Wheat disease resistance gene taLOX and its application in regulating resistance to stripe rust

Abstract

The application discloses a wheat disease-resistant gene TaLOX and application thereof in regulating resistance to stripe rust. TaLOX The application overexpresses the wheat gene in wheat, inoculates the overexpressed wheat plants with stripe rust, and observes the resistance phenotype of the overexpressed wheat plants to the stripe rust (Puccinia striiformis f. Puccinia striiformis sp. tritici ). TaLOX The results show that, compared with a control group, the amount of spores of the stripe rust on the surface of the overexpressed wheat plants is significantly reduced, the disease index and the fungal biomass are significantly reduced, and the mycelium length is significantly shorter than that of the control group, indicating that the gene positively regulates the resistance of the wheat to the stripe rust. TaLOX The application has application prospects in breeding of wheat varieties resistant to stripe rust and improvement of the resistance level of the wheat to the stripe rust.

Description

Technical Field

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

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

[0003] A series of stripe rust resistance genes play a crucial role in wheat's resistance to the disease. With the continuous advancement of wheat genome research, numerous stripe rust resistance genes have been discovered and identified; to date, no single gene has been found to be particularly effective against stripe rust. TaLOX Reports indicate that genes have the function of regulating resistance to stripe rust. Summary of the Invention

[0004] The main objective of this invention is to provide wheat TaLOX The role of genes in regulating crop resistance to stripe rust.

[0005] To achieve the above objectives, the technical solution adopted by the present invention includes:

[0006] This invention provides wheat TaLOX The application of genes in regulating plant resistance to stripe rust includes: by altering wheat... TaLOX The transcriptional or expression levels of genes in plants regulate plant resistance to stripe rust; wherein, the alteration of wheat... TaLOX The level of gene transcription or expression in plants can enhance wheat TaLOX The transcription or expression level of genes in plants can also reduce or weaken wheat. TaLOX The transcriptional or expression level of genes in plants; correspondingly, by altering wheat TaLOXThe gene can be used to regulate the transcription or expression level of the plant to achieve the resistance to the stripe rust, or to change the wheat TaLOX The gene can be used to regulate the transcription or expression level of the plant to achieve the resistance to the stripe rust, or to change the wheat

[0007] As to how to increase or decrease the resistance of the wheat TaLOX The gene can be used to regulate the transcription or expression level of the plant to achieve the resistance to the stripe rust, or to change the wheat TaLOX The gene can be used to regulate the transcription or expression level of the plant to achieve the resistance to the stripe rust, or to change the wheat TaLOX The gene can be used to regulate the transcription or expression level of the plant to achieve the resistance to the stripe rust, or to change the wheat TaLOX The gene can be used to regulate the transcription or expression level of the plant to achieve the resistance to the stripe rust, or to change the wheat TaLOX The gene can be used to regulate the transcription or expression level of the plant to achieve the resistance to the stripe rust, or to change the wheat TaLOX The gene can be used to regulate the transcription or expression level of the plant to achieve the resistance to the stripe rust, or to change the wheat

[0008] The gene can be used to regulate the transcription or expression level of the plant to achieve the resistance to the stripe rust, or to change the wheat TaLOX The gene can be used to regulate the transcription or expression level of the plant to achieve the resistance to the stripe rust, or to change the wheat TaLOX The gene can be used to regulate the transcription or expression level of the plant to achieve the resistance to the stripe rust, or to change the wheat TaLOX The gene can be used to regulate the transcription or expression level of the plant to achieve the resistance to the stripe rust, or to change the wheat TaLOX The gene can be used to regulate the transcription or expression level of the plant to achieve the resistance to the stripe rust, or to change the wheat

[0009] In the present application, any plant transformation method can be used to transform the constructed overexpression plant vector or gene editing vector of the present application into the tissue or cell of the target plant to obtain transformants, and then the transformants are regenerated into complete plants and their clones or offspring by plant tissue culture method. The transformation method includes Agrobacterium-mediated genetic transformation, protoplast transformation, plant virus vector, microinjection method, electroporation method, etc.

[0010] As a preferred embodiment of the present application, the present application provides a method for improving the resistance of plants to stripe rust, comprising: constructing an overexpression vector of the wheat TaLOX gene or a homologous gene thereof; overexpressing the wheat TaLOX gene or the homologous gene thereof in plants, and obtaining an overexpression plant with improved resistance to stripe rust.

[0011] As another preferred embodiment of the present application, the present application provides a method for creating a plant variety resistant to stripe rust, comprising: constructing an overexpression vector of the wheat TaLOX gene or a homologous gene thereof; overexpressing the wheat TaLOX gene or the homologous gene thereof in plants; and screening an overexpression plant with improved resistance to stripe rust from the obtained overexpression positive plants.

