Application of stripe rust effector proteins and their encoding genes in plant breeding or creation

Abstract

The present invention belongs to the field of genetic engineering technology, and relates to the application of stripe rust effector proteins and their encoding genes in plant breeding or creation. The present invention discloses wheat stripe rust effector protein Pst9653, which has the ability to inhibit plant immune response. <h2 style=";text-align:left;direction:ltr">Pst9653 The present invention utilizes Agrobacterium-mediated genetic transformation to obtain genes <h2 style=";text-align:left;direction:ltr"> Pst9553 Overexpressed and silenced transgenic plants, and verified the disease resistance phenotype of transgenic plants to stripe rust. The present invention also proves that the wheat stripe rust effector protein Pst9653 interacts with the host catalase TaCAT3. The present invention enriches the knowledge of those skilled in the art on the regulation of plant immunity by wheat stripe rust, provides ideas for subsequent research on wheat stripe rust resistant varieties and sustainable regulation, and has application value for the design of drug targets for biological control.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering, and relates to the application of stripe rust effector proteins and encoding genes thereof in plant breeding or creation. Background Art

[0002] Wheat stripe rust caused by Puccinia striiformis is a serious disease that threatens wheat production. Therefore, improving wheat stripe rust prevention and control measures and ensuring food safety production have become urgent needs that need to be addressed in agricultural production. Wheat stripe rust forms a highly differentiated specific infection structure-haustorium in the process of infecting wheat. Transcriptome sequencing analysis of wheat stripe rust haustorium, spores and germ tubes has found a large number of effector proteins and proved that haustorium plays an indispensable role in the growth and pathogenicity of wheat stripe rust. Wheat stripe rust can use haustorium to obtain the nutrients it needs from the host. For example, studies have found that the sucrase secreted by the haustorium can decompose the host sucrose to provide hexose for its own sugar absorption process to meet its own growth and development needs, and can secrete toxic factors into the host to affect the plant's immune response. Studies have found that Pst-milR1 secreted by wheat stripe rust has the function of plant miRNA, which regulates the host's immune response by interfering with important disease resistance genes in the host wheat. At present, planting resistant varieties is the most economical, environmentally friendly and effective method to prevent and control wheat stripe rust. However, due to the large-scale planting of a single variety and the easy mutation of wheat stripe rust, the disease-resistant wheat varieties have "lost" their resistance, resulting in a pandemic of wheat stripe rust, which continues to threaten the safety of global wheat production. Therefore, clarifying the variation of wheat stripe rust and its pathogenic mechanism is of great significance for the formulation of a sustainable strategy for the prevention and control of stripe rust. Designing new pest-resistant breeding materials will greatly reduce the spraying of chemical pesticides and lay the foundation for the development of green agriculture. Wheat stripe rust will secrete effector proteins into wheat with the help of its unique haustorium structure to enhance its own infection and colonization ability. Identifying important or key effector protein genes of pathogens and analyzing their participation in regulating the immune response mechanism of the host are hot scientific issues. Studying the effector proteins of wheat stripe rust can deepen the understanding of the interaction between wheat stripe rust and wheat by those skilled in the art, and at the same time can lay the foundation for those skilled in the art to understand the variation mechanism and pathogenic mechanism of wheat stripe rust, which is of great significance for the development of new sustainable and effective prevention and control strategies for wheat stripe rust. Summary of the invention

[0003] During the process of wheat stripe rust infection, wheat stripe rust secretes effector proteins into the host through haustoria to promote its infection. The purpose of the present invention is to provide the application of stripe rust effector proteins and their encoding genes in plant breeding or creation, aiming to enrich the new cognition of those skilled in the art on the inhibition of plant immune response by wheat stripe rust effector proteins, and provide theoretical cognition and gene selection for the breeding and creation of excellent germplasm materials resistant to stripe rust.

[0004] In order to fully understand the technical solution of the present invention without objection, it is necessary to add that the coding gene of the stripe rust effector protein Pst9653 of the present invention is represented by italic font “ Pst9653 " indicates that the stripe rust effector protein Pst9653 is represented by the non-italic font "Pst9653". Of course, a person skilled in the art can clearly and completely understand the meaning and expression of the relevant genes and their encoded proteins based on the description of the present invention.

[0005] On the one hand, the present invention relates to the application of stripe rust effector protein Pst9653 in plant breeding or creation, the amino acid sequence of the stripe rust effect protein Pst9653 is shown in SEQ ID NO: 1, the signal peptide of the stripe rust effect protein Pst9653 has secretion function, and the stripe rust effect protein Pst9653 inhibits the immune function of the host plant.

