Wheat cysteine-rich receptor kinase taCRK25-1b and its coding gene in regulating plant resistance to stripe rust

Abstract

The application provides a wheat cysteine receptor kinase TaCRK25-1B and an application of a coding gene thereof in regulating plant resistance to stripe rust, and belongs to the technical field of plant genetic engineering.The TaCRK25-1B provided by the application is induced to express in wheat under the infection of a stripe rust fungus, and a VIGS technology transient silencing experiment shows that silencing the TaCRK25-1B can enhance the resistance of the wheat to the stripe rust fungus CYR31, while a genetic transformation technology makes the TaCRK25-1B overexpress in the wheat, and the overexpression weakens the resistance of the wheat to the stripe rust, and a TaCRK25-1B RNAi interference plant created by an RNAi technology is used for a pathogenic bacteria infection experiment, and the experiment shows that the TACRK25-1B plays a negative regulation role in the wheat resistance to the stripe rust.Therefore, the application provides a new means for the wheat resistance to the stripe rust and wheat breeding with resistance to the stripe rust.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering technology and relates to the application of a wheat cysteine ​​receptor kinase TaCRK25-1B and its encoding gene in regulating plant resistance to stripe rust. Background Technology

[0002] Wheat stripe rust is a major disease of wheat, caused by the pathogenic fungus *Streptococcus stripe* wheat-specific strain (…). Puccinia striiformis f. sp. Tritici This is caused by [the pathogen's] pathogenic mechanism. Extensive research and production practices have shown that different wheat varieties exhibit significant differences in resistance to stripe rust. Breeding and planting resistant varieties is the safest, most economical, and effective method for controlling wheat stripe rust. However, the rapid variation of stripe rust pathogenic races can easily lead to the "loss" of resistance in resistant varieties, resulting in continuous disease outbreaks and damage. Therefore, accelerating research into the pathogenic mechanism of stripe rust is of great significance for developing long-lasting resistance in wheat.

[0003] Cysteine-rich receptor-like kinases (CRKs) are the largest subfamily of RLKs. Most CRKs contain two conserved DUF-26 domains in their extracellular domains, which possess antibacterial activity. The conserved cysteine ​​residues within these domains can form disulfide bonds, serving as potential sites for thiol redox reactions and playing a crucial role in plant sensing of external redox changes. However, there are currently no reports on whether cysteine-rich receptor-like kinases play a role in interactions with stripe rust fungi. Summary of the Invention

[0004] The purpose of this invention is to provide a wheat cysteine ​​receptor kinase or its encoding gene. TaCRK25-1B Its application in regulating plant resistance to stripe rust is to promote plant resistance to stripe rust through negative regulation.

[0005] This invention provides a wheat cysteine ​​receptor kinase or its encoding gene. TaCRK25-1B Application in regulating plant resistance to stripe rust.

[0006] Preferably, the wheat cysteine ​​receptor kinase or its encoding gene is... TaCRK25-1B Application of negative regulation in enhancing plant resistance to stripe rust.

[0007] Preferably, the wheat cysteine ​​receptor kinase or its encoding gene is... TaCRK25-1B Application in positive regulation to enhance plant susceptibility to stripe rust.

[0008] This invention provides knockout or knockdown of wheat cysteine ​​receptor kinases or their encoding genes. TaCRK25-1BThe application of the reagent in improving plant resistance to stripe rust, cultivating stripe rust-resistant plant varieties, or breeding stripe rust-resistant plants.

[0009] Preferably, the knockdown of wheat cysteine ​​receptor kinase or its encoding gene is... TaCRK25-1 B's reagents include VIGS technology for transient silencing. TaCRK25-1B Gene interference reagents and RNAi technology TaCRK25 Reagents for gene expression.

[0010] Preferably, the VIGS technology is momentarily silent. TaCRK25-1B The gene reagents include primer pairs for amplifying silent fragments; the primer pairs for amplifying silent fragments include a forward primer with a nucleotide sequence as shown in SEQ ID NO:3 and a reverse primer with a nucleotide sequence as shown in SEQ ID NO:4;

[0011] The RNAi technology interferes with TaCRK25-1B The reagents for gene expression include primer pairs for constructing RNAi vectors; the primer pairs for constructing RNAi vectors include a forward primer with a nucleotide sequence as shown in SEQ ID NO:14 and a reverse primer with a nucleotide sequence as shown in SEQ ID NO:15.

[0012] Preferably, the amino acid sequence of the wheat cysteine ​​receptor kinase is shown in SEQ ID NO:1;

[0013] The encoding gene of wheat cysteine ​​receptor kinase TaCRK25-1B The nucleotide sequence is shown in SEQ ID NO:2.

