Application of stripe rust effect protein gene Ps13167 or encoded protein thereof in regulation and control of stripe rust resistance of plants

Abstract

The invention belongs to the technical field of plant genetic engineering, and particularly relates to application of a stripe rust effect protein gene Ps13167 or an encoding protein thereof in regulating and controlling the stripe rust resistance of plants. A silent segment and a full-length segment of a stripe rust effect protein gene Ps13167 are transformed into a wheat immature embryo by utilizing an agrobacterium-mediated wheat genetic transformation system, a gene silent plant and an overexpression plant are respectively obtained, and phenotypic identification finds that the silent plant shows remarkable resistance to wheat stripe rust; the over-expressed plant shows weakened resistance to stripe rust. Furthermore, the invention proves that the puccinia striiformis effect protein gene Ps13167 plays an important role in wheat infected by puccinia striiformis and can be used for improving the disease resistance of wheat, so that the puccinia striiformis effect protein gene Ps13167 is used for culturing a rust-resistant genetic material.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of plant genetic engineering, and particularly relates to a stripe rust effector protein gene Ps13167 or application of the encoded protein thereof in regulating plant resistance to stripe rust. BACKGROUND

[0002] Wheat stripe rust is a kind of serious fungal disease caused by Puccinia striiformis f. sp. tritici (Pst) (hereinafter referred to as stripe rust fungus) which seriously endangers the safety of wheat food production. Puccinia striiformis f. sp. tritici, Pst Wheat stripe rust is a typical representative of air-borne diseases. Spores of the stripe rust fungus can be rapidly and long-distance transmitted by air flow, leading to the widespread spread of the disease. In addition, the stripe rust fungus and wheat have a co-evolution relationship, and new strains of the stripe rust fungus that evolve through continuous variation are prone to cause large-scale prevalence of wheat stripe rust.

[0003] The life history of the stripe rust fungus is divided into two stages, namely the sexual and asexual stages, and has five spore morphologies. Among them, the urediniospores cause serious damage to wheat through primary infection and several re-infections. The urediniospores germinate into a germ tube within 2-3 hours under suitable temperature and humidity. The germ tube extends to the stomata and invades the wheat through the stomata, forming a substomatal vesicle. Then, the substomatal vesicle further develops and produces primary hyphae, the hyphae proliferate between cells to form haustorial mother cells, and the haustorial mother cells penetrate the cell wall of mesophyll cells to form haustoria.

[0004] The haustorium is a key place for the stripe rust fungus to carry out "molecular dialogue" and material exchange with host cells. The stripe rust fungus, through the haustorium, continuously draws nutrients from the host cells on the one hand, and secretes effector proteins on the other hand, which target different plant disease resistance pathways to change the immune response of the host plant cells, thereby promoting the infection process of the wheat. Therefore, the effector protein plays an extremely important role in the successful infection of the pathogen to the host.

[0005] Therefore, it is of great significance to identify and utilize the stripe rust effector protein to study the pathogenic mechanism of the stripe rust fungus and create wheat materials resistant to stripe rust and improve the resistance of wheat to the stripe rust fungus. SUMMARY

[0006] The purpose of the present application is to provide a stripe rust effector protein gene Ps13167 or application of the encoded protein thereof in regulating plant resistance to stripe rust, which can regulate the resistance of plants to stripe rust and increase the biological use of the stripe rust effector protein gene Ps13167 . Ps13167

[0007] The present application provides a stripe rust effector protein gene Ps13167 ​or the use of the encoded protein in regulating the resistance of plants to stripe rust, the stripe rust effector protein gene Ps13167 The amino acid sequence of the encoded protein is shown as SEQ ID NO: 2.

[0008] Preferably, the stripe rust effector protein gene Ps13167 or the use of the expression of the encoded protein in improving the resistance of plants to stripe rust, or the use of the stripe rust effector protein gene Ps13167 or the use of the expression of the encoded protein in reducing the resistance of plants to stripe rust.

[0009] The present application also provides a biological material for inhibiting the expression of the stripe rust effector protein gene Ps13167 or the expression of the encoded protein in improving the resistance of plants to stripe rust and / or cultivating plant varieties resistant to stripe rust; The stripe rust effector protein gene Ps13167 The amino acid sequence of the encoded protein is shown as SEQ ID NO: 2.

