CN110684114A - Application of plant stress tolerance associated protein TaBAKL in regulation and control of plant stress tolerance - Google Patents

Abstract

The invention discloses application of plant stress tolerance related protein TaBAKL in regulation and control of plant stress tolerance. The plant stress tolerance associated protein TaBAKL 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 TaBAKL is expressed under the induction of SA, and the TaBAKL gene is introduced into a wild plant to obtain a transgenic plant, the resistance of the transgenic plant to the pseudomonas syringae DC3000 is higher than that of the wild plant, a foundation is provided for artificially controlling the expression of genes related to stress resistance and stress tolerance, and the transgenic plant plays an important role in cultivating plants with enhanced stress resistance and stress tolerance.

Description

Application of plant stress tolerance associated protein TaBAKL in regulation and control of plant stress tolerance

Technical Field

The invention relates to the application of plant stress tolerance associated protein TaBAKL in regulation and control of plant stress tolerance in the field of biotechnology.

Background

Adversity stress is a barrier factor affecting the growth and development of wheat. 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, plants can be correspondingly adjusted on molecular, cellular and overall levels to minimize the damage caused by the environment 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 disease course related protein, ion channel protein, aquaporin and other gene products which directly participate in plant stress response; 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, protein kinases play an important role in the regulation of the perception and transmission of plant stress signals.

To date, about 300 protein kinases have been discovered, with a homologous catalytic domain consisting of about 270 amino acid residues. In systems of cell signaling, cell cycle regulation, and the like, protein kinases form criss-cross networks. Such enzymes catalyze the transfer of phosphate from ATP and covalently bind to the hydroxyl groups of certain serine, threonine, or tyrosine residues in a particular protein molecule, thereby altering the conformation and activity of the protein, enzyme.

The disease resistance response mediated by the plant immune system is an important means for the plant to defend itself. The presence of pathogenic bacteria induces the synthesis of Salicylic Acid (SA), activating the SA signal-mediated defense response. This defense response does not only act on the site affected by the pathogenic bacteria, but it protects the entire plant. This long-lasting, wide-range resistance is called System Acquired Resistance (SAR).

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 TaBAKL 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 TaBAKL protein in A2) above is a protein having 75% or more identity to 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 TaBAKL protein in A2) can be synthesized artificially, or can be obtained by synthesizing the coding gene and then performing biological expression.

The gene encoding the TaBAKL protein in A2) above can be obtained by deleting one or several codons of amino acid residues from the DNA sequence shown in positions 63 to 926 of the sequence No. 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 sequence 2 encodes TaBAKL protein shown in sequence 1.

The invention also provides the application of the biological material related to the TaBAKL 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 TaBAKL 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) -B4):

b1) the coding sequence is cDNA molecule or DNA molecule from 63 rd to 926 th site of sequence 2 in the sequence table;

b2) a cDNA molecule or a DNA molecule of a sequence 2 in a sequence table;

b3) a cDNA molecule or a genomic DNA molecule having 75% or more identity with the nucleotide sequence defined in b1) or b2) and encoding a TaBAKL protein;

b4) a cDNA molecule or a genomic DNA molecule which hybridizes under stringent conditions with the nucleotide sequence defined in b1) or b2) or b3) and encodes a TaBAKL 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 a TaBAKL protein of the present invention may be readily mutated by one of ordinary skill in the art using known methods, such as directed evolution and point mutation. Those nucleotides which are artificially modified and have 75% or more identity to the nucleotide sequence of the TaBAKL protein isolated in the present invention are derived from the nucleotide sequence of the present invention and are identical to the sequence of the present invention as long as they encode the TaBAKL protein and have the function of the TaBAKL 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 applications, the expression cassette containing a nucleic acid molecule encoding a TaBAKL protein (TaBAKL gene expression cassette) described in B2) refers to a DNA capable of expressing a TaBAKL protein in a host cell, and the DNA may include not only a promoter for initiating transcription of the TaBAKL gene, but also a terminator for terminating transcription of the TaBAKL 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: wound-induced starter from tomatoesThe mover, leucine aminopeptidase ("LAP", Chao et al (1999) plantaPhysiol 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 existing expression vector can be used for constructing a recombinant vector containing the TaBAKL gene expression cassette. 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-TaBAKL. The pCAMBIA1302-TaBAKL is a recombinant vector obtained by introducing a DNA molecule shown in a sequence 2 in a sequence table into a pCAMBIA1302 vector, and the recombinant vector can express TaBAKL 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 applications of the TaBAKL 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) a TaBAKL protein or the biological material;

x2) product for improving stress tolerance of plants, containing TaBAKL protein or said biological material.

