Protein TaNPR3 and application thereof in regulation and control of plant disease resistance

Abstract

The invention discloses protein TaNPR3 and application thereof in regulation and control of plant disease resistance, and belongs to the technical field of biology. The invention aims to solve the technical problem of how to regulate and control the disease resistance of plants. The protein TaNPR3 disclosed by the invention is a protein of which the amino acid sequence is SEQ ID No. 3. Experiments show that compared with a wild type plant, the powdery mildew resistance of an overexpressed TaNPR3 strain (TaNPR3-OE) is reduced. Therefore, the TaNPR3 or the substance for regulating and controlling the expression of the coding gene of the TaNPR3 or the substance for regulating and controlling the activity or content of the TaNPR3 can be used for regulating and controlling the powdery mildew resistance of the plant. The plants with changed powdery mildew resistance can be used as breeding resources, and the cultivation process of the plants with powdery mildew resistance is accelerated.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to the protein TaNPR3 and its application in regulating plant disease resistance. Background Technology

[0002] Wheat powdery mildew is an airborne fungal disease that severely damages wheat production. The causative agent is *Erysiphe brucellae*, a member of the Poaceae family. Powdery mildew occurs in wheat-growing areas worldwide, causing yield losses of up to one-fifth annually, making it a significant global wheat disease. In China, powdery mildew mainly occurs in the wheat-growing regions of the middle and lower reaches of the Yangtze River, southwestern China, and the Huang-Huai-Hai Plain. *Erysiphe brucellae* can infect wheat throughout its entire growth cycle, primarily affecting the leaves, and in severe cases, infecting the stems and ears. This impairs photosynthesis and carbohydrate accumulation and transport, resulting in shriveled grains, reduced thousand-grain weight, and consequently, yield and quality losses. Therefore, continuously exploring and effectively utilizing new disease-resistant genes in wheat and its closely related species and genera, enriching the diversity of disease-resistant gene resources, expanding the genetic basis for disease-resistant breeding, and achieving disease-resistant gene diversification are of great significance for cultivating and planting wheat varieties with long-lasting and broad-spectrum disease resistance.

[0003] Powdery mildew infection in wheat not only triggers local defense responses but also induces the production of signals such as salicylic acid (SA). SA is an important plant defense hormone that promotes immunity against biotic and hemitrophic pathogens. It plays a crucial role in basic defense, amplification of local immune responses, and the establishment of systemic acquired resistance. After synthesis, SA requires specific signal transduction to transmit information about pathogen infection to the terminal immune response. Summary of the Invention

[0004] The technical problem to be solved by this invention is how to regulate the disease resistance of plants.

[0005] To address the aforementioned technical problems, the present invention first provides at least one of the following applications of proteins or substances that regulate the content or activity of said proteins: D1) Regulates plant disease resistance; D2) Prepare products that regulate plant disease resistance; D3) Cultivating plants with altered disease resistance; D4) Prepare products from plants with altered disease resistance; The protein is derived from wheat ( Triticum aestivum L.), its name is TaNPR3 protein, TaNPR3 protein is as follows A1), A2) or A3): A1) The amino acid sequence of this protein is SEQ ID No. 3; A2) A protein that has more than 98% identity with A1) and has the same function as the amino acid sequence shown in SEQ ID No. 3 in the sequence listing, after substitution and / or deletion and / or addition of amino acid residues; A3) is a fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of A1) or A2).

[0006] In the TaNPR3 protein mentioned in A2) above, the "more than 98% identity" refers to 98% or 99% identity. Identity refers to the identity of the amino acid sequence. The identity of the amino acid sequence can be determined using homology search sites on the Internet, such as the BLAST page on the NCBI homepage. For example, in Advanced BLAST 2.1, using blastp as the program, setting the Expect value to 10, setting all filters to OFF, using BLOSUM62 as the matrix, setting the Gap existence cost, Per residue gap cost, and Lambda ratio to 11, 1, and 0.85 (default values) respectively, and performing an identity search on a pair of amino acid sequences, the identity value (%) can then be obtained.

[0007] The TaNPR3 protein in A2 above can be synthesized artificially, or its encoding gene can be synthesized first and then expressed biologically.

[0008] The gene encoding the TaNPR3 protein in A2) above can be obtained by deleting one or more amino acid residues from the codons in the DNA sequence shown in SEQ ID No. 2, and / or by performing a missense mutation of one or more base pairs, and / or by attaching a tag coding sequence to its 5′ and / or 3′ ends. The DNA molecule shown in SEQ ID No. 2 encodes the TaNPR3 protein shown in SEQ ID No. 3.

