Wheat disease-resistant gene TaLOX and application thereof in regulation and control of stripe rust resistance

Abstract

The invention discloses a wheat disease-resistant gene TaLOX and application thereof in regulation and control of stripe rust resistance. The wheat TaLOX gene is subjected to overexpression in wheat, after an overexpressed wheat plant is inoculated with puccinia striiform f. Sp. Tritici, the resistance phenotype of the overexpressed wheat plant to the puccinia striiform f. Sp. Tritici is observed, and the result shows that compared with a control group, the amount of puccinia striiform f. Sp. Tritici spores on the surface of the wheat plant with the overexpressed TaLOX gene is remarkably reduced, the disease index and the fungal biomass are remarkably reduced, and compared with the control group, the puccinia striiform f. Sp. Tritici is remarkably reduced. The hypha length is obviously shorter than that of a control group, which indicates that the TaLOX gene positively regulates the resistance of wheat to puccinia striiformis. The method has application prospects in the aspects of breeding stripe rust resistant wheat varieties, improving the resistance level of wheat to stripe rust and the like.

Description

Technical Field

[0001] The present invention relates to disease resistance-related genes separated from wheat and uses thereof, and in particular to disease resistance-related genes separated from wheat. TaLOX Gene and its use in regulating plant resistance to stripe rust, belonging to TaLOX Areas of application of genes. Background Art

[0002] wheat( Triticum aestivum L.) is a major crop worldwide, consumed as a staple food by 35% of the world's population (FAOSTAT 2024). Stripe rust is caused by the fungal pathogen Puccinia striiformis f.sp. tritici Stripe rust (Pst) is a serious disease widespread in wheat-producing areas worldwide. It typically causes yield losses of 10-70%, and in severe cases, up to 100%. Pst is highly genetically diverse, resulting in different races with varying levels of pathogenicity. Increased virulence leads to a weakening of existing plant resistance, and wheat varieties typically lose their innate resistance to stripe rust after only 3-5 years of field use. Artificially breeding varieties with durable and broad-spectrum resistance is considered the most effective method for disease control. Therefore, identifying new wheat stripe rust-resistant genes and studying their molecular regulatory mechanisms will aid in the selection and breeding of disease-resistant varieties and are of great significance for the long-term and effective prevention and control of wheat stripe rust.

[0003] In the process of wheat coping with stripe rust, a series of stripe rust resistance genes play a key role. With the continuous deepening of wheat genome research, many stripe rust resistance genes have been discovered and identified; TaLOX Reports on genes that regulate resistance to stripe rust. Summary of the Invention

[0004] The main purpose of the present invention is to provide wheat TaLOX The use of genes in regulating crop resistance to stripe rust.

[0005] To achieve the above objectives, the technical solutions adopted by the present invention include: The present invention provides wheat TaLOX The application of genes in regulating plant resistance to stripe rust, including: TaLOX The transcription or expression level of the gene in the plant is used to regulate the plant's resistance to stripe rust; wherein the altered wheat TaLOX The transcription or expression level of a gene in a plant can be enhanced TaLOX The transcription or expression level of the gene in the plant can also be reduced or weakened TaLOX Gene transcription or expression levels in plants; accordingly, by changing the wheat TaLOXGene transcription or expression level at the plant to achieve stripe rust resistance regulation or by changing wheat TaLOX Regulating stripe rust resistance at the transcription and translation levels of genes in plants should fall within the scope of protection of the present invention.

[0006] How to increase or decrease wheat TaLOX The transcription or expression level of genes in plants can be achieved by various conventional technical means by those skilled in the art; for example, by constructing wheat TaLOX Gene overexpression vector, transformed into plants through Agrobacterium-mediated genetic transformation, and obtained wheat TaLOX Overexpression of genes in plants can improve the resistance of overexpressing plants to stripe rust; or by knocking out or interfering with genes in plants through CRISPR and VIGS methods. TaLOX The transcription or expression level of genes in plants TaLOX Genes or their homologous genes are deleted or mutated, ultimately silencing or reducing the expression of TaLOX The transcription or expression level of the gene or its homologous gene is reduced, and the resulting transgenic plant has reduced resistance to stripe rust.

[0007] Those skilled in the art can construct wheat overexpression gene vectors by conventional methods for constructing overexpression gene vectors. TaLOX Overexpression plant vector of gene or its homologous gene; or construct wheat according to conventional gene editing technology or gene knockout vector construction method in the field TaLOX Gene or its homologous gene editing vector, these methods are all well known to those skilled in the art; for example, the wheat TaLOX The gene or its homologous gene is operably connected to an expression control element to obtain an overexpression plant vector that can express the gene in plants; the overexpression plant vector comprises a promoter, a wheat TaLOX The vector may also contain a CDS sequence and terminator of a gene or its homologous gene; the promoter may be constitutive, inducible, or tissue- or organ-specific, and the terminator sequence may be derived from the Ti-plasmid of Agrobacterium tumefaciens, such as the octopine synthase and nopaline synthase terminator regions. The vector may also contain a selectable marker gene for selecting transformed cells or tissues. Such marker genes include genes encoding antibiotic resistance and genes conferring herbicide resistance. Furthermore, such marker genes include phenotypic markers such as β-galactosidase and fluorescent protein.

