CN110885365B - Application of ATAF2 protein and related biological materials thereof in regulation and control of disease resistance of plants to rubber tree powdery mildew - Google Patents

Abstract

The invention discloses application of ATAF2 protein and related biological materials thereof in regulating and controlling disease resistance of plants to rubber tree powdery mildew. According to the invention, firstly, the wild type of arabidopsis thaliana inoculated with powdery mildew is analyzed, the fact that the transcription factor ATAF2 of arabidopsis thaliana is remarkably up-regulated and expressed 4 days after inoculation is found, the fact that ATAF2 is induced and expressed by powdery mildew is presumed, then, the experiments of inoculating the powdery mildew to arabidopsis thaliana mutant ATAF2 are further verified, the fact that ATAF2 mutant shows the phenotype of the powdery mildew of the rubber tree, the disease resistance of arabidopsis thaliana to the powdery mildew of the rubber tree can be positively regulated and controlled, GST pull-down and co-immunoprecipitation experiments are utilized to find that ATAF2 and EDS1 can directly interact, and a good foundation is laid for further discussing a plant disease resistance signal path involved in EDS1 in the future.

Description

Application of ATAF2 protein and related biological materials thereof in regulation and control of disease resistance of plants to rubber tree powdery mildew

Technical Field

The invention belongs to the technical field of biology, and particularly relates to application of ATAF2 protein and related biological materials thereof in regulation and control of disease resistance of plants to rubber powdery mildew.

Background

In nature, plants use their own 2 lines of immunity to defend against infestation by pathogenic microorganisms. The 1 st line of immune defense is a pathogen-associated molecular pattern or an immune response (PTI) elicited by microbial-associated factors (PAMPs/MAMPs). The 2 nd line of immune defense is that after the plant disease resistance gene (R gene, resistance) recognizes the effector protein of pathogenic microorganism, an immune Response (ETI) is triggered, which produces a large amount of cell death at the site of infection, also known as Hypersensitivity (HR).

EDS1(enhanced disease susceptibility 1) plays a very important role in the 2-way line of immunity of plants. Especially in the 2 nd line of immune defense, EDS1 is needed for the normal function of most TIR-NB-LRR (Toll-Interleukin1Receptor-nucleotide binding-leucoine-rich repeat) disease-resistant genes of plants. EDS1 can interact with pathogenic bacteria effector protein and can form a complex with TIR-NB-LRR protein in plants, thereby realizing normal transmission of disease-resistant signal paths. Meanwhile, two other proteins structurally homologous to EDS1, PAD4(Phytoalexin Deficient 4) and SAG101(Senescence Associated Gene 101), both containing an acyl esterase domain and an EP (EDS1-PAD4) domain, are present in plants. EDS1 can interact with two other proteins to mediate different disease-resistant signal pathways. Recent studies have found that the three components can also form a complex to function. Currently, less is known about the downstream signaling pathway of EDS1, and EDS1 is regulating the synthesis of salicylic acid, but what the specific acting elements are, is to be further studied.

Rubber tree powdery mildew (Oidium Heveae) is an obligate living parasitic fungus, and causes the rubber tree powdery mildew caused by infecting rubber trees, thereby causing huge loss of the yield of natural rubber. Recent studies have found that powdery mildew can also infect arabidopsis thaliana, provoking a disease-resistant response in arabidopsis thaliana wild type Col-0, and that the disease-resistant response is totally dependent on EDS1 and PAD4, and partially dependent on SAG101 and the SA signaling pathway.

Disclosure of Invention

The invention aims to improve the disease resistance of plants to rubber tree powdery mildew (Oidium Heveae).

The invention firstly provides a new application of the ATAF2 protein.

The invention provides application of ATAF2 protein in regulation and control of plant resistance to powdery mildew.

The invention also provides application of the ATAF2 protein in interaction with EDS1 protein (the amino acid sequence is shown as a sequence 4 in a sequence table).

In the application, the ATAF2 protein is a protein shown in a) or b) or c) or d) as follows:

a) the amino acid sequence is a protein shown in a sequence 2;

b) a fusion protein obtained by connecting a label to the N end and/or the C end of the protein shown in the sequence 2;

c) the protein with the same function is obtained by replacing and/or deleting and/or adding one or more amino acid residues of the amino acid sequence shown in the sequence 2;

d) and (b) a protein having a homology of 75% or more than 75% with the amino acid sequence shown in the sequence 2 and having the same function.

In order to facilitate the purification of the protein in a), the amino terminus or the carboxyl terminus of the protein shown in sequence 2 in the sequence listing may be linked with a tag shown in table 1.

TABLE 1 tag sequences

Label (R)	Residue of	Sequence of
			Poly-Arg	5-6 (typically 5)	RRRRR
Poly-His	2-10 (generally 6)	HHHHHH
			FLAG	8	DYKDDDDK
Strep-tag II	8	WSHPQFEK
			c-myc	10	EQKLISEEDL

In the above c), the substitution and/or deletion and/or addition of one or several amino acid residues is a substitution and/or deletion and/or addition of not more than 10 amino acid residues.

In the above c), the protein may be artificially synthesized, or may be obtained by synthesizing the coding gene and then performing biological expression.

In the above c), the gene encoding the protein can be obtained by deleting one or several codons of amino acid residues from the DNA sequence shown in the sequence No. 1, and/or performing missense mutation of one or several base pairs, and/or connecting the coding sequence of the tag shown in Table 1 to the 5 'end and/or 3' end.

In the above d), "homology" includes an amino acid sequence having 75% or more, or 80% or more, or 85% or more, or 90% or more, or 95% or more homology with the amino acid sequence represented by the sequence 2 of the present invention.

The invention also provides a new application of the biological material related to the ATAF2 protein.

The invention provides application of biological materials related to ATAF2 protein in regulation and control of plant resistance to powdery mildew of rubber trees;

the biomaterial is any one of the following A1) to A12):

A1) a nucleic acid molecule encoding an ATAF2 protein;

A2) an expression cassette comprising the nucleic acid molecule of a 1);

A3) a recombinant vector comprising the nucleic acid molecule of a 1);

A4) a recombinant vector comprising the expression cassette of a 2);

A5) a recombinant microorganism comprising the nucleic acid molecule of a 1);

A6) a recombinant microorganism comprising the expression cassette of a 2);

A7) a recombinant microorganism comprising a3) said recombinant vector;

A8) a recombinant microorganism comprising a4) said recombinant vector;

A9) a transgenic plant cell line comprising the nucleic acid molecule of a 1);

A10) a transgenic plant cell line comprising the expression cassette of a 2);

A11) a transgenic plant cell line comprising the recombinant vector of a 3);

A12) a transgenic plant cell line comprising the recombinant vector of a 4).

In the above application, the nucleic acid molecule of A1) is a gene as shown in 1) or 2) or 3) below:

1) the coding sequence is cDNA molecule shown in sequence 1 or genome DNA molecule shown in 1501-2532 site of sequence 7;

2) a cDNA molecule or a genome DNA molecule which has 75 percent or more than 75 percent of identity with the nucleotide sequence defined by 1) and codes ATAF2 protein;

3) a cDNA molecule or a genome DNA molecule which is hybridized with the nucleotide sequence limited by 1) or 2) under strict conditions and codes ATAF2 protein.

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 nucleotide sequence encoding ATAF2 protein of the invention can be easily mutated by one of ordinary skill in the art using known methods, such as directed evolution and point mutation. Those nucleotides which have been artificially modified to have 75% or more identity to the nucleotide sequence encoding ATAF2 protein are derived from and identical to the nucleotide sequence of the present invention as long as they encode ATAF2 protein and have the same function.