X2) the product can use TaBAKL protein or the biological material as the active component, and can also combine the TaBAKL protein or the biological material with other substances with the same function as the active component.

The invention also provides a method for cultivating the stress-tolerant plant, which comprises the following steps: improving the activity and/or content of TaBAKL protein in a target plant, or promoting the expression of a coding gene of the TaBAKL protein to obtain the stress-tolerant plant with enhanced stress tolerance compared with the target plant.

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

In the above method, the coding gene of the TaBAKL protein may be B1).

In the above method, the coding gene of TaBAKL may be modified as follows, and then introduced into a target plant, so as to achieve a better expression effect:

1) modifying and optimizing according to actual needs to enable the gene to be efficiently expressed; for example, according to the codon preferred by the target plant, the codon of the coding gene of TaBAKL can be changed to conform to the plant preference while the amino acid sequence of the coding gene of TaBAKL is maintained; 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 coding gene of TaBAKL can be introduced into a target plant by using a recombinant expression vector containing the coding gene of TaBAKL. The recombinant expression vector can be specifically pCAMBIA 1302-TaBAKL.

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 disease resistance. The disease resistance may be particularly manifested in resistance to Pst DC 3000.

Experiments prove that the TaBAKL gene discovered by the invention is expressed under the induction of SA, and the TaBAKL gene is introduced into a transgenic plant obtained from a wild type plant, so that the resistance of the TaBAKL gene to Pseudomonas syringae (Pseudomonas syringa. tomato) DC3000 is higher than that of the wild type plant, and the TaBAKL protein and the coding gene thereof provided by the invention provide a basis for artificially controlling the expression of genes related to stress resistance and stress tolerance, and can play an important role in cultivating plants with enhanced stress resistance and stress tolerance.

Drawings

FIG. 1 shows the results of the identification of disease resistance of transgenic plants. A is a negative control group; b is the phenotype inoculated with Pst DC 3000; c is the percentage of the chlorosis area of different leaves in the whole area of the leaves; d is the amount of bacteria at 0dpi and 3dpi after the leaves were inoculated with Pst DC3000, indicating that the difference reached a significant level compared to Col-0 (p <0.05) and that the difference reached a very significant level compared to Col-0 (p < 0.01).

FIG. 2 shows the growth and index detection of transgenic Arabidopsis under SA treatment. A is the mutation position of AtBAKL gene in AtBAKL; b and C are the expression conditions of AtBAKL and TaBAKL respectively; D-G is the plant phenotype under different SA concentration treatment; H-O is the root length and the fresh weight of the plant under different SA concentrations.

FIG. 3 is the expression pattern of TaBAKL.

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 (Liu J, Sun N, Liu M, et al. Authoragetorya loop controlling Arabidopsis HsfA2expression: roll of heatshell-induced organic catalysis [ J ] Plant Physiology,2013,162(1):512-521) in the following examples is publicly available from the Applicant for use only in repeating experiments related to the present invention and not for other uses.

Pseudomonas syringae pv. tomato in the examples below DC3000(Yang L, Li B, Zheng X, et al Corrigengdum: Salicic acid biosynthes allowed and connected to involved biological phosphorus bacteria [ J ] Nature Communications 2015,6:7309), was publicly available from the applicant for use only in experiments relating to the repetition of the present invention, but not for other uses.

