CN111606986B

CN111606986B - Drought-resistant salt-tolerant associated protein, and related biological material and application thereof

Info

Publication number: CN111606986B
Application number: CN202010523202.XA
Authority: CN
Inventors: 张黎黎; 李瑞芬; 冯浩; 江颖; 张海纹
Original assignee: Beijing Academy of Agriculture and Forestry Sciences
Current assignee: Beijing Academy of Agriculture and Forestry Sciences
Priority date: 2020-06-10
Filing date: 2020-06-10
Publication date: 2021-12-28
Anticipated expiration: 2040-06-10
Also published as: CN111606986A

Abstract

The invention discloses a drought and salt resistant related protein, a related biological material and application thereof, wherein the amino acid sequence of the drought and salt resistant related protein is shown as a sequence 2, and the coding gene thereof is shown as the 1 st-1062 nd position of the sequence 1. The drought-resistant salt-tolerant related protein can improve the drought-resistant salt tolerance of a transgenic plant by over-expressing the HbDSWLP in wild arabidopsis thaliana, and shows that the member HbDSWLP of the transcription factor of the WRKY family participates in regulating the drought-resistant salt tolerance of the plant.

Description

Drought-resistant salt-tolerant associated protein, and related biological material and application thereof

Technical Field

The invention relates to the field of plant regulation, in particular to a drought-resistant salt-tolerant related protein, a related biological material and application thereof.

Background

The problems of soil salinization and drought are important factors for restricting the rapid and sustainable development of agricultural production in China. At present, it is relatively difficult to increase crop yields on a large scale on extremely limited arable land. In addition, the existence of various problems of ecological environment destruction, sea level rise, urbanization and the like also enables the availability ratio of cultivated land to be continuously reduced. Besides the protection of the existing cultivated land, the improvement of crops and the cultivation of new stress-resistant varieties by a biotechnology means are effective ways for improving and utilizing salinized wasteland and urban high-dry areas. Key genes of stress-resistant regulation and control ways are excavated from wild resources, and main functions and stress response mechanisms of the genes are analyzed, so that the method is a hot problem for scientific researchers to pay attention to and research.

Wild barley (Hordeum brevisubulus (Trin.) Link) is a wild related species of the barley genus of the wheat family, grows in saline-alkali and drought natural environments for a long time, and excavation and research of stress resistance genes of the wild barley are of great significance for researching plant stress resistance mechanisms and breeding new stress resistance varieties (Li et al, 2012). Research shows that the HsCBL8 gene of annual wild barley is over-expressed in rice, can obviously enhance the water retention capacity of plant somatic plasma membranes, increase the proline content and reduce the sodium ion intake (Guo et al, 2016). The perennial wild barley CBL interacting protein kinase HbCIPK2 can interact with HbFd1 and participate in regulating the photosynthetic steady state under the response of salt stress, thereby affecting the salt tolerance of wild barley (Zhang et al, 2015). Therefore, it is important to fully utilize the function of wild resources as an excellent gene library.

With the growing understanding of the plant stress-resistance pathway, the important role of transcription factors in this immediate and efficient regulatory process is becoming increasingly recognized. Transcription factors exert their expression regulation function by binding to cis-acting elements in the promoter region to activate or inhibit transcription of target genes (Liu et al, 1999). Transcription factors containing WRKY structural domains are verified to play an important role in response processes of plant growth, development, disease resistance, aging and the like from discovery to now (Tao et al, 2011; Qiu et al, 2009; Zhou et al, 2008; Huang et al, 2013; Chen et al, 2017), but related researches on functions of the transcription factors of wild germplasm resources are not reported so far.

Disclosure of Invention

The technical problem to be solved by the invention is how to regulate and control the drought resistance and/or salt tolerance of the plant.

In order to solve the technical problems, the invention provides a protein derived from Drought-and Salt-tolerant WRKY-Like protein (HbDSWLP) of wild barley (Trin. Link), wherein the protein is A1), A2) or A3:

A1) the amino acid sequence is protein of a sequence 2 in a sequence table;

A2) protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues of the amino acid sequence shown in the sequence 2 in the sequence table, has more than 90 percent of identity with the protein shown in A1), and is related to the salt tolerance and drought resistance of plants;

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

In the protein, the sequence 2 in the sequence table consists of 353 amino acid residues.

The protein can be artificially synthesized, or can be obtained by synthesizing the coding gene and then carrying out biological expression.

In the above protein, the protein tag (protein-tag) refers to a polypeptide or protein that is expressed by fusion with a target protein using in vitro recombinant DNA technology, so as to facilitate expression, detection, tracking and/or purification of the target protein. The protein tag may be a Flag tag, a His tag, an MBP tag, an HA tag, a myc tag, a GST tag, and/or a SUMO tag, among others.

In the above proteins, identity refers to the identity of amino acid sequences. The identity of the amino acid sequences can be determined using homology search sites on the Internet, such as the BLAST web pages of the NCBI home website. For example, in the advanced BLAST2.1, by using blastp as a program, setting the value of Expect to 10, setting all filters to OFF, using BLOSUM62 as a Matrix, setting Gap existence cost, Per residual Gap cost, and Lambda ratio to 11, 1, and 0.85 (default values), respectively, and performing a calculation by searching for the identity of a pair of amino acid sequences, a value (%) of identity can be obtained.

In the above protein, the 90% or more identity may be at least 91%, 92%, 95%, 96%, 98%, 99% or 100% identity.

Among the above proteins, the HbDSWLP may be derived from Hordeum brachisuubulus (Trin.) Link.

