Application of aegilops tauschii AetDR1 protein and coding gene thereof in regulating drought resistance of wheat

Abstract

The invention belongs to the technical field of biology, and particularly relates to application of aegilops tauschii AetDR1 protein and a coding gene thereof in regulation and control of drought resistance of wheat. The invention provides the application of the aegilops tauschii AetDR1 protein in any one of the following items: (1) regulating the drought resistance of plants; (2) regulating and controlling the sizes of plant seeds; (3) regulating and controlling the plant yield under the drought condition; (4) cultivating a transgenic plant with improved drought resistance; (5) cultivating a transgenic plant with increased yield under a drought condition; the aegilops tauschii AetDR1 protein is a protein with an amino acid sequence as shown in SEQ ID NO: 1. The aegilops tauschii AetDR1 protein and the coding gene thereof have important significance in molecular breeding for regulating drought resistance and yield of wheat.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to application of Aegilops tauschii AetDR1 protein and a coding gene thereof in regulating drought resistance of wheat. Background Art

[0002] wheat( Triticum aestivum L.) is one of the three major staple foods in my country. Drought stress can seriously threaten wheat yield. Wheat is an allohexaploid species (AABBDD, 2 n =6 x =42), the genome is derived from three diploid ancestral species. Advances in modern breeding technology have enabled more adaptable wheat varieties to be widely planted around the world, but at the same time, driven by long-term directional selection for yield and artificial domestication, the genetic diversity of modern cultivated wheat varieties has gradually declined, and many excellent stress resistance genes have been lost. Aegilopstauschii ,DD,2 n = 14) is the donor species of the wheat D subgenome. As a wild weed, the genome of Aegilops tauschii contains rich stress-resistant gene resources. Therefore, exploring the excellent stress-resistant gene resources in Aegilops tauschii and utilizing them in wheat is of great significance for breeding new high-yield and stress-resistant wheat varieties.

[0003] The protein AetDR1 (Aegilops tauschii Drought Related 1) in Aegilops tauschii is a type of RNA binding protein that can maintain transcription by binding to the precursor RNA generated during transcription and opening its secondary structure. Since its discovery, RNA binding proteins have been a hot topic in life science research because these proteins have standardized regulatory functions in plant life activities and play a very important role in gene expression, growth and development, and stress response. Summary of the invention

[0004] The invention proposes the application of Aegilops tauschii AetDR1 protein and its encoding gene in regulating the drought resistance of wheat.

[0005] In a first aspect, the present invention protects new uses of Aegilops tauschii AetDR1 protein.

[0006] The invention provides a method for protecting the Aegilops tauschii AetDR1 protein in any one of the following (1) to (5): (1) Regulating plant drought resistance; (2) Regulating plant seed size; (3) Regulating plant yields under drought conditions; (4) Cultivating transgenic plants with improved drought resistance; (5) Cultivate transgenic plants with improved yield under drought conditions.

[0007] The Aegilops tauschii AetDR1 protein is: (1) The amino acid sequence is the protein shown in SEQ ID NO: 1; (2) A fusion protein obtained by connecting a tag to the N-terminus or / and the C-terminus of the protein shown in SEQ ID NO: 1; (3) A protein related to plant yield and / or grain size obtained by replacing and / or deleting and / or adding one or more amino acid residues of the amino acid sequence shown in SEQ ID NO: 1; (4) A protein having 90% identity with the amino acid sequence shown in SEQ ID NO: 1, which is derived from Aegilops tauschii and is associated with plant yield and / or grain size.

[0008] Wherein, the above SEQ ID NO: 1 consists of 146 amino acid residues.

[0009] In the protein described in (2) above, the tag refers to a polypeptide or protein that is fused and expressed together with the target protein using DNA in vitro recombination technology to facilitate the expression, detection, tracing and / or purification of the target protein. The tag may be a Flag tag, a GFP tag, a His tag, an MBP tag, a HA tag, a myc tag, a GST tag and / or a SUMO tag, etc.

[0010] In the protein described in (3) above, the substitution and / or deletion and / or addition of one or more amino acid residues is a substitution and / or deletion and / or addition of no more than 10 amino acid residues.

[0011] In the protein described in (4) above, "identity" includes an amino acid sequence having 80% or more of the amino acid sequence shown in SEQ ID NO: 1 of the present invention.