Example 1 TaBAKL Gene can improve disease resistance in Arabidopsis

This example provides a wheat variety Triticum aestivum cv. Xiaobaimai, publicly available from the institute of crop science, the national germplasm resources repository (No. ZM242), among others, wheat TaDREB6 transcription factor interaction protein screening, Chinese agricultural science 2011,44(22) 4740. 4747. among others, wheat protein isolate and molecular characterization of the Triticum aestivum L.ethidium-responsive factor 1(TaERF1) which is derived from wheat variety Qin yellow barley simple strain, Plant Mol Biol (2007)65: Gao 732, Shi yellow wine Qin yellow wine 719, yellow wine, yellow wine, yellow wine, yellow wine, the name of the TaBAKL gene is TaBAKL gene, the sequence of cDNA is sequence 2 in a sequence table, an open reading frame is 1-1101 th site of the 5' end of the sequence 2 in a self-sequence table, and TaBAKL protein shown in the sequence 1 in the sequence table is coded. The TaBAKL protein consists of 366 amino acid residues. The TaBAKL gene and the protein coded by the same can improve the disease resistance of plants, and the specific detection steps are as follows:

construction of recombinant expression vector

The TaBAKL gene sequence (with terminator removed) was constructed on pCAMBIA1302 by 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:

TaBAKLF：5'-GGGACTCTTGACCATGATGCACCACAGGCTCTCG-3'；

TaBAKLR：5'-TCAGATCTA C CCATGGAGCCCCAGCCAGCCCT-3'。

carrying out PCR amplification on the wheat leaf cDNA by utilizing TaBAKLF and TaBAKLR 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 correct sequence is named pCAMBIA1302-TaBAKL and can express TaBAKL protein.

Second, obtaining transgenic plants

Introducing the pCAMBIA1302-TaBAKL obtained in the step one into agrobacterium GV3101 to obtain a recombinant strain A-pCAMBIA1302-TaBAKL, and then transforming Arabidopsis thaliana by using a dipping method

2. Transformation of Arabidopsis thaliana by flower dipping 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-TaBAKL 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 Positive strains

The seeds obtained after agrobacteria infection are marked as T0 generations, sterilized, cleaned and spread 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 T2 generation strains until homozygous strains are obtained, and verifying the homozygous transgenic strains through PCR. The PCR primer sequences were as follows:

TaBAKL F：5'-GGGAAGAGTAAGGCACAAAA-3'；

TaBAKL R：5'-CGCCGCCAGTCAAGAAG-3'。

the empty vector pCAMBIA1302 is transferred into Col-0 by the same method to obtain the empty-transferred vector Arabidopsis thaliana.

Thirdly, identifying stress tolerance of transgenic plants

1. Germ inoculation and counting of colony numbers

Fresh Pst DC3000 was seeded on King's medium B (KB), and after 48h of growth at 28 ℃ single clones were picked and treated with 10mM MgCl₂The solution was resuspended to an OD of 0.002 to obtain a suspension of the cells. Inoculating the thallus suspension to the leaves of homozygous transgenic lines of four-week-old with a syringe without a needle, injecting 5 plants into each line, and injecting 4-5 leaves into each line, wherein the number of leaves is 20-25. After 3d, the leaf blade injected with the bacterial solution was photographed by observation. 0.25cm each was excised from 5 leaves of each genotype²The leaves of (4) are ground into powder and then treated with 10mM MgCl₂The solution was diluted, applied to KB medium containing rifampicin antibiotics, grown at 28 ℃ for 2 days and counted under a microscope, and the number of colonies was measured by taking the number of counts. The experiment was set up in 3 replicates, using empty vector Arabidopsis thaliana and Arabidopsis thaliana mutant atbakl as controls, and no Pst DC3000 injected (only an equal volume of 10mM MgCl was injected)₂Solution) as a negative control. Arabidopsis mutant atbakl is a T-DNA insertion mutant of Arabidopsis Biological Resource Center (ABRC) SALK-085834C.