[0008] In the present invention, any plant transformation method can be used to transform the overexpression plant vector or gene editing vector constructed by the present invention into the tissues and cells of the target plant to obtain a transformant, and then the transformant is regenerated by plant tissue culture to obtain a complete plant and its asexual line or its offspring. The transformation method includes Agrobacterium-mediated genetic transformation, protoplast transformation, plant virus vector, microinjection, electric shock method, etc.

[0009] As a preferred embodiment of the present invention, the present invention provides a method for improving plant resistance to stripe rust, comprising: constructing wheat TaLOX gene or its homologous gene overexpression vector; the wheat TaLOX The gene or its homologous gene is overexpressed in a plant, and the resulting overexpressed plant has improved resistance to stripe rust.

[0010] As another preferred embodiment of the present invention, the present invention provides a method for creating a plant variety resistant to stripe rust, comprising: constructing wheat TaLOX Overexpression vector of gene or its homologous gene; TaLOX The gene or its homologous gene is overexpressed in plants; overexpression plants with improved resistance to stripe rust are screened from the obtained overexpression positive plants.

[0011] The plants described in the present invention include but are not limited to monocotyledonous plants or dicotyledonous plants. Most preferably, the plant is wheat.

[0012] The wheat of the present invention TaLOX The nucleotide sequence of the gene (XM_044587023.1, PREDICTED: Triticumaestivum probable lipoxygenase 8, chloroplastic (LOC123169172)) is selected from any one of (a) to (e): (a) the polynucleotide sequence shown in SEQ ID No. 1; (b) a polynucleotide that can hybridize to 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 in which one or more bases are deleted, substituted or inserted, 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.

[0013] In addition, those skilled in the art can optimize the nucleotide sequence shown in SEQ ID No. 1 to enhance expression efficiency in plants; for example, the preferred codons of the target plant can be used for optimization to synthesize the polynucleotide to enhance expression efficiency in the target plant.

[0014] Chimeric genes or expression cassettes obtained by chimerizing or connecting the gene shown in SEQ ID No. 1 of the present invention with other genes fall within the scope of protection of the present invention; recombinant expression vectors containing the chimeric gene or expression cassette also fall within the scope of protection of the present invention.

[0015] The following described in the present invention TaLOX The amino acid sequence of the protein encoded by the gene is shown in SEQ ID No. 2.

[0016] The present invention uses the dynamic samples of Liangchun 1723 after inoculation as materials, and screens out the key genes regulating wheat stripe rust resistance through combined transcriptome and metabolome analysis. TaLOX Through transient overexpression and gene silencing technology, we conducted a preliminary study on the function of this gene in wheat stripe rust resistance and found that TaLOX The gene plays a key role in wheat stripe rust resistance and is overexpressed in wheat TaLOX The gene can significantly improve wheat's resistance to stripe rust. TaLOX Gene silencing significantly weakens wheat's resistance to stripe rust. TaLOX The gene participates in the wheat stripe rust resistance response process as a positive regulatory factor, and has application prospects in improving plant resistance to stripe rust or cultivating stripe rust-resistant plant varieties.

[0017] Term definitions involved in the present 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 belongs. Although any methods, devices, and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods, devices, and materials are now described.

[0018] The term "polynucleotide" or "nucleotide" means deoxyribonucleotides, deoxyribonucleosides, ribonucleosides, or ribonucleotides and polymers thereof in single-stranded or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogs of natural nucleotides that have binding properties similar to the 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 PNA (peptide nucleic acid), DNA analogs used in antisense technology (phosphorothioates, phosphamidates, etc.). Unless otherwise specified, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (including but not limited to degenerate codon substitutions) and complementary sequences as well as explicitly specified sequences. In particular, degenerate codon substitutions can be achieved by generating a sequence in which position 3 of one or more selected (or all) codons is substituted with mixed bases and / or deoxyinosine residues.

[0019] The term "homology" refers to the level of similarity or percent identity between polynucleotide sequences in terms of percent nucleotide positional identity (i.e., sequence similarity or identity). As used herein, 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 hybridize specifically under specific conditions to form duplex molecules. Under these conditions, referred to as stringent hybridization conditions, a polynucleotide molecule can be used as a probe or primer to identify another polynucleotide molecule that shares homology.