[0012] The proteins described in (1) or (2) or (3) or (4) above can be synthesized artificially, or their encoding genes can be synthesized first and then expressed biologically.

[0013] In a second aspect, the present invention provides novel uses of biological materials related to Aegilops tauschii AetDR1 protein.

[0014] The present invention protects the use of biological materials related to Aegilops tauschii AetDR1 protein in any of the following (1) to (5): (1) Regulating plant drought resistance; (2) Regulating plant seed size; (3) Regulating plant yields under drought conditions; (4) Cultivating transgenic plants with improved drought resistance; (5) Cultivating transgenic plants with increased yield under drought conditions; The biological material is any one of the following (1) to (12): (1) a nucleic acid molecule encoding AetDR1 protein from Aegilops tauschii; (2) an expression cassette containing the nucleic acid molecule described in (1); (3) a recombinant vector containing the nucleic acid molecule described in (1); (4) a recombinant vector containing the expression cassette described in (2); (5) A recombinant microorganism containing the nucleic acid molecule described in (1); (6) A recombinant microorganism containing the expression cassette described in (2); (7) A microorganism containing the recombinant vector described in (3); (8) A microorganism containing the recombinant vector described in (4); (9) A transgenic plant cell line containing the nucleic acid molecule described in (1); (10) A transgenic plant cell line containing the expression cassette described in (2); (11) A transgenic plant cell line containing the recombinant vector described in (3); (12) A transgenic plant cell line containing the recombinant vector described in (4).

[0015] In the above application, the nucleic acid molecule is a gene as shown in (1) or (2) or (3) or (4) below: (1) the genomic DNA molecule shown in SEQ ID NO: 3; (2) the cDNA molecule shown in SEQ ID NO: 2; (3) a cDNA molecule or genomic DNA molecule that has 90% or more identity with the nucleotide sequence defined in (1) or (2) and encodes the Aegilops tauschii AetDR1 protein; (4) A cDNA molecule or genomic DNA molecule that hybridizes with the nucleotide sequence defined in (1) or (2) or (3) under stringent conditions and encodes the above-mentioned Aegilops tauschii AetDR1 protein.

[0016] 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.

[0017] A person skilled in the art can easily mutate the nucleotide sequence encoding the Aegilops tauschii AetDR1 protein of the present invention by using known methods, such as directed evolution and point mutation. Those artificially modified nucleotides having 80% or higher identity with the nucleotide sequence encoding the Aegilops tauschii AetDR1 protein, as long as they encode the Aegilops tauschii AetDR1 protein and have the same function, are derived from the nucleotide sequence of the present invention and are equivalent to the sequence of the present invention.

[0018] The term "identity" as used herein refers to sequence similarity to a natural nucleic acid sequence. "Identity" includes nucleotide sequences that have 80% or more, or 85% or more, or 90% or more, or 95% or more identity to the nucleotide sequence of the present invention encoding the protein consisting of the amino acid sequence shown in SEQ ID NO:1. Identity can be evaluated by the naked eye or by computer software. Using computer software, the identity between two or more sequences can be expressed as a percentage (%), which can be used to evaluate the identity between related sequences.

[0019] The above-mentioned 80% or more identity may be 80%, 85%, 90% or 95% or more identity.

[0020] In the above application, the stringent conditions are hybridization and washing at 68°C in a 2×SSC, 0.1% SDS solution for 5 min each time, and hybridization and washing at 68°C for 15 min each time in a 0.5×SSC, 0.1% SDS solution for 15 min each time; or hybridization and washing at 65°C in a 0.1×SSPE (or 0.1×SSC), 0.1% SDS solution.

