CN118256519B

CN118256519B - Application of disease-resistant related protein TaMYB22 in regulation and control of plant stripe rust resistance

Info

Publication number: CN118256519B
Application number: CN202410657082.0A
Authority: CN
Inventors: 郭军; 王策; 何付新; 孙慧琳; 柏星轩; 郭嘉; 高月圆
Original assignee: Shenzhen Research Institute Of Northwest University Of Agriculture And Forestry Science And Technology
Current assignee: Shenzhen Research Institute Of Northwest University Of Agriculture And Forestry Science And Technology
Priority date: 2024-05-25
Filing date: 2024-05-25
Publication date: 2024-08-23
Anticipated expiration: 2044-05-25
Also published as: CN118256519A

Abstract

The invention discloses application of disease-resistant related protein TaMYB22 in regulation and control of plant stripe rust resistance, and belongs to the technical field of genetic engineering. The disease resistance related protein is TaMYB22, and the amino acid sequence is shown in SEQ ID NO:2, the TaMYB22 gene for encoding the disease-resistant related protein is shown as SEQ ID NO: 1. The gene TaMYB22 of the invention shows a down-regulating expression trend in the interaction process of wheat and stripe rust bacteria, and the encoded protein is distributed in the cell nucleus. The inhibition of the expression of the TaMYB22 protein can weaken the disease resistance of the plant, and the disease resistance of the TaMYB22 transient silencing plant obtained by utilizing the VIGS to the stripe rust is reduced. The disease resistance related protein TaMYB22 and the coding gene thereof play an important role in cultivating plant breeding with enhanced stripe rust resistance.

Description

Application of disease-resistant related protein TaMYB22 in regulation and control of plant stripe rust resistance

Technical Field

The invention belongs to the technical field of genetic engineering, and particularly relates to application of disease resistance related protein TaMYB22 in regulation and control of plant stripe rust resistance.

Background

Wheat stripe rust is one of the most important diseases in wheat production, and has strong popularity, wide distribution and serious harm. For a long time, because the stripe rust bacteria can not be cultivated in vitro and can not be genetically transformed, the wheat heterohexaploid genome is huge and complex, and the like, the research of the stripe rust disease is relatively lagged, and the disease prevention and control are in a passive state. The reasonable utilization of disease-resistant varieties is the most economical and environment-friendly measure for preventing and controlling stripe rust. However, the problems of frequent toxicity variation of the rust bacteria, lack of disease-resistant resources of wheat and the like lead to great challenges of traditional rust-resistant breeding. In recent years, with the appearance and development of the wheat species 32, 33 and V26 in wheat stripe rust, most of the main cultivars in wheat production have lost stripe rust resistance, so that the wheat is again in danger of stripe rust pandemic hazard. Therefore, the development of new yellow rust resistance gene resources of wheat, the development of disease resistance mechanism research mediated by disease resistance factors and the important significance for the durable green prevention and control of the yellow rust of wheat are urgently needed.

MYB transcription factors, as one of a family of transcription factors widely distributed in plants, are involved in plant growth and stress response by binding to cis-acting elements in target gene promoters to regulate gene expression. MYB transcription factors have highly conserved DNA binding domains, termed MYB domains. Typically, the MYB domain comprises 1-4 amino acid sequence repeats (IMPERFECT AMINO ACID SEQUENCE REPEATS, rs) that are not perfectly repeated in tandem. 393R 2R3-MYB and 12 3R MYB were identified in wheat. The identification of MYB transcription factors in plants greatly motivates the study of MYB transcription factor function.

MYB plays an important role in coping with abiotic stresses such as drought, salt and cold stresses, including: through regulating the synthesis of flavonoid compounds and horny layer, the plant is involved in drought tolerance related to leaf permeability; regulation of salt tolerance by regulation of stratum corneum formation and antioxidant defenses; the regulation of salt tolerance is participated by regulating genes in ABA signals; the expression level of the key transcription factor CBF (C-repeat-binding factors) involved in regulating the response of cold stress plays a role in cold resistance. Functional studies of MYB transcription factors in plants responding to biotic stress have become a new hot spot in recent years. MYB can improve the defense level against pathogenic bacteria by regulating the accumulation of various metabolites such as anthocyanin, wax, ferulic acid, lignin and the like. In wheat, a number of MYB transcription factors have been identified as being involved in the wheat's defensive response to pathogenic bacteria. Although a number of MYB transcription factors have been identified in wheat that regulate wheat resistance to stripe rust, the mechanism of action of MYB transcription factors in wheat interaction with stripe rust is not clear.

Disclosure of Invention

The invention aims to provide application of an anti-disease related protein TaMYB22 in regulating and controlling plant stripe rust resistance.

The invention provides a stripe rust disease-resistant related protein gene TaMYB22, which is derived from a wheat variety water source 11 (Triticum aestivum). The nucleotide sequence of the gene TaMYB22 is shown in SEQ ID NO:1, the gene TaMYB22 is induced to express by the rust bacteria.

Further, the invention provides an anti-disease related protein TaMYB22, wherein the protein TaMYB2 is encoded by the gene TaMYB22 and has the amino acid sequence shown in SEQ ID NO:2, and a polypeptide having the amino acid sequence shown in 2.

Further, the invention relates to an expression vector, which comprises the steps of constructing an expression vector containing a gene TaMYB22 specific gene fragment, wherein the sequence of the gene TaMYB22 specific gene fragment is shown in SEQ ID NO:3 or SEQ ID NO: 4.

Furthermore, the invention utilizes plant genetic engineering technology to transform the specific gene fragment of the disease-resistant related protein gene TaMYB22 into wheat cells to obtain the wheat variety for silencing the gene TaMYB 22.

The invention also provides the gene TaMYB22 and the coding protein thereof, or the application of the expression vector in the cultivation of rust-resistant plants.