[0012] The plant in the present application includes but is not limited to monocotyledonous plants or dicotyledonous plants. Most preferably, the plant is wheat.

[0013] The nucleotide sequence of the wheat TaLOX gene (XM_044587023.1, PREDICTED: Triticum aestivum probable lipoxygenase 8, chloroplastic (LOC123169172)) in the present application is selected from any one of (a)-(e):

[0014] (a) the polynucleotide sequence shown in SEQ ID No. 1; (b) a polynucleotide capable of hybridizing to the complement of SEQ ID No. 1 under stringent hybridization conditions; (c) a polynucleotide having at least 90% or more homology with the polynucleotide shown in SEQ ID No. 1; (d) a mutant having one or more deletions, substitutions or insertions of bases based on the polynucleotide shown in SEQ ID No. 1, and the mutant still has the function or activity of regulating resistance to stripe rust; (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] The following is described in this invention TaLOX The amino acid sequence of the protein encoded by the gene is shown in SEQ ID No. 2.

[0018] This invention uses dynamic samples of Liangchun 1723 wheat after inoculation as material, and through combined transcriptomic and metabolomic analysis, screens out key genes regulating wheat resistance to stripe rust. TaLOX The function of this gene in wheat resistance to stripe rust was preliminarily explored using transient overexpression and gene silencing technologies. TaLOX The gene plays a crucial role in wheat resistance to stripe rust and is overexpressed in wheat. TaLOX Genes can significantly improve wheat's resistance to stripe rust by inhibiting the growth of certain pathogens in wheat. TaLOX Gene silencing significantly weakens the resistance response of wheat to stripe rust, indicating that... TaLOX Genes, as positive regulators, participate in the wheat's resistance to stripe rust and have promising applications in improving plant resistance to stripe rust or breeding stripe rust-resistant plant varieties.

[0019] Definitions of terms involved in the present invention

[0020] 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.

[0021] The term "polynucleotide" or "nucleotide" means deoxyribonucleotides, deoxyribonucleosides, ribonucleosides or ribonucleotides in either single- or double-stranded form, and polymers thereof. Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless specifically indicated otherwise, the term also means oligonucleotide analogs, including PNAs (peptide nucleic acids), DNA analogs used in antisense technology (phosphorothioates, phosphoramidates, etc.). Unless otherwise specified, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (including, but not limited to, simplifying degenerate codon substitutions) and complementary sequences as well as the sequence explicitly indicated. Specifically, a degenerate codon variant can be generated by generating the sequence of a parent nucleic acid and then substituting one or more codons with their degenerate counterparts. For example, a degenerate codon variant of a parent nucleic acid can be created by substituting one or more codons of the parent nucleic acid with a different codon that encodes the same amino acid.

[0022] The term "homology" refers to the level of similarity or percent identity in percent nucleotide position identity (i.e., sequence similarity or identity) between polynucleotide sequences. The term homology as used herein also refers to the concept of similar functional properties between different polynucleotide molecules, for example, promoters with similar functions can have homologous cis-elements. Polynucleotide molecules are homologous when they specifically hybridize under certain conditions to form a duplex molecule. Under these conditions (referred to as stringent hybridization conditions) one polynucleotide molecule can be used as a probe or primer to identify another polynucleotide molecule that shares homology.

[0023] "Stringent hybridization conditions" as used in the present invention means conditions of low ionic strength and high temperature known in the art. Typically, under stringent conditions, a probe will hybridize to its target sequence to a detectable higher degree than to other sequences (e.g., at least 2-fold over background. Stringent hybridization conditions are sequence dependent, and will be different in varying environmental conditions, with longer sequences being specifically hybridized at higher temperatures. By controlling the stringency or wash conditions of the hybridization, one can identify target sequences that are 100% complementary to the probe. For an extensive guide to nucleic acid hybridization, see 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 stringent conditions will ordinarily be selected to be less than about 5-10°C below the thermal melting point (Tm) for the specific sequences at a defined ionic strength pH. T m )m temperature at which 50% of the probes are occupied in equilibrium (because of the excess of target sequence, at T m Stringent conditions can be conditions in which 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 can also be achieved with the addition of destabilizing agents such as formamide. For selective or specific hybridization, a positive signal can be at least twice background hybridization, and in some cases 10 times background hybridization. Exemplary stringent hybridization conditions can be as follows: 50% formamide, 5 x SSC, and 1% SDS, at 42°C, or 5 x SSC, 1% SDS, at 65°C, with wash in 0.2 x SSC and 0.1% SDS at 65°C. The wash can be for 5, 15, 30, 60, 120 minutes or more.