[0006] SEQ ID NO: 1 is specifically as follows,

[0007] MNTTLYALLSLAVATLSVGAVGQTNQCQFYASVGVKGYTCNERPDIICSEGCKSFVTMTGCVLTQYPKKPATTELCTVGYGRDTAAFKACLTSQGAFRCNGTSTGQATCSGCVPRGDVTWAN.

[0008] Further, in the application provided by the present invention, the stripe rust effector protein Pst9653 is produced by gene Pst9653 coding.

[0009] Furthermore, in the application provided by the present invention, the gene Pst9653 The nucleotide sequence is shown in SEQ ID NO:2.

[0010] SEQ ID NO: 2 is specifically as follows,

[0011] ATGAACACCACTCTTTACGCTCTCCTTTCGCTTGCTGTCGCTACCCTCAGCGTCGGAGCTGTTGGTCAGACCAACCAATGCCAATTCTACGCTTCAGTAGGAGTTAAAGGATACACCTGCAACGAACGACCCGACATCATCTGCTCCGAAGGTTGCAAGTCATTCGTTACCATGACCGGATGCGT TCTTACTCAGTACCCTAAGAAGCCGGCCACCACCGAGCTTTGCACCGTTGGCTACGGACGTGACACTGCCGCTTTCAAGGCCTGTCTCACTAGCCAAGGCGCCTTCCGTTGCAACGGTACTTCCACCGGACAAGCGACTTGCTCCGGTTGCGTTCCCAGAGGCGATGTCACCTGGGCTAATTGA.

[0012] Furthermore, in the application provided by the present invention, the gene Pst9653 The expression is upregulated after wheat is infected by stripe rust.

[0013] Furthermore, in the application provided by the present invention, the gene is overexpressed Pst9653 The host plant's resistance to stripe rust is reduced.

[0014] Furthermore, in the application provided by the present invention, silencing the gene Pst9653 The host plant's resistance to stripe rust is improved.

[0015] Furthermore, in the application provided by the present invention, silencing the gene Pst9653 The specific silencing fragment has a nucleotide sequence as shown in SEQ ID NO:3.

[0016] SEQ ID NO: 3 is specifically as follows,

[0017] GGATGCGTTCTTACTCAGTACCCTAAGAAGCCGGCCACCACCGAGCTTTGCACCGTTGGCTACGGACGTGACACTGCCGCTTTCAAGGCCTGTCTCACTAGCCAAGGCGCCTTCCGTTGCAACGGTACTTCCACCGGACAAGCGA.

[0018] Furthermore, in the application provided by the present invention, the stripe rust effector protein Pst9653 inhibits the cell necrosis reaction induced by the mouse pro-apoptotic factor BAX.

[0019] Furthermore, in the application provided by the present invention, the host plant is wheat.

[0020] Furthermore, in the application provided by the present invention, the stripe rust effector protein Pst9653 interacts with the catalase TaCAT3 of the host plant.

[0021] Those skilled in the art can easily use known methods, such as directed evolution and point mutation methods, to mutate the nucleotide sequence encoding the stripe rust effector protein Pst9653 of the present invention. Pst9653 As long as the nucleotides with 75% or higher identity to the nucleotide sequence of the present invention encode the stripe rust effector protein Pst9653 and have the same function, they are derived from the nucleotide sequence of the present invention and are equivalent to the sequence of the present invention.

[0022] The term "identity" as used herein refers to sequence similarity to a natural nucleic acid sequence. "Identity" includes nucleotide sequences that have 75% or more, or 85% or more, or 90% or more, or 95% or more identity to the nucleotide sequence of the present invention encoding the protein consisting of the amino acid sequence shown in SEQ ID NO: 1. Identity can be evaluated by the naked eye or by computer software. Using computer software, the identity between two or more sequences can be expressed as a percentage (%), which can be used to evaluate the identity between related sequences.

[0023] The above-mentioned 75% or more identity may be 80%, 85%, 90% or 95% or more identity.

[0024] Those skilled in the art can easily use known methods, such as gene Pst9653 Gene silencing cassette Pst9653 The product, the expression cassette (gene Pst9653 Expression cassette) refers to DNA that can express stripe rust effector protein Pst9653 in host cells, which can include not only the promoter gene Pst9653 The promoter of transcription may also include the terminator gene Pst9653 The expression cassette may further include an enhancer sequence. The promoters that can be used in the present invention include, but are not limited to, constitutive promoters; tissue, organ and development-specific promoters and inducible promoters. Suitable transcription terminators include, but are not limited to, Agrobacterium nopaline synthase terminator (NOS terminator), cauliflower mosaic virus CaMV 35S terminator, tml terminator, pea rbcS E9 terminator and nopaline and octopine synthase terminators.