[0010] Preferably, the biological material comprises at least one of the following: 1) a host-induced gene silencing stripe rust effector protein gene Ps13167 nucleic acid molecule; 2) an interference stripe rust effector protein gene Ps13167 nucleic acid molecule; 3) a recombinant vector containing the nucleic acid molecule of 1) or 2); 4) an engineered bacterium containing the nucleic acid molecule of 1) or 2); 5) an engineered bacterium containing the recombinant vector of 3).

[0011] Preferably, the 1) host-induced gene silencing stripe rust effector protein gene Ps13167 nucleic acid molecule comprises nucleic acid molecule Ps13167-1 amplified by a primer pair shown as SEQ ID NO: 9 and SEQ ID NO: 10, and / or nucleic acid molecule Ps13167-2 amplified by a primer pair shown as SEQ ID NO: 11 and SEQ ID NO: 12.

[0012] Preferably, the 2) interference stripe rust effector protein gene Ps13167 nucleic acid molecule is amplified by a primer pair shown as SEQ ID NO: 13 and SEQ ID NO: 14.

[0013] The present application also provides a biological material for promoting the expression of the stripe rust effector protein gene Ps13167 or the encoded protein in reducing the resistance of plants to stripe rust. The said stripe rust effector protein gene Ps13167 The amino acid sequence of the encoded protein is shown as SEQ ID NO: 2.

[0014] Preferably, the biological material comprises 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 of A); D) an engineered bacterium containing the stripe rust effector protein gene Ps13167 ; F) an engineered bacterium containing the expression cassette of A); G) an engineered bacterium containing the recombinant vector of B) or C).

[0015] Preferably, the plant comprises wheat.

[0016] The present application also provides a method for cultivating a stripe rust resistant wheat variety, comprising the following steps: inhibiting the expression of a stripe rust effector protein gene Ps13167 in a receptor wheat or reducing the expression amount of a stripe rust effector protein Ps13167 in a receptor wheat, to obtain a stripe rust resistant wheat variety; the stripe rust effector protein gene Ps13167 encodes a stripe rust effector protein Ps13167, and the amino acid sequence of the stripe rust effector protein Ps13167 is shown as SEQ ID NO: 2.

[0017] Beneficial effects: The present application provides the application of a stripe rust effector protein gene Ps13167 or its encoded protein in regulating the stripe rust resistance of a plant. The present application determines the expression of a stripe rust effector protein gene Ps13167 in the urediniospore, germ tube, and the early stage of infection, the biotrophic stage, the late stage of infection, and the sporulation stage of a wheat infected by a stripe rust fungus by using qRT-PCR; further determines the pathogenic function of a stripe rust effector protein gene Ps13167 in a wheat infected by a stripe rust fungus by using a host-induced transient gene silencing system; finally, a specific fragment of a stripe rust effector protein gene Ps13167 is cloned into an RNAi expression vector, and a gene fragment of a stripe rust effector protein gene Ps13167 after removing the signal peptide is cloned into an overexpression vector, a wheat immature embryo is transformed by using an Agrobacterium-mediated wheat genetic transformation system to obtain a gene silencing plant and an overexpression plant, and phenotype identification finds that the gene silencing plant shows significant resistance to a wheat stripe rust fungus, and the overexpression plant shows weakened resistance to the stripe rust fungus. Furthermore, the present application proves that a stripe rust effector protein gene Ps13167The important role in the infection of wheat by Puccinia striiformis and the use of Puccinia striiformis effector protein genes Figure 1 Improving the disease resistance of wheat to breed genetic materials resistant to rust. BRIEF DESCRIPTION OF DRAWINGS

[0018] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed in the embodiments will be briefly introduced below.