The term "identity" as used herein refers to sequence similarity to a native nucleic acid sequence. "identity" includes a nucleotide sequence that is 75% or greater, or 85% or greater, or 90% or greater, or 95% or greater identical to the nucleotide sequence of a protein consisting of the amino acid sequence set forth in coding sequence 2 of the present invention. 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 assess the identity between related sequences.

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

In the above application, the stringent conditions are hybridization and membrane washing 2 times at 68 ℃ for 5min in a solution of 2 XSSC, 0.1% SDS, and hybridization and membrane washing 2 times at 68 ℃ for 15min in a solution of 0.5 XSSC, 0.1% SDS; alternatively, hybridization was carried out at 65 ℃ in a solution of 0.1 XSSPE (or 0.1 XSSC), 0.1% SDS, and the membrane was washed.

In the above application, the vector may be a plasmid, a cosmid, a phage, or a viral vector.

In the above application, the microorganism may be yeast, bacteria, algae or fungi, such as Agrobacterium.

In the above applications, the transgenic plant cell line does not comprise propagation material.

In the above application, the modulation is an increase or a decrease. The regulation is embodied in: when the expression level and/or activity of ATAF2 protein in a plant is reduced, the resistance of the plant to rubber powdery mildew is reduced; when the expression level and/or activity of the ATAF2 protein in a plant is increased, the resistance of the plant to powdery mildew is increased.

The invention also provides application of the ATAF2 protein or the biological material in culturing transgenic plants with improved resistance to powdery mildew of rubber trees.

The invention also provides application of the ATAF2 protein or the biological material in culturing transgenic plants with reduced resistance to powdery mildew of rubber trees.

The application of the ATAF2 protein or the biological material in plant breeding also belongs to the protection scope of the invention.

The invention also provides a method for cultivating the transgenic plant with reduced resistance to rubber tree powdery mildew.

The method for cultivating the transgenic plant with the reduced resistance to the rubber tree powdery mildew comprises the steps of reducing the expression quantity and/or activity of ATAF2 protein in a receptor plant to obtain the transgenic plant; the transgenic plant has lower resistance to Blumeria necator than the recipient plant.

Further, the method for reducing the expression amount and/or activity of the ATAF2 protein in the receptor plant is realized by silencing or inhibiting the expression and/or activity of the gene coding for the ATAF2 protein in the genome of the receptor plant or knocking out the gene coding for the ATAF2 protein.

Furthermore, the silencing or inhibiting the expression and/or activity of the gene coding for the ATAF2 protein in the genome of the receptor plant or knocking out the gene coding for the ATAF2 protein is to mutate the gene coding for the ATAF2 protein in the genome of the receptor plant, so that the expression level of the gene coding for the ATAF2 protein in the genome of the receptor plant is reduced or the gene coding for the ATAF2 protein in the genome of the receptor plant is subjected to deletion mutation or insertion mutation or base substitution;

the method for generating deletion mutation or insertion mutation or base substitution of the gene encoding ATAF2 protein in the genome of the receptor plant can be CRISPR/Cas9 or TELLEN or T-DNA insertion or EMS mutagenesis.

The nucleotide sequence of the gene encoding the ATAF2 protein is a cDNA molecule shown in sequence 1 or a genomic DNA molecule shown in 1501-2532 th site of sequence 7.

In a specific embodiment of the invention, the method for insertionally mutating the gene encoding ATAF2 protein in the genome of the recipient plant is a T-DNA insertion.

The resistance of the transgenic plant to the powdery mildew is lower than that of the receptor plant specifically represented by: the transgenic plant produces the conidia of the powdery mildew of the rubber tree after being inoculated with the powdery mildew of the rubber tree, and the receptor plant does not produce the conidia of the powdery mildew of the rubber tree after being inoculated with the powdery mildew of the rubber tree; and/or the transgenic plant generates weak powdery mildew disease spots after being inoculated with rubber tree powdery mildew, and the receptor plant generates obvious disease-resistant yellowing phenotype after being inoculated with rubber tree powdery mildew.

The invention also provides a method for cultivating the transgenic plant with improved resistance to the rubber tree powdery mildew.

The method for cultivating the transgenic plant with improved resistance to the rubber tree powdery mildew provided by the invention comprises the steps of improving the expression quantity and/or activity of ATAF2 protein in a receptor plant to obtain the transgenic plant; the transgenic plant has higher resistance to Blumeria necator than the recipient plant.

Further, the method for improving the expression amount and/or activity of the ATAF2 protein in the receptor plant is to over-express the ATAF2 protein in the receptor plant.

Furthermore, the overexpression method is to introduce a gene encoding the ATAF2 protein into a recipient plant.

The nucleotide sequence of the gene encoding the ATAF2 protein is a cDNA molecule shown in sequence 1 or a genomic DNA molecule shown in 1501-2532 th site of sequence 7.

In one embodiment of the invention, the gene encoding the ATAF2 protein is introduced into the recipient plant via a recombinant expression vector. The recombinant expression vector is a recombinant expression vector pCAMBIA1300-ATAF 2. The recombinant expression vector pCAMBIA1300-ATAF2 is a vector obtained by inserting a DNA molecule (1.5Kb promoter region + ATAF2 genome DNA) shown in a sequence 7 between Xho I enzyme cutting sites and BstB I enzyme cutting sites of the pCAMBIA1300 vector and keeping other sequences of the pCAMBIA1300 vector unchanged.

The resistance of the transgenic plant to the rubber powdery mildew is higher than that of the receptor plant specifically represented by the following characteristics: the transgenic plant does not produce the conidia of the powdery mildew of the rubber tree after being inoculated with the powdery mildew of the rubber tree, and the receptor plant produces the conidia of the powdery mildew of the rubber tree after being inoculated with the powdery mildew of the rubber tree; and/or the transgenic plant has an obvious disease-resistant yellowing phenotype after being inoculated with the rubber tree powdery mildew, and the receptor plant has weak powdery mildew lesion after being inoculated with the rubber tree powdery mildew.

In the above application or method, the powdery mildew can be powdery mildew o.

In the above application or method, the plant may be a monocotyledon or a dicotyledon; the dicotyledonous plant can be specifically arabidopsis thaliana; the Arabidopsis thaliana may specifically be wild type Arabidopsis thaliana (Col-0 ecotype Columbia) or Arabidopsis thaliana mutant ataf2 (Arabidopsis thaliana mutants ataf2-1 and ataf 2-2).

RNA-seq data analysis is firstly carried out on Arabidopsis thaliana wild type Col-0 inoculated with rubber tree powdery mildew Oidium heveae HN1106, and the fact that the Arabidopsis thaliana transcription factor ATAF2 is severely up-regulated and expressed 4 days after inoculation is found, and the fact that ATAF2 is induced and expressed by the rubber tree powdery mildew is presumed. Then, the inoculation experiment of rubber powdery mildew on an arabidopsis mutant ATAF2 is further verified, the ATAF2 mutant is found to show the phenotype of rubber powdery mildew infection, the disease resistance of arabidopsis to rubber powdery mildew can be positively regulated, and GST pull-down and co-immunoprecipitation experiments are utilized to find that ATAF2 and EDS1 can directly interact with each other, so that a good foundation is laid for further discussing a plant disease-resistant signal path in which EDS1 participates in the future.

Drawings

FIG. 1 shows that ATAF2 gene is up-regulated by Erysiphe hevea.

FIG. 2 shows that ATAF2 is regulating the disease resistance of Arabidopsis thaliana to Blumeria rubber fungus. A is the leaf inoculation result. And B is the result of counting conidia.

Fig. 3 shows that ATAF2 can interact directly with EDS 1.A is the interaction of ATAF2 with EDS1 in arabidopsis protoplasts. B is the direct interaction of ATAF2 and EDS1 in vitro.