[0025] Existing expression vectors can be used to construct Pst9653 Recombinant vector of expression cassette. The plant expression vector includes binary Agrobacterium vector and vector that can be used for plant microprojectile bombardment, etc. Such as pAHC25, pBin438, pCAMBIA1302, pCAMBIA2300, pCAMBIA2301, pCAMBIA1305, pCAMBIA1300, pBI121, pCAMBIA1391-Xa or pCAMBIA1391-Xb (CAMBIA Company), etc. The plant expression vector may also contain the 3' non-translated region of the foreign gene, i.e., a polyadenylic acid signal and any other DNA fragments involved in mRNA processing or gene expression. The polyadenylic acid signal can guide the addition of polyadenylic acid to the 3' end of the mRNA precursor, such as the Agrobacterium crown gall induction (Ti) plasmid gene (such as the nopaline synthase gene Nos), the 3' non-translated region of the plant gene (such as the soybean storage protein gene) has similar functions. When using the gene construction plant expression vector of the present invention, enhancers can also be used, including translation enhancers or transcription enhancers. These enhancer regions can be ATG start codons or adjacent region start codons, etc., but must be identical to the reading frame of the coding sequence to ensure the correct translation of the entire sequence. The source of the translation control signal and the start codon is extensive and can be natural or synthetic. The translation initiation region can come from a transcription initiation region or a structural gene. In order to facilitate the identification and screening of transgenic plant cells or plants, the plant expression vectors used can be processed, such as adding genes that can be expressed in plants and encode enzymes or luminescent compounds that can produce color changes (GUS gene, luciferase gene, etc.), antibiotic marker genes (such as nptII gene that confers resistance to kanamycin and related antibiotics, bar gene that confers resistance to the herbicide phosphinothricin, hph gene that confers resistance to the antibiotic hygromycin, dhfr gene that confers resistance to methotrexate, EPSPS gene that confers resistance to glyphosate), or chemical resistance marker genes (such as herbicide resistance genes), mannose-6-phosphate isomerase gene that provides the ability to metabolize mannose. Considering the safety of transgenic plants, no selective marker genes can be added, and transformed plants can be directly screened by adversity.

[0026] The above-mentioned vector or expression vector can be a plasmid, cosmid, phage or virus vector.

[0027] The microorganisms may be yeast, bacteria, algae or fungi, such as Agrobacterium.

[0028] The above transgenic plant cell lines do not include propagation materials.

[0029] The above disease resistance is resistance to wheat stripe rust.

[0030] Compared with the prior art, the technical solution provided by the present invention has at least the following beneficial effects or advantages:

[0031] (1) The stripe rust effector protein Pst9653 provided by the present invention is the first reported effector protein in wheat stripe rust that can directly target the host catalase TaCAT3 gene related to active oxygen scavenging to regulate plant immunity. By creating a silent plant expressing the stripe rust effector protein Pst9653 with the help of host wheat, the expression of the effector protein is inhibited when the stripe rust infects wheat, and the inoculation of the stripe rust compatible physiological race CYR31 will lead to a significant reduction in the spore production of the silent plant and an increase in immune necrotic cells.

[0032] (2) Pst9653 The transgenic plants showed resistance to wheat stripe rust. Therefore, the stripe rust effector protein Pst9653 can be used to create rust-resistant wheat lines, providing excellent materials for the breeding of wheat stripe rust-resistant varieties.

[0033] (3) The effector protein gene provided by the present invention is highly conserved in different subspecies, laying the foundation for the creation of persistent and broad-spectrum disease-resistant wheat varieties. At the same time, it has important reference significance for the target research of new biological control drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0035] Picture 1 The stripe rust effector protein gene provided by the embodiment of the present invention Pst9653 Analysis of expression levels at different times after stripe rust race CYR31 infected wheat. Picture 1 The data are the mean ± SEM of three biological replicates.

[0036] Picture 2 The stripe rust effector protein gene provided by the embodiment of the present invention Pst9653 Signal peptide secretion function validation diagram. Among them, CMD-W and YPRAA represent different culture media, YTK12, pSUC2, Mg87, Avr1b and Pst9653 represent control combination experimental groups, TPF represents 1,3,5-tribenzaldehyde, and TTC represents 2,3,5-triphenyltetrazolium chloride.

[0037] Picture 3 Effector protein gene Pst9653Figure 2. Inhibition of cell necrosis caused by BAX. BAX indicates mouse pro-apoptotic factor, GFP indicates green fluorescent protein, and + indicates co-injection.