[0019] Figure 2 is a flowchart of the method for identifying Puccinia striiformis effectors provided by the embodiments of the present application; Ps13167 is an effector protein gene secreted by Puccinia striiformis during the infection of wheat by Puccinia striiformis in Example 1 of the present application Figure 3 schematic diagram of expression profile analysis, wherein represents P <0.01, represents P <0.001 and represents P <0.0001; Ps13167 is a specific transient silencing effector protein gene in Example 2 of the present application Figure 4 schematic diagram of phenotype results, wherein B in represents P <0.05; Ps13167 is a Puccinia striiformis effector protein gene provided in Example 3 of the present application Ps13167 schematic diagram of phenotype results of overexpression wheat plants inoculated with avirulent races of Puccinia striiformis and effector protein genes Ps13167 schematic diagram of phenotype results of silencing plants inoculated with major epidemic races of Puccinia striiformis, silencing efficiency detection, biomass and ROS detection, wherein represents P <0.05, represents P <0.01, represents P <0.001 and represents P <0.0001. DETAILED DESCRIPTION

[0020] The present application provides a Puccinia striiformis effector protein gene Ps13167 or its encoded protein for use in regulating the resistance of plants to stripe rust, the Puccinia striiformis effector protein gene Ps13167 The amino acid sequence of the encoded protein is shown in SEQ ID NO: 2.

[0021] The amino acid sequence represented by SEQ ID NO: 2 is specifically: MSFISSVIISSSLLVLLSVKLVLANWDPATGHLHNYGPSQHWISQHKKGQSCYNAIQVSECAQNTRLAYPNVQLFATFQVDHSDDNYHGCPYGTCCAYTQLPSPSDMEADFTNHHSFFWHGLGGQPGPGTNPIANPQTGGFGYESSDGKFHEGKPDVSVQQKGHDSNYPGFKLPHAWPRVNYPGSQPTQPKCGTASGKNLDPGQVRGSYGNYKPAPASSYKAPPARLV.

[0022] As an embodiment, the gene of the effector protein of the G. foriformis described in the present application Ps13167atgagttttataagcagcgtgatcatctcttcatccttgttagtgttgttatccgtcaaacttgtgttggcgaattgggatccagcgacgggacatcttcataactatggaccttcccaacattggatcagtcagcacaaaaagggccaaagttgttacaatgcaattcaagtctcggaatgtgcacagaacactaggttagcatacccgaatgttcaattgtttgcaactttccaagtcgatcactctgatgacaactaccacggctgcccttacggaacctgctgtgcatacactcagttgccgtcgccatccgacatggaagccgacttcacgaatcatcattcattcttttggcatggtctcggggggcaacccggacctggtacgaatccgatcgccaatccacagacgggggggtttgggtacgagagtagtgatgggaagtttcacgaaggcaaaccggacgtctccgtacaacaaaagggtcacgattctaattatcctggctttaaattaccacacgcctggcctagagtcaactatcctggatctcaaccgactcaacctaaatgtggaacagctagcggaaagaatttggaccctggacaggttcgtggaagctatggaaattacaaacctgccccggctagctcttacaaggctccaccagcccgtctcgtttag.

[0023] As an embodiment, the Puccinia effector protein gene Ps13167 or the protein encoded thereby is used for increasing the resistance of a plant to Puccinia and / or for breeding a Puccinia-resistant plant variety. Ps13167 As an embodiment, the plant can be a plant of the family Poaceae or a wheat plant.

[0024] The present application also provides a biological material for inhibiting the Puccinia effector protein gene Ps13167 or the protein encoded thereby for use in increasing the resistance of a plant to Puccinia and / or for breeding a Puccinia-resistant plant variety. The Puccinia effector protein genePs13167 The amino acid sequence of the encoded protein is shown in SEQ ID NO:2.

[0025] In one embodiment, the biomaterial includes at least one of the following: 1) Host-induced gene silencing of stripe rust effector protein genes Ps13167 Nucleic acid molecules; 2) Interference with stripe rust effector protein genes Ps13167 Expressing nucleic acid molecules; 3) A recombinant vector containing the nucleic acid molecules described in 1) or 2); 4) Engineered bacteria containing the nucleic acid molecules described in 1) or 2); 5) Engineered bacteria containing the recombinant vector described in 3).

[0026] 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.

[0027] 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.

[0028] In one embodiment, the biomaterial includes at least one of the following: A) Contains the stripe rust effector protein gene Ps13167 The expression box; B) Contains the stripe rust effector protein gene Ps13167 Recombinant vectors; C) A recombinant vector containing the expression cassette described in A); D) Contains the stripe rust effector protein gene Ps13167 Engineered bacteria; F) Engineered bacteria containing the expression cassette described in A); G) Engineered bacteria containing the recombinant vector described in B) or C).