Biomaterials related to HbDSWLP are also within the scope of the invention.

The biomaterial related to the protein HbDSWLP provided by the invention is any one of the following B1) to B9):

B1) a nucleic acid molecule encoding the protein HbDSWLP;

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

B3) a recombinant vector containing the nucleic acid molecule of B1) or a recombinant vector containing the expression cassette of B2);

B4) a recombinant microorganism containing B1) the nucleic acid molecule, or a recombinant microorganism containing B2) the expression cassette, or a recombinant microorganism containing B3) the recombinant vector;

B5) a transgenic plant cell line comprising B1) the nucleic acid molecule or a transgenic plant cell line comprising B2) the expression cassette;

B6) transgenic plant tissue comprising the nucleic acid molecule of B1) or transgenic plant tissue comprising the expression cassette of B2);

B7) a transgenic plant organ containing the nucleic acid molecule of B1), or a transgenic plant organ containing the expression cassette of B2);

B8) a nucleic acid molecule that reduces the expression of the protein of claim 1;

B9) an expression cassette, a recombinant vector, a recombinant microorganism or a transgenic plant cell line comprising the nucleic acid molecule according to B8).

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.

In the above-mentioned related biological material, the nucleic acid molecule of B1) is the gene encoding the protein as shown in B1) or B2) below:

b1) the coding sequence is cDNA molecule or DNA molecule of nucleotide of sequence 1 in the sequence table;

b2) the nucleotide is a cDNA molecule or a DNA molecule of a sequence 1 in a sequence table.

Wherein, the sequence 1 in the sequence table is composed of 1465 nucleotides, the coding sequence is 1-1062 nucleotides in the sequence 1 in the sequence table, and the coding sequence encodes the protein shown in the sequence 2 in the sequence table.

The biomaterial according to the above, wherein the HbD-containing code of B2)An expression cassette for a nucleic acid molecule of SWLP (HbDSWLP gene expression cassette), refers to DNA capable of expressing TaRHP1 in a host cell, which DNA may include not only a promoter that initiates transcription of the HbDSWLP gene, but also a terminator that terminates transcription of the HbDSWLP. 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: the constitutive promoter of cauliflower mosaic virus 35S; the wound-inducible promoter from tomato, leucine aminopeptidase ("LAP", Chao et al (1999) Plant Physiology 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 jasmonic acid ester); 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 expression vector containing the HbDSWLP gene expression cassette can be constructed by using the conventional plant expression vector. The plant expression vector comprises a binary agrobacterium vector, a vector for plant microprojectile bombardment and the like. Such as pAHC25, pWMB123, pBin438, pCAMBIA1302, pCAMBIA2301, pCAMBIA1301, pCAMBIA1300, pBI121, pCAMBIA1391-Xa or pCAMBIA1391-Xb (CAMBIA Corp.) and the like. 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 identification and screening of transgenic plant cells or plants, plant expression vectors to be used may be processed, for example, by adding genes encoding enzymes or luminescent compounds which produce a color change (GUS gene, luciferase gene, etc.), marker genes for antibiotics which are expressible in plants (e.g., nptII gene which confers resistance to kanamycin and related antibiotics, bar gene which confers resistance to phosphinothricin which is a herbicide, hph gene which confers resistance to hygromycin which is an antibiotic, dhS gene which confers resistance to methatrexate, EPSPS gene which confers resistance to glyphosate), or marker genes for chemical resistance (e.g., herbicide resistance), mannose-6-phosphate isomerase gene which provides the ability to metabolize mannose, etc. 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 biological material, the recombinant microorganism may be specifically yeast, bacteria, algae and fungi.

In order to solve the technical problems, the invention also provides a plant drought-resistant agent and/or a plant salt-tolerant agent.

The plant drought-resistant agent and/or the plant salt-tolerant agent contain the protein or/and the biological material.

The active ingredients of the plant drought-resistant agent and/or the plant salt-tolerant agent can be the protein or biological materials related to the protein, and the active ingredients of the plant drought-resistant agent and/or the plant salt-tolerant agent can also contain other biological ingredients or/and non-biological ingredients, and the other active ingredients of the agent can be determined by a person skilled in the art according to the drought-resistant and/or salt-tolerant effects of plants.

The protein or the biological material can be applied to any one of the following P1-P9:

the use of P1, the protein, or the biological material for modulating drought resistance in a plant;

the use of P2, the protein, or the biological material in the manufacture of a product for increasing drought resistance in a plant;

the use of P3, the protein, or the biological material in the cultivation of drought resistant plants;

the use of P4, the protein, or the biological material in the preparation of a plant drought resistant product;

use of P5, the protein, or the biological material for modulating salt tolerance in a plant;

use of P6, the protein, or the biological material for the manufacture of a product for increasing salt tolerance in plants;

the use of P7, said protein, or said biological material for growing salt tolerant plants;

the use of P8, the protein, or the biomaterial in the preparation of a plant salt tolerant product;

use of P9, the protein, or the biological material in plant breeding.

In order to solve the technical problems, the invention also provides a method for cultivating salt-tolerant and/or drought-resistant plants.

The method for cultivating the salt-tolerant and/or drought-resistant plant comprises the steps of improving the expression level of the protein or the coding gene thereof in the target plant to obtain the salt-tolerant and/or drought-resistant plant; the salt tolerance and/or drought resistance of the salt-tolerant and/or drought-resistant plant is higher than that of the target seed plant.