[0024] "substitutions" mean replacement of one or more amino acid residues by different amino acid residues; "deletions" mean reduction in the number of amino acid residues, i.e., absence of one or more amino acid residues; and "insertions" mean changes in the sequence of amino acid residues, i.e., addition of one or more amino acid residues relative to the native molecule.

[0025] The term "promoter" refers to a polynucleotide molecule which, in its natural state, is located upstream or 5' of the translation initiation codon of an open reading frame (or protein coding region) and is involved in the recognition and binding of RNA polymerase II and other proteins (trans-acting transcription factors) to initiate transcription.

[0026] The term "operably linked" refers to linkage of a first polynucleotide molecule (e.g., a promoter) to a second transcribable polynucleotide molecule (e.g., a gene of interest) in which the polynucleotide molecules are arranged so 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 are more preferably adjacent. For example, a promoter is operably linked to a gene of interest if the promoter regulates or mediates transcription of the gene of interest in a cell.

[0027] The term "overexpression vector": one or more DNA vectors used to effect plant transformation; these vectors are often referred to in the art as binary vectors. Binary vectors, along with vectors having helper plasmids, are most often used for Agrobacterium-mediated transformation. Binary vectors typically include: cis-acting sequences required for T-DNA transfer, a selectable marker engineered to be expressible in plant cells, a heterologous DNA sequence to be transcribed, etc.

[0028] The term "transformation": a process of introducing a heterologous DNA sequence into a host cell or organism.

[0029] The term "expression": transcription and / or translation of an endogenous gene or transgene in a plant cell.

[0030] The term "recombinant host cell strain" or "host cell" means a cell which contains a polynucleotide of the present application, whether or not the polynucleotide is integrated into the host genome, or maintained as a non-integrated vector, such as a plasmid, or otherwise. The host cell can be a prokaryotic or eukaryotic cell, and the host cell can be a monocot or dicot plant cell. BRIEF DESCRIPTION OF DRAWINGS

[0031] Figure 1 Figure 1 is the results of bioinformatics analysis of TaLOX protein; (a): domain prediction of TaLOX; (b): transmembrane structure prediction of TaLOX; (c): secondary structure prediction of TaLOX; (d): tertiary structure prediction of TaLOX.

[0032] Figure 2 Figure 2 is the results of quantitative real-time PCR of TaLOX gene; significance analysis using t-test, ***: P < 0.001; ****: P < 0.0001.

[0033] Figure 3 Figure 3 is the results of subcellular localization of TaLOX protein in tobacco leaves.

[0034] Figure 4 Figure 4 is TaLOX Figure 5 is construction of gene pYBA-1132-GFP vector; (a): schematic diagram of pYBA-1132-GFP vector; (b): TaLOX Figure 6 is amplification of gene PCR product; (c): colony PCR; (d): 5000 DNA Marker.

[0035] Figure 5 Figure 7 is TaLOX Figure 8 is analysis of disease resistance of gene transient overexpression plants; (a): TaLOX Figure 9 is phenotype observation of gene transient overexpression plants 14 d after inoculation; (b):TaLOX Detection of transient overexpression efficiency of genes; (c) : TaLOX Disease index of plants with transient overexpression of genes 14 d after inoculation; (d) : TaLOX Phenotype observation of WGA staining of plants with transient overexpression of genes 48 h after inoculation; SV, substomatal vesicles; HMC, haustorial mother cells; IH, infection hyphae; (e) : TaLOX Statistical analysis of Puccinia striiformis hyphal length of plants with transient overexpression of genes 48 h after inoculation, with 30 samples; (f) : TaLOX Detection of fungal DNA amount of plants with transient overexpression of genes 2 d and 7 d after inoculation; T test was used for significance analysis, **: P < 0.01; ****: P < 0.0001.

[0036] Figure 6 To 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.

[0037] Figure 7 To TaLOX Detection results of gene silencing efficiency; (a) : Phenotype of positive control plants; (b) : TaLOX Detection of gene silencing efficiency; T test was used for significance analysis, **: P < 0.01; ****: P < 0.0001.

[0038] Figure 8 To TaLOX Phenotype of plants with gene silencing; (a) : TaLOX Phenotype observation of plants with gene silencing 14 d after inoculation; (b) : TaLOX Disease index of plants with gene silencing 14 d after inoculation; (c) : TaLOX Fungal DNA amount of plants with gene silencing 2 d after inoculation; (d) : TaLOX Phenotype observation of DAB staining of plants with gene silencing 24 h and 48 h, SV, substomatal vesicles; H2O2 appears brown after DAB staining. bar = 20 um; (e) : TaLOX Statistical analysis of H2O2 area of plants with gene silencing 24 h and 48 h, with 30 samples; (f) : TaLOX Phenotype observation of WGA staining of plants with gene silencing 24 h and 48 h, SV, substomatal vesicles; HMC, haustorial mother cells; IH, infection hyphae; (g) : TaLOXThe hypha length of the gene-silenced plants was counted at 24 h and 48 h, and the sample number was 30; (h): ELISA detection of JA content in leaves; t test was used for significance analysis, *: P<0.05; ****: P<0.0001. DETAILED DESCRIPTION

[0039] The advantages and features of the present application will become more apparent with the following specific examples. However, it should be understood that the examples are only exemplary, and do not constitute any limitation on the scope of the present application. Those skilled in the art should understand that the details and forms of the technical solutions of the present application can be modified or replaced without departing from the spirit and scope of the present application, and such modifications or replacements all fall within the protection scope of the present application.