As a preference for the application, silencing the gene TaMYB22, or silencing a specific gene fragment of the gene TaMYB22, or inhibiting expression of effector protein TaMYB22, can reduce the resistance of plants to Rhizoctonia.

Compared with the prior art, the technical scheme provided by the invention has at least the following beneficial effects or advantages:

The invention provides application of disease-resistant related protein TaMYB22 in regulation and control of plant stripe rust resistance. According to the invention, RT-PCR is utilized to detect the induction expression condition of the gene TaMYB22 by the rust stripe, and the fact that the TaMYB22 shows up-regulation expression trend in the early stage (6-24 h) and the later stage (72-120 h) of rust stripe infection in the non-affinity and affinity combination is found, so that the gene TaMYB22 is induced to be expressed by the rust stripe of wheat.

The invention suppresses the expression of the disease-resistant related protein TaMYB22 by silencing the gene TaMYB22 by means of genetic engineering technology, and obviously reduces the stripe rust resistance of plants. The protein and the gene provided by the invention provide a foundation for controlling the stripe rust, and play an important role in cultivating plants with enhanced stripe rust disease resistance.

The invention provides a wheat stripe rust resistant variety cultivation method. The method is to inhibit or weaken the transfer of a plant disease-resistant related protein gene TaMYB22 (specific fragment) into a wheat material to obtain a wheat variety for silencing the plant disease-resistant related protein gene TaMYB 22. Proved by verification, the transgenic wheat has reduced resistance to the main epidemic race CYR32 of the rust.

Drawings

FIG. 1 shows the expression profile of TaMYB22 in wheat and Rhizoctonia cerealis interaction combinations.

FIG. 2 shows the localization of TaMYB22 in wheat protoplasts.

FIG. 3 is a block diagram of the alpha, beta and gamma chains in the BSMV-VIGS vector system. Wherein TaMYB22-1S (2S) represents a TaMYB22 silencing sequence.

FIG. 4 shows that transiently silencing TaMYB22 reduces wheat resistance to Rhizoctonia cerealis. Wherein, the A graph shows the relative expression quantity of TaMYB22 in the silent plant and the control plant after the inoculation of the rust bacteria; panel B shows the 15d leaf phenotype of the silencing plants and the control plants after leaf inoculation with Rumex sp CYR 23; panel C shows the number of spore stacks per unit area of Rumex tigrinus in TaMYB22 silenced leaves, normalized to 1 for Rumex tigrinus biomass in control leaves; taEF-1α gene was used for normalization; values represent mean ± standard error (n=3); significance analysis was performed using one-way anova followed by Tukey test, with asterisks indicating the presence of significant differences (< 0.05;, < 0.01).

FIG. 5 is a histological observation of wheat inoculated with Rumex rhzomorphus CYR23 leaf after transient silencing of TaMYB 22. Wherein, the graph A shows that the growth condition of the hyphae of the rust bacteria is observed in the inoculation period of 120 hours, the SV is the air hole lower sac, and the IH is the infection hyphae; the diagram B is statistical analysis of the bacterial colony area of the rust bacteria inoculated for 120h, and the diagram C is observation of the accumulation condition of the rust bacteria inoculated for 72h H ₂O₂; panel D is cumulative area statistics for inoculated rust 72h H ₂O₂; e, observing the necrosis of the cells inoculated with the stripe rust for 72 hours; panel F is statistical analysis of necrotic areas; the statistic w value represents the mean ± standard error (n=3); significance analysis was performed using one-way anova followed by Tukey test, with asterisks indicating the presence of significant differences (< 0.05;, < 0.01).

Detailed Description

The following describes the technical aspects of the present invention with reference to examples, but the present invention is not limited to the following examples. The experimental methods and the detection methods in each embodiment are conventional methods unless otherwise specified; the reagents and materials are commercially available unless otherwise specified.

The 16318hGFP (green fluorescent protein) vector in the following examples is described in "molecular characterization of millet WRKY36 transcription factor and functional identification [ J ]. Chinese agricultural science, 2015, 48 (5): 851-860, "the public may obtain the above-mentioned biological materials from the applicant, and the above-mentioned biological materials were obtained only for repeated experiments of the present invention.

The BSMV viral vectors (including the alpha, beta and gamma plasmids) in the examples described below are described in "Hein I, Barciszewska-Pacak M, Hrubikova K, et al. Virus-induced gene silencing-based functional characterization of genes associated with powdery mildew resistance in barley[J]. Plant Physiology, 2005. 138, 2155-2164.", and the above biological materials are publicly available from the applicant and are only used in duplicate experiments of the present invention.

The reagents used in the examples below were: cellulase R10 (YaKult Honsha) cellulase (Yakult, C6270-1 g); mecerozyme R10 (YaKult Honsha) pectase (Rongxing organism, RX-L0042-100 mg), mannitol mannitol (Beijing dream Yimei commercial center, M0122-500 g), MES (Beijing Bayer Di Biotechnology Co., ltd., DE-E169-100 g), PEG4000 (Beijing Bayer Di Biotechnology Co., ltd., BR-0084), BSA bovine serum albumin (Beijing Zeping technology Co., 0219989980.); beta-Mercaptoethanol mercaptoethanol (Beijing Ruidebaiao Biotechnology Co., ltd., 0482-100 ML).

The reagent formulations used in the following examples were as follows:

TABLE 1 cellulase enzymatic hydrolysate formulations

TABLE 2 PEG4000 solutions

Note that: the solution is prepared in situ (i.e. one preparation can be stored for 5 days), 100 mu L of PEG4000 solution is needed for each sample, and the total preparation amount of the solution can be adjusted according to the amount of the experimental sample.

