Application of TaCSP-H protein and coding gene thereof in regulating drought resistance and / or yield of wheat

Abstract

The invention belongs to the technical field of biology, and particularly relates to application of TaCSP-H protein and a coding gene thereof to regulation and control of drought resistance and / or yield of wheat. The invention provides application of TaCSP-H protein and a biological material related to the TaCSP-H protein to more than one of the following applications: p1, regulating the drought resistance of wheat; p2, regulating and controlling the wheat yield; p3, cultivating transgenic wheat with improved drought resistance; and p4, cultivating the transgenic wheat with improved yield under the drought condition. It is found that the survival rate of TaCSP-H gene overexpressed wheat strains under the drought condition is remarkably higher than that of wild type wheat, and the yield is remarkably increased. The invention has important significance in molecular breeding for regulating and controlling the drought resistance and yield of wheat.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to the application of TaCSP-H protein and its encoding gene in regulating the drought resistance and / or yield of wheat. Background Art

[0002] Cold shock proteins (CSPs) are a class of nucleic acid-binding proteins whose encoding genes are induced in response to abiotic stresses such as low temperature, drought, and high salinity. They bind to precursor RNAs generated during transcription and unwind their secondary structures, maintaining transcription. CSPs are widely involved in the response of bacteria and plants to abiotic stresses.

[0003] wheat( Triticum aestivum The yield of wheat (L.) is crucial for ensuring food security. Drought stress threatens wheat yields. As plants adapt to terrestrial environments, they have evolved a series of self-protective mechanisms to combat drought stress. Therefore, identifying genes involved in regulating wheat drought stress responses and elucidating their molecular mechanisms is crucial for clarifying the molecular regulatory networks underlying wheat drought responses and developing new high-yield, drought-resistant wheat varieties. Summary of the Invention

[0004] The present invention discovers the application of wheat cold shock protein TaCSP-H in regulating wheat drought resistance and / or yield.

[0005] In a first aspect, the present invention protects a new use of TaCSP-H protein.

[0006] The present invention protects the use of TaCSP-H protein in one or more of the following: p1. Regulate plant drought resistance; p2. Regulate plant yield; p3. Cultivate transgenic plants with improved drought resistance; p4. Cultivate transgenic plants with increased yield; or cultivate transgenic plants with increased yield under drought conditions.

[0007] The TaCSP-H protein is a1, a2, a3 or a4: a1. A protein having an amino acid sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 4; wherein the protein shown in SEQ ID NO: 1 is TaCSP-H3 protein, and the protein shown in SEQ ID NO: 4 is TaCSP-H4 protein.

[0008] a2. A fusion protein obtained by connecting a tag to the N-terminus and / or C-terminus of the protein shown in SEQ ID NO: 1 or SEQ ID NO: 4; a3. A protein related to plant yield and / or drought resistance obtained by substituting and / or deleting and / or adding one or more amino acid residues of the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 4; a4. A protein having the same amino acid sequence as SEQ ID NO: 1 or SEQ ID NO: 4, derived from wheat and associated with plant yield and / or drought resistance.

[0009] Wherein, the plant is wheat. SEQ ID NO: 1 consists of 71 amino acid residues. SEQ ID NO: 4 consists of 70 amino acid residues.

[0010] In the protein described in a2 above, the tag refers to a polypeptide or protein that is fused and expressed with the target protein using in vitro DNA recombination technology to facilitate the expression, detection, tracing, and / or purification of the target protein. The tag can be a Flag tag, GFP tag, His tag, MBP tag, HA tag, myc tag, GST tag, and / or SUMO tag, etc.

[0011] In the protein described in a3 above, the substitution and / or deletion and / or addition of one or several amino acid residues is a substitution and / or deletion and / or addition of no more than 10 amino acid residues.

[0012] In the protein described in a4 above, "identity" includes an amino acid sequence having 80% or higher identity with the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 4 of the present invention.

