Application of the puccinia effector protein gene ps13167 or its encoded protein in regulating plant resistance to stem rust

Abstract

This invention belongs to the field of plant genetic engineering technology, specifically relating to stripe rust fungus effector protein genes. Ps13167 The invention relates to the application of its encoded proteins in regulating plant resistance to stripe rust. This invention utilizes an Agrobacterium-mediated wheat genetic transformation system to transfer stripe rust effector protein genes... Ps13167 The silent and full-length fragments of the gene were transformed into wheat embryos, resulting in gene-silenced plants and overexpressing plants, respectively. Phenotypic identification revealed that the silent plants exhibited significant resistance to wheat stripe rust, while the overexpressing plants showed weakened resistance to stripe rust. Furthermore, this invention demonstrates the effectiveness of the stripe rust effector protein gene... Ps13167 Its important role in wheat infection by stripe rust and the ability to utilize stripe rust effector protein genes Ps13167 Improve the disease resistance of wheat, so as to breed genetic material resistant to rust.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering technology, specifically relating to stripe rust fungus effector protein genes. Ps13167 Or the application of its encoded proteins in regulating plant resistance to stripe rust. Background Technology

[0002] Wheat stripe rust is caused by the wheat-specific strain of *Strombus styracifolius* (Strombus styracifolius). Puccinia striiformis f. sp. tritici, Pst Stripe rust (hereinafter referred to as stripe rust) is a major fungal disease that seriously threatens wheat grain production. Wheat stripe rust is a typical example of an airborne disease. The spores of stripe rust can be spread rapidly and over long distances through air currents, leading to the widespread spread of the disease. In addition, stripe rust and wheat have a co-evolutionary relationship, and the new strains that evolve from the continuous mutation of stripe rust can easily cause large-scale epidemics of wheat stripe rust.

[0003] Stripe rust fungi have a life cycle consisting of both sexual and asexual stages, exhibiting five spore morphologies. Among them, urediniospores cause severe damage to wheat through primary infection and multiple reinfections. Under suitable temperature and humidity conditions, urediniospores germinate into germ tubes within 2-3 hours. The germ tubes extend to the stomata, invading the wheat through the stomata and forming hypostomatal vesicles below the stomata. Subsequently, the hypostomatal vesicles further develop and produce primary hyphae. The hyphae proliferate between cells, forming haustoria mother cells, which penetrate the mesophyll cell walls to form haustoria.

[0004] The haustorium is a crucial site for molecular dialogue and material exchange between stripe rust fungi and host cells. Through the haustorium, stripe rust fungi continuously absorb nutrients from host cells while simultaneously secreting effector proteins. These effector proteins target different disease resistance pathways in the plant, altering the immune response of host plant cells and thus promoting the infection process in wheat. Therefore, effector proteins play a vital role in the successful infection of the host by pathogens.

[0005] Therefore, the identification and utilization of stripe rust effector proteins are of great significance for studying the pathogenic mechanism of stripe rust, creating wheat materials resistant to stripe rust, and improving wheat resistance to stripe rust. Summary of the Invention

[0006] The purpose of this invention is to provide a stripe rust bacteria effector protein gene. Ps13167 Or the application of its encoded protein in regulating plant stripe rust resistance, the stripe rust effector protein gene Ps13167 It can regulate plant resistance to stripe rust and increase the gene pool of stripe rust fungus effector proteins. Ps13167 Biological applications.

[0007] This invention provides a stripe rust bacteria effector protein gene. Ps13167The application of its encoded protein in regulating plant stripe rust resistance, the stripe rust effector protein gene Ps13167 The amino acid sequence of the encoded protein is shown in SEQ ID NO:2.

[0008] Preferred gene for inhibiting stripe rust effector proteins Ps13167 Or the application of its encoded protein expression in improving plant resistance to stripe rust, or promoting the expression of stripe rust fungus effector protein genes. Ps13167 Or the application of its encoded protein expression in reducing plant resistance to stripe rust.