TABLE 3 W5 solution

TABLE 4 MMG solutions

TABLE 5 WI solution

The disease-resistant related protein TaMYB22 provided in the following examples is derived from a wheat variety water source 11 (Triticum aestivum) and is (1) to (3):

(1) As set forth in SEQ ID NO:2, and a protein consisting of an amino acid sequence shown in the formula 2;

(2) A protein comprising the amino acid sequence terminal of the protein of (1) and a tag sequence;

(3) And (3) the protein derived from the protein (1) with the same function is obtained by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence of the protein (1).

In order to facilitate purification of TaMYB22 in (1), it can be found in the sequence set forth in SEQ ID NO:2, and a tag shown in table 6 is attached to the amino-terminal or carboxyl-terminal of the protein consisting of the amino acid sequence shown in table 2.

TABLE 6 sequence of tags

The TaMYB22 in the above (3) can be synthesized artificially or obtained by synthesizing the coding gene and then biologically expressing. The coding gene of TaMYB22 in (2) above can be obtained by expression of the gene of SEQ ID NO:1, and/or by making missense mutations of one or more base pairs, and/or by ligating the coding sequences of the tags shown in Table 1 at the 5 'and/or 3' ends thereof.

Nucleic acid molecules encoding the above proteins are also within the scope of the present invention.

The nucleic acid molecule for encoding the protein TaMYB22 is a DNA molecule of any one of the following (1) - (3):

(1) As set forth in SEQ ID NO:1, a DNA molecule shown in fig. 1;

(2) A DNA molecule which hybridizes under stringent conditions to the DNA sequence defined in (1) and which encodes a protein having the same function;

(3) A DNA molecule having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% homology to the DNA sequence defined in (1) and encoding a protein having the same function.

Wherein the nucleic acid molecule may be DNA, such as cDNA, genomic DNA, or recombinant DNA; the nucleic acid molecule may also be RNA, such as mRNA or hnRNA, etc.

The mutation of the nucleotide sequence encoding TaMYB22 of the invention can be easily performed by a person skilled in the art using known methods, such as directed evolution and point mutation. Those artificially modified nucleotides having 75% or more identity to the nucleotide sequence of TaMYB22 isolated by the present invention are derived from the nucleotide sequence of the present invention and are equivalent to the sequence of the present invention as long as they encode TaMYB22 and have the same function.

The term "identity" as used herein refers to sequence similarity to a native nucleic acid sequence. "identity" includes reference to the nucleotide sequence of the invention encoding SEQ ID NO:2 has 75% or more of the nucleotide sequence of a protein consisting of the amino acid sequence shown in FIG. 2, or 85% or more, or 90% or more, or 95% or more. Identity can be assessed visually or by computer software. Using computer software, the identity between two or more sequences can be expressed in percent (%), which can be used to evaluate the identity between related sequences.

The 75% or more identity may be 80%, 85%, 90% or 95% or more identity.

Recombinant vectors, expression cassettes or recombinant microorganisms or recombinant transgenic plant cell lines containing the above-mentioned nucleic acid molecules are also within the scope of the invention.

The expression cassette containing the TaMYB22 nucleic acid molecule (TaMYB 22 gene expression cassette) refers to DNA capable of expressing TaMYB22 in a host cell, and the DNA can comprise a promoter for promoting transcription of the TaMYB22 and a terminator for stopping transcription of the TaMYB 22. Further, the expression cassette may also include an enhancer sequence. Promoters useful in the present invention include, but are not limited to: a constitutive promoter; 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 terminator.

The recombinant vector containing the TaMYB22 gene expression cassette can be constructed by using the existing expression vector. The plant expression vector comprises a binary agrobacterium vector, a vector which can be used for plant microprojectile bombardment and the like. Such as pAHC25, pBin438, pCAMBIA1302, pCAMBIA2300, pCAMBIA2301, pCAMBIA1305, pCAMBIA1300, pBI121, pCAMBIA1391-Xa, or pCAMBIA1391-Xb (CAMBIA Co.). The plant expression vector may also comprise the 3' -untranslated region of a foreign gene, i.e., comprising a polyadenylation signal and any other DNA segments involved in mRNA processing or gene expression. The polyadenylation signal may direct the addition of polyadenylation to the 3 'end of the mRNA precursor and may function similarly to the 3' transcribed untranslated regions of Agrobacterium tumefaciens induction (Ti) plasmid genes (e.g., nopaline synthase gene Nos) and plant genes (e.g., soybean storage protein genes). When the gene of the present invention is used to construct a plant expression vector, enhancers, including translational or transcriptional enhancers, may be used, and these enhancers may be ATG initiation codon or adjacent region initiation codon, etc., but must be identical to the reading frame of the coding sequence to ensure proper translation of the entire sequence. The sources of the translational control signals and initiation codons are broad, and can be either natural or synthetic. The translation initiation region may be derived from a transcription initiation region or a structural gene. To facilitate identification and selection of transgenic plant cells or plants, the plant expression vectors used may be processed, for example by adding genes encoding enzymes or luminescent compounds which produce a color change (GUS gene, luciferase gene, etc.), antibiotic marker genes (such as nptII gene conferring resistance to kanamycin and related antibiotics, bar gene conferring resistance to the herbicide phosphinothricin, hph gene conferring resistance to antibiotic hygromycin, dhfr gene conferring resistance to methotrexate, EPSPS gene conferring resistance to glyphosate) or chemical marker genes, etc. (such as herbicide resistance genes), mannose-6-phosphate isomerase gene providing mannose metabolization ability, etc. From the safety of transgenic plants, transformed plants can be screened directly in stress without adding any selectable marker gene.

The vector may be a plasmid, cosmid, phage or viral vector.

The microorganism can be yeast, bacteria, algae or fungi, such as Agrobacterium.

None of the transgenic plant cell lines described above include propagation material.

The use of the above proteins, the above nucleic acid molecules or the above recombinant vectors, expression cassettes or recombinant microorganisms or recombinant transgenic plant cell lines for regulating plant disease resistance is also within the scope of the present invention.