[0038] Picture 4 The phenotypes of wild-type control and transgenic silenced plants inoculated with stripe rust race CYR31, respectively. Fielder is the wild-type control, and L23 and L24 represent the two transgenic lines 23 and 24 of the T3 generation.

[0039] Picture 5 The interaction map of Pst9653 and TaCAT3 was verified by yeast two-hybrid experiment. SV40 large T-antigen (T) and murine p53 (53) were used as positive controls, SV40 large T-antigen (T) and humanlamin C (Lam) were used as negative controls, L represents leucine, W represents tryptophan, H represents histidine, A represents adenine, and X-α-gal represents 5-bromo-4-chloro-3-indole-α-D-galactopyranoside. DETAILED DESCRIPTION

[0040] The technical scheme of the present invention is described below in conjunction with the examples, but the present invention is not limited to the following examples. The experimental methods and detection methods described in each example are conventional methods unless otherwise specified; the reagents and materials are available on the market unless otherwise specified, and the % in the following examples are all mass percentages unless otherwise specified.

[0041] Example 1

[0042] This example provides the stripe rust effector protein gene after the stripe rust race CYR31 infects wheat. Pst9653 Expression analysis at different time points.

[0043] 1. Acquisition of Stripe Rust Real-time Fluorescence Quantitative PCR Samples

[0044] Wheat seeds (water source 11) were planted in a 10 cm diameter pot and cultured under a 16 / 8 h light / dark cycle and a temperature of 16 ℃. When the wheat seedlings grew to the one-leaf-one-heart stage, fresh wheat stripe rust summer spores were diluted with electronic fluoride liquid, and 10 μL of the diluted spore solution was evenly smeared and inoculated on the wheat leaves with a pipette. The inoculated wheat seedlings were kept in the dark at 12 ℃ for 36 h and then continued to be cultured under normal light conditions. After the wheat leaves were inoculated, they were sampled at 6 h, 18 h, 24 h, 36 h, 48 h, 72 h, 120 h, 168 h, 216 h, and 264 h as samples for real-time fluorescence quantitative PCR. Each group of samples selected leaves from at least three different wheat seedlings, and the middle area of ​​the inoculated leaves was mainly collected. After sampling, they were immediately frozen in liquid nitrogen and then transferred to an ultra-low temperature refrigerator for storage.

[0045] 2. Preparation of Stripe Rust Template

[0046] The collected samples were used to prepare cDNA templates for real-time fluorescence quantitative detection of stripe rust using RNA extraction kit and reverse transcription kit (Quanshijin Biotechnology Co., Ltd.) according to the operating instructions.

[0047] 3. Real-time fluorescence quantitative PCR detection

[0048] Elongation factor gene of Puccinia striiformis PstEF As an internal reference gene, the relative quantitative method was used to analyze the gene expression.

[0049] The sequences of the internal reference gene primers are:

[0050] PstEF-F: TTCGCCGTCCGTGATATGAACAA;

[0051] PstEF-R:ATGCGTATCATGGTGGTGGAGTGA.

[0052] Pst9653-specific primers were designed using Primer Premier 5.0 software. The sequences of Pst9653-specific primers are as follows:

[0053] DL-9653-F: CTTGCTGTCGCTACCCTCA;

[0054] DL-9653-R: AGTAAGAACGCATCCGGTCAT.

[0055] UltraSYBR One Step RT-qPCR Kit (Kangwei Century Biotechnology Co., Ltd.) was used according to the instructions, and the relevant detection was completed using the Bio-Rad iQ5 real-time fluorescence quantitative PCR instrument. The relative expression of different genes was based on 2 -ΔΔCt law, PstEF The expression level was calculated as a reference.

[0056] The results are as follows Picture 1 As shown in the figure, when the stripe rust race CYR31 infected wheat for 6 h, the effector protein gene Pst9653 A large amount of expression, effector protein gene at 48 h Pst25662 The highest expression level. Pst9653 It is secreted into the host to exert its effects in the early stages of stripe rust infection.

[0057] Example 2

[0058] This example provides a test to verify the signal peptide secretion function of the effector protein Pst25662 in cells.