[0009] This invention also provides a gene for inhibiting stripe rust effector proteins. Ps13167 The application of biological materials encoding or expressing proteins thereof in improving plant resistance to stripe rust and / or breeding stripe rust-resistant plant varieties;

[0010] The stripe rust effector protein gene Ps13167 The amino acid sequence of the encoded protein is shown in SEQ ID NO:2.

[0011] Preferably, the biomaterial includes at least one of the following:

[0012] 1) Host-induced gene silencing of stripe rust effector protein genes Ps13167 Nucleic acid molecules;

[0013] 2) Interference with stripe rust effector protein genes Ps13167 Expressing nucleic acid molecules;

[0014] 3) A recombinant vector containing the nucleic acid molecules described in 1) or 2);

[0015] 4) Engineered bacteria containing the nucleic acid molecules described in 1) or 2);

[0016] 5) Engineered bacteria containing the recombinant vector described in 3).

[0017] Preferably, 1) the host-induced gene silencing of the stripe rust effector protein gene Ps13167 The nucleic acid molecules include nucleic acid molecules Ps13167-1 and / or nucleic acid molecules Ps13167-2, wherein nucleic acid molecule Ps13167-1 is obtained by amplification using the primer pairs shown in SEQ ID NO:9 and SEQ ID NO:10, and nucleic acid molecule Ps13167-2 is obtained by amplification using the primer pairs shown in SEQ ID NO:11 and SEQ ID NO:12.

[0018] Preferably, 2) the interference stripe rust effector protein gene Ps13167 The nucleic acid molecules were obtained by amplification using the primer pairs shown in SEQ ID NO:13 and SEQ ID NO:14.

[0019] This invention also provides a gene that promotes the effector protein of stripe rust fungi. Ps13167 The application of biological materials encoding or expressing proteins thereof in reducing plant resistance to stripe rust;

[0020] The stripe rust effector protein gene Ps13167 The amino acid sequence of the encoded protein is shown in SEQ ID NO:2.

[0021] Preferably, the biomaterial includes at least one of the following:

[0022] A) Contains the stripe rust effector protein gene Ps13167 The expression box;

[0023] B) Contains the stripe rust effector protein gene Ps13167 Recombinant vectors;

[0024] C) A recombinant vector containing the expression cassette described in A);

[0025] D) Contains the stripe rust effector protein gene Ps13167 Engineered bacteria;

[0026] F) Engineered bacteria containing the expression cassette described in A);

[0027] G) Engineered bacteria containing the recombinant vector described in B) or C).

[0028] Preferably, the plant includes wheat.

[0029] This invention also provides a method for breeding wheat varieties resistant to stripe rust, comprising the following steps: inhibiting the stripe rust effector protein gene in recipient wheat. Ps13167 By expressing or reducing the expression level of the stripe rust effector protein Ps13167 in recipient wheat, stripe rust-resistant wheat varieties can be obtained; the stripe rust effector protein gene Ps13167 The stripe rust effector protein Ps13167 is encoded, and the amino acid sequence of the stripe rust effector protein Ps13167 is shown in SEQ ID NO:2.

[0030] Beneficial effects:

[0031] This invention provides a stripe rust bacteria effector protein gene. Ps13167 The application of its encoded proteins in regulating plant stripe rust resistance. This invention uses qRT-PCR to identify stripe rust effector protein genes. Ps13167 The expression patterns of stripe rust bacteria in wheat during the early infection, in vivo parasitism, late infection, and sporulation stages were investigated using urediniospores, germ tubes, and in wheat. Furthermore, a host-induced transient gene silencing system was used to preliminarily determine the stripe rust effector protein genes. Ps13167 The pathogenic function of stripe rust fungus in wheat infection; finally, the stripe rust effector protein gene was identified. Ps13167 Cloning specific fragments into RNAi expression vectors and stripe rust effector protein genes Ps13167 The gene fragment after removing the signal peptide was cloned into an overexpression vector, and wheat embryos were transformed using an Agrobacterium-mediated wheat genetic transformation system to obtain gene-silenced plants and overexpression plants. Phenotypic identification revealed that the silenced plants showed significant resistance to wheat stripe rust, while the overexpression plants showed weakened resistance to stripe rust. Therefore, this invention demonstrates the stripe rust effector protein gene... Ps13167 Its important role in wheat infection by stripe rust and the ability to utilize stripe rust effector protein genes Ps13167 To improve the disease resistance of wheat, which can then be used to breed genetic material resistant to rust. Attached Figure Description