In the above application, the disease resistance is stripe rust resistance.

In the application, the stripe rust resistance is embodied as any one of the following (1) to (5):

(1) Under the condition of stripe rust infection, the severity of stripe rust of a wheat plant with the expression of TaMYB22 protein inhibited is stronger than that of a control plant, and the generation of summer spore piles on wheat leaves is obviously increased;

(2) Under the condition of stripe rust infection, the number of stripe rust absorbers of wheat plant infection points with inhibited TaMYB22 protein expression is obviously larger than that of control plants;

(3) Under the condition of stripe rust infection, the number of stripe rust absorber parent cells of a wheat plant infection point with inhibited TaMYB22 protein expression is obviously larger than that of a control plant;

(4) Under the condition of rust infection, the length of the rust hyphae of the wheat plant infection point with the TaMYB22 protein expression being inhibited is obviously larger than that of a control plant;

(5) Under the condition of rust infection, the area of rust colony of wheat plant infection points with inhibited TaMYB22 protein expression is obviously larger than that of control plants. The result shows that the disease resistance of wheat plants with inhibited expression of TaMYB22 protein is reduced, and the TaMYB22 gene has the disease resistance function of stripe rust.

In the above application, the plant is a monocotyledonous plant. The monocot plant may specifically be wheat; the wheat may specifically be a water source 11.

In the following examples, an agent capable of reducing the activity or amount of the protein TaMYB22 or inhibiting the expression of a nucleic acid molecule encoding the protein TaMYB22 is provided, including any one of the following:

(1) A silencing sequence;

the silencing sequence is SEQ ID NO:3 or SEQ ID NO:4 is shown in the figure;

(2) A recombinant vector comprising the silencing sequence;

The recombinant vector is a viral vector containing the silencing sequence; the recombinant vector comprises SEQ ID NO:3 or a BSMV viral vector comprising the DNA molecule shown in SEQ ID NO:4, a BSMV viral vector gamma of the DNA molecule shown in fig. 4;

(3) A system comprising the recombinant vector, comprising: BSMV viral vector α, BSMV viral vector β and a vector comprising SEQ ID NO:3, a BSMV viral vector gamma of the DNA molecule shown in fig. 3; BSMV viral vector α, BSMV viral vector β and a vector comprising SEQ ID NO:4, and a BSMV viral vector gamma of the DNA molecule shown in fig. 4.

The following examples provide for the use of agents that reduce the activity or amount of the protein TaMYB22, including any of the following:

(1) Use of a substance that reduces the activity or content of the protein TaMYB22 for reducing disease resistance of a plant or for culturing a transgenic plant;

(2) Use of a substance that inhibits expression of a nucleic acid molecule encoding the protein TaMYB22 to reduce disease resistance in plants or to culture transgenic plants.

The disease resistance is stripe rust resistance by the application.

The following examples provide a method of breeding disease resistant transgenic plants.

The method provided by the invention comprises the following specific steps:

(1) Inhibiting or reducing the content and/or activity of the protein TaMYB22 in the target plant to obtain a transgenic plant;

(2) Inhibiting or reducing expression of a nucleic acid molecule encoding the protein TaMYB22 in the plant of interest to obtain a transgenic plant;

The transgenic plant has a disease resistance lower than that of the plant of interest.

The plant with disease resistance lower than the aim is specifically embodied as any one of the following:

(1) Under the condition of stripe rust infection, the number of infection point sucking devices of the transgenic plant is larger than that of the target plant;

(2) Under the condition of stripe rust infection, the colony area of the transgenic plant is larger than that of the target plant;

(3) Under the condition of rust bacteria infection, the hypha length of the transgenic plant is longer than that of the target plant.

The plant is double in the above method cotyledon plants or monocot plants.

The pathogenic bacteria of the stripe rust are the physiological race CYR23 of the stripe rust in the embodiment of the invention.

Example 1

This example provides the acquisition of the TaMYB22 protein and its encoding gene.

1. Isolation of mRNA and amplification of TaMYB22 Gene

Taking 11 seedlings of wheat water source which normally grow to 7 days, quick-freezing the seedlings with liquid nitrogen, and preserving the seedlings at-80 ℃ for later use. Total RNA from wheat leaves was extracted by Trizol method (TianGen), and first strand cDNA was synthesized using reverse transcriptase XL (AMV). cDNA was synthesized by SMART method, and the PCR products were detected by 1.0% agarose gel electrophoresis. The amplification primers are as follows:

TaMYB22-F：5’-ATGTCACGGATCGGAGATTTGA-3’；

TaMYB22-R：5’-TATTGAGTTACCATCATGTGGAGAACC-3’。

A1194 bp PCR product was obtained.

The PCR product has the sequence shown in SEQ ID NO:1, the gene of the nucleotide is named as TaMYB22 gene, and the amino acid sequence of the protein encoded by the gene is SEQ ID NO:2, the protein was designated TaMYB22.

2. RT-PCR detection of induction expression condition of TaMYB22 gene by rust

1. Preparation of the Experimental Material

The wheat is inoculated with the rust bacteria in a leaf-heart period. Wheat water source 11 leaves were inoculated with non-affinity combination and affinity combination micro CYR23 and CYR31, respectively, and sterile water was inoculated as a control.

Samples were taken at 0h, 6h, 12h, 24h, 48h, 72h, 120h, respectively, after inoculation, and control sampling time points were consistent with treatment. During sampling, fresh leaves are cut, wrapped by tin-platinum paper, put into liquid nitrogen for quick freezing, and then placed at-80 ℃ for standby. Total RNA from wheat leaves was extracted by Trizol method (TianGen), and first strand cDNA was synthesized using reverse transcriptase XL (AMV). cDNA was synthesized by SMART method.