Detailed Description

The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged.

Mutant ataf2-1, referred to in the examples below, was purchased from the ABRC seed center and was numbered SALK-136355.

Mutant ataf2-2, referred to in the examples below, was purchased from the ABRC seed center and was numbered SALK-015750.

The Erysiphe hevea strain O.Heveae HN1106, referred to in the examples below, is described in the literature "Mei, S.Hou, S.S., Cui, H.and Rong W.Characterisation of the interaction between Oideum hevea and Arabidopsis thaliana [ J ]. Molecular plant Pathology,2016,17(9): 1331-.

The amino acid sequence of the ATAF2 protein in the following examples is shown in sequence 2, and the coding gene sequence of the ATAF2 protein is shown in sequence 1.

The amino acid sequence of the EDS1 protein in the following examples is shown in sequence 4, and the coding gene sequence of the EDS1 protein is shown in sequence 3.

The amino acid sequence of XopD protein in the following examples is shown in sequence 6, and the coding gene sequence of XopD protein is shown in sequence 5.

Example 1 upregulation of the ATAF2 Gene by Induction of Blumeria rubber Tree powdery mildew

First, discovery that ATAF2 gene is up-regulated by induction of Erysiphe hevea

Oidium heveae HN1106 was inoculated into wild type Arabidopsis thaliana Col-0 and RNA-seq data analysis was performed 4 days after inoculation.

The analysis result of the RNA-seq data shows that: after inoculation of Oidium heveae HN 11064 days on wild type Arabidopsis thaliana Col-0, the Arabidopsis thaliana NAC family transcription factor ATAF2 was up-regulated by 6 times.

Second, verification that ATAF2 gene is up-regulated by induction of Erysiphe hevea

1. Wild type Arabidopsis thaliana Col-0 cultured for about 5 weeks was inoculated with Oidium heveae HN 1106.

2. Leaves of wild type Arabidopsis thaliana Col-0 at 0, 2, 4 and 8 days after inoculation were respectively cut.

3. Total RNA of leaves inoculated with the wild type Arabidopsis thaliana Col-0 at different times was extracted separately. The method comprises the following specific steps: precooling the mortar with liquid nitrogen, putting the Arabidopsis leaves, repeatedly adding the liquid nitrogen, and grinding for 3 times. 1mL of Invitrogen Trizol (Lot No.66223) solution was added to extract the total RNA from Arabidopsis thaliana leaves.

4. DNase was added to the extracted RNA, and the resulting mixture was digested at 37 ℃ for 1 hour, followed by reverse transcription using RNA reverse transcription kit III (Lot No.696045) from Invitrogen to obtain cDNA.

5. Using cDNA as template

Premix Ex Taq II (Lot No. AK2702) kit for fluorometryQuantitative PCR. ACTIN was used as an internal reference gene. PCR procedure: 40s at 95 ℃; 6s at 95 ℃, 40s at 62 ℃ and 40 cycles. The primer sequences are as follows:

ACTIN-F：5′-TGGTGGAAGCACAGAAGTTG-3′；

ACTIN-R：5′-GATCCATGTTTGGCTCCTTC-3′；

ATAF2-F：5′-ACGGACGAGGAGCTTGTGAA-3′；

ATAF2-R：5′-CTCCGGTAGCTTTCCAATAAC-3′。

the results are shown in FIG. 1. The results show that: the ATAF2 gene was up-regulated approximately 5-fold 2 days after inoculation (FIG. 1), up-regulated approximately 20-fold 4 days after inoculation (FIG. 1), up-regulated approximately 15-fold 8 days after inoculation (FIG. 1), and the expression trend was consistent with the RNA-seq data, indicating that the ATAF2 gene was up-regulated by Erysiphe hevea induction.

Example 2 application of ATAF2 in the Positive control of resistance of Arabidopsis thaliana to Blumeria rubber Tree

First, obtaining of mutant

The ataf 2T-DNA insertion pure and line mutants, ataf2-1 and ataf2-2, were purchased from the ABRC seed center.

Compared with the wild Arabidopsis thaliana Col-0, only the ATAF2 gene in the mutant ATAF2-1 has T-DNA insertion mutation between the 212 th and 213 th positions of the second exon of the ATAF2 gene.

Compared with the wild Arabidopsis thaliana Col-0, only the ATAF2 gene in the mutant ATAF2-2 has T-DNA insertion mutation at the position between the 42 th position and the 43 th position of the second intron of the ATAF2 gene.

Second, acquisition of ataf2 complementary Arabidopsis plants

1. Construction of recombinant expression vectors

The DNA molecule shown in the sequence 7 (1.5Kb promoter region + ATAF2 genome DNA molecule) is inserted between the Xho I and BstB I enzyme cutting sites of pCAMBIA1300 vector (Beijing Bainovei Biotech Co., Ltd.), and other sequences of the pCAMBIA1300 vector are kept unchanged, so that the recombinant expression vector pCAMBIA1300-ATAF2 is obtained. Wherein the 1501-2532 site of the sequence 7 is a genomic DNA molecule of the ATAF2 gene.

2. Construction of recombinant bacterium

And (2) transforming the recombinant expression vector pCAMBIA1300-ATAF2 obtained in the step (1) into agrobacterium GV1301 (Beijing Bainovei Biotech limited), and obtaining a positive transformant through PCR identification, wherein the positive transformant is used for infecting an arabidopsis thaliana plant.

3. Obtaining of ATAF 2-transgenic Arabidopsis plants

The recombinant bacteria (agrobacterium GV1301 containing the recombinant expression vector pCAMBIA1300-ATAF 2) constructed in the step 2 are adopted to infect the ATAF2-1 mutant inflorescence by a flower dipping method. Harvesting of T₁Seeds are generated and screened on a screening culture medium. Screening the next generation of seedlings obtained by screening, and repeating the steps to finally obtain T₂Transgenic ATAF2 Arabidopsis plants.

4. Identification of ATAF 2-transferred Arabidopsis plants

The T obtained in step 3 was subjected to western blot analysis using an anti-FLAG antibody (purchased from Sigma)₂And (3) transferring an ATAF2 Arabidopsis plant for detection, and obtaining a positive ATAF2 Arabidopsis plant which is an ATAF2 complementary Arabidopsis plant and is used for detecting the disease resistance of rubber powdery mildew.

Third, ATAF2 positively regulates the disease resistance of Arabidopsis to rubber tree powdery mildew

The wild type Arabidopsis thaliana Col-0, mutant ataf2(ataf2-1 and ataf2-2) and positive ataf2 complementary Arabidopsis thaliana plants were inoculated with the Erysiphe hevea Oidium heveae HN 1106. 10 days after inoculation, leaf phenotype was observed and conidia were counted. The method comprises the following specific steps:

arabidopsis thaliana grown for 5 weeks was placed in a nylon membrane inoculation chamber of 40 cm. times.40 cm. times.50 μm, and rubber powdery mildew was brushed on the nylon membrane with a brush for inoculation. After 10 days of inoculation, leaf disease symptoms were observed and photographed. Cutting leaves, and decolorizing overnight (decolorizing solution, ethanol: phenol: H)₂O: lactic acid 2: 1: 1: 1). The decolorized leaves were stained in Coomassie brilliant blue staining solution (G250, 6G/L, ethanol solution) for 30s, and observed microscopically, and the number of conidia generated from a single spore was counted.