[0032] 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.

[0033] Figure 1 This is a flowchart of the wheat stripe rust fungus effect identification method provided in the embodiments of the present invention;

[0034] Figure 2 This refers to the effector protein gene secreted during wheat stripe rust infection by wheat in Example 1 of this invention. Ps13167 Schematic diagram of expression profile analysis, in which express P <0.01, express P <0.001 and express P <0.0001;

[0035] Figure 3 This is the specific transient silencing effector protein gene in Example 2 of the present invention. Ps13167 A schematic diagram of the phenotypic results, where B is... express P <0.05;

[0036] Figure 4 The stripe rust effector protein gene provided in Example 3 of this invention. Ps13167 Schematic diagram of the phenotype of wheat plants overexpressing a non-virulent race of stripe rust and the effector protein gene. Ps13167 A schematic diagram illustrating the phenotypic results of the main prevalent races of stripe rust inoculated into silent plants, silencing efficiency detection, biomass, and ROS detection. express P <0.05, express P <0.01, express P <0.001 and express P <0.0001. Detailed Implementation

[0037] This invention provides a stripe rust bacteria effector protein gene. Ps13167 The application of its encoded protein in regulating plant stripe rust resistance, the stripe rust effector protein gene Ps13167 The amino acid sequence of the encoded protein is shown in SEQ ID NO:2.

[0038] SEQ ID The amino acid sequence shown in NO:2 is specifically: MSFISSVIISSSLLVLLSVKLVLANWDPATGHLHNYGPSQHWISQHKKGQSCYNAIQVSECAQNTRLAYPNVQLFATFQVDHSDDNYHGCPYGTCCAYTQLPSPSD MEADFTNHHSFFWHGLGGQPGPGTNPIANPQTGGFGYESSDGKFHEGKPDVSVQQKGHDSNYPGFKLPHAWPRVNYPGSQPTQPKCGTASGKNLDPGQVRGSYGNYKPAPASSYKAPPARLV.

[0039] As one embodiment, the stripe rust fungus effector protein gene of the present invention Ps13167The nucleotide sequence is as shown in SEQ ID NO:1, specifically:

[0040] As one implementation method, inhibiting the stripe rust effector protein gene Ps13167 Or the application of its encoded protein expression in improving plant resistance to stripe rust, or promoting the expression of stripe rust fungus effector protein genes. Ps13167 Or the application of its encoded protein expression in reducing plant resistance to stripe rust. As one embodiment, the plant can be a grass or wheat.

[0041] This invention also provides a gene for inhibiting stripe rust effector proteins. Ps13167 The application of biological materials encoding or expressing proteins thereof in improving plant resistance to stripe rust and / or breeding stripe rust-resistant plant varieties;

[0042] The stripe rust effector protein gene Ps13167 The amino acid sequence of the encoded protein is shown in SEQ ID NO:2.

[0043] In one embodiment, the biomaterial includes at least one of the following:

[0044] 1) Host-induced gene silencing of stripe rust effector protein genes Ps13167 Nucleic acid molecules;

[0045] 2) Interference with stripe rust effector protein genes Ps13167 Expressing nucleic acid molecules;

[0046] 3) A recombinant vector containing the nucleic acid molecules described in 1) or 2);

[0047] 4) Engineered bacteria containing the nucleic acid molecules described in 1) or 2);

[0048] 5) Engineered bacteria containing the recombinant vector described in 3).