Wherein the increase in the expression level of the protein of claim 1 or a gene encoding the protein in a plant is achieved by introducing a gene encoding the protein into the plant.

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

1) 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;

2) 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;

3) 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;

4) 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 the protein can be introduced into Plant cells by conventional biotechnological methods using Ti plasmids, Plant virus vectors, 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).

In the above method, the heat-resistant and/or disease-resistant plant may be a transgenic plant, or may be a plant obtained by a conventional breeding technique such as crossing.

In the above methods, the transgenic plant is understood to include not only the first to second generation transgenic plants but also the progeny thereof. 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 transgenic plants include seeds, callus, whole plants and cells.

As described above, the plant and the plant of interest are both monocotyledonous or dicotyledonous plants. The monocotyledon can be wheat, and the dicotyledon can be cruciferous plants such as Arabidopsis thaliana.

The HbDSWLP can regulate and control the drought resistance and/or salt tolerance of plants; the drought resistance test of transgenic arabidopsis thaliana expressed by HbDSWLP can show that the water loss rates of the HbDSWLP overexpression strain and the WT plant leaf under the control treatment are detected, the result shows that the water loss rate of the WT plant leaf is high, the obvious difference exists after 2h, the water loss rates of the 3 HbDSWLP overexpression strain plant leaves are very similar and approximate, the water loss rates are extremely obviously lower than that of the WT after 2h, and the drought resistance of arabidopsis thaliana can be improved through the HbDSWLP overexpression. As can be seen from the salt tolerance test of transgenic arabidopsis thaliana expressed by HbDSWLP, the growth state and root development of seedlings of a strain over-expressing HbDSWLP are obviously superior to those of a wild type strain under the stress of NaCl with different concentrations, and particularly the strain is more obvious under the stress of 150mM NaCl.

Drawings

FIG. 1 shows the analysis of gene expression level of overexpression Arabidopsis homozygous lines HbDSWLP, wherein WT is wild type, and 2-7, 6-11, 7-8, 13-8, 18-1, 19-6, 20-1, 22-7 and 25-6 are transgenic lines.

FIG. 2 shows that the overexpression of HbDSWLP enhances the salt tolerance of Arabidopsis thaliana, wherein WT is a wild type, and W38-ox1, 2 and 3 are transgenic lines.

FIG. 3 shows that HbDSWLP overexpression improves drought resistance of Arabidopsis, wherein WT is a wild type, and W38-ox1, 2 and 3 are transgenic lines.

FIG. 4 shows that the overexpression of HbDSWLP enhances ABA adaptation of Arabidopsis thaliana, wherein WT is a wild type, and W38-ox1, 2 and 3 are transgenic lines.

FIG. 5 shows the analysis of HbDSWLP overexpression transcriptome and ABA dependent pathway drought response gene expression, wherein WT is wild type, and W38-ox1, 2 and 3 are transgenic lines.

Detailed Description

Wild barley (Hordeum brevisubulus (Trin.) Link) in the following examples was treated in Li, Ruifen; zhang, Junwen; wu, Guingyu et al.2012 HbCIPK2, a novel CBL-interacting protein kinase from halophyte Hordeum brevisubulatum, company salt and ecological stress tolerance PLANT CELLAND ENVIRONMENT,35(9): 1582-.

Yeast strain NMY51 in the following example, Dualmemabrane kitP01001, Beijing Davidae, Biotechnology Inc.

Arabidopsis wild type (Arabidopsis thaliana, ecotype Columbia) in the following examples is disclosed in the literature Zhang HW, Zhao FG, Tang RJ, Yu YX, Song JL, Wang Y, Li LG, Luan S. Two toplast MATE proteins functions as transmitter-regulating chloride channels in Arabidopsis proceedings of the National Academy of Sciences of the USA,2017,114(10): E2036-E2045 publicly available from the Academy of agriculture and forestry, Beijing.

The plant expression vector pYBA1104 in the following examples is disclosed in the documents Lili Zhang, Yunxiao Wang, Qike Zhang, Ying Jiang, Haiwen Zhang, Ruifen Li, Overexpression of HbMBF1a, encoding multiprotein cleaving factor 1from the halophile Hordeum breviatilis, company saline tolerance and sensitivity in transport agricultural Biology,2020,102(1-2):1-17, publicly available from the agroforestry academy of Beijing.

The plasmid pDH 1-HbCIPK2 in the examples described below is disclosed in the documents Chao Zhang, Rongcha Ge, Junwen Zhang, Yajuan Chen, Hongzhi Wang, Jianhua Wei, Ruifen Li, Identification and Expression Analysis of a Novel HbCIPK 2-interaction Ferredoxin from halogen H.breviaultem.2015, PlosONE DOI: 10.1371/journel.0144132, publicly available from the agroforestrial academy of sciences of Beijing.

Agrobacterium strain Gv3101, obtained commercially from Shanghai dimension Biotech, Inc., in the examples described below.

ABA, Abscisic Acid (ABA), in the following examples was obtained from Sorbox technologies, Inc. of Beijing.

