CN116333076A - Effector protein PST-8853 containing iron-sulfur cluster and application thereof - Google Patents

Abstract

The invention discloses an effector protein PST-8853 containing iron-sulfur clusters, which plays a toxic role in the interaction process of stripe rust and wheat. The amino acid sequence of effector protein PST-8853 containing iron-sulfur cluster is shown as SEQ ID NO.1, and the toxic effector protein is encodedPST‑8853The nucleotide sequence of the open reading frame of the gene is shown as SEQ ID NO. 2.PST‑8853The gene is up-regulated in the process of the infection of the stripe rust bacteria. Will bePST‑8853Cloning the gene specific fragment into an interference vector, transforming young wheat embryo by using agrobacterium-mediated transgenic technology, and obtaining transgenic wheat plant against wheat stripe rustExhibit resistance. The invention provides a technical idea for creating wheat disease-resistant materials by utilizing effector protein PST-8853 and encoding genes thereof and cultivating durable stripe rust-resistant wheat varieties.

Description

Effector protein PST-8853 containing iron-sulfur cluster and application thereof

Technical Field

The invention belongs to the technical field of agricultural biology, relates to anti-strip rust effector protein, and particularly relates to effector protein PST-8853 containing iron-sulfur clusters and application thereof.

Background

Wheat, one of three world crops, contributes to about half of the world's population. The wheat special shape is characterized by bar-shaped rust bacteriaPuccinia striiformis f. sp. Tritici，Pst) Wheat stripe rust caused by the method is a serious fungal disease in wheat production, the epidemic year of the disease can lead to 20% -40% yield reduction of the wheat, and serious harm can even lead to harvest failure. Disease-resistant breeding is one of the most economical and effective measures for preventing and treating wheat stripe rust, but the excavating period of disease-resistant genes is long, the breeding difficulty of disease-resistant varieties is high, and the toxicity of stripe rust is fast changed, so that the disease is difficult to realize lasting control in preventing and treating. Therefore, the creation of durable disease-resistant materials is a fundamental way to control wheat stripe rust.

Effector proteins are a class of exocrine protein molecules secreted by pathogenic bacteria, which can change the structure and defense pathways of host plant cells through plant pathogen interaction, thereby promoting the successful infection and colonization of the host plants by the pathogenic bacteria or triggering host defense reaction. Pathogenic bacteria enter host plant cells through secretion of effector proteins to influence different plant disease resistance related genes so as to inhibit the immune response of the host, thereby helping the colonization and infection of the pathogenic bacteria. In recent years, research on iron-sulfur proteins has been mainly carried out on mitochondria, chloroplasts and iron-element-binding related iron-sulfur proteins, and at present, effects of iron-sulfur cluster-containing effector proteins in interaction between plants and pathogenic bacteria have been freshly reported.

Disclosure of Invention

The invention aims to provide an effector protein PST-8853 containing iron-sulfur clusters, which lays a theoretical foundation for creating wheat disease-resistant materials by using pathogenic bacteria pathogenic factors by analyzing pathogenic mechanisms, and has important significance for cultivating durable wheat stripe rust resistant varieties.

In order to achieve the above purpose, the invention adopts the following technical scheme: an effector protein PST-8853 containing iron-sulfur clusters, wherein the amino acid sequence of the effector protein PST-8853 is shown in SEQ ID NO. 1.

Based on the description of the invention, the invention provides the application of the effector protein PST-8853 containing the iron-sulfur cluster in the cultivation of the wheat stripe rust resistant variety for the first time, and the cultivation work of the stripe rust resistant wheat variety is not difficult to realize by combining the prior art with the common technical personnel in the field. As a preferred embodiment for the application of cultivating the rust-resistant wheat variety, the expression of the effector protein PST-8853 is inhibited through gene silencing, so that the wheat resistance to rust bacteria is improved.

The invention adopts a reverse genetics method to obtain the coding gene of the effector protein PST-8853PST-8853. The genePST-8853Has a nucleotide sequence shown as SEQ ID NO. 2. It was confirmed that the number of the cells,PST- 8853the gene is up-regulated in the process of the infection of the stripe rust bacteria. Silencing using host-mediated gene silencing techniquesPST-8853Gene determinationPST-8853Toxic function of genes during stripe rust infection.