The results are shown in FIG. 2. As a result, it was found that: wild type arabidopsis and ataf2 complemented the development of a clear disease-resistant yellowing phenotype in arabidopsis plants (fig. 2A), and the mutants ataf2-1 and ataf2-2 developed faint powdery mildew plaques (fig. 2A). Further analysis by conidium count showed that: blumeria rubber powdery mildew did not produce conidia on wild type Arabidopsis thaliana and ataf2 complementary Arabidopsis thaliana plants, and produced a large number of conidia on mutant ataf2 (FIG. 2B), with an average number of conidia formed on the ataf2-1 and ataf2-2 mutants of 28 and 26, respectively, per mature spore. Taken together, the results show that the ATAF2 mutant exhibits a phenotype of susceptibility to powdery mildew, suggesting that ATAF2 may positively regulate plant resistance to powdery mildew.

Example 3 direct interaction of ATAF2 with EDS1

First, co-immunoprecipitation test verifies the interaction of ATAF2 and EDS1

An ATAF2 protoplast expression vector with a FLAG label (a vector obtained by connecting an ATAF2 gene sequence into a position between Xho I and BstB I enzyme cutting sites of a PUC18 vector (Beijing Bainovei Biotechnology Co., Ltd.) driven by a 35S promoter) and an EDS1 protoplast expression vector with an HA label (a vector obtained by connecting an EDS1 gene sequence into a position between Kpn I and Sal I enzyme cutting sites of a PUC18 vector (Beijing Bainovei Biotechnology Co., Ltd.) driven by a 35S promoter) are constructed, and after plasmids are extracted by cesium chloride density gradient centrifugation, arabidopsis thaliana protoplasts are transformed together, and a Xanthomonas XopD protein is used as a control for an immune coprecipitation test. The method comprises the following specific steps:

XopD-FLAG, ATAF2-FLAG and EDS1-HA plasmids were extracted by cesium chloride density gradient centrifugation. Protoplasts of wild type Arabidopsis thaliana Col-0 were prepared, and 60. mu.g each of the mixed plasmids of ATAF2-FLAG and EDS1-HA and 60. mu.g each of the mixed plasmids of XopD-FLAG and EDS1-HA were transformed into the protoplasts, respectively, and cultured overnight under a weak light. After centrifugation at 100g for 2min, the supernatant was discarded, and 1mL of protein extraction buffer (0.15mol/L KCl, 50mM HEPES-KOH (pH 7.5), 1mM EDTA, 1mM DTT, 0.2% Triton-X100, cocktail protease inhibitor) was added to the protoplast pellet to extract the total protein of the protoplasts. Then 20. mu.L of anti-Flag M2 agarose (sigma) was added. Incubate at 4 ℃ for 4 hours. After centrifugation at 500g for 1min, the supernatant was discarded, and the pellet was rinsed 6 times with 1mL of rinsing buffer (0.15mol/L KCl, 50mM HEPES-KOH (pH 7.5), 1mM EDTA, 1mM DTT, 0.5% Triton-X100, cocktail protease inhibitor), 50. mu.L of elution buffer (0.5mM3XFLAG polypeptide) was added and incubated at 4 ℃ for 1 hour. Centrifuging, taking the supernatant, carrying out SDA-PAGE electrophoresis, and detecting protein interaction by using FLAG antibody and HA antibody respectively.

The results are shown in FIG. 3A. The results show that: ATAF2 interacted with EDS1, whereas the control protein XOPD did not.

Second, pull down test verifies the interaction of ATAF2 with EDS1

Constructing a GST-ATAF2 protein expression vector (a vector obtained by inserting an ATAF2 gene sequence into enzyme cutting sites of BamH I and Xho I of a pGEX6p-1 vector (Beijing Bainovei Biotech Co., Ltd)) and a HIS-EDS1 protein expression vector (a vector obtained by inserting an EDS1 gene sequence into enzyme cutting sites of EcoR I and Sal I of a pET30a vector (Beijing Bainovei Biotech Co., Ltd)) to transform escherichia coli BL21, inducing protein expression by IPTG, purifying GST, GST-ATAF2 and HIS-EDS1 proteins respectively, and performing pull down experiments. The method comprises the following specific steps:

GST-ATAF2 and GST and HIS-EDS1 were mixed in 50. mu.g each in 2 1.5mL centrifuge tubes, and the mixture was brought to 1mL with a buffer (0.025mol/L Tris-HCl (pH 7.5), 0.1mol/L NaCl, 0.001mol/L DTT), and incubated at 4 ℃ for half an hour. Then adding 50 μ L of Glutathione Sepharose^TM4B, incubation at 4 ℃ for 1 hour. 500g for 1min, discard the supernatant, precipitate with 1mL rinsing buffer (0.025mol/L Tris-HCl (pH 7.5), 0.1mol/L NaCl, 0.001mol/L DTT, 0.2% Triton-X100) repeatedly washing centrifugation, washing 5 times. Finally, 100. mu.L of elution buffer (10mM reduced glutathione, 50mM Tris-HCl pH 8.0) was added and incubated at 4 ℃ for 10 min. Centrifuging, taking the supernatant, carrying out SDS-PAGE electrophoresis, and detecting the interaction by using lithopone red staining and an HIS antibody respectively.

The results are shown in FIG. 3B. The results show that: GST-ATAF2 could interact with HIS-EDS1, whereas control GST did not, indicating that ATAF2 interacts directly with EDS 1.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the technical principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Sequence listing

<110> university of Hainan

Application of <120> ATAF2 protein and related biological materials thereof in regulation and control of disease resistance of plants to powdery mildew of rubber trees

<160>7

<170>PatentIn version 3.5

<210>1

<211>852

<212>DNA

<213>Artificial Sequence

<400>1

atgaagtcgg agctaaattt accagctggg ttccgattcc atccaacgga cgaggagctt 60

gtgaaattct acttgtgccg gaaatgtgct tccgagcaga tctcggctcc ggttatcgcc 120

gagattgatc tctacaagtt caatccttgg gagcttccag agatgtctct gtacggagag 180

aaagagtggt acttcttctc acctagagat cggaaatacc caaacggttc gcgtcctaac 240

cgggcagcag gaaccggtta ttggaaagct accggagcag ataaaccgat tggtaaaccg 300

aagacgttgg gtatcaagaa agcactcgtc ttctacgcag ggaaagctcc aaaagggatt 360

aagaccaatt ggataatgca tgagtatcgt ctcgctaatg ttgatagatc agcttctgtt 420

aacaaaaaga acaacctacg acttgatgat tgggttttat gtcgaatata caacaagaaa 480

ggaaccatgg agaagtattt ccccgcggat gagaagccga ggaccacgac aatggctgaa 540

cagtcatcat caccttttga tacatcagac tcgacttacc cgacattgca agaggatgat 600

tccagcagct caggtggtca cggtcacgtg gtgtcaccgg atgttttgga ggttcagagc 660

gagcctaaat ggggagagct tgaggatgct ttggaagctt ttgatacttc aatgtttggt 720

agttccatgg agttgttgca gcctgacgct tttgtccctc agttcttgta tcagtctgat 780

tatttcactt ccttccagga tccgcctgag cagaaaccat tcttgaattg gagttttgct 840

ccacaggggt aa 852

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Met Lys Ser Glu Leu Asn Leu Pro Ala Gly Phe Arg Phe His Pro Thr