[0021] In the above application, the expression cassette containing the nucleic acid molecule encoding the Aegilops tauschii AetDR1 protein ( AetDR1 Gene expression cassette refers to a gene that can be expressed in host cells AetDR1 The DNA of the gene may include not only the promoter AetDR1 The promoter of gene transcription may also include the terminator AetDR1The terminator of gene transcription. Further, the expression cassette may also include an enhancer sequence. Promoters that can be used 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 35S of cauliflower mosaic virus; a wound-inducible promoter from tomato, leucine aminopeptidase ("LAP", Chao et al. (1999) Plant Physiol 120:979-992); a chemically inducible promoter from tobacco, pathogenesis-related 1 (PR1) (induced by salicylic acid and BTH (benzothiadiazole-7-thiocarboxylic acid S-methyl ester)); a tomato proteinase inhibitor II promoter (PIN2) or a LAP promoter (both inducible by methyl jasmonate); a heat shock promoter (U.S. Pat. No. 5,187,267); a tetracycline-inducible promoter (U.S. Pat. No. 5,057,422); a seed-specific promoter, such as millet seed-specific promoter pF128 (CN101063139B (Chinese Patent No. 200710099169.7)), a promoter specific for seed storage proteins (e.g., phaseolin, napin, oleosin and soybean beta conglycin promoters (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 cited in their entirety. Suitable transcription terminators include, but are not limited to, the Agrobacterium nopaline synthase terminator (NOS terminator), the cauliflower mosaic virus CaMV 35S terminator, the tml terminator, the pea rbcS E9 terminator, and the nopaline and octopine synthase terminators (see, for example, Odell et al. (1996) EMBO J. 4: 3047-3053). 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).

[0022] Existing expression vectors can be used to construct AetDR1Recombinant vector of gene expression cassette. The plant expression vector includes binary Agrobacterium vector and vector that can be used for plant microprojectile bombardment, etc. Such as pAHC25, pBin438, pCAMBIA1302, pCAMBIA2301, pCAMBIA1301, pCAMBIA1300, pBI121, pCAMBIA1391-Xa or pCAMBIA1391-Xb (CAMBIA Company), etc. The plant expression vector may also contain the 3' non-translated region of the foreign gene, i.e., a polyadenylic acid signal and any other DNA fragment involved in mRNA processing or gene expression. The polyadenylic acid signal can guide the addition of polyadenylic acid to the 3' end of the mRNA precursor, such as the Agrobacterium crown gall induction (Ti) plasmid gene (such as the nopaline synthase gene Nos ), the non-translated regions transcribed at the 3' end of plant genes (such as soybean storage protein genes) all have similar functions. When using the gene of the present invention to construct a plant expression vector, enhancers can also be used, including translation enhancers or transcription enhancers. These enhancer regions can be ATG start codons or adjacent region start codons, etc., but must be the same as the reading frame of the coding sequence to ensure the correct translation of the entire sequence. The sources of the translation control signals and start codons are wide-ranging and can be natural or synthetic. The translation start region can come from the transcription start region or the structural gene. In order to facilitate the identification and screening of transgenic plant cells or plants, the plant expression vector used can be processed, such as adding genes that can be expressed in plants and encode enzymes or luminescent compounds that can produce color changes ( GUS genes, luciferase genes, etc.), antibiotic marker genes (such as those that confer resistance to kanamycin and related antibiotics nptII Gene that confers resistance to the herbicide phosphinothricin bar Gene that confers resistance to the antibiotic hygromycin hph genes, and those conferring resistance to methotrexate dhfr Genes, such as EPSPS genes that confer resistance to glyphosate) or chemical resistance marker genes (such as herbicide resistance genes), mannose-6-phosphate isomerase genes that provide the ability to metabolize mannose. Considering the safety of transgenic plants, no selective marker genes can be added, and transformed plants can be directly screened by adversity.

[0023] In the above applications, the vector may be a plasmid, a cosmid, a phage or a viral vector.

[0024] In the above applications, the microorganism may be yeast, bacteria, algae or fungi, such as Agrobacterium.

[0025] In a third aspect, the present invention provides a method for cultivating transgenic plants with improved drought resistance and / or increased yield under drought conditions.

[0026] The method for cultivating transgenic plants with improved drought resistance and / or increased yield under drought conditions provided by the present invention comprises the following steps: increasing the content and / or activity of AetDR1 protein in a recipient plant to obtain a transgenic plant; the drought resistance and / or yield of the transgenic plant are greater than those of the recipient plant.

[0027] Furthermore, the method for increasing the content and / or activity of AetDR1 protein in the recipient plant is: overexpressing AetDR1 protein in the recipient plant; the overexpression method is introducing a nucleic acid molecule encoding AetDR1 protein into the recipient plant.

[0028] The yield of the transgenic plant is greater than that of the recipient plant, which is specifically embodied in that: the yield of a single transgenic plant is greater than that of the recipient plant; Furthermore, the nucleic acid molecule encoding the AetDR1 protein is a cDNA molecule shown in SEQ ID NO: 2 or a DNA molecule shown in SEQ ID NO: 3 in the sequence listing.

