC: haustorium mother cells; IH: infected hyphae); B: Observation of hyphal length at 24 hpi and 48 hpi after inoculation; C: Observation of hyphal area at 24 hpi after inoculation; D: Observation of hyphal area at 48 hpi after inoculation; E: Observation of hyphal area at 120 hpi after inoculation.

[0033] Figure 6 A schematic diagram of the pYBA-1132 vector (containing a GFP tag).

[0034] Figure 7 for TaALDH7 Functional results of transient gene overexpression validation; Note: A: TaALDH7 Identification of overexpression vector construction; B: Overexpression TaALDH7 Phenotypic diagram 14 days after CYR32 inoculation; C: Relative fungal biomass 14 days after CYR32 inoculation; D: TaALDH7 Transient overexpression efficiency detection; E: Disease index results 14 days after CYR32 vaccination.

[0035] Figure 8 for TaALDH7 Results of DAB staining and reactive oxygen species (ROS) content detection in leaves after transient gene overexpression; Note: A: H2O2 accumulation in wheat leaves at 24 hpi and 48 hpi after inoculation; B: H2O2 area measurement at 24 hpi and 48 hpi after inoculation.

[0036] Figure 9 for TaALDH7 Results of WGA staining of leaves and detection of hyphal length and area after transient gene overexpression; Note: A: Growth of stripe rust fungus in wheat leaves at 24 hpi and 48 hpi after inoculation (SV: substomatal vesicles; HMC: haustorium mother cells; IH: infected hyphae); B: Observation of hyphal length at 24 hpi and 48 hpi after inoculation; C: Observation of hyphal area at 24 hpi after inoculation; D: Observation of hyphal area at 48 hpi after inoculation.

[0037] Figure 10 for TaALDH7 Genotoxicity verification results; Note: A: TaALDH7 PCR amplification results of the gene; B:pGBKT7- TaALDH7 Plasmid (left); pGBKT7 empty vector (right).

[0038] Figure 11 pGBKT7- TaALDH7 Self-activation test results.

[0039] Figure 12 This is the result of point-to-point verification of yeast two-hybrids. Detailed Implementation

[0040] The present invention will be further described below with reference to specific experimental examples, and the advantages and features of the present invention will become clearer with the description. However, it should be understood that the experimental examples are merely exemplary and do not constitute any limitation on the scope of the present invention. Those skilled in the art should understand that modifications or substitutions can be made to the details and form of the technical solutions of the present invention without departing from the spirit and scope of the present invention, but such modifications or substitutions all fall within the protection scope of the present invention.

[0041] Experimental materials, vectors and strains The experimental materials, wheat varieties Liangchun 1723 and pYBA1132 subcellular localization vectors, were provided by the Institute of Plant Protection, Xinjiang Academy of Agricultural Sciences / Key Laboratory of Integrated Pest Management for Crops in Northwest Desert Oasis, Ministry of Agriculture and Rural Affairs; Escherichia coli strain DH5α and Agrobacterium strain GV3101 competent cells were purchased from TransGen Biotech, Beijing. The experimental materials used in this invention include various plasmids, among which the pGBKT7 and pGADT7 plasmid systems, as well as the vectors pYFPNE / pYFPCE required for the YFP bimolecular fluorescence complementary system and the matching vectors pLUCN / pLUCC for the LCI luciferase reporter system, were all obtained from the Crop Functional Genomics and Molecular Improvement Laboratory of the College of Life Sciences, Xinjiang Agricultural University.

[0042] Test reagents Plant total RNA mini-extraction kit was purchased from Guangzhou Magen Biotechnology Co., Ltd.; plasmid mini-extraction kit was purchased from Tiangen Biotech (Beijing) Co., Ltd.; DNA Marker and reverse transcription kit MonScript™ RTIII all-in-one Mix (with dsDNase) were purchased from Monad Biotechnology Co., Ltd.; 2×Taq PCR Mix was purchased from Beijing Adley Biotechnology Co., Ltd.; restriction endonucleases... EcoR I , BamHI、SpeI Purchased from NEB (Beijing) Co., Ltd.; 2×Assembly Mix; 2×ChamQ Universal SYBR qPCR Master Mix was purchased from Nanjing Novizan Biotechnology Co., Ltd.; X-α-Gal chromogenic substrate and yeast screening medium used in this invention were purchased from Biomed.