[0009] The tag described in A3) can be a polypeptide or protein that is fused with the target protein using in vitro DNA recombination technology, so as to facilitate the expression, detection, tracing and / or purification of the target protein.

[0010] Specifically, the substance that regulates the content or activity of TaNPR3 protein is any one of the following B1) to B7): B1) Nucleic acid molecules encoding the TaNPR3 protein; B2) An expression cassette containing the nucleic acid molecule described in B1); B3) A recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2); B4) Recombinant microorganisms containing the nucleic acid molecules described in B1), or recombinant microorganisms containing the expression cassette described in B2), or recombinant microorganisms containing the recombinant vector described in B3); B5) A transgenic plant cell line containing the nucleic acid molecule described in B1), or a transgenic plant cell line containing the expression cassette described in B2); B6) Transgenic plant tissue containing the nucleic acid molecules described in B1), or transgenic plant tissue containing the expression cassette described in B2); B7) A transgenic plant organ containing the nucleic acid molecule described in B1), or a transgenic plant organ containing the expression cassette described in B2).

[0011] 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 mRNA or hnRNA.

[0012] Those skilled in the art can readily mutate the nucleotide sequence encoding the TaNPR3 protein of this invention using known methods, such as directed evolution and point mutation. Any artificially modified nucleotides that possess 75% or higher identity to the nucleotide sequence of the TaNPR3 protein isolated according to this invention, provided they encode and function the TaNPR3 protein, are derived from and equivalent to the nucleotide sequence of this invention.

[0013] Furthermore, B1) The nucleic acid molecule described is a DNA molecule whose coding sequence is SEQ ID No. 2 in the sequence listing.

[0014] Specifically, B1) refers to the nucleic acid molecule shown in SEQ ID No. 2, which is the DNA molecule.

[0015] Furthermore, the expression cassette (TaNPR3 gene expression cassette) containing a nucleic acid molecule encoding the TaNPR3 protein described in B2) refers to DNA capable of expressing the TaNPR3 protein in a host cell. This DNA may include not only a promoter to initiate TaNPR3 gene transcription but also a terminator to terminate TaNPR3 gene transcription. Furthermore, the expression cassette may also include an enhancer sequence.

[0016] Recombinant vectors containing the TaNPR3 gene expression cassette can be constructed using existing expression vectors.

[0017] Furthermore, the vector can be a plasmid, a granule, a bacteriophage, or a viral vector. Specifically, the plasmid can be the pTCK303 vector.

[0018] B3) The recombinant vector may specifically be pTCK303-TaNPR3. pTCK303-TaNPR3 is a vector that carries the double-stranded DNA molecule shown in SEQ ID No. 2 (i.e., TaNPR3 A circular recombinant plasmid containing a double-stranded DNA molecule as shown in SEQ ID No. 2 was obtained by inserting the gene into plasmid pTCK303.

[0019] Furthermore, the microorganisms may be yeast, bacteria, algae, or fungi. Among them, bacteria may be Agrobacterium, such as Agrobacterium EHA105.

[0020] Furthermore, the transgenic plant cell lines, transgenic plant tissues, and transgenic plant organs do not include propagation material.

[0021] Furthermore, the substance that regulates the content or activity of TaNPR3 protein is a substance that increases the content or activity of TaNPR3 protein, the substance that regulates plant disease resistance is a substance that reduces the plant disease resistance, and the substance that cultivates plants with altered disease resistance is a substance that cultivates plants with reduced disease resistance.

[0022] The present invention also provides any of the following methods: X1) A method for reducing plant disease resistance, comprising: expressing the protein described in claim 1 in the plant, or increasing the content or activity of TaNPR3 protein in the plant to reduce plant disease resistance; X2) Methods for cultivating plants with reduced disease resistance include: expressing TaNPR3 protein in plants, or increasing the content or activity of TaNPR3 protein in plants to obtain plants with reduced disease resistance.

[0023] Furthermore, the methods described in X1) and X2) are achieved by introducing the gene encoding the TaNPR3 protein into the plant and expressing the gene.

[0024] Specifically, the encoding gene may be the nucleic acid molecule described in B1).

[0025] The gene encoding the TaNPR3 protein can be introduced into the plant using a recombinant expression vector containing the gene encoding the TaNPR3 protein. Specifically, the recombinant expression vector can be pTCK303-TaNPR3.