Example 1 acquisition of HbDSWLP Gene

The protein kinase HbCIPK2 is used as bait protein, a wild barley salt stress yeast cDNA library is screened by a yeast two-hybrid technology of a membrane system, and the nucleotide sequence of the transcription factor HbDSWLP is obtained and identified, as shown in a sequence 1. The sequence has 1465 nucleotides, an open reading frame containing 1062 nucleotides in the sequence 1 is shown as 1-1062 of the sequence 1, and codes 353 amino acids shown as the sequence 2, and a 3' UTR containing 303 nucleotides is shown as 1063-1366 of the sequence 1. The method comprises the following specific steps:

1. wild barley material culture: wild barley (Hordeum brevisubulatum (Trin.) Link) seeds are collected from inner Mongolia salinized grassland, the wild barley seeds are soaked in water at room temperature, placed at 4 ℃ for vernalization for 2d, placed in an incubator with 22-25 ℃ (day and night temperature threshold) light period of 16h/8h for pregermination for 2-3d, observed to sprout about 1cm, transplanted to a 250ml beaker containing Hoagland nutrient solution, the seedling roots are immersed in the nutrient solution, and the sprouts float on a gauze. Continuing culturing for about 2 weeks, performing stress treatment such as NaCl, PEG, ABA, etc. and sampling when the wild barley plants grow to the two-leaf one-heart stage, repeating the biological treatment for 3 times for the above-ground part (shot) and the below-ground part (root) samples, storing the quick-frozen liquid nitrogen in the quick-frozen liquid nitrogen-80 ℃ refrigerator. The formula of the Hoagland nutrient solution comprises the following components: 0.51g/L KNO₃、0.82g/L Ca(N0₃)₂、0.49g/L MgSO₄.7H₂0、0.136g/L KH₂PO₄(ii) a Then adding lmL Fe EDTA solution (preparation method: respectively dissolving 7.45g Na)₂EDTA、5.57g FeSO₄.7H₂0 in 200mL of distilled water, and heating. Constantly stirring Na₂EDTA solution and FeS0₄Mixing the solutions, and fixing the volume to 1L); then adding the lmLA-Z solution (preparation method: H)₃B0₃ 2.80mg/L，CuS0₄·5H ₂0 0.08mg/L，ZnSO₄·7H ₂0 0.22mg/L，MgCl₂·6H ₂0 81mg/L，HMoO·4H ₂0 0.09mg/L)。

2. Yeast competence preparation and transformation: reference ProQuest^TMMethods in the Two-Hybrid System. Streaking yeast strain NMY51 frozen at-80 deg.C on YPDA plate, culturing at 30 deg.C for 2-3 d; selecting yeast with diameter of 2-3mm, and culturing in 5mL YPDA liquid culture medium at 30 deg.C overnight; a small amount of bacterial liquid is sucked into 100ml of preheated YPDA liquid culture medium to be expanded and cultured to OD₆₀₀0.6-0.8; transferring the bacterial liquid into a 50mL centrifugal tube, centrifuging at 3500g and 5min at 4 ℃, and removing the supernatant; after the precipitate is evenly washed by 20mL of sterilized ultrapure water, 3500g is carried out for 5min, the centrifugation is carried out at 4 ℃, and the supernatant is discarded; resuspending the precipitate with 10ml LiAc/TE solution, 3500g, 5min, centrifuging at 4 ℃, and discarding the supernatant; then 1ml LiAc/TE solution is used for resuspending the cells, and the cells are subpackaged in a 1.5ml centrifuge tube and placed for 30min at room temperature, thus obtaining the yeast competence.

3. Reference ProQuest^TMThe Two-Hybrid System adopts a LiAc conversion method to boil salmon sphere DNA in boiling water bath for 10min, and ice bath is carried out for 5min and 2 times to denature the salmon sphere DNA; plasmid DNA (pDH 1-HbCIPK2) (pDH 1 vector purchased from Beijing Dake Biotech Co., Ltd., pDH 1-HbCIPK2 vector constructed by the laboratory) 2. mu.g, 100. mu.g salmon sperm DNA, 300. mu.l of PEG/LiAc mixed solution (freshly prepared) were added to 100. mu.l of competent cells, respectively, and mixed; shaking with shaking table at 200rpm at 30 deg.C for 30-45 min; add 20. mu.l DMSO, mix by inversion (not vortexable); heat shock at 42 deg.C for 15min, strictly controlling time, and reversing every 5 min; after ice bath for at least 2min, 12000rpmCentrifuging for 1min, removing supernatant, and resuspending with 200 μ l sterilized ultrapure water; spread on SD/-Leu-Trp plates, cultured in an inverted manner at 30 ℃ for 2-3d and observed for growth to obtain pDHB1-HbCIPK2 bait strain. In addition, a bait protein self-activation test is carried out to determine the screening concentration of the 3-AT. Empty vector pPR3-N (pPR3-N from Biotech, Inc. of Beijing Davidae) of the bait vector and component library was co-transformed with yeast strain NMY51, which was plated on SD/-Trp-Leu-His-Ade screening medium supplemented with 3-AT (3-Amino-1,2, 4-triazole) AT different concentrations of 0, 0.5 and 1mM, respectively, to observe its growth.