[0040] Test materials and general test methods

[0041] 1. Test materials

[0042] The test wheat was Liangchun 1723 (high resistance), Xinchun 34 (high susceptibility) and Xinchun 35 (medium resistance), and the stripe rust physiological race was CYR32.

[0043] 2. Test strains and vectors

[0044] DH5α E. coli competent cells and GV3101 (pSoup) Agrobacterium competent cells were purchased from Shanghai Generay Biotech Co., Ltd. The overexpression vector pYBA-1132-GFP and the silencing vectors BSMV-α, BSMV-β, BSMV-γ and BSMV-PDS were preserved in the laboratory of the present inventors.

[0045] 3. Test reagents and instruments

[0046] Kanamycin (Kan), rifampicin (Rif) and acetyl-piperitone (AP) were purchased from Sigma-Aldrich. Kan Rif AS ​​) were purchased from Beijing Solabio Biotech Co., Ltd.; total RNA extraction kit, fluorescent quantitative kit and reverse transcription kit were purchased from Fujian Biotech Co., Ltd.; β-mercaptoethanol, anhydrous ethanol and jasmonic acid (JA) enzyme-linked immunoassay kit were purchased from Shanghai Meiji Biotech Co., Ltd.; high-fidelity DNA polymerase was purchased from Beijing NEB Biotech Co., Ltd.; high-fidelity endonuclease and T4 DNA ligase were purchased from Thermo Fisher Scientific, USA; agarose electrophoresis product purification kit and circular DNA separation and purification reagent were from Jiangsu Novizen Biotech Co., Ltd. Ligase and DNA molecular weight marker were purchased from Beijing Quanshi Gold Co., Ltd. Chloroform, isopropyl alcohol, glycerol, anhydrous ethanol, 2-morpholinoethanesulfonic acid, magnesium chloride and LB medium were prepared by domestic analytical pure reagents; primers for PCR and qRT-PCR were synthesized and sequenced by Xinjiang Youkang Biotech Co., Ltd.

[0047] -80℃ ultra-low temperature refrigerator, constant temperature incubator, high-speed variable temperature centrifuge, ABI7500 Fast fluorescence quantitative instrument, pipettor, electrophoresis instrument, ultraviolet gel imaging instrument, super clean laboratory workbench, electric heating constant temperature water bath, high pressure steam sterilization pot, ultraviolet spectrophotometer, visible spectrophotometer, etc.

[0048] 4 Wheat planting and inoculation treatment of stripe rust

[0049] Select seeds of Laimian 1723 with good grain development and consistent color and shape, sow in 7 cm x 7 cm small flowerpots after germination, cultivate under a light cycle of 16 h light and 8 h darkness, inoculate stripe rust when the wheat grows to one leaf one heart, and use distilled water to simulate inoculation of wheat plants as a blank control. Take wheat samples at 0 h, 6 h, 12 h, 18 h, 24 h, 36 h, 48 h, 72 h and 96 h after inoculation, freeze in liquid nitrogen and store in a -80℃ refrigerator.

[0050] 5 Extraction of total RNA of wheat and synthesis of cDNA

[0051] Take the stored samples from the -80℃ refrigerator and grind them into fine powder. Then, use the total RNA extraction kit to extract RNA according to the standard procedure. After extraction, detect the quality of RNA by electrophoresis to determine whether the RNA meets the requirements of subsequent experiments. After confirming that the RNA quality is qualified, synthesize cDNA using the reverse transcription kit, and the reaction system setting and reaction program in the reverse transcription process are performed according to Table 1 and Table 2.

[0052] Table 1 Reverse transcription system

[0053]

[0054] Table 2 Reverse transcription PCR reaction program

[0055]

[0056] 6 qRT-PCR detection of target gene expression

[0057] Using cDNA as template, wheat elongation factor TaEF was used as the internal reference gene, and three technical repeats were performed. 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 Gossypium hirsutum small GTP-binding protein gene GhROP3 [J]. Biotechnology Bulletin, 2021, 37(09): 106-113.) According to the Ct value of the target gene and the internal reference gene, the expression amount of the gene was calculated using 2 -ΔΔCt Method (Livak, K J, and T D 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.). TaLOX The primers are shown in Table 3.