1 5 10 15

Asp Glu Glu Leu Val Lys Phe Tyr Leu Cys Arg Lys Cys Ala Ser Glu

20 25 30

Gln Ile Ser Ala Pro Val Ile Ala Glu Ile Asp Leu Tyr Lys Phe Asn

35 40 45

Pro Trp Glu Leu Pro Glu Met Ser Leu Tyr Gly Glu Lys Glu Trp Tyr

50 55 60

Phe Phe Ser Pro Arg Asp Arg Lys Tyr Pro Asn Gly Ser Arg Pro Asn

65 70 75 80

Arg Ala Ala Gly Thr Gly Tyr Trp Lys Ala Thr Gly Ala Asp Lys Pro

85 90 95

Ile Gly Lys Pro Lys Thr Leu Gly Ile Lys Lys Ala Leu Val Phe Tyr

100 105 110

Ala Gly Lys Ala Pro Lys Gly Ile Lys Thr Asn Trp Ile Met His Glu

115 120 125

Tyr Arg Leu Ala Asn Val Asp Arg Ser Ala Ser Val Asn Lys Lys Asn

130 135 140

Asn Leu Arg Leu Asp Asp Trp Val Leu Cys Arg Ile Tyr Asn Lys Lys

145 150 155 160

Gly Thr Met Glu Lys Tyr Phe Pro Ala Asp Glu Lys Pro Arg Thr Thr

165 170 175

Thr Met Ala Glu Gln Ser Ser Ser Pro Phe Asp Thr Ser Asp Ser Thr

180 185 190

Tyr Pro Thr Leu Gln Glu Asp Asp Ser Ser Ser Ser Gly Gly His Gly

195 200 205

His Val Val Ser Pro Asp Val Leu Glu Val Gln Ser Glu Pro Lys Trp

210 215 220

Gly Glu Leu Glu Asp Ala Leu Glu Ala Phe Asp Thr Ser Met Phe Gly

225 230 235 240

Ser Ser Met Glu Leu Leu Gln Pro Asp Ala Phe Val Pro Gln Phe Leu

245 250 255

Tyr Gln Ser Asp Tyr Phe Thr Ser Phe Gln Asp Pro Pro Glu Gln Lys

260 265 270

Pro Phe Leu Asn Trp Ser Phe Ala Pro Gln Gly

275 280

<210>3

<211>1872

<212>DNA

<213>Artificial Sequence

<400>3

atggcgtttg aagctcttac cggaatcaat ggtgatctaa tcaccagatc atggtcagcc 60

tcgaagcaag cttacctaac cgagcgctat cacaaggaag aagcaggagc agtcgtaatc 120

ttcgctttcc aaccatcttt ctcagagaaa gatttcttcg atccggacaa taaatcttcc 180

tttggagaaa tcaagttgaa ccgtgttcag tttccttgta tgaggaaaat cggtaaaggt 240

gatgtagcta ctgttaacga agctttcctc aagaatcttg aagctatcat tgatccaaga 300

acctcatttc aagcttctgt ggaaatggct gtgaggagta gaaaacagat agtgttcaca 360

ggacattcct caggaggtgc aactgcaatc ttagcaacag tttggtattt ggagaaatac 420

ttcatacgca atccaaatgt ttaccttgag cctcgttgtg tgacatttgg agctcctttg 480

gttggtgact ctatcttcag tcacgcactt gggagagaaa aatggagccg gttctttgtg 540

aactttgtct caagattcga tattgtccct cggattatgc ttgctcgaaa ggcgtctgta 600

gaggaaactt tgcctcatgt tcttgcccaa ttggatccca gaaagtcttc cgtccaagag 660

agtgaacaga gaataacaga gttttacaca agggtgatgc gagacacatc aactgttgca 720

aaccaagctg tttgtgaatt gactggaagc gcagaggcgt ttttagagac cctttctagt 780

ttccttgagc taagtcctta tagacccgcc ggtacttttg ttttctctac agagaagaga 840

ttggttgcag tgaacaactc ggacgccatt cttcaaatgc tgttttacac ttctcaagcc 900

agcgatgaac aagaatggtc tctaattcca tttcgaagta tcagagatca tcatagctat 960

gaggaactgg tacagtcgat gggaaagaag ttgtttaatc atttggatgg agaaaactca 1020

atagagtcta cgctcaatga ccttggagtg agcacaagag gcagacagta cgttcaagct 1080

gcattagagg aagagaagaa acgagtagag aatcagaaga agattattca ggtgatcgag 1140

caagagaggt ttttaaagaa actagcatgg atagaagatg aatacaagcc aaagtgtcaa 1200

gcccataaaa atgggtatta tgattccttc aaagtttcaa atgaagagaa tgacttcaaa 1260

gcaaacgtca agagagctga gttagccggt gtttttgacg aggtgcttgg tttaatgaag 1320

aaatgtcaac ttccagatga gttcgaaggg gacatagatt ggatcaagtt agcaactcga 1380

taccgcagat tagttgagcc tcttgatatt gcaaactacc atcgacattt aaagaacgaa 1440

gacacagggc cgtacatgaa aagaggaaga ccaacccgct acatatatgc tcagagaggc 1500

tacgaacatt atatactgaa gccaaacgga atgattgcag aagatgtatt ttggaacaag 1560

gtaaatggtc ttaacttagg gttacagcta gaagaaattc aagagactct aaagaattcg 1620

ggatccgagt gcggatcatg cttttgggct gaggttgaag aactcaaagg aaagccatac 1680

gaggaagttg aggtaagagt taagacatta gaagggatgc ttggagaatg gatcacagac 1740

ggggaggtag atgataagga aatatttctg gagggttcaa cgtttagaaa gtggtggatt 1800

acgcttccca aaaatcacaa atcgcattct cctctgcgag actatatgat ggatgaaata 1860

acagatacct ga 1872

<210>4

<211>623

<212>PRT

<213>Artificial Sequence

<400>4

Met Ala Phe Glu Ala Leu Thr Gly Ile Asn Gly Asp Leu Ile Thr Arg

1 5 10 15

Ser Trp Ser Ala Ser Lys Gln Ala Tyr Leu Thr Glu Arg Tyr His Lys

20 25 30

Glu Glu Ala Gly Ala Val Val Ile Phe Ala Phe Gln Pro Ser Phe Ser

35 40 45

Glu Lys Asp Phe Phe Asp Pro Asp Asn Lys Ser Ser Phe Gly Glu Ile

50 55 60

Lys Leu Asn Arg Val Gln Phe Pro Cys Met Arg Lys Ile Gly Lys Gly

65 70 75 80

Asp Val Ala Thr Val Asn Glu Ala Phe Leu Lys Asn Leu Glu Ala Ile

85 90 95

Ile Asp Pro Arg Thr Ser Phe Gln Ala Ser Val Glu Met Ala Val Arg

100 105 110

Ser Arg Lys Gln Ile Val Phe Thr Gly His Ser Ser Gly Gly Ala Thr

115 120 125

Ala Ile Leu Ala Thr Val Trp Tyr Leu Glu Lys Tyr Phe Ile Arg Asn

130 135 140

Pro Asn Val Tyr Leu Glu Pro Arg Cys Val Thr Phe Gly Ala Pro Leu

145 150 155 160

Val Gly Asp Ser Ile Phe Ser His Ala Leu Gly Arg Glu Lys Trp Ser

165 170 175

Arg Phe Phe Val Asn Phe Val Ser Arg Phe Asp Ile Val Pro Arg Ile

180 185 190

Met Leu Ala Arg Lys Ala Ser Val Glu Glu Thr Leu Pro His Val Leu

195 200 205

Ala Gln Leu Asp Pro Arg Lys Ser Ser Val Gln Glu Ser Glu Gln Arg

210 215 220

Ile Thr Glu Phe Tyr Thr Arg Val Met Arg Asp Thr Ser Thr Val Ala

225 230 235 240

Asn Gln Ala Val Cys Glu Leu Thr Gly Ser Ala Glu Ala Phe Leu Glu

245 250 255

Thr Leu Ser Ser Phe Leu Glu Leu Ser Pro Tyr Arg Pro Ala Gly Thr

260 265 270

Phe Val Phe Ser Thr Glu Lys Arg Leu Val Ala Val Asn Asn Ser Asp

275 280 285

Ala Ile Leu Gln Met Leu Phe Tyr Thr Ser Gln Ala Ser Asp Glu Gln

290 295 300

Glu Trp Ser Leu Ile Pro Phe Arg Ser Ile Arg Asp His His Ser Tyr

305 310 315 320

Glu Glu Leu Val Gln Ser Met Gly Lys Lys Leu Phe Asn His Leu Asp