[0029] In any of the above applications or methods, the plant is a dicotyledon or a monocotyledon; further, the monocotyledon is a grass plant; further, the grass plant is wheat. In a specific embodiment of the present invention, the wheat variety is Fielder.

[0030] The beneficial effects of the present invention are: The present invention found that the sequence shown in SEQ ID NO: 2 in the sequence list AetDR1 The recombinant vector p110UBI-DR1 carrying the CDS sequence of the gene was transformed into wheat, and the resulting T 3 The survival rate of the transgenic lines under drought conditions was significantly higher than that of wild-type wheat, and the yield increased by about 18%, which was statistically significantly higher than that of wild-type wheat. Therefore, the AetDR1 protein of Aegilops tauschii and its encoding gene are of great significance in molecular breeding for regulating wheat drought resistance and yield. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 The p110UBI-DR1 vector was introduced into different strains of wheat. AetDR1 The expression level of the gene.

[0032] Figure 2 For the overexpression of the present invention AetDR1 Phenotypes of genetically different wheat lines before and after drought treatment.

[0033] Figure 3 For the overexpression of the present invention AetDR1 Statistics of the survival rates of genetically different wheat lines after drought treatment.

[0034] Figure 4 For the overexpression of the present invention AetDR1 Seed size phenotypes under normal and drought conditions in genetically distinct wheat lines.

[0035] Figure 5 For the overexpression of the present invention AetDR1 Statistics of seed length (A) and width (B) of genetically different wheat lines under normal and drought conditions.

[0036] Figure 6 For the overexpression of the present invention AetDR1 Yield statistics of genetically different wheat lines under normal and drought conditions. DETAILED DESCRIPTION

[0037] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution in the embodiment of the present invention will be clearly described below in conjunction with the drawings in the embodiment of the present invention. Obviously, the described embodiment is a part of the embodiment of the present invention, not all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0038] The test materials used in the following examples, unless otherwise specified, were purchased from conventional biochemical reagent stores. The quantitative tests in the following examples were repeated three times, and the results were averaged.

[0039] The biological material information in the following examples is as follows: The p110UBI-GFP / Flag vector was independently modified by the applicant. The vector is based on the commercial vector pCAMBIA and contains the conservative gene in monocotyledons. UBI Gene promoter, multiple cloning site and GFP / Flag protein tag are connected between the left and right borders of the vector by two restriction endonucleases, PstI and EcoRI. The vector also contains kanamycin and rifamycin resistance genes that can be used for resistance screening. The public can obtain it from the School of Life Sciences of Henan University.

[0040] Agrobacterium tumefaciens EHA105 strain is described in the literature “Qu LQ, Xing YP, Liu WX, Xu XP, Song YR. (2008) Expression pattern and activity of six glutelin gene promoters intransgenic rice. Journal of Experimental Botany 59: 2417–2424.” and is available to the public from the School of Life Sciences, Henan University; The wild type wheat material is the variety "Fielder" ( Triticum aestivum L. cv. Fielder) is recorded in the literature "Xing, HL et al. (2014) A CRISPR / Cas9 toolkit for multiplex genome editing in plants. BMC Plant Biology 14: 327-335.” Available to the public from the College of Life Sciences, Henan University.

[0041] Example 1. Obtaining protein AetDR1 and its encoding gene 1. Take wheat Fielder seeds, soak them in water at room temperature for 48 hours, and then place them in a 4℃ refrigerator for germination for 24 hours. Transfer the germinated wheat seeds to nutrient soil and culture them for two weeks. Quickly freeze the whole plant in liquid nitrogen, grind it to extract total RNA, perform reverse transcription, and obtain cDNA.

[0042] 2. Using the cDNA obtained in step 1 as a template, PCR amplification was performed using 5'-CGACTCTAGAGGATCCCCGGGATGCAGATTACTGGT-3' as a forward primer and 5'-GGTCTTTGTAGTCCATTAATTTGCTCGAAA-3' as a reverse primer to obtain a PCR product.

[0043] 3. The amplified PCR product was subjected to agarose gel electrophoresis to separate and purify a DNA fragment of about 450 bp and sequence it. The sequencing results showed that the nucleotide sequence of the DNA fragment was as shown in SEQ ID NO: 2, positions 1 to 441.