[0013] The proteins described in a1, a2, a3 or a4 above can be synthesized artificially, or their encoding genes can be synthesized first and then expressed biologically.

[0014] In a second aspect, the present invention protects novel uses of biomaterials related to TaCSP-H proteins.

[0015] The present invention protects the use of biomaterials related to TaCSP-H protein in one or more of the following: p1. Regulate plant drought resistance; p2. Regulate plant yield; p3) Cultivating transgenic plants with improved drought resistance; p4. Cultivate transgenic plants with increased yield; or cultivate transgenic plants with increased yield under drought conditions.

[0016] The biological material is any one of the following A1 to A12: A1, nucleic acid molecule encoding TaCSP-H protein; A2, an expression cassette containing the nucleic acid molecule described in A1; A3, a recombinant vector containing the nucleic acid molecule described in A1; A4, a recombinant vector containing the expression cassette described in A2; A5, a recombinant microorganism containing the nucleic acid molecule described in A1; A6, a recombinant microorganism containing the expression cassette described in A2; A7, a recombinant microorganism containing the recombinant vector described in A3; A8, a recombinant microorganism containing the recombinant vector described in A4; A9, a transgenic plant cell line containing the nucleic acid molecule described in A1; A10, a transgenic plant cell line containing the expression cassette described in A2; A11, a transgenic plant cell line containing the recombinant vector described in A3; A12. A transgenic plant cell line containing the recombinant vector described in A4.

[0017] In the above application, the nucleic acid molecule described in A1 is any one of the following B1, B2, B3 or B4: B1, the cDNA molecule shown in SEQ ID NO: 2 or SEQ ID NO: 5; the cDNA shown in SEQ ID NO: 2 encodes TaCSP-H3 protein, and the cDNA shown in SEQ ID NO: 5 encodes TaCSP-H4 protein; B2, a genomic DNA molecule represented by SEQ ID NO:3 or SEQ ID NO:6; SEQ ID NO:3 is a genomic DNA encoding TaCSP-H3 protein, and SEQ ID NO:6 is a genomic DNA encoding TaCSP-H4 protein; B3, a cDNA molecule or genomic DNA molecule having the same nucleotide sequence as defined in B1 or B2 and encoding the TaCSP-H protein; B4, a cDNA molecule or genomic DNA molecule that hybridizes with the nucleotide sequences defined by B1, B2, and B3 under stringent conditions and encodes the above-mentioned TaCSP-H protein.

[0018] The nucleic acid molecule may be the DNA shown in B1, B2, B3 or B4 above, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule may also be the RNA transcribed from B1, B2, B3 or B4 above, such as mRNA or hnRNA.

[0019] Those skilled in the art can readily mutate the nucleotide sequence encoding the TaCSP-H protein of the present invention using known methods, such as directed evolution and point mutagenesis. Artificially modified nucleotide sequences that share 80% or greater identity with the nucleotide sequence encoding the TaCSP-H protein are derived from and equivalent to the nucleotide sequence of the present invention, as long as they encode the TaCSP-H protein and have the same function.

[0020] As used herein, the term "identity" refers to sequence similarity to a naturally occurring nucleic acid sequence. "Identity" includes nucleotide sequences that are 80% or greater, 85% or greater, 90% or greater, or 95% or greater identical to a nucleotide sequence of the present invention encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 4. Identity can be assessed visually or using computer software. Using computer software, the identity between two or more sequences can be expressed as a percentage (%), which can be used to assess identity between related sequences.

[0021] The aforementioned 80% or greater identity may be 80%, 85%, 90% or 95% or greater identity.

[0022] In the above applications, the stringent conditions are hybridization and washing in a 2×SSC, 0.1% SDS solution at 68°C twice for 5 minutes each, and hybridization and washing in a 0.5×SSC, 0.1% SDS solution at 68°C twice for 15 minutes each; or hybridization and washing in a 0.1×SSPE (or 0.1×SSC), 0.1% SDS solution at 65°C.