[0043] Experimental Example 1 TaALDH7 Gene expression pattern identification 1. Experimental Methods Ten plump, uniformly sized wheat seeds were sown evenly in 7 cm diameter plastic pots. After sowing, the pots were transferred to a climate chamber at 16 °C with 16 h of light and 8 h of darkness for cultivation. When the seedlings reached the stage of one leaf and one bud, a suspension of CYR32 urediniospores of stripe rust fungus was evenly applied to the upper surface of the leaf using a pipette. The seedlings were then treated in a 10 °C, high-humidity, dark environment for 24 h, and then transferred to a climate chamber at 8–12 °C for normal cultivation. Samples of inoculated wheat and control were collected at 0, 6, 12, 18, 24, 48, 72, and 96 hpi. Roots, stems, and leaves were also collected at the seedling stage for tissue-specific expression analysis. All samples were immediately flash-frozen in liquid nitrogen and stored at –80 °C. Each treatment was performed in triplicate.

[0044] Wheat tissue was thoroughly homogenized using a tissue homogenizer, and total RNA was extracted using the RNAprep Pure Polysaccharide-Polyphenol Plant Total RNA Extraction Kit. 1 µL of RNA sample was taken, and its integrity was assessed by 1% agarose gel electrophoresis. After quality verification, the RNA was reverse transcribed into cDNA using an M-MLV reverse transcriptase kit. The resulting cDNA sample was stored at -20 °C for later use. TaEF- RT-F / R and TaALDH7 qRT-PCR reactions were performed using the qRT-F / R method (Table 1), with three technical replicates per sample. After the reaction, the Ct values ​​of the target gene and internal reference gene were used to determine the optimal parameters. -ΔΔCt The method calculates the expression level of the target gene.

[0045] Table 1 Primer information used

[0046] 2. Experimental Results The expression level of TaALDH7 during the pathogen infection stage was detected using qRT-PCR. Wheat seedlings at the one-leaf-one-heart stage were inoculated with 5 µl of stripe rust fungal suspension. Infected leaves and fresh urediniospores were collected at eight time points, and RNA was extracted to detect TaALDH7 gene expression. The results showed that TaALDH7 was highly induced in wheat during the early stage of pathogen infection (12 hpi). Figure 1 A).

[0047] To clarify the role of genes in wheat tissues TaALDH7To investigate gene-specific expression patterns, this study performed qRT-PCR analysis on wheat root, stem, and leaf tissues. The results showed that, compared to leaves and stems, [the specific expression patterns of these genes were observed]. TaALDH7 The gene was expressed at the highest level in wheat roots. Figure 1 B).

[0048] Experimental Example 2: TaALDH7 Subcellular Localization Assay 1. Experimental Methods TaALDH7 is predicted to be localized on the cell membrane. By constructing the pYBA1132-TaALDH7 vector and transforming tobacco leaves with Agrobacterium, TaALDH7:: GFP Fusion genes and GFP The empty vector was transiently expressed in tobacco leaf cells, and the localization of TaALDH7 was determined by co-fusion of nuclear localization and cell membrane localization proteins (red light).

[0049] Combining the subcellular localization results predicted by the online platform WoLFPROST (https: / / wolfpsort.hgc.jp / ), primer p1132- TaALDH7 -F / R (Table 1) Cloning TaALDH7 The CDS region sequence of the gene was inserted into an expression vector containing the green fluorescent protein (GFP) gene. pYBA1132 ( Bam HI and Eco The plasmid was successfully constructed and transformed into Agrobacterium GV3101, where it was transiently expressed in tobacco. 48 h after injection into the lower epidermis of tobacco, the fluorescent subcellular localization was observed under an LSM980 confocal laser scanning microscope (Zeiss, Jena, Germany).

[0050] 2. Experimental Results Fluorescent subcellular localization results observed under confocal laser scanning microscopy (Zeiss, Jena, Germany) are as follows: Figure 2 As shown: TaALDH7:: GFP The cell membrane of tobacco leaf epidermal cells containing the fusion gene showed green fluorescence, which coincided with the red light indicating cell membrane localization, resulting in yellow light. This demonstrates that TaALDH7 is localized on the cell membrane. Figure 2 ).