[0058] Table 3 Primer information used

[0059]

[0060] Note: F represents forward primer, R represents reverse primer, and lowercase letters represent homologous arms. The same below.

[0061] Example 1 TaLOX protein bioinformatics analysis

[0062] TaLOX protein structure analysis was performed using NCBI online tools; TaLOX protein physicochemical properties, transmembrane structure analysis, secondary structure analysis, and tertiary structure prediction were analyzed using ExPASy-ProtScale, TMHMM, PSIPRED, and SWISS-MODEL online tools. The tool websites used are shown in Table 4.

[0063] Table 4 Bioinformatics analysis tools

[0064]

[0065] Wheat TaLOX protein is a lipoxygenase. Analysis of TaLOX's conserved domains and motifs using NCBI revealed that TaLOX contains typical lipoxygenase and PLAT_LH2 domains. Figure 1 (a) The TaLOX protein sequence was analyzed using ProtScale. The TaLOX protein is a protein containing 932 amino acids, with the molecular formula C0. 4557 H 7145 N 1281 O 1355 S 29 The protein has a total of 14,367 atoms, a molecular weight of 10.248737 kDa, an isoelectric point of 7.35, a total of 109 negatively charged residues (Asp+Glu), a total of 109 positively charged residues (Arg+Lys), an instability index of 46.45, an average water solubility of -0.321, and a lipid solubility index of 79.58. Subcellular localization prediction of TaLOX indicates that the protein is located in the cell membrane. TMHMM prediction of the transmembrane helical structure shows that the TaLOX protein does not possess a transmembrane helical structure. Figure 1 (b)).

[0066] The secondary and tertiary structures of the TaLOX protein were predicted using the SOPMA online platform and the SWISS-MODEL modeling system. Structural analysis of the secondary structure components revealed that the TaLOX protein consists of 35.84% α-helices, 12.23% extended strands, 2.15% β-turns, and 49.79% random coils. Figure 1 (c)). Its tertiary structure predictions are consistent with the predicted secondary structure predictions. Figure 1 (d)).

[0067] Experimental Example 2 TaLOX Gene expression pattern identification test

[0068] In the transcriptome sequencing results, TaLOX Gene expression was significantly upregulated, in order to further identify TaLOX To investigate the gene expression patterns during wheat infection by pathogens, this experiment used real-time quantitative qRT-PCR to detect gene expression levels in wheat leaves at 0 h, 6 h, 12 h, 18 h, 24 h, 36 h, 48 h, 72 h, and 96 h.

[0069] The test results found TaLOX The gene was significantly upregulated at 12 h, 18 h, and 24 h after infection with stripe rust, and the trend of quantitative fluorescence results was consistent with that of transcriptome results. Figure 2 This result indicates that... TaLOXGenes may play a key role in the early stages of stripe rust infection.

[0070] Experimental Example 3: Subcellular localization of TaLOX protein

[0071] The pYBA-1132-GFP vector contains the green fluorescent protein (GFP) gene and 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 prediction results, the corresponding marker protein (red fluorescence) was selected as a control. Tobacco leaves were transformed using Agrobacterium-mediated transformation. TaLOX -GFP was transiently expressed in tobacco, thereby determining the subcellular localization of the TaLOX protein.

[0072] PSORT was used to predict the localization of TaLOX protein in the cell membrane. Tobacco leaves were transformed with Agrobacterium to induce pYBA- TaLOX -GFP is transiently expressed in tobacco, and experimental results show that ( Figure 3 ): The fluorescence of empty GFP is uniformly distributed throughout the cell, while the expression of GFP is... TaLOX Genes TaLOX The -GFP fusion protein exhibited significant green fluorescence at the cell membrane of tobacco leaf epidermal cells, and this green fluorescence overlapped with the red fluorescence of the cell membrane localization marker, forming a yellow fluorescence, which further proves that the TaLOX protein is localized in the cell membrane.

[0073] Test Example 4 TaLOX Construction and transformation of gene overexpression and silencing vectors, and phenotypic identification of transgenic plants.

[0074] 1. Experimental Methods

[0075] 1.1 TaLOX Construction of gene overexpression and silencing vectors

[0076] according to TaLOX Based on the CDS sequence of the gene, specific amplification primers were designed (Table 3). Using wheat cDNA as a template, the target fragment was cloned. Homologous recombination technology was used to insert the target fragment into the corresponding restriction site of the pYBA-1132-GFP vector, resulting in pYBA- TaLOX-GFP gene overexpression vector. The vector was transformed into DH5a E. coli competent cells, and positive clones were screened in LB solid medium containing antibiotics (Kan 50 ug / mL), and then the vector construction was confirmed by liquid PCR detection and sequencing of positive clones using pYBA-1132-GFP universal primers 1132-F / R. The recombinant plasmid with correct sequencing was transformed into GV3101 (pSoup) Agrobacterium competent cells, and cultured in double-antibiotic LB medium (Kan 50 ug / mL + Rif 50 ug / mL) for 48 h for subsequent experiments.