As shown in FIG. 1, there was no significant difference between leaves of atbakl, Col-0 and homozygous transgenic lines in the negative control group. In an experimental group, diseases of different degrees appear on leaves inoculated with Pst DC3000, the diseases on the leaves of atbakl are the most serious, compared with atbakl and Col-0, the diseases on the leaves of a homozygous transgenic plant are lighter, and part of the leaves are still green; the leaf disease degree of the wild type Col-0 is between that of the Arabidopsis mutant and that of the homozygous transgenic line. The chlorosis area of the leaves is counted, and the proportion of the leaves of the homozygous transgenic plant in the range of 0-25% and 75-100% is obviously higher than that of Col-0. The amount of the cells at 0dpi and 3dpi after inoculating the leaves with Pst DC3000 was measured. At 0dpi, there was no significant difference between the three plants; at 3dpi, the amount of bacteria on the leaves of the homozygous transgenic lines was significantly less than Col-0, and the amount of bacteria on the leaves of Col-0 was significantly less than atbakl. The results indicate that transgenic plants have enhanced resistance to PstDC 3000.

2. Root length experiment of transgenic Arabidopsis

In MS Medium (NH)₄NO₃1650mg/L，KNO₃1900mg/L，CaCl₂·2H₂O 440mg/L，MgSO₄·7H₂O 370mg/L，KH₂PO4 170mg/L，KI 0.83mg/L，H₃BO₃6.2mg/L，MnSO₄·4H₂O 22.3mg/L，ZnSO₄·7H₂O 8.6mg/L，Na₂MoO₄·2H₂O 0.25mg/L，CuSO₄·5H₂O 0.025mg/L，CoCl₂·6H₂O0.025mg/L，FeSO₄·7H₂O 27.8mg/L，Na₂-EDTA·2H₂O37.3 mg/L, inositol 100mg/L, nicotinic acid 0.5mg/L, vitamin B60.5 mg/L, vitamin B10.1 mg/L, glycine 2mg/L, sucrose 20g/L) 4d Col-0, Arabidopsis mutant atbakl and homozygous transgenic line (35S:: TaBAKL-1 and 35S:: TaBAKL-2) seedlings were transferred to MS medium containing different concentrations of SA (the concentrations of SA were 30. mu.M, 50. mu.M and 80. mu.M respectively), plant phenotype was observed after 13d growth, fresh weight of the parts above the roots was weighed, total root length was counted by root-sweeping, and photography was performed. As shown in FIG. 2, there was no significant difference in total root length and fresh plant weight between any two of Col-0, atbakl, 35S:: TaBAKL-1 and 35S:: TaBAKL-2 on MS medium. On the culture medium containing SA, the stress on the plant is increased along with the increase of the concentration of SA, the leaves of the plant become smaller, and the color of the plant becomes light green or yellow. TaBAKL-1 has a significantly higher root length and plant fresh weight than Col-0 in a 50 μ M SA medium. The result shows that high-concentration SA can cause stress on plants, and influence the size, color, root length and fresh weight of leaves, and TaBAKL transgenic Arabidopsis can reduce stress damage caused by high-concentration SA.

Detecting the expression conditions of AtBAKL (homologous gene of TaBAKL in Arabidopsis thaliana, identity is 54.45%) and TaBAKL in Col-0, AtBAKL, 35S, and TaBAKL-1 and 35S in TaBAKL-2, respectively using Actin2 and Actin as references, and detecting the expression levels of the AtBAKL genes by using the following primers:

AtBAKL F：5'-CAGCCACCAACAGTTTCAA-3'；

AtBAKL R：5'-CTTCAGCACAGTAGCCACG-3'。

the primers used for detecting the expression of TaBAKL are as follows:

TaBAKL F：5'-GGGAAGAGTAAGGCACAAAA-3'；

TaBAKL R：5'-CGCCGCCAGTCAAGAAG-3'。

as a result, as shown in B and C in FIG. 2, the AtBAKL gene was expressed in Col-0 and not in AtBAKL; TaBAKL was not expressed in both Col-0 and atbakl, and was expressed in both 35S:: TaBAKL-1 and 35S:: TaBAKL-2.