[0044] SEQ ID NO:2 is the full-length coding region sequence of the protein AetDR1 in wheat Fielder encoding SEQ ID NO:1. The gene encoding the protein AetDR1 is named gene AetDR1 .

[0045] Example 2: Application of AetDR1 in regulating drought resistance of wheat 1. Construction of recombinant overexpression vector p110UBI-DR1 and recombinant Agrobacterium R1 1. Construction of p110UBI-DR1 recombinant overexpression vector The DNA fragment shown in positions 1 to 441 of SEQ ID NO: 2 was cloned into the vector p110UBI-GFP / Flag Bm I restriction site, and then after sequencing and alignment, the recombinant overexpression vector p110UBI-DR1 was obtained.

[0046] The recombinant overexpression vector p110UBI-DR1 is obtained by inserting the DNA fragment shown in positions 1 to 441 of SEQ ID NO: 2 into the vector p110UBI-GFP / Flag. Bm I restriction site and keeping other sequences of the vector p110UBI-GFP / Flag unchanged.

[0047] 2. Obtaining p110UBI-DR1 recombinant Agrobacterium tumefaciens The recombinant overexpression vector p110UBI-DR1 was transformed into the Agrobacterium tumefaciens EHA105 strain, and after PCR detection, the recombinant Agrobacterium DR1 containing the recombinant vector p110UBI-DR1 was obtained.

[0048] 2. Overexpression AetDR1 Obtaining transgenic wheat lines The recombinant Agrobacterium tumefaciens DR1 was transformed into wild-type wheat Fielder by embryogenic callus infection, and T 0 Generation of wheat lines and harvesting of T 1 Generation of seeds; 1 After the first generation of seeds germinate, the resistant seedlings are obtained for subsequent planting and harvesting to obtain T 2 Generation of seeds; 2 After the seeds of the first generation germinated, different transgenic lines were randomly selected for qRT-PCR detection to determine the overexpression AetDR1 Select wheat lines. AetDR1 The two transgenic lines with higher gene expression levels were tested for drought resistance and yield and were named OE-4 and OE-13, respectively.

[0049] T 0 Generation refers to the plants of the current generation, T 1 Representative T 0 The seeds produced by self-pollination and the plants grown from them, T 2 Representative by T 1 The seeds produced by self-pollination and the plants grown from them.

[0050] The specific operation steps of the above-mentioned Agrobacterium-mediated wheat transgenic process are as follows: (1) Obtaining callus induced by mature wheat embryos: Take mature wheat seeds and dry them, remove the shells and place them in a sterile 100 mL Erlenmeyer flask. Add 70% alcohol in a clean bench and disinfect the surface for 45 seconds, shaking the flask continuously. Then transfer the seeds to a 2.5% sodium hypochlorite solution, add a drop of Triton X-100, place the seeds in a 28°C shaker, and shake at 200 rpm for 15 minutes. Then transfer the seeds to a 2.5% sodium hypochlorite solution and shake at 200 rpm in a 28°C shaker for 15 minutes. After discarding the disinfectant, rinse repeatedly with sterile distilled water until the rinsed solution is clear and free of foreign matter. Pour the seeds out and place them on a culture dish covered with sterile filter paper, and dry them in a clean bench for 45 minutes. Place the seeds on N6D solid culture medium in a 28°C incubator and culture for 4 weeks. Pick the tender yellow and smooth embryonic callus and subculture it on new N6D solid medium. After culturing for 5 days, it can be used for Agrobacterium transformation.

[0051] (2) Cultivation of Agrobacterium: Take the transformed Agrobacterium stored at -80℃, spread it on YEB solid medium containing the corresponding antibiotics for streak activation, and then culture the Agrobacterium in a 28℃ incubator for 3 days. Pick the monoclonal strain with good growth and streak it again on YEB solid medium containing the corresponding antibiotics. After culturing at 28℃ for 1 day, use a sterile medicine spoon to scrape the solid strain the size of a match head and suspend it in 30 mL AAM (the final concentration of acetosyringone is 40 mg / L) liquid medium for transformation.

[0052] (3) Transformation and co-culture of Agrobacterium: Scrape the small-grain callus tissue that grows vigorously after subculture from the N6D medium and place it in a 100 mL Erlenmeyer flask. Suspend the callus in AAM medium suspended with Agrobacterium strains, soak for 20 min, and then discard the AAM medium. Place the callus on a culture dish covered with sterile filter paper, blow dry it in a clean bench for 30 min, and then place it on N6D+AAM co-culture medium covered with a layer of filter paper. Place the callus in a 23°C incubator and culture it in the dark for 4 days.