325 330 335

Gly Glu Asn Ser Ile Glu Ser Thr Leu Asn Asp Leu Gly Val Ser Thr

340 345 350

Arg Gly Arg Gln Tyr Val Gln Ala Ala Leu Glu Glu Glu Lys Lys Arg

355 360 365

Val Glu Asn Gln Lys Lys Ile Ile Gln Val Ile Glu Gln Glu Arg Phe

370 375 380

Leu Lys Lys Leu Ala Trp Ile Glu Asp Glu Tyr Lys Pro Lys Cys Gln

385 390 395 400

Ala His Lys Asn Gly Tyr Tyr Asp Ser Phe Lys Val Ser Asn Glu Glu

405 410 415

Asn Asp Phe Lys Ala Asn Val Lys Arg Ala Glu Leu Ala Gly Val Phe

420 425 430

Asp Glu Val Leu Gly Leu Met Lys Lys Cys Gln Leu Pro Asp Glu Phe

435 440 445

Glu Gly Asp Ile Asp Trp Ile Lys Leu Ala Thr Arg Tyr Arg Arg Leu

450 455 460

Val Glu Pro Leu Asp Ile Ala Asn Tyr His Arg His Leu Lys Asn Glu

465 470 475 480

Asp Thr Gly Pro Tyr Met Lys Arg Gly Arg Pro Thr Arg Tyr Ile Tyr

485 490 495

Ala Gln Arg Gly Tyr Glu His Tyr Ile Leu Lys Pro Asn Gly Met Ile

500 505 510

Ala Glu Asp Val Phe Trp Asn Lys Val Asn Gly Leu Asn Leu Gly Leu

515 520 525

Gln Leu Glu Glu Ile Gln Glu Thr Leu Lys Asn Ser Gly Ser Glu Cys

530 535 540

Gly Ser Cys Phe Trp Ala Glu Val Glu Glu Leu Lys Gly Lys Pro Tyr

545 550 555 560

Glu Glu Val Glu Val Arg Val Lys Thr Leu Glu Gly Met Leu Gly Glu

565 570 575

Trp Ile Thr Asp Gly Glu Val Asp Asp Lys Glu Ile Phe Leu Glu Gly

580 585 590

Ser Thr Phe Arg Lys Trp Trp Ile Thr Leu Pro Lys Asn His Lys Ser

595 600 605

His Ser Pro Leu Arg Asp Tyr Met Met Asp Glu Ile Thr Asp Thr

610 615 620

<210>5

<211>2406

<212>DNA

<213>Artificial Sequence

<400>5

atggacagat tatttaattt tgactataaa aactatctcg aaatgacgga ggcggcggat 60

gatcaccgca attctccgcc ccctgaagaa cacagggaaa actacagcac gggttacaac 120

atgcatccgc tgcttgaatc cctcccgcga agaaatccca cgcaagtgcc tgccgatagc 180

tcggtccatc agatgcgcgc agccgcgccg acctccaatc gccataggag ttactcaaga 240

ttattagatc taagtggcgc ctatggtgat ggcaaaaaaa tgtcagaact gctaagaagc 300

tttccaactt ttgattttta tttgaaaacc gacggcctat cacatcgggc tggcatgcag 360

atgcttcggg aattgagcgg agatcaatgc gaccatgtca taggtcaaat aataagacga 420

gttgagaatt gcctgggtcc tgcaaagcga gaaatcgcgc tgagcaacct ggagttggat 480

tgcagtcaaa acattgtgct gagccaactg actttagatc gcattgccaa agccaaagcc 540

aaagtcaaag gcaaagccaa agccaaagcc gaagccgaag ctaaagccga agtcgcagtc 600

gcagtcgaag tcgcagccaa attcgcagcc gaagacgcag ccacagccgc agccgccgct 660

gcagcccaga gtgtgatcag taaaattatc gaacatctac caatatatga aacattacag 720

aaactgccgc gccacgcgga gggtgtgagc gggttccaga attacttgtg taaaaatggc 780

tcatttggcc aagcgttaat caacatcctt gaatacgcca ccccagatca gaaggaaaga 840

ttgaagcagg caattcagaa gcgccgacag gccttggctg cacataacgc cgtggctgca 900

actaagagta atgtgcccag aactggagta ttccggaccc tttataaaaa tccaaagtta 960

attcttggaa tctcaggcaa attaagcaaa agaacatgca gtattaacga tgcaagcagc 1020

ggatatttat cacaagcaga cctcgaaaaa atagtcgacg agaaaaccgg cgaactgact 1080

cctctgggtg aagaagtgat ttcaggtgca tccgaaagca tccaggatgc aattcgcgcc 1140

aatttcagaa tgcgttacca acaaccggaa ctgcccccca acagcccccc tcaggccttt 1200

tatccgcagg aagaaaatcc gccgccctca tcgttcttcc agggggtgag ctcatattgg 1260

agacatgtgg gctgcgagcc ggaaccggac actccgcaat acccccctca aagcccagct 1320

tcgactttta gtggcctctc ctccatgagc cattacggcc gcgaattcga cctcaatacg 1380

ccccagcaga tcgagcagcc agggtggcag ggcgccacgc ccgcccaaag cacggactcg 1440

accttcggcg gcctctcctc catgagccat tacggccgcg aattcgacct caatacgccc 1500

cagcagatcg agcaaccagg gtggcagggc gccacgcccg cccaaagcac ggactcgacc 1560

ttcgattgcc tctcctccat gagccattac ggccgcgaat tcgacctcaa tacgccccgg 1620

gaagagcagc tttggagcac tgatggggac tatggcactc aggctcccgt ggggcgctcg 1680

gccacccccg ccatgagtcc cgagaggatt gatgtggaca atctgccgtc gccccaggac 1740

gtcccagact ccgagcttcc tccagcgacg gccacttcgt ggctgctaga cggccatttg 1800

cgcgcctaca ccgacgacct cgcccgccga ttgcgagggc agcccaatgc ccatttactc 1860

cactttgccg actcgcaggt agtgaccatg ctgagctcca cagatccaga ccaacaggcc 1920

cgcgcacggc gccttcttgt tggagacgac gtcccaccta tcgtgttcct gccgatcaat 1980

cagcgcgaca ctcattggtc attgctcgtc gtcgaccgtc gtaacaagga cgctgttgcg 2040

acctaccact acgattccat ggcacagatg cagcaacagc aacgctacct tgggcagatg 2100

gcggtctatc acctagacct tgattaccaa caaattcatg aaatgcccac cgcgatacag 2160

cgggatggtt attcctgcgg cgatcatgtg ctgaccggaa tagagatgct ggctcacagg 2220

gtgatcgacg gcaccttcga cgatgcggac ggcagggacc tgcgcggtat cacaccagac 2280

cgcggcctca tcaggggtcg tcttgcccaa gcggagcaag ctccggcaga aagcagcgtc 2340

aggcaagctc ccgaactgcc catcgaacag aagaaaaaga aaagcaagtg gtggaagttc 2400

cagtaa 2406

<210>6

<211>801

<212>PRT

<213>Artificial Sequence

<400>6

Met Asp Arg Leu Phe Asn Phe Asp Tyr Lys Asn Tyr Leu Glu Met Thr

1 5 10 15

Glu Ala Ala Asp Asp His Arg Asn Ser Pro Pro Pro Glu Glu His Arg

20 25 30

Glu Asn Tyr Ser Thr Gly Tyr Asn Met His Pro Leu Leu Glu Ser Leu

35 40 45

Pro Arg Arg Asn Pro Thr Gln Val Pro Ala Asp Ser Ser Val His Gln

50 55 60

Met Arg Ala Ala Ala Pro Thr Ser Asn Arg His Arg Ser Tyr Ser Arg

65 70 75 80

Leu Leu Asp Leu Ser Gly Ala Tyr Gly Asp Gly Lys Lys Met Ser Glu

85 90 95

Leu Leu Arg Ser Phe Pro Thr Phe Asp Phe Tyr Leu Lys Thr Asp Gly

100 105 110

Leu Ser His Arg Ala Gly Met Gln Met Leu Arg Glu Leu Ser Gly Asp

115 120 125

Gln Cys Asp His Val Ile Gly Gln Ile Ile Arg Arg Val Glu Asn Cys

130 135 140

Leu Gly Pro Ala Lys Arg Glu Ile Ala Leu Ser Asn Leu Glu Leu Asp

145 150 155 160

Cys Ser Gln Asn Ile Val Leu Ser Gln Leu Thr Leu Asp Arg Ile Ala

165 170 175