[0020] As used herein, "stringent hybridization conditions" refer to conditions of low ionic strength and high temperature as known in the art. Typically, under stringent conditions, the detectable degree of hybridization of a probe to its target sequence is greater than the detectable degree of hybridization to other sequences (e.g., at least 2-fold above background). Stringent hybridization conditions are sequence-dependent and will vary under different environmental conditions, with longer sequences hybridizing specifically at higher temperatures. By controlling the stringency of hybridization or washing conditions, target sequences that are 100% complementary to the probe can be identified. For detailed guidance on nucleic acid hybridization, reference can be made to the relevant literature (Tijssen, Techniques in biochemistry and molecular biology hybridization with nucleic acid probes, "Overview of principles of hybridization and the strategy of nucleic acid assays," 1993). More specifically, the stringent conditions are typically selected to be below the thermal melting point (T) of the specific sequence at a specified ionic strength pH. m ) about 5-10℃. Tm The temperature (under specified ionic strength, pH, and nucleic acid concentration) at which 50% of the probes complementary to the target hybridize to the target sequence at equilibrium (because the target sequence is present in excess, the temperature at T m Stringent conditions may include conditions where the salt concentration is less than about 1.0 M sodium ion concentration, typically about 0.01 to 1.0 M sodium ion concentration (or other salts), at pH 7.0 to 8.3, and the temperature is at least about 30°C for short probes (including but not limited to, 10 to 50 nucleotides) and at least about 60°C for long probes (including but not limited to, greater than 50 nucleotides). Stringent conditions may also be achieved by the addition of destabilizing agents such as formamide. For selective or specific hybridization, a positive signal may be at least two times background hybridization, optionally 10 times background hybridization. Exemplary stringent hybridization conditions may be as follows: 50% formamide, 5× SSC, and 1% SDS, incubation at 42°C; or 5× SSC, 1% SDS, incubation at 65°C, wash in 0.2× SSC, and wash in 0.1% SDS at 65°C. The washing may be performed for 5, 15, 30, 60, 120 minutes or longer.

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

[0022] The term "promoter" refers to a polynucleotide molecule that is located upstream or 5' of the translation start codon of the open reading frame (or protein coding region) in its natural state and is involved 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 connection of a first polynucleotide molecule (e.g., a promoter) to a second transcribable polynucleotide molecule (e.g., a gene of interest), wherein the polynucleotide molecules are arranged such that the first polynucleotide molecule affects the function of the second polynucleotide molecule. Preferably, the two polynucleotide molecules are part of a single contiguous polynucleotide molecule, and more preferably, are adjacent. For example, a promoter is operably linked to a gene of interest if it regulates or mediates transcription of the gene of interest within a cell.

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

[0025] The term "transformation" refers to a process by which a heterologous DNA sequence is introduced into a host cell or organism.

[0026] The term "expression" refers to the transcription and / or translation of an endogenous gene or a transgene in a plant cell.

[0027] The term "recombinant host cell strain" or "host cell" refers to a cell comprising a polynucleotide of the present invention, regardless of the method used for insertion to produce the recombinant host cell, such as direct uptake, transduction, mating, or other methods known in the art. The exogenous polynucleotide may be maintained as a non-integrating vector, such as a plasmid, or may be integrated into the host genome. The host cell may be a prokaryotic cell or a eukaryotic cell, and the host cell may also be a monocotyledonous or dicotyledonous plant cell. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 Results of bioinformatics analysis of TaLOX protein; (a): domain structure prediction of TaLOX; (b): transmembrane structure prediction of TaLOX; (c): secondary structure prediction of TaLOX; (d): tertiary structure prediction of TaLOX.

[0029] Figure 2 The results of fluorescence quantitative expression of TaLOX gene were analyzed by t test, ***: P < 0.001; ****: P < 0.0001.

[0030] Figure 3 The subcellular localization results of TaLOX protein in tobacco leaves.

[0031] Figure 4 for TaLOX Construction of gene pYBA-1132-GFP vector; (a): Schematic diagram of pYBA-1132-GFP vector; (b): TaLOX Gene PCR product amplification; (c): colony PCR; (d): 5000 DNA Marker.

[0032] Figure 5 for TaLOX Disease resistance analysis of plants with transient gene overexpression; (a): TaLOX Phenotypic observation of plants with transient gene overexpression 14 days after inoculation; (b):TaLOX Detection of gene transient overexpression efficiency; (c): TaLOX Disease index of plants with transient gene overexpression 14 days after inoculation; (d): TaLOX Phenotypic observation of transiently overexpressed gene plants 48 h after inoculation by WGA staining; SV, substomatal sac; HMC, haustorial mother cell; IH, infective hyphae; (e): TaLOX Statistics of stripe rust hypha length in plants with transient gene overexpression 48 hours after inoculation, with 30 samples collected. (f) TaLOX The amount of fungal DNA in plants with transient gene overexpression was detected 2 and 7 days after inoculation. T-test was used for significance analysis, **: P < 0.01; ****: P < 0.0001.

[0033] Figure 6 for TaLOX Gene BSMV- TaLOX Vector construction; (a): BSMV- PDS Vector schematic diagram; (b): TaLOX Gene PCR product amplification; (c): colony PCR; (d): 2000 DNA Marker.