[0053] (4) Removal and screening of Agrobacterium: Scrape the callus tissue after co-cultivation into a sterile conical flask with a sterile medicine spoon and rinse it several times with sterile distilled water until the water is clear. Add a certain volume of sterile distilled water again, let it stand in the clean bench for 15 minutes, and then soak it twice with sterile distilled water containing a final concentration of 400 mg / L carbenicillin, each time for 15 minutes. Pour the soaked callus tissue into a culture dish covered with sterile filter paper, place it in the clean bench for 3 hours to blow and dry, and then spread the callus on N+ culture medium containing the corresponding antibiotics. Place it in a 30℃ incubator for dark culture, subculture once every two weeks, and subculture twice.

[0054] (5) Redifferentiation of resistant calli after infection: The callus tissues after screening and subculture were transferred to hyperosmotic medium according to different transgenic strains, placed in a 30℃ light incubator, and cultured under light for 2 weeks until the callus turned green. The green callus was transferred to hypotonic medium again until tender shoots grew. The tender shoots were transferred to rooting medium in triangular bottles according to different clones, placed in a 30℃ light incubator, and cultured under light for 2 weeks to obtain resistant seedlings.

[0055] (6) Hydroponics and transplanting of resistant seedlings: When the resistant seedlings grow to the top of the triangular bottle, remove the filter membrane on the top and place the seedlings in the air. At the same time, add sterile distilled water to the triangular bottle and acclimate it in a light incubator. After the leaves of the seedlings are straight, pull the seedlings out of the triangular bottle, wash off the culture medium at the roots of the seedlings, and transplant them into the greenhouse for cultivation and management. After the plants bloom and pollinate under normal greenhouse conditions, harvest the seeds.

[0056] The medium formula used in the process of Agrobacterium-mediated wheat callus transformation is: N6D solid culture medium 1L: sucrose, 30 g; NB Basal Medium, 4.1 g; Casein, 0.3 g; L-Proline, 2.875 g; 2,4-D, 0.2 g; Gelrite, 4 g; pH=5.8.

[0057] AAM Agrobacterium activation medium 1 L: AA-1, 1 mL; AA-2, 1 mL; AA-3, 1 mL; AA-4, 10 mL; AA-5, 1 mL; AA-6, 5 mL; AA-Sol, 10 mL; Casein, 0.5 g; glucose, 36 g; sucrose, 68.5 g; aspartic acid, 0.3 g; L-glutamine, 0.9 g; inositol, 0.1 g; KCl, 3 g; acetosyringone, 40 mg; pH = 5.2.

[0058] AA-1 100 mL: MnSO 4 6H 2 O, 1 g; H 3 BO 4 , 300 mg; ZnSO 4 7H 2 O, 200 mg; KI, 75 mg; NaMoO 4 ·2H 2 O, 25 mg; CuSO 4 ·5H 2 O, 2.5 mg; CoCl 2 6H 2 O, 2.5 mg.

[0059] AA-2 100 mL: CaCl 2 ·2H 2 O, 15 g.

[0060] AA-3 100 mL: MgSO 4 7H 2 O, 25 g.

[0061] AA-4 100 mL: FeSO 4 7H 2 O, 278 mg; Na 2 EDTA, 373 mg.

[0062] AA-5 100 mL: NaH 2 PO4·2H 2 O, 15 g.

[0063] AA-6 100 mL: niacin, 20 mg; vitamin B1, 20 mg; vitamin B6, 20 mg; inositol, 2 g.

[0064] AA-sol 100 mL: Arginine, 176.67 mg; Glycine, 75 mg.

[0065] N6D screening medium 1L: sucrose, 30g; NB Basal Medium, 4.1g; Casein, 0.3g; L-Proline, 2.875g; 2,4-D, 0.2 g; Gelrite, 4g; pH=5.8; hygromycin 50 mg or bipropylamine 2mg; carbobenzyl 200mg; cephalexin 250mg.

[0066] Hypertonic differentiation medium 1 L: sucrose, 30 g; sorbitol, 30 g; MS Medium, 4.43 g; Casein, 0.5 g; Gelrite, 4 g; pH = 5.8; hygromycin, 50 mg or bialaphos, 2 mg; carbobenzyl, 200 mg; cephalexin, 250 mg; NAA, 0.3 mg; 6-BA, 3 mg.