[0049] As one implementation method, 1) the host-induced gene silencing of the stripe rust effector protein gene Ps13167 The nucleic acid molecules include nucleic acid molecules Ps13167-1 and / or nucleic acid molecules Ps13167-2. Ps13167-1 is obtained by amplification using the primer pairs shown in SEQ ID NO:9 and SEQ ID NO:10, and Ps13167-2 is obtained by amplification using the primer pairs shown in SEQ ID NO:11 and SEQ ID NO:12. As one embodiment, 2) the interference stripe rust effector protein gene... Ps13167 The nucleic acid molecules were amplified using the primer pairs shown in SEQ ID NO:13 and SEQ ID NO:14. As one embodiment, the initial vector in the recombinant vector can be a γ-retroviral vector or an RNAi vector. As one embodiment, the initial strain in the engineered bacteria is Agrobacterium. This invention does not specifically limit the construction method of the recombinant vector and engineered bacteria; conventional methods for constructing recombinant vectors and engineered bacteria in the art can be used. As one embodiment, the plant can be a gramineous plant or wheat.

[0050] This invention also provides a gene that promotes the effector protein of stripe rust fungi. Ps13167 The application of biological materials encoding or expressing the protein of the aforementioned fungus in reducing plant resistance to stripe rust; the stripe rust effector protein gene Ps13167 The amino acid sequence of the encoded protein is shown in SEQ ID NO:2.

[0051] In one embodiment, the biomaterial includes at least one of the following:

[0052] A) Contains the stripe rust effector protein gene Ps13167 The expression box;

[0053] B) Contains the stripe rust effector protein gene Ps13167 Recombinant vectors;

[0054] C) A recombinant vector containing the expression cassette described in A);

[0055] D) Contains the stripe rust effector protein gene Ps13167 Engineered bacteria;

[0056] F) Engineered bacteria containing the expression cassette described in A);

[0057] G) Engineered bacteria containing the recombinant vector described in B) or C).

[0058] As one implementation, the stripe rust effector protein gene in the expression cassette Ps13167 This involves removing the gene fragment after the signal peptide (the 24 amino acids of the N segment) from the stripe rust effector protein. As one embodiment, the initial vector in the recombinant vector is a plasmid vector, or alternatively, the pANIC6E vector. As one embodiment, the initial strain in the engineered bacteria is Agrobacterium. This invention does not specifically limit the construction method of the recombinant vector and engineered bacteria; conventional methods for constructing recombinant vectors and engineered bacteria in the art can be used. As one embodiment, the plant can be a gramineous plant or wheat.

[0059] This invention also provides a method for breeding wheat varieties resistant to stripe rust, comprising the following steps: inhibiting the stripe rust effector protein gene in recipient wheat. Ps13167 By expressing or reducing the expression level of the stripe rust effector protein Ps13167 in recipient wheat, stripe rust-resistant wheat varieties can be obtained; the stripe rust effector protein gene Ps13167 The gene encodes the stripe rust effector protein Ps13167, the amino acid sequence of which is shown in SEQ ID NO:2. As one embodiment, the gene inhibiting the receptor wheat stripe rust effector protein... Ps13167 The methods for expressing or reducing the expression level of the stripe rust effector protein Ps13167 in wheat can include transient silencing, RNAi interference, knockout, or knockdown. The specific process is not particularly limited and can be any conventional procedure in the art. For example, in this invention, the method involves using a gene containing transient silencing or RNAi interference to express the stripe rust effector protein. Ps13167 The recombinant vector expressing the nucleotide molecule was transferred into recipient wheat via Agrobacterium-mediated transformation to achieve the expression of the stripe rust effector protein gene. Ps13167 Knockout or silencing of the virus resulted in genetically modified wheat with enhanced resistance to stripe rust.

[0060] 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.