[0026] The recombinant expression vector can be introduced into plant cells using conventional biotechnological methods such as Ti plasmids, plant virus vectors, direct DNA transformation, microinjection, and electroporation (Weissbach, 1998, Method for Plant Molecular Biology VIII, Academy Press, New York, pp.411-463; Geiserson and Corey, 1998, Plant Molecular Biology (2nd Edition)).

[0027] The target plant is understood to include not only first-generation plants containing the TaNPR3 protein or its encoding gene, but also their progeny. For the target plant, the gene can be propagated within the species, or it can be transferred into other varieties of the same species, particularly commercial varieties, using conventional breeding techniques. The target plant includes seeds, callus tissue, intact plants, and cells.

[0028] TaNPR3 protein or substances that regulate the content or activity of TaNPR3 protein are also within the scope of protection of this invention.

[0029] In this invention, the disease resistance is powdery mildew resistance. Further, the powdery mildew resistance is wheat powdery mildew fungus resistance.

[0030] The plant is M1, M2, or M3. M1) Monocotyledons; M2) Gramineae plants; M3) wheat.

[0031] Experimental results show that the TaNPR3 overexpression line (TaNPR3-OE) exhibits decreased powdery mildew resistance compared to wild-type plants. This indicates that TaNPR3, substances regulating the expression of the TaNPR3-encoding gene, or substances regulating TaNPR3 activity or content can be used to regulate powdery mildew resistance in plants. The powdery mildew-resistant plants produced by this invention can serve as breeding resources, accelerating the cultivation of powdery mildew-resistant plants. These powdery mildew-resistant plants can also be used to study which genes participate in the powdery mildew resistance response after TaNPR3 overexpression. This invention provides new germplasm resources for genetic breeding work, new materials for plant variety selection, and plays a positive role in accelerating the improvement of plant varieties. Attached Figure Description

[0032] Figure 1Figure 1: Yeast two-hybrid validation results of TaNPR3 and Pm41. DDO: Yeast two-lost plate (SD / -Trp-Leu solid selection medium); QDO: Yeast four-lost plate (DO / -His-Leu-Met-Trp solid medium).

[0033] Figure 2 Leaf phenotypes of each strain.

[0034] Figure 3 Statistical results of plaque area for each strain. Detailed Implementation

[0035] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0036] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials, reagents, instruments, etc., used in the following examples are commercially available.

[0037] In the quantitative experiments described below, at least three replicates were performed. Data from the following examples were processed using SPSS 11.5 statistical software, and a one-way ANOVA test was used. P < 0.05 (*) indicates a significant difference, and P < 0.01 (**) indicates a highly significant difference.

[0038] The wheat powdery mildew strain E09 described in the following examples (LU et al., A wheat tandem kinase and NLR pair confers resistance to multiple fungal pathogens, SCIENCE, 27 Mar 2025, Vol 387, Issue 6741, pp. 1418-1424, DOI: 10.1126 / science.adp5469) is available to the public from the applicant. This biological material is for the purpose of repeating the relevant experiments of the present invention only and shall not be used for any other purpose.

[0039] Table 1 Primers

[0040] Example 1: Interaction verification between Pm41 and TaNPR3 (1) Construction of recombinant vector The Pm41 and TaNPR3 genes were amplified by PCR. The amplified fragments were recovered using a PCR product purification and recovery kit from Beijing Adley Company and stored at -20℃ for later use. The Pm41 and TaNPR3 fragments and the target vectors pGBK-T7 and pGAD-T7 were double-digested at 37℃ using restriction endonucleases from NEB Company. The target fragments and vector fragments were purified and recovered. The Pm41 and TaNPR3 fragments and the vector backbone were ligated using T4 ligase to form recombinant plasmid vectors pGBK-T7-Pm41 and pGAD-T7-TaNPR3 (abbreviated as BD-Pm41 and AD-TaNPR3).

[0041] pGBK-T7-Pm41 is a recombinant vector obtained by inserting the Pm41 gene shown in SEQ ID No. 1 into a DNA fragment between the NdeⅠ recognition sequences in the pGBK-T7 vector.

[0042] pGAD-T7-TaNPR3 is a recombinant vector obtained by inserting the TaNPR3 gene shown in SEQ ID No. 2 into the DNA fragment between the NdeⅠ recognition sequences in the pGBK-T7 vector.

