Application of ta jaz9 protein and its coding gene in resistance to wheat stripe rust

Abstract

The application discloses TaJAZ9 protein and an application of a coding gene thereof in resistance to wheat stripe rust, and belongs to the technical field of genetic engineering. TaJAZ9 The application value of the gene in improving the resistance of wheat to stripe rust is found for the first time. TaJAZ9 By improving the expression level of the gene or enhancing the function of the TaJAZ9 protein, the disease degree of wheat after being infected by the stripe rust can be reduced, the leaf spot expansion and the uredium formation can be reduced, and thus the resistance of the wheat plant to the stripe rust is improved, and the application value for cultivating the wheat variety resistant to the stripe rust is important.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, specifically to the application of TaJAZ9 protein and its encoding gene in resistance to wheat stripe rust. Background Technology

[0002] Wheat stripe rust is caused by the wheat stripe rust fungus. Puccinia striiformis f. sp. tritici This is a type of airborne fungal disease that primarily affects wheat leaves, but can also infect above-ground tissues such as leaf sheaths, stems, and ears. Under suitable conditions of low temperature and high humidity, this disease spreads rapidly and widely. Severe outbreaks can significantly reduce wheat's photosynthetic capacity and grain-filling efficiency, leading to decreased yield and deteriorated quality.

[0003] Breeding disease-resistant varieties is an economical, effective, and environmentally friendly way to reduce stripe rust losses. However, because common wheat is an allohexaploid crop with a large genome and abundant homologous sequences, screening and functional confirmation of candidate genes are challenging. Currently, the number of cloned wheat stripe rust resistance genes is still limited, and most are NBS-LRR genes. Therefore, discovering new wheat stripe rust resistance gene resources is of great significance for breeding new wheat varieties with stable resistance to stripe rust. Summary of the Invention

[0004] In view of the above-mentioned prior art, the purpose of this invention is to provide the application of TaJAZ9 protein and its encoding gene in resistance to wheat stripe rust.

[0005] To achieve the above objectives, the present invention adopts the following technical solution: A first aspect of the present invention provides the application of TaJAZ9 protein in resistance to wheat stripe rust, wherein the TaJAZ9 protein is as shown in (A1) or (A2): (A1) A protein with the amino acid sequence shown in SEQ ID NO.1; (A2) The protein obtained by attaching a protein tag to the N-terminus and / or C-terminus of the protein defined in (A1).

[0006] In the aforementioned proteins, the protein tag refers to a polypeptide or protein sequence fused with the TaJAZ9 protein or its functional fragment using in vitro DNA recombination technology. Its function is to facilitate the expression, detection, localization, tracing, and / or purification of the TaJAZ9 protein or its functional fragment. The protein tag can be linked to the N-terminus and / or C-terminus of the TaJAZ9 protein or its functional fragment. If necessary, a linker peptide can also be provided between the protein tag and the TaJAZ9 protein or its functional fragment to reduce the tag's influence on the spatial conformation or biological function of the TaJAZ9 protein.

[0007] In some embodiments, the protein tag includes, but is not limited to, one or more of the following: Flag tag, His tag, MBP tag, HA tag, Myc tag, GST tag, and SUMO tag.

[0008] In the above applications, wheat resistance to stripe rust can be improved by overexpressing TaJAZ9 protein in plants or by applying TaJAZ9 protein exogenously. The concentration of TaJAZ9 protein applied exogenously can be adjusted according to factors such as the wheat growth stage and the severity of the disease.

[0009] A second aspect of the present invention provides the application of the gene encoding the TaJAZ9 protein in resistance to wheat stripe rust, wherein the gene encoding the TaJAZ9 protein is... TaJAZ9 A gene is a nucleic acid molecule as shown in (i) or (ii) below: (i) Nucleic acid molecules with nucleotide sequences as shown in SEQ ID NO.2; (ii) Nucleic acid molecules other than (i) that encode the amino acid sequence shown in SEQ ID NO.1.

[0010] In the above applications, the nucleic acid molecule can be DNA, such as cDNA, genomic DNA, or recombinant DNA; the nucleic acid molecule can also be RNA, such as gRNA or mRNA.

