Application of the wheat TaHCT5 gene in improving crop resistance to stripe rust and breeding disease-resistant varieties

Abstract

This invention discloses wheat TaHCT5 Application of genes in improving crop resistance to stripe rust and breeding disease-resistant varieties. This invention uses combined transcriptomic and metabolomic analysis of wheat with qRT-PCR technology to screen and identify candidate genes that are significantly upregulated in the process of disease resistance. TaHCT5 By constructing overexpression and silencing vectors and combining transient transformation and virus-induced gene silencing technologies, the functional roles of candidate genes in wheat stripe rust resistance were systematically analyzed. It was found that overexpression of these genes in wheat... TaHCT5 Genes can significantly improve wheat's resistance to stripe rust by inhibiting the growth of certain pathogens in wheat. TaHCT5 Gene silencing significantly reduces the resistance response of wheat to stripe rust, indicating that... TaHCT5 Genes, as positive regulators, participate in the wheat's resistance to stripe rust. This invention has promising applications in improving wheat's resistance to stripe rust or breeding stripe rust-resistant wheat varieties.

Description

Technical Field

[0001] This invention relates to disease resistance-related genes isolated from wheat and their uses, particularly to genes isolated from wheat. TaHCT5 Genes and their role in regulating wheat resistance to stripe rust belong to TaHCT5 Applications of genes in disease resistance. Background Technology

[0002] Wheat stripe rust is one of the most serious diseases in wheat cultivation, caused by the fungus *Stripetra rust*. Puccinia striiformis f. sp tritici , Pst Stripe rust, characterized by its rapid infection, frequent variation in virulent races, and wide spread, poses a serious threat to wheat yield and quality. The main harms of this disease are significantly reduced photosynthetic efficiency in infected plants, insufficient grain filling, and hindered nutrient accumulation. In severe cases, it can lead to premature aging and even crop failure. The high incidence, widespread nature, and difficulty in controlling stripe rust make it an undeniable challenge in wheat production. Studying the pathogenic biological characteristics and transmission patterns of stripe rust, as well as developing superior resistant varieties, based on its distribution patterns and damage characteristics, is crucial for achieving sustainable wheat production.

[0003] Research has found clones RLP1.1 The gene is a wheat receptor protein gene involved in the defense response to stripe rust infection of wheat (Zhang G, Wang X, Wang X, et al.). RLP1.1 , a novel wheat receptor-likeprotein gene, is involved in the defense response against Puccinia striiformis f. sp. tritici [J]. Journal of experimental botany, 2013, 64 (12):3735-3746.). A calcium-dependent protein kinase (CDPK) gene identified from the wheat reference genome was detected by qRT-PCR and showed a significant induction effect during wheat-strip rust interactions. Knockdown of CDPK expression using virus-induced gene silencing (VIGS) resulted in impaired wheat resistance to stripe rust, accompanied by reduced hydrogen peroxide (H2O2) accumulation, increased fungal biomass ratio, decreased expression of defense-related genes, and enhanced pathogen hyphal growth, indicating... TaCDPK7It plays a regulatory role in wheat resistance to stripe rust (Goher F, Bai X, Liu S, et al. The Calcium-Dependent Protein Kinase). TaCDPK7 Positively Regulates Wheat Resistanceto Puccinia striiformis f. sp tritici [J]. International Journal of Molecular Sciences, 2024, 25 (02): 1048. A team from Northwest A&F University identified and characterized a wheat receptor-like cytoplasmic kinase gene. TaPsIPK1 This gene indicates susceptibility to the pathogen. PsSpg1 is a secretory fungal effector crucial to the virulence of stripe rust fungi, capable of binding... TaPsIPK1 This enhances its kinase activity and promotes its nuclear localization, thereby phosphorylating the transcription factor TaCBF1d for gene regulation. TaCBF1d Phosphorylation of this compound alters its transcriptional activity in downstream genes. In a two-year field trial in wheat... TaPsIPK1 CRISPR-Cas9 inactivation conferred broad-spectrum resistance to stripe rust without affecting important agronomic traits (Alberto M, Pengcheng W, Kang JZ. Modification of the susceptibility gene TaPsIPK1-awin-win for wheat disease resistance and yield[J].Stress Biology,2022,2(1):40-40.DOI:10.1007 / S44154-022-00060-3).