[0043] (2) Detection of the interaction between Pm41 and TaNPR3 ①Using plasmids PGBKT7-Lam Vector and PGADT7-T Vector as negative controls, and plasmids PGBKT7-53 Vector and PGADT7-T Vector as positive controls. Streak Y2H Gold yeast on YPDA plates and incubate upside down at 30℃ for about 3 days until the clone size is 2-3 mm; ② Pick one yeast clone and place it in 5 mL of YPDA liquid medium (50 mL sterile centrifuge tube); incubate overnight at 30°C with shaking. ③ Pipette 1 mL of bacterial culture into different 1.5 mL EP tubes, centrifuge at 4000-5000 rpm for 5 min to collect the bacterial cells, remove the supernatant, add 5-10 μL each of the plasmid containing the target fragment, the positive control plasmid, and the negative control plasmid, mix well, add 500 μL of yeast transformation mixture, pipette mix well, let stand at room temperature for 1 day, heat shock at 45℃ for 15 min, and place on ice for 15 min; ④ Centrifuge the transformed yeast culture at 4000 rpm for 5 min, remove 400 μL of supernatant, mix the remaining 100 μL by pipetting and spreading it on the corresponding defective culture medium, and incubate at 30℃ upside down for about 3 days. ⑤ Pick several single clones containing the target fragment and empty vector plasmid into 1.5 mL EP tubes containing 200 μL SD / -Trp-Leu liquid selection medium, seal with sealing film, and incubate overnight at 30℃ and 250 rpm. ⑥ Mix each bacterial culture by pipetting, then spot 1 μL onto a plate and incubate for 2-3 days before taking a photo and recording the results.

[0044] The results are as follows Figure 1 As shown, PGBKT7-Lam +PGADT7-T, PGBKT7-53+PGADT7-T, and BD-Pm41+AD-TaNPR3 were cloned on SD / -Trp-Leu solid selection medium; while BD-Pm41+AD-TaNPR3 and the positive control combination PGBKT7-53+PGADT7-T were cloned on DO / -His-Leu-Met-Trp solid medium. The experiment shows that Pm41 and TaNPR3 have an interaction relationship.

[0045] Example 2: Identification of powdery mildew resistance in TaNPR3 transgenic wheat 1. Construction of recombinant vectors Construction of pTCK303-TaNPR3: The double-stranded DNA molecule shown in SEQ ID No. 2 (i.e., TaNPR3 The gene was inserted into plasmid pTCK303 to obtain a circular recombinant plasmid with a double-stranded DNA molecule as shown in SEQ ID No. 2, which was named plasmid pTCK303-TaNPR3.

[0046] The plasmid pTCK303-TaNPR3 has been validated by sequencing. The plasmid pTCK303-TaNPR3 is started by the Ubi promoter. TaNPR3 Gene expression.

[0047] 2. Construction of the TaNPR3 overexpression line (TaNPR3-OE) 2.1 Plasmid pTCK303-TaNPR3 was introduced into Agrobacterium tumefaciens EHA105 to obtain recombinant Agrobacterium.

[0048] 2.2 The recombinant Agrobacterium obtained in step 2.1 was used to perform Agrobacterium-mediated genetic transformation on the embryogenic callus of wheat Fielder (the steps were: infection, co-culture, recovery culture, kanamycin resistance selection culture and rooting culture in sequence) to obtain rooted regenerated plants, which are the T0 generation plants.

[0049] 2.3. Screening transgenic plants from seedling T0 generation plants.

[0050] Screening method: Take leaves from plants with 4-5 leaves and extract genomic DNA; use the genomic DNA as a template and perform PCR amplification using primer pair composed of TaNPR3-OE-F and TaNPR3-OE-R (target product is about 440bp), and then perform agarose gel electrophoresis; if the electrophoresis shows the target band, the plant is a positive transgenic plant, and the obtained positive transgenic plant is the TaNPR3 overexpressing line TaNPR3-OE.

[0051] TaNPR3-OE-F (corresponding to) TaNPR3 Gene): 5'-AAGCAGACGTCACACCAGAA-3'; TaNPR3-OE-R (corresponding vector sequence): 5'-gtgtgcgcaatgaaactgat-3'.

[0052] 3. Phenotypic detection The wheat samples tested included: TaNPR3 overexpression lines (TaNPR3-OE), Pm41-COM, and wild-type wheat (Fielder). Pm41-COM was the Pm41 gene expression line. Following steps 1 and 2, the wheat was... TaNPR3 Positive lines were obtained by replacing the gene with the Pm41 gene.

[0053] To investigate whether there is a difference in resistance to powdery mildew between TaNPR3-overexpressing wheat lines (TaNPR3-OE) and wild-type wheat, leaves of wheat plants with good and consistent growth were inoculated with powdery mildew strain E09. Disease incidence was observed and recorded. The specific steps are as follows: ① The wheat to be tested was planted in culture pots in advance and cultured at 22℃ for 18 hours during the day and 20℃ for 8 hours at night.

