CN110746498B - Application of plant stress tolerance-related protein TaANTL7A.2 in regulation and control of plant stress tolerance - Google Patents

Abstract

The invention discloses an application of a plant stress tolerance related protein TaANTL7A.2 in regulating and controlling plant stress tolerance. The plant stress tolerance-related protein TaANTL7A.2 disclosed by the invention is A1) or A2) or A3): A1) a protein with an amino acid sequence of sequence 1; A2) protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence of the sequence 1 and has the same function and is derived from A1); A3) a fusion protein obtained by connecting a label to the N-terminal or/and the C-terminal of A1) or A2). The TaANTL7A.2 can improve the stress tolerance of plants, can be used for preparing products for improving the stress tolerance of the plants and directly used for the stress tolerance, provides a basis for artificially controlling the expression of genes related to the stress tolerance and the stress tolerance, and plays an important role in breeding plants with enhanced stress tolerance and stress tolerance.

Description

Application of plant stress tolerance-related protein TaANTL7A.2 in regulation and control of plant stress tolerance

Technical Field

The invention relates to the field of biotechnology, and discloses application of a plant stress tolerance-related protein TaANTL7A.2 in regulation and control of plant stress tolerance.

Background

Adversity stresses such as drought and high temperature are barrier factors affecting plant growth and development. Therefore, understanding the response and signal transduction mechanism of wheat to stress conditions and improving the stress resistance of wheat varieties become one of the important tasks of wheat genetic research and wheat variety improvement.

Under the stress of adversity, a series of response reactions are generated in plants, and a plurality of physiological, biochemical and developmental changes are accompanied. The reaction mechanism of the plant to the stress is determined, and scientific data is provided for the research and application of the stress-resistant gene engineering. At present, the research on plant stress resistance has been advanced to the cellular and molecular level, and combined with the research on genetics and genetic engineering, the research on improving the growth characteristics of plants by biotechnology is aimed at improving the adaptability of plants to stress.

Under the adverse conditions of environmental stress such as drought, high temperature and the like, the plant can be correspondingly adjusted on molecular, cellular and overall levels so as to reduce the damage caused by the environment to the maximum extent and survive. Many genes are induced to express by stress, and the products of the genes not only can be directly involved in the stress response of plants, but also can regulate the expression of other related genes or be involved in signal transduction pathways, so that the plants can avoid or reduce damage, and the resistance to the stress environment is enhanced. Stress-related gene products can be divided into two broad categories: the products coded by the first gene comprise gene products directly participating in plant stress response, such as ion channel protein, aquaporin, osmotic regulatory factor (sucrose, proline, betaine and the like) synthetase and the like; the second class of genes encodes products including protein factors involved in stress-related signaling and regulation of gene expression, such as protein kinases, transcription factors, and the like. Among them, transcription factors play an important role in gene expression regulation of plant stress response.

Transcription factors, also known as trans-acting factors, are DNA binding proteins that specifically interact with cis-acting elements in the promoter region of eukaryotic genes, and through their interactions with other related proteins, activate or inhibit transcription. The DNA binding region of a transcription factor determines its specificity of binding to cis-acting elements, while the transcriptional regulatory region determines its activation or inhibition of gene expression. In addition, its own activity is also affected by nuclear localization and oligomerization. AP2/ERF is a plant-specific family of transcription factors that contain a DNA binding domain of the AP2/ERF type. The AP2/ERF domain was first discovered in the APETALA 2 gene of Arabidopsis thaliana. The AP2/ERF family has a total of 145 members in Arabidopsis, divided into four subfamilies, AP2, RAV, ERF and depression-responsive element-binding protein (DREB). The subfamily AP2 contains two AP2/ERF domains, has 14 members in Arabidopsis thaliana, and is further divided into two small subfamilies AP2 and AINTEGUMENTA (ANT). The RAV subfamily contains an AP2/ERF domain and a B3DNA binding domain, with 6 members in Arabidopsis. The remaining 125 members contained only one AP2/ERF domain, and were divided into two large subfamilies based on the similarity of the AP2/ERF domain, DREB (56 members, subfamily a) and ERF (65 members, subfamily B), and 4 additional members were not included in this class. The subfamily AP2 contains two domains AP2, a connecting sequence is arranged between the two domains AP2, and the two domains are divided into two subfamilies AP2 and ANT according to the characteristics of the sequence. In the small subfamily of AP2, the AP2 domain near the 3' end contains a miR172 binding site; in the ANT subfamily, two AP2 contain a specific insertion sequence.

The adversity stress severely restricts the normal growth of wheat and reduces the quality and yield of wheat. As the second major food crop in China, the development of stress-resistant breeding research on wheat has important significance on food safety and national economy in China. The dry hot air is high-temperature and low-humidity agricultural disastrous weather accompanied by certain wind power, and is a meteorological disaster frequently suffered by winter wheat in the later growth stage. It can accelerate the filling speed of wheat, leading to early completion of filling, reduction of dry matter accumulation and grain weight reduction. Extreme high temperatures also stop photosynthesis, leading to plant death. Therefore, the identification and separation of the high-temperature resistance gene, the analysis of the molecular mechanism of the high-temperature stress and the explanation of the regulation and control network of the high-temperature stress have important theoretical guidance significance and practical application value for the breeding of the wheat anti-adversity molecules.

Disclosure of Invention

The invention aims to provide an application of a protein from wheat in regulating and controlling plant stress tolerance; the protein is named TaANTL7A.2 and is A1) or A2) or A3) as follows:

A1) a protein with an amino acid sequence of sequence 1;

A2) protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence of the sequence 1 and has the same function and is derived from A1);

A3) a fusion protein obtained by connecting a label to the N-terminal or/and the C-terminal of A1) or A2).

In order to facilitate the purification of the protein of A1), the amino terminal or the carboxyl terminal of the protein consisting of the amino acid sequence shown in sequence 1 in the sequence listing may be labeled as shown in the following table.

Table: sequence of tags

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

The TaANTL7A.2 protein in A2) above is a protein having an identity of 75% or more to 75% or more of the amino acid sequence of the protein shown in SEQ ID NO. 1 and having the same function. The identity of 75% or more than 75% is 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity.

The TaANTL7A.2 protein in A2) can be synthesized by human, or obtained by synthesizing the coding gene and performing biological expression.

The gene encoding the taantl7a.2 protein in a2) above may be obtained by deleting one or several codons for amino acid residues from the DNA sequence shown in positions 129 to 1292 of the sequence 2, and/or by carrying out missense mutation of one or several base pairs, and/or by attaching a coding sequence of the tag shown in the above table to the 5 'end and/or 3' end thereof. Wherein, the DNA molecule shown in the 129 th site to the 1292 th site of the sequence 2 codes the TaANTL7A.2 protein shown in the sequence 1.