[0034] Figure 7 for TaLOX Gene silencing efficiency test results; (a): positive control plant phenotype; (b): TaLOX Gene silencing efficiency detection; significance analysis was performed using t-test, **: P < 0.01; ****: P < 0.0001.

[0035] Figure 8 for TaLOX Resistance phenotype of gene silenced plants; (a): TaLOX Phenotypic observation of gene silenced plants 14 days after inoculation; (b): TaLOX Disease index of gene silenced plants 14 days after inoculation; (c): TaLOX Fungal DNA amount in gene-silenced plants 2 days after inoculation; (d): TaLOX Phenotypic observation of gene-silenced plants after 24 and 48 h of DAB staining. SV, substomatal sac; brown after DAB staining indicates H2O2. Bar = 20 μm; (e): TaLOX H2O2 area statistics of gene silenced plants at 24 h and 48 h, with 30 statistical samples; (f): TaLOX Phenotypic observation of gene silenced plants after 24 h and 48 h after WGA staining, SV, substomatal sac; HMC, haustorial mother cell; IH, infected hyphae; (g): TaLOXMycelial length of gene-silenced plants at 24 and 48 h post-transplantation, with a total of 30 samples. (h): JA content in leaves was detected by ELISA. Significance analysis was performed using t-test, *: P < 0.05; ****: P < 0.0001. DETAILED DESCRIPTION

[0036] The present invention will be further described below in conjunction with specific test examples, and the advantages and features of the present invention will become clearer as the description proceeds. However, it should be understood that the test examples are merely exemplary and do not limit the scope of the present invention in any way. It should be understood by those skilled in the art that the details and forms of the technical solutions of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, but such modifications or replacements fall within the scope of protection of the present invention.

[0037] Test materials and general test methods 1 Test materials The tested wheat varieties were Liangchun 1723 (highly resistant), Xinchun 34 (highly susceptible), and Xinchun 35 (moderately resistant), and the stripe rust race was CYR32.

[0038] 2 Test strains and vectors DH5α competent Escherichia coli and GV3101 (pSoup) competent Agrobacterium cells were purchased from Shanghai Sangon Biotechnology Co., Ltd. The overexpression vector pYBA-1132-GFP and the silencing vectors BSMV-α, BSMV-β, BSMV-γ, and BSMV-PDS were kept in the laboratory of the present inventors.

[0039] 3 Test reagents and instruments Kanamycin ( Kan ), Rifampicin ( Rif ) and acetosyringone ( AS) were purchased from Beijing Solaibao Biological Co., Ltd.; plant total RNA extraction kits, fluorescence quantification kits, and reverse transcription kits were purchased from Fuji Biotechnology Co., Ltd.; β-mercaptoethanol, anhydrous ethanol, and jasmonic acid (JA) enzyme-linked immunosorbent assay kits were purchased from Shanghai Meiji Biotechnology Co., Ltd.; high-fidelity DNA polymerase was purchased from Beijing NEB Biotechnology Co., Ltd.; high-fidelity endonuclease and T4 DNA ligase were purchased from Thermo Fisher Scientific, USA; agarose gel electrophoresis product purification kit and circular DNA isolation and purification reagents were from Jiangsu Novizan Biotechnology Co., Ltd. Ligase and DNA molecular weight marker were purchased from Beijing Quanshijin Co., Ltd. Chloroform, isopropanol, glycerol, anhydrous ethanol, 2-morpholinoethanesulfonic acid, magnesium chloride, LB medium preparation, MES, acetosyringone, MgCl2, potassium dihydrogen phosphate, potassium hydrogen phosphate, potassium hydroxide, and anhydrous sodium sulfate were all domestically produced analytical grade chemicals. Primers used for PCR and qRT-PCR were synthesized and sequenced by Xinjiang Youkang Biotechnology Co., Ltd.

[0040] -80℃ ultra-low temperature refrigerator, constant temperature incubator, high-speed variable temperature centrifuge, ABI7500 Fast fluorescence quantitative instrument, pipette, electrophoresis instrument, UV gel imager, ultra-clean laboratory workbench, electric constant temperature water bath, high-pressure steam sterilizer, UV spectrophotometer, visible spectrophotometer, etc.

[0041] 4 Wheat planting and stripe rust inoculation treatment Liangchun 1723 seeds with well-developed grains and consistent color and shape were selected. After germination, they were sown in small 7 cm x 7 cm pots and cultured under a photoperiod of 16 h light and 8 h dark. When the wheat had grown to one leaf and one heart, they were inoculated with stripe rust. Wheat plants simulated inoculated with distilled water served as blank controls. Wheat samples were collected at 0, 6, 12, 18, 24, 36, 48, 72, and 96 hours after inoculation, quickly frozen in liquid nitrogen, and stored at -80°C.