[0051] Experimental Example 3: Silent Wheat TaALDH7 Phenotypic identification test of gene resistance to stripe rust 1. Experimental Methods Design specific primers TaALDH7 -VIGS-F / R (Table 1). PCR amplification TaALDH7The VIGS gene fragment was inserted into the BSMV-PDS multiple cloning site Spe I and Bam HI room. The vectors BSMV-α, BSMV-β, BSMV-γ, BSMV- PDS Following the method described in the literature (Wu Hongyan. Identification of the function of SA pathway-related genes in resistance to wheat scab using BSMV-VIGS technology [D]. Shandong Agricultural University. 2016), the recombinant plasmid BSMV-VIGS was constructed and then... TaALDH7 (Target design based on the SGN-VIGS website (https: / / vigs.solgenomics.net / )) TaALDH7 The specific fragment 300 bp primers were ligated into the BSMV-PDS vector using a seamless cloning kit (Novozymes, Nanjing) to construct the recombinant plasmid BSMV-. TaALDH7 The bacteria were transformed with Agrobacterium, and the mixed vector was injected into tobacco leaves at the 5-6 leaf stage via Agrobacterium-mediated transformation. After 3-5 days, tobacco leaf juice was extracted and rubbed onto wheat leaves (Xinchun 34) that had grown to the two-leaf-one-heart stage, following the method of Wu Hongyan et al., to induce silencing. Three groups of mixed resuspensions (BSMV-α, BSMV-β, and BSMV-...) were then used to induce silencing. PDS (Positive control, BSMV:) PDS ); BSMV-α, BSMV-β and BSMV-γ (negative control, BSMV: 00); BSMV-α, BSMV-β and BSMV- TaALDH7 (Target gene experimental group, BSMV:) TaALDH7 Wheat plants infected with the virus were used as positive controls, negative controls, and the target gene experimental group. When the positive control plants (BSMV: PDS After the leaves of the genus *Bacillus leuciscus* were bleached, they were divided into three groups: the Mock group (wild-type control group without any bacterial infection), the negative control group (BSMV: 00), and the target gene experimental group (BSMV: 00). TaALDH7 The fourth wheat leaf was inoculated with stripe rust fungus CYR34. Samples were collected at 24 hpi, 48 hpi, and 120 hpi post-inoculation to assess silencing efficiency. Tissue samples were also collected at 24 hpi and 48 hpi post-inoculation for histological processing and observation. Sporulation was observed approximately two weeks after inoculation, and disease index was statistically analyzed (Table 2).

[0052] Table 2 Grading and Identification Criteria for Wheat Stripe Rust Reactivity Type

[0053] Disease index = [Σ (number of diseased leaves at each level × corresponding severity level) / (total number of leaves surveyed × highest severity level)] × 100.

[0054] 2. Experimental Results 2.1 Construction of VIGS Recombinant Vector Instantaneous silencing of wheat using VIGS technology TaALDH7 Genes, including BSMV:PDS silent wheat TaPDS The gene that causes the whitening phenotype in wheat was used as a positive control (BSMV:PDS). Negative control plants (BSMV:PDS) were extracted. 00 ) and silence TaALDH7 Genes (that are silenced) TaALDH7 Gene-silenced plants (BSMV) following the nucleotide sequence of the gene shown in SEQ ID No. 14. TaALDH7 Total RNA from wheat leaves, detected by qPCR TaALDH7 Gene expression levels, results TaALDH7 Gene expression levels in gene-silenced plants (BSMV: TaALDH7 The values ​​were significantly lower than those in the control group. Figure 3 B). After the positive control (BSMV:PDS) showed an albino phenotype, the Mock and BSMV:PDS were then subjected to further treatment. 00 Phenotypic observation was conducted on wheat plants inoculated with BSMV:TaALDH7. Figure 3 A) Samples were collected at three time points (24 hpi, 48 hpi, and 120 hpi) after CYR34 inoculation to detect silencing efficiency. The results showed that... TaALDH7 Gene expression levels were significantly lower in gene-silenced plants than in the control group. Figure 3 E- Figure 3 F). Compared with BSMV:00 wheat plants, BSMV:TaALDH7 wheat showed a significant increase in disease index and fungal biomass. Figure 3 C Figure 3 D). Indicates TaALDH7 Genes play an important role in wheat's resistance to stripe rust infection.

