Wheat calcium-dependent protein kinase TaCDPK1 gene and application of wheat calcium-dependent protein kinase TaCDPK1 gene in wheat powdery mildew resistance

Abstract

The invention discloses a wheat calcium-dependent protein kinase TaCDPK1 gene and application thereof in wheat powdery mildew resistance, and belongs to the technical field of plant genetic engineering. The invention verifies that the wheat TaCDPK1 gene has a clear and obvious positive regulation effect on wheat powdery mildew through functional experiments from a CDPK gene family with complex functions and numerous members for the first time. A real-time fluorescent quantitative PCR (Polymerase Chain Reaction) result shows that the TaCDPK1 gene is rapidly and specifically up-regulated and expressed after being infected by powdery mildew bacteria; a further overexpression genetic transformation experiment directly proves that the resistance of a susceptible wheat material to powdery mildew can be remarkably enhanced by improving the gene expression. The data fully verifies the effectiveness of the gene function and application, and lays a solid foundation for the subsequent practical application of the gene in molecular marker-assisted selection, transgenic breeding or gene editing breeding.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering technology, specifically relating to a wheat calcium-dependent protein kinase. TaCDPK1 Genes and their application in wheat resistance to powdery mildew. Background Technology

[0002] Wheat powdery mildew is caused by the wheat powdery mildew fungus ( Blumeria graminis f. sp. tritici Diseases caused by pathogens severely impact wheat yield and quality. Currently, production relies mainly on chemical fungicides and disease-resistant varieties for control. However, chemical control easily leads to environmental pollution and pathogen resistance, while traditional disease-resistant breeding often results in the loss of varietal resistance due to rapid mutation of pathogen races. Therefore, identifying key genes with broad-spectrum and durable disease resistance potential at the molecular level and using biotechnology for breeding improvement is an important strategy to ensure safe wheat production.

[0003] When plants face pathogen infection, they activate a complex network of immune signals. Among these, calcium ions (Ca²⁺) play a crucial role. + As a ubiquitous second messenger, transient changes in intracellular concentration are among the earliest events triggering defense responses. Calcium-dependent protein kinases (CDPKs or CPKs) are a unique class of plant protein kinases. Their structure contains both a serine / threonine kinase domain and a calcium-binding domain similar to calmodulin, enabling them to directly sense and decode specific calcium signals, which are then transmitted downstream through phosphorylation of target proteins. Studies have shown that the CDPK family plays a central role in plant responses to various biotic and abiotic stresses.

[0004] It is worth noting that CDPK is a large gene family. For example, in model plants such as rice and Arabidopsis thaliana, the CDPK family has dozens of members. Although these members are structurally similar, their gene expression exhibits high tissue specificity, developmental stage specificity, and response specificity to different stimuli, resulting in a high degree of functional diversity and specificity. Different members of the same CDPK family may participate in different signaling pathways: some have been shown to play a key role in drought stress response; some function in salt stress or low temperature stress response; and some have been reported to participate in immune responses to specific pathogens (such as bacteria, fungi, or oomycetes).

[0005] In wheat, although some studies suggest that CDPKs may be involved in the disease resistance process, accurately identifying key genes with significant positive regulatory functions against powdery mildew from the large and functionally diverse wheat CDPK family, and elucidating their application value, still presents significant uncertainties and technical obstacles. Therefore, accurately identifying key genes with clear positive resistance to wheat powdery mildew is not only of great scientific significance, but also a key technical problem that must be solved before applying them to molecular breeding. Summary of the Invention

[0006] In view of the above-mentioned prior art, the object of the present invention is a wheat calcium-dependent protein kinase. TaCDPK1 Genes and their application in wheat resistance to powdery mildew.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides wheat TaCDPK1 The application of genes in the following (1) or (2): (1) Improve wheat's resistance to powdery mildew; (2) Develop wheat varieties resistant to powdery mildew; The wheat TaCDPK1 A gene is a DNA molecule as shown in any of the following (I)-IV): I) The nucleotide sequence is the DNA molecule shown in SEQ ID NO.1, as follows: SEQ ID NO.1:

[0008] II) The nucleotide sequence is the DNA molecule (CDS coding region) shown in SEQ ID NO.2, as follows: SEQ ID NO.2:

[0009] III) DNA molecules encoding the amino acid sequence shown in SEQ ID NO.3, except for i); IV) A DNA molecule that has 80% or more identity with the DNA fragment defined in i) or ii) and encodes a protein that is functionally equivalent to the protein shown in SEQ ID NO.3.

[0010] Preferably, by promoting wheat TaCDPK1 Gene expression is used to enhance wheat's resistance to powdery mildew.