The invention also provides the application of the biological material related to the TaANTL7A.2 protein in regulating and controlling the stress tolerance of plants; the biomaterial is any one of the following B1) to B14):

B1) a nucleic acid molecule encoding a taantl7a.2 protein;

B2) an expression cassette comprising the nucleic acid molecule of B1);

B3) a recombinant vector comprising the nucleic acid molecule of B1);

B4) a recombinant vector comprising the expression cassette of B2);

B5) a recombinant microorganism comprising the nucleic acid molecule of B1);

B6) a recombinant microorganism comprising the expression cassette of B2);

B7) a recombinant microorganism containing the recombinant vector of B3);

B8) a recombinant microorganism containing the recombinant vector of B4);

B9) a transgenic plant cell line comprising the nucleic acid molecule of B1);

B10) a transgenic plant cell line comprising the expression cassette of B2);

B11) transgenic plant tissue comprising the nucleic acid molecule of B1);

B12) transgenic plant tissue comprising the expression cassette of B2);

B13) a transgenic plant organ containing the nucleic acid molecule of B1);

B14) a transgenic plant organ containing the expression cassette according to B2).

In the above application, the nucleic acid molecule of B1) may be any one of the following B1) -B5):

b1) the coding sequence is cDNA molecule or DNA molecule from 129 th site to 1292 th site of sequence 2 in the sequence table;

b2) a cDNA molecule or DNA molecule from 129 th site to 1292 th site of a sequence 2 in a sequence table;

b3) a cDNA molecule or a DNA molecule of a sequence 2 in a sequence table;

b4) a cDNA molecule or a genomic DNA molecule having 75% or more identity to the nucleotide sequence defined in b1) or b2) or b3) and encoding taantl7 a.2;

b5) hybridizes under stringent conditions with the nucleotide sequence defined in b1) or b2) or b3) or b4) and encodes a cDNA molecule or a genomic DNA molecule of taant l7 a.2.

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 the TaANTL7A.2 protein of the invention can be easily mutated by a person skilled in the art using known methods, such as directed evolution and point mutation. Those nucleotides which have been artificially modified to have an identity of 75% or more with the nucleotide sequence of the tankl 7a.2 protein isolated according to the invention are derived from the nucleotide sequence of the invention and are identical to the sequence of the invention, as long as they encode the tankl 7a.2 protein and have the function of the tankl 7a.2 protein.

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 coding sequence 1 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.

In the above application, the stringent conditions may be as follows: 50 ℃ in 7% Sodium Dodecyl Sulfate (SDS), 0.5M NaPO₄Hybridization with 1mM EDTA, rinsing in2 XSSC, 0.1% SDS at 50 ℃; also can be: 50 ℃ in 7% SDS, 0.5M NaPO₄Hybridization with 1mM EDTA, rinsing at 50 ℃ in 1 XSSC, 0.1% SDS; also can be: 50 ℃ in 7% SDS, 0.5M NaPO₄Hybridization with 1mM EDTA, rinsing in 0.5 XSSC, 0.1% SDS at 50 ℃; also can be: 50 ℃ in 7% SDS, 0.5M NaPO₄Hybridization with 1mM EDTA, rinsing in 0.1 XSSC, 0.1% SDS at 50 ℃; also can be: 50 ℃ in 7% SDS, 0.5M NaPO₄Hybridization with 1mM EDTA, rinsing in 0.1 XSSC, 0.1% SDS at 65 ℃; can also be: hybridization in a solution of 6 XSSC, 0.5% SDS at 65 ℃ followed by washing the membrane once with each of 2 XSSC, 0.1% SDS and 1 XSSC, 0.1% SDS; can also be: hybridization and washing of membranes 2 times, 5min each, at 68 ℃ in a solution of 2 XSSC, 0.1% SDS, and hybridization and washing of membranes 2 times, 15min each, at 68 ℃ in a solution of 0.5 XSSC, 0.1% SDS; can also be: 0.1 XSSPE (or 0.1 XSSC), 0.1% SDS at 65 ℃ and washing the membrane.

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

In the above-mentioned applications, the expression cassette containing a nucleic acid molecule encoding a taantl7a.2 protein (taantl7a.2 gene expression cassette) according to B2) is a DNA capable of expressing the taantl7a.2 protein in a host cell, and the DNA may include not only a promoter which initiates transcription of the taantl7a.2 gene but also a terminator which terminates transcription of the taantl7a.2 gene. Further, the expression cassette may also include an enhancer sequence. Promoters useful in the present invention include, but are not limited to: constitutive promoters, tissue, organ and development specific promoters, and inducible promoters. Examples of promoters include, but are not limited to: constitutive promoter of cauliflower mosaic virus 35S: the wound-inducible promoter from tomato, leucine aminopeptidase ("LAP", Chao et al (1999) Plant Physiol 120: 979-992); chemically inducible promoter from tobacco, pathogenesis-related 1(PR1) (induced by salicylic acid and BTH (benzothiadiazole-7-carbothioic acid S-methyl ester)); tomato proteinase inhibitor II promoter (PIN2) or LAP promoter (both inducible with methyl jasmonate); heat shock promoters (U.S. patent 5,187,267); tetracycline-inducible promoters (U.S. Pat. No. 5,057,422); seed-specific promoters, such as the millet seed-specific promoter pF128(CN101063139B (Chinese patent 200710099169.7)), seed storage protein-specific promoters (e.g., the promoters of phaseolin, napin, oleosin, and soybean beta conglycin (Beachy et al (1985) EMBO J.4: 3047-3053)). They can be used alone or in combination with other plant promoters. All references cited herein are incorporated by reference in their entirety. Suitable transcription terminators include, but are not limited to: agrobacterium nopaline synthase terminator (NOS terminator), cauliflower mosaic virus CaMV 35S terminator, tml terminator, pea rbcS E9 terminator and nopaline and octopine synthase terminators (see, e.g., Odell et al (I)⁹⁸⁵) Nature 313: 810; rosenberg et al (1987) Gene,56: 125; guerineau et al (1991) mol.gen.genet,262: 141; proudfoot (1991) Cell,64: 671; sanfacon et al Genes Dev.,5: 141; mogen et al (1990) Plant Cell,2: 1261; munroe et al (1990) Gene,91: 151; ballad et al (1989) Nucleic Acids Res.17: 7891; joshi et al (1987) Nucleic Acid Res, 15:9627)。