4. Salt stressed yeast cDNA library screening: streaking activated pDHB1-HbCIPK2 bait strain (plasmid DNA (pDHB1-HbCIPK2) -transformed strain obtained in step 3); precooling a 50mL sterile centrifuge tube, and then sequentially adding: mixing 10-20 μ g of plasmid DNA (linearized Bait vector) and 20 μ L, pDHB1-HbCIPK2 Bait yeast competent cell 600 μ L, PEG/LiAc 2.5mL of Carrier DNA by using thumb gently; mixing, culturing at 30 deg.C for 45min, and mixing gently once every 15 min; add 160. mu.L DMSO, mix well (gentle motion) taking care not to vortex; standing in water bath at 42 deg.C for 20min (time critical), and mixing twice; performing instantaneous centrifugation for 15s, sucking and removing upper-layer liquid, adding 3mL YPD Plus Medium, and uniformly mixing bacterial cells; culturing at 30 deg.C and 250rpm in incubator for 60-90 min; centrifuging at room temperature of 700g for 15s after the culture is finished, and removing a supernatant; adding 15mL of 0.9% NaCl Solution to resuspend the thalli; taking 500 mu L of the mixed bacterial liquid, coating the mixed bacterial liquid on an SD/-Leu-Trp (containing 1mM3-AT) plate, and carrying out inverted culture AT constant temperature of 30 ℃ for 3-5 d; single colonies (small amount gently, too much not detectable) were picked with a tip from plates of SD/-Leu-Trp (containing 1mM3-AT), and 50. mu.L of autoclaved ddH was used₂Diluting with O, mixing with 25 μ l bacterial liquid template and 25 μ l Matchmaker Insert Check PCR Mix II, centrifuging instantly, and performing reaction amplification in a PCR instrument according to the following procedures. Pre-denaturation: 1min at 94 ℃; denaturation: 98 ℃ for 10s, annealing: 68 ℃ for 3min, extension: 1min at 72 ℃ for 30 cycles; and (3) complete extension: storing the reaction product at 72 deg.C for 7min and 4 deg.C, and spotting the reaction product on 1.0% agarose gel electrophoresis for detection.

5. Detecting the single bacterium with the insert by PCR in the step 4Dropping, taking 10 μ L of the residual bacteria liquid corresponding to the serial number, spotting on a YPDA plate, and performing inverted culture at 30 ℃ for 3 d; scraping 1/2 yeast with a gun head, dissolving in 500 μ L, 10mM EDTA, and mixing; centrifuging at room temperature and 11000g for 1min, and pouring out the upper layer liquid; adding 200. mu.L ZYM Buffer to resuspend the pellet; adding 20 mu L of Zymolyase, shaking gently and mixing uniformly, and suspending the thalli; culturing at 30 deg.C and 250rpm for 1h, centrifuging at 2000g for 10min, and removing supernatant to complete yeast cell lysis; adding 250 mu LY1 Buffer (containing RNaseA) into the yeast cells after the lysis is finished, and shaking to mix fully; adding 250 mu LY2 lysine Buffer (the solution is determined to be evenly mixed before use and no precipitate is generated) to the step (1), and mixing lightly for about 6 times without vortex; adding 300 mu LY3 neural lysis Buffer, reversing the upside down for about 6 times, and acting gently and without violence; centrifuging at room temperature of 11000g for 5min, taking supernatant, transferring to an adsorption column, and repeating the step for 1 time; the adsorption column is sleeved in a 2mL collection pipe, centrifuged for 1min at the room temperature of 11000g, and waste liquid is discarded; dripping 450 mu L Y4 Wash Buffer into the middle position of the adsorption column, keeping the rotating speed of the centrifuge unchanged, centrifuging for 3min, and discarding the waste liquid; continuously sleeving the adsorption column into a collecting pipe, adding no solution, and carrying out idle rotation centrifugation for 3min at the same rotating speed; discarding the collection tube, replacing with a new sterilized centrifuge tube, adding 50 μ L YE precipitation Buffer to the adsorption column, covering with a cover, standing for 1min, and centrifuging at 11000g for 1 min. Converting yeast plasmids into escherichia coli, and adding Amp resistance into the LB culture medium coated on the plate; coli monoclonals were picked from Amp-resistant LB plates and diluted in 20. mu.L of sterile ddH₂And in O, taking 2 mu L of the mixture as a template of the PCR high-fidelity amplification reaction, detecting the PCR result by agarose gel electrophoresis, and sequencing the bacterial liquid with a proper band size.

And (3) performing sequence comparison analysis on a sequencing result on an NCBI website to obtain a coding gene of HbDSWLP (Dry-and Salt-tolerantWRKY-Like Protein), determining the full length of the gene by DNAMAN comparison, performing multiple sequence comparison analysis by BioEdit and ClustalW software, and analyzing a phylogenetic evolutionary tree by using MEGA4 software. The HbDSWLP gene is shown as a sequence 1, an open reading frame containing 1062 nucleotides in the sequence 1 is shown as 1-1062 of the sequence 1, the open reading frame codes 353 amino acids shown as the sequence 2, and a 3' UTR containing 303 nucleotides in the sequence 1 is shown as 1063-1366 of the sequence 1.

Example 2 obtaining and identification of drought-resistant and salt-tolerant HbDSWLP transgenic Arabidopsis thaliana

Construction of recombinant expression vector

1. The coding sequence of HbDSWLP is constructed on a plant expression vector pYBA1104, and the specific operation is as follows: taking the sequence of the HbDSWLP gene obtained in the embodiment 1 as a template, and adopting a primer HbDSWLP-EcoR 1-Forward: 5' -AGAATTCATGGATCCATGGATGGGCAGC-3' (the underlined part is the sequence of the cleavage site), HbDSWLP-Sal 1-Reverse: 5' -TGTCGACTTAATTGATGTCCCTGGTCGGCGAG-3' (the underlined part is a sequence of enzyme cutting sites), carrying out PCR amplification, recovering an amplification product, cloning the gene of the HbDSWLP between the EcoRI/SalI enzyme cutting sites of the pYBA1104 through the EcoRI/SalI enzyme cutting sites after correct sequencing, and obtaining an over-expression vector pYBA1104-HbDSWLP, wherein the expression vector pYBA1104-HbDSWLP is a recombinant expression vector which replaces the sequence between the EcoRI and SalI recognition sites of the pYBA1104 with the 1 st-1062 nucleotides (protein of the sequence 2 in the coding sequence table) in the sequence 1 and keeps other sequences unchanged.