[0059] 1. Recombinant plasmid pSUC2- Pst9653 sp Construction

[0060] According to the predicted signal peptide sequence of Pst9653, the amplification was designed using Primer 5.0 software. Pst9653 sp According to the principle of one-step cloning, a homology arm CGGAATTTTAATTAA was added to the 5' end of the designed forward primer. GATTC ( Eco RI) and the designed reverse primer 5' end with homology arm CACTATAGGGAGAAC CTCGAG ( Xho I) The primer sequences are:

[0061] Pst9653-pSUC2-F CGGAATTTTAATTAAGAATTCATGAACACCACTCTTTACG

[0062] Pst9653-pSUC2-R CACTATAGGGAGAACCTCGAGACCAACAGTCCCGACGCT

[0063] Using the cDNA of wheat leaves infected with stripe rust fungus CYR31 as a template, PCR amplification was performed in a PCR instrument according to the following system and program:

[0064]

[0065] 98℃ for 10 seconds, 58℃ for 15 seconds, 72℃ for 5 seconds, 40 cycles; 72℃ for 10 minutes.

[0066] The amplified PCR products were detected by agarose gel electrophoresis and recovered.

[0067] First, follow the following system

[0068]

[0069] pSUC2 was digested on a PCR instrument at 37°C for 2 to 5 hours, and the digested vector plasmid was detected by electrophoresis and recovered on gel.

[0070] Then according to the following system

[0071]

[0072] At 37°C for 30 minutes, the gel-recovered product of the gene was connected to the enzyme-cut pSUC2 (ampicillin resistance) on a PCR instrument.

[0073] 2. Verification of secretion function

[0074] The recombinant plasmid was transformed into the mutant yeast strain YTK12 as follows:

[0075] In the clean bench, a small amount of the preserved YTK12 yeast solution was taken and added to 4 mL of YPD liquid medium containing 2% glucose. The shaking incubator was set to 30°C, 220 rpm, and cultured for more than 24 hours to activate the YTK12 strain.

[0076] When the yeast liquid OD 600 =2, transfer the activated yeast solution to YPD culture medium containing 2% glucose at a ratio of 1:10, set the shaker at 30˚C, 220 rpm, and culture for 4-5 hours until the yeast solution concentration reaches OD 600 =0.6~0.8.

[0077] Use a sterile 2 mL centrifuge tube to collect the yeast solution, centrifuge at 7000 rpm for 3 minutes, discard the supernatant, wash the bacteria once with sterile ddH2O, repeat this step once, and wash the YPD culture solution on the bacteria as clean as possible.

[0078] Add 100 μL of PEG-LiAc transformation solution to the centrifuge tube and gently resuspend the bacteria, and let it stand for 15 minutes. The above steps are for the preparation of yeast competent cells.

[0079] Add the following components to the centrifuge tube in order:

[0080]

[0081] The shaker was set at 30°C, 220 rpm for 30 minutes, and then incubated in a 42°C water bath for 15 minutes. The culture was removed from the water bath and gently inverted several times to mix.

[0082] Centrifuge at 5500 rpm for 45 seconds, discard the supernatant, add 500 μL sterile ddH2O to resuspend the cells, centrifuge at 4500 rpm for 30 seconds, discard the supernatant, repeat once. Add 100 μL sterile ddH2O to resuspend the cells, and spread on the corresponding solid culture dish.

[0083] The culture dish was placed upside down in a 30°C constant temperature incubator and cultured for more than 36 hours.

[0084] According to the growth of bacterial plaques, a number of positive monoclonal colonies were selected for PCR amplification and agarose gel electrophoresis identification. Positive clones that met the expected size were selected and propagated with yeast culture medium, and then the strains were preserved with 20% glycerol.

[0085] The same method was used to transfer pSUC2, the signal peptide of the positive control soybean blast fungus protein PsAvR1b, and the first 25 amino acids of the negative control rice blast fungus protein MoMg87 into the YTK12 strain, and the same method was used to expand and culture and store them for future use.

[0086] 3. The secretion function was verified by the plate coating method and TTC colorimetric method. The steps are as follows:

[0087] Will transfer Pst9653 sp -pSUC2, PsAvR1b sp -pSUC2, MoMg87 sp -pSUC2 and pSUC2 plasmids and YTK12 yeast strain were activated and cultured with corresponding culture medium.

[0088] The activated bacterial solution was collected in 2 mL sterile centrifuge tubes, centrifuged at 7000 rpm for 3 minutes, and washed with sterile ddH2O several times.

[0089] Divide into 2 portions, measure the concentration of one of them, and dilute it to the same concentration with sterile ddH2O, spread it on the culture dishes of CMD-W and YPRAA, put the culture dishes upside down in a constant temperature incubator at 30℃, culture for more than 48 hours, and take pictures to record the growth conditions.

[0090] In the other portion, add the following components in sequence and resuspend:

[0091]

[0092] Incubate in a 37°C water bath for 15 minutes.