2. RT-PCR detection of expression level of TaMYB22 Gene

Specific quantitative PCR primers were designed based on the sequences of the wheat TaMYB22 gene and the elongation factor TaEF-1. Alpha. Gene (GenBank accession number: U76744).

The RT-PCR primer sequences are as follows:

Qmyb22-F：5’-TGATGTTCATTCTGCGGGAAC-3’；

Qmyb22-R：5’-ATCACCTTTTCTGCCAAAAGAGTG-3’。

QTaEF-F：5’-TGGTGTCATCAAGCCTGGTATGGT-3’；

QTaEF-R：5’-ACTCATGGTGCATCTCAACGGACT-3’。

The specificity and the amplification efficiency (more than or equal to 90%) of the amplified product of the quantitative PCR primer need to be detected before the quantitative PCR primer is used, and TaEF-1 alpha is used as an internal reference gene in Real-time PCR analysis. Real-time quantitative PCR amplification was performed using AceQ Universal SYBR QPCR MASTER Mix (Vazyme, nanj, china) and Bio-Rad CFX Manager quantitative PCR instruments (Bio-Rad, hercules, california) with reference to the instructions, using cDNA at each treatment sampling point as a template. At least 3 replicates per reaction were made and the Ct values for each replicate and their mean and standard deviation were generated by a quantitative PCR instrument by manually adjusting the baseline. 3 repeats of each reaction are carried out, ct values are averaged, experimental data are analyzed by DELTA DELTA CT method, and the relative expression quantity of genes is determined.

The qRT-PCR results are shown in figure 1, wherein the expression patterns of TaMYB22 genes are respectively inoculated in wheat water source 11 for 0h,6h,12h,24h,48h,72h and 120h after the wheat water source 11 is inoculated with the non-affine race CYR23 and the wheat water source is inoculated with the affine race CYR31, the TaMYB22 genes show up-regulation expression trend in the early stage (6-24 h) and the later stage (72-120 h) of the rust infection in the non-affine and affine combination, the 48h expression quantity after the inoculation in the non-affine combination is high as 2.846 times of that of the control, and the 48h expression quantity after the inoculation in the affine combination is highest as 3.5000 times of the control. Where "×" represents significance at p <0.05 levels compared to 0h, respectively.

The results show that the TaMYB22 gene is induced to express by the rust bacteria.

3. TaMYB22 subcellular localization analysis

1. Vector construction

The PCR product is amplified by using the PCR product TaMYB22 fragment as a template and a primer with a BamHI enzyme cutting site, and the amplified product is connected with 16318hGFP (green fluorescent protein) carrier subjected to the same enzyme cutting after the BamHI enzyme cutting, so as to obtain a recombinant carrier 16318hGFP-TaMYB22 and express fusion protein.

The amplification primers used for construction of the TaMYB22 subcellular localization vector were (underlined to indicate cleavage sites):

TaMYB22-GFP-F：5’-GACGATATCTCTAGAGGATCCATGTCACGGATCG GAGATTTG-3’；

TaMYB22-GFP-R：5’-GCCCTTGCTCACCATGGATCCTATTGAGTTACCAT CATGTGGA-3’。

2. protoplast preparation

(1) The preparation and transformation method of the wheat protoplast is as follows:

The soil cultivation room is used for sowing and planting a wheat variety water source 11. Under good growth, protoplasts were prepared by taking leaves before flowering. Cutting the leaves with good growth in the middle, and cutting the leaves into strips with the width of 0.5-1 mm by using a blade. The cut leaves were placed into the cellulase solutions shown in Table 1 (about 10-20 leaves per 5-10 mL of solution). The leaves were completely immersed in the enzymatic hydrolysate with forceps. The vacuum pump was evacuated in the dark (tinfoil wrapping) for 30 minutes. PEG4000 solutions, 200. Mu.L and 1000. Mu.L tips were prepared as shown in Table 2 to ease the sucking during handling. The enzymatic hydrolysis was continued in the dark for at least 3h (50 rpm for wheat 28 ℃) at room temperature without shaking. Gently shaking the dish when the enzymatic hydrolysate turns green causes the protoplasts to release. A certain amount of W5 solution was precooled. And examining the protoplast in the solution under a microscope, wherein the size of the wheat mesophyll protoplast is about 30-50 mu m. The enzyme solution containing protoplasts was diluted with an equal amount of the W5 solution shown in Table 3 before removing undissolved leaves by filtration. A nylon membrane or a 60-100 mesh sieve with a size of 35-75 mu m is wetted with a W5 solution, and then the enzymolysis solution containing protoplasts is filtered. The protoplasts were precipitated by centrifugation at 1-2 min at 4℃using a 30mL round bottom centrifuge tube with 100g, and the supernatant was removed as much as possible. Protoplasts were then gently resuspended with 10mL of pre-chilled W5 solution on ice. The protoplasts were allowed to stand on ice for 30 minutes.

The following operations are carried out at room temperature of 23 ℃,100g is centrifuged for 8-10 min, and protoplasts are precipitated. The W5 solution was removed as much as possible without touching the protoplast pellet. The protoplasts were then resuspended in an appropriate amount of MMG solution (1M) to a final concentration of 2X 10 ⁵/mL. mu.L or 20. Mu.L of DNA (10-20. Mu.g of recombinant vector 16318 hGFP-10 kb, taMYB 22) was added to a 2mL EP tube. 100. Mu.L of protoplasts (2X 10 ⁴) were added and gently mixed. 110. Mu.L of PEG solution was added and the centrifuge tube was gently tapped to mix thoroughly (about 6-10 samples were converted each time). The transformation mixture is induced for 20-30 min (transformation time is determined by experiment, and longer transformation time may be needed for higher expression level). Diluting the conversion mixed solution with 400-440 mu L W solution at room temperature, and then gently reversing the shaking centrifuge tube to ensure complete mixing so as to terminate the conversion reaction. 100g was centrifuged for 2min at room temperature, and the supernatant was removed. Then, 1mL of the W5 solution was added and the mixture was washed once in suspension, and 100g of the mixture was centrifuged for 2min to remove the supernatant. Protoplasts were gently resuspended in a multi-well tissue culture dish with 1mL WI solution. And inducing the protoplast for more than 18 hours at room temperature (20-25 ℃). The vector transferred 16318hGFP is used as a control. GFP tag expression was then observed under a confocal laser microscope.

