Application of wheat receptor-like cytoplasmic kinase taRLCK118 or its coding gene in improving crop resistance to stripe rust

Abstract

This invention belongs to the field of plant genetic engineering technology, specifically relating to the application of wheat receptor cytoplasmic kinase TaRLCK118 or its encoding gene in improving crop stripe rust resistance. This invention was verified using reverse genetics methods. TaRLCK118 Genes play a positive regulatory role in wheat resistance to stripe rust, and can further enhance resistance in recipient crops. TaRLCK118 The gene expression increases the expression level of wheat receptor cytoplasmic kinase TaRLCK118, thereby enhancing the crop's resistance to stripe rust and providing genetic resources and biological materials for the breeding of stripe rust-resistant wheat varieties.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering technology, specifically relating to the application of wheat receptor cytoplasmic kinase TaRLCK118 or its encoding gene in improving crop stripe rust resistance. Background Technology

[0002] Wheat stripe rust is caused by the rust fungus *Strombus styracifolius* (Styracifolius styracifolius). Puccinia striiformis f. sp. tritici, Pst Stripe rust is one of the fungal diseases affecting wheat production. However, due to the high variability of stripe rust fungi, wheat often loses its resistance, leading to frequent and devastating outbreaks. Gene-assisted breeding of wheat rust is an important means to improve wheat rust resistance. Therefore, it is urgent to explore and discover new types of resistance-related genes, analyze their mediated rust resistance mechanisms, and provide important gene resources and theoretical basis for the rational utilization of disease resistance and wheat disease-resistant breeding.

[0003] Protein kinases (RLCKs) are a class of enzymes that catalyze the phosphorylation of specific substrate proteins. By transferring the γ-phosphate group of adenosine triphosphate (ATP) to a specific amino acid of the substrate protein, they influence the substrate protein's localization, structure, and activity, thereby transducing and regulating corresponding signals. They play important roles in many processes in plants, including growth, metabolism, differentiation, and immunity. Cytoplasmic receptor kinases (RLCKs) in plants are a class of protein kinases containing only intracellular kinase domains and lacking extracellular and transmembrane domains. They play important functions in growth, development, and various stress responses. RLCKs act as switches for intracellular signal regulation through phosphorylation modification. Phosphorylation of substrate proteins may affect their activity, stability, or subcellular localization, thereby precisely regulating various biological processes. However, there are no reports on cytoplasmic receptor kinases that exert resistance to stripe rust in wheat. Summary of the Invention

[0004] The purpose of this invention is to provide the application of wheat receptor cytoplasmic kinase TaRLCK118 or its encoding gene in improving crop resistance to stripe rust, and to provide genetic resources and technical support for the breeding of stripe rust resistant crop varieties.

[0005] This invention provides the application of wheat receptor cytoplasmic kinase TaRLCK118 or its encoding gene in improving crop resistance to stripe rust and / or breeding of stripe rust-resistant crops, wherein the amino acid sequence of wheat receptor cytoplasmic kinase TaRLCK118 is shown in SEQ ID NO:1.

[0006] Preferably, the method for improving crop resistance to stripe rust and / or breeding stripe rust-resistant crops includes: introducing the encoding gene of the wheat receptor cytoplasmic kinase TaRLCK118 into the recipient crop.

[0007] Preferably, the method for introducing the coding gene of wheat receptor cytoplasmic kinase TaRLCK118 into crops includes transgenic technology.

[0008] Preferably, the biological materials used in the transgenic technology include: a recombinant vector containing the encoding gene of the wheat receptor cytokinase TaRLCK118, and / or a transformant containing the encoding gene of the wheat receptor cytokinase TaRLCK118 or the recombinant vector.

[0009] Preferably, the transformant includes engineered bacteria, transgenic plant cell lines, or transgenic plant tissues.

[0010] Preferably, the base vector in the recombinant vector includes a plasmid vector.

[0011] Preferably, the basic strain of the engineered bacteria is Agrobacterium.

[0012] This invention also provides a method for breeding stripe rust-resistant crops, comprising: increasing the expression level of wheat receptor cytokinase TaRLCK118 in the recipient crop or increasing the expression level of... TaRLCK118 Gene expression;

[0013] The TaRLCK118 The gene encodes wheat receptor cytokinase TaRLCK118, the amino acid sequence of which is shown in SEQ ID NO:1.

[0014] Preferably, the method involves increasing the expression level of wheat receptor cytokinase TaRLCK118 in the recipient crop or increasing the expression level of the recipient crop... TaRLCK118 Methods of gene expression include: TaRLCK118 Genes are introduced into recipient crops using transgenic technology.