The recombinant vector containing the TaANTL7A.2 gene expression cassette can be constructed by using the existing expression vector. The plant expression vector comprises a binary agrobacterium vector, a vector for plant microprojectile bombardment and the like. Such as pAHC25, pBin438, pCAMBIA1302, pCAMBIA2301, pCAMBIA1301, pCAMBIA1300, pBI121, pCAMBIA1391-Xa, PSN1301, or pCAMBIA1391-Xb (CAMBIA Corp.), etc. The plant expression vector may also comprise the 3' untranslated region of the foreign gene, i.e., a region comprising a polyadenylation signal and any other DNA segments involved in mRNA processing or gene expression. The poly A signal can lead poly A to be added to the 3 'end of mRNA precursor, and the untranslated regions transcribed at the 3' end of Agrobacterium crown gall inducible (Ti) plasmid genes (such as nopaline synthase gene Nos) and plant genes (such as soybean storage protein gene) have similar functions. When the gene of the present invention is used to construct a plant expression vector, enhancers, including translational or transcriptional enhancers, may be used, and these enhancer regions may be ATG initiation codon or initiation codon of adjacent regions, etc., but must be in the same reading frame as the coding sequence to ensure correct translation of the entire sequence. The translational control signals and initiation codons are widely derived, either naturally or synthetically. The translation initiation region may be derived from a transcription initiation region or a structural gene. In order to facilitate the identification and screening of transgenic plant cells or plants, the plant expression vector to be used may be processed, for example, by adding a gene encoding an enzyme or a luminescent compound capable of producing a color change (GUS gene, luciferase gene, etc.), a marker gene for antibiotics (e.g., nptII gene conferring resistance to kanamycin and related antibiotics, bar gene conferring resistance to phosphinothricin as an herbicide, hph gene conferring resistance to hygromycin as an antibiotic, dhfr gene conferring resistance to methotrexate, EPSPS gene conferring resistance to glyphosate) or a marker gene for chemical resistance (e.g., herbicide resistance), a mannose-6-phosphate isomerase gene providing the ability to metabolize mannose, which can be expressed in plants. From the safety of transgenic plants, the transgenic plants can be directly screened and transformed in a stress environment without adding any selective marker gene.

In the above application, the vector may be a plasmid, a cosmid, a phage, or a viral vector. The plasmid may be specifically a pCAMBIA1302 vector.

B3) The recombinant vector can be pCAMBIA 1302-TaANTL7A.2. The pCAMBIA1302-TaANTL7A.2 is a recombinant vector obtained by introducing a DNA molecule represented by 129 th to 1292 th positions of a sequence 2 in a sequence table into a pCAMBIA1302 vector, and the recombinant vector can express a TaANTL7A.2 protein.

In the above application, the microorganism may be yeast, bacteria, algae or fungi. Wherein the bacterium can be Agrobacterium, such as GV 3101.

In the above application, the transgenic plant cell line, the transgenic plant tissue and the transgenic plant organ do not comprise propagation material.

The invention also provides any one of the following uses of the taantl7a.2 protein or the biological material:

C1) the application of the plant stress tolerance improvement;

C2) the application in the preparation of products for improving the stress tolerance of plants;

C3) the application in cultivating the stress tolerance-enhanced plant;

C4) the application in the preparation of plant products with enhanced stress tolerance;

C5) application in plant breeding.

The invention also provides the following products of X1) or X2):

x1) taantl7a.2 protein or the biological material;

x2) product for improving stress tolerance of plants, containing TaANTL7A.2 protein or the biological material.

The product can use TaANTL7A.2 protein or the biological material as its active component, and can also use TaANTL7A.2 protein or the biological material and other substances with the same function as its active component.

The invention also provides a method for cultivating the stress-tolerant plant, which comprises the following steps: increasing the activity and/or content of a TaANTL7A.2 protein in a plant of interest, or promoting the expression of a gene encoding a TaANTL7A.2 protein, to obtain a stress tolerant plant with increased stress tolerance as compared to the plant of interest.

In the above method, the stress-tolerant plant may be a transgenic plant having an increased expression of taantl7a.2 protein as compared to the target plant, which is obtained by introducing a gene encoding the taantl7a.2 protein into the target plant.

The non-stress tolerant plant may be a transgenic plant having reduced expression of taantl7a.2 protein compared to the plant of interest, obtained by introducing B16) the recombinant vector into the plant of interest.

In the above method, the gene encoding taantl7a.2 protein may be the nucleic acid molecule of B1).

In the above method, the gene encoding taantl7a.2 may be modified as follows and then introduced into a target plant to achieve a better expression effect:

1) modifying and optimizing according to actual needs to enable the gene to be efficiently expressed; for example, the amino acid sequence of the gene encoding TaANTL7A.2 of the present invention may be changed to conform to plant preferences, while maintaining the amino acid sequence, according to the codons preferred by the plant of interest; during the optimization, it is desirable to maintain a GC content in the optimized coding sequence to best achieve high expression levels of the introduced gene in plants, wherein the GC content can be 35%, more than 45%, more than 50%, or more than about 60%;

2) modifying the sequence of the gene adjacent to the initiating methionine to allow efficient initiation of translation; for example, modifications are made using sequences known to be effective in plants;

3) linking with promoters expressed by various plants to facilitate the expression of the promoters in the plants; such promoters may include constitutive, inducible, time-regulated, developmentally regulated, chemically regulated, tissue-preferred, and tissue-specific promoters; the choice of promoter will vary with the time and space requirements of expression, and will also depend on the target species; for example, tissue or organ specific expression promoters, depending on the stage of development of the desired receptor; although many promoters derived from dicots have been demonstrated to be functional in monocots and vice versa, desirably, dicot promoters are selected for expression in dicots and monocot promoters for expression in monocots;

4) the expression efficiency of the gene of the present invention can also be improved by linking to a suitable transcription terminator; tml from CaMV, E9 from rbcS; any available terminator which is known to function in plants may be linked to the gene of the invention;

5) enhancer sequences, such as intron sequences (e.g., from Adhl and bronzel) and viral leader sequences (e.g., from TMV, MCMV, and AMV) were introduced.

The gene encoding TaANTL7A.2 can be introduced into a target plant using a recombinant expression vector containing the gene encoding TaANTL 7A.2. The recombinant expression vector can be specifically pCAMBIA 1302-TaANTL7A.2.

The recombinant expression vector can be introduced into Plant cells by using conventional biotechnological methods such as Ti plasmid, Plant virus vector, direct DNA transformation, microinjection, electroporation, etc. (Weissbach,1998, Method for Plant Molecular Biology VIII, academic Press, New York, pp.411-463; Geiserson and Corey,1998, Plant Molecular Biology (2nd Edition)).

Said stress tolerant plant and said stress-tolerant plant are understood to comprise not only the first generation transgenic plant but also its progeny. For transgenic plants, the gene can be propagated in the species, and can also be transferred into other varieties of the same species, including particularly commercial varieties, using conventional breeding techniques. The cold-resistant plants include seeds, callus, whole plants and cells.

In the present invention, the plant may be m1) or m2) or m 3):

m1) a monocotyledonous or dicotyledonous plant;

m2) a graminaceous plant or a cruciferous plant;

m3) wheat (such as triticale) or Arabidopsis thaliana;

the plant of interest may be m1) or m2) or m 3):

m1) a monocotyledonous or dicotyledonous plant;

m2) a graminaceous plant or a cruciferous plant;

m3) wheat (such as Triticum aestivum L.) or Arabidopsis thaliana.

In the present invention, the stress tolerance may be heat resistance. The heat resistance may be manifested in resistance to an environment greater than 40 ℃. The heat resistance may be embodied in resistance to an environment of 42-45 ℃.