Example 2expression Pattern of TaBAKL Gene

In order to analyze the possible functions of TaBAKL in pathogenic bacteria defense reaction, the expression pattern of TaBAKL in the Xiaobaimai under the SA treatment is analyzed by utilizing qRT-PCR (quantitative reverse transcription-polymerase chain reaction), and the TaBAKL is up-regulated by the SA treatment, reaches the peak after 36h and is up-regulated by about 4 times (figure 3). The specific operation method comprises the following steps:

the SA treatment is to soak the roots of the seedlings in 2mM SA, and the wheat material obtained in different time periods is stored in a refrigerator at-80 ℃ for later use after being quickly frozen in liquid nitrogen.

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

<120> application of plant stress tolerance associated protein TaBAKL in regulation and control of plant stress tolerance

<160>2

<170>PatentIn version 3.5

<210>1

<211>366

<212>PRT

<213> Triticum aestivum L.)

<400>1

Met His His Arg Leu Ser Arg Val Ala His Arg Val Leu Cys Cys Gly

1 5 10 15

Arg Gln Ala Ser Gly Asp Asp Leu Asn Asp Glu Arg Asn Gly Ser Ile

20 25 30

Arg Trp Val Phe Ser Leu Arg Glu Leu Gln Ser Ala Thr Asn Arg Phe

35 40 45

Asn Tyr Asp Asn Lys Ile Gly Glu Gly Ser Leu Gly Ser Val Tyr Trp

50 55 60

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

65 70 75 80

Ala Arg Asn Gly Thr Glu Met Glu Phe Ala Ser Glu Val Glu Ser Leu

85 90 95

Gly Arg Val Arg His Lys Asn Leu Leu Ser Leu Arg Gly Tyr Cys Ala

100 105 110

Asp Gly Pro Glu Arg Ile Leu Val Tyr Asp Tyr Met Pro Asn Ser Ser

115 120 125

Leu Phe Ala His Leu His Gly Thr His Ser Ser Glu Cys Leu Leu Asp

130 135 140

Trp Arg Arg Arg Thr Phe Ile Ala Ile Gly Ala Ala Arg Ala Ile Ala

145 150 155 160

Tyr Leu His His His Glu Thr Pro Pro Ile Ile His Gly Ser Ile Lys

165 170 175

Ser Thr Asn Val Leu Leu Asp Ser Asp Phe Gln Ala His Val Gly Asp

180 185 190

Phe Gly Leu Met Lys Leu Ile Ser Asp Glu Ile Asp His Asp Lys Ile

195 200 205

Ile Gly Glu Asn Gln Arg Gly Tyr His Ala Pro Glu Tyr Val Met Phe

210 215 220

Gly Lys Pro Thr Thr Gly Cys Asp Val Tyr Ser Phe Gly Ile Ile Leu

225 230 235 240

Leu Glu Leu Thr Ser Gly Arg Lys Pro Val Glu Lys Ser Gly Ser Gln

245 250 255

Lys Met Leu Gly Val Arg Asn Trp Met Leu Pro Leu Ala Lys Glu Gly

260 265 270

Arg Tyr Asp Glu Ile Ala Asp Ser Lys Leu Asn Asp Lys Tyr Ser Glu

275 280 285

Ser Glu Leu Lys Arg Val Val Leu Ile Gly Leu Ala Cys Thr His Arg

290 295 300

Glu Pro Asp Lys Arg Pro Thr Met Leu Glu Val Val Ser Leu Leu Lys

305 310 315 320

Gly Glu Ser Lys Glu Met Leu Leu Arg Leu Glu Lys Glu Glu Leu Phe

325 330 335

Arg Pro Asp Ser Met Ala Ser Ser Val Gly Thr Thr Pro Glu Gly Ser

340 345 350

Thr Asp Cys Ile Leu Lys Asn Asp Glu Gly Leu Ala Gly Ala

355 360 365

<210>2

<211>1101

<212>DNA

<213> Triticum aestivum L.)