[0014] Preferably, the plant includes grasses (Poaceae).

[0015] This invention provides a method for improving plant resistance to stripe rust by knocking out cysteine ​​receptor kinases or their encoding genes in plants. TaCRK25-1B Or knock down cysteine ​​receptor kinases or their encoding genes in plants. TaCRK25-1B The level of expression.

[0016] This invention provides a wheat cysteine ​​receptor kinase or its encoding gene. TaCRK25-1B Application in regulating plant resistance to stripe rust. This invention utilizes VIGS technology for transient silencing. TaCRK25-1B Genes, and their expression patterns are analyzed to determine silencing. TaCRK25-1B This invention enhances wheat resistance to CYR31 and induces its expression during the interaction between the plant and stripe rust fungus. Simultaneously, it creates transgenic interference materials, which, after inoculation with CYR31, significantly reduce the yield of urediniospores on the leaf surface of the interference materials. Furthermore, this invention also creates overexpression... TaCRK25-1B Genetically modified materials have been clearly identified as promoting TaCRK25- 1B Gene expression increases plant susceptibility to stripe rust fungi; overexpression TaCRK25-1B The gene weakened wheat's resistance to stripe rust. The above experimental results indicate that... TaCRK25-1B Genes play a negative regulatory role in plant defense responses against stripe rust. Compared with traditional disease-resistant breeding techniques, this invention, based on molecular biology, opens up a new avenue for plant disease-resistant breeding through plant disease-resistant genetic engineering. It achieves breakthroughs in reproductive isolation between species and incompatibility in distant hybridization, and can efficiently achieve targeted improvement of target traits in a relatively short period of time, providing theoretical guidance for breeding stable and heritable disease-resistant materials. Attached Figure Description

[0017] Figure 1 for TaCRK25- 1B Transcriptome data from the wheat-striped rust interaction;

[0018] Figure 2 VIGS Instant Silence TaCRK25 The disease resistance identification diagram shows that the experimental group was BMSV:TaCRK25 and the negative control group was BMSV:γ; CYR23 is the physiological race CYR23 of stripe rust; CYR31 is the physiological race CYR31 of stripe rust.

[0019] Figure 3 To detect gene mutations in silent plants inoculated with stripe rust fungi CYR23 and CYR31, qRT-PCR was used. TaCRK25 Silent efficiency results; among which express P Value < 0.01; express P Value < 0.005;

[0020] Figure 4 To investigate the effects of wheat cysteine ​​receptor kinase on inoculation of the Fielder wheat variety with the non-toxic race CYR23 (incompatible interaction) and the toxic race CYR31 (affinity interaction) of stripe rust fungus at the two-leaf stage. TACRK25-1B Gene expression profile analysis diagram; among which, express P Value < 0.05, express P Value < 0.01; express P Value < 0.005;

[0021] Figure 5 qRT-PCR technology was used to detect T1 generation transgenic plants. TaCRK25 -OE#L37 and TaCRK25 -OE#L52 TaCRK25The diagram shows the expression level results; where L37 and L52 are the overexpressing plant lines L37 and L52, respectively. express P Value < 0.01; express P Value < 0.005;

[0022] Figure 6 T1 generation wheat TaCRK25 Schematic diagram of phenotype results of overexpressing plants inoculated with stripe rust fungus CYR23; in the figure, Fielder is a wild-type wheat variety; CYR23 is the physiological race CYR23 of stripe rust fungus; L37: TaCRK25 Overexpression plants: L37 line; L52: TaCRK25 The L52 strain was overexpressed.

[0023] Figure 7 T1 generation wheat TaCRK25 A schematic diagram showing the biomass changes of overexpressing plants 10 days after inoculation with stripe rust fungus CYR23 and the wild-type plant Fielder; where, express P Value < 0.01;

[0024] Figure 8 qRT-PCR technology was used to detect T1 generation RNAi plants. TaCRK25 -RNAi#L5 and TaCRK25 -RNAi#L7 TaCRK25 The diagram shows the expression level results; where L5 and L7 are the L5 and L7 RNAi plant lines, respectively. express P Value < 0.005;

[0025] Figure 9 T1 generation wheat TaCRK25 Schematic diagram of the phenotypic results of RNAi plants inoculated with stripe rust fungus CYR31. In the figure, Fielder is a wild-type wheat variety; CYR31 is the physiological race CYR31 of stripe rust fungus; L5 represents RNAi plant line L5 and RNAi plant line L7; L7 represents RNAi plant line L7.