[0004] The study identified wheat NLR protein resistance gene analog 3 ( TaRGA3 This gene was significantly upregulated during stripe rust infection. Nucleotide-binding and leucine-rich repeat receptor (NLR) is a protein encoded by a disease resistance gene (R gene) that activates the innate immune response upon sensing pathogen attack. Virus-induced gene silencing (VIGS) and overexpression demonstrated that… TaRGA3 Promoting wheat resistance to stripe rust by promoting the accumulation of reactive oxygen species (ROS) (Fang N, Jia C, Chen R, et al. The wheat CC-NBS-LRR protein) TaRGA3Confers resistance to stripe rust by suppressing Ascorbate peroxidase 6 activity[J]. Plant physiology, 2024). coding AP2 transcription factors TaAP2-10 It was identified from a cDNA library of wheat-strip rust incompatible interactions. Transient silencing of TaAP2-10 by barley stripe mosaic virus (BSMV)-induced gene silencing (VIGS) impaired wheat resistance to stripe rust. The results indicate... TaAP2-10 As a transcription factor, it positively regulates resistance to wheat stripe rust (Hu Z, Wang X, Wei L, Wansee S, et al.). TaAP2-10 , an AP2 / ERF transcription factor, contributes to wheat resistance against striperust[J]. Journal of Plant Physiology, 2023, 288 (09): 154078). In summary, the genes and signal transduction pathways involved in wheat stripe rust that have been cloned so far mainly involve receptor proteins, protein kinases, and transcription factor regulation. While some progress has been made in the research of wheat stripe rust-related genes, the discovery and application of resistance gene resources remain scarce and require further in-depth research.

[0005] Wheat is an important food crop, and its safe, stable, and high-quality production is crucial for food security. However, wheat stripe rust is one of the major diseases threatening wheat production. In environments suitable for stripe rust fungus growth, highly susceptible varieties may experience severe yield reductions or even crop failure. Stripe rust is spread by air currents, and its virulent races mutate rapidly, exhibiting strong adaptability and rapid disease development, making its control difficult. Traditional chemical control measures, while able to suppress the disease in the short term, suffer from environmental pollution and high costs. In contrast, planting disease-resistant varieties is currently the most economical and environmentally friendly control strategy. Therefore, discovering disease-resistant genes and screening plants with stable resistance is key to solving the wheat stripe rust problem at its source and is also an important pathway to achieving green control. Summary of the Invention

[0006] The main objective of this invention is to provide wheat TaHCT5 The use of genes and their expression vectors in regulating crop resistance to stripe rust.

[0007] To achieve the above objectives, the main technical solutions adopted by the present invention include: This invention provides wheat TaHCT5 The application of genes in regulating plant resistance to stripe rust includes: by altering wheat... TaHCT5 The transcriptional or expression levels of genes in plants regulate plant resistance to stripe rust; wherein, the alteration of wheat... TaHCT5 The level of gene transcription or expression in plants can enhance wheat TaHCT5 The transcription or expression level of genes in plants can also reduce or weaken wheat. TaHCT5 The transcriptional or expression level of genes in plants; correspondingly, by altering wheat TaHCT5 Genes regulate stripe rust resistance at the transcriptional or expression level in plants or by altering wheat... TaHCT5 The regulation of stripe rust resistance by gene transcription and translation in plants should fall within the scope of protection of this invention.

[0008] As for how to increase or decrease wheat TaHCT5 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... TaHCT5 Gene overexpression vectors are used to transform plants via Agrobacterium-mediated genetic transformation to obtain wheat. TaHCT5 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. TaHCT5 The level of gene transcription or expression affects the levels of gene transcription or expression in plants. TaHCT5 Deletion or mutation of a gene or its homologous gene can ultimately silence or reduce its effectiveness in plants. TaHCT5 The transcription or expression level of the gene or its homologous gene reduces the resistance of the resulting transgenic plants to stripe rust.

[0009] Those skilled in the art can construct wheat overexpression gene vectors using conventional methods. TaHCT5 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. TaHCT5 The editing vectors of genes or their homologous genes are methods well-known to those skilled in the art; for example, the wheat... TaHCT5 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 TaHCT5The 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.

[0010] 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.

[0011] As a preferred embodiment of the present invention, the present invention provides a method for improving plant resistance to stripe rust, comprising: constructing wheat TaHCT5 The overexpression vector of the gene or its homologous gene; the wheat TaHCT5 Overexpression of the gene or its homologous gene in plants results in overexpressed plants with increased resistance to stripe rust.