[0077] According to TaLOX the nucleotide sequence of the gene, the silencing fragment was designed using Premier 5.0 software, and the specificity of the designed silencing fragment was determined using the online website NCBI Blastn. The designed gene fragment (Table 5) was amplified by PCR and connected to the BSMV-PDS vector (pYBA-1132-GFP) by one-step enzyme digestion method, and the correct vector was obtained by E. coli DH5a transformation and the like. The plasmid with correct sequencing was transformed into GV3101 (pSoup) Agrobacterium competent cells and plated on LB plates containing Figure 6 and Kan antibiotics (Kan 50 ug / mL + Rif 50 ug / mL) for 48 h for subsequent experiments. Rif

[0078] Table 5 TaLOX Silencing sequence

[0079]

[0080] 1.2 TaLOX Gene overexpression plant construction and disease resistance identification

[0081] LB broth (Kan 50 ug / mL + Rif 50 ug / mL) was added to Agrobacterium containing pYBA- TaLOX -GFP plasmid, and the bacteria were shaken at 200 rpm. When the bacterial concentration was OD600=0.8-1.0, the bacteria were centrifuged (4000 rpm / 10 min) using a centrifuge, and the supernatant was discarded. The bacteria were resuspended in LB medium (Kan 50 ug / mL + Rif 50 ug / mL) to a final concentration of OD600=0.8-1.0, and then used for subsequent experiments. As ​The concentration was diluted with buffer to OD600 = 1.0. Wheat plants (Xinchun 34) at the two-leaf-one-heart stage were selected. The second leaf was injected onto the underside of the leaf, and the injection area was marked with a marker. Sixty wheat plants were injected, with the same number of plants injected as a control (pYBA-1132-GFP empty vector). After all injections were completed, the plants were placed in the dark at 23℃ for 24 h. Thirty-six h after injection, freshly harvested highly virulent wheat stripe rust race CYR32 was inoculated onto the upper surface of the injected leaf area. An additional number of wild-type plants were inoculated as mock plants. After all inoculations were completed, the plants were placed in the dark at 16℃ for 24 h. Samples were collected at 24 h and 48 h after inoculation to detect overexpression efficiency. Tissue samples were also collected at these two time points for histological observation. Sporulation was observed approximately two weeks after inoculation with stripe rust, and disease index was statistically analyzed according to the method of Zhang Hongchang (Zhang Hongchang. Histological and cytological study on resistance of mature wheat plants to stripe rust and study on the mechanism of resistance of wheat non-host to broad bean rust [D]. Northwest A&F University. 2012.).

[0082] 1.3 TaLOX Construction of gene-silenced plants and identification of disease resistance

[0083] The vectors BSMV-α, BSMV-β, BSMV-γ, BSMV- PDS (The BSMV-α, BSMV-β, BSMV-γ, and BSMV-PDS were constructed using the methods disclosed in the reference (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- TaLOX Agrobacterium was transformed separately, 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 35) that had grown to the two-leaf stage, following the method described by Wu Hongyan et al. (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.) to induce silencing. Three groups of mixed resuspensions (BSMV-α, BSMV-β, and BSMV-...) were then... PDS (positive), BSMV-α, BSMV-β and BSMV-γ (BSMV: γ), BSMV-α, BSMV-β and BSMV- TaLOX (BSMV: TaLOX Infected wheat plants served as positive and negative controls (BSMV: γ) and the target gene experimental group (BSMV: γ). TaLOX The same number of wild-type plants were used as mocks. When the positive control plants BSMV- PDSWhen the leaves of the transgenic plants showed obvious albino phenotype, the plants were inoculated with Puccinia striiformis f. sp. tritici race CYR32 on the fourth leaf. The samples were collected at 0 h, 24 h and 48 h after inoculation for detecting the silencing efficiency. Meanwhile, the tissue samples were collected at 24 h and 48 h after inoculation for histological observation. The sporulation was observed about two weeks after inoculation with Puccinia striiformis f. sp. tritici race CYR32, and the disease index was calculated. In addition, the samples were collected at 24 h after inoculation with Puccinia striiformis f. sp. tritici race CYR32 for detecting the content of jasmonic acid (JA) in the leaves of the transgenic plants. TaLOX The content of JA in the leaves of the transgenic plants was determined according to the operation steps in the instruction manual of the plant jasmonic acid (JA) enzyme-linked immunoassay kit.