II, obtaining of HbDSWLP gene-transferred plant

1. And introducing the recombinant expression vector pYBA1104-HbDSWLP into competent cells of agrobacterium GV3101 to obtain recombinant agrobacterium GV3101/pYBA 1104-HbDSWLP.

2. Preparation of transformed bacterial liquid of recombinant Agrobacterium GV3101/pYBA1104-HbDSWLP (5% sucrose, 0.05% Silwet L-77, OD₆₀₀0.8 to 1.2); arabidopsis thaliana wild-type (Arabidopsis thaliana, ecotype Columbia) plants were dip-transformed according to the dipping method (floral dip) proposed by Clough and Bent (1998) to give Arabidopsis thaliana-pYBA 1104-HbDSWLP. Culturing Arabidopsis thaliana-pYBA 1104-HbDSWLP, and harvesting T₀Seeds of Arabidopsis thaliana-pYBA 1104-HbDSWLP. Will T₀Seeds of arabidopsis thaliana-pYBA 1104-HbDSWLP are screened on an MS plate (containing 4.3g/L MS powder, 20g/L sucrose, 6g/L plant gel and pH5.8) containing 50mg/L Kan, and a positive single plant is subjected to selfing and passage to obtain T₁Seeds of arabidopsis thaliana-pYBA 1104-HbDSWLP are generated; repeating the above steps for further selfing passage to obtain T₂Generation of Arabidopsis thaliana-pYBA 1Seeds of 104-HbDSWLP and T₃Seeds of Arabidopsis thaliana-pYBA 1104-HbDSWLP.

3. Screening and obtaining of HbDSWLP overexpression arabidopsis homozygous strain

pYBA1104-HbDSWLP was transferred to wild type Arabidopsis thaliana T₀Sterilizing seed generation with chlorine, vernalizing at 4 deg.C for two days, dibbling on 1/2MS culture substrate containing Kan (concentration 50mg/L) to grow 7 days later, making yellow and green seedlings be clearly visible, transplanting green seedlings to grow in culture soil in greenhouse for 2 weeks, selecting 30 robust transgenic seedlings, and harvesting seeds (T)₁) After drying for one week, sterilizing with chlorine, vernalizing for two days at 4 ℃, dibbling on 1/2MS culture medium plate containing Kan (concentration 50mg/L) to grow for 7 days, and selecting green seedlings from the strain with the statistical ratio of yellow-green seedlings being 1:3 to continue transplanting in culture soil for greenhouse growth. Selecting 6 sister lines of each strain to harvest seeds (T)₂) After drying for one week, sterilizing with chlorine, after vernalizing for two days at 4 ℃, dibbling on 1/2MS culture medium plate containing Kan (concentration 50mg/L) to grow for 7 days, observing that the transgenic arabidopsis thaliana strains which are all green seedlings are the HbDSWLP overexpression arabidopsis thaliana homozygous strains.

The expression quantity of the HbDSWLP gene of the 10 obtained over-expression homozygous strains is detected, and the qRT-PCR result shows that the HbDSWLP gene in the WT plant is not expressed, which indicates that the specificity of the primer is good, and other members of a WRKY gene family cannot be amplified. In 10 over-expressed homozygous lines, the HbDSWLP gene is expressed to different degrees, wherein the expression level of 19-6 lines is the highest, and the expression level of 13-8 lines is the lowest (as shown in FIG. 1). We determined 3 over-expression homozygous lines, 19-6, 20-1 and 22-7 respectively, based on the difference of the expression levels of the HbDSWLP genes and combined with pre-experiments of the stress phenotype of the over-expression homozygous lines, and labeled them as W38-ox1, W38-ox2 and W38-ox3 in subsequent studies.

Third, salt tolerance test of transgenic arabidopsis thaliana expressed by HbDSWLP

And (2) sterilizing and vernalizing seeds of wild Arabidopsis (WT) and 3 HbDSWLP overexpression homozygous strains (W38-ox 1, W38-ox2 and W38-ox3) obtained in the second step, respectively, dibbling the seeds on a normal MS culture medium by using a sterile gun head, vertically growing for 3 days, selecting Arabidopsis seedlings with consistent growth state and root length from an MS plate in a super-clean workbench, respectively transferring seedlings of different strains to MS plates containing 0mM, 125mM, 150mM and 175mM NaCl by using pointed tweezers, observing the growth condition and root length change of the seedlings after 10 days, and photographing and measuring the root length and fresh weight. The results are shown at A, B in FIG. 2.

Seeds of wild type Arabidopsis thaliana (WT for short) and 3 HbDSWLP over-expression homozygous lines (W38-ox 1, W38-ox2 and W38-ox3 for short) obtained in example 1 are sterilized and vernalized, and are directly sown in soil soaked by 0mM and 150mM NaCl solutions, the difference of germination rates of the seeds in salt stress soil is observed, and the germination rate is photographed and counted for 14d after sowing. The results are shown at C, D in FIG. 2.

