Application of ATAF2 protein and related biological materials thereof in regulation and control of disease resistance of plants to rubber tree powdery mildew
阅读说明：本技术 Ataf2蛋白及其相关生物材料在调控植物对橡胶树白粉菌的抗病性中的应用 (Application of ATAF2 protein and related biological materials thereof in regulation and control of disease resistance of plants to rubber tree powdery mildew ) 是由 梅双双 戎伟 于 2019-12-11 设计创作，主要内容包括：本发明公开了ATAF2蛋白及其相关生物材料在调控植物对橡胶树白粉菌的抗病性中的应用。本发明首先对接种橡胶树白粉菌的拟南芥野生型进行分析,发现接种4天后拟南芥转录因子ATAF2发生显著上调表达,推测ATAF2受橡胶树白粉菌的诱导表达,接下来通过对拟南芥突变体ataf2进行橡胶树白粉菌接种实验进一步验证,发现ataf2突变体表现出对橡胶树白粉菌感病的表型,可正调控拟南芥对橡胶树白粉菌的抗病性,并且利用GST pull-down和免疫共沉淀实验发现ATAF2与EDS1二者可以直接发生相互作用,为将来进一步探讨EDS1参与的植物抗病信号通路奠定了很好的基础。(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 experiment of inoculating the powdery mildew to arabidopsis thaliana mutant ATAF2 is further verified, the fact that the ATAF2 mutant shows the phenotype of the powdery mildew infection of the rubber tree is found, the disease resistance of arabidopsis thaliana to the powdery mildew can be positively regulated, 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.)
Use of the ATAF2 protein for modulating the resistance of plants to powdery mildew.
Use of the ATAF2 protein for interacting with the EDS1 protein.
3. Use according to claim 1 or 2, characterized in that: 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 substituting and/or deleting and/or adding one or more amino acid residues in 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.
4. Use of a biological material related to the ATAF2 protein for modulating the resistance of a plant to powdery mildew;
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).
5. Use according to claim 4, characterized in that: A1) the nucleic acid molecule is a gene shown in the following 1) or 2) or 3):
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 genomic DNA molecule having 75% or more identity to the nucleotide sequence defined in 1) and encoding the ATAF2 protein described in claim 1;
3) a cDNA molecule or a genomic DNA molecule which hybridizes under stringent conditions to the nucleotide sequence defined in 1) or 2) and encodes the ATAF2 protein of claim 1.
6. Use of the ATAF2 protein of any one of claims 1-3 or the biological material of claim 4 or 5 for breeding transgenic plants with increased resistance to powdery mildew of hevea brasiliensis;
or, use of the ATAF2 protein according to any one of claims 1 to 3 or the biological material according to claim 4 or 5 for breeding transgenic plants with reduced resistance to powdery mildew of hevea brasiliensis;
or, use of the ATAF2 protein according to any one of claims 1 to 3 or the biological material according to claim 4 or 5 in plant breeding.
7. A method for breeding a transgenic plant with reduced resistance to powdery mildew of rubber tree comprising the step of reducing the expression level and/or activity of ATAF2 protein according to any one of claims 1 to 3 in a recipient plant to obtain a transgenic plant; the transgenic plant has lower resistance to Blumeria necator than the recipient plant.
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 realized by silencing or inhibiting the expression and/or activity of a gene encoding ATAF2 protein or knocking out a gene encoding ATAF2 protein in the genome of the receptor plant;
or, the gene for silencing or inhibiting the expression and/or activity of the gene for coding the ATAF2 protein or knocking out the ATAF2 protein in the genome of the receptor plant is a gene for coding the ATAF2 protein in the genome of the mutant receptor plant, so that the expression level of the gene for coding the ATAF2 protein in the genome of the receptor plant is reduced, or the gene for coding the ATAF2 protein in the genome of the receptor plant is subjected to deletion mutation, insertion mutation or base substitution;
alternatively, the method of making a deletion mutation, an insertion mutation or a base substitution in the gene encoding ATAF2 protein in the genome of the recipient plant is a T-DNA insertion.
9. A method for breeding a transgenic plant with improved resistance to powdery mildew of rubber tree, comprising the step of increasing the expression level and/or activity of ATAF2 protein according to any one of claims 1 to 3 in a recipient plant to obtain a transgenic plant; the transgenic plant has higher resistance to Blumeria necator than the recipient plant.
10. The method of claim 9, wherein: the method for increasing the expression amount and/or activity of ATAF2 protein described in any one of claims 1 to 3 in a recipient plant comprises the steps of over-expressing ATAF2 protein described in any one of claims 1 to 3 in the recipient plant;
alternatively, the overexpression method is carried out by introducing the gene encoding ATAF2 protein according to any one of claims 1 to 3 into a recipient plant.
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 an immune response (PTI) elicited by pathogenic bacteria-associated molecular patterns or microorganism-associated factors (PAMPs/MAMPs, pathogenic or microbiased molecular patterns). 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 additional 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, were 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
In the above application, the ATAF2 protein is a protein represented by a) or b) or c) or d) as follows:
a) the amino acid sequence is a protein shown in a
b) a fusion protein obtained by connecting a label to the N end and/or the C end of the protein shown in the
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
d) and (b) a protein having a homology of 75% or more than 75% with the amino acid sequence shown in the
In order to facilitate the purification of the protein in a), the amino terminus or the carboxyl terminus of the protein shown in
TABLE 1 tag sequences
Label (R)
Residue of
Sequence of
Poly-Arg
5-6 (typically 5)
RRRRR
Poly-His
2-10 (generally 6)
HHHHHH
8
DYKDDDDK
Strep-tag II
8
WSHPQFEK
c-
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
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 nucleotide sequences that are 75% or more, or 85% or more, or 90% or more, or 95% or more identical to the nucleotide sequence of a protein consisting of the amino acid sequence shown in
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
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-
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 strain referred to in the examples below is described in the references "Mei, S.Hou, S.S., Cui, H.and Rong W.characteristics of the interaction between Oidum hevea and Arabidopsis thaliana [ J ] Molecular plant technology, 2016,17(9): 1331-1343", publicly available from the Applicant, and this biomaterial is only used for the repetition of the experiments related to the present invention and is not used for other purposes.
The amino acid sequence of the ATAF2 protein in the following examples is shown in
The amino acid sequence of the EDS1 protein in the following examples is shown in
The amino acid sequence of XopD protein in the following examples is shown in sequence 6, and the gene sequence encoding XopD protein is shown in
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