[0084] 1.4 Histological treatment

[0085] Active oxygen staining treatment and observation: To explore the histopathological changes of the silencing wheat leaves after inoculation with Puccinia striiformis f. sp. tritici, the DAB (3,3'-diamino-bezidine, Sigma-Aldrich, St. Louis, USA) staining solution was used to stain the wheat leaf tissues at 24 h and 48 h after inoculation with Puccinia striiformis f. sp. tritici, and 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 the wheat leaves at 24 h and 48 h after inoculation were cut into leaf segments with a length of about 1 cm and placed in the DAB staining solution, and a vacuum pump was used to vacuum for 30 min; (2) the staining was poured off, and the solution was replaced with decolorizing solution (glacial acetic acid: anhydrous ethanol = 1:1), and the decolorizing solution was replaced whenever it became turbid until the leaf segments were completely decolorized; (3) the decolorizing solution was poured off, and the solution was replaced with chloral hydrate, and the samples were fixed for 24 h and then stored in sample storage solution; (4) the infection was observed using an inverted fluorescence microscope in bright field, and the stomata forming haustorial mother cells were used as infection points, and the active oxygen area around the infection points was counted using ImageJ.

[0086] Hyphae staining and observation of stripe rust: In order to observe the infection structure and hyphae expansion of stripe rust during the infection process of wheat leaves, WGA-Alexa488 dye was used to stain the leaves of wheat 24 and 48 h after inoculation. The method is as follows: (1) The wheat samples stored in the sample storage solution were washed with 50% ethanol, 50% ethanol, Tris-Hcl (0.05 M), and Tris-Hcl (0.05 M) in sequence, each for 15 min; (2) Add 1 M KOH solution, immerse the sample, and boil the sample for 10 min; (3) Wash the leaf sample with Tris-Hcl (0.05 M) for 5 min for 3 times (4) Absorb the washing solution, add appropriate amount of 20 ug / mL WGA-Alexa488 fluorescent dye, avoid light staining for 2 h, wash the leaf sample with Tris-Hcl (0.05 M) for 5 min for 3 times; (5) Store with Tris-Hcl (0.05 M); (6) Observe the appressoria, hyphae and other structures of stripe rust under the GFP filter of confocal microscope, and measure the hyphae length and expansion area of hyphae using ImageJ (Hu Zeyu. Functional research of transcription factor TaNF-YC1 in wheat-stripe rust interaction [D]. Northwest A&F University, 2022.).

[0087] 2 Test results

[0088] 2.1 TaLOX Construction of gene overexpression vector

[0089] TaLOX The construction strategy of the overexpression vector of the gene is shown in Figure 4 (a). The coding region (CDS) sequence of the target gene was amplified by directional amplification. TaLOX Electrophoresis verification showed that the size of the target product was consistent with the expected 2799 bp ( Figure 4 (b)). The purified target fragment was connected with the linearized pYBA-1132-GFP vector by seamless cloning technology and transformed into DH5a. The recombinant plasmid was verified by colony PCR, and the fragment size was about 2799 bp ( Figure 4 (c)) and sequencing comparison analysis, to determine that the pYBA- TaLOX- GFP vector construction was successful.

[0090] 2.2 TaLOX Resistance detection of gene transient overexpression plants

[0091] To preliminarily verify TaLOX the potential function of the gene in resistance to wheat stripe rust, the pYBA- TaLOX -GFP overexpression vector was constructed, and it was transfected into the mesophyll tissue of Xinchun 34 (highly susceptible to stripe rust) by Agrobacterium mediation, to realize the transient expression of pYBA-TaLOX Transient overexpression of GFP in wheat (TaLOX-GFP); meanwhile, pYBA-1132-GFP vector was transfected into mesophyll tissue of new 34 (highly susceptible to stripe rust) by Agrobacterium-mediated, achieving transient overexpression of pYBA-1132-GFP in wheat (GFP0).

[0092] In the transient overexpression plants TaLOX The expression of the gene was 3.48 times higher than that of the control group at 24 h, and 4.73 times higher than that of the control group at 48 h Figure 5 (b), indicating that the transient overexpression plants of the gene were successfully obtained TaLOX These results laid a foundation for further research on the function of the gene in the mechanism of resistance to stripe rust. TaLOX

[0093] After inoculating the overexpression plants with stripe rust for 14 d, it was found that compared with the control group, TaLOX The amount of spores of stripe rust on the surface of the transient overexpression plants of the gene was significantly reduced, and the disease index and the amount of fungal DNA were significantly reduced Figure 5 (a), Figure 5 (c), Figure 5 (f). And TaLOX The hypha length of the transient overexpression plants of the gene was significantly shorter than that of the control group Figure 5 (e). Further supporting its inhibitory effect on pathogen infection. Figure 5 (d) was TaLOX Phenotype observation of WGA staining of the transient overexpression plants of the gene inoculated with stripe rust for 48 h; the above results showed that overexpression of the gene in wheat significantly enhanced the resistance of wheat to stripe rust. TaLOX