[0055] 2.2 TaALDH7 Results of DAB staining and reactive oxygen species content detection in leaves after gene silencing Samples were collected at 24 hpi, 48 hpi, and 120 hpi after CYR34 inoculation, stained with DAB, and then observed under a fluorescence microscope to examine the stomatal vesicles formed by stripe rust infection. The area of ​​reactive oxygen species generated around these vesicles was also counted. Results showed that, from the perspective of reactive oxygen species emission, compared with the control plants, the transient silencing of reactive oxygen species was significantly reduced. TaALDH7 Plants with the gene produced less reactive oxygen species around the infection site, with the most significant differences observed at 24 hpi, 48 hpi, and 120 hpi time points. Figure 4 ).

[0056] 2.3 TaALDH7 Leaf WGA staining and mycelial length and area detection after gene silencing Samples were collected at 24 hpi, 48 hpi, and 120 hpi after inoculation with CYR34. Staining was performed using WGA-Alexa Fluor488, followed by observation under a fluorescence microscope to determine the colony area and hyphal length of the stripe rust fungus at different time points. Results showed that the colony area of ​​stripe rust fungus in BSMV:TaALDH7 was significantly larger than that in the control plant BSMV:00 at 48 hpi and 120 hpi, but the difference was not significant at 24 hpi. The hyphal length of stripe rust fungus in BSMV:TaALDH7 was significantly longer than that in the control plant BSMV:00 at 24 hpi and 48 hpi. Figure 5 ).

[0057] Example 4: Transient overexpression in wheat TaALDH7 Gene function verification test 1. Experimental Methods To verify TaALDH7 Using wheat 1723 cDNA as a template, specific primer pairs were designed to achieve the desired function. TaALDH7 To clone and construct the gene in pYBA1132 After the vector was sequenced correctly, it was transformed into Agrobacterium. When wheat reached the two-leaf-one-heart stage (Mingxian 169), the second leaf was injected using the dorsal leaf injection method. The injection area was marked with a marker. A total of 30 wheat plants were injected. A pYBA-1132-GFP empty vector control and a wild-type control (Mock) were also included. After sample processing, the samples were transferred to a 23℃ incubator for 24 h of humidity control. 36 h after injection, the vitreous surface of the injected leaf area was inoculated with the highly virulent wheat stripe rust race CYR32. After inoculation, the samples were placed in a 16℃ incubator for 24 h of humidity control. Samples were collected at 24 hpi and 48 hpi for transient overexpression efficiency detection and fungal biomass assessment. The 48 hpi leaf was also used for WGA staining and observation. Sporulation was observed approximately two weeks after stripe rust inoculation, and disease index was calculated.

[0058] 2. Experimental Results 2.1 Analysis using wheat transient overexpression technology TaALDH7 function of genes Cloning in one step TaALDH7 The gene was constructed into the pYBA-1132 vector (containing a GFP tag) (see the diagram of this vector). Figure 6The GFP:TaALDH7 recombinant vector was identified and transformed into wheat MX169 using Agrobacterium. Inoculation was performed 36 h after inoculation, and samples were collected at 24 hpi and 48 hpi to detect overexpression efficiency. The results showed... TaALDH7 Gene expression levels were significantly higher in gene-overexpressing plants than in controls. Tissue samples were collected at both time points for histochemical observation (DAB staining and WGA staining). The phenotype 14 days after CYR32 inoculation is shown in [Figure number missing]. Figure 7 Sporulation was observed approximately two weeks after inoculation with stripe rust. Compared to GFP:00 wheat plants, the disease index and fungal biomass of GFP:TaALDH7 wheat were significantly reduced. Figure 7 The results showed that... TaALDH7 Genes play an important role in the process of stripe rust infecting wheat.

[0059] 2.2 TaALDH7 Leaf DAB staining and reactive oxygen species content detection after transient gene overexpression Samples were collected at 24 hpi and 48 hpi after CYR32 inoculation, stained with DAB, and then observed under a fluorescence microscope to examine the stomatal vesicles formed by stripe rust infection. The area of ​​reactive oxygen species (ROS) generated around these vesicles was also counted. The results showed that, from the perspective of ROS bursts, compared with the control plants (GFP:00), plants transiently overexpressing TaALDH7 produced more ROS around the infection site, with the most significant differences observed at 24 hpi and 48 hpi. Figure 8 ).