[0011] Preferably, the wheat-promoting TaCDPK1 The substance used for gene expression is any one of the following: C1) contains TaCDPK1 Gene expression cassettes; C2) contains TaCDPK1 Recombinant vectors of genes, or recombinant vectors containing the expression cassette described in C1); C3) contains TaCDPK1 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 TaCDPK1 Transgenic plant cell lines containing the gene, or transgenic plant cell lines containing the expression cassette described in C1); C5) contains TaCDPK1 Transgenic plant tissue containing the gene, or transgenic plant tissue containing the expression cassette described in C1); C6) contains TaCDPK1 Transgenic plant organs containing genes, or transgenic plant organs containing the expression cassette described in C1).

[0012] In a second aspect, the present invention provides wheat TaCDPK1 The application of gene-encoded proteins in improving wheat resistance to powdery mildew, wherein the wheat TaCDPK1 The proteins encoded by the gene are shown in (A1) or (A2) below: (A1) A protein consisting of the amino acid sequence shown in SEQ ID NO.3 of the sequence listing, as follows: SEQ ID NO.3: MEDVRATYTFGRELGRGQFGVTYLATHKSTGARYACKSIAARKLARADDVEDARREVQIMHHLTGHRSIVELRGAYEDRHSVNLVMELCEGGELFDRIIARGHYSERAAATLCRE VVSVVHSCHSMGVMHRDLKPENFLFLNKREDSPLKATDFGLSVFFKPGEQFRDLVGSAYYVAPEVLKRRYGAEADIWSAGVILYILLSGVPPFWAENEEGIFDAVLKGHIDFSSD PWPSISNGAKDLVKRMLRQDPKERLTAAEILNHPWIREDGEAPDKPLDITVISRMKQFRAMNKLKKVALKIVAESLSEEEIVGLREMFKSLDTDNSGTITLDELRAGLPKLGTKI TESEIRQLMEAADVDGNGTIDYVEFISATMHMNRLEKEDHIFKAFEYFDKDHSGYITVDELEEALKKYDMGDEATIKDIIAEVDTDHDGKINYQEFVAMMKNNSPEIVPNRRRLF.

[0013] (A2) The protein obtained by attaching a protein tag to the N-terminus and / or C-terminus of the protein defined in (A1).

[0014] In a third aspect, the present invention provides a product containing wheat TaCDPK1 Application of recombinant gene expression vectors in improving wheat resistance to powdery mildew, the wheat TaCDPK1 The gene nucleotide sequence is shown in SEQ ID NO: 1 or SEQ ID NO: 2.

[0015] In a fourth aspect, the present invention provides a method for improving wheat resistance to powdery mildew by increasing the concentration of certain components in the target wheat material. TaCDPK1 Gene expression levels may increase TaCDPK1 The activity of gene-encoded proteins is used to enhance wheat's resistance to powdery mildew.

[0016] Preferably, the improvement in the target wheat material TaCDPK1 The gene expression level is achieved through gene editing, which targets the promoter or coding region of the gene to enhance its transcriptional activity.

[0017] In a fifth aspect, the present invention provides a method for breeding wheat varieties or lines resistant to powdery mildew, comprising the following steps: (a) Obtaining through genetic transformation or gene editing technologies TaCDPK1 underlying causeWheat plants with increased expression levels or protein activity; (b) Identify the powdery mildew resistance phenotype of the wheat plants obtained in step (a); (c) Select plants with significantly enhanced resistance to powdery mildew and use them for breeding to obtain wheat varieties or lines resistant to powdery mildew.

[0018] The beneficial effects of this invention are: This invention is the first to functionally validate wheat genes from the complex and numerous CDPK gene family through experiments. TaCDPK1 The gene has a clear and significant positive regulatory effect on wheat powdery mildew. Real-time quantitative PCR results show that... TaCDPK1 The gene was rapidly and specifically upregulated after infection with powdery mildew fungus; further overexpression genetic transformation experiments directly demonstrated that increasing gene expression significantly enhanced the resistance of susceptible wheat materials to powdery mildew. These data fully validated the gene's function and the effectiveness of its application, laying a solid foundation for its subsequent practical application in molecular marker-assisted selection, transgenic breeding, or gene editing breeding. Attached Figure Description

[0019] Picture 1 After inoculating wheat with powdery mildew virus race E09 TaCDPK1 Analysis of the relative transcriptional level of genes.

[0020] Picture 2 For overexpression TaCDPK1 The gene enhanced Fielder's resistance to powdery mildew. Detailed Implementation

[0021] 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.