[0094] 2.3 TaLOX Construction of gene silencing vector

[0095] TaLOX The schematic diagram of the construction of the silencing vector of the gene is shown in Figure 6 (a), and agarose electrophoresis detection showed that the length of the product was completely matched with the expected 300 bp Figure 6 (b). The purified fragment was directionally connected to the enzyme-digested BSMV-PDS linearized vector by using seamless cloning technology, the recombinant plasmid was verified by colony PCR, the fragment size was about 500 bp (including a part of the vector sequence) Figure 6 (c), Figure 6 (d), and sequencing comparison analysis determined that the BSMV- TaLOX vector was successfully constructed.

[0096] 2.4 TaLOX Detection of gene silencing efficiency ​​

[0097] To further verify TaLOX The role of genes in stripe rust resistance was analyzed using virus-induced gene silencing (VIGS) technology. When the positive control group BSMV: PDS Infected wheat plants exhibited significant albinism. Figure 7 (a) indicates that the VIGS vector can function normally in plants. To evaluate... TaLOX The effect of gene silencing was detected by qRT-PCR in silenced plants. TaLOX Gene expression levels. Results showed that in silent plants... TaLOX Gene expression levels were significantly lower than those in the control group ( Figure 7 (b) verified TaLOX Gene-silencing plants were successfully constructed.

[0098] 2.5 TaLOX Resistance detection in gene-silenced plants

[0099] In order to evaluate TaLOX The effect of gene silencing on wheat stripe rust resistance was investigated by examining the resistance of silencing plants 14 days after inoculation with stripe rust fungus. Results showed that, compared to the control group, TaLOX The number of stripe rust spores was significantly increased in gene-silenced plants. Figure 8 (a)), the disease index increased significantly ( Figure 8 (b)), and the amount of fungal DNA increased significantly ( Figure 8 (c)). These results indicate that TaLOX Gene silencing weakens wheat's resistance to stripe rust. Simultaneously, DAB staining was performed on plant samples 24 h and 48 h after stripe rust inoculation to observe the accumulation of reactive oxygen species (H2O2). The results showed that, compared with the control group, TaLOX The area of ​​H2O2 accumulation in gene-silenced plants was significantly reduced. Figure 8 (d), Figure 8 (e)), indicating TaLOX Gene silencing may have suppressed the plant's rapid defense response to stripe rust infection. Furthermore, observation of hyphal length at 24 h and 48 h post-inoculation using WGA staining showed that, compared to the control group, TaLOX The hyphal length of gene-silenced plants was significantly increased. Figure 8 (g) further indicates TaLOX Gene silencing may have promoted the growth and development of stripe rust fungi in wheat; silencing TaLOX After gene administration, the JA content in the leaves of silenced plants was significantly lower than that in control plants. Figure 8 (h)); Figure 8 (f) is TaLOXGene silencing plants 24 h and 48 h WGA staining phenotype observation results.

[0100] The above results show TaLOX The gene plays a key role in the process of wheat resistance to stripe rust, and its silencing can significantly weaken the resistance response of wheat to stripe rust, in addition TaLOX The gene may play a positive regulation role on stripe rust by synthesizing JA.

Claims

1. Wheat TaLOX The application of genes in improving wheat resistance to stripe rust is characterized by, The wheat TaLOX The nucleotide sequence of the gene is shown in SEQ ID No. 1; The aforementioned improvement in wheat resistance to stripe rust is to enhance wheat... TaLOX The transcription or expression level of genes in wheat can improve wheat's resistance to stripe rust.

2. The application according to claim 1, characterized in that, include: Building wheat TaLOX Gene overexpression vectors; The wheat TaLOX The gene was overexpressed in wheat, and the resulting transgenic wheat plants with overexpressed genes showed increased resistance to stripe rust.

3. A method for obtaining wheat resistant to stripe rust, characterized in that, include: Building wheat TaLOX Gene overexpression vectors; wheat TaLOX The gene was overexpressed in wheat; Wheat plants with enhanced resistance to stripe rust were screened from the obtained overexpression-positive wheat plants; the wheat TaLOX The nucleotide sequence of the gene is shown in SEQ ID No.

1.

4. A method for improving wheat resistance to stripe rust, characterized in that, include: Building wheat TaLOX Gene overexpression vectors; The wheat TaLOX Overexpression of the gene in wheat resulted in wheat plants with increased resistance to stripe rust; the wheat described TaLOX The nucleotide sequence of the gene is shown in SEQ ID No. 1.

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