[0026] Figure 10 T1 generation wheat TaCRK25- A schematic diagram showing the biomass changes of RNAi plants 10 days after inoculation with stripe rust fungus CYR31 and the results of wild-type Fielder plants; among which, express P Value < 0.01. Detailed Implementation

[0027] This invention provides a wheat cysteine ​​receptor kinase or its encoding gene. TaCRK25-1B Application in regulating plant resistance to stripe rust.

[0028] In this invention, the preferred amino acid sequence of the wheat cysteine ​​receptor kinase is as shown in SEQ ID NO:1 (MRYSDKRFFPANATVDLAAYLRSGKSRGKGAIIGGILGGVAFLLLLGLL)

[0029] The encoding gene for the wheat cysteine ​​receptor kinase is shown in the figure. TaCRK25-1B

[0030] In this invention, the plant preferably includes plants of the Poaceae family. The Poaceae family preferably includes species such as *Triticum* and *Oat*. In this embodiment of the invention, wheat is used as an example to illustrate the coding gene. TaCRK25-1B The relationship between regulation of plant resistance to stripe rust.

[0031] In this invention, the wheat cysteine ​​receptor kinase or its encoding gene is described. TaCRK25-1B The preferred application is in negative regulation to enhance plant resistance to stripe rust. The pathogen causing the stripe rust is preferably a stripe rust fungus. The stripe rust fungus is preferably the affinity race CYR31.

[0032] In one embodiment of the present invention, VIGS technology is used for transient silencing. TaCRK25-1B Genes, and their expression patterns are analyzed to determine silencing. TaCRK25-1B This enhances wheat's resistance to CYR31. In another embodiment of the invention, this is achieved by creating a repressor gene. TaCRK25-1B Inoculation of the interference material with CYR31 significantly reduced the yield of urediniospores on the leaf surface, indicating that the repressor gene... TaCRK25-1B The expression enhances the plant's resistance to stripe rust.

[0033] In this invention, the wheat cysteine ​​receptor kinase or its encoding gene is described. TaCRK25-1B Application in positive regulation to enhance plant susceptibility to stripe rust. The pathogen of stripe rust is preferably a stripe rust fungus. The stripe rust fungus is preferably the incompatible race CYR23.

[0034] In one embodiment of the present invention, through overexpression TaCRK25-1B Genetically modified materials have been clearly identified as promoting TaCRK25-1B Gene expression increases plant susceptibility to stripe rust fungi; overexpression TaCRK25-1B The gene weakened wheat's resistance to the stripe rust fungus CYR23. This indicates that overexpression... TaCRK25-1B It increased the susceptibility of wheat to stripe rust.

[0035] This invention provides knockout or knockdown of wheat cysteine ​​receptor kinases or their encoding genes. TaCRK25-1B The application of the reagent in improving plant resistance to stripe rust, cultivating stripe rust-resistant plant varieties, or breeding stripe rust-resistant plants.

[0036] In this invention, the knockdown of wheat cysteine ​​receptor kinase or its encoding gene is described. TaCRK25-1 The preferred reagents for B include VIGS technology for transient silencing. TaCRK25-1B Gene interference reagents and RNAi technology TaCRK25 Reagents for gene expression.

[0037] In this invention, the VIGS technology is momentarily silent. TaCRK25-1B The reagents for gene amplification preferably include primer pairs for silencing fragment amplification; said primer pairs include a forward primer with a nucleotide sequence as shown in SEQ ID NO:3 and a reverse primer with a nucleotide sequence as shown in SEQ ID NO:4. The reagents also include transient silencing agents. TaCRK25-1B Recombinant viral vectors of genes. The transient silencing... TaCRK25-1B The recombinant viral vector of the gene preferably utilizes the primer pair for amplification of the silent fragment to obtain the silent fragment, which is then cloned into BSMV: γ In the middle, we obtain BSMV: γ-TaCRK25. Knockdown of wheat cysteine ​​receptor kinase or its encoding gene using the reagent described above. TaCRK25-1B The preferred method is to use BSMV: γ-TaCRK25 After linearization, the product was transcribed in vitro and then inoculated into wheat plants. Positive plants were identified, and silencing was then achieved. TaCRK25-1B The plant.