[0093] After centrifugation at 13,000 rpm for 15 minutes, aspirate the supernatant and add it to a graduated test tube containing 0.1% TTC, wait for a few minutes, and take a photo to record the color change.

[0094] The results are as follows Picture 2 The conversion shown Pst9653 sp -pSUC2 and PsAvR1b sp -pSUC2 YTK12 yeast strain can grow in YPRAA and CMD-W medium and can reduce TTC to red insoluble TPF. MoMg87 sp -pSUC2, pSUC2-derived YTK12 strain, and YTK12 strain could not grow on CMD-W and YPRAA medium, respectively, and could not reduce TTC either. These results indicate that the signal peptide of Pst9653 has secretion function.

[0095] Example 3

[0096] This example provides a validation test for the effector function of protein Pst9653.

[0097] 1. Recombinant plasmid pGR107- Pst9653 ∆sp Construction

[0098] Design Amplification Pst9653 ∆sp The primers of , and the subsequent construction steps refer to the content of Example 2. The primer sequences are:

[0099] Pst9653-PVX-F CACCAGCTAGCATCGATTCCCGGGATGCAGACCAACCA

[0100] Pst9653-PVX-R GGAACATCGTATGGGTACGCGGCCGCATTAGCCCAGGTGACAT

[0101] 2. Tobacco Overexpression Pst9653

[0102] The recombinant plasmid is transformed into Agrobacterium, and Agrobacterium infects tobacco.

[0103] After 24 hours, the same method was used to treat Bax Inject the Agrobacterium solution at the marked positions. After 3 to 5 days, observe the phenotype and take photos to record.

[0104] The tobacco leaf samples from the previous step were collected, first decolorized, and then dyed with trypan blue dye, the steps are as follows:

[0105] Prepare a 4% solution of trypan blue powder and mix it with anhydrous ethanol in equal proportions before use.

[0106] Place the decolorized tobacco leaves in the trypan blue dye solution prepared in the previous step and perform dyeing on a horizontal shaker at room temperature.

[0107] After staining, replace the trypan blue dye solution with ddH2O to clean the surface. After staining, the dead cells of the tobacco leaves are dyed a distinct blue, while the living cells remain colorless and transparent. Take pictures to record the phenotype.

[0108] The results are as follows Picture 3 As shown, the injection contains Pst9653 ∆sp Bacterial liquid and Bax The bacterial solution did not produce cell necrosis. GFP Bacterial liquid and Bax Partial and separate injection of bacterial solution containing Bax Part of the bacterial liquid produced necrosis, and Pst9653 had the function of inhibiting cell necrosis caused by BAX, which indicated that Pst9653 had the ability to inhibit plant immune response.

[0109] Example 4

[0110] This embodiment provides Pst9653 Identification experiment of gene silenced transgenic plants' resistance to wheat stripe rust.

[0111] 1. Construction of transgenic vector

[0112] The ORF region of the stripe rust effector protein Pst9653 gene sequence was compared with the homologous genes of wheat and wheat stripe rust, and the Pst9653 specific fragment sequence was selected for the construction of the transgenic vector, and the nucleotide sequence of the Pst9653 gene silencing fragment was (as shown in SEQ ID NO: 3). The selected target fragments were respectively inserted into two positions of the pC336 vector by homologous recombination (one fragment was inserted forward and the other was inserted in reverse complementation, so as to form a hairpin structure), and the pC336-9653 plasmid was obtained.

[0113] The primer sequence containing the attB site is:

[0114] attB-9653-F:

[0115] GGGGACAAGTTTGTACAAAAAAGCAGGCTATATGCAGACCAACCAATGC;

[0116] attB-9653-R:

[0117] GGGGACCACTTTGTACAAGAAAGCTGGGTATCAATTAGCCCAGGTGACAT.

[0118] 2. Transformation of Agrobacterium

[0119] Take out the EHA105 competent cells stored in the ultra-low temperature refrigerator and melt them on ice, add the correctly sequenced pC336-Pst9653 plasmid, ice bath for 30 min, liquid nitrogen for 2 min, 37 ℃ water bath for 3 min, ice bath for 5 min, add appropriate amount of liquid LB medium, and shake culture for 3 h; take 150 μL of the transformation bacterial liquid and spread it on a solid plate (rifampicin and Kana antibiotics), invert and culture in the dark until positive transformants grow.

[0120] Using Agrobacterium-mediated wheat transformation technology, the recombinant silencing vector pC336-Pst9653 was transferred into the wheat variety Fielder material to obtain RNAi silenced plants. DNA testing was performed on the obtained plants, and positive plants were retained for subsequent verification.