[0012] As another preferred embodiment of the present invention, the present invention provides a method for breeding stripe rust-resistant plant varieties, comprising: constructing wheat... TaHCT5 overexpression vectors of genes or their homologs; wheat TaHCT5 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.

[0013] The plants described in this invention include, but are not limited to, monocotyledonous or dicotyledonous plants, with wheat being the most preferred plant.

[0014] The wheat described in this invention TaHCT5 The nucleotide sequence of the gene (XM_044564034) is selected from any 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.

[0015] 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.

[0016] 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.

[0017] Based on previous combined transcriptomic and metabolomic analyses of wheat, this invention, combined with qRT-PCR technology, screened and identified candidate genes that were significantly upregulated during disease resistance. TaHCT5 By constructing candidate genes TaHCT5 Overexpression and silencing vectors were used, combined with transient transformation and virus-induced gene silencing (VIGS) technology, to systematically analyze the functional roles of candidate genes in wheat stripe rust resistance. Simultaneously, yeast two-hybrid (Y2H), luciferase complementation imaging (LCI), and bimolecular fluorescence complementation (BiFC) techniques were employed to screen and validate interacting proteins of candidate genes, further analyzing their expression patterns and regulatory mechanisms to reveal their specific functions in the disease resistance network. The results showed... TaHCT5 Genes play a crucial role in wheat resistance to stripe rust; among them, overexpression in wheat TaHCT5 Genes can significantly improve wheat's resistance to stripe rust by inhibiting the growth of certain pathogens in wheat. TaHCT5 Gene silencing significantly reduces the resistance response of wheat to stripe rust; the results indicate that... TaHCT5 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.

[0018] Definitions of terms involved in this 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.

[0019] 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.

[0020] 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.

[0021] 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.

[0022] 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.

[0023] 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.

[0024] 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.

[0025] 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.

[0026] The term "conversion" refers to the method of introducing a heterologous DNA sequence into a host cell or organism.

[0027] The term "expression" refers to the transcription and / or translation of endogenous genes or transgenes in plant cells.

[0028] 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

[0029] Figure 1 is TaHCT5Expression pattern identification and tissue expression analysis results; A: TaHCT5 Results of highly induced gene expression in wheat during the early stage of pathogen infection (12 hpi); B: TaHCT5 Results of qRT-PCR analysis of genes in wheat root, stem, and leaf tissues.

[0030] Figure 2 shows the subcellular localization results of TaHCT5 in tobacco leaves.

[0031] Figure 3 for TaHCT5 Results of transient gene silencing verification function; Note: A: Albinism phenotype and phenotype of CYR34 inoculation 14d; B: Electrophoresis gel image of silenced fragment amplification (left image shows silenced fragment amplification, right image shows identification after vector construction); C: Disease index of CYR34 inoculation 14d; D: Fungal biomass detection 14d after inoculation; E: Silencing efficiency detection before inoculation; F: Silencing efficiency detection results after inoculation.

[0032] Figure 4 for TaHCT5 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.

[0033] Figure 5 for TaHCT5 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.

[0034] Figure 6 for TaHCT5 Functional results of transient gene overexpression validation; Note: A: TaHCT5 A: Identification results of the overexpression vector construction (left electrophoresis image shows CDS fragment amplification, right image shows identification after vector construction); B: Phenotype after CYR32 inoculation for 14 days; C: Relative fungal biomass after CYR32 inoculation for 14 days; D: TaHCT5 Transient overexpression efficiency detection; E: Disease index and severity of disease after CYR3214 days of vaccination.

[0035] Figure 7 for TaHCT5 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, 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.

[0036] Figure 8 for TaHCT5 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 9 for TaHCT5 Genotoxicity verification results; Note: A: TaHCT5 PCR amplification results of the gene; B:pGBKT7- TaHCT5 Plasmid (left); pGBKT7 empty vector (right).

[0038] Figure 10 pGBKT7- TaHCT5 Self-activation test results.