[0023] In the above application, the expression cassette containing the nucleic acid molecule encoding TaCSP-H protein described in A2 ( TaCSP-H Gene( TaCSP-H3 Gene or TaCSP-H4 Gene) expression cassette), refers to a DNA capable of expressing TaCSP-H protein in a host cell, which may include not only a promoter TaCSP-H The promoter of gene transcription may also include the terminator TaCSP-HA terminator for gene transcription. Furthermore, 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 from 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)); tomato proteinase inhibitor II promoter (PIN2) or LAP promoter (both induced by methyl jasmonate); heat shock promoters (U.S. Patent No. 5,187,267); tetracycline-inducible promoters (U.S. Patent No. 5,057,422); seed-specific promoters, such as millet seed-specific promoter pF128 (CN101063139B (Chinese Patent No. 200710099169.7)), and promoters specific for seed storage proteins (e.g., the promoters for phaseolin, napin, oleosin, and soybean beta-conglycin (Beachy et al. (1985) EMBO J. 4: 3047-3053)). These 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, the Agrobacterium nopaline synthase terminator (NOS terminator), the cauliflower mosaic virus CaMV 35S terminator, the tml terminator, the pea rbcSE9 terminator, and the nopaline and octopine synthase terminators (see, e.g., Odell et al. (1985) 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).

[0024] Existing expression vectors can be used to construct TaCSP-HRecombinant vectors containing gene expression cassettes. These plant expression vectors include binary Agrobacterium vectors and vectors that can be used for plant microprojectile bombardment. Examples include pAHC25, pBin438, pCAMBIA1302, pCAMBIA2301, pCAMBIA1301, pCAMBIA1300, pBI121, pCAMBIA1391-Xa, or pCAMBIA1391-Xb (CAMBIA). These plant expression vectors may also contain the 3' untranslated region of the foreign gene, i.e., a polyadenylation signal and any other DNA fragments involved in mRNA processing or gene expression. The polyadenylation signal can direct the addition of polyadenylic acid to the 3' end of the mRNA precursor, such as genes in the Agrobacterium crown gall-inducing (Ti) plasmid (e.g., 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 initiation region can come from the transcription initiation 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 that confer resistance to methotrexate dhfr Genes that confer resistance to glyphosate EPSPS Genes (e.g., herbicide resistance genes), or genes that mark chemical resistance (e.g., herbicide resistance genes), or the mannose-6-phosphate isomerase gene that provides the ability to metabolize mannose. For safety reasons, transgenic plants can be screened directly for transformed plants using stress without adding any selectable marker genes.

[0025] In the above applications, the vector may be a plasmid, cosmid, phage or viral vector.

[0026] In the above application, the microorganism may be bacteria, algae or fungi, and the bacteria may be Agrobacterium.

[0027] 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. The method comprises the steps of increasing the content and / or activity of the TaCSP-H protein in a recipient plant to obtain a transgenic plant; the transgenic plant exhibiting greater drought resistance and / or yield than the recipient plant.

[0028] Furthermore, the method for increasing the content and / or activity of TaCSP-H protein in the recipient plant is: overexpressing TaCSP-H protein in the recipient plant; the overexpression method is to introduce a nucleic acid molecule encoding TaCSP-H protein into the recipient plant.

[0029] 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 the yield of a single recipient plant.

[0030] In any of the above applications or methods, the plant is a dicotyledonous plant or a monocotyledonous plant; further, the monocotyledonous plant is a grass plant; further, the grass plant is wheat.