[0121] 3. Phenotypic Identification of Transgenic Plants

[0122] Two T3 transgenic homozygous strains were selected for disease resistance testing and inoculated with wheat stripe rust CYR31 in the same manner as in Example 1. Phenotypic identification was performed after sporulation of wheat leaves, and photographs were taken for record.

[0123] The results are as follows Picture 4 As shown, the spore production of stripe rust races on the silenced plants inoculated with wheat stripe rust CYR31 was significantly reduced, indicating that the stripe rust effector protein Pst9653 proposed in the present invention plays a role as a key effector protein in the infection process.

[0124] Example 5

[0125] This embodiment provides Pst9653 Genetic screening for interacting proteins.

[0126] 1. Pst9653 Construction of bait vector

[0127] Design Amplification Pst9653 ∆sp The primers of the invention are used, and the joints need to be changed to the homology arms corresponding to the vector to be connected. The remaining construction steps refer to the contents of Example 2. The primer sequences are:

[0128] Pst9653-pBD-FGAGGACCTGCATATGATGCAGACCAACCA;

[0129] Pst9653-pBD-RGATCCCGGGAATTCTCAATTAGCCCAGGTGA.

[0130] 2. The recombinant plasmid was transformed into yeast strain AH109. The transformation steps were as described in Example 2.

[0131] 3. Pst9653 ∆sp -Verification of self-activation of pBD bait vector

[0132] will contain Pst9653 ∆sp -pBD (pGBKT7) AH109 yeast strain was prepared into competent state, transformed with pAD (pGADT7), and finally spread on two-deficiency (-LW) and three-deficiency (-LWH) culture medium, respectively. After cultivation, the growth of colonies on the two-deficiency and three-deficiency plates was observed to determine whether there was self-activation.

[0133] 4. Yeast two-hybrid screening library

[0134] The constructed bait vector was used to screen the yeast library for the interaction between wheat and stripe rust. The steps are as follows:

[0135] Perform sterile operation in a clean bench and transfer the Pst9653 ∆sp -pBD yeast solution was activated with corresponding liquid culture medium plus glucose. The shaker was set at 30°C and 220 rpm. After culturing for 2 days, the activated bacterial solution was transferred to 50 mL of liquid culture medium at a ratio of 1:500 and continued to be cultured until the bacterial solution concentration OD 600 ≈0.2.

[0136] The bacterial suspension obtained in the previous step was transferred to 100 mL of liquid culture medium at a ratio of 1:100 and cultured at 30°C and 220 rpm until OD 600 ≈0.6, enrich the yeast solution in a sterile 50 mL centrifuge tube, centrifuge at 5000 rpm for 5 minutes, and gently resuspend in 25 mL sterile ddH2O until there is no precipitation.

[0137] Centrifuge the yeast solution at 5000 rpm for 5 minutes, gently suspend the cells with 1.5 mL PEG-LiAc conversion solution until there is no precipitation, then transfer the yeast solution to a sterile 2 mL centrifuge tube and centrifuge at 5000 rpm for 1 minute. Suspend the cells with 500 μL PEG-LiAc conversion solution until there is no precipitation, then transfer the yeast solution to a sterile 15 mL centrifuge tube and let it stand for 10 minutes.

[0138] Add the following components to a 15 mL centrifuge tube in order:

[0139]

[0140] The shaker was set at 30°C, 220 rpm, and incubated for 40 minutes. 150 μL of DMSO was added and placed in a 42°C water bath for 20 minutes. Every 10 minutes, the tube was taken out of the water bath and gently inverted several times to mix. Centrifuged at 5000 rpm for 5 minutes, the supernatant was discarded, 4 mL of liquid culture medium was added, and the shaker was set at 30°C, 220 rpm, and incubated for 90 minutes.

[0141] 5000 rpm, centrifuge for 5 minutes, discard the supernatant, add 3 mL of sterile 0.9% NaCl solution to resuspend the bacteria. Take a little of the treated bacterial solution and dilute it 10 times and 100 times, respectively, and spread it on the two-deficient solid culture medium (-LW) to observe the success rate of this yeast transformation experiment. Spread the remaining bacterial solution on the three-deficient solid culture medium (-LWH) at a volume of 100 μL per plate, and invert the culture dish in a 30℃ constant temperature incubator and culture for more than 48 hours.

[0142] Take out all the plates, first observe the transformation efficiency of the two two-deficiency plates, then pick all the yeast colonies growing on the three-deficiency plates, streak them all onto the four-deficiency solid culture medium (-LWHA), invert them in a 30℃ constant temperature incubator, and culture for more than 48 hours.