[0015] Preferably, the crop includes wheat.

[0016] Beneficial effects:

[0017] This invention provides the application of wheat receptor cytoplasmic kinase TaRLCK118 or its encoding gene in improving crop stripe rust resistance. This invention verifies the results using reverse genetics methods. TaRLCK118 Genes play a positive regulatory role in wheat resistance to stripe rust, and can further enhance resistance in recipient crops. TaRLCK118 The gene expression increases the expression level of wheat receptor cytoplasmic kinase TaRLCK118, thereby enhancing the crop's resistance to stripe rust and providing genetic resources and biological materials for the breeding of stripe rust-resistant wheat varieties. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0019] Figure 1 This is a flowchart illustrating the application of wheat receptor cytoplasmic kinase TaRLCK118 and its encoding gene in the improvement of wheat rust-resistant varieties.

[0020] Figure 2 For example, qRT-PCR detection in Example 2 TaRLCK118 The induced expression of genes during wheat infection by stripe rust fungus, among which express P ≤0.001, express P ≤0.01;

[0021] Figure 3 The Agrobacterium-mediated transformation into wheat embryos in Example 3 includes TaRLCK118 Gene expression vectors;

[0022] Figure 4 The T0 generation obtained in Example 3 TaRLCK118 -OE wheat plants;

[0023] Figure 5 In Example 3 TaRLCK118 Structural diagram for molecular identification of gene-overexpressing plants;

[0024] Figure 6 In Example 4 TaRLCK118 - Results of stripe rust biomass assay 14 days after inoculation with CYR31 by OE and Fielder, including express P ≤0.05;

[0025] Figure 7 In Example 4 TaRLCK118 Phenotypic results of OE and Fielder 14 days after inoculation with stripe rust fungus CYR31;

[0026] Figure 8 VIGS-mediated in Example 5 TaRLCK118 The momentary silence weakened wheat's resistance to stripe rust. (Results diagram) Detailed Implementation

[0027] This invention provides the application of wheat receptor cytoplasmic kinase TaRLCK118 or its encoding gene in improving crop resistance to stripe rust and / or breeding of stripe rust-resistant crops, wherein the amino acid sequence of wheat receptor cytoplasmic kinase TaRLCK118 is shown in SEQ ID NO:1.

[0028] As one implementation method, the gene for the wheat receptor cytokinase TaRLCK118 is... TaRLCK118 The TaRLCK118 The nucleotide sequence of the gene is shown in SEQ ID NO:2.

[0029] In this invention, the sequences represented by SEQ ID NO:1 to SEQ ID NO:2 are as follows:

[0030] SEQ ID NO:1:MGNCWGTRIKDGSTHPGASGMFSRGSGKDGSRLSACSSRASSASMPPSAKTECEILQSANVKVFSYNDLRLATRNFRPDSVLGEGGFGSVYKGWIDEHTLSA CKPGTGIPVAVKRLNLEGLQGHREWLAEVNYLGQFCHTNLVKLIGYCLEDEHRLLVYECMPRGSLENHLFRRGSHFQPLSWNLRMKVALGAAKGLAYLHSAEAKVIYR DFKTSNILLDTDYTAKLSDFGLAKDGPVGEKSHVSTRVMGTHGYAAPEYLSTGHLTAKSDIYSFGVVLLEMLSGRRAIDKNRPQGEHNLVEWARPYLTHKRKIFRVLD TRLEGQYSLNGAQTIAALAVECLSFEAKMRPSMDAVVSILEGIQDSSDPARRPADPTRRPAAERPQDPKSGSKTAPGASNSGKGRRKSSGDLLKEPGRDPKPSAYSS;

[0031]

[0032] As one embodiment, the method for improving crop resistance to stripe rust and / or breeding stripe rust-resistant crops according to the present invention includes: introducing the coding gene of wheat receptor cytokinase TaRLCK118 into a recipient crop; further, the coding gene of wheat receptor cytokinase TaRLCK118 can be introduced into the recipient crop through transgenic technology; even further, a recombinant vector containing the coding gene of wheat receptor cytokinase TaRLCK118 can be constructed, and the recombinant vector can be transferred into the recipient crop via Agrobacterium-mediated transformation to obtain a transgenic crop containing the coding gene of wheat receptor cytokinase TaRLCK118. As one embodiment, the crop can be a monocotyledonous plant, a gramineous plant, or wheat; when the crop is wheat, the recombinant vector containing the coding gene of wheat receptor cytokinase TaRLCK118 is transferred into the wheat embryo to obtain transgenic wheat.