[0038] In this invention, the RNAi technology interferes with... TaCRK25-1B The reagents for gene expression preferably include primer pairs for constructing RNAi vectors; the primer pairs for constructing RNAi vectors include a forward primer with a nucleotide sequence as shown in SEQ ID NO:14 and a reverse primer with a nucleotide sequence as shown in SEQ ID NO:15. The method for constructing the RNAi vector utilizes the primer pairs to amplify the gene. TaCRK25-1B The specific fragment was cloned into the backbone vector pDONR221, and the resulting first recombinant vector was linearized and then ligated to an RNAi vector via LR recombination to obtain... TaCRK25-1B -RNAi. The interference TaCRK25-1B The gene expression method, which will... TaCRK25-1B RNAi was introduced into Agrobacterium, and the recombinant Agrobacterium was transferred into plants. After multiple generations, T1 generation interference material was obtained. After inoculation with stripe rust, the T1 generation interference material showed a significant reduction in not only the production of urediniospores on the leaf surface but also a significant decrease in fungal biomass. TaCRK25-1B It plays a negative regulatory role in wheat's resistance to stripe rust infection.

[0039] This invention provides a method for improving plant resistance to stripe rust by knocking out cysteine ​​receptor kinases or their encoding genes in plants. TaCRK25-1B Or knock down cysteine ​​receptor kinases or their encoding genes in plants. TaCRK25-1B The level of expression.

[0040] In this invention, cysteine ​​receptor kinases or their encoding genes in plants are knocked out. TaCRK25-1BThe preferred method utilizes sgRNA or a recombinant gene editing vector containing sgRNA to introduce the gene into plants, thereby enabling the encoding gene to... TaCRK25-1B Mutations in this protein cause frameshift mutations in the sequence of cysteine ​​receptor kinase proteins.

[0041] The following examples illustrate the application of the wheat cysteine ​​receptor kinase TaCRK25-1B and its encoding gene in regulating plant resistance to stripe rust. However, these examples should not be construed as limiting the scope of protection of this invention.

[0042] Example 1

[0043] To preliminarily verify the genes TaCRK25-1B To investigate whether it plays a role in wheat's resistance to stripe rust infection, transcriptome data of wheat interactions with stripe rust, leaf rust, stem rust, powdery mildew, and Fusarium graminearum were first analyzed. A receptor-like protein kinase gene was identified and named after a gene that was significantly upregulated only in the affinity interaction between wheat and stripe rust. TaCRK25- 1B Meanwhile, the expression patterns of these three genes were analyzed in the transcriptome.

[0044] The results are as follows Figure 1 As shown, TaCRK25- 1B is most significantly expressed during the affinity interaction between wheat and stripe rust fungi; therefore, chromosome 1B was selected. TaCRK25-1B It is being studied as a candidate gene.

[0045] Example 2

[0046] VIGS technology instantly silences wheat TaCRK25-1B Genetic methods

[0047] First, use amplification primers. TaCRK25-1B -VIGS-F: 5'-TTATATGAAAATGAGAACTT-3' (SEQ IDNO: 3); TaCRK25-1B-VIGS-R:5'-CCTGGCTGAAAACTGTGAAAT-3' (SEQ ID NO:4) was subjected to PCR amplification of a specific fragment and sequencing. The correctly sequenced sequence (TTATATGAAAATGAGAACTTGATTAGCTTGGTGGACGAATCGTTGGATCGTGAAGAATACAAGCCAGAAGAGGTAAAGAAAATAATAGAGATAGCACTTCTGTGCACTCAATCAGTGGTTGCTTCAAGGCCAACGATGTCAGAGGTGGTTGTGTTGTTGTTGTCAAGAAATTCTTCAGAATTACAGCCCAAGGCCCACATTTATTGATTCAACAAGTAGAGTGCGAGGTGAAACATCCTCCTCAAGTTCATCCTCTGCATCCAGGGCCACTGTCTCTATTTCACAGTTTTCAGCCAGG, SEQ ID NO:5) was ligated into the BSMV:γ-PDS vector using a one-step cloning method to obtain BSMV: γ-TaCRK25-1B. At the same time, the viral vector plasmid BSMV: α、 BSMV: γ use Mlu Enzymes undergo vector linearization; BSMV: β use Spe I. Enzyme vector linearization treatment, BSMV: γ-PDS And the recombinant plasmid BSMV: γ-TaCRK25-1B use BssH Enzyme II was linearized into a vector; the in vitro transcription kit (RiboMAX) was used. TM The Large Scale RNA Production System-T7 was used to perform in vitro transcription of the linearized vector to obtain the in vitro transcription product. Wheat plants were inoculated with the recombinant virus. 270 μL of prepared FES buffer was pipetted onto a PE glove, and then BSMV was added to the FES buffer. α BSMV: β BSMV: γ、 BSMV: γ-PDS And BSMV: γ-TaCRK25 15 μL of each of the in vitro transcription products were thoroughly mixed and divided into several equal portions for virus inoculation. Finally, the viral load on wheat leaves was observed 14 days later.