Lys Ala Lys Ala Lys Val Lys Gly Lys Ala Lys Ala Lys Ala Glu Ala

180 185 190

Glu Ala Lys Ala Glu Val Ala Val Ala Val Glu Val Ala Ala Lys Phe

195 200 205

Ala Ala Glu Asp Ala Ala Thr Ala Ala Ala Ala Ala Ala Ala Gln Ser

210 215 220

Val Ile Ser Lys Ile Ile Glu His Leu Pro Ile Tyr Glu Thr Leu Gln

225 230 235 240

Lys Leu Pro Arg His Ala Glu Gly Val Ser Gly Phe Gln Asn Tyr Leu

245 250 255

Cys Lys Asn Gly Ser Phe Gly Gln Ala Leu Ile Asn Ile Leu Glu Tyr

260 265 270

Ala Thr Pro Asp Gln Lys Glu Arg Leu Lys Gln Ala Ile Gln Lys Arg

275 280 285

Arg Gln Ala Leu Ala Ala His Asn Ala Val Ala Ala Thr Lys Ser Asn

290 295 300

Val Pro Arg Thr Gly Val Phe Arg Thr Leu Tyr Lys Asn Pro Lys Leu

305 310 315 320

Ile Leu Gly Ile Ser Gly Lys Leu Ser Lys Arg Thr Cys Ser Ile Asn

325 330 335

Asp Ala Ser Ser Gly Tyr Leu Ser Gln Ala Asp Leu Glu Lys Ile Val

340 345 350

Asp Glu Lys Thr Gly Glu Leu Thr Pro Leu Gly Glu Glu Val Ile Ser

355 360 365

Gly Ala Ser Glu Ser Ile Gln Asp Ala Ile Arg Ala Asn Phe Arg Met

370 375 380

Arg Tyr Gln Gln Pro Glu Leu Pro Pro Asn Ser Pro Pro Gln Ala Phe

385 390 395 400

Tyr Pro Gln Glu Glu Asn Pro Pro Pro Ser Ser Phe Phe Gln Gly Val

405 410 415

Ser Ser Tyr Trp Arg His Val Gly Cys Glu Pro Glu Pro Asp Thr Pro

420 425 430

Gln Tyr Pro Pro Gln Ser Pro Ala Ser Thr Phe Ser Gly Leu Ser Ser

435 440 445

Met Ser His Tyr Gly Arg Glu Phe Asp Leu Asn Thr Pro Gln Gln Ile

450 455 460

Glu Gln Pro Gly Trp Gln Gly Ala Thr Pro Ala Gln Ser Thr Asp Ser

465 470 475 480

Thr Phe Gly Gly Leu Ser Ser Met Ser His Tyr Gly Arg Glu Phe Asp

485 490 495

Leu Asn Thr Pro Gln Gln Ile Glu Gln Pro Gly Trp Gln Gly Ala Thr

500 505 510

Pro Ala Gln Ser Thr Asp Ser Thr Phe Asp Cys Leu Ser Ser Met Ser

515 520 525

His Tyr Gly Arg Glu Phe Asp Leu Asn Thr Pro Arg Glu Glu Gln Leu

530 535 540

Trp Ser Thr Asp Gly Asp Tyr Gly Thr Gln Ala Pro Val Gly Arg Ser

545 550 555 560

Ala Thr Pro Ala Met Ser Pro Glu Arg Ile Asp Val Asp Asn Leu Pro

565 570 575

Ser Pro Gln Asp Val Pro Asp Ser Glu Leu Pro Pro Ala Thr Ala Thr

580 585 590

Ser Trp Leu Leu Asp Gly His Leu Arg Ala Tyr Thr Asp Asp Leu Ala

595 600 605

Arg Arg Leu Arg Gly Gln Pro Asn Ala His Leu Leu His Phe Ala Asp

610 615 620

Ser Gln Val Val Thr Met Leu Ser Ser Thr Asp Pro Asp Gln Gln Ala

625 630 635 640

Arg Ala Arg Arg Leu Leu Val Gly Asp Asp Val Pro Pro Ile Val Phe

645 650 655

Leu Pro Ile Asn Gln Arg Asp Thr His Trp Ser Leu Leu Val Val Asp

660 665 670

Arg Arg Asn Lys Asp Ala Val Ala Thr Tyr His Tyr Asp Ser Met Ala

675 680 685

Gln Met Gln Gln Gln Gln Arg Tyr Leu Gly Gln Met Ala Val Tyr His

690 695 700

Leu Asp Leu Asp Tyr Gln Gln Ile His Glu Met Pro Thr Ala Ile Gln

705 710 715 720

Arg Asp Gly Tyr Ser Cys Gly Asp His Val Leu Thr Gly Ile Glu Met

725 730 735

Leu Ala His Arg Val Ile Asp Gly Thr Phe Asp Asp Ala Asp Gly Arg

740 745 750

Asp Leu Arg Gly Ile Thr Pro Asp Arg Gly Leu Ile Arg Gly Arg Leu

755 760 765

Ala Gln Ala Glu Gln Ala Pro Ala Glu Ser Ser Val Arg Gln Ala Pro

770 775 780

Glu Leu Pro Ile Glu Gln Lys Lys Lys Lys Ser Lys Trp Trp Lys Phe

785 790 795 800

Gln

<210>7

<211>2532

<212>DNA

<213>Artificial Sequence

<400>7

gtgcacgaaa attatttagc taaaagccta aaagatgtgt atgaaatgat acgacttata 60

taacttgcat tcttatatcg ataaagaatc cttattgaca acaacaaaaa gtcatgtttg 120

accacaaata tttcgtatta taattaattt ggctactaag ggacttttaa tagtaaaagg 180

aaaagaccat gatcttataa aacattacgt tgaatcgatc ataattagtt tataatatga 240

tagaaaacgt aagtgatgat gatcataaaa cggggaacaa gcccacgtca tgcttgcata 300

cacacctacc tcaactttac atttaaagat tttaaaatag aagaggattc atgggcatta 360

tttgctagtg aatcaaactt atgaattata aattaagtct gtaatgaaag cggctcaatg 420

ggtacttaat taaggattaa gctagtggtt aaatagttgt ccttaacaca tatttttatg 480

taacggggaa actcaaacta aagcgcaaac gctttctttg cgaactccaa tgtttttatt 540

tatgtgaatg tagtattatt agtcgagagt atttataatt agtgactatg attggcgatt 600

gcaactttaa atgacgtcat ccatcatgac gcaaagcttc gcctgaagaa ggaaattaat 660

cagctgcgga gatgctcacc gtagtttcat caaactgtcc aatcaccagt caaaactaca 720

acttccgccc caagaaatgc tgacttatct tcagcaaggt tacaataatg tgtcaatttc 780

aatatcataa aaaagtagtg gctgctgaga tttttctttt taaatcttag tggtaaatct 840

tagtggtaag taaaggaaca aaagcgataa gctacactga gaagcaggag attacggttt 900

gacatataaa ggcatttcca tctacgagaa atccacaaaa tttctcaaac aaaaaaaata 960

ttaatattat attataattt aattatttaa ttaaaaaagt atttttgtta aaaaattgaa 1020

ccaatagtaa gatgagaatt gtcatgatgg gttgtaaagt atctcaaagt ttctcaagta 1080

agaaactttc tacactctct ttcccaatgg agatggtcta aagtcggtca acaattcaca 1140

aaaggtttaa gtgacgcaag caatgacaac atctaagtcg actatccaat aatattacgc 1200

cacgtatata tccagctggc tgcggtcagc gtagaaaaga aaacggtctg ttttgccgca 1260

cgtgcttgaa aatcccctcg tgtcatctct tttctgtttt ttgtgttttt tcaatttaaa 1320

ttaattaaaa catttcggcg attttcgtcc ttataaaacc ttcgtcgcct cactcacata 1380

tcctcgatta caatcttgag cagacgaaga aaacccaaaa aaaatccaga tccagctgaa 1440

attgaatttt caaccaacga agaagagatt tttccaagag caacagacaa gaagaagaga 1500

atgaagtcgg agctaaattt accagctggg ttccgattcc atccaacgga cgaggagctt 1560

gtgaaattct acttgtgccg gaaatgtgct tccgagcaga tctcggctcc ggttatcgcc 1620

gagattgatc tctacaagtt caatccttgg gagcttccag gtacacctat gggcttcttc 1680

taattatttt tcgcgaaatc atgtgtcttc gagaatcgtt gtttattgag atgtttgttc 1740

ttctttcaga gatgtctctg tacggagaga aagagtggta cttcttctca cctagagatc 1800