[0042] 5. Extraction of total RNA and synthesis of cDNA from wheat Remove the sample from the -80°C freezer and grind it into a fine powder. Subsequently, use a total RNA extraction kit according to standard procedures to extract RNA. After extraction, test the RNA quality by electrophoresis to determine whether it meets the requirements of subsequent experiments. After confirming the RNA quality, use a reverse transcription kit to synthesize cDNA. Refer to Tables 1 and 2 for the reaction system settings and reaction procedures during reverse transcription.

[0043] Table 1 Reverse transcription system

[0044] Table 2 Reverse transcription PCR reaction procedure

[0045] 6. Detection of target gene expression by qRT-PCR Using cDNA as template, wheat elongation factor TaEF The reaction conditions and system were carried out according to the method of Hu Ziyao et al. (Hu Ziyao, Dai Peihong, Liu Chao, et al. Cloning, expression and VIGS vector construction of small GTP-binding protein gene GhROP3 in upland cotton [J]. Biotechnology Bulletin, 2021, 37(09):106-113.). According to the Ct value of the target gene and the internal reference gene, 2 -ΔΔCt Methods (Livak, KJ, and TD Schmittgen. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T))Method [J]. Methods (San Diego, Calif.), 2001, 25(4): 402-408.) Calculation TaLOX The expression levels of genes were measured. The primers are shown in Table 3.

[0046] Table 3 Primer information used

[0047] Note: F represents forward primer, R represents reverse primer, and lowercase letters represent homology arms. Same below.

[0048] Experimental Example 1 Bioinformatics Analysis of TaLOX Protein TaLOX protein structure analysis was performed using NCBI online tools. Physicochemical properties, transmembrane structure analysis, secondary structure analysis, and tertiary structure prediction for TaLOX protein were analyzed using ExPASy-ProtScale, TMHMM, PSIPRED, and SWISS-MODEL online tools. The websites for the tools used are listed in Table 4.

[0049] Table 4 Bioinformatics analysis tools

[0050] Wheat TaLOX protein is a lipoxygenase. The conserved domains and motifs of TaLOX were analyzed using NCBI. The results showed that TaLOX contains typical lipoxygenase and PLAT_LH2 domains ( Figure 1(a)). The TaLOX protein sequence was analyzed using ProtScale. The TaLOX protein contains 932 amino acids with a molecular formula of C 4557 H 7145 N 1281 O 1355 S 29 The total number of atoms is 14,367, the molecular weight is 10.248737 kDa, the isoelectric point is 7.35, the total number of negatively charged residues (Asp+Glu) is 109, the total number of positively charged residues (Arg+Lys) is 109, the instability index is 46.45, the average water solubility is -0.321, and the lipid solubility index is 79.58. Subcellular localization prediction of TaLOX shows that the protein is located in the cell membrane. The transmembrane helical structure prediction by TMHMM shows that the TaLOX protein does not have a transmembrane helical structure ( Figure 1 (b)).

[0051] The secondary and tertiary structures of TaLOX protein were predicted using the SOPMA online platform and the SWISS-MODEL modeling system. The secondary structural component analysis showed that TaLOX protein is composed of 35.84% α-helix, 12.23% extended chain, 2.15% β-turn and 49.79% random coil ( Figure 1 (c)). Its tertiary structure prediction is consistent with the secondary structure prediction ( Figure 1 (d)).

[0052] Test Example 2 TaLOX Gene expression pattern identification assay In the transcriptome sequencing results, TaLOX Gene expression was significantly upregulated, and for further identification TaLOX The expression pattern of genes during pathogen infection of wheat was determined by real-time fluorescence quantitative qRT-PCR in wheat leaves at 0 h, 6 h, 12 h, 18 h, 24 h, 36 h, 48 h, 72 h and 96 h.

[0053] The test results found TaLOX The gene was significantly induced and up-regulated at 12 h, 18 h, and 24 h after stripe rust infection, and the fluorescence quantitative results were consistent with the transcriptome results ( Figure 2 ), the results show that TaLOX The gene may play a key role in the early stages of stripe rust infection.

[0054] Experimental Example 3 TaLOX protein subcellular localization The pYBA-1132-GFP vector contains the green fluorescent protein gene (GFP), which can be used for subcellular localization experiments of TaLOX protein. The subcellular localization of TaLOX protein was predicted using the online software PSORT (https: / / www.genscript.com / psort.html). Based on the predicted results, the corresponding marker protein (red fluorescence) was selected as a control. The tobacco leaves were transformed using the Agrobacterium-mediated method. TaLOX -GFP was transiently expressed in tobacco to determine the subcellular localization of TaLOX protein.

[0055] PSORT was used to predict that TaLOX protein was located at the cell membrane. TaLOX -GFP was transiently expressed in tobacco, and the experimental results showed that ( Figure 3 ): The fluorescence of empty GFP is evenly distributed throughout the cell, while the expression TaLOX genetic TaLOX -GFP fusion protein showed obvious green fluorescence at the cell membrane of tobacco leaf epidermal cells, and the green fluorescence overlapped with the red fluorescence of the cell membrane localization marker to form yellow fluorescence, which further proved that the TaLOX protein was localized at the cell membrane.