[0033] In one embodiment, the biological material used in the transgenic technology includes: a recombinant vector containing the coding gene for the wheat receptor cytokinase TaRLCK118, and / or a transformant containing the coding gene for the wheat receptor cytokinase TaRLCK118 or the recombinant vector; in another embodiment, the transformant can be an engineered bacterium, a transgenic plant cell line, or a transgenic plant tissue; the transgenic plant cell line and transgenic plant tissue are non-stable genetically derived transgenic plant cell lines or tissues. In one embodiment, the base vector is a plasmid vector, more specifically, the pANIC6E vector. In one embodiment, the base strain of the engineered bacterium is Agrobacterium. This invention does not specifically limit the method for constructing the recombinant vector, and employs conventional methods for constructing recombinant vectors in the art.

[0034] This invention also provides a method for breeding stripe rust-resistant crops, comprising: increasing the expression level of wheat receptor cytokinase TaRLCK118 in the recipient crop or increasing the expression level of... TaRLCK118 Gene expression; the aforementioned TaRLCK118 The gene encodes wheat receptor cytokinase TaRLCK118, the amino acid sequence of which is shown in SEQ ID NO:1.

[0035] As one implementation method, the method involves increasing the expression level of wheat receptor cytokinase TaRLCK118 in the recipient crop or increasing the expression level of the recipient crop... TaRLCK118 Methods of gene expression include: TaRLCK118The gene is introduced into the recipient crop via transgenic technology, specifically by introducing the nucleotide sequence shown in SEQ ID NO:2 into the recipient crop. As one implementation, the crop can be a monocotyledonous plant, a grass, or more specifically, wheat.

[0036] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0037] The present invention is carried out in accordance with the following embodiments Figure 1 Methodological Research in Flowcharts TaRLCK118 The function of genes, and at the same time, the creation of transgenic wheat materials resistant to stripe rust.

[0038] Example 1

[0039] TaRLCK118 Gene cloning and sequence analysis

[0040] Cloning was performed on wheat variety “Suwon 11” using primers shown in SEQ ID NO:3 and SEQ ID NO:4. TaRLCK118 The full-length encoded sequence is shown in SEQ ID NO:2.

[0041] Forward primer: TaRLCK118-ORF-F: 5'-ATGGGGAACTGCTGGGGC-3' (SEQ ID NO:3) and reverse primer: TaRLCK118-ORF-R: 5'-TACGACGAGTAGGCCGACGG-3' (SEQ ID NO:4).

[0042] Sequence alignment showed that the above gene had the highest similarity to the gene with accession number TraesCS5D02G493800.1 in the EnsemblPlants (https: / / plants.ensembl.org / index.html) database, which encodes a typical receptor-like cytoplasmic kinase, and the amino acid sequence is shown in SEQ ID NO:1.

[0043] Example 2

[0044] TaRLCK118 Gene expression patterns during wheat infection by stripe rust fungus

[0045] Using the wheat variety "Fielder" as material, wheat was inoculated with stripe rust-compatible race CYR31 and incompatible race CYR23 (both from the Plant Immunology Research Team of Northwest A&F University) at the two-leaf-one-heart stage. Samples were taken at 0 hpi (hour post inoculation), 6 hpi, 12 hpi, 24 hpi, 48 hpi, 72 hpi, 96 hpi and 120 hpi for analysis.

[0046] Based on qRT-PCR technology, using the elongation factor gene TaEF1-α For internal reference, using TaRLCK118 Real-time quantitative PCR was performed using gene-specific primers, combined with 3 biological replicates and 2... -△△Ct Legal analysis TaRLCK118 Gene expression patterns at different time points in wheat infected with stripe rust fungus.

[0047] in TaRLCK118 Quantitative primers and internal reference genes for gene sequencing TaEF1-α The quantitative primer sequences are as follows:

[0048] Quantitative primers: Forward primers: TaRLCK118 -qRT-F: 5'-GCAAAGGTCGCCGTAA-3' (SEQ ID NO: 5), reverse primer: TaRLCK118 -qRT-R: 5'-CTCCAATGCCAATAGTTCA-3' (SEQ ID NO: 6).

[0049] Internal reference primer: Forward primer: TaEF1α -F: 5'-TGGTGTCATCAAGCCTGGTATGGT-3' (SEQ ID NO: 7), reverse primer: TaEF1α -R: 5'-ACTCATGGTGCATCTCAACGGACT-3' (SEQ ID NO: 8).

[0050] qRT-PCR analysis showed that ( Figure 2 ), TaRLCK118 Gene expression was significantly upregulated 18 hpi after infection with the incompatible race (CYR23) of stripe rust, peaking at a 15-fold increase. Expression changes were not significant in infection with the compatible race (CYR31) of stripe rust, indicating its involvement in specific disease resistance responses.