[0048] The results are as follows Figure 2 As shown, the experimental group (BMSV: γ-TaCRK25-1BThe presence of pale green stripes and chlorosis on the leaves of the control group (BMSV:γ) and the negative control group (BMSV:γ) proved that barley stripe mosaic virus had successfully infected wheat leaf tissue. Afterwards, the wheat leaves were inoculated with the stripe rust-compatible race CYR31 and the non-compatible race CYR23, and the sporulation of wheat leaves was observed after 14 days.

[0049] The results are as follows Figure 2 As shown, compared with the control, the amount of sporulation on the leaves of silent plants after inoculation with CYR31 was significantly reduced. These preliminary results indicate that silencing... TaCRK25-1B This enhances wheat's resistance to CYR31.

[0050] Wheat leaf samples were collected at 24 h, 48 h, and 120 h after inoculation with stripe rust fungi CYR23 and CYR31, respectively. RNA was extracted and reverse transcribed into cDNA for RT-qPCR analysis. TaCRK25-1B Expression levels at various time points. The reverse transcription system consisted of: 4.0 μL 5×Reaction Buffer, 0.5 μL RNase inhibitor, 2.0 μL dNTP, 1.5 μL RevertAidM-Mul V RT, and 12.0 μL RNase-free ddH2O; the reverse transcription reaction program was: 42℃, 60 min; 25℃, 5 min; 70℃, 5 min. TaEF Internal reference gene primers: TaEF -F: 5'-TGGTGTCATCAAGCCTGGTATGGT-3' (SEQ ID NO: 6); TaEF -R: 5'-ACTCATGGTGCATCTCAACGGACT-3' (SEQ ID NO:7). Wheat cysteine ​​receptor kinase encoding gene. TaCRK25-1B Specific fragment primers: TaCRK25-1B -qRT-F: 5'- CCATGGCCAACTATCAGAGAAG-3' (SEQ ID NO: 8); TaCRK25-1B -qRT-R: 5'-TTCAGCTCCAGCTTTGTATC-3' (SEQ ID NO: 9). Quantitative PCR reaction system: 10.0 μL 2×ChamQ SYBR Mixture, 0.5 μL forward primer, 0.5 μL reverse primer, 2.0 μL template DNA, 7.0 μL ddH2O; Quantitative PCR reaction program: 95℃ for 10 minutes; 95℃ for 10 seconds; 60℃ for 30 seconds; 72℃ for 30 seconds; 40 cycles.

[0051] The results are as follows Figure 3 The results showed that in transiently silent plants TaCRK25-1B The expression level was significantly reduced, and transient silencing of CYR23 in plants was observed. TaCRK25-1B The expression levels at 24 h, 48 h, and 120 h were 0.47, 0.25, and 0.46 times that of the control group, respectively; after inoculation with CYR31, the expression levels in transiently silenced plants were significantly lower. TaCRK25-1B The expression levels at 24 h, 48 h, and 120 h were 0.45-fold, 0.50-fold, and 0.52-fold higher than those of the control group, respectively. The experimental results indicate that in transiently silenced plants… TaCRK25-1B The expression of is suppressed.

[0052] Example 3

[0053] wheat cysteine ​​receptor kinase encoding gene TaCRK25-1B Expression profile analysis

[0054] Using Fielder wheat cultivars grown in soil as material, stripe rust physiological races CYR23 and CYR31 were inoculated onto the two leaves at the two-leaf-one-heart stage of wheat using the smear method and kept in the dark and moist for 24 h. Water was set up as a control group. Inoculated leaves were collected at 0 h, 6 h, 12 h, 24 h, 48 h, 72 h and 120 h after inoculation and frozen at -80℃.

[0055] Total RNA was extracted from the collected samples and reverse transcribed into cDNA first strand using reverse transcriptase. Using the cDNA as a template, RT-qPCR was performed according to the method in Example 2. TaCRK25-1B Gene expression status.

[0056] The results are as follows Figure 4 As shown, by Figure 4 It is known that wheat cysteine ​​receptor kinase TaCRK25-1B The gene is induced to express by stripe rust infection during the interaction between wheat and stripe rust.