[0056] Test Example 4 TaLOX Construction and transformation of gene overexpression and silencing vectors and phenotypic identification of transgenic plants 1 Test method 1.1 TaLOX Construction of gene overexpression and silencing vectors according to TaLOX The CDS sequence of the gene was determined, and specific amplification primers were designed (Table 3). The target fragment was cloned using wheat cDNA as a template. The target fragment was inserted into the corresponding restriction site of the pYBA-1132-GFP vector using homologous recombination technology to obtain pYBA- TaLOX -GFP gene overexpression vector. The vector was transformed into DH5α E. coli competent cells, and positive clones were screened in LB solid medium containing antibiotics (Kan 50 ug / mL). The successful vector construction was confirmed by bacterial liquid PCR detection and sequencing of positive clones using the pYBA-1132-GFP universal primers 1132-F / R. The correctly sequenced recombinant plasmid was transformed into GV3101 (pSoup) Agrobacterium competent cells and cultured in double-resistance LB medium (Kan 50 ug / mL + Rif 50 ug / mL) for 48 hours before use in subsequent experiments.

[0057] according to TaLOXThe nucleotide sequence of the gene was determined. Silencing fragments were designed using Premier 5.0 software. The specificity of the designed silencing fragments was confirmed using the online website NCBI Blastn. The designed gene fragments (Table 5) were amplified by PCR and ligated into the BSMV-PDS vector ( Figure 6 ) and obtained the correct vector through E. coli DH5α transformation and other steps. The correctly sequenced plasmid was transformed into GV3101 (pSoup) Agrobacterium competent cells and spread on Kan and Rif The cells were cultured on LB plates with resistance for 48 h and used in subsequent experiments.

[0058] Table 5 TaLOX Silent sequence

[0059] 1.2 TaLOX Construction of gene overexpression plants and identification of disease resistance Add LB broth (Kan 50 ug / mL + Rif 50 ug / mL) containing pYBA- TaLOX -GFP plasmid Agrobacterium, shake the bacteria at 200 rpm, and when the bacterial solution concentration is OD600 = 0.8-1.0, centrifuge the bacterial solution (4000 rpm / 10 min) and use As Dilute with buffer to an OD600 concentration of 1.0. Wheat (Xinchun 34) plants grown to the two-leaf, one-heart stage were selected. The second leaf was injected on the underside, and the injection area was marked with a marker. Sixty wheat plants were injected. The same number of plants, carrying the empty pYBA-1132-GFP vector, were injected as a control. After all injections were completed, the plants were kept in the dark at 23°C for 24 hours. Thirty-six hours after injection, the leaves in the injected area were inoculated with freshly harvested virulent wheat stripe rust race CYR32. An equal number of wild-type plants were also inoculated as mock plants. After all inoculations were completed, the plants were kept in the dark at 16°C for 24 hours. Samples were collected at 24 and 48 hours after inoculation to assess overexpression efficiency. Tissue samples were also collected at these two time points for histocytological analysis. The spore production was observed about two weeks after the inoculation of stripe rust, and the disease index was calculated according to the method of Zhang Hongchang (Zhang Hongchang. Histological and cytological studies on the resistance of adult wheat plants to stripe rust and the mechanism of wheat non-host resistance to broad bean rust [D]. Northwest Agriculture and Forestry University. 2012.).

[0060] 1.3 TaLOX Construction of gene-silenced plants and identification of disease resistance The vectors BSMV-α, BSMV-β, BSMV-γ, BSMV-PDS (The construction of BSMV-α, BSMV-β, BSMV-γ and BSMV-PDS was constructed according to the method disclosed in the literature (Wu Hongyan. Identification of the function of SA pathway related genes in resisting wheat scab by BSMV-VIGS technology [D]. Shandong Agricultural University. 2016.)) and BSMV- TaLOX Agrobacterium was transformed separately and the mixed vectors were injected into tobacco leaves at the 5-6 leaf stage through Agrobacterium-mediated infection. After 3-5 days, the tobacco leaf juice was extracted and rubbed on wheat leaves (Xinchun 35) that had grown to two leaves and one heart according to the method of Wu Hongyan et al. (Wu Hongyan. Identification of the function of SA pathway-related genes in wheat scab resistance using BSMV-VIGS technology [D]. Shandong Agricultural University. 2016.) to induce silencing. PDS (positive), BSMV-α, BSMV-β and BSMV-γ (BSMV: γ), BSMV-α, BSMV-β and BSMV- TaLOX (BSMV: [[ID=1 )) were used as positive and negative controls (BSMV: γ) and the target gene test group (BSMV: ​ ), the same number of wild-type plants were used as Mock. ​ When the leaves showed obvious white phenotype, the stripe rust fungus CYR32 was inoculated on the fourth wheat leaf, and samples were collected at 0 h, 24 h and 48 h after inoculation to detect the silencing efficiency. At the same time, tissue samples were collected at 24 h and 48 h after inoculation for histocytological observation. The spore production was observed about two weeks after inoculation of stripe rust, and the disease index was calculated. In addition, 24 hours after inoculation of stripe rust, samples were collected. ​ 0.1 g wheat leaf samples from gene-silenced plants were used to determine the JA content according to the instructions of the plant jasmonic acid (JA) enzyme-linked immunosorbent assay kit.