ggaaataccc aaacggttcg cgtcctaacc gggcagcagg aaccggttat tggaaagcta 1860

ccggagcaga taaaccgatt ggtaaaccga agacgttggg tatcaagaaa gcactcgtct 1920

tctacgcagg gaaagctcca aaagggatta agaccaattg gataatgcat gagtatcgtc 1980

tcgctaatgt tgatagatca gcttctgtta acaaaaagaa caacctacga gtaagttctg 2040

tttcttatta acttatgtat catcttttcg tgaatgatga tttagattat taactttgtt 2100

gtggtgggtt ttgctgtaca gcttgatgat tgggttttat gtcgaatata caacaagaaa 2160

ggaaccatgg agaagtattt ccccgcggat gagaagccga ggaccacgac aatggctgaa 2220

cagtcatcat caccttttga tacatcagac tcgacttacc cgacattgca agaggatgat 2280

tccagcagct caggtggtca cggtcacgtg gtgtcaccgg atgttttgga ggttcagagc 2340

gagcctaaat ggggagagct tgaggatgct ttggaagctt ttgatacttc aatgtttggt 2400

agttccatgg agttgttgca gcctgacgct tttgtccctc agttcttgta tcagtctgat 2460

tatttcactt ccttccagga tccgcctgag cagaaaccat tcttgaattg gagttttgct 2520

ccacaggggt aa 2532

Claims (13)

1. Use of the ATAF2 protein for regulating the resistance of plants to powdery mildew of rubber trees;

   the ATAF2 protein is a protein shown in a sequence 2;

   the plant is Arabidopsis thaliana.
2. Use of the ATAF2 protein for interacting with the EDS1 protein;

   the ATAF2 protein is a protein shown in a sequence 2.
3. 3. The application of biological materials related to ATAF2 protein in regulating and controlling the resistance of plants to the powdery mildew of rubber trees;

   the ATAF2 protein is a protein shown in a sequence 2;

   the plant is Arabidopsis thaliana;

   the biomaterial is any one of the following A1) to A12):

   A1) a nucleic acid molecule encoding an ATAF2 protein;

   A2) an expression cassette comprising the nucleic acid molecule of a 1);

   A3) a recombinant vector comprising the nucleic acid molecule of a 1);

   A4) a recombinant vector comprising the expression cassette of a 2);

   A5) a recombinant microorganism comprising the nucleic acid molecule of a 1);

   A6) a recombinant microorganism comprising the expression cassette of a 2);

   A7) a recombinant microorganism comprising a3) said recombinant vector;

   A8) a recombinant microorganism comprising a4) said recombinant vector;

   A9) a transgenic plant cell line comprising the nucleic acid molecule of a 1);

   A10) a transgenic plant cell line comprising the expression cassette of a 2);

   A11) a transgenic plant cell line comprising the recombinant vector of a 3);

   A12) a transgenic plant cell line comprising the recombinant vector of a 4).
4. 4. Use according to claim 3, characterized in that: A1) the nucleic acid molecule is a cDNA molecule shown in a sequence 1 or a genomic DNA molecule shown in the 1501-2532 th site of a sequence 7.
5. 5. Use of the ATAF2 protein of claim 1 or the biological material of claim 3 for breeding transgenic plants with increased resistance to powdery mildew of rubber; the plant is Arabidopsis thaliana.
6. 6. Use of the ATAF2 protein of claim 1 or the biological material of claim 3 in plant breeding; the plant is Arabidopsis thaliana.
7. 7. A method for breeding a transgenic plant with reduced resistance to hevea brasiliensis, comprising the step of reducing the expression level and/or activity of ATAF2 protein according to claim 1 in a recipient plant to obtain a transgenic plant; the transgenic plant has lower resistance to Blumeria necator than the recipient plant; the plant is Arabidopsis thaliana.
8. 8. The method of claim 7, wherein: the method for reducing the expression amount and/or activity of ATAF2 protein in a receptor plant, which is described in claim 1, is realized by silencing or inhibiting the expression and/or activity of a gene encoding ATAF2 protein in the genome of the receptor plant or knocking out a gene encoding ATAF2 protein.
9. 9. The method of claim 8, wherein: the coding gene for silencing or inhibiting the expression and/or activity of the coding gene of the ATAF2 protein or knocking out the ATAF2 protein in the genome of the receptor plant is the coding gene of the ATAF2 protein in the genome of the mutation receptor plant, so that the expression level of the coding gene of the ATAF2 protein in the genome of the receptor plant is reduced, or the coding gene of the ATAF2 protein in the genome of the receptor plant is subjected to deletion mutation or insertion mutation or base substitution.
10. 10. The method of claim 9, wherein: the method for generating deletion mutation or insertion mutation or base substitution of the gene coding for the ATAF2 protein in the genome of the receptor plant is T-DNA insertion.
11. 11. A method for producing a transgenic plant having improved resistance to Erysiphe hevea, comprising the step of increasing the expression level and/or activity of ATAF2 protein of claim 1 in a recipient plant to obtain a transgenic plant; the transgenic plant has higher resistance to Blumeria necator than the recipient plant; the plant is Arabidopsis thaliana.
12. 12. The method of claim 11, wherein: the method for increasing the expression level and/or activity of ATAF2 protein described in claim 1 in a recipient plant is to overexpress ATAF2 protein described in claim 1 in the recipient plant.
13. 13. The method of claim 12, wherein: the method for overexpression, which comprises introducing a gene encoding ATAF2 protein according to claim 1 into a recipient plant.

Images