[0060] 2.3 TaALDH7 WGA staining of leaves and detection of hyphal length and area after transient gene overexpression Samples were collected at 24 hpi and 48 hpi after CYR32 inoculation, stained with WGA-Alexa Fluor 488, and then observed under a fluorescence microscope to count the colony area and hyphal length of the stripe rust fungus at different time points. Results showed that overexpression... TaALDH7 The colony area of ​​*Strombus stripe rust* in the overexpressing plants was significantly smaller at 24 hpi and 48 hpi compared to the control plants (GFP:00), and the hyphal length of *Strombus stripe rust* in the overexpressing plants was significantly shorter at 24 hpi and 48 hpi compared to the control plants (GFP:00). Figure 9 ).

[0061] Experimental Example 5 TaALDH7 Screening and validation of gene-interacting proteins 1. Experimental Methods 1.1 TaALDH7 screening of interacting proteins TaALDH7A gene encoding an aldehyde dehydrogenase was successfully constructed. TaALDH7 Recombinant bait vectors were used, and candidate interacting proteins were identified and validated using yeast library screening technology. The functional localization of this interaction module in the wheat stripe rust resistance regulatory network was preliminarily elucidated. The research results contribute to clarifying... TaALDH7 This provides experimental evidence for the mediated disease-fighting molecular mechanism.

[0062] Will TaALDH7 Transfected into AH109 yeast strain, with pGBKT7 as a control, yeast toxicity and self-activation were tested. After completion, TaALDH7 interacting proteins in cotton were screened. First, BD- TaALDH7 The vector was co-transformed with the yeast library plasmid into AH109 competent cells and inoculated into SD / -Trp / -Leu dual-deficient medium. After white, moist single colonies grew, the single colonies were streaked to SD / -Trp / -Leu / -His triple-deficient medium. After single colonies grew, PCR amplification and sequencing were performed.

[0063] 1.2 Verification of yeast two-hybrid point-to-point interaction To address the false positive phenomenon in yeast two-hybrid interaction screening, TaATPas29 was selected as a candidate interaction factor for point-to-point verification based on the NCBI annotation database and existing literature information. Its CDS sequence was obtained using targeted amplification technology (primer information is shown in Table 3), and pGADT7- was constructed using homologous recombination. TaATPas29 Recombinant vector. pGBKT7- TaALDH7 With pGADT7- TaATPas29 AH109 yeast competent cells were co-transformed and inoculated in stages into SD / -Trp / -Leu dual-deficient medium and SD / -Trp / -His / -Ade triple-deficient medium supplemented with X-α-Gal. Positive / negative controls were set up. Colony growth status and β-galactosidase activity (blue color reaction) were used to determine whether TaALDH7 protein could interact with... TaATPas29 Protein-protein interactions.

[0064] Table 3 Primers used in this experiment

[0065] 2. Experimental Results 2.1 TaALDH7 Gene plasmid construction, toxicity and self-activation verification Using cDNA from Liangchun 1723 wheat as a template, specific primers were designed for amplification. TaALDH7 The CDS sequence was obtained. Using In-Fusion technology, the corresponding CDS sequence was inserted into the MCS site of the pGBKT7 vector (linearized by EcoRI / BamHI digestion), ultimately yielding the corresponding pGBKT7- TaALDH7 Plasmid. The already constructed pGBKT7- TaALDH7 The plasmid and the empty pGBKT7 vector were transformed into Saccharomyces cerevisiae Y2HGold competent cells, respectively, and evenly spread on SD / -Trp solid medium plates. After incubation at 28℃ for 2-3 days, the morphology, size, and number of yeast single colonies were observed to be basically consistent, indicating that... TaALDH7 It is not toxic to yeast. Figure 10 ).