[0031] The present invention has the following beneficial effects: Experiments have shown that the TaCSP-H3 The recombinant vector p110UBI-R1 containing the CDS sequence of the gene was transformed into wheat. The resulting T3 transgenic lines survived significantly longer under drought conditions than wild-type wheat, and their yield increased by an average of about 16%, which was statistically significantly higher than that of wild-type wheat. TaCSP-H4 The recombinant vector p110UBI-R2 containing the gene CDS sequence was used to transform wheat. The resulting T3 transgenic lines survived significantly longer under drought conditions than wild-type wheat, and their yield increased by an average of about 18%, which was statistically significantly higher than that of wild-type wheat.

[0032] The invention has important significance in molecular breeding for regulating wheat drought resistance and yield. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 The p110UBI-R1 vector was introduced into different wheat strains in the embodiment of the present invention. TaCSP-H3 The expression level of the gene (A) and the expression of different wheat lines after the p110UBI-R2 vector was introduced TaCSP-H4 Gene expression level.

[0034] Figure 2 For overexpression in the embodiment of the present invention TaCSP-H3 Phenotypes of wheat lines with different genes before and after drought treatment (A) and overexpression TaCSP-H4Phenotypes of genetically different wheat lines before and after drought treatment (B).

[0035] Figure 3 For overexpression in the embodiment of the present invention TaCSP-H3 Statistics of survival rates of wheat lines with different genes after drought treatment (A) and overexpression TaCSP-H4 Statistics of the survival rates of genetically different wheat lines after drought treatment (B).

[0036] Figure 4 For overexpression in the embodiment of the present invention TaCSP-H3 Yield phenotypes of wheat lines with different genes under normal and drought conditions (A) and overexpression TaCSP-H4 Yield phenotypes of genetically different wheat lines under normal and drought conditions (B).

[0037] Figure 5 For overexpression in the embodiment of the present invention TaCSP-H3 Yield statistics of wheat lines under normal and drought conditions (A) and overexpression of different genes TaCSP-H4 Yield statistics of genetically different wheat lines under normal and drought conditions (B). DETAILED DESCRIPTION

[0038] To make the objectives, technical solutions, and advantages of the present invention more clear, the technical solutions in the embodiments of the present invention will be clearly described below in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0039] The experimental methods in the following examples, unless otherwise specified, are conventional methods. The experimental materials used in the following examples, unless otherwise specified, were purchased from conventional biochemical reagent stores. The quantitative experiments in the following examples were all repeated three times, and the results were averaged.

[0040] The biological material information in the following examples is as follows: The p110UBI-GFP / Flag vector is based on the commercial vector pCAMBIA, which contains the UBI gene promoter conserved in monocots, multiple cloning sites and GFP / Flag protein tags. Pst I and EcoR Two restriction endonucleases are linked between the left and right borders of the vector and it contains kanamycin and rifamycin resistance genes that can be used for resistance selection.

[0041] Agrobacterium tumefaciens EHA105 strain is a commercially available product; 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 Biology14: 327-335.” is a commonly used recipient variety for wheat transgenics in this field.

[0042] Example 1. Obtaining the protein TaCSP-H and its encoding gene 1. Soak wheat Fielder seeds in water at room temperature for 48 hours to allow them to imbibe. Then, place them in a 4°C refrigerator for 24 hours to germinate. Transfer the germinated wheat seeds to nutrient soil and incubate for two weeks. Quickly freeze the whole plant in liquid nitrogen, grind it, extract total RNA, perform reverse transcription, and obtain cDNA.

[0043] 2. Using the cDNA obtained in step 1 as a template, PCR amplification was performed with 5'-ATGGCGCCGAGGCAGGTAGGG-3' as a forward primer and 5'-TTACTCCTCAGTGATGACTTCATTG-3' as a reverse primer to obtain PCR product 1; PCR amplification was performed with 5'-ATGGCGCCGAGGCAGGTAGGG-3' as a forward primer and 5'-TTACTCAGTGATGACTTCATTGGCTTG-3' as a reverse primer to obtain PCR product 2.