[0061] The present invention is carried out in accordance with the following embodiments Figure 1 Methods using flowcharts to study secretory protein genes in stripe rust fungi Ps13167 It has the function of creating wheat materials resistant to stripe rust.

[0062] Example 1

[0063] Based on qRT-PCR, the gene of the secreted (effective) protein of stripe rust fungus was identified. Ps13167 Whether a fungus participates in the process of stripe rust fungus infecting the host depends on the following steps:

[0064] RNA was extracted from wheat stripe rust urediniospores, germ tubes, and Fielder wheat at inoculation times of CYR32 12, 24, 48, 72, and 120 hpi and then reverse transcribed into cDNA. Using the cDNA as a template, the stripe rust elongation factor gene was... PsEF1 As an internal control, the stripe rust effector protein gene was utilized. Ps13167 qRT-PCR was performed using primers with specific fragments to identify the stripe rust effector protein gene. Ps13167 Expression levels of stripe rust in wheat at different infection stages (early infection, in vivo parasitism, late infection, and sporulation).

[0065] Stripe rust elongation factor gene PsEF1 The internal reference primers and gene quantification primers are as follows.

[0066] Internal reference primer:

[0067] forward primer PsEF1-F : 5'-ttcgccgtccgtgatatgagacaa-3' (SEQ ID NO:3); reverse primer PsEF1-R :5'-atgcgtatcatggtggtggagtga-3' (SEQ ID NO: 4).

[0068] Gene quantification primers:

[0069] forward primer Ps13167 -1-qRT-F: 5'-ttcaagtctcggaatgtg-3' (SEQ ID NO:5); reverse primer Ps13167-1-qRT-R: 5'-ggcagccgtggtagtt-3' (SEQ ID NO:6).

[0070] Forward primer Ps13167-2-qRT-F: 5'-ttgccgtcgccatcc-3' (SEQ ID NO:7); Reverse primer Ps13167 -2-qRT-R: 5'-tgaaacttcccatcactactct-3' (SEQ ID NO: 8).

[0071] The results are as follows Figure 2 As shown, U and GT on the horizontal axis represent the urediniospores and germ tubes of stripe rust fungus, respectively.

[0072] Depend on Figure 2 It can be concluded that: stripe rust bacteria effector protein gene Ps13167 The relative expression level of stripe rust fungus reached its peak 12 hours after infection with wheat, with an induction fold as high as 52.7-fold, indicating that... Ps13167 It plays a role in the early stages of stripe rust infection.

[0073] Example 2

[0074] Using host-induced gene silencing technology, the effector protein gene of stripe rust fungus was preliminarily identified. Ps13167 The pathogenic function of stripe rust fungus in wheat infection proceeds through the following steps:

[0075] First, BLASTn analysis was performed in the stripe rust fungus database (Ensembl Fungi database, accessed at http: / / fungi.ensembl.org / Puccinia_striiformis_gca_002920065 / Info / Index) and the wheat database (Ensembl Plants database, accessed at https: / / plants.ensembl.org / Triticum_aestivum / Info / Index) to obtain... Ps13167 Designed using unique gene fragments Ps13167 -γ primers and construct Ps13167 -γ carrier, in which Ps13167 -γ primers are as follows:

[0076] forward primer Ps13167 -1-vigs-F: 5'-tttttagctagctgattaattaattcaagtctcggaatgtg-3' (SEQ ID NO: 9); Reverse primer: Ps13167 -1-vigs-R: 5'-tccgttgctagctgagcggccgcggcagccgtggtagtt-3' (SEQ ID NO:10); forward primer Ps13167-2-vigs-F: 5'-tttttagctagctgattaattaattgccgtcgccatcc-3' (SEQ ID NO:11); Reverse primer: Ps13167 -2-vigs-R: 5'-tccgttgctagctgagcggccgctgaaacttcccatcactactct-3' (SEQ ID NO: 12).