[0061] 1.4 Histological processing Reactive oxygen staining and observation: To investigate the histopathological changes of silent wheat leaves after stripe rust inoculation, DAB (3,3'-diamino-bezidine, Sigma-Aldrich, St. Louis, USA) staining solution was used to stain wheat leaf tissue 24 and 48 h after stripe rust inoculation. The method was as follows: (1) 1.5 mL of DAB staining solution (1.5 mg / mL) was taken into a 10 mL centrifuge tube, and wheat leaves 24 and 48 h after inoculation were cut into about 1 cm long leaf segments and placed in the DAB staining solution. Vacuum was used for 30 min; (2) The staining solution was discarded and the solution was replaced with decolorizing solution (glacial acetic acid: anhydrous ethanol = 1:1). Whenever the decolorizing solution became turbid, the decolorizing solution was replaced until the leaf segment was completely decolorized; (3) The decolorizing solution was discarded and the solution was replaced with chloral hydrate. The solution was fixed for 24 h. h. Use sample preservation solution to preserve the sample; (4) Use an inverted fluorescence microscope to observe the infection situation in the bright field, using the stomata of the haustorial mother cells as the infection points, and use ImageJ to count the area of ​​active oxygen around the infection points.

[0062] Stripe rust hyphae staining and observation: In order to observe the infection structure and hyphae expansion of wheat leaves during the stripe rust infection process, wheat leaves were stained with WGA-Alexa488 dye solution 24 and 48 h after inoculation. The method is as follows: (1) The wheat samples stored in the sample preservation solution were washed with 50% ethanol, 50% ethanol, Tris-HCl (0.05M), and Tris-HCl (0.05M) in sequence, each for 15 min; (2) 1 M KOH solution was added to submerge the sample, and the sample was treated with boiling water for 10 min; (3) The leaf sample was washed with Tris-HCl (0.05M) for 5 min / 3 times; (4) The washing solution was aspirated, and an appropriate amount of 20 ug / mL WGA-Alexa488 fluorescent dye solution was added. The leaves were stained in the dark for 2 h, and the leaf sample was washed with Tris-HCl (0.05M) for 5 min / 3 times; (5) The leaf sample was washed with Tris-HCl (0.05M) for 5 min / 3 times. M) preservation; (6) Observe the structures of stripe rust fungi such as the substomatal capsule and hyphae under the GFP filter of a confocal microscope, and use ImageJ to measure the hyphae length and hyphae expansion area (Hu Zeyu. Study on the function of transcription factor TaNF-YC1 in the interaction between wheat and stripe rust fungi [D]. Northwest Agriculture and Forestry University. 2022.).

[0063] 2 Test results 2.1 ​ Gene overexpression vector construction ​ Strategies for constructing gene overexpression vectors ​ As shown in (a), by directed amplification ​The coding region (CDS) sequence of the gene was analyzed. Electrophoresis verification showed that the target product size was consistent with the expected 2799 bp ( ​ (b)). The purified target fragment was connected to the pYBA-1132-GFP linearized vector using seamless cloning technology and transformed into DH5α. The recombinant plasmid was verified by colony PCR, and the fragment size was about 2799 bp ( ​ (c)) and sequencing and comparative analysis confirmed that pYBA- ​ The GFP vector was constructed successfully.

[0064] 2.2 ​ Resistance detection in plants with transient gene overexpression For preliminary verification ​ The potential function of genes in wheat stripe rust resistance was investigated. In this study, pYBA- ​ -GFP overexpression vector was transfected into the mesophyll tissue of wheat Xinchun 34 (highly susceptible to stripe rust) via Agrobacterium-mediated transfection to achieve pYBA- ​ -GFP was transiently overexpressed in wheat (TaLOX-GFP); at the same time, the pYBA-1132-GFP vector was transfected into the mesophyll tissue of Xinchun 34 (highly susceptible to stripe rust) wheat through Agrobacterium-mediated transfection to achieve transient overexpression of pYBA-1132-GFP in wheat (GFP0).

[0065] Transient overexpression in plants ​ The gene expression level was 3.48 times higher than that of the control group at 24 h and 4.73 times higher at 48 h ( ​ (b)), indicating that the ​ Transient gene overexpression plants. These results provide a basis for further research. ​ The function of the gene in the mechanism of resistance to stripe rust lays the foundation.