Experiments prove that the TaANTL7A.2 can improve the stress tolerance of plants, can be used for preparing products for improving the stress tolerance of the plants and directly used for the stress tolerance, provides a basis for artificially controlling the expression of stress-resistant and stress-tolerant related genes, and plays an important role in breeding plants with enhanced stress tolerance and stress tolerance.

Drawings

FIG. 1 shows the phenotype of TaANTL7A.2 transgenic Arabidopsis under high temperature stress treatment.

FIG. 2 shows the results of detecting the expression characteristics of TaANTL7A.2 gene under high temperature stress.

FIG. 3 shows the results of a subcellular localization analysis of TaANTL7 A.2. In the figure, the scale is 10 μm.

FIG. 4 shows the verification of the transcriptional activation activity of TaANTL 7A.2.

Detailed Description

The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The experimental procedures in the following examples are conventional unless otherwise specified. Materials, reagents, instruments and the like used in the following examples are commercially available unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged. In the following examples, unless otherwise specified, the 1 st position of each nucleotide sequence in the sequence listing is the 5 'terminal nucleotide of the corresponding DNA, and the last position is the 3' terminal nucleotide of the corresponding DNA.

The pCAMBIA1302 vector (described in the literature as pCAMBIA1302 vector) in the following examples is Liu J, Sun N, Liu M, et al, an animal experimental loop controlling Arabidopsis HsfA2 expression, roll of heat shock-induced organic partitioning [ J ] Plant Physiology,2013,162(1):512-521), which is publicly available from the applicant and is used only for repeating the experiments related to the present invention and is not usable for other purposes.

Example 1 TaANTL7A.2 increasing the thermotolerance of Arabidopsis thaliana

This example provides a wheat variety Triticum aestivum L.derived from Triticum aestivum cv. Xiaobaimai, publicly available from the institute of crop science of the Chinese academy of agricultural sciences, publicly available from the national germplasm resources repository (No. ZM242), Sun Hao et al, screening of the wheat TaDREB6 transcription factor interaction protein, Chinese agricultural science, 2011,44 (22): 4740-, the name is TaANTL7A.2 gene, the cDNA sequence is sequence 2 in the sequence table, the open reading frame is from 129 th site to 1292 th site of 5' end of sequence 2 in the sequence table, and the TaANTL7A.2 protein shown in sequence 1 in the sequence table is coded. The tanthl7a.2 protein consists of 387 amino acid residues, all with a conserved double AP2 domain. The TaANTL7A.2 gene and the protein coded by it can improve the heat resistance of plants, and the detection steps are as follows:

construction of recombinant expression vector

The TaANTL7A.2 gene sequence (with terminator removed) was constructed on pCAMBIA1302 by the In-Fusion HD Cloning Kit (Takara Bio Inc.) using the principle of homologous recombination, and the primer sequences for adding the linker were as follows:

TaANTL7A.2-1302F：5'-GGGACTCTTGACCATGATGGCCACCACCGTCC-3'

TaANTL7A.2-1302R：5'-TCAGATCTACCCATGGTTGTCCAGCTAACTTTGTGACT-3'

performing PCR amplification on the wheat leaf cDNA by using TaANTL7A.2-1302F and TaANTL7A.2-1302R to obtain a PCR product;

the pCAMBIA1302 vector was digested with Nco I cleavage sites, and the vector backbone was recovered.

Reaction system for attaching the fragment of interest to the vector: 10 μ l. 10-200ng of PCR product, 50-200ng of carrier backbone, 2. mu.l of 5 XIn-Fusion HD Enzyme Premix, ddH were added to a 0.2mL centrifuge tube₂The amount of O was adjusted to 10. mu.l. After ligation, TOP10 competent cells were transformed, and the recombinant plasmid was detected by bacterial liquid PCR and sequenced. The recombinant plasmid with the correct sequence is named pCAMBIA1302-TaANTL7A.2 and can express the protein of TaANTL7 A.2.

Second, obtaining transgenic plants

Introducing pCAMBIA1302-TaANTL7A.2 obtained in step one into Agrobacterium GV3101 to obtain recombinant strain A-pCAMBIA1302-TaANTL7A.2, and transforming Arabidopsis thaliana by dipping flower method

Immersing a dye solution: adding sucrose and Silwet-77 into 1/2MS culture medium, wherein the mass percentages of sucrose and Silwet-77 in the staining solution are 5% and 0.05%, respectively, and the pH value is 5.7.

Preparation of transformation solution: A-pCAMBIA1302-TaANTL7A.2 was inoculated into 200mL of LB liquid medium containing 50mg/L kanamycin and 50mg/L rifampicin antibiotic, cultured overnight at 28 ℃ and 200rpm until OD600 became about 2.0, and the cells were collected by centrifugation, resuspended in an invader solution, and adjusted to OD 600. about.0.6-0.8 to obtain a transformant.

Transformation of Arabidopsis by dipping flower method: soaking the inflorescence of arabidopsis thaliana (Col-0) with good growth condition in the transformation solution, infecting for 3min, keeping flat and culturing in dark place for 1d, placing under normal growth condition for continuous growth, infecting for the second time after one week, and infecting for the third time if more inflorescences exist, thus obtaining T0 generation plants.

3. Acquisition of transgenic Arabidopsis Positive strains

T0 generation seeds obtained after agrobacterium infection are sterilized and cleaned, and then are sown on MS culture medium containing hygromycin resistance (40mg/L) for screening. The seeds can normally grow after germination, the primary screened positive seedlings of the T1 generation can not normally grow, and the roots of the non-transgenic seedlings can be yellowed and died. And (3) transplanting the T1 generation positive seedlings to soil to obtain harvested seeds, continuously screening the seeds by using a hygromycin resistance-containing culture medium, planting the seeds to obtain a T2 generation strain until a homozygous strain is obtained, and obtaining the homozygous transgenic strain through PCR verification. The PCR primer sequences were as follows:

F：5'-GAAGGCTCATCCGAGGTAGT-3'；

R：5'-CCATCTTCACAGCAGTTGCT-3'。

third, obtaining empty vector control plants

And transforming agrobacterium with the plasmid pCAMBIA1302 to obtain recombinant agrobacterium, transforming Arabidopsis with the recombinant agrobacterium to obtain a transgenic empty vector control plant, and the method is the same as the second step.

Fourth, phenotype of transgenic Arabidopsis under high temperature stress

1. Survival rate under high temperature treatment

High-temperature treatment of flat seedlings: wild type arabidopsis thaliana (Columbia ecotype, Col-0) growing for 7d and homozygous transgenic lines are placed at 45 ℃ for treatment for 3.5h, the wild type arabidopsis thaliana is used as a control, the wild type arabidopsis thaliana is transferred to 22 ℃ to recover growth for 8d, the survival rate is counted, three times of experiments are set, and 36 arabidopsis thaliana plants of each type are tested in each time. Non-hyperthermophiled plants were used as controls.