[0039] Figure 11 The results validated the interaction between TaHCT5 and TaRubisco. Note: A: In the yeast two-hybrid experiment, pGBKT7+pGADT7 was used as a negative control, and pGBKT7-53+pGADT7-T was used as a positive control. The growth status of the experimental group and the control group on SD / -Trp / -Leu / -His / -Ade auxotrophic medium was observed after 3-5 days; B: In vivo bimolecular fluorescence complementation experiments were performed using cYFP+nYFP, cYFP+nYFP-TaHCT5, and cYFP-TaRubisco. + nYFP was used as the control, and cYFP-TaRubisco+nYFP-TaHCT5 was used as the experimental group. After 3 days, the yellow fluorescence signal was observed by laser confocal microscopy. 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, and the subcellular localization vector 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 types of 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 pLUCN / pLUCC required 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 , BamH I, Spe I 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: Wheat TaHCT5 Gene expression pattern identification test 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 hours of light and 8 hours of darkness. Once the seedlings had developed 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 at 10°C, high humidity, and darkness for 24 hours, before being 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 TaHCT5 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] 2. Experimental Results Based on the combined analysis of previous transcriptomic and metabolomic data from our laboratory, and using qRT-PCR technology, we screened out key genes that showed a significant response to wheat stripe rust induction. The results indicated... TaHCT5 It was highly induced to express ( ) in the early stage of pathogen infection in wheat (12 hpi) Figure 1 A), this result indicates that, TaHCT5 It plays an important role in the early stages of pathogen infection in wheat.

[0046] To clarify the role of genes in wheat tissues TaHCT5 To investigate the specific expression pattern of the gene, this study performed qRT-PCR analysis on wheat root, stem, and leaf tissues. The results showed that, compared to roots and stems, the gene... TaHCT5 The highest expression level was observed in wheat leaves. Figure 1 B).

[0047] Experimental Example 2 TaHCT5 Subcellular localization assay 1. Experimental Methods Combining the subcellular localization results predicted by the online platform WoLFPROST (https: / / wolfpsort.hgc.jp / ), p1132- TaHCT5 -F / R (Table 1) Cloning TaHCT5 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).

[0048] 2. Experimental Results TaHCT5 Predicted to be located on the cell membrane, through the construction of pYBA1132- TaHCT5 The carrier utilizes Agrobacterium to transform tobacco leaves, enabling... TaHCT5 :: GFP The fusion gene and empty GFP vector were transiently expressed in tobacco leaf cells. Simultaneously, nuclear localization and cell membrane localization proteins (red light) were co-fused to determine... TaHCT5 The localization. Subcellular localization results are as follows: Figure 2 As shown, TaHCT5 :: GFP The cell membranes of tobacco leaf epidermal cells containing the fused gene exhibited green fluorescence, which coincided with the red light localized to the cell membrane, resulting in yellow light. These results demonstrate... TaHCT5 It is located on the cell membrane.

[0049] Experimental Example 3: Transient Silencing of Wheat Using VIGS Technology TaHCT5 Gene analysis TaHCT5 function of genes 1. Experimental Methods Design specific primers TaHCT5 -VIGS-F / R (Table 1).

[0050] Table 1 Primer Sequences

[0051] PCR amplification TaHCT5 The 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(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.) and BSMV- TaHCT5 (BSMV-) TaHCT5 Construction method ： Target design based on the SGN-VIGS website (https: / / vigs.solgenomics.net / ) TaHCT5 Primers containing a specific 300 bp fragment were used to ligate the fragment into the BSMV-PDS vector using a seamless cloning kit (Novozymes, Nanjing) to construct the recombinant plasmid BSMV-. TaHCT5 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 , BSMV-α, BSMV-β and BSMV-γ, BSMV-α, BSMV-β and BSMV- TaHCT5 Infected wheat plants were used as positive and negative controls, as well as the target gene experimental group. When the positive control plant BSMV- PDS After the leaves turned white, the fourth wheat leaf was inoculated with stripe rust fungus CYR34. Samples were collected at three time points: 0 h, 24 h, and 48 h post-inoculation to determine the silencing efficiency. Simultaneously, tissue samples were collected at 24 h and 48 h post-inoculation for histological processing and observation. Sporulation was observed approximately two weeks after inoculation, and disease index statistics were performed (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 TaHCT5 Results of TaHCT5 expression level, wheat disease index, and fungal biomass detection after gene silencing. Instantaneous silencing of wheat using VIGS technology TaHCT5 The gene (the nucleotide sequence of the silenced wheat TaHCT5 gene is SEQ ID No. 14), in which BSMV:PDS silences wheat TaPDSThe gene, which causes the whitening phenotype in wheat, was used as a positive control. BSMV was extracted from the control plants. 00 And silence TaHCT5 Total RNA from wheat leaves after gene administration, detected by qPCR TaHCT5 The expression level, experimental results are shown in Figure 3 ;according to Figure 3 The results show TaHCT5 Gene expression levels were significantly lower in gene-silenced plants than in the control. After the positive control developed an albino phenotype, Mock, BSMV:00, and BSMV: TaHCT5 Wheat plants were inoculated with CYR34 to observe phenotype. Samples were collected at three time points (24 hpi, 48 hpi, and 120 hpi) after inoculation to detect silencing efficiency. The results showed that... TaHCT5 Gene expression levels were significantly lower in gene-silenced plants than in the control. Compared to BSMV:00 wheat plants, BSMV:TaHCT5 wheat showed significantly increased disease index and fungal biomass. This indicates... TaHCT5 Genes play an important role in wheat's resistance to stripe rust infection.