[0067] Hypotonic differentiation medium 1 L: sucrose, 30 g; MS Medium, 4.43 g; Casein, 0.5 g; Gelrite, 4 g; pH = 5.8; hygromycin, 50 mg or bialaphos, 2 mg; carbobenzyl, 200 mg; cephalexin, 250 mg; NAA, 0.3 mg; 6-BA, 3 mg.

[0068] Rooting medium 1L: sucrose, 10g; MS Medium, 2.215g; Gelrite, 4g; pH=5.8.

[0069] The above qRT-PCR method for detecting transgenic wheat is as follows: Take T 2 Total RNA from wheat leaves of transgenic wheat seedlings 14 days after germination and wild-type wheat seedlings under the same germination conditions was extracted using the Tiangen Plant RNA Miniprep Kit, and DNA was digested with DNase I. After the concentration was measured by NanoDrop2000 (Thermo Fisher, USA), 2 μg was taken and cDNA was synthesized using the Invitrogen reverse transcription kit with Oligo d(T) as the primer. AetDR1 Gene-specific quantitative detection primer pair QF and QR AetDR1 The cDNA of the gene was amplified and the Actin The gene was used as an internal reference, and its primers were AF and AR. After quantitative analysis, the different transgenic wheat lines AetDR1 Gene expression levels Figure 1 As shown: The sequences of the above primers are as follows: QF: 5'- GAGAGAAAATGCAGATTACTGG-3'; QR: 5'- CCTTTTTGGCCGTCTTGAATTTC-3'; AF: 5'- GACCGTATGAGCAAGGAGAT-3'; AR: 5'- CAATCGCTGGACCTGACTC-3'.

[0070] The results showed that compared with the wild type, the overexpression AetDR1 In two transgenic lines (OE-4 and OE-13), AetDR1 The gene expression level increased significantly ( Figure 1 ).

[0071] 3. Phenotypic analysis of drought resistance in transgenic wheat Take T 3 Homozygous AetDR1 The seeds of overexpressing wheat transgenic lines (OE-4 and OE-13) and wild-type wheat (Fielder) were cultured in a greenhouse after germination. After 20 days of growth under normal conditions, watering was stopped and drought treatment was started. After another 2 weeks of growth, the phenotype and plant survival rate were counted.

[0072] The results show that AetDR1The growth of the overexpressing wheat transgenic lines (OE-4 and OE-13) under drought conditions was significantly better than that of the wild type (Fielder), as shown by more stretched leaves and less wilting ( Figure 2 ), and the overall survival rate of the plants after drought treatment was significantly higher than that of the wild type ( Figure 3 The above results show that AetDR1 The gene and its encoded protein have the function of positively regulating wheat drought resistance and are overexpressed in wheat AetDR1 Genes can improve the growth status and increase the survival rate of wheat under drought stress conditions.

[0073] 4. Phenotypic analysis of transgenic wheat seeds and yield per plant Take T 3 Homozygous AetDR1 The seeds of the overexpressing wheat transgenic lines (OE-4 and OE-13) and wild-type wheat (Fielder) were cultured in a greenhouse after germination. After 30 days of growth under normal conditions, watering was restricted to maintain the soil moisture content at about 8%. The seeds continued to grow until maturity, and the mature seeds were selected for statistical analysis of seed size and other phenotypes.

[0074] The results showed that wild-type wheat Fielder, AetDR1 The average seed lengths of the overexpression wheat transgenic lines OE-4 and OE-13 under normal conditions were 0.6340 cm, 0.6274 cm, and 0.6263 cm, respectively, and the average seed lengths under drought conditions were 0.5980 cm, 0.6320 cm, and 0.6430 cm, respectively. Figure 4 Compared with the wild type, the two AetDR1 The average seed length of the overexpressing wheat transgenic lines (OE-4 and OE-13) increased by 5.69% and 7.53%, respectively, under drought conditions ( Figure 5 A); wild-type wheat Fielder, AetDR1 The average seed widths of the two overexpressing wheat transgenic lines (OE-4 and OE-13) under normal conditions were 0.3158 cm, 0.3246 cm, and 0.3201 cm, respectively, and the average seed widths under drought conditions were 0.2759 cm, 0.2952 cm, and 0.3000 cm, respectively. AetDR1 The average seed width of the overexpressing wheat transgenic lines (OE-4 and OE-13) increased by 7.00% and 8.74%, respectively, under drought conditions ( Figure 5 B).