[0057] Example 4

[0058] overexpression TaCRK25-1B The creation of genetically modified wheat and its disease resistance identification

[0059] Genes TaCRK25-1B The fragment was constructed into an overexpression vector via a Gateway reaction. First, the amplified target gene was recombinated with the pDONR221 vector using a BP reaction to complete the BP cloning reaction, yielding the recombinant vector. The primers used to construct the overexpression recombinant vector are as follows: TaCRK25 -OE-F:TTAGCCCTGCCTTCATACGATGAGGTACTCTGATAAGCG (SEQ ID NO:10); TaCRK25-OE-R: CTATCATAGATGTCGCTATAAACCTTACCTGGCTGAAAACT

[0060] GTGAAA (SEQ ID NO:11). BP reaction system: 2.0 μL 1×TE Buffer, 0.5 μL BP enzyme, 1.0 μL pDONR221 vector, 1.5 μL gene fragment. BP reaction program: overnight incubation at 25℃ for 18 h. Linearization reaction system: 2.0 μL recombinant vector, 3.0 μL... NRU I enzyme, 2.0 μL 10×Buffer, 33.0 μL ddH2O.

[0061] The recombinant vector was transformed into *E. coli*. Positive clones were subjected to colony PCR detection and sent to a sequencing company for sequencing. The correctly sequenced vector plasmids were linearized, and the linearized fragments were then ligated to the pANIC-6E vector via a logistic regression (LR) reaction. The LR reaction mixture consisted of 2.0 μL 1×TE buffer, 0.5 μL LR enzyme, 1.0 μL pANIC-6E vector, and 1.5 μL linearized plasmid. The LR reaction program was: overnight incubation at 25°C for 18 hours. Finally, the ligation product was transformed into *E. coli*, and positive clones were selected and sent to a sequencing company for sequencing. The correctly sequenced single clones were amplified, and plasmids were extracted to obtain the overexpressing gene. TaCRK25-1B Recombinant vector TaCRK25-1B -OE. This will lead to the successful construction of the gene. TaCRK25-1B Overexpression vector TaCRK25-1B -OE is introduced into Agrobacterium to obtain the gene. TaCRK25-1B Overexpressing plants.

[0062] The overexpressing transgenic plants were multiplied to the T1 generation, and RNA was extracted from wheat leaves. The expression level of the transgenic plants was detected using qRT-PCR. The expression level was found to be [specifically, the wheat cysteine ​​receptor kinase encoding gene]. TaCRK25-1B Specific fragment primers: TaCRK25-1B -qRT-F: 5'- CCATGGCCAACTATCAGAGAAG-3' (SEQ ID NO: 8); TaCRK25-1B -qRT-R: 5'- TTCAGGCTCCAGCTTTGTATC -3' (SEQ ID NO: 9); TaEF Internal reference gene primers: TaEF -F: 5'-TGGTGTCATCAAGCCTGGTATGGT-3' (SEQ ID NO: 6); TaEF-R: 5'-ACTCATGGTGCATCTCAACGGACT-3' (SEQ ID NO: 7). Quantitative PCR reaction system: 10.0 μL 2×ChamQ SYBR Mixture, 0.5 μL forward primer, 0.5 μL reverse primer, 2.0 μL template DNA, 7.0 μL ddH2O; Quantitative PCR reaction program: 95℃ for 10 minutes; 95℃ for 10 seconds; 60℃ for 30 seconds; 72℃ for 30 seconds; 40 cycles.

[0063] The results are as follows Figure 5 As shown, compared to the wild-type plant Fielder, TaCRK25 exist TaCRK25 -OE#L37 and TaCRK25 The expression levels in -OE#L52 were upregulated by 6.3-fold and 3.9-fold, respectively, indicating that... TaCRK25 -OE#L37 and TaCRK25 -OE#L52 is a transgenic positive plant.

[0064] Meanwhile, when the material grew to the two-leaf stage, the incompatible race CYR23 was inoculated at the two-leaf stage, and the biomass changes of the overexpressed material and the wild-type Fielder plant 10 days after inoculation with CYR23 were quantitatively analyzed using qRT-PCR technology.

[0065] The results are as follows Figure 6 and Figure 7 The results showed that after inoculation with CYR23, the overexpression material produced a large number of urediniospores on its leaves, while the control group, fielder leaves, exhibited a typical hypersensitive necrosis reaction and did not produce urediniospores. The fungal biomass of both materials was analyzed using qRT-PCR. The method for detecting fungal biomass was as follows:

[0066] (1) First, draw a standard curve and amplify the internal reference gene. PstEF Genes and TaEF Genes were measured and serially diluted (100 ng / μL - 1 × 10⁻⁶). -5 The copy number can be obtained by inputting the DNA sample concentration and base pair length into the tool website (https: / / agbio.com.cn / softtools / copy / ).