[0044] 3. The amplified PCR product 1 was subjected to agarose gel electrophoresis to separate and purify a DNA fragment of approximately 216 bp in length and sequenced. The sequencing results showed that the nucleotide sequence of the DNA fragment was as shown in SEQ ID NO: 2, positions 1-216 in the sequence listing. SEQ ID NO: 2 in the sequence listing is the full-length coding region sequence of the protein TaCSP-H3 shown in SEQ ID NO: 1 in the coding sequence listing of wheat Fielder. The gene encoding the protein TaCSP-H3 was named gene TaCSP-H3 , SEQ ID NO: 3 is TaCSP-H3 gene sequence (including promoter).

[0045] The amplified PCR product 2 was subjected to agarose gel electrophoresis to separate and purify a DNA fragment of approximately 213 bp in length and sequenced. The sequencing results showed that the nucleotide sequence of the DNA fragment was as shown in SEQ ID NO: 5, positions 1-213 in the sequence listing. SEQ ID NO: 5 in the sequence listing is the full-length coding region sequence of the protein TaCSP-H4 shown in SEQ ID NO: 4 in the coding sequence listing of wheat Fielder. The gene encoding the protein TaCSP-H4 was named gene TaCSP-H4 , SEQ ID NO: 6 is TaCSP-H4 gene sequence (including promoter).

[0046] Example 2: Application of TaCSP-H in regulating wheat drought resistance 1. Construction of recombinant overexpression vector p110UBI-R and recombinant Agrobacterium R 1. Construction of p110UBI-R recombinant overexpression vector The DNA fragments shown at positions 1-216 of SEQ ID NO: 2 and the DNA fragments shown at positions 1-213 of SEQ ID NO: 5 in the sequence table were cloned into the vector p110UBI-GFP / Flag. BamH I restriction enzyme cutting site, and then after sequencing and alignment, the recombinant overexpression vectors p110UBI-R1 and p110UBI-R2 were obtained.

[0047] The recombinant overexpression vector p110UBI-R1 is obtained by inserting the DNA fragment shown at positions 1-216 of SEQ ID NO: 2 in the sequence table into the vector p110UBI-GFP / Flag. BamH I restriction enzyme cutting site, and keep the other sequences of the vector p110UBI-GFP / Flag unchanged. The recombinant overexpression vector p110UBI-R2 is obtained by inserting the DNA fragment shown in SEQ ID NO: 5 at positions 1-213 into the vector p110UBI-GFP / Flag. BamH I restriction enzyme cutting site, and keep the other sequences of the vector p110UBI-GFP / Flag unchanged.

[0048] 2. Obtaining p110UBI-R recombinant Agrobacterium tumefaciens The recombinant overexpression vectors p110UBI-R1 and p110UBI-R2 were transformed into Agrobacterium tumefaciens EHA105 strain, and after PCR detection, recombinant Agrobacterium R1 and recombinant Agrobacterium R2 containing the recombinant vectors p110UBI-R1 and p110UBI-R2 were obtained, respectively.

[0049] 2. Overexpression TaCSP-HObtaining transgenic wheat lines Recombinant Agrobacterium R1 and recombinant Agrobacterium R2 were transformed into wild-type wheat Fielder by embryogenic callus infection, and T0 wheat lines and T1 seeds were harvested. After the T1 seeds germinated, resistant seedlings were obtained for subsequent planting and harvesting to obtain T2 seeds. After the T2 seeds germinated, different transgenic lines were randomly selected for RT-qPCR detection to determine overexpression TaCSP-H3 、 TaCSP-H4 Wheat strains. TaCSP-H3 The two transgenic lines with higher gene expression levels were tested for drought resistance and yield and named OE-1 and OE-2, respectively. TaCSP-H4 The two transgenic lines with higher gene expression levels were tested for drought resistance and yield and named OE-1' and OE-2', respectively.

[0050] The T0 generation represents plants of the transformed generation, the T1 generation represents seeds produced by self-pollination of the T0 generation and plants grown therefrom, and the T2 generation represents seeds produced by self-pollination of the T1 generation and plants grown therefrom.