[0055] 2.2 TaHCT5 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 determine the stomatal vesicles formed by stripe rust infection. The area of ​​reactive oxygen species generated around these vesicles was also counted. The results are shown below. Figure 4 According to the experimental results, from the perspective of reactive oxygen species (ROS) emission, compared with the control plants, plants that transiently silenced TaHCT5 produced less ROS around the infection site, with the most significant differences observed at 24 hpi, 48 hpi, and 120 hpi. Figure 4 ).

[0056] 2.3 TaHCT5 Results of WGA staining of leaves and detection of hyphal length and area after gene silencing Samples were collected at 24 hpi, 48 hpi, and 120 hpi after CYR34 inoculation, stained with WGA-Alexa Fluor488, and then observed under a fluorescence microscope. The colony area and hyphal length of the stripe rust fungus at different time points were recorded. The experimental results are shown in […]. Figure 5The experimental results show that the colony area of ​​stripe rust fungus in the silent plant BSMV:TaHCT5 was significantly larger than that in the control plant BSMV:00 at 24 hpi and 120 hpi, but the difference was not significant at 48 hpi; the hyphal length of stripe rust fungus in the silent plant BSMV:TaHCT5 was significantly longer than that in the control plant BSMV:00 at 24 hpi and 48 hpi.

[0057] Experimental Example 4: Analysis using wheat transient overexpression technology TaHCT5 function of genes 1. Experimental Methods To verify TaHCT5 Using wheat 1723 cDNA as a template, specific primer pairs were designed to achieve the desired function. TaHCT5 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 h and 48 h after inoculation to detect transient overexpression efficiency and fungal biomass. Leaves inoculated for 48 h were 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 TaHCT5 Results of TaHCT5 expression level, wheat disease index, and fungal biomass detection after transient gene overexpression. The candidate gene was constructed into the pYBA-1132 vector (containing a GFP tag) using a one-step cloning method to obtain the GFP:TaHCT5 recombinant vector. This vector was transformed into wheat MX169 using Agrobacterium, and inoculation was performed 36 h later. Samples were collected at 24 hpi and 48 hpi to detect overexpression efficiency. The results are shown below. Figure 6 The test results show that TaHCT5Gene expression levels were significantly higher in gene-overexpressing plants than in the control. Tissue samples were collected at both time points for histochemical observation (DAB staining and WGA staining). Sporulation was observed approximately two weeks after stripe rust inoculation. Compared to GFP:00 wheat plants, GFP:TaHCT5 wheat showed significantly lower disease index and fungal biomass, indicating that the TaHCT5 gene plays a crucial role in stripe rust infection of wheat.

[0059] 2.2 TaHCT5 Results of DAB staining and reactive oxygen species (ROS) content detection in leaves after transient gene overexpression Samples were collected at 24 hpi, 48 hpi, and 120 hpi after CYR32 inoculation, stained with DAB, and then observed under a fluorescence microscope for the stomatal vesicles formed by stripe rust infection. The area of ​​reactive oxygen species generated around these vesicles was also counted. The experimental results are shown in [Figure number missing]. Figure 7 The experimental results showed that, from the perspective of reactive oxygen species emission, compared with the control plants, transient overexpression of [specific reactive oxygen species] was significantly higher. TaHCT5 The plants produced a significant amount of reactive oxygen species around the infection site, with the most significant difference observed at 48 hpi time point, while the difference was negligible at 24 hpi time point.