[0067] (2) Using diluted DNA of different concentrations as templates, quantitative primers were used for amplification to obtain the Ct values ​​of each group. PstEFInternal reference gene primer sequences: PstEF1-F: TTCGCCGTCCGTGATATGAGACAA (SEQ IN NO:12), PstEF1-R: ATGCGTATCATGGTGGTGGAGTGA (SEQ IN NO:13); TaEF Internal reference gene primers: TaEF -F: 5'-TGGTGTCATCAAGCCTGGTATGGT-3' (SEQ ID NO: 6); TaEF -R: 5'-ACTCATGGTGCATCTCAACGGACT-3' (SEQ ID NO: 7). Quantitative PCR reaction system: 10.0 μL 2×ChamQ SYBR Mixture, 0.5 μL forward primer, 0.5 μL reverse primer, 2.0 μL template DNA, 7.0 μL ddH2O; Quantitative PCR reaction program: 95℃ for 10 minutes; 95℃ for 10 seconds; 60℃ for 30 seconds; 72℃ for 30 seconds; 40 cycles.

[0068] (3) Take the logarithm of the DNA copy number and use it as the x-axis, and plot the Ct value as the y-axis to draw a regression curve. If the coefficient of determination of the regression curve is greater than 0.99, it can be used.

[0069] (4) After scanning the phenotype, leaf DNA was extracted, and the concentration was measured and then diluted to 200 ng / mL. It was used as a template for qPCR experiments to obtain the Ct value.

[0070] (5) Substitute the Ct value into the standard curve to calculate the logarithm of the copy number of PstEF gene and TaEF gene in the template. The ratio of the two is the biomass of wheat stripe rust in the sample.

[0071] The results show that TaCRK25-1B Overexpression plants TaCRK25 -OE#L37 and TaCRK25 Fungal biomass increased by 73% and 53% in -OE#L52, respectively. This indicates that the TaCRK25-1B gene plays a negative regulatory role in the wheat stripe rust resistance response.

[0072] Example 5

[0073] Gene TaCRK25-1B Creation of RNAi materials and identification of their disease resistance

[0074] Genes TaCRK25-1BThe interfering fragment was constructed into the pC336 vector via a Gateway reaction, consistent with the method used to construct the overexpression vector. First, the amplified specific fragment was recombinated with the pDONR221 vector using a BP reaction to complete the BP cloning reaction, yielding the recombinant vector. The gene... TaCRK25-1B The primers used for amplifying the specific interfering fragment are as follows: TaCRK25- RNAi-F: GGGGACAAGTTTGTACAAAAAAGCAGGCTTTTTATATGAAAATGAG (SEQ ID NO: 14); TaCRK25- RNAi-R: AGAAAGCTGGGTTCCTGGCTGAAAACTG (SEQ ID NO:15). BP reaction system: 2.0 μL 1×TEBuffer, 0.5 μL BP enzyme, 1.0 μL pDONR221 vector, 1.5 μL specific interference fragment; BP reaction program: overnight incubation at 25℃ for 18 h. Vector linearization reaction system: 2.0 μL recombinant vector, 3.0 μL... NRU I enzyme, 2.0 μL 10×Buffer, 33.0 μL ddH2O.

[0075] The recombinant vector was transformed into *E. coli*. Positive clones were subjected to colony PCR detection and sent to a sequencing company for sequencing. The plasmids of correctly sequenced vectors were linearized, and the linearized fragments were then ligated to the pC336 vector via a logistic regression (LR) reaction. The LR reaction mixture consisted of 2.0 μL 1×TE buffer, 0.5 μL LR enzyme, 1.0 μL pC336 vector, and 1.5 μL linearized plasmid. The LR reaction program was: overnight incubation at 25°C for 18 hours. Finally, the ligation product was transformed into *E. coli*, and positive clones were selected and sent to a sequencing company for sequencing. The plasmids from correctly sequenced single clones were then extracted to obtain the gene. TaCRK25- 1B RNAi vectors TaCRK25-1B -RNAi. The successfully constructed gene will then be... TaCRK25-1B RNAi vectors TaCRK25-1B -RNAi introduction into Agrobacterium to obtain genes TaCRK25-1B RNAi plants were developed. Simultaneously, the RNAi plants were passaged to the T1 generation, and RNA was extracted from wheat leaves and detected using qRT-PCR. TaCRK25 -RNAi#L5 and TaCRK25 -RNAi#L 7 TaCRK25-1B The level of expression. Among them TaCRK25- RNAi-qRT-F:CCATGGCCAACTATCAGAGAAG-3' (SEQ ID NO:8); TaCRK25-RNAi-qRT-R: TTCAGGCTCCAGCTTTGTATC -3' (SEQ ID NO:9); TaEF Internal reference gene primers: TaEF -F: 5'-TGGTGTCATCAAGCCTGGTATGGT-3' (SEQ ID NO: 6); TaEF -R: 5'-ACTCATGGTGCATCTCAACGGACT-3' (SEQ ID NO:7); Reverse transcription system: 4.0 μL 5×Reaction Buffer, 0.5 μL RNase inhibitor, 2.0 μL dNTP, 1.5 μL RevertAid M-Mμl V RT, 12.0 μL RNase-free ddH2O; Reverse transcription reaction program: 42℃, 60 min; 25℃, 5 min; 70℃, 5 min. Quantitative PCR reaction system: 10.0 μL 2×ChamQ SYBRMixture, 0.5 μL forward primer, 0.5 μL reverse primer, 2.0 μL template DNA, 7.0 μL ddH2O. Quantitative PCR reaction program: 95℃ for 10 min; 95℃ for 10 s; 60℃ for 30 s; 72℃ for 30 s; 40 cycles.