High-temperature treatment of potted seedlings: the wild type arabidopsis thaliana (Columbia ecotype, Col-0) and the homozygous transgenic line which grow for 28d are placed at 45 ℃ for treatment for 5h, the wild type arabidopsis thaliana is used as a control, the wild type arabidopsis thaliana is transferred to 22 ℃ to recover growth for 9d, the survival rate is counted, three times of experiments are set, and 36 arabidopsis thaliana plants of each type are obtained in each time of repeated experiments. Non-hyperthermophiled plants were used as controls.

2. Determination of conductivity at high temperature treatment

After treating 28d wild Arabidopsis (Columbia ecotype, Col-0) and homozygous transgenic lines at 45 ℃ for 3h, the cut leaves were washed clean with deionized water, 8 leaves were placed in each test tube with a 5mm diameter punch, 10mL of deionized water was added, and 4 replicates were set. 10mL of deionized water was added to the blank control tube. The mixture was shaken on a shaker for 24 hours, after which the initial conductance E1 and the blank conductance E01 were measured with a conductivity meter. Placing the test tube in a water bath kettle, boiling for 30min, shaking on a shaker for 24 hr after tissue death and electrolyte release stability, and measuring final conductance value E2 and blank control conductance value E02. The relative conductivity was calculated. Relative conductivity ═ (E1-E01)/(E2-E02) × 100%. Plants that were not subjected to high temperature treatment were used as controls.

The results of high temperature identification of the transgenic lines are shown in FIG. 1, and the transgenic lines of homozygous 35S:: TaANTL7A.2#1 and 35S:: TaANTL7A.2#2 Arabidopsis thaliana were analyzed for heat resistance of seedlings at different stages (plate seedlings and pot seedlings) using 7d large Col-0 with good and consistent growth vigor. The results show that the survival rates of 35S:: TaANTL7A.2#1 and 35S:: TaANTL7A.2#2 of the plants at the flat seedling stage and the pot seedling stage after high temperature treatment are both significantly higher than that of Col-0. The results of the measurement of the electrical conductivity of the plant leaves under the high temperature treatment showed that under normal growth conditions, there was no significant difference between 35S:: TaANTL7A.2#1 and 35S:: TaANTL7A.2#2 and Col-0; after the high temperature treatment, the electrical conductivity of 35S:: TaANTL7A.2#1 and 35S:: TaANTL7A.2#2 was significantly lower than in Col-0. It was demonstrated that the TaANTL7A.2 gene and the protein encoded by it can improve the thermotolerance of Arabidopsis thaliana.

Example 2 analysis of the expression characteristics of TaANTL7A.2 under high temperature stress by real-time fluorescent quantitative PCR

Stress management

The method comprises the following steps of taking small white wheat seedlings growing at room temperature for about 10 days to perform the following treatment:

(1) high temperature treatment (fig. 2): the young wheat seedlings are placed at 42 ℃, are cultured for 30 minutes, 1 hour, 2 hours, 4 hours, 8 hours, 12 hours and 24 hours under illumination, are respectively taken out and are quickly frozen by liquid nitrogen, and are stored at-80 ℃ for later use.

(2) Treatment of the control: the young white wheat seedlings without any treatment were directly frozen at-80 ℃ as a control (0 hour).

II, isolation of mRNA

Total RNA from wheat leaves was extracted and purified by Trizol method (Tianggen).

Third, reverse transcription into cDNA

The purified RNA was reverse transcribed to cDNA.

Four, real-time fluorescent quantitative PCR

The cDNA was diluted 50-fold and used as a template for Q-RT-PCR. Q-RT-PCR amplification is carried out on the sample by using a specific primer of a non-coding region at the 3' end of the gene, actin is used as an internal reference, and the response condition of the gene to high-temperature treatment is analyzed. The primer sequences are as follows:

TaANTL7A.2 F：5'-GAAGAAACTAGAAGGCTC-3'；

TaANTL7A.2 R：5'-GATGAGCCTTCTAGTTTCT-3'。

Q-RT-PCR in ABI

7500 real-time fluorescence quantitative PCR, 3 times of repetition of one parallel test. The method reported by Livak KJ and Schmittgen TD (2001), 2^-ΔΔCTAnd calculating the relative expression amount.

ΔΔC_T＝(C_T.Target－C_T.Actin)_{Time x}－(C_T.Target－C_T.Actin)_{Time 0}

Time x denotes an arbitrary Time point, Time₀Represents the expression of a 1-fold amount of the target gene after actin correction.

The results are shown in FIG. 2. Under high temperature treatment, TaANTL7A.2 is rapidly induced to express, and reaches the maximum point at 2h, and compared with 0h, the expression amount is increased by about 6 times. Indicating that TaANTL7A.2 is expressed under the induction of high temperature stress.

Example 3 subcellular localization analysis of TaANTL7A.2

Construction of recombinant expression vector

The sequence of wheat TaANTL7A.2 (terminator removed) is constructed into 16318GFP vector (BamH I restriction site, reference 16318GFP vector is published: cloning and functional verification of Wudy. wheat anti-stress related gene TaFKBP62c-2B [ D ]. northwest agroforestrial science and technology university, 2016), and primer sequence of linker connecting vector is added as follows:

TaANTL7A.2-16318GFP F：5'-TATCTCTAGAGGATCCATGGCCACCACCGTCC-3'

TaANTL7A.2-16318GFP R：5'-TGCTCACCATGGATCCTTGTCCAGCTAACTTTGTGACT-3'

performing PCR amplification on cDNA of the small white wheat by using TaANTL7A.2-16318GFP F and TaANTL7A.2-16318GFP R to obtain a PCR product, and performing enzyme digestion on the obtained PCR product by using BamH I to obtain a fragment 1; carrying out enzyme digestion on the 16318GFP vector by using BamH I, and recovering to obtain a vector framework; the fragment 1 was ligated to the vector backbone and the resulting recombinant vector with the correct sequence was named 16318-TaANTL7 A.2-GFP. 16318-TaANTL7A.2-GFP can express TaANTL7A.2-GFP, which is a fusion protein of TaANTL7A.2 and GFP.