<400>2

atgcaccaca ggctctcgcg ggtcgcccac agggtcctct gctgcggcag gcaagcctcc 60

ggggacgatc tgaacgacga gcggaatgga tccatcaggt gggtgttctc gctgagggag 120

ctccagtcgg cgacaaatag attcaattac gacaacaaga tcggagaagg ctcgcttggg 180

agcgtctact ggggacaagt ttgggatggc tctcagattg ctgttaaaaa gttaaagaat 240

gcaagaaatg ggacagaaat ggagtttgct tcagaagtcg aatctttggg aagagtaagg 300

cacaaaaacc tcctgagttt gcggggatat tgtgctgatg ggcctgaacg cattctggtg 360

tatgactata tgccgaactc aagtcttttt gcacatctcc acggaacaca ctcttcggag 420

tgccttcttg actggcggcg gagaacattt attgccattg gtgctgctcg ggctattgcg 480

tatcttcacc accatgagac acctccgata atccatggaa gcatcaaatc gaccaatgtg 540

ttacttgatt cggatttcca agcacacgtc ggtgactttg gtcttatgaa gctcatctca 600

gatgaaatag atcatgataa gattatcggt gaaaaccaac gaggctatca tgctcctgag 660

tacgtcatgt ttggcaagcc tacaacaggt tgtgatgtct acagctttgg cataatactt 720

ctggagctca ctagtgggag aaagccagta gaaaaatcag gctcccagaa aatgctcggg 780

gtccgaaact ggatgctccc gctggcgaaa gagggtagat acgatgaaat cgcggactca 840

aaactcaatg ataagtattc tgagtctgaa ctaaagaggg tggtgctgat tggactagcg 900

tgcacacaca gagaacccga taagagaccg acgatgcttg aggtagtgtc cctgctgaaa 960

ggtgaatcga aagagatgct tctgaggctt gaaaaggagg agctatttag gccagactcg 1020

atggctagtt ccgtcggaac gactccggaa gggagcaccg actgcattct taagaatgat 1080

gaagggctgg ctggggcttg a 1101

Claims (10)

1. The application of protein in regulating and controlling plant stress tolerance; the protein 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).

2. Use of a biological material related to a protein according to claim 1 for modulating stress tolerance in a plant; the biomaterial is any one of the following B1) to B14):

B1) a nucleic acid molecule encoding the protein of claim 1;

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).

3. Use according to claim 2, characterized in that: B1) the nucleic acid molecule is any one of the following b1) -b 4):

b1) the coding sequence is cDNA molecule or DNA molecule of sequence 2 in the sequence table;

b2) a cDNA molecule or a DNA molecule of a sequence 2 in a sequence table;

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

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

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

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.

5. The following X1) or X2):

x1) the protein of claim 1 or the biomaterial of claim 2 or 3;

x2) product for increasing stress tolerance in plants, comprising a protein according to claim 1 or a biological material according to claim 2 or 3.

6. A method of growing stress tolerant plants comprising: increasing the activity and/or content of the protein of claim 1 in a plant of interest, or promoting the expression of a gene encoding the protein of claim 1, to obtain a stress-tolerant plant with increased stress tolerance compared to the plant of interest.

7. The method of claim 6, wherein: the stress-tolerant plant is a transgenic plant having the expression of the protein of claim 1 increased as compared with the target plant, which is obtained by introducing the gene encoding the protein into the target plant.

8. The method of claim 7, wherein: the gene encoding the protein according to claim 1 is the nucleic acid molecule according to B1) of claim 3.

9. Use according to claim 4, or a product according to claim 5, or a method according to any one of claims 6-8, wherein:

the plant is m1) or m2) or m 3):

m1) a monocotyledonous or dicotyledonous plant;

m2) a graminaceous plant or a cruciferous plant;

m3) wheat or Arabidopsis;

the target plant is m1) or m2) or m 3):

m1) a monocotyledonous or dicotyledonous plant;

m2) a graminaceous plant or a cruciferous plant;

m3) wheat or Arabidopsis thaliana.

10. The use according to claim 4, or the product according to claim 5, or the method according to any one of claims 6 to 8, or the use, product or method according to claim 9, wherein: the stress tolerance is disease resistance.

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