[0054] ② One week later, the powdery mildew mycelium was transferred to the test wheat and cultured.

[0055] ③ After 4 days, phenotypic identification was performed and the area of ​​infected leaves by the pathogen was counted and analyzed using ImageJ.

[0056] See results Figures 2-3 .

[0057] The results showed that wild-type wheat exhibited mild disease four days after inoculation, Pm41-COM showed no disease, but TaNPR3-OE showed more severe disease. The leaf area infected by the pathogen differed significantly among the three types. The experimental results indicate that TaNPR3-OE exhibits decreased powdery mildew resistance compared to wild-type wheat. These results suggest that TaNPR3, or substances regulating the expression of the gene encoding TaNPR3, or substances regulating TaNPR3 activity or content, can be used to regulate powdery mildew resistance in wheat. The powdery mildew-resistant wheat produced by this invention can serve as a breeding resource, accelerating the development of powdery mildew-resistant wheat. The powdery mildew-resistant wheat produced by this invention can also be used to study which genes participate in the powdery mildew resistance response after TaNPR3 overexpression. This invention provides new germplasm resources for genetic breeding work, new materials for wheat variety selection, and plays a positive role in accelerating the improvement of wheat varieties.

[0058] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.

Claims

1. Use of at least one of the following: D1) modulating disease resistance of a plant; D2) producing a product for modulating disease resistance of a plant; D3) breeding a plant with altered disease resistance; D4) producing a product for breeding a plant with altered disease resistance; of a protein or a substance that modulates the content or activity of the protein, the protein being A1), A2) or A3): A1) a protein whose amino acid sequence is SEQ ID No. 3; A2) a protein having 98% or more identity with A1) and having the same function, by substitution and / or deletion and / or addition of amino acid residues to the amino acid sequence represented by SEQ ID No. 3 in the sequence listing; A3) a fusion protein obtained by linking a tag to the N terminus or / and C terminus of A1) or A2). the substance that modulates the content or activity of the protein being any one of B1) to B7):

2. Use according to claim 1, characterized in that: B1) a nucleic acid molecule encoding the protein; B2) an expression cassette containing the nucleic acid molecule of B1); B3) a recombinant vector containing the nucleic acid molecule of B1) or containing the expression cassette of B2); B4) a recombinant microorganism containing the nucleic acid molecule of B1) or containing the expression cassette of B2) or containing the recombinant vector of B3); B5) a transgenic plant cell line containing the nucleic acid molecule of B1) or containing the expression cassette of B2); B6) a transgenic plant tissue containing the nucleic acid molecule of B1) or containing the expression cassette of B2); B7) a transgenic plant organ containing the nucleic acid molecule of B1) or containing the expression cassette of B2). B1) the nucleic acid molecule is a DNA molecule whose coding sequence is SEQ ID No. 2 in the sequence listing.

3. Use according to claim 2, characterized in that: the substance that modulates the content or activity of the protein being a substance that increases the content or activity of the protein, the modulating disease resistance of a plant being decreasing disease resistance of the plant, the breeding a plant with altered disease resistance being breeding a plant with decreased disease resistance.

4. Use according to any one of claims 1 to 3, characterized in that: the disease resistance being powdery mildew resistance; 5. Use according to any one of claims 1 to 4, characterized in that: and / or, the plant being M1) or M2) or M3): M1) a monocotyledonous plant; M2) a plant of the family Poaceae; M3) wheat.

6. Any one of the following methods: X1) a method for decreasing disease resistance of a plant by expressing in the plant a protein as claimed in claim 1 or increasing the content or activity of a protein as claimed in claim 1 in the plant; X1 ) A method for reducing the disease resistance of a plant, comprising: X2) a method for breeding a plant with decreased disease resistance, comprising: expressing in the plant a protein as claimed in claim 1 or increasing the content or activity of a protein as claimed in claim 1 in the plant, to obtain a plant with decreased disease resistance. the method of X1) and X2) being achieved by introducing into the plant a gene encoding the protein and allowing the gene to be expressed.

7. The method of claim 6, wherein: the gene being the nucleic acid molecule of B1) of claim 2 or 3.

8. The method of claim 7, wherein: the disease resistance being powdery mildew resistance; 9. The method of any one of claims 6-8, wherein: and / or, the plant being M1) or M2) or M3): M1) a monocotyledonous plant; M2) a plant of the family Poaceae; M3) wheat. ​ 10. The protein of claim 1 or the agent modulating the content or activity of the protein of any one of claims 1-3.

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