TaANTL7A.2 was fragmented according to position double AP2 and the fragment sequence was constructed into 16318GFP vector (BamH I site) and the primer sequences for ligation of the vector were added as follows:

TaA1-16318GFP F：5'-TATCTCTAGAGGATCCATGGCCACCACCGTCC-3'

TaA1-16318GFP R：5'-TGCTCACCATGGATCCTTTTGCATAATCAGAAATAT-3'

TaA2-16318GFP F：5'-TATCTCTAGAGGATCCATGGCCACCACCGTCC-3'

TaA2-16318GFP R：5'-TGCTCACCATGGATCCCGATGTTCCTCTTGAGAAAC-3'

TaA3-16318GFP F：5'-TATCTCTAGAGGATCCCGCTACCACGGCGTG-3'

TaA3-16318GFP R：5'-TGCTCACCATGGATCCTTCGGAGTAGTTGCTGATAT-3'

TaA4-16318GFP F：5'-TATCTCTAGAGGATCCGAAATTGAGATCATGAAGAGC-3'

TaA4-16318GFP R：5'-TGCTCACCATGGATCCCTATTGTCCAGCTAACTTTG-3'

TaA5-16318GFP F：5'-TATCTCTAGAGGATCCTCATACAGGGGTGTAACA-3'

TaA5-16318GFP R：5'-TGCTCACCATGGATCCTTCGTCTTGTTACACCCCT-3'

carrying out PCR amplification on cDNA of the wheat barley by utilizing TaA1-16318GFP F and TaA1-16318GFP R to obtain a PCR product, and carrying out enzyme digestion on the obtained PCR product by utilizing BamH I to obtain a fragment 2; carrying out enzyme digestion on the 16318GFP vector by using BamH I, and recovering to obtain a vector framework; the fragment 2 was ligated to the vector backbone and the resulting recombinant vector with the correct sequence was named 16318-TaA 1-GFP. 16318-TaA1-GFP expresses a fusion protein TaA1-GFP of the first AP2 sequence of TaANTL7A.2 at the N-terminus and GFP.

Carrying out PCR amplification on cDNA of the wheat barley by utilizing TaA2-16318GFP F and TaA2-16318GFP R to obtain a PCR product, and carrying out enzyme digestion on the obtained PCR product by utilizing BamH I to obtain a fragment 3; carrying out enzyme digestion on the 16318GFP vector by using BamH I, and recovering to obtain a vector framework; the fragment 3 was ligated to the vector backbone and the resulting recombinant vector with the correct sequence was named 16318-TaA 2-GFP. 16318-TaA2-GFP expresses a fusion protein TaA2-GFP of the N-terminal, first AP2 sequence of TaANTL7A.2 and a linker sequence between two AP2 sequences with GFP.

Carrying out PCR amplification on cDNA of the wheat barley by utilizing TaA3-16318GFP F and TaA3-16318GFP R to obtain a PCR product, and carrying out enzyme digestion on the obtained PCR product by utilizing BamH I to obtain a fragment 4; carrying out enzyme digestion on the 16318-GFP vector by using BamH I, and recovering to obtain a vector framework; the fragment 4 was ligated to the vector backbone and the resulting recombinant vector with the correct sequence was named 16318-TaA 3-GFP. 16318-TaA3-GFP can express the fusion protein TaA3-GFP of two AP2 sequences of TaANTL7A.2 and the connecting sequence between them and GFP.

Carrying out PCR amplification on cDNA of the wheat barley by utilizing TaA4-16318GFP F and TaA4-16318GFP R to obtain a PCR product, and carrying out enzyme digestion on the obtained PCR product by utilizing BamH I to obtain a fragment 5; carrying out enzyme digestion on the 16318GFP vector by using BamH I, and recovering to obtain a vector framework; the fragment 5 was ligated to the vector backbone and the resulting recombinant vector with the correct sequence was named 16318-TaA 4-GFP. 16318-TaA4-GFP expresses a fusion protein TaA4-GFP of the C-terminal, second AP2 sequence of TaANTL7A.2 and a linker sequence between the two AP2 sequences with GFP.

Carrying out PCR amplification on cDNA of the wheat barley by utilizing TaA5-16318GFP F and TaA5-16318GFP R to obtain a PCR product, and carrying out enzyme digestion on the obtained PCR product by utilizing BamH I to obtain a fragment 6; carrying out enzyme digestion on the 16318GFP vector by using BamH I, and recovering to obtain a vector framework; the fragment 6 was ligated to the vector backbone and the resulting recombinant vector with the correct sequence was named 16318-TaA 5-GFP. 16318-TaA5-GFP expresses a fusion protein TaA5-GFP of the C-terminal and second AP2 sequence of TaANTL7A.2 with GFP.

Preparation and transformation of protoplast

(1) Cutting leaves of small white wheat about one week into 0.5cm pieces²The small segments of (a).

(2) Preparing an enzymolysis liquid:

mixing the components in the table, heating in a water bath at 55 ℃ for 10min, cooling to room temperature, adding the following reagents, and uniformly mixing to obtain an enzymolysis solution:

(3) and putting the cut leaves into the enzymolysis liquid, vacuumizing for about 30min in the dark, and then putting the leaves on a shaker at 50rpm for enzymolysis for 3h in the dark at room temperature in a dark place.

(4) The enzyme solution containing protoplasts was diluted with an equal amount of W5 solution pre-cooled on ice, the 100 mesh sieve was wetted with W5 solution, the solution was then sieved off the leaf debris, and the green liquid containing protoplasts was dispensed into 2.0mL centrifuge tubes and centrifuged at 100g for 2min at 4 ℃ with an acceleration of 2. The supernatant was removed as much as possible and the protoplasts were gently resuspended in 1mL of W5 solution. Standing on ice for 30 min.

W5 solution

Note: the pH was adjusted to 5.8 with KOH.

The following steps were carried out at room temperature.

(5) The protoplasts were pelleted at the bottom of the tube by centrifugation at 100g for 5 min. The W5 solution was removed as much as possible without touching the protoplast pellet. Resuspend protoplast with appropriate amount of MMG solution to give final concentration of 2X 10⁵And (4) obtaining a protoplast solution.

MMG solution

Note: the pH was adjusted to 5.6 with KOH.

(6) Add 20. mu.g of the recombinant vector from step one to a 2.0mL centrifuge tube, and then add 200. mu.l of the protoplast solution to the centrifuge tube. One recombinant vector per centrifuge tube.

(7) Preparing a PEG solution:

(8) and (4) adding 110 mu l of PEG solution into the centrifuge tube after the completion of the step (6), gently beating the centrifuge tube, uniformly mixing, and inducing and converting at room temperature for 20-30min to obtain a conversion solution.

(9) The transformation was stopped by diluting the solution with 400. mu. l W5 and gently inverting the tube to mix well.

(10) The acceleration was set at 2, 100g centrifugation for 2min, and then the supernatant was removed. Then 1mL of W5 solution was added to the suspension and washed once, and the supernatant was centrifuged at 100g for 2 min.

(11) The protoplasts were gently resuspended in 300. mu. l W5 solution and induced for more than 18h at room temperature (20-25 ℃).

(12) GFP green fluorescence was observed under a confocal laser microscope.

The subcellular localization results are shown in FIG. 3. The fusion protein of TaANTL7A.2-GFP emits green fluorescence in the nucleus under a confocal laser microscope. In contrast to protoplasts transformed with 16318GFP empty vector, GFP signals appear throughout the cell, including the nucleus, cytoplasm, and cell membrane.

TaA1-GFP, A2-GFP, TaA4-GFP and TaA5-GFP fusion proteins were sub-cellular localized in the whole cell in accordance with the pJIT16318 empty vector control (FIG. 3), and only the fluorescence signal of the TaA3-GFP fusion protein was localized in the nucleus in accordance with the sub-cellular localization of TaANTL7A.2-GFP (FIG. 3). It was demonstrated that two complete AP2 structures and linker sequences are necessary for nuclear localization of taantl7 a.2.