[0075] At the same time, wild-type wheat Fielder, AetDR1The yield per plant of the overexpression wheat transgenic lines OE-4 and OE-13 under normal conditions was 8.71 g, 9.51 g, and 10.47 g, respectively, and the yield per plant under drought conditions was 3.33 g, 4.21 g, and 3.68 g, respectively. AetDR1 The yield per plant of the overexpressing wheat transgenic lines (OE-4 and OE-13) increased by 26.4% and 10.5% respectively under drought conditions ( Figure 6 ). AetDR1 Overexpression of the gene significantly increased the yield per plant of wheat under drought stress. AetDR1 The gene and its encoded protein have the function of positively regulating wheat seed size and single plant yield, and are overexpressed in wheat AetDR1 Gene that could improve wheat yield under drought conditions.

[0076] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. Use of Aegilops tauschii AetDR1 protein in any of the following (1) to (5), It is characterized in that (1) Regulating plant drought resistance; (2) Regulating plant seed size; (3) Regulating plant yields under drought conditions; (4) Cultivating transgenic plants with improved drought resistance; (5) Cultivating transgenic plants with increased yield under drought conditions; The Aegilops tauschii AetDR1 protein is a protein whose amino acid sequence is shown in SEQ ID NO:

1.

2. Use of biological materials related to Aegilops tauschii AetDR1 protein in any of the following (1) to (5), It is characterized in that (1) Regulating plant drought resistance; (2) Regulating plant seed size; (3) Regulating plant yields under drought conditions; (4) Cultivating transgenic plants with improved drought resistance; (5) Cultivating transgenic plants with increased yield under drought conditions; The biological material is any one of the following (1) to (12): (1) a nucleic acid molecule encoding AetDR1 protein from Aegilops tauschii; (2) an expression cassette containing the nucleic acid molecule described in (1); (3) a recombinant vector containing the nucleic acid molecule described in (1); (4) a recombinant vector containing the expression cassette described in (2); (5) A recombinant microorganism containing the nucleic acid molecule described in (1); (6) A recombinant microorganism containing the expression cassette described in (2); (7) A microorganism containing the recombinant vector described in (3); (8) A microorganism containing the recombinant vector described in (4); (9) A transgenic plant cell line containing the nucleic acid molecule described in (1); (10) A transgenic plant cell line containing the expression cassette described in (2); (11) A transgenic plant cell line containing the recombinant vector described in (3); (12) A transgenic plant cell line containing the recombinant vector described in (4).

3. The use according to claim 2, It is characterized in that The nucleic acid molecule is: (1) the cDNA molecule shown in SEQ ID NO: 2; (2) The genomic DNA molecule shown in SEQ ID NO:

3.

4. The use according to claim 2, It is characterized in that The vector is a plasmid, cosmid, phage or virus vector.

5. The use according to claim 2, It is characterized in that The microorganism is yeast, bacteria, algae or fungi.

6. A method for cultivating transgenic plants with improved drought resistance and / or increased yield under drought conditions, It is characterized in that The steps include: The content and / or activity of Aegilops tauschii AetDR1 protein in a recipient plant is increased to obtain a transgenic plant; the drought resistance and / or yield of the transgenic plant are greater than those of the recipient plant.

7. The method according to claim 6, It is characterized in that The method for increasing the content and / or activity of Aegilops tauschii AetDR1 protein in a recipient plant is: The Aegilops tauschii AetDR1 protein is overexpressed in a recipient plant; the overexpression method is to introduce a nucleic acid molecule encoding the Aegilops tauschii AetDR1 protein into the recipient plant.

8. The method according to claim 6, It is characterized in that The yield of the transgenic plant is greater than that of the recipient plant, which is specifically embodied in that the yield of a single transgenic plant is greater than that of the recipient plant.

9. The method according to claim 6, It is characterized in that The nucleic acid molecule encoding the Aegilops tauschii AetDR1 protein is a cDNA molecule shown in SEQ ID NO:2 or a DNA molecule shown in SEQ ID NO:

3.

10. The method according to claim 6, It is characterized in that The plant is wheat.

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