[0143] Take out all plates, pick up all yeast colonies still growing on the four-deficient plates, streak all of them onto the four-deficient solid culture medium with the addition of a chromogenic substrate (20 μg / mL X-α-gal), invert them in a 30°C constant temperature incubator, and culture for more than 48 hours.

[0144] All plates were taken out, and all blue colonies were potential interacting target genes of Pst9653. Single yeast colonies were picked for sequencing, and the sequencing results were compared by BLAST in the NCBI database, and the results were further analyzed and verified.

[0145] 3. Reversion Verification of Yeast Two-hybrid System

[0146] According to the BLAST results of the potential interaction target genes, specific primers for amplifying the ORF of the potential interaction target genes were designed using Primer 5.0, and a homology arm GGAGGCCAGT was added to the 5' end of the designed forward primer. GATTC ( Eco RI) and the reverse primer 5' end with a homology arm CACCCGGGTG GATTC ( Eco RI), and the remaining construction steps refer to the contents of Example 2. The primer sequences are:

[0147] TaCAT3-pAD-F:

[0148] GAGGCCAGTGAATTCATGGATCCCTACAAGCACCGG;

[0149] TaCAT3-pAD-R:

[0150] ACCCGGGTGGAATTCCTACATGCTCGGCTTAATTCTGA.

[0151] will contain Pst9653 ∆sp -pBD (pGBKT7) AH109 yeast strain was prepared into competent cells, and the recombinant plasmid containing the candidate target gene constructed in the previous step was transformed into the prepared competent cells. The preparation and transformation steps of competent cells refer to the above part. They were spread on two-deficient (-LW) and three-deficient (-LWH) solid culture media respectively. After cultivation, the colony growth on the two-deficient and three-deficient plates was observed, and it was decided whether to streak on the four-deficient culture media and the solid culture media with chromogenic substrates according to the situation.

[0152] The results are as follows Picture 5 As shown, the negative control group could not grow on the three-deficient and four-deficient plates, while the positive control group and the experimental group could grow normally on the three-deficient and four-deficient plates and could make the color substrate X- α -gal turns blue, and the experiment finally confirms that Pst9653 interacts with TaCAT3 in the yeast two-hybrid system.

[0153] In summary, the stripe rust effector protein Pst9653 proposed in the present invention plays a key role in the process of stripe rust infection of wheat, which is mainly reflected in its ability to suppress plant immunity and maintain high expression throughout the infection stage. By constructing transgenic RNAi plants to silence the expression of this gene, a significant disease resistance response is produced to wheat stripe rust, which has a reference significance for the creation of durable disease-resistant materials. At the same time, the stripe rust effector protein Pst9653 interacts with the host catalase TaCAT3, clarifying the mechanism of wheat stripe rust effector protein Pst9653 regulating host immunity, providing a theoretical basis and new ideas for the prevention and control of wheat stripe rust.

[0154] As described above, the basic principle, main features and advantages of the present invention are well described. The above embodiments and descriptions are only descriptions of the preferred implementation modes of the present invention, and the present invention is not limited by the above embodiments. Without departing from the spirit and scope of the present invention, various changes and improvements made by ordinary technicians in this field to the technical solution of the present invention should fall within the protection scope determined by the present invention.

Claims

1. Application of stripe rust effector protein Pst9653 in plant breeding or creation, characterized in that: The expression of the stripe rust effect protein Pst9653 is inhibited to improve the resistance of plants to stripe rust. The amino acid sequence of the stripe rust effect protein Pst9653 is shown in SEQ ID NO:

1. The signal peptide of the stripe rust effect protein Pst9653 has a secretion function. The stripe rust effect protein Pst9653 inhibits the immune function of the plant. The plant is wheat.

2. The use according to claim 1, characterized in that: The stripe rust effector protein Pst9653 is expressed by the gene Pst9653 Encoding, the gene Pst9653 The nucleotide sequence is shown in SEQ ID NO:

2.

3. The use according to claim 2, characterized in that: Silencing the gene Pst9653 The resistance of the plant to stripe rust is improved.

4. The use according to claim 3, characterized in that: Silencing the gene Pst9653 The specific silencing fragment has a nucleotide sequence as shown in SEQ ID NO:

3.

5. The use according to claim 1, characterized in that: The stripe rust effector protein Pst9653 inhibits the cell necrosis response induced by the mouse pro-apoptotic factor BAX.

6. The use according to claim 1, characterized in that: The stripe rust effector protein Pst9653 interacts with the plant's catalase TaCAT3.

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