The study found that the proportion of over-expressed line seeds that germinated and formed seedlings was significantly higher than WT under 150mM NaCl treatment (C, D in fig. 2). WT seeds were also able to germinate, but soon became yellow and dry and could not continue to grow, with no significant difference in the time of germination between the two seeds. Also shown in FIG. 2 are the differences of seedling growth and root length between WT and overexpression strains W38-ox1, W38-ox2 and W38-ox3 under different concentrations of NaCl, and the results show that the seedling growth state and root development of the overexpression strains are obviously better than that of WT under different concentrations of NaCl stress, and particularly show more remarkable performance under 150mM NaCl stress (A, B in FIG. 2). The plants of the over-expressed lines were significantly greener and still in a growing state, while the WT plants appeared dry, yellow-shrunken and the root system essentially stopped growing.

Fourth, drought resistance test of transgenic arabidopsis thaliana expressed by HbDSWLP

And (2) planting seeds of wild Arabidopsis (WT) and 3 HbDSWLP overexpression homozygous lines (W38-ox 1, W38-ox2 and W38-ox3 respectively) obtained in the second step in a greenhouse, vernalizing the seeds of each line, directly sowing the vernalized seeds in a rectangular tray of 35.5cm multiplied by 28cm multiplied by 7.6cm for growing for 7d, thinning out, keeping 21 wild Arabidopsis and 3 HbDSWLP transgenic Arabidopsis homozygous lines with consistent growth states, and watering a small amount to maintain root growth. After continuing to grow for 7 days, carrying out drought treatment, namely continuously not watering for about 20 days, rehydrating for one week, photographing and counting the survival rate; taking leaves of wild type and HbDSWLP overexpression strains which are normally cultured in a greenhouse for 20d to grow, carrying out in-vitro dehydration treatment (10 leaves/strain), weighing the fresh weight of the leaves every 1h from 0h, weighing the fresh weight for 9 times in total, calculating the water loss rate of the leaves of the wild type and the HbDSWLP overexpression strains, and repeating the experiment for at least 3 times. The results are shown in FIG. 3.

As can be seen from FIG. 3, overexpression of HbDSWLP improves drought resistance in Arabidopsis. After the arabidopsis seedlings are transplanted, the seedlings are revived for 10 days, watering is not carried out after the seedlings are watered for the first time, and drought water control is about 20 days, so that the drought resistance of the plant of the HbDSWLP overexpression strain is obviously higher than that of the WT, and after one week of rehydration, the recovery state and the survival rate of the plant of the overexpression strain are obviously better than that of the WT (A and B in a picture 3). Meanwhile, by detecting the water loss rates of the leaves of the HbDSWLP overexpression strain and the WT plant under the control treatment, the results show that the water loss rate of the leaves of the WT plant is high and is remarkably different after 2h, while the water loss rates of the leaves of 3 HbDSWLP overexpression strain plants are very similar and close and are remarkably lower than that of the WT after 2h (C in FIG. 3).

Fifthly, HbDSWLP overexpression enhances ABA insensitivity of arabidopsis thaliana

After seeds of arabidopsis wild type (abbreviated as WT) and 3 HbDSWLP overexpression homozygous lines (abbreviated as W38-ox1, W38-ox2 and W38-ox3 respectively) obtained in the second step are sterilized and vernalized, the seeds are respectively dibbled on MS plates containing 0mM, 0.5 mu M and 1 mu MABA by using sterile tips, and the MS plates are placed in a culture room for 12d, and then photographing is carried out, and the green leaf rate of seed cotyledons is counted, and the result is shown in FIG. 4. From FIG. 4, it can be seen that HbDSWLP overexpression can enhance insensitivity of Arabidopsis thaliana to exogenous ABA, and shows that the seed germination rate and seedling growth state of the overexpression strain are obviously superior to WT (A and B in FIG. 4) under the stress of different exogenous ABA. The over-expressed line plants appeared yellow-green with a significantly greater lateral root number than WT, whereas WT plants dried up and whitened under 30. mu. MABA treatment and the root system essentially stopped growing (C, D in FIG. 4).

In order to further understand the molecular mechanism of adjusting drought stress tolerance by HbDSWLP, a series of differential genes are obtained by analyzing RNA-seq data under mannitol stress, and genes which change by more than 2 times in an over-expressed plant are selected as main research objects. According to this criterion, we performed wien map analysis on all differentially expressed genes in comparison with wild type in the over-expressed plants under control and in comparison with wild type in the over-expressed plants treated with mannitol, and found that 78 differentially expressed genes were obtained in total in leaves of the over-expressed plants compared with WT, of which 48 up-regulated genes and 30 down-regulated genes were present. And 266 differentially expressed genes were obtained from the roots of the over-expressed plants, 164 up-regulated genes and 102 down-regulated genes (A, B in FIG. 5). According to the screening result of the Wien diagram, the difference genes are identified to be caused by the overexpression of the HbDSWLP gene and the mannitol stress together.

Through detecting the expression quantity of several Marker genes in an ABA-dependent drought response pathway, the expression of AtRAB18, AtRD22 and AtRD29B genes in HbDSWLP overexpression Arabidopsis plants is induced to be remarkably up-regulated under the stress of 200mM mannitol (C-E in figure 5), and the fact that the HbDSWLP participates in Arabidopsis drought response regulation and control is achieved through an ABA-dependent signal response regulation and control pathway is shown.

The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific embodiments, it will be appreciated that the invention can be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is possible within the scope of the claims attached below.