[0066] After 14 days of inoculation of stripe rust in the overexpression plants, the results showed that compared with the control group, ​ The amount of stripe rust spores on the surface of plants with transient gene overexpression was significantly reduced, and the disease index and fungal DNA content were significantly reduced ( ​ (a) ​ (c) ​ (f)). ​ The hyphae length of the plants with transient gene overexpression was significantly shorter than that of the control group ( ​ (e)). This further supports its inhibitory effect on pathogen infection. ​ (d) ​ WGA staining phenotype was observed 48 h after transient gene overexpression inoculation in wheat plants. ​ The gene significantly enhances wheat's resistance to stripe rust.

[0067] 2.3 ​ Gene silencing vector construction ​ Schematic diagram of gene silencing vector construction ​ As shown in (a), agarose electrophoresis detection showed that the product length was exactly the same as the expected 300 bp ( ​ (b)). The purified fragment was ligated to the restriction endonuclease of the BSMV-PDS linearized vector using seamless cloning technology. The recombinant plasmid was verified by colony PCR, and the fragment size was about 500 bp (including part of the vector sequence) ( ​ (c) ​ (d)) and sequencing and comparative analysis were performed to determine BSMV- ​ The vector was constructed successfully.

[0068] 2.4 ​ Gene silencing efficiency detection To further verify ​ The role of genes in stripe rust resistance was analyzed using virus-induced gene silencing (VIGS) technology. ​ Infected wheat plants showed a marked bleaching phenomenon ( ​ (a)), indicating that the VIGS vector can function normally in plants. ​ The silencing effect of the gene was detected by qRT-PCR in the silenced plants. ​ The results showed that the expression level of the gene in the silenced plants ​ The gene expression level was significantly lower than that in the control group ( ​ (b)), verified ​ The gene-silenced plants were successfully constructed.

[0069] 2.5 ​ Resistance detection in gene-silenced plants To evaluate ​ The effect of gene silencing on wheat stripe rust resistance was investigated. The disease resistance of silenced plants was tested 14 days after inoculation with stripe rust. The results showed that compared with the control group, ​ The number of stripe rust spores in gene-silenced plants increased significantly ( ​ (a)), the disease index increased significantly ( ​ (b)), and the amount of fungal DNA increased significantly ( ​ (c)). These results indicate that ​ Gene silencing weakened wheat's resistance to stripe rust. Plant samples taken 24 and 48 hours after inoculation with stripe rust were subjected to DAB staining to observe the accumulation of reactive oxygen species (H2O2). The results showed that compared with the control group, ​The H2O2 accumulation area in gene-silenced plants was significantly reduced ( ​ (d) ​ (e)), indicating ​ Gene silencing may have inhibited the plant's rapid defense response to stripe rust infection. In addition, WGA staining was used to observe the hyphae length 24 h and 48 h after docking. The results showed that compared with the control group, ​ The hyphae length of gene silenced plants increased significantly ( ​ (g)), further indicating ​ Gene silencing may promote the growth and development of stripe rust in wheat; silencing ​ After the gene was silenced, the JA content in the leaves of the silenced plants was significantly lower than that of the control plants ( ​ (h)); ​ (f) is ​ WGA staining phenotype observation results of gene silenced plants at 24 h and 48 h.

[0070] The above results show that ​ The gene plays a key role in wheat resistance to stripe rust. Its silencing will significantly weaken the resistance response of wheat to stripe rust. ​ The gene may play a positive regulatory role on stripe rust by synthesizing JA.

Claims

1. Wheat TaLOX Application of genes in regulating wheat resistance to stripe rust; the wheat TaLOX The nucleotide sequence of the gene is selected from any one of (a) or (b): (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.

2. The use according to claim 1, characterized in that include: Changing wheat TaLOX The transcription or expression level of the gene in wheat regulates wheat's resistance to stripe rust.

3. The use according to claim 2, characterized in that The altered wheat TaLOX The transcription or expression level of genes in plants is to enhance wheat TaLOX The transcription or expression level of the gene in wheat improves the resistance of wheat to stripe rust.

4. The use according to claim 3, characterized in that include: Building wheat TaLOX Gene overexpression vector; the wheat TaLOX The gene is overexpressed in wheat, and the resulting overexpressed wheat has improved resistance to stripe rust.

5. The use according to claim 2, characterized in that The altered wheat TaLOX The transcription or expression level of the gene in wheat is reduced or weakened TaLOX The transcription or expression level of the gene in wheat reduces wheat's resistance to stripe rust.

6. A method for obtaining a plant resistant to stripe rust, characterized in that: include: Building wheat TaLOX Gene overexpression vector; Wheat TaLOX Genes are overexpressed in plants; Screening the obtained overexpression positive plants to obtain overexpression plants with improved resistance to stripe rust; the wheat TaLOX The nucleotide sequence of the gene is shown in SEQ ID No. 1; the plant is wheat.

7. A method for improving plant resistance to stripe rust, characterized in that: include: Building wheat TaLOX Gene overexpression vector; the wheat TaLOX The gene is overexpressed in plants, and the resulting overexpressed plants have increased resistance to stripe rust; the wheat TaLOX The nucleotide sequence of the gene is shown in SEQ ID No. 1; the plant is wheat.

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