Further, the present invention claims the genePST-8853The application in the cultivation of wheat stripe rust resistant varieties. As a preferred embodiment of said application, silencing saidPST-8853The gene improves the resistance of wheat to the stripe rust. The silencing can be obtained by genetic engineering techniques combined with the prior art by one of ordinary skill in the artPST-8853The transgenic wheat material expressed by the gene can obviously improve the resistance of wheat plants to the stripe rust.

The invention aims to create a rust-resistant wheat strain by utilizing a toxic effector protein PST-8853 and a coding gene thereof, and provides an excellent wheat material for cultivating the rust-resistant wheat strain. To this end, the present invention provides a method for breeding wheat against diseasePST-8853Transferring the gene silencing fragment into wheat material to obtain silencing productPST-8853Gene of geneWheat variety, saidPST-8853The nucleotide sequence of the gene silencing fragment is shown in SEQ ID NO: 3.

The method for breeding wheat with disease resistance comprises the steps of constructing a strain containingPST-8853An expression vector for the gene; agrobacterium tumefaciens-mediated transfer into wheat young embryo; obtaining the saidPST-8853Genetically silenced transgenic wheat. The expression vector contains the following componentsPST-8853A gene silencing fragment.

Further, transforming the gene specific fragment of the wheat stripe rust effector protein PST-8853 into wheat cells by using a plant genetic engineering technology to obtain the wheat stripe rust effector proteinPST-8853Wheat variety of RNAi. Further, constructs comprising wheat stripe rust effector proteinPST-8853A plant interference vector of the gene; transforming the plant interference vector into wheat young embryo by using agrobacterium-mediated genetic transformation method; obtainingPST-8853RNAi transgenic wheat.

For complete and unobjectionable understanding of the technical scheme of the invention, it is required to supplement that the toxic effector protein is represented by a non-inclined font "PST-8853", and the encoding gene of the effector protein PST-8853 is represented by an inclined font'PST-8853"means. Of course, the meaning and expression of the relevant genes and their encoded proteins can be clearly and completely understood by those skilled in the art from the description of the present invention.

By implementing the technical scheme of the invention, the following beneficial effects can be achieved:

(1) The invention discloses an effector protein PST-8853 which plays a toxic function in the interaction process of the stripe rust and wheat for the first time, and is a key toxic effector protein of the wheat stripe rust. Effector protein PST-8853 has the sequence as set forth in SEQ ID NO:1, and a polypeptide having the amino acid sequence shown in 1. The invention lays a theoretical foundation for creating wheat disease-resistant materials by utilizing the effector protein PST-8853 and the encoding genes thereof by analyzing the pathogenesis of wheat stripe rust, and has important significance for cultivating durable stripe rust-resistant wheat varieties.

(2) The invention provides a new technical idea for creating the wheat disease-resistant material. By means of genetic engineering technology, wheat stripe rust bacteria effector proteinPST-8853The invention provides a new solution for developing cultivation of wheat stripe rust resistant varieties from the aspect of molecular biology.

(3) The invention discloses a cultivation method of a wheat stripe rust resistant variety. The method is to make wheat stripe rust bacteria effector proteinPST-8853Transferring the gene silencing fragment into wheat material to obtain the silencing effector proteinPST-8853Wheat variety of the gene.PST-8853The RNAi transgenic plants exhibited resistance to Leptosphaeria gracilis CYR 31.

Drawings

FIG. 1 is a flow chart of a verification method for the application of wheat stripe rust effector protein PST-8853 in toxicity function, cultivation and improvement of wheat rust resistant varieties.

FIG. 2 is a schematic diagram showing the analysis of the expression profile of effector protein PST-8853 gene provided by the embodiment of the invention.

FIG. 3 is a schematic diagram showing that effector protein PST-8853 contains iron-sulfur clusters according to an embodiment of the present invention.