[0051] Example 3

[0052] Creation and Disease Resistance Verification of Transgenic Plants

[0053] The pANIC6E vector (published in the following literature [Mann et al. 2012, PlantBiotechnology Journal, Gateway-compatible vectors for high-throughput genefunctional analysis in switchgrass)) was used. Panicum virgatum L.) and other monocotspecies) as the skeletal carrier, will TaRLCK118 The coding sequence of the gene (nucleotide sequence shown in SEQ ID NO.2) is inserted into the multiple cloning site of the vector to construct... TaRLCK118 -pANIC6E overexpression vector, such as Figure 3 As shown.

[0054] Specific methods for constructing recombinant plasmids:

[0055] use TaRLCK118 Primers for overexpression vector construction to amplify gene fragments: Primers for overexpression vector construction are as follows: TaRLCK118 -6E-F: 5'-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGGGGAACTGCTGGGGC-3' (SEQ IDNO: 9), reverse primer: TaRLCK118 -6E-R: 5'-GGGGACCACTTTGTACAAGAAAGCTGGGTCCTACGACGAGTAGGCCGACGG-3' (SEQ ID NO: 10).

[0056] Using the BP reaction in Gateway cloning technology, TaRLCK118 The coding sequence of the gene was ligated into the pDONR221 intermediate vector. The reaction mixture consisted of 4.0 μL of the gene fragment, 0.5 μL of BP enzyme, 2.0 μL of the pDONR221 intermediate vector, and buffer 1×TE to a final volume of 10 μL. After ligation at 25°C for 12 h, the ligation was performed on *E. coli* DH5α. The successfully ligated sequence was then... TaRLCK118 -pDONR221 is linked to the final loading pANIC6E via an LR reaction, and the reaction system is as follows: TaRLCK1181.5 μL of pDONR221, 1.0 μL of pANICE6E vector, 0.5 μL of LR enzyme, and 1×TE buffer were added to bring the total volume to 5 μL. After ligation at 25°C for 12 h, the mixture was transformed into *E. coli* DH5α. The pDONR221 vector used in this invention is the standard pDonor221 plasmid from the Gateway recombinant cloning system of Thermo Fisher Scientific, and is commercially available.

[0057] Through the Agrobacterium-mediated genetic transformation system TaRLCK118 -pANIC6E overexpression vector ( Figure 3 (As shown) Overexpression lines were obtained by transforming wheat embryos. TaRLCK118 -OE). Select positive single clones and place them in 10 mL of MGL (with added biotin) medium, incubate at 180 rpm for 24 h. The treatment method for Agrobacterium is as described in [Ishida, Y.; Tsunashima, M.; Hiei, Y.; Komari, T. Wheat ( Triticum aestivum [L.) Transformation Using Immature Embryos. Methods Mol Biol. 2015. 1223, 189-198.] This study describes Agrobacterium-mediated transformation of immature wheat embryos. Ears of the Fielder wheat variety, 14 days after flowering, were carefully removed from the ears. Immature embryos were then selected after surface sterilization. The transformation method and culture medium preparation followed the methods described in the aforementioned literature. After screening and differentiation using herbicides, the embryos were rooted and then transplanted into the substrate to obtain the transformed embryos. TaRLCK118 Gene-regenerated plants, Figure 4 Phenotypic diagram of the obtained transgenic wheat T0 generation plants.

[0058] Molecular analysis was performed on the obtained transgenic wheat T0 generation, and the results are as follows: Figure 5 As shown, lane 1 on the right is designated as lane 1, and so on from right to left. Lane 1 represents Marker DL2000; lanes 2-3 represent the wild-type control and plasmid-positive control, respectively; lanes 4-22 represent... TaRLCK118-OE Amplification results using wheat DNA from different transgenic lines as templates yielded OE lines (L4, L10).

[0059] Example 4

[0060] right TaRLCK118 The OE strain was inoculated with the stripe rust-compatible race CYR31 on the leaf surface of wild-type "Fielder" using the smear method, and its resistance was evaluated. The results are as follows: Figure 7 As shown.

[0061] Fourteen days after inoculation with the stripe rust-affinity race CYR31, RT-qPCR was used for detection. TaRLCK118 -OE strains TaRLCK118 The expression situation was as follows: the primers were the same as in Example 2, and the results were as follows: Figure 6 As shown, it can be seen that TaRLCK118 -OE strains TaRLCK118 The expression level was significantly upregulated, with the L4 strain being 3 times that of the wild type and the L10 strain being 2 times that of the wild type.