[0022] The specific embodiments of the present invention will be described in further detail below with reference to examples. The following detailed descriptions are illustrative and intended to provide further explanation of this application, rather than limiting the scope of the invention.

[0023] Example 1: Wheat TaCDPK1 Gene cloning and sequence analysis Young leaves of the wheat variety 'Chinese Spring' were used as material. Genomic DNA was extracted using the CTAB method. Based on publicly available wheat genome database information and homology alignment, specific primers were designed and synthesized for amplification. TaCDPK1 underlying cause .

[0024] Primer sequences: TaCDPK1-F (SEQ ID NO: 4): ATGGAGGACGTCCGCGCCAC; TaCDPK1 -R (SEQ ID NO: 5): AAATAGGCGCCGTCGATTTGGAACAATC.

[0025] PCR products were detected by 1% agarose gel electrophoresis, and the target band was recovered by gel extraction using a DNA gel extraction kit. The sequenced sequences were compared with reference sequences in a database to ultimately determine the sequence involved in this invention. TaCDPK1 The full-length nucleotide sequence of the gene is shown in SEQ ID NO: 1; its coding region (CDS) sequence is shown in SEQ ID NO: 2; and the amino acid sequence of the protein it encodes is shown in SEQ ID NO: 3.

[0026] Example 2: Powdery mildew infection-induced TaCDPK1 underlying cause Expression pattern analysis After the second leaf of wheat Fielder seedlings unfolds, inoculate with the powdery mildew pathogenic race E09. The specific steps are as follows: Gently brush fresh conidia from the diseased wheat leaves with a sterile brush, collect them in sterile water containing 0.05% Tween-20, and magnetically stir for 5 minutes to prepare a homogeneous spore suspension. Count the spores using a hemocytometer, and adjust the spore concentration of the suspension to 3 × 10⁻⁶. 6 Apply the solution at spores / mL and then evenly onto the first leaf of the seedling. After inoculation, place the seedlings in an incubator at 18–22℃.

[0027] Samples were taken at 0, 12, 24, 48, 72, and 96 hours after inoculation. For sampling, approximately 2 cm long leaves inoculated with powdery mildew were cut, wrapped in aluminum foil, flash-frozen in liquid nitrogen, and then stored at -80°C for later use. Total RNA was extracted from wheat leaves using the Trizol method (TianGen) and reverse transcribed using First-strand cDNA Synthesis Mix. qRT-PCR was then used for detection. TaCDPK1 The amount of expression.

[0028] The qRT-PCR primer sequences are: TaCDPK1 -RT-F (SEQ ID NO: 6):GGAAGAGATCGTGGGCTTAAGAGA; TaCDPK1 -RT-R (SEQ ID NO: 7): CATCATGATCTGTATCCACTTCAGCAATG.

[0029] Ta18S-rRNA-RT-F (SEQ ID NO: 8): GTGACGGGGTGACGGAGAATT; Ta18S-rRNA-RT-R (SEQ ID NO: 9): GACACTAATGCGCCCGGTAT.

[0030] Ta18S-rRNA was used as an internal reference gene in real-time PCR analysis. The results of qRT-PCR are as follows: Picture 1 As shown, the results indicate that compared to 0 hpi, TaCDPK1 Gene expression began to significantly upregulate 12 hours after inoculation with powdery mildew fungus E09, peaking between 24 and 48 hours, then slightly decreasing but remaining at a high level. This result demonstrates... TaCDPK1 The gene can be specifically and rapidly induced to express by powdery mildew, suggesting its involvement in the early defense response of wheat against powdery mildew.

[0031] Example 3: Overexpression TaCDPK1 underlying cause Functional verification of enhancing wheat resistance to powdery mildew 1. Construction of plant overexpression vector: using the vector obtained in Example 1. TaCDPK1 Using the gene CDS sequence (SEQ ID NO:2) as a template, specific primers with recognition sites for the restriction endonucleases BamHI and HindIII were designed and synthesized. The target fragment was obtained by PCR amplification, purified, and then double-digested with BamHI and HindIII on both the target fragment and the pCambia3301 empty vector. The digestion products were then purified. TaCDPK1 The CDS gene was directionally cloned downstream of the ZmUBI promoter in the pCambia3301 vector, successfully constructing a plant overexpression recombinant vector.

[0032] The primer sequences involved are as follows: 3301-CDPK1-BamHI-F (SEQ ID NO: 10): TCTGCAGCCCGGGGATCCATGGAGGACGTCCGCGCCAC; 3301-CDPK1-HindIII-R (SEQ ID NO: 11): TGTAATTCACACGTGTCAAAATAGGCGCCGTCGATTTGGAACAATC.