Sequence listing

<110> agriculture and forestry academy of sciences of Beijing City

<120> drought-resistant salt-tolerant related protein, related biological material and application thereof

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caggcgccag ccacgcctca ccactcgcag cagcacaaac aggaagcggc agcggtcgtc 900

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Val Gly Leu Pro Pro Met Gly His Pro His His His His Ser Gln Gln

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Gln Ala Ala Pro Met Ile Ala Leu Ala Lys Pro Lys Ile Leu Val Glu

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Glu Asn Phe Met Pro Leu Lys Lys Asp Pro Lys Val Ala Val Leu Glu

50 55 60

Ser Glu Leu Gln Arg Val Ser Glu Glu Asn Arg Arg Leu Gly Glu Met

65 70 75 80

Leu Arg Glu Val Ala Ser Lys Tyr Glu Ala Leu Gln Gly Gln Phe Thr

85 90 95

Asp Met Val Thr Ala Gly Ala Asn Cys Gly Gly Asn Asn Ser His Tyr

100 105 110

Asn Asn Gln Pro Ser Ser Ala Ser Glu Gly Gly Ser Val Ser Pro Ser

115 120 125

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

130 135 140

Gln Gln Gln His Tyr Ala Ala Gly Leu Ala Tyr Ala Val Ala Pro Asp

145 150 155 160

Gln Ala Glu Cys Thr Ser Gly Glu Pro Cys Lys Arg Ile Arg Glu Glu

165 170 175

Cys Lys Pro Val Ile Ser Lys Arg Tyr Val His Ala Asp Pro Ser Asp

180 185 190

Leu Ser Leu Val Val Lys Asp Gly Tyr Gln Trp Arg Lys Tyr Gly Gln

195 200 205

Lys Val Thr Lys Asp Asn Pro Cys Pro Arg Ala Tyr Phe Arg Cys Ser

210 215 220

Phe Ala Pro Gly Cys Pro Val Lys Lys Lys Val Gln Arg Ser Ala Glu

225 230 235 240

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

245 250 255

Gln Pro Pro Pro Ser Gln Pro Gln Gln Gln Asn Asp Gly Ser Gly Ala

260 265 270

Gly Lys Asn Ala Gly Lys Pro Pro Gln Ala Pro Ala Thr Pro His His

275 280 285

Ser Gln Gln His Lys Gln Glu Ala Ala Ala Val Val Val Ser Gly Glu

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Ser Thr Ala Ala Ala Ser Glu Leu Ile Arg Arg Asn Leu Ala Glu Gln

305 310 315 320

Met Ala Met Thr Leu Thr Arg Asp Pro Ser Phe Lys Ala Ala Leu Val

325 330 335

Thr Ala Leu Ser Gly Arg Ile Leu Glu Leu Ser Pro Thr Arg Asp Ile

340 345 350

Asn

Claims

1. A protein, which is a protein of A1) or A2) as follows:

A1) the amino acid sequence is protein of a sequence 2 in a sequence table;

A2) a1) at the N-terminus or/and C-terminus.

2. The biomaterial related to the protein of claim 1, which is any one of the following B1) to B9):

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

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

3. The related biological material according to claim 2, wherein: B1) the nucleic acid molecule is a cDNA molecule or DNA molecule of which the coding sequence is the nucleotides from 1 st to 1062 nd in the sequence 1 in the sequence table.

4. A plant drought-resistant agent and/or plant salt-tolerant agent, wherein the plant drought-resistant agent and/or plant salt-tolerant agent comprises the protein of claim 1, or/and the biological material of claim 2 or 3.

5. The plant drought-resistant agent and/or plant salt-tolerant agent according to claim 4, wherein the active ingredient of the plant drought-resistant agent and/or plant salt-tolerant agent is the protein according to claim 1 or/and the biological material according to claim 2 or 3.

6. The protein of claim 1, or the biomaterial of claim 2 or 3 for use in any one of the following P1-P9:

use of P1, a protein according to claim 1, or a biomaterial according to claim 2 or 3 for modulating drought resistance in a plant;

use of P2, a protein according to claim 1, or a biological material according to claim 2 or 3, for the manufacture of a product for increasing drought resistance in a plant;

use of P3, a protein according to claim 1, or a biological material according to claim 2 or 3 for breeding drought resistant plants;

use of P4, a protein according to claim 1, or a biological material according to claim 2 or 3 for the preparation of a plant drought resistant product;

use of P5, the protein of claim 1, or the biomaterial of claim 2 or 3 for modulating salt tolerance in a plant;

use of P6, a protein according to claim 1, or a biomaterial according to claim 2 or 3 for the manufacture of a product for increasing salt tolerance in plants;

use of P7, the protein of claim 1, or the biomaterial of claim 2 or 3 for growing salt tolerant plants;

use of P8, the protein of claim 1, or the biomaterial of claim 2 or 3 for the preparation of a plant salt-tolerant product;

the plant is arabidopsis thaliana or wheat.

7. A method for cultivating a salt-tolerant and/or drought-resistant plant, comprising increasing the expression level of the protein or its coding gene of claim 1 in a target plant to obtain a salt-tolerant and/or drought-resistant plant; the salt tolerance and/or drought resistance of the salt-tolerant and/or drought-resistant plant is higher than that of the target seed plant, and the target plant is arabidopsis thaliana or wheat.

8. The method of claim 7, wherein: the improvement of the expression level of the protein of claim 1 or a gene encoding the protein in a plant of interest is achieved by introducing the gene encoding the protein of claim 1 into the plant of interest, which is Arabidopsis thaliana or wheat.

9. The drought resistant agent according to claim 4 or 5, or the salt tolerant agent according to claim 4 or 5, wherein: the plant is a monocotyledon or a dicotyledon.

10. The drought resistant agent according to claim 4 or 5, or the salt tolerant agent according to claim 4 or 5, wherein: the plant is arabidopsis thaliana or wheat.