[0029] 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.

[0030] 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. Figure 1 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.

[0031] 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.

[0032] The present invention is carried out in accordance with the following embodiments Ps13167 Methods using flowcharts to study secretory protein genes in stripe rust fungi PsEF1the function of the host plant, while creating anti-striper wheat materials.

[0033] Example 1 Based on the qRT-PCR method, it is clear that the stripe rust secreted (effector) protein gene Ps13167 participates in the process of stripe rust infection of the host, the steps are as follows: Extract the wheat stripe rust urediniospores, germ tubes and Fielder wheat at 12, 24, 48, 72 and 120 hpi after inoculation with wheat stripe rust CYR32 12, 24, 48, 72 and 120 hpi. Reverse transcription of RNA to cDNA, using cDNA as template, using stripe rust elongation factor gene Ps13167 as an internal reference, using the specific fragment primer of stripe rust effector protein gene PsEF1 qRT-PCR to determine the expression of stripe rust effector protein gene PsEF1-F in different infection stages of wheat by stripe rust (early infection, living parasitic stage, late infection and sporulation stage).

[0034] Stripe rust elongation factor gene PsEF1-R Internal reference primer and gene quantification primer are as follows.

[0035] Internal reference primer: Forward primer Ps13167 : 5'-ttcgccgtccgtgatatgagacaa-3' (SEQ ID NO: 3); Reverse primer Ps13167 : 5'-atgcgtatcatggtggtggagtga-3' (SEQ ID NO: 4).

[0036] Gene quantification primer: Forward primer Figure 2 -1-qRT-F: 5'-ttcaagtctcggaatgtg-3' (SEQ ID NO: 5); Reverse primer Ps13167-1-qRT-R: 5'-ggcagccgtggtagtt-3' (SEQ ID NO: 6).

[0037] Forward primer Ps13167-2-qRT-F: 5'-ttgccgtcgccatcc-3' (SEQ ID NO: 7); Reverse primer Figure 2 -2-qRT-R: 5'-tgaaacttcccatcactactct-3' (SEQ ID NO: 8).

[0038] The results are shown in Ps13167 , in which U and GT in the abscissa represent urediniospores and germ tubes of stripe rust, respectively.

[0039] ​Ps13167 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.

[0040] Example 2 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: 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: 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: TaPDS -2-vigs-R: 5'-tccgttgctagctgagcggccgctgaaacttcccatcactactct-3' (SEQ ID NO: 12).

[0041] 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.

[0042] 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. Figure 3 (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.

[0043] The results are as follows Ps13167 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 Figure 3 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.

[0044] 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: Ps13167 As 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.

[0045] Example 3 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: 1. Stripe rust effector protein gene Ps13167-RNAi Construction of Silent Transgenic Plants: First, the effector protein gene was transmitted via the Gateway method. Ps13167-RNAiThe specific fragment of the silencing construct was constructed into the RNAi vector PC336 (the RNAi vector PC336 is disclosed in Han X. Cloning and analysis of barley salicylic acid synthesis genes ICS and PAL [D]. Shandong Agricultural University, 2013.).

[0046] Silencing construct primers: forward primer Ps13167 -F: 5'-ggggacaagtttgtacaaaaaagcaggcttcttgccgtcgccatcc-3' (SEQ ID NO: 13), reverse primer Ps13167 RNAi -R: 5'-ggggaccactttgtacaagaaagctgggtctgaaacttcccatcactactct-3' (SEQ ID NO: 14).

[0047] The recombinant plasmid was constructed by using a BP reaction to connect Ps13167RNAi The silencing fragment was connected to the pDONR221 intermediate vector, and the reaction system was as follows: gene fragment 4.0 μL, BP enzyme 0.5 μL, intermediate vector 2.0 μL, buffer 1×TE supplemented to 10 μL. After 25°C connection for 12 h, Escherichia coli DH5α was transformed. The pDONR221 vector used in the present application is a standard pDONR221 plasmid in the Gateway recombination cloning system of Thermo Fisher Scientific Company, which can be publicly purchased from a commercial company.