[0066] Positive plasmid pGBKT7-P53 / pGADT7-SV40-T, negative plasmid pGBKT7 / pGADT7, and experimental plasmid pGBKT7-TaALDH7 / pGADT7 were transformed into *Saccharomyces cerevisiae* Y2HGold competent cells, respectively. The cells were then evenly spread on SD / -Trp / -Leu solid medium and incubated at 28°C for 2-3 days until single colonies appeared. Colony PCR was performed on the single colonies grown on SD / -Trp / -Leu solid medium to observe whether the positive band matched the target band. If they matched, it indicated that the plasmids of the three combinations had been successfully transformed into Y2HGold. Positive single colonies were picked and spotted onto SD / -Trp / -Leu and SD / -Trp / -Leu / -His / -Ade solid medium using a serial dilution method, and incubated at 28°C for 3-4 days to observe their growth status. On SD / -Trp / -Leu medium, positive, negative, and experimental groups all showed growth. However, on SD / -Trp / -Leu / -His / -Ade medium, only positive cells showed growth; negative cells and experimental groups did not show colony growth. This indicates that TaALDH7 cannot activate the expression of downstream reporter genes and there is no self-activation phenomenon. Figure 11 ).

[0067] 2.2 TaALDH7 Screening and validation of interacting proteins Using cDNA from Liangchun 1723 wheat as a template, specific primers were designed for amplification. TaALDH7 The CDS sequence was obtained. In-Fusion technology was used to extract the CDS sequence from the pGADT7 vector. Eco R Ⅰ / Bam The corresponding CDS sequence was inserted into the MCS site of the linearized (H Ⅰ) enzyme digestion, resulting in the pGADT7-TaATPs29 plasmid. The constructed pGADT7-TaATPs29 plasmid was co-transformed into yeast with the pGBKT7-TaALDH7 plasmid, plated on a two-deficient plate, and cultured. After clones grew on the two-deficient plate, spots were streaked onto a new three-deficient plate. Yeast point-to-point results showed an interaction between TaALDH7 and TaATPs29 (…). Figure 12 ).

Claims

1. Wheat TaALDH7 The application of genes in improving wheat resistance to stripe rust is characterized by, include: Enhanced wheat TaALDH7 The transcriptional or expression level of genes in wheat can improve wheat's resistance to stripe rust; the wheat TaALDH7 The nucleotide sequence of the gene is shown in SEQ ID No.

1.

2. The application according to claim 1, characterized in that, include: Building wheat TaALDH7 Gene overexpression vector; wheat TaALDH7 Overexpression of the gene in wheat resulted in wheat plants with increased resistance to stripe rust.

3. The application according to claim 2, characterized in that, The overexpression vector is a plant overexpression vector.

4. Contains wheat TaALDH7 Application of gene expression cassettes in improving wheat resistance to stripe rust; the wheat TaALDH7 The nucleotide sequence of the gene is shown in SEQ ID No.

1.

5. The application according to claim 4, characterized in that, include: The wheat-containing TaALDH7 Gene expression cassettes are genetically transformed into wheat, thus transforming wheat TaALDH7 The gene is overexpressed in wheat.

6. Contains wheat TaALDH7 Application of gene overexpression vectors in improving wheat resistance to stripe rust; the wheat TaALDH7 The nucleotide sequence of the gene is shown in SEQ ID No.

1.

7. The application according to claim 6, characterized in that, The overexpression vector is a plant overexpression vector.

8. The application according to claim 6, characterized in that, include: The wheat-containing TaALDH7 Gene overexpression vectors genetically transform wheat, turning wheat TaALDH7 The gene is overexpressed in wheat.

9. A method for breeding wheat varieties resistant to stripe rust, characterized in that, include: Building wheat TaALDH7 Gene expression cassettes or overexpression vectors; wheat TaALDH7 Gene expression cassettes or overexpression vectors are used to genetically transform wheat, enabling wheat to... TaALDH7 The gene was overexpressed in wheat, and wheat varieties resistant to stripe rust with improved resistance were screened from the obtained overexpression-positive wheat plants; the wheat TaALDH7 The nucleotide sequence of the gene is shown in SEQ ID No.

1.

10. A method for improving wheat resistance to stripe rust, characterized in that, include: Building wheat TaALDH7 Gene expression cassettes or overexpression vectors; wheat TaALDH7 Gene expression cassettes or overexpression vectors are used to genetically transform wheat, enabling wheat to... TaALDH7 The gene was overexpressed in wheat, resulting in transgenic wheat with increased resistance to stripe rust; the wheat described above... TaALDH7 The nucleotide sequence of the gene is shown in SEQ ID No. 1.

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