[0077] Using the primers described above, the fragment was amplified using wheat stripe rust cDNA as a template, and then constructed into the barley stripe mosaic virus (BSMV) vector BSMV:γ via homologous recombination. Sequencing was then used to confirm the presence of the fragment. Ps13167 The fragment was successfully constructed into the BSMV:γ vector.

[0078] Next, RNA products for inoculation were prepared by in vitro transcription. In vitro transcription was performed according to the steps on the Promega T7 in vitro transcription kit (T7 RiboMAX™ Express Large Scale RNA Production System, Promega). The samples were inoculated into wheat two-leaf rubbing inoculation. The silenced phytoene dehydrogenase gene was detected approximately 7 days after inoculation. TaPDS (A key enzyme in the carotenoid synthesis pathway; silencing it causes photobleaching in the leaves, used to detect whether silencing was successful.) Whether wheat leaves exhibit a bleached appearance after silencing and the results of the silencing process. Ps13167 After inoculation, check whether the wheat leaves show signs of chlorosis. If so, it indicates that the virus inoculation was successful. Inoculate the 4-leaf stage of the infected plant with fresh spores of wheat stripe rust fungus CYR31. Collect leaves at 24, 48, and 120 h after inoculation to extract RNA and reverse transcribe it into cDNA. Detect the silencing efficiency at different time points after inoculation by qRT-PCR. The primers and methods used for qRT-PCR detection are the same as in Example 1.

[0079] The results are as follows Figure 3 As shown in Figure B, the fragments were observed 24 hours after inoculation. Ps13167 -1 and Ps13167 The silencing efficiencies of -2 were 45% and 84%, respectively, and the inoculation fragment was 48 h old. Ps13167 -1 and Ps13167 The silencing efficiencies of -2 were 48% and 88%, respectively, and the inoculation fragment was 120 h old. Ps13167 -1 and Ps13167 The silencing efficiencies of -2 were 42% and 82%, respectively, and the expression levels of both were significantly reduced, indicating that the effector protein genes... Ps13167 Effectively silenced momentarily.

[0080] On day 14 post-inoculation, the sporulation of stripe rust fungus on the leaf surface was observed and statistically analyzed. The results are as follows: Figure 3As shown in Figure A, it can be seen that the effector protein gene Ps13167 After being silenced, the sporulation rate of the leaves decreased significantly, indicating that the effector protein Ps13167 plays a toxic role in the process of stripe rust infecting wheat.

[0081] Example 3

[0082] Using a wheat genetic transformation system, a stripe rust effector protein gene was constructed. Ps13167 The RNAi and overexpression plants were used to further clarify the pathogenic function of stripe rust fungus in wheat infection, following the steps below:

[0083] 1. Stripe rust effector protein gene Ps13167 Construction of Silent Transgenic Plants: First, the effector protein gene was transmitted via the Gateway method. Ps13167 The specific fragment was constructed into the RNAi vector PC336 (RNAi vector PC336 was published in Han Xiuli. Cloning and analysis of barley salicylic acid synthesis genes ICS and PAL [D]. Shandong Agricultural University, 2013.).

[0084] Silent primer construction: forward primer Ps13167-RNAi -F: 5'-ggggacaagtttgtacaaaaaagcaggcttcttgccgtcgccatcc-3' (SEQ ID NO:13), reverse primer Ps13167-RNAi -R: 5'-ggggaccactttgtacaagaaagctgggtctgaaacttcccatcactactct-3' (SEQ ID NO: 14).

[0085] The method for constructing recombinant plasmids utilizes the BP reaction to... Ps13167 The silenced fragment was ligated into the pDONR221 intermediate vector. The reaction mixture consisted of 4.0 μL of gene fragment, 0.5 μL of BP enzyme, 2.0 μL of intermediate vector, and 1×TE buffer to a final volume of 10 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 pDONR221 plasmid from the Gateway recombinant cloning system of Thermo Fisher Scientific, and is commercially available.