[0011] In the above applications, overexpression in plants TaJAZ9 Genes can be used to improve a plant's resistance to stripe rust.

[0012] In some preferred embodiments of the present invention, overexpression in plants TaJAZ9 Genes are generated through the following substances: C1) contains TaJAZ9 Gene expression cassettes; C2) contains TaJAZ9 Recombinant vectors of genes, or recombinant vectors containing the expression cassette described in C1); C3) contains TaJAZ9 Recombinant microorganisms containing genes, or recombinant microorganisms containing the expression cassette described in C1), or recombinant microorganisms containing the recombinant vector described in C2); C4) contains TaJAZ9 Transgenic plant cell lines containing the gene, or transgenic plant cell lines containing the expression cassette described in C1); C5) contains TaJAZ9 Transgenic plant tissue containing the gene, or transgenic plant tissue containing the expression cassette described in C1); C6) contains TaJAZ9Genetically modified plant organs, or transgenic plant organs containing the expression cassette described in C1).

[0013] In the above applications, the resistance to wheat stripe rust specifically refers to: overexpression of [a specific substance] in wheat. TaJAZ9 Genes were used to obtain plant materials resistant to wheat stripe rust.

[0014] Preferably, the plant material resistant to wheat stripe rust includes: seedlings, callus tissue, embryogenic tissue, plant cells, transgenic lines, gene-edited lines, or breeding intermediates.

[0015] A third aspect of the present invention provides a method for improving wheat resistance to stripe rust, comprising the following steps: Improve wheat TaJAZ9 Increase gene expression levels; or increase the content or activity of TaJAZ9 protein in wheat.

[0016] The beneficial effects of this invention are: (1) This invention is the first to discover TaJAZ9 The application value of genes in improving wheat stripe rust resistance. By improving... TaJAZ9 Improving gene expression levels or enhancing TaJAZ9 protein function can reduce the severity of wheat disease after stripe rust infection, reduce leaf spot expansion and uredinia formation, thereby increasing wheat plant resistance to stripe rust.

[0017] (2) This invention provides a new source of resistance genes for breeding wheat varieties resistant to stripe rust. The resistance genes can be increased in wheat materials through transgenic methods, gene editing, promoter regulation, marker-assisted selection, hybridization, or other molecular breeding techniques. TaJAZ9 Investigating gene expression levels or functional activity to obtain wheat varieties, lines, breeding intermediates, or their progeny with enhanced resistance to stripe rust is of significant application value for breeding stripe rust-resistant wheat varieties. Attached Figure Description

[0018] Figure 1 : Schematic diagram of the structure of plant expression vector SVPC0349.

[0019] Figure 2 Schematic diagram of the overexpression vector PC1439.

[0020] Figure 3 PCR identification results of T1 generation transgenic plants; In the figure, Fielder#9, Fielder#7, Fielder#14, Fielder#4 and Fielder#17 represent the T1 generation identification results of 5 independent T0 generation transgenic plants.

[0021] Figure 4 Overexpression TaJAZ9Results of resistance study of transgenic wheat lines to stripe rust; in the figure, A represents phenotypic identification after inoculation with CYR32; B represents statistical results of disease severity. Detailed Implementation

[0022] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of this application. Unless otherwise specified, 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 application pertains.

[0023] As mentioned earlier, breeding disease-resistant varieties is an economical, effective, and environmentally friendly way to reduce stripe rust losses. However, because common wheat is an allohexaploid crop with a large genome and abundant homologous sequences, it is quite difficult to screen functional genes with stripe rust resistance from the wheat genome.