[0076] The results are as follows Figure 8 As shown, TaCRK25-1B exist TaCRK25 -RNAi#L5 and TaCRK25 -RNAi#L7 expression levels were downregulated by 61% and 74% respectively compared to the wild-type Fielder plant, indicating that TaCRK25 -RNAi#L5 and TaCRK25 -RNAi#L7 is a transgenic positive plant that can undergo... TaCRK25-1B Subsequent functional verification.

[0077] Meanwhile, after inoculation with the stripe rust-affinity race CYR31, phenotypic identification was performed, and the biomass changes of the interference material and wild-type wheat Fielder 10 days after inoculation with CYR31 were quantitatively analyzed using qRT-PCR technology.

[0078] result Figure 9 and Figure 10 As shown, the yield of urediniospores on the leaf surface of the transgenic interference material was significantly reduced after inoculation with CYR31. Quantitative analysis of the fungal biomass of both materials yielded the following results. TaCRK25-1B The overexpression reduced fungal biomass in the plants by 43% and 51%, respectively, further validating the results. TaCRK25-1B It plays a negative regulatory role in wheat's resistance to stripe rust infection.

[0079] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Encoding gene for wheat cysteine ​​receptor kinase TaCRK25-1B The application of negative regulation in enhancing plant resistance to stripe rust, wherein the negative regulation is the knockout of the gene encoding cysteine ​​receptor kinase in plants. TaCRK25-1B Alternatively, knock down the gene encoding cysteine ​​receptor kinases in plants. TaCRK25-1B The expression level of the plant is wheat, and the amino acid sequence of the wheat cysteine ​​receptor kinase is shown in SEQ ID NO:

1.

2. Knockout or knockdown of the gene encoding wheat cysteine ​​receptor kinase TaCRK25-1B The application of the reagent in improving plant resistance to stripe rust or in breeding stripe rust-resistant plant varieties, wherein the amino acid sequence of the wheat cysteine ​​receptor kinase is shown in SEQ ID NO:1, and the plant is wheat.

3. The application according to claim 2, characterized in that, The gene encoding the knockdown of wheat cysteine ​​receptor kinase TaCRK25-1 B's reagents include VIGS technology for transient silencing. TaCRK25-1B Gene interference by reagents or RNAi technology TaCRK25 Reagents for gene expression.

4. The application according to claim 3, characterized in that, The VIGS technology provides instantaneous silence. TaCRK25-1B The gene reagents include primer pairs for amplifying silent fragments; the primer pairs for amplifying silent fragments include a forward primer with a nucleotide sequence as shown in SEQ ID NO:3 and a reverse primer with a nucleotide sequence as shown in SEQ ID NO:4; The RNAi technology interferes with TaCRK25-1B The reagents for gene expression include primer pairs for constructing RNAi vectors; the primer pairs for constructing RNAi vectors include a forward primer with a nucleotide sequence as shown in SEQ ID NO:14 and a reverse primer with a nucleotide sequence as shown in SEQ ID NO:

15.

5. The application according to claim 1 or 2, characterized in that, The encoding gene of wheat cysteine ​​receptor kinase TaCRK25-1B The nucleotide sequence is shown in SEQ ID NO:

2.

6. A method for improving plant resistance to stripe rust, characterized in that, Knockout of the gene encoding cysteine ​​receptor kinase in wheat TaCRK25-1B Alternatively, knock down the gene encoding cysteine ​​receptor kinase in wheat. TaCRK25-1B The expression level of the cysteine ​​receptor kinase is shown in SEQ ID NO:1.

Images