[0033] 2. Wheat genetic transformation: The recombinant overexpression vector was introduced into embryogenic callus of the susceptible wheat variety 'Fielder' using Agrobacterium-mediated transformation. The callus was induced to differentiate into seedlings, and the transformed wheat plants were planted in a greenhouse. Overexpression lines with significantly higher expression levels than WT were screened by PCR amplification and sequencing. TaCDPK1 -OE).

[0034] Will TaCDPK1 Overexpression lines ( TaCDPK1 -OE) and the control Fielder were grown in a greenhouse under 16 hours of light and 8 hours of darkness, and cultured at 22°C for 10 days. They were inoculated with powdery mildew E09 using the same method as in Example 2. Disease resistance phenotypes were observed after 14 days. Picture 2 As shown, compared to WT'Fielder', overexpression TaCDPK1 The number of spore masses on the leaves of the gene-overexpressing strains was significantly reduced, and mycelial growth was markedly inhibited. This indicates that overexpression... TaCDPK1 The gene can significantly enhance the resistance of susceptible wheat varieties to powdery mildew.

[0035] The above description is merely a preferred embodiment of this application and is not intended to limit the application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications made within the spirit and principles of this application are not permitted. Equivalent substitutions and improvements should all be included within the scope of protection of this application.

Claims

1. Wheat TaCDPK1 Use of the gene in (1) or (2) below: (1) improving the resistance of wheat to powdery mildew; (2) breeding wheat varieties resistant to powdery mildew; The wheat TaCDPK1 The gene is a DNA molecule according to any one of I) to IV): I) a DNA molecule with the nucleotide sequence shown in SEQ ID NO. 1; II) a DNA molecule with the nucleotide sequence shown in SEQ ID NO. 2; III) a DNA molecule encoding the amino acid sequence shown in SEQ ID NO. 3, except for I); IV) a DNA molecule with 80% or more identity to the DNA fragment defined in I) or II), and encoding a protein functionally equivalent to the protein shown in SEQ ID NO.

3.

2. Use according to claim 1, characterized in that, By promoting expression of the wheat TaCDPK1 gene, the resistance of the wheat to powdery mildew is increased.

3. Use according to claim 2, characterized in that, The promoting wheat TaCDPK1 The substance for promoting gene expression is any one of the following: C1) contains TaCDPK1 expression cassette of a gene; C2) comprises TaCDPK1 a recombinant vector of a gene, or a recombinant vector comprising the expression cassette of C1). C3) a recombinant microorganism comprising TaCDPK1 a recombinant microorganism comprising the expression cassette of C1), or a recombinant microorganism comprising the recombinant vector of C2). C4) a transgenic plant cell line comprising TaCDPK1 a transgenic plant cell line of a gene, or a transgenic plant cell line comprising C1) the expression cassette; C5) a transgenic plant tissue comprising TaCDPK1 a transgenic plant tissue of a gene, or a transgenic plant tissue comprising C1) the expression cassette; C6) a transgenic plant organ comprising TaCDPK1 a transgenic plant organ of a gene, or a transgenic plant organ comprising C1) the expression cassette.

4. Wheat TaCDPK1 The use of a gene encoding a protein in increasing the resistance of wheat to powdery mildew, characterized in that, The wheat TaCDPK1 The protein encoded by the gene is a protein as shown in (Al) or (A2) below: (A1) a protein consisting of the amino acid sequence shown in SEQ ID NO. 3; (A2) a protein obtained by linking a protein tag to the N-terminus and / or C-terminus of the protein defined in (A1).

5. A wheat plant comprising TaCDPK1 The use of a recombinant expression vector comprising a wheat The wheat TaCDPK1 The nucleotide sequence of the gene is set forth in SEQ ID NO: 1 or SEQ ID NO:

2.

6. A method of increasing the resistance of wheat to powdery mildew, characterized in that, By increasing the content of target wheat materials TaCDPK1 Gene expression levels may increase TaCDPK1 The activity of gene-encoded proteins is used to enhance wheat's resistance to powdery mildew.

7. The method of claim 6, wherein, in a target wheat material TaCDPK1 in a manner that increases the expression level of a gene, the gene editing being directed to a promoter region or a coding region of the gene to enhance its transcriptional activity.

8. A method of breeding a wheat variety or line that is resistant to powdery mildew, characterized by, comprising the following steps: (a) obtaining by genetic transformation or gene editing techniques TaCDPK1 gene wheat plants with increased expression levels or protein activity; (b) identifying the phenotype of the wheat plants obtained in step (a) for resistance to powdery mildew; (c) selecting plants with significantly enhanced resistance to powdery mildew, and using them for breeding to obtain wheat varieties or lines resistant to powdery mildew.

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