[0062] Phenotypic observation revealed that the wild-type "Fielder" produces a large number of urediniospores on the leaf surface, while TaRLCK118 -OE strains (L4, L10) showed only minor chlorosis and necrosis on their leaves, with a significant reduction in sporulation; thus, TaRLCK118 -OE strains showed enhanced resistance to all tested races, with a significant reduction in leaf lesion area and sporulation; further confirming... TaRLCK118 Genes positively regulate wheat resistance to stripe rust.

[0063] Example 5

[0064] Gene silencing in wild-type Fielder plants mediated by barley stripe mosaic virus TaRLCK118 Using URGI (https: / / wheat-urgi.versailles.inrae.fr / ) TaRLCK118 The sequence was BLASTed in the wheat genome and aligned to... TaRLCK118 Gene-specific fragments were selected, and the silenced fragments were analyzed and evaluated using Si-Fi software with reference to the wheat genome. Primer 5.0 software was used to design... TaRLCK118 VIGS silencing primers for gene A.

[0065] SEQ ID NO:5: VIGS primers: TaRLCK118 -vigs1as-F: 5'-CCTTAATTAAGCTTCTTCGGCTTCCA-3' (SEQ ID NO: 11); TaRLCK118 -vigs1as-R: 5'-ATAAGAATGCGGCCGCTCCAAACCCTCCTTCG-3' (SEQ ID NO: 12); TaRLCK118 -vigs2as-F: 5'-CCTTAATTAAGCAAAGGTCGCCGTAA-3' (SEQ ID NO: 13); TaRLCK118 -vigs2as-R: 5'-ATAAGAATGCGGCCGCCTCCAATGCCAATAGTTCA-3' (SEQ ID NO: 14).

[0066] Inoculation with CYR23 and CYR31 was performed, and their resistance was assessed. The results are as follows: Figure 8 As shown. Phenotypic observation revealed that BSMV:γ-PDS exhibited significant photobleaching 12 days after viral inoculation. BSMV:γ and BSMV: TaRLCK118 All wheat leaves showed some degree of striped chlorosis, indicating successful virus inoculation. All plants were then inoculated with stripe rust fungi CYR23 and CYR31 for disease resistance assessment. Fourteen days later, it was found that, compared with Mock and BMSV:γ inoculated plants, the leaves of plants inoculated with CYR23 and CYR31 showed significantly higher resistance to BMSV:γ. TaRLCK118 The amount of spores produced on the surface of the plant leaves increased significantly.

[0067] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. The application of increasing the expression of wheat receptor cytoplasmic kinase TaRLCK118 or its encoding gene in improving wheat stripe rust resistance and / or breeding wheat resistant to stripe rust, wherein the amino acid sequence of wheat receptor cytoplasmic kinase TaRLCK118 is shown in SEQ ID NO:1; the pathogen of stripe rust is wheat stripe rust-affinity race CYR31.

2. The application according to claim 1, characterized in that, The method for improving wheat stripe rust resistance and / or breeding wheat resistant to stripe rust includes: introducing the encoding gene of the wheat receptor cytoplasmic kinase TaRLCK118 into recipient wheat.

3. The application according to claim 2, characterized in that, Methods for introducing the gene encoding the wheat receptor cytokinase TaRLCK118 into wheat include transgenic techniques.

4. The application according to claim 3, characterized in that, The biological materials used in the transgenic technology include: a recombinant vector containing the encoding gene of the wheat receptor cytokinase TaRLCK118, and / or a transformant containing the encoding gene of the wheat receptor cytokinase TaRLCK118 or the recombinant vector.

5. The application according to claim 4, characterized in that, The transformants include engineered bacteria, transgenic plant cell lines, or transgenic plant tissues.

6. The application according to claim 4, characterized in that, The basic vector in the recombinant vector includes a plasmid vector.

7. The application according to claim 5, characterized in that, The basic strain of the engineered bacteria is Agrobacterium.

8. A method for breeding wheat resistant to stripe rust, characterized in that, include: Increasing the expression level of wheat receptor cytokinase TaRLCK118 in recipient wheat or increasing the expression level of recipient wheat TaRLCK118 Gene expression; The TaRLCK118 The gene encodes wheat receptor cytokinase TaRLCK118, the amino acid sequence of which is shown in SEQ ID NO:1; the pathogen of the stripe rust is wheat stripe rust-affinity race CYR31.

9. The method according to claim 8, characterized in that, The method of increasing the expression level of wheat receptor cytokinase TaRLCK118 in recipient wheat or increasing the expression level of recipient wheat TaRLCK118 Methods of gene expression include: TaRLCK118 The gene was introduced into the recipient wheat through transgenic technology.

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