[0048] Then, the successfully connected Ps13167 -pDONR221 was connected to the final vector PC336 by an LR reaction, and the reaction system was as follows: Ps13167- -pDONR221 1.5 μL, PC336 vector 1.0 μL, LR enzyme 0.5 μL, buffer 1×TE supplemented to 5 μL. After 25°C connection for 12 h, Escherichia coli DH5α was transformed.

[0049] Using Agrobacterium-mediated genetic transformation, transgenic plants of Figure 4 -RNAi were obtained. The silencing efficiency of the transgenic plants of Figure 4 RNAi was detected by qRT-PCR (primers and methods are the same as in Example 1) after inoculation with the stripe rust virulence physiology race CYR32 for 24 h, and the active oxygen content of the plant leaves was determined using a plant active oxygen (ROS) ELISA kit (FANKEW, Fankewei). The results are shown in Ps13167- .

[0050] The transgenic plants of Figure 4From B, it can be concluded that: 14 days after inoculation, compared with Fielder, Ps13167- RNAi-inoculated plants showed leaf necrosis, significantly reduced sporulation, and a significant decrease in stripe rust biomass. Ps13167 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%. Figure 4 RNAi-L5 plants Ps13167- The silencing efficiency is 68%. (By...) Ps13167 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.

[0051] 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.

[0052] 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 Panicum virgatum nsp -6e-R: 5'-ggggaccactttgtacaagaaagctgggtcctaagcgtagtctgggacgtcgtatgggtaaacgagacgggctggtgg-3' (SEQ ID NO: 16).

[0053] 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. Ps13167The 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"). Ps13167- L.) and other monocotspecies”, using Agrobacterium-mediated genetic transformation, obtained Ps13167- -OE transgenic plants. In wild-type Fielder and Figure 4 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]). Ps13167 (A) indicates overexpression of the stripe rust effector protein gene. ​ It significantly reduced wheat resistance to stripe rust, further demonstrating the effectiveness of stripe rust effector protein genes. ​ It exerts its toxic function during the infection of wheat by stripe rust fungi.

[0054] 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. Stripe rust 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.

2. The application according to claim 1, characterized in that, Inhibit stripe rust effector protein genes 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.

3. Inhibition of stripe rust effector protein genes 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; The stripe rust effector protein gene Ps13167 The amino acid sequence of the encoded protein is shown in SEQ ID NO:

2.

4. The application according to claim 3, characterized in that, The biomaterial includes at least one of the following: 1) Host-induced gene silencing of stripe rust effector protein genes Ps13167 Nucleic acid molecules; 2) Interference with stripe rust effector protein genes Ps13167 Expressing nucleic acid molecules; 3) A recombinant vector containing the nucleic acid molecules described in 1) or 2); 4) Engineered bacteria containing the nucleic acid molecules described in 1) or 2); 5) Engineered bacteria containing the recombinant vector described in 3).

5. The application according to claim 4, characterized in that, 1) The host-induced gene silencing of stripe rust effector protein genes 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.

6. The application according to claim 4, characterized in that, 2) The gene interfering with the stripe rust effector protein Ps13167 The nucleic acid molecules were obtained by amplification using the primer pairs shown in SEQ ID NO:13 and SEQ ID NO:

14.

7. Promotes the growth of stripe rust effector protein genes Ps13167 The application of biological materials encoding or expressing proteins thereof 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.

8. The application according to claim 7, characterized in that, The biomaterial includes at least one of the following: A) Contains the stripe rust effector protein gene Ps13167 The expression box; B) Contains the stripe rust effector protein gene Ps13167 Recombinant vectors; C) A recombinant vector containing the expression cassette described in A); D) Contains the stripe rust effector protein gene Ps13167 Engineered bacteria; F) Engineered bacteria containing the expression cassette described in A); G) Engineered bacteria containing the recombinant vector described in B) or C).

9. The application according to any one of claims 1 to 8, characterized in that, The plant mentioned includes wheat.

10. A method for breeding wheat varieties resistant to stripe rust, characterized in that, The steps include: inhibiting the receptor wheat stripe rust effector protein gene. 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.

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