[0060] 2.3 TaHCT5 Results of 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. The colony area and hyphal length of the stripe rust fungus at different time points were counted. The experimental results are shown in […]. Figure 8 The experimental results show that: overexpression plants TaHCT5 The colony area of ​​stripe rust in the gene was significantly smaller than that of the control plant at 24 hpi and 48 hpi; the hyphal length of stripe rust in the overexpressing plant was significantly shorter than that of the control plant at 24 hpi and 48 hpi.

[0061] Experimental Example 5 TaHCT5 Screening and validation experiments of gene-interacting proteins 1. Experimental Methods 1.1 TaHCT5 Screening interacting proteins TaHCT5 A hydroxycinnamoyltransferase has been successfully constructed. TaHCT5 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... TaHCT5 This provides experimental evidence for the mediated disease-fighting molecular mechanism. TaHCT5 Transfected into AH109 yeast strain, with pGBKT7 as a control, yeast toxicity and self-activation were tested. After completion, TaHCT5 interacting proteins in cotton were screened. First, BD- TaHCT5 The vector was co-transformed with the yeast library plasmid into AH109 competent cells and inoculated into SD / -Trp / -Leu. After white, moist single colonies grew, the single colonies were streaked to SD / -Trp / -Leu / -His. After the single colonies grew, PCR amplification and sequencing were performed.

[0062] 1.2 Verification of yeast two-hybrid point-to-point interaction To address the false positive phenomenon in yeast two-hybrid interaction screening, Ta was selected based on the NCBI annotation database and existing literature information. Rubisco Point-to-point validation was performed as a candidate interaction factor. The CDS sequence was obtained using targeted amplification technology (primer information is shown in Table 2), and pGADT7- was constructed via homologous recombination. TaRubisco Recombinant vector. pGBKT7- TaHCT5 With pGADT7- TaRubisco AH109 yeast competent cells were co-transformed and inoculated in stages into SD / -Trp / -Leu double-deficient medium and X-α-Gal-supplemented quadruple-deficient medium (SD / -Trp / -Leu / -His / -Ade). Positive and negative controls were set up, and the interaction between TaRubisco protein and TaHCT5 protein in vitro was determined by colony growth status and β-galactosidase activity (blue color reaction).

[0063] 1.3 Bimolecular fluorescence complementation to verify protein interactions Based on bimolecular fluorescence complementarity (BiFC) technology, pYFPNE- TaHCT5 With interaction factor pYFPCE- TaRubisco The recombinant vector (primers are shown in Table 2) was introduced into GV3101 Agrobacterium competent cells via freeze-thaw transformation. The engineered bacteria carrying the pYFPNE and pYFPCE vectors were mixed in a 1:1 ratio and transiently co-expressed in *Nicotiana benthamiana* epidermal cells via leaf injection. The fluorescence reaction was observed under a confocal microscope.

[0064] Table 3 Primer sequence information

[0065] 2. Experimental Results 2.1 TaHCT5 Gene plasmid construction, toxicity and self-activation verification Using cDNA from Liangchun 1723 wheat as a template, specific primers were designed for amplification. TaHCT5 CDS sequence ( Figure 9 A). Using In-Fusion technology, the corresponding CDS sequence was inserted into the MCS site of the pGBKT7 vector (linearized by EcoRI / BamHI digestion), ultimately obtaining the corresponding pGBKT7- TaHCT5 Plasmid.

[0066] The already constructed pGBKT7- TaHCT5 The plasmid and the empty pGBKT7 vector were transformed into *Saccharomyces cerevisiae* Y2HGold competent cells, respectively. The cells were then evenly spread on SD / -Trp solid medium plates and cultured at 28°C for 2-3 days. Afterward, the yeast clones were observed to be morphologically consistent in size and number, indicating that... TaHCT5 It is not toxic to yeast. Figure 9 B).

[0067] Positive plasmid pGBKT7-P53 / pGADT7-SV40-T, negative plasmid pGBKT7 / pGADT7, and experimental plasmid pGBKT7-TaHCT5 / 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, while on SD / -Trp / -Leu / -His / -Ade medium, only positive cells showed growth; negative and experimental groups showed no colony growth. This indicates that TaHCT5 cannot activate the expression of downstream reporter genes and does not exhibit self-activation. Figure 10 ).