Example 4 verification of the transcriptional activation Activity of TaANTL7A.2

Construction of recombinant expression vector

TaANTL7A.2 is constructed on a pGBKT7 vector (EcoR I restriction site) to form a recombinant plasmid TaANTL7A.2-pGBKT7, and a primer sequence added with a joint connecting vector is as follows:

TaANTL7A.2-pGBKT7F：5'-CATGGAGGCCGAATTCATGGCCACCACCGTCC-3'

TaANTL7A.2-pGBKT7R：5'-GGATCCCCGGGAATTCCTATTGTCCAGCTAACTTTGTG A-3'

carrying out PCR amplification on cDNA of the small white wheat by using TaANTL7A.2-pGBKT7F and TaANTL7A.2-pGBKT7R to obtain a PCR product, and carrying out enzyme digestion on the obtained PCR product by using EcoR I to obtain a DNA fragment 1; carrying out enzyme digestion on the pGBKT7 vector by using EcoR I, and recovering to obtain a vector framework; the DNA fragment 1 was ligated to the vector backbone and the resulting recombinant vector with the correct sequence was named pGBKT7-TaANTL7 A.2.

Two, two-hybrid yeast

1. Preparation of Yeast competent cells

(1) Inoculating yeast AH109 to YPDA culture medium, culturing at 30 deg.C for 2-3 days to obtain white clone about 2 mm. Yeast single clone was picked and cultured at 30 ℃ and 230-.

(2) Inoculating the newly activated yeast liquid into 50mL YPDA liquid culture medium, culturing at 30 deg.C and 230-250rpm for 11-12h to make OD₆₀₀To 0.15-0.30.

(3) Centrifuging at room temperature at 700g for 5min, removing supernatant, resuspending the thallus with 100mL YPDA liquid culture medium, and culturing for about 3-5 hr to obtain OD₆₀₀To 0.4-0.5.

(4) Centrifugation was carried out at 700g for 5min at room temperature, the supernatant was decanted, and the cells were resuspended in 3mL of 1.1 XTE/LiAC and dispensed into two 2.0mL centrifuge tubes.

(5) After centrifugation at 8000-.

The YPDA medium and the 1.1 XTE/LiAC solution were formulated as follows:

YPDA medium

Note: 20g of agar (w/v 2.0%) was added to the solid medium.

1.1 XTE/LiAC solution

Note: 10 XTE buffer (composition 100mM Tris-HCl, 10mM EDTA).

2. Yeast sensitive cell transformation

(1) Salmon sperm DNA was boiled in advance for 5min, placed on ice, and the following ingredients were added to a 5mL EP tube in order.

Note: the bait plasmid was TaANTL7A.2-pGBKT7, and the library plasmid vector was pGADT7 vector (restriction site was EcoR I).

(2) Add 600. mu.l of freshly prepared AH109 competed with 2.5mL PEG/LiAc, mix gently and place in a 30 ℃ water bath for 45 min.

(3) Adding 200 μ l DMSO, mixing, and water bathing at 42 deg.C for 20min, mixing once every 10 min.

(4) After centrifugation at 8000-.

(5) Centrifuging at 8000-10000rpm for 15s, discarding the supernatant, suspending the thallus in 2mL of 0.9% NaCl, gently mixing uniformly, coating onto different types of defect plates, and performing inverted culture at 30 ℃ for 3-6d until colonies appear.

PEG/LiAc solution

Defective Medium SD/-Trp

Self-activation validation defective culture medium SD/-Trp-His

Self-activation validation defective culture medium SD/-Trp-Ade

The growth of the recombinant vectors on the deficient yeast medium is shown in FIG. 4, in which both plasmid-transformed yeast cells were able to grow on the SD/-Trp deficient medium control; neither yeast cells transformed with BD-TaANTL7A.2 grew on SD/-Trp-His nor SD/-Trp-Ade medium, indicating that TaANTL7A.2 had no transcriptional activation activity.

<110> institute of crop science of Chinese academy of agricultural sciences

Application of <120> plant stress tolerance related protein TaANTL7A.2 in regulation and control of plant stress tolerance

<160> 2

<170> PatentIn version 3.5

<210> 1

<211> 387

<212> PRT

<213> Triticum aestivum L.)

<400> 1

Met Ala Thr Thr Val Gln Pro His Ser Pro Asp Pro Thr Ala Ile Thr

1 5 10 15

Thr Thr Pro Ala Pro Pro Pro Pro Pro Ser Pro Pro Arg Gln Glu Asn

20 25 30

Pro Thr Ala Ala Gly Glu Gly Val Glu Ile Ala Ala Leu Asp Glu Gln

35 40 45

Pro Ala Ala Val Ala Val Ala Asp Lys Gly Lys Thr Ala Pro Gly Gly

50 55 60

Gly Lys Leu Val Ala Glu Ala Met Arg Lys Cys Ala Ala Pro Arg Ser

65 70 75 80

Ser Arg Tyr His Gly Val Thr Arg Leu Lys Trp Ser Gly Lys Tyr Glu

85 90 95

Ala His Leu Trp Asp Asn Thr Ser Gln Val Glu Gly Arg Lys Arg Lys

100 105 110

Gly Lys His Val Tyr Leu Gly Ser Tyr Val Thr Glu Glu Asn Ala Ala

115 120 125

Arg Ala His Asp Leu Ala Ala Leu Lys Tyr Trp Gly Ile Thr Gln Pro

130 135 140

Thr Lys Leu Asn Phe Asn Ile Ser Asp Tyr Ala Lys Glu Ile Glu Ile

145 150 155 160

Met Lys Ser Met Asn Gln Asp Glu Phe Val Ala Tyr Ile Arg Arg Gln

165 170 175

Ser Ser Cys Phe Ser Arg Gly Thr Ser Ser Tyr Arg Gly Val Thr Arg

180 185 190

Arg Lys Asp Gly Lys Trp Gln Ala Arg Ile Gly Arg Ile Gly Glu Ser

195 200 205

Arg Asp Thr Lys Asp Ile Tyr Leu Gly Thr Phe Glu Thr Glu Val Glu

210 215 220

Ala Ala Glu Ala Tyr Asp Leu Ala Ala Ile Gln Leu Arg Gly Val His

225 230 235 240

Ala Val Thr Asn Phe Asp Ile Ser Asn Tyr Ser Glu Glu Gly Leu Lys

245 250 255

Lys Leu Glu Gly Ser Ser Glu Val Val Asn Leu Glu Asp Gln Ser Glu

260 265 270

Val Thr Lys Leu Ala Val Thr Asn Leu Asp Ile Ser Gln His Cys Glu

275 280 285

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

290 295 300

Asp Gln Ser Glu Val Thr Lys Leu Ser Val Thr Asn Phe Asp Ile Ser

305 310 315 320

Asn Cys Cys Glu Asp Gly Leu Lys Lys Leu Asp Gly Ala Ser Gln Ile

325 330 335

Val Asn Leu Glu Asp Gln Ser Glu Val Thr Lys Leu Ser Val Thr Asn

340 345 350

Phe Asp Ile Ser Asn Cys Cys Glu Asp Gly Leu Lys Lys Leu Glu Gly

355 360 365

Ser Ser Glu Val Ala Asn Leu Glu Asp Gln Ser Glu Val Thr Lys Leu

370 375 380

Ala Gly Gln

385

<210> 2

<211> 1753

<212> DNA

<213> Triticum aestivum L.)