[0024] Through years of research, this invention has screened a new wheat stripe rust resistance gene from the wheat genome, and named it... TaJAZ9 The gene encodes the TaJAZ9 protein. Among its components: The amino acid sequence of the TaJAZ9 protein is shown in SEQ ID NO.1, as follows: MEGAGSGGKMSGGEGMAGRAGAGAASAAKEKSGFAATCSLLSRYMKEKKGGALQGLVGLDMSPPAAVVVGEGAFQPPTTMNLLSGLEEPNAADVELPLEKSSVGQFLKATTDNQD AGEDAHQLTIFYGGKVVVVDNFPSTKVKDLLQMADGAGDKTGSSSLAQQSPPQPAQNALPDLPIARRNSLHRFLEKRKGRIVAKAPYQINSSSAAPSKQANGDNSWLGLGQEVTN. TaJAZ9 The nucleotide sequence of the gene is shown in SEQ ID NO.2, as follows: .

[0025] This invention constructs TaJAZ9 The gene overexpression vector was transferred into common wheat Fielder via Agrobacterium-mediated transformation to obtain overexpression. TaJAZ9 Transgenic wheat lines containing the gene were then used to identify stripe rust resistance. Overexpression of the gene was found in these lines. TaJAZ9 The gene can significantly improve wheat resistance to stripe rust and can serve as a novel stripe rust resistance gene, hence this invention.

[0026] To enable those skilled in the art to better understand the technical solution of this application, the technical solution of this application will be described in detail below with reference to specific embodiments.

[0027] The test materials used in the embodiments of this invention are all conventional test materials in the art and can be purchased through commercial channels. Experimental methods without specified detailed conditions are performed according to conventional test methods or the supplier's recommended operating instructions. Wherein: The plant expression vector SVPC0349 uses the existing PC186 vector as its backbone, with a GFP tag (linked to UniProtKB / Swiss-Prot: P42212.1) added to the backbone, and is named SVPC0349. A schematic diagram of the structure of the plant expression vector SVPC0349 is shown below. Figure 1 As shown.

[0028] wheat stripe rust ( Puccinia striiformis f. sp. tritici The physiological race is CYR32, described in the journal article "Sequencing trait-associated mutations to clone wheat rust-resistancegene YrNAM, Nature Communications, 2023".

[0029] Example 1: Overexpression TaJAZ9 Construction of transgenic wheat lines 1. Constructing overexpression vectors: Using cDNA obtained by reverse transcription of RNA from common wheat Fielder leaves as a template, the cDNA was amplified using primers SVPC-JAZ9-GFP-F / SVPC-JAZ9-GFP-R. TaJAZ9 The encoding sequence was obtained, and homologous arms were added to both ends of the primers for subsequent homologous recombination.

[0030] SVPC-JAZ9-GFP-F: ATCTTAGATCACAAGTTTTGTACTAGTATGGAAGGGGCGGGG; (SEQ IDNO.3) SVPC-JAZ9-GFP-R:CGCCCGAGCCACCGCCACCACTAGTGTTTGTCACTTCTTGGCCTA. (SEQ IDNO.4) The plant expression vector SVPC0349 was linearized by digesting it with the SpeI restriction endonuclease to obtain a linearized vector.

[0031] Linkage via homologous recombination TaJAZ9 The fragment and linearized vector SVPC0349 were transformed into E. coli. After PCR and sequencing verification, the plasmid was extracted to obtain the successfully constructed overexpression vector, which was named PC1439. Its structural diagram is shown below. Figure 2As shown.

[0032] 2. Genetic transformation: The successfully constructed overexpression vector PC1439 was transformed into Agrobacterium tumefaciens EHA105 competent cells, and the wheat variety Fielder was transformed using Agrobacterium-mediated transformation to obtain T0 generation transgenic plants.

[0033] T0 generation transgenic plants were self-pollinated to obtain T1 generation transgenic plants. DNA from some T1 generation transgenic plants was extracted and PCR was performed using A26 / JIANDING-JAZ9-R. The expected amplified fragment size was 407 bp.

[0034] A26: TTTAGCCCTGCCTCATACG; (SEQ ID NO.5) JIANDING-JAZ9-R:CGTGCGCGGCGTTT. (SEQ ID NO.6) The results are as follows Figure 3 As shown, the results indicate that some plants amplified specific bands, suggesting that exogenous [genes / genes]... TaJAZ9 The gene has been successfully integrated into the wheat genome. Based on the identification results, three positive lines, Fielder#4, Fielder#9, and Fielder#14, were selected for subsequent disease resistance analysis.