[0086] Next, the connection will be successful. Ps13167 RNAi -pDONR221 is linked to the final loading PC336 via an LR reaction, and the reaction system is as follows: Ps13167RNAi1.5 μL of pDONR221, 1.0 μL of PC336 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.

[0087] Using Agrobacterium-mediated genetic transformation, we obtained... Ps13167 -RNAi transgenic plants. In wild-type Fielder and Ps13167- Two leaves of RNAi transgenic plants were inoculated with the physiologically virulent race CYR32 of stripe rust fungus. Leaves of the transgenic plants were collected 24 h after inoculation. The silencing efficiency after inoculation was detected by qRT-PCR (primers and methods were the same as in Example 1), and the reactive oxygen species (ROS) content of the plant leaves was determined using a plant reactive oxygen species (ROS) ELISA kit (FANKEW). The results are as follows. Figure 4 As shown.

[0088] Depend on Figure 4 From B, it can be concluded that after 14 days of inoculation, compared with Fielder, Ps13167- RNAi-inoculated plants showed leaf necrosis, significantly reduced sporulation, and a significant decrease in stripe rust biomass. Figure 4 From C, it can be concluded that: qRT-PCR showed that 24 h after inoculation, compared with wild-type Fielder, Ps13167- RNAi-L3 plants Ps13167 The silence efficiency is 85%. Ps13167- RNAi-L5 plants Ps13167 The silencing efficiency is 68%. Figure 4 From D, it can be concluded that: Leaves collected 24 hours after inoculation showed that, compared to wild-type Fielder, Ps13167- RNAi plants show increased reactive oxygen species content in their leaves.

[0089] The above results demonstrate the ability to silence stripe rust effector protein genes. Ps13167 It significantly enhanced wheat's resistance to stripe rust, and the stripe rust effector protein Ps13167 played a toxic role in the process of stripe rust infecting wheat.

[0090] 2. Stripe rust effector protein gene Ps13167 Construction of transgenic plants overexpressing the gene: Design of stripe rust effector protein gene Ps13167 The full-length primers for the fragment after removing the signal peptide, and the primers for constructing the vector of the overexpression fragment are as follows: forward primer. Ps13167 nsp-6e-F: 5'-ggggacaagtttgtacaaaaaagcaggcttcagcgtagtctgggacgtcgtatgggtaatgaattggggatccagcgac-3' (SEQ ID NO: 15); reverse primer Ps13167 nsp -6e-R: 5'-ggggaccactttgtacaagaaagctgggtcctaagcgtagtctgggacgtcgtatgggtaaacgagacgggctggtgg-3' (SEQ ID NO: 16).

[0091] The stripe rust effector protein gene was amplified by PCR and then recovered by agarose gel electrophoresis. The Gateway method was used to isolate the gene. Ps13167 The vector was constructed into the pANIC6E vector (published in the following literature: "Mann et al. 2012, PlantBiotechnology Journal, Gateway-compatible vectors for high-throughput genefunctional analysis in switchgrass"). Panicum virgatum L.) and other monocotspecies”, using Agrobacterium-mediated genetic transformation, obtained Ps13167 -OE transgenic plants. In wild-type Fielder and Ps13167- When the two leaves of OE plants were inoculated with the non-toxic physiological race CYR23 of stripe rust, significant necrosis was observed in the leaves of wild-type Fielder plants 14 days after inoculation. Ps13167- OE plants showed obvious uredinia on their leaves, and the biomass of stripe rust fungus was significantly increased (see [link]). Figure 4 (A) indicates overexpression of the stripe rust effector protein gene. Ps13167 It significantly reduced wheat resistance to stripe rust, further demonstrating the effectiveness of stripe rust effector protein genes. Ps13167 It exerts its toxic function during the infection of wheat by stripe rust fungi.