<400> 2

cgggcggaaa cacaacgacg cagtcacgca ccggctcttt cctccacccc tcgtagggct 60

agctagggtt tctccgccca aatcgtgtaa taataaccca cttgaagatt cgcgcgaacg 120

ctgcggcgat ggccaccacc gtccagcccc actccccgga cccaacagcc atcaccacca 180

cccctgcccc accccctccc ccttctccac ctcgccagga gaacccgacc gccgcggggg 240

aaggcgtaga gatcgcggcg ctcgatgagc agcctgccgc cgtcgccgtc gccgacaagg 300

ggaagacggc ccccggcggc gggaagctgg tggcggaggc catgcgcaag tgcgcggcgc 360

cccggtcgtc gcgctaccac ggcgtgacga ggctcaagtg gagcggcaag tacgaggcac 420

acctctggga caacaccagc caggttgagg ggcgcaagcg caagggcaag catgtgtact 480

tgggaagcta tgttactgaa gagaatgctg caagggcaca tgaccttgca gccctgaaat 540

attggggcat aactcaaccc accaaactaa acttcaatat ttctgattat gcaaaagaaa 600

ttgagatcat gaagagcatg aatcaagatg aatttgtggc ctacataagg aggcagagta 660

gttgtttctc aagaggaaca tcgtcataca ggggtgtaac aagacgaaag gatggtaaat 720

ggcaagcacg tattggtagg attggtgaga gtagagacac taaagacatc tatcttggga 780

cctttgaaac tgaagtggag gcagctgaag cgtatgacct agcagcaatt cagctccgtg 840

gtgttcatgc tgtgaccaac tttgatatca gcaactactc cgaagaaggt ttgaagaaac 900

tagaaggctc atccgaggta gtgaacctgg aggaccaatc agaagtcact aagttagctg 960

tgaccaactt ggatattagc caacactgcg aagatggttt gaagaaacta gatggcgcat 1020

cccagatagt gaacctggag gaccaatcag aagtcaccaa gttatctgtg accaactttg 1080

atattagcaa ctgctgtgaa gatggtttga agaaactaga tggcgcatcc cagatagtga 1140

acctggagga ccaatcagaa gtcaccaagt tatctgtgac taactttgat attagcaact 1200

gctgtgaaga tggtttgaag aaactagaag gctcatccga ggtagcgaac ctggaggacc 1260

aatcagaagt cacaaagtta gctggacaat agatattaga atagcaacat gtaaattatt 1320

tatttcttcc tttttatctt ttctctgatc gtcagtctct ccacctttct cttttcttga 1380

ttgatgatga gagtgtcttt tcttcgtcca cctttcctga aacttttttt tccccaagga 1440

atttcccttc gctggttgcc aactctatta tttaccagct ccagagtagg atcgtgtgat 1500

gctgtactct gtattctcat gtacactgat tacaaaccat tgatagtatt tgtagacatt 1560

acaacacaaa ggaagttcat tcttctattt ttgtatattt gtttttatcc tcctaactcc 1620

agttttgaat gctggtttgt tccactgtcc tcatgaacag aatactctga tatagtgcat 1680

gatagaaaaa ttcactcttt gcgcttcaaa tgggacaaca gacggtggtt ctgaataaca 1740

ggacaggttt gat 1753

Claims (7)

1. The application of protein-related biological materials in regulating and controlling the heat resistance of wheat or arabidopsis; the protein is A1) or A2) as follows:

A1) a protein with an amino acid sequence of sequence 1;

A2) a fusion protein obtained by connecting a label to the N-terminal or/and the C-terminal of A1);

the biomaterial is any one of the following B1) to B8):

B1) a nucleic acid molecule encoding the protein;

B2) an expression cassette comprising the nucleic acid molecule of B1);

B3) a recombinant vector comprising the nucleic acid molecule of B1);

B4) a recombinant vector comprising the expression cassette of B2);

B5) a recombinant microorganism comprising the nucleic acid molecule of B1);

B6) a recombinant microorganism comprising the expression cassette of B2);

B7) a recombinant microorganism containing the recombinant vector of B3);

B8) a recombinant microorganism comprising the recombinant vector of B4).

2. Use according to claim 1, characterized in that: B1) the nucleic acid molecule is any one of the following b1) -b 5):

b1) the coding sequence is DNA molecule from 129 th site to 1292 th site of sequence 2 in the sequence table;

b2) DNA molecules from 129 th site to 1292 th site of a sequence 2 in a sequence table;

b3) DNA molecule of sequence 2 in the sequence table;

b4) a DNA molecule having 75% or more identity to the nucleotide sequence defined in b1) or b2) or b3) and encoding the protein of claim 1;

b5) a cDNA molecule or a genomic DNA molecule which hybridizes under stringent conditions with the nucleotide sequence defined in b1) or b2) or b3) or b4) and encodes the protein as claimed in claim 1.

3. Use of a protein according to claim 1 or a biomaterial according to claim 1 or 2 for any of the following applications:

C1) the application of the compound in improving the heat resistance of wheat or arabidopsis thaliana;

C2) the application in preparing products for improving the heat resistance of wheat or arabidopsis thaliana;

C3) the application in cultivating heat-resistant enhanced wheat or arabidopsis thaliana;

C4) the application in the preparation and cultivation of heat-resistant enhanced wheat or arabidopsis products;

C5) the application in wheat or Arabidopsis breeding.

4. The following X1) or X2):

x1) the protein of claim 1 or the biomaterial of claim 1 or 2;

x2) product for increasing the heat resistance of wheat or Arabidopsis thaliana comprising a protein according to claim 1 or a biomaterial according to claim 1 or 2.

5. A method of growing heat tolerant wheat or arabidopsis comprising: increasing the expression level of the protein in claim 1 in the target wheat or arabidopsis thaliana to obtain the heat-resistant wheat or arabidopsis thaliana with enhanced heat resistance compared with the target wheat or arabidopsis thaliana.

6. The method of claim 5, wherein: the heat-resistant wheat or Arabidopsis thaliana is transgenic wheat or Arabidopsis thaliana which is obtained by introducing the coding gene of the protein in claim 1 into the target wheat or Arabidopsis thaliana and has the protein expression higher than that of the target wheat or Arabidopsis thaliana.

7. The method of claim 6, wherein: the gene encoding the protein of claim 1 is the nucleic acid molecule of B1) of claim 2.

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