3. Wheat protoplast microscopy

Protoplasts after dark culture for 18h were pelleted, and GFP (green fluorescent protein) fluorescence was observed under a laser scanning confocal microscope (Bio-Rad MicroRadiance) (LASER SCANNING confocal microscopy, LSMC) and scanned. The operating parameters of the LSCM are: ex=488nm, em=525±15nm, power=10%, zoom7, medium speed scan, frame512×512. The software is TIME-COURSE and PHOTOSHOP5.0.

The results are shown in FIG. 2, above, the control of empty vector protoplasts transformed with recombinant vector 16318 hGFP; the following is a map of TaMYB22 localization in protoplasts transformed with recombinant vector 16318hGFP-TaMYB22, from which it is known that TaMYB22 is distributed in the nucleus.

Example 2

The present example provides the use of inhibiting expression of the TaMYB22 gene in regulating stripe rust resistance in plants.

1. Acquisition of TaMYB22 Gene silencing plants

1. Construction of TaMYB22 gene BSMV-VIGS vector system

(1) Acquisition of silencing sequences

Acquisition of the silencing sequence TaMYB 22-S1:

The 1194bpTaMYB fragment (SEQ ID NO: 1) amplified in example 1 was used as template for PCR amplification using the primer pairs TaMYB22-S1F and TaMYB22-S1R to give a 258bp PCR amplification product (SEQ ID NO: 3), which was designated as silencing sequence TaMYB22-S1.

The nucleotide sequences of the primer pairs TaMYB22-S1F and TaMYB22-S1R are as follows (the sequences shown under the underline are restriction enzyme cleavage recognition sites for PacI enzyme and NotI enzyme):

TaMYB22-S1F：5’-TTAATTAACTGGGAGGACGAATGGTGC-3’；

TaMYB22-S1R：5’-GCGGCCGCCTGTGAAGCCTACTTCAGATATGGA-3’。

Acquisition of the silencing sequence TaMYB 22-S2:

The 1194bp TaMYB22 fragment amplified in example 1 was used as a template, and the primer pairs TaMYB22-S2F and TaMYB22-S2R were used for PCR amplification to obtain a PCR amplification product of 244bp (SEQ ID NO: 4), which was designated as the silencing sequence TaMYB22-S2.

The nucleotide sequences of the primer pairs TaMYB22-S2F and TaMYB22-S2R are as follows (the sequences shown under the underline are PacI enzyme and NotI enzyme restriction enzyme cleavage recognition sites):

TaMYB22-S2F：5’-TTAATTAAGCTAGAGTGGCATCAGCCAAAG-3’；

TaMYB22-S2R：5’-GCGGCCGCCCGGCTTTGCACATTCTGATAG-3’。

(2) Construction of silencing vector:

construction of gamma-TaMYB 22-S1 silencing vector:

And (3) respectively carrying out enzyme digestion on the silencing sequence TaMYB22-S1 and the BSMV-VIGS virus vector gamma obtained in the step (1) by PacI and NotI, and then connecting the digested silencing sequence TaMYB22-S1 with the digested BSMV-VIGS virus vector gamma vector skeleton to obtain a recombinant vector gamma-TaMYB 22-S1.

The recombinant vector gamma-TaMYB 22-S1 is obtained by replacing DNA molecules between PacI and NotI cleavage sites of the BSMV-VIGS virus vector gamma with the silencing sequence TaMYB22-S1 obtained in the step (1) and keeping other sequences of the BSMV-VIGS virus vector gamma unchanged, wherein the TaMYB22-S1 is reversely inserted into the BSMV-VIGS virus vector gamma.

And (3) carrying out PCR amplification and sequencing identification on the recombinant vector gamma-TaMYB 22-S1 by using primer pairs gamma-F and gamma-R, wherein positive cloning is to insert the TaMYB22-S1 (SEQ ID NO: 3) between PacI and NotI enzyme cutting sites of a gamma chain of the BSMV-VIGS virus vector according to the opposite direction of gene expression, and keeping other sequences of the BSMV-VIGS virus vector gamma unchanged.

The sequences of the gamma-F and gamma-R primers are as follows:

γ-F：5’-AAAGTGAGGTTAACGCAATACG-3’；

γ-R：5’-TCAGGCATCGTTTTCAAGTT-3’。

construction of gamma-TaMYB 22-S2 silencing vector:

And (3) respectively carrying out enzyme digestion on the silencing sequence TaMYB22-S2 and the BSMV-VIGS virus vector gamma obtained in the step (1) by PacI and NotI, and then connecting the digested silencing sequence TaMYB22-S2 with the digested BSMV-VIGS virus vector gamma vector skeleton to obtain a recombinant vector gamma-TaMYB 22-S2.

The recombinant vector gamma-TaMYB 22-S2 is obtained by replacing DNA molecules between PacI and NotI cleavage sites of the BSMV-VIGS virus vector gamma with the silencing sequence TaMYB22-S2 obtained in the step (1) and keeping other sequences of the BSMV-VIGS virus vector gamma unchanged, wherein the TaMYB22-S2 is reversely inserted into the BSMV-VIGS virus vector gamma.