FIG. 4 is a schematic illustration of specific silencing provided by an embodiment of the present inventionPST-8853Schematic representation of gene phenotype results, in the figure:

a is the phenotype of the VIGS silenced PST-8853 gene, BSMV in A represents virus inoculation, mock represents buffer solution as negative control, taPDS represents silent PDS gene as positive control, gamma represents empty virus as negative control, PST-8853 represents silent Pst-8853 gene;

b is the relative expression quantity of the target gene in the silent plant, wherein, gamma represents a control plant, PST-8853 represents the silent plant;

and C is spore density statistics of the silent plants and the control plants after 13 days of inoculation, wherein BSMV: gamma in C represents the control plants, and BSMV: PST-8853 represents the silent plants.

FIG. 5 is a schematic diagram of PCR detection results of T1 generation PST-8853 silenced transgenic plants provided by an embodiment of the present invention; in the figure, M represents a DNA Marker, WT represents a wild-type plant, P represents a plasmid carrying a gene of interest, L1 represents line 1 of a positive plant, and L2 represents line 2 of a positive plant.

FIG. 6 is a schematic representation of the phenotype results of T1 generation PST-8853 silenced transgenic plants inoculated with Rumex striolatus major circulating race CYR31 provided by the example of the invention; in the figure, WT represents a wild-type plant, L1 represents line 1 of a positive plant, and L2 represents line 2 of a positive plant.

Detailed Description

The technical solutions of the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

FIG. 1 shows a technical scheme of the invention "effector protein PST-8853 containing iron-sulfur clusters and its use". The embodiment of the invention provides a method for verifying the toxicity function of effector protein PST-8853 containing iron-sulfur clusters, which comprises the following steps:

s101, performing real-time fluorescence quantitative PCR detection and determiningPST-8853The expression quantity of the gene at different infection times of the stripe rust bacteria infection wheat;

s102, expressing and purifying effector protein PST-8853 in vitro by using escherichia coli, observing the color characteristics of the protein, and culturing by using mutant cysteine and a deletion iron element to find that the color of the protein changes and that the effector protein PST-8853 contains iron-sulfur clusters;

s103, utilizing virus-induced gene silencing technique specific transient silencing effector genePST-8853The toxicity function is clear;

s104, obtainingPST-8853The silent transgenic wheat is subjected to molecular detection on the obtained T1 generation transgenic wheat;

s105, inoculating the T1 generation transgenic plant with the main epidemic race CYR31 of the rust fungus, identifying the resistance of the transgenic plant to the epidemic race CYR31 of the rust fungus, and determiningPST-8853The anti-rust properties of the silenced transgenic wheat.

The embodiment of the invention further provides a method for identifying the toxic function of effector protein PST-8853 containing iron-sulfur clusters, which comprises the following steps:

qRT-P basedCR to extend factor genesTaEF1-αUse of effector proteins as internal controlsPST-8853Real-time quantitative PCR (polymerase chain reaction) is carried out on specific primers of genes, and determination is carried outPST-8853The expression quantity of the gene at different infection times of the stripe rust bacteria infection wheat;

expressing and purifying in vitro by using escherichia coli to express effector protein PST-8853, observing the color characteristics of the protein, and culturing by using mutant cysteine and iron-deficiency elements to find that the color of the protein changes, wherein the effector protein PST-8853 contains iron-sulfur clusters;

transient silencing effector genes specific for gene silencing techniques using virus inductionPST-8853Detection of silencing on day 10 of two-leaf inoculation with virusTaPDSWhether the wheat leaves are bleached or not, if so, the virus inoculation is successful; inoculating 4 leaves with fresh spores of wheat stripe rust, detecting the number of spore piles on the surfaces of the leaves on 12 th day after inoculation, and checking whether necrosis occurs on the inoculated leaves;

effector proteinsPST-8853The silent gene segment is constructed to a plant interference vector, and agrobacterium-mediated genetic transformation is utilized to obtainPST-8853And (3) silencing the transgenic plant and verifying the resistance condition of the transgenic plant to the epidemic race CYR31 of the rust fungus of China.

The method for verifying the toxic function of effector protein PST-8853 containing iron-sulfur clusters and the method for identifying the same will now be described in detail with reference to examples 1 to 5.

Example 1

The effector protein PST-8853 which is up-regulated in the infection stage of the wheat stripe rust is obtained by screening from an interaction transcriptome of the wheat and the stripe rust, and the effector protein PST-8853 containing iron-sulfur clusters is obtained by utilizing an NCBI database (https:// www.ncbi.nlm.nih.gov /) and has an amino acid sequence shown as SEQ ID NO. 1.