[0035] Example 2: Overexpression TaJAZ9 Investigation of resistance of transgenic wheat lines to stripe rust 1. Test method: Fielder wheat and the overexpression constructed in Example 1 TaJAZ9 Transgenic wheat lines (Fielder#4, Fielder#9, and Fielder#14) were cultured in an artificial climate incubator under conditions of 15 ℃, 16 h light / 8 h dark. When the seedlings reached the 2-3 leaf stage, they were inoculated with wheat stripe rust fungus. Puccinia striiformis f. sp. tritici Physiological race CYR32. The inoculation method is as follows: Before inoculation, thoroughly mix stripe rust urediniospores with pine nut powder at a ratio of 1:15 (w / w). Use a small brush to apply the diluted spore mixture evenly to the surface of the first leaf of wheat seedlings for artificial inoculation, ensuring a consistent inoculation amount.

[0036] After inoculation, the plants were placed in an artificial climate chamber for 24 hours under the following conditions: 12 °C, darkness, and 100% relative humidity. After 24 hours, the conditions were adjusted to 12 °C, 16 hours of light / 8 hours of darkness, and the relative humidity was maintained at 100% until the inoculated leaves showed signs of chlorosis, at which point the humidity could be appropriately reduced. Phenotypic assessment was conducted 14 days after inoculation, and the phenotypic grade was evaluated using the internationally accepted nine-level classification standard (Table 1).

[0037] Table 1: Phenotypic Grades for Stripe Rust Identification 2. Test Results: The results are as follows Figure 4 As shown, Fielder plants exhibit a large number of uredinia on the leaf surface, displaying typical symptoms of infection, with a disease severity level of 9; while TaJAZ9 The number of spore masses on leaves of the overexpressing strains was significantly reduced, accompanied by a marked disease resistance response, and the disease severity was reduced to level 3-4. The results indicate that TaJAZ9 overexpression significantly improved wheat resistance to stripe rust (CYR32).

[0038] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. Overexpression in wheat TaJAZ9 The application of protein to enhance wheat resistance to stripe rust is characterized by, The TaJAZ9 The protein is shown in either (A1) or (A2) below: (A1) A protein with the amino acid sequence shown in SEQ ID NO.1; (A2) The protein obtained by attaching a protein tag to the N-terminus and / or C-terminus of the protein defined in (A1).

2. Overexpression in wheat TaJAZ9 The application of genes to enhance wheat resistance to stripe rust is characterized by, The TaJAZ9 Genes are nucleic acid molecules as shown in (i) or (ii) below: (i) Nucleic acid molecules with nucleotide sequences as shown in SEQ ID NO.2; (ii) Nucleic acid molecules other than (i) that encode the amino acid sequence shown in SEQ ID NO.

1.

3. The application according to claim 2, characterized in that, Overexpression in wheat TaJAZ9 Genes are generated through the following substances: C1) contains TaJAZ9 Gene expression cassettes; C2) contains TaJAZ9 Recombinant vectors of genes, or recombinant vectors containing the expression cassette described in C1); C3) contains TaJAZ9 Recombinant microorganisms containing genes, or recombinant microorganisms containing the expression cassette described in C1), or recombinant microorganisms containing the recombinant vector described in C2); C4) contains TaJAZ9 Transgenic wheat cell lines containing the gene, or transgenic wheat cell lines containing the expression cassette described in C1); C5) contains TaJAZ9 Transgenic wheat tissue containing the gene, or transgenic wheat tissue containing the expression cassette described in C1); C6) contains TaJAZ9 Wheat organs containing the gene, or transgenic wheat organs containing the expression cassette described in C1).

4. The application according to claim 2, characterized in that, Overexpression in wheat TaJAZ9 Genes were used to obtain plant materials resistant to wheat stripe rust.

5. The application according to claim 4, characterized in that, The plant materials resistant to wheat stripe rust include: seedlings, callus tissue, embryogenic tissue, plant cells, or transgenic lines.

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