And (3) carrying out PCR amplification and sequencing identification on the recombinant vector gamma-TaMYB 22-S2 by using primer pairs gamma-F and gamma-R, wherein positive cloning is to insert the TaMYB22-S2 (SEQ ID NO: 3) between PacI and NotI enzyme cutting sites of a gamma chain of the BSMV-VIGS virus vector according to the opposite direction of gene expression, and keeping other sequences of the BSMV-VIGS virus vector gamma unchanged.

(3) BSMV-VIGS vector system

The BSMV-VIGS viral vectors α, β and γ together constitute the viral vector system BSMV: gamma.

The BSMV-VIGS virus vectors alpha and beta and the recombinant vector gamma-TaMYB 22-S1 together form a virus silencing vector system BSMV capable of silencing the TaMYB22 gene: taMYB22-S1.

The BSMV-VIGS system vectors alpha, beta and the recombinant vector gamma-TaMYB 22-S2 together form a viral silencing vector system BSMV capable of silencing the TaMYB22 gene: taMYB22-S2.

The structure of the vector system for inducing gene silencing (BSMV-VIGS) by the barley streak mosaic virus constructed above is shown in figure 3, and is respectively the structure of alpha, beta and gamma chains in the vector system; the silencing fragments TaMYB22-S1 and TaMYB22-S2 of the TaMYB22 gene are reversely inserted into restriction enzyme NotI and PacI restriction enzyme cutting sites of a gamma chain respectively, and two BSMV-VIGS vector systems BSMV of the TaMYB22 gene are respectively constructed and obtained: taMYB22-S1as and BSMV: taMYB22-S2as.

2. BSMV in vitro transcription

(1) Linearization of the vector: the MluI is used for respectively cutting BSMV virus vector alpha and gamma vectors, the BssHII is used for cutting recombinant vector gamma-TaMYB 22-S1 and recombinant vector gamma-TaMYB 22-S2, and the SpeI is used for cutting BSMV virus vector beta chain, so that linearization plasmids are respectively obtained.

(2) And (3) carrying out in vitro transcription by taking the linearized plasmid obtained in the step (1) as a template to respectively obtain in vitro transcribed BSMV virus vectors alpha, beta, gamma-TaMYB 22-S1 and gamma-TaMYB 22-S2.

The above-mentioned in vitro transcription reaction was carried out according to the instructions of RiboMAXTMLarge Scale RNA Production System-T7 (product of Promega company, cat# P1300).

The transcription reaction system and conditions are respectively as follows: the total reaction volume was 20.0. Mu.L, including 6.5. Mu.L, 5X Transcription Buffer 4.0. Mu.L, 1.5. Mu.L Cap (Promega Co., ltd.: P1718), 6.0. Mu.L rNTP Premix, 2.0. Mu.L Enzyme Mix, 37℃for 2 hours, 1. Mu.L of the measured concentration was taken, and the in vitro transcript concentration was required to be not less than 0.125. Mu.g/. Mu.L to ensure successful inoculation of wheat leaf virus, and the remaining transcripts were kept at-70℃for later use.

3. BSMV vaccination

Sowing a wheat water source 11 into nutrient soil, and taking 10 mu L of in vitro transcribed products BSMV respectively after the wheat water source grows to a two-leaf period: gamma recombinant virus solution, BSMV: taMYB22-S1 recombinant virus solution and BSMV: the TaMYB22-S2 recombinant virus solution is smeared and inoculated on a second flat leaf of wheat, and a part of plants are smeared with 1 XFES Buffer to obtain simulated inoculated plants as a control. After 10min, the mixture was sprayed with DEPC ddH ₂ O and the temperature was adjusted to 25℃for 24h. Then culturing at 25 ℃ under normal conditions to obtain the transformed BSMV respectively: gamma plants, BSMV transformed: taMYB22-S1as plants and transformed BSMV: taMYB22-S2as plants.

The above BSMV: the TaMYB22-S1 recombinant viral vector solution is obtained by diluting in vitro transcribed BSMV-VIGS vector alpha, beta and gamma-TaMYB 22-S1 with DEPC water for 3 times, mixing in equal volume, and adding 1 XFES Buffer for 6 times. Wherein, BSMV: the concentration of alpha, beta and gamma-TaMYB 22-S1 in vitro transcription products in the TaMYB22-S1 recombinant virus vector solution is not less than 1.98 ng/. Mu.L.

The above BSMV: the TaMYB22-S2 recombinant viral vector solution is obtained by diluting in vitro transcribed BSMV-VIGS vector alpha, beta and gamma-TaMYB 22-S2 with DEPC water for 3 times, mixing the same volume, and adding 1 XFES Buffer for 6 times. Wherein, BSMV: the concentration of alpha, beta and gamma-TaMYB 22-S2 in vitro transcription products in the TaMYB22-S2 recombinant virus vector solution is not less than 1.98 ng/. Mu.L.

The above BSMV: the gamma recombinant virus vector solution is obtained by diluting in vitro transcribed BSMV-VIGS vectors alpha, beta and gamma with DEPC water for 3 times, mixing the same amount, and adding 1 XFES Buffer with 6 times of volume. Wherein, BSMV: the concentration of alpha, beta and gamma in vitro transcription products in the gamma recombinant virus vector solution is not less than 1.98 ng/. Mu.L.

The above-described BSMV: taMYB22-S1as plants and transformed BSMV: the plant of TaMYB22-S2as is the wheat plant for silencing the TaMYB22 gene, and the BSMV is transferred: the gamma plants are negative control plants, and the simulated inoculated plants smeared with 1 XFES Buffer are blank control plants.