Example 2

This example describes genes encoding effector proteins containing iron-sulfur clustersPST-8853Is described herein).

The wheat variety water source 11 of normal soil culture is used as a model material, and in the two-leaf and one-heart period of wheat seedlings, a brush pen is adopted to inoculate the stripe rust epidemic race CYR31 and to preserve moisture, and the total RNA of the sample is sampled and extracted in 24 hours, 48 hours, 72 hours, 96 hours, 120 hours and 176 hours. Total RNA was extracted using Trizol reagent (purchased from Vietnam Beijing Hua) and RNA was purified.

Total RNA was reverse transcribed into cDNA first strand using reverse transcriptase M-MLV (available from Thermo Fisher) under the following reaction conditions: 65 ℃ for 5min; preserving at-20deg.C at 42deg.C for 60 min. Then, a certain amount of cDNA was taken out from the sample at each time point and mixed into a total template, and the total template was usedPST-8853PCR amplification of specific primers PST-8853-cDNA-F (5'-ATGAACATCACGTCTTTGGGT-3') and PST-8853-cDNA-R (5'-CTAGCGTTTTTTGATGGCCTT-3') to give a kit containingPST-8853A DNA fragment of the full-length coding frame of the gene under the following reaction conditions: operating at 95 ℃ for 10 minutes; operating at 95 ℃ for 30 s,58 ℃ for 30 s,72 ℃ for 30 s, and circulating 40 times; extending at 72℃for 10 min.

And (3) carrying out T connection on the obtained PCR amplification product, constructing into a pGEM-T vector (purchased from Takara), transforming escherichia coli DH5 alpha competent cells (purchased from Shanghai microorganism), screening and detecting positive clones, shaking overnight, extracting plasmids named as T-A subclones, screening positive clones with correct sequencing, and amplifying to obtain a nucleotide sequence shown as SEQ ID NO. 2.

Example 3

This example describes genesPST-8853Is described. Based on examples 1 and 2, analysis was performed using real-time fluorescent quantitative PCR techniquesPST-8853Expression profiling of genes under rust infection.

Wheat-stripe rust interaction cDNA of samples at different time points is taken as a template, and a wheat extension gene is taken as an internal reference gene (PsEF-1-F: 5'-TTCGCCGTCCGTGATATGAGACAA-3' and PsEF-1-R: 5'-ATGCGTATCATGGTGGTGGAGTGA-3') and is utilizedPST-8853The gene specific primers (PST-8853-qRT-F: 5'-TGGGTACATTCTTCGTGGTT-3' and PST-8853-qRT-R: 5'-CTTTCGGGACAATCTTCTTTAT-3') were subjected to real-time fluorescent quantitative PCR under the following reaction conditions: operating at 95 ℃ for 10 minutes; the method is circulated for 40 times by operating at 95 ℃ for 10 s,55 ℃ for 20 s and 72 ℃ for 30 s; extension was performed at 72℃for 10min, followed by dissolution profile analysis. Reactions were carried out 3 per sampleThe CT values were averaged over replicates.

FIG. 2 shows the expression profile of effector protein PST-8853 containing iron-sulfur clusters at various time points after inoculation of wheat variety water source 11 with the rust epidemic race CYR 31. As can be appreciated from the view of figure 2,PST-8853the gene up-regulates expression in the infected wheat water source 11, and is a gene related to wheat stripe rust toxicity.

Example 4

In this example, effector protein PST-8853 was expressed by E.coliPST-8853The gene is constructed on a prokaryotic expression vector pGEX4T-1, and is transformed into the competent E.coli expression strain BL21 to detect positive colonies. Culturing E.coli OD at 37℃in M9 inorganic salt medium ₆₀₀ Adding IPTG with final concentration of 0.5mM to 0.6-0.8, culturing at 18deg.C for 12-14hours at 600 rpm for 10min, collecting bacteria, and observing color of bacteria with naked eyes. And (3) setting effector protein PST-8853 wild type protein, and culturing the mutant cysteine and iron-deficiency element culture medium.