[0092] 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. Application of biomaterials that inhibit the expression of the stripe rust effector protein gene Ps13167 in improving wheat stripe rust resistance and / or breeding stripe rust-resistant wheat varieties; The nucleotide sequence of the stripe rust effector protein gene Ps13167 is shown in SEQ ID NO:1; The biological material includes a host-induced gene silencing gene of stripe rust effector protein Ps13167 and / or an RNAi stripe rust effector protein gene of Ps13167. The host-induced gene silencing gene of stripe rust effector protein Ps13167 includes nucleic acid molecules Ps13167-1 and / or nucleic acid molecules Ps13167-2. The nucleic acid molecule Ps13167-1 is obtained by amplification using the primer pairs shown in SEQ ID NO:9 and SEQ ID NO:10, and the nucleic acid molecule Ps13167-2 is obtained by amplification using the primer pairs shown in SEQ ID NO:11 and SEQ ID NO:

12. The nucleic acid molecule of the RNAi stripe rust effector protein gene Ps13167 was obtained by amplification using the primer pairs shown in SEQ ID NO:13 and SEQ ID NO:

14.

2. Application of biomaterials that promote the expression of the stripe rust effector protein gene Ps13167 or its encoded protein in reducing wheat stripe rust resistance; The amino acid sequence of the protein encoded by the stripe rust effector protein gene Ps13167 is shown in SEQ ID NO:2; The biomaterial includes at least one of the following: A) An expression cassette containing the stripe rust effector protein gene Ps13167; B) A recombinant vector containing the stripe rust effector protein gene Ps13167; C) A recombinant vector containing the expression cassette described in A); D) Engineered bacteria containing the stripe rust effector protein gene Ps13167; E) Engineered bacteria containing the expression cassette described in A); F) Engineered bacteria containing the recombinant vector described in B) or C).

3. A method for breeding wheat varieties resistant to stripe rust, characterized in that, The process includes the following steps: introducing the biological material that inhibits the expression of the stripe rust effector protein gene Ps13167 as described in claim 1 into recipient wheat to obtain a stripe rust-resistant wheat variety; the nucleotide sequence of the stripe rust effector protein gene Ps13167 is shown in SEQ ID NO:1; The biological material includes a host-induced gene silencing gene of stripe rust effector protein Ps13167 and / or an RNAi stripe rust effector protein gene of Ps13167. The host-induced gene silencing gene of stripe rust effector protein Ps13167 includes nucleic acid molecules Ps13167-1 and / or nucleic acid molecules Ps13167-2. The nucleic acid molecule Ps13167-1 is obtained by amplification using the primer pairs shown in SEQ ID NO:9 and SEQ ID NO:10, and the nucleic acid molecule Ps13167-2 is obtained by amplification using the primer pairs shown in SEQ ID NO:11 and SEQ ID NO:

12. The nucleic acid molecule of the RNAi stripe rust effector protein gene Ps13167 was obtained by amplification using the primer pairs shown in SEQ ID NO:13 and SEQ ID NO:

14.

4. A wheat material resistant to stripe rust, characterized in that, The stripe rust-resistant wheat material contains or expresses the biological material described in claim 1 that inhibits the expression of the stripe rust effector protein gene Ps13167; The biological material includes a host-induced gene silencing gene of stripe rust effector protein Ps13167 and / or an RNAi stripe rust effector protein gene of Ps13167. The host-induced gene silencing gene of stripe rust effector protein Ps13167 includes nucleic acid molecules Ps13167-1 and / or nucleic acid molecules Ps13167-2. The nucleic acid molecule Ps13167-1 is obtained by amplification using the primer pairs shown in SEQ ID NO:9 and SEQ ID NO:10, and the nucleic acid molecule Ps13167-2 is obtained by amplification using the primer pairs shown in SEQ ID NO:11 and SEQ ID NO:

12. The nucleic acid molecule of the RNAi stripe rust effector protein gene Ps13167 was obtained by amplification using the primer pairs shown in SEQ ID NO:13 and SEQ ID NO:14; The nucleotide sequence of the stripe rust effector protein gene Ps13167 is shown in SEQ ID NO:1.

Images