[0068] 2.2 TaHCT5 Screening and validation of interacting proteins Using cDNA from the Liangchun 1723 wheat variety as a template, specific primers were designed to amplify the CDS sequence of TaRubisco. In-Fusion technology was then used to amplify the sequence in the pGADT7 vector (…). Eco R Ⅰ / BamThe corresponding CDS sequence was inserted into the MCS site of the linearized H I enzyme digestion, ultimately yielding the corresponding pGADT7-TaRubisco plasmid; the constructed pGADT7-TaRubisco plasmid was then compared with pGBKT7- TaHCT5 The plasmid was co-transformed into yeast, plated onto two-deficient plates, and cultured. After clones grew on the two-deficient plates, spots were picked and streaked onto new three-deficient and four-deficient plates. Point-to-point results showed that there is an interaction between the proteins TaHCT5 and TaRubisco. Figure 11 A).

[0069] To verify the interaction site of TaHCT5 and TaRubisco in plants, the encoding gene of the interacting protein TaRubisco obtained from yeast two-hybrid synthesis was amplified by PCR and constructed into BiFC expression vectors pSAT1-nEYFP and pSAT1-cEYFP. The pSAT1-nEYFP-TaHCT5 and pSAT1-cEYFP-TaRubisco expression vectors were then co-transformed into tobacco. The results showed that yellow fluorescence was observed when pSAT1-nEYFP-TaHCT5 was co-transformed with pSAT1-cEYFP-TaRubisco, with the fluorescence mainly concentrated on the cell membrane, indicating that pSAT1-nEYFP-TaHCT5 and pSAT1-cEYFP-TaRubisco interact at the cell membrane. Figure 11 B).

[0070] To confirm the reliability of the protein-protein interaction results, this study used a luciferase complementation assay for verification. TaHCT5 and TaRubisco were recombined into the pLUCC / pLUCN vector system, respectively. After gene introduction via a transient tobacco transformation system, fluorescence signals were detected under specific excitation conditions. As shown in the figure, characteristic fluorescence phenomena were detected in the experimental group samples, confirming the specific interaction between TaHCT5 and TaRubisco.

Claims

1. Wheat TaHCT5 The application of genes in improving wheat resistance to stripe rust is characterized by, include: wheat TaHCT5 Overexpression of the gene in wheat enhances its resistance to stripe rust; the wheat TaHCT5 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 TaHCT5 Gene overexpression vector; wheat TaHCT5 Overexpression of the gene in wheat resulted in wheat 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 TaHCT5 The application of gene expression cassettes in improving wheat resistance to stripe rust includes: The wheat-containing TaHCT5 Gene expression cassettes are genetically transformed into wheat, thus transforming wheat TaHCT5 The gene is overexpressed in wheat; the wheat TaHCT5 The nucleotide sequence of the gene is shown in SEQ ID No.

1.

5. Contains wheat TaHCT5 The application of gene overexpression vectors in improving wheat resistance to stripe rust includes: The wheat-containing TaHCT5 Gene overexpression vectors genetically transform wheat, turning wheat TaHCT5 The gene is overexpressed in wheat; the wheat TaHCT5 The nucleotide sequence of the gene is shown in SEQ ID No.

1.

6. The application according to claim 5, characterized in that, The overexpression vector is a plant overexpression vector.

7. A method for breeding wheat varieties resistant to stripe rust, characterized in that, include: Building wheat TaHCT5 Gene expression cassettes or overexpression vectors; wheat TaHCT5 Gene expression cassettes or overexpression vectors are used to genetically transform wheat, enabling wheat to... TaHCT5 The gene was overexpressed in plants, and transgenic wheat with enhanced resistance to stripe rust was obtained by screening the obtained overexpression-positive plants; the wheat TaHCT5 The nucleotide sequence of the gene is shown in SEQ ID No.

1.

8. A method for improving wheat resistance to stripe rust, characterized in that, include: Building wheat TaHCT5 Gene expression cassettes or overexpression vectors; wheat TaHCT5 Gene expression cassettes or overexpression vectors are used to genetically transform wheat, enabling wheat to... TaHCT5 The gene was overexpressed in the plant, resulting in transgenic wheat with increased resistance to stripe rust; the wheat described above TaHCT5 The nucleotide sequence of the gene is shown in SEQ ID No. 1.

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