4. QRT-PCR verification of TaMYB22 gene silencing wheat

And (3) transferring the BSMV obtained in the step (3): taMYB22-S1as and transfer BSMV: taMYB22-S2as plants and BSMV transformed: after culturing gamma plants and MOCK-inoculated plants (MOCK) under normal conditions for 10 days, the physiological race CYR31 of the rust bacteria is inoculated, the inoculation method is described in literature "Kang Zhensheng, li Zhenqi. Discovery of new pathogenic bacterial lines at normal temperature of lovulin 10 [ J ]. Journal of northwest university of agriculture and forestry science and technology (natural science edition), 1984 (04): 18-28'. Samples were taken 24h, 48h, 120h after inoculation, respectively, for RNA extraction and cDNA synthesis by reverse transcription. RT-PCR was performed using the synthesized cDNA as a template, and the relative expression level of TaMYB22 was measured by the method of example 1.

The results of the detection of the relative expression amounts of genes are shown in FIG. 4A, in which MOCK: simulating inoculated blank control plants; BSMV: gamma is vaccinated BSMV: negative control plants of γ; BSMV: taMYB22-S1 and BSMV: taMYB22-S2 represents plants that down-regulate expression of the gene TaMYB22 using the silencing fragments S1 and S2, respectively; as can be seen from fig. 4A, BSMV was transferred 0h, 48h, 72h and 120h after inoculation with rust: taMYB22-S1as and transfer BSMV: the expression level of the gene TaMYB22 in the TaMYB22-S2as plant is that the BSMV is transformed simultaneously: 50% -80% of gamma control plants show that the expression level of TaMYB22 is successfully inhibited in the silent plants, and the two silencing sequences TaMYB22-S1 and TaMYB22-S2 selected in the experiment are effective, so that the expression of the gene TaMYB22 can be significantly reduced by the silencing fragments S1 and S2 (p <0.05 is represented by ").

2. Stripe rust resistance analysis of TaMYB22 Gene-silenced plants

BSMV transfer to effectively silence: taMYB22-S1as plants and BSMV transformed: plants of TaMYB22-S2as, negative control plants (BSMV: gamma), and MOCK plants (MOCK) were inoculated with the physiological race CYR23 of Rumex striolatus after expansion of the fourth leaf. Sampling is carried out at 48h and 120h after inoculation, WGA staining is carried out, and the samples are used for observing the development condition of the rust bacteria after rust bacteria infection. The onset of disease was observed 14d after inoculation.

The identification of the reaction type was based on the nine-type classification standard of Hovmøller（Hovmøller M.S., Rodriguez-Algaba J., Thach T., Sørensen C.K. (2017) Race Typing of Puccinia striiformis on Wheat. In: Periyannan S. (eds) Wheat Rust Diseases. Methods in Molecular Biology, vol 1659. Humana Press, New York, NY）.

The results are shown in FIG. 4B, MOCK: simulating inoculated blank control plants; BSMV: gamma is vaccinated BSMV: negative control plants of γ; BSMV: taMYB22-S1 and BSMV: taMYB22-S2 represents the transformed BSMV: taMYB22-S1as plants and transformed BSMV: plants of TaMYB22-S2 as; under rust infection conditions, visible summer spore piles were observed on all treated leaves, and the severity of stripe rust was stronger for TaMYB22 gene-silenced plants than for control plants.

Statistical analysis of leaf spore bulk showed a significant increase in spore bulk per 0.5cm ² of inoculated TaMYB22 gene-silenced plants (FIG. 4C).

The wheat leaf which is used for down regulating the expression of the TaMYB22 gene is shown to reduce the disease resistance to the physiological race CYR23 of the Phlebopus.

Histological observation shows that, as shown in fig. 5A and 5B, the infected mycelium area of the rust bacteria at the infection point is counted by microscopic observation 120 hours after the rust bacteria are inoculated; FIG. 5C, FIG. 5D, microscopic observation of the statistical H ₂O₂ area 72H after inoculation with rust; FIG. 5E, FIG. 5F, after 72 hours from inoculation of rust, microscopic observation of the necrotic area at the affected site; each biological experiment counted 50 infection points and 3 biological replicates were performed. Error bars in the graph are shown as standard deviations, where "×" indicates significance at p <0.05 levels compared to 0h, respectively. The graph shows that under the rust infection condition, the number of infection point absorbers, colony area and hypha length of the TaMYB22 silent plants are lower than those of control plants, which indicates that the disease resistance of wheat is reduced by reducing the expression of the TaMYB 22.

Thus, taMYB22 is an important gene related to rust disease resistance, and inhibition of the expression of the gene can reduce the rust resistance of plants.

The embodiments described above are some, but not all, embodiments of the invention. The detailed description of the embodiments of the invention is not intended to limit the scope of the invention, as claimed, but is merely representative of selected embodiments of the invention. All other embodiments obtained without inventive effort by a person skilled in the art, which are related deductions and substitutions made by the person skilled in the art under the condition of the inventive concept, are within the scope of protection of the present invention.

Claims

1. The application of the disease-resistant related protein gene TaMYB22 in the cultivation of low stripe rust resistant wheat varieties is characterized in that the disease-resistant related protein gene TaMYB22 is silenced or the expression of the disease-resistant related protein TaMYB22 is inhibited, so that the resistance of wheat to stripe rust is reduced;

The nucleotide sequence of the disease resistance related protein gene TaMYB22 is shown in SEQ ID NO:1 is shown in the specification;

the amino acid sequence of the disease-resistant related protein TaMYB22 is shown in SEQ ID NO: 2.

2. The use according to claim 1, wherein the resistance of wheat to stripe rust is reduced by using a specific gene fragment of the disease-resistant related protein gene TaMYB 22;

the specific gene fragment of the disease resistance related protein gene TaMYB22 has a sequence shown in SEQ ID NO:3 or SEQ ID NO: 4.

3. A method of breeding transgenic wheat with reduced stripe rust resistance, wherein expression of the disease resistance-associated protein gene TaMYB22 of claim 1 is inhibited or reduced.

4. A method according to claim 3, comprising constructing a recombinant expression vector, transforming the recombinant expression vector into wheat; the recombinant expression vector contains the specific gene fragment of the disease resistance related protein gene TaMYB22 in claim 2.