As can be seen from FIG. 3, after the mutation of cysteine in effector protein and the lack of iron medium, effector protein was cultured, and the cells were collected to observe the color change, thus confirming that effector protein PST-8853 contains iron-sulfur clusters.

Example 5

By sequence alignment (http:// blast. Ncbi. Nlm. Nih. Gov/blast. Cgi) and specificity analysis,PST-8853the gene has a segment of gene with higher sequence specificity (the nucleotide sequence is shown as SEQ ID NO. 3). The present example is constructed based on this specific gene fragmentPST-8853A gene silencing vector.

PST-8853The construction of the gene silencing vector is described in example 2. The cDNA library of the stripe rust-wheat interaction is used as a template, andPST-8853gene-specific primers amplified to representPST-8853The specific silencing fragment of the gene (the nucleotide sequence is shown as SEQ ID NO. 3). Constructing a gamma vector by a homologous recombination mode, and silencing a specific fragment of the wheat stripe rust effector protein PST-8853 by utilizing a virus-induced transient silencing technology.

Is specifically implemented in wheat growthArtificial friction inoculation is carried out for 16 days, and the inoculated strain is placed in a growth incubator with a light period of 8 hours and a light period of 16 hours at 24 ℃ after the strain is placed in the dark of 100% at 24 ℃ for moisturizing for 24 hours. Obvious striped chlorosis on the virus-receiving leaves can be observed after 10 days of virus inoculation, and the success of virus inoculation is proved. Inoculating a rust virulent race CYR31, placing the strain into a moisturizing box, moisturizing the strain for 24 hours in the dark at the temperature of 16 ℃, and then transferring the strain into a growth incubator with a photoperiod of 8 hours in the dark at the temperature of 14 ℃ after illumination at the temperature of 16 ℃ for 16 hours. Silencing can be observed 14 days after inoculationPST-8853As shown in FIG. 4, the necrotic area of the leaf increases, demonstrating that PST-8853 plays a toxic role in wheat infection by wheat rust, and was created based on the experimental resultsPST-8853Transgenic wheat expressed by gene silencing.

Example 6

Description of the present embodimentPST-8853Cultivation of gene silencing expression transgenic wheat and identification of disease resistance thereof, and the flow of the method is shown in figure 1.

After the transgenic plants obtained by agrobacterium-mediated genetic transformation, DNA of the transgenic plants was extracted and PCR detection was performed using specific primers for the effector protein PST-8853 gene, as shown in fig. 5. The two positive strains L1 and L2 of the T1 generation are respectively inoculated with main epidemic minispecies CYR31, and the result is shown in figure 6, and the transgenic strain can be seen to have less spore production than a wild plant, which shows that the transgenic plant with PST-8853 silence shows resistance to the Phlebia trolley CYR 31.

The foregoing is merely illustrative of specific embodiments of the present invention, and the scope of the invention is not limited thereto, but any modifications, equivalents, improvements and alternatives falling within the spirit and principles of the present invention will be apparent to those skilled in the art within the scope of the present invention.

Claims (4)

1. GenePST-8853Application of the gene in breeding of wheat stripe rust resistant variety is characterized in that the genePST-8853Is a gene encoding effector protein PST-8853;

the amino acid sequence of the effector protein PST-8853 is shown as SEQ ID NO. 1;

the genePST-8853The nucleotide sequence of (2) is shown as SEQ ID NO. 2;

the genePST-8853Up-regulating expression under the mediation of stripe rust bacteria;

silencing the genePST-8853The resistance of wheat to the stripe rust is improved.

2. A method for breeding wheat with disease resistance is characterized in thatPST-8853Transferring the gene silencing fragment into wheat material to obtain silencing productPST-8853A wheat variety of a gene, saidPST-8853The nucleotide sequence of the gene silencing fragment is shown as SEQ ID NO. 3.

3. The method of claim 2, wherein constructing comprisesPST-8853An expression vector for the gene; agrobacterium tumefaciens-mediated transfer into wheat young embryo; obtaining the saidPST-8853Genetically silenced transgenic wheat.

4. The method of claim 3, wherein said expression vector comprises saidPST-8853A gene silencing fragment.

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