[0051] The specific operation steps of the above-mentioned Agrobacterium-mediated wheat genetic modification are as follows: (1) Obtaining callus induced by mature wheat embryos: Take mature wheat seeds that have been dried, shell them, and place them in a sterile 100 mL triangular flask. Add 70% alcohol to the triangular flask in a clean bench and disinfect the surface for 45 seconds, shaking the triangular flask continuously. Then transfer the seeds to a 2.5% sodium hypochlorite solution, add a drop of Triton X-100, and place the seeds in a 28°C shaker at 200 rpm for 15 minutes. Then transfer the seeds to a 2.5% sodium hypochlorite solution and place them in a 28°C shaker at 200 rpm 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 calli and subculture them on new N6D solid medium. After culturing for 5 days, they can be used for Agrobacterium transformation.

[0052] (2) Cultivation of Agrobacterium: Take the transformed Agrobacterium stored at -80℃ and spread it on YEB solid medium containing the corresponding antibiotics for streak activation. Then, place the Agrobacterium in a 28℃ incubator for 3 days. Pick out the well-growing monoclonal strain and streak it again on YEB solid medium containing the corresponding antibiotics. After culturing at 28℃ for 1 day, scrape the solid strain the size of a match head with a sterile spoon and suspend it in 30 mL AAM liquid medium (final concentration of acetosyringone is 40 mg / L) for transformation.

[0053] (3) Transformation and co-culture of Agrobacterium: Scrape the small-grain callus tissue that grows vigorously after subculture from the N6D solid medium and place it in a 100 mL Erlenmeyer flask. Suspend the callus in AAM liquid medium containing Agrobacterium strains and soak it for 20 minutes. Discard the AAM liquid medium. Place the callus on a culture dish covered with sterile filter paper. After air drying in a clean bench for 30 minutes, place it on a N6D+AAM co-culture medium covered with a layer of filter paper (place the filter paper soaked in AAM liquid medium on the N6D solid medium). Place the callus in a 23°C incubator and culture in the dark for 4 days.

[0054] (4) Removal and screening of Agrobacterium: Scrape the callus tissue after co-cultivation with a sterile medicine spoon into a sterile triangular flask and rinse it several times with sterile distilled water until the water is clear. Add a certain volume of sterile distilled water again and let it stand in the clean bench for 15 minutes. 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 and blow it for 3 hours to dry, then spread the callus on N6D screening medium containing the corresponding antibiotics. Place it in a 30℃ incubator for dark culture and subculture once every two weeks.

[0055] (5) Redifferentiation of resistant calli after infection: The calli after screening and subculture were transferred to hypertonic differentiation medium according to different transgenic lines, placed in a 30℃ light incubator, and cultured under light for 2 weeks until the calli turned green. The green calli were transferred to hypotonic differentiation medium again until shoots grew. The shoots were transferred to rooting medium in triangular flasks according to different clones, placed in a 30℃ light incubator, and cultured under light for 2 weeks to obtain resistant seedlings.

[0056] (6) Hydroponics and transplanting of resistant seedlings: When the resistant seedlings grow to the top of the triangular flask, remove the filter membrane at the top and place the seedlings in the air. At the same time, add sterile distilled water to the triangular flask and acclimate in a light incubator. After the leaves of the seedlings are straight, pull the seedlings out of the triangular flask, wash off the culture medium at the roots of the seedlings, and transplant them into a greenhouse for cultivation and management. After the plants bloom and pollinate under normal greenhouse conditions, harvest the seeds.

[0057] The culture medium formula used in the Agrobacterium-mediated wheat callus transformation process is as follows: 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.

[0058] AAM liquid 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.

[0059] AA-1 100 mL: MnSO4·6H2O, 1 g; H3BO4, 300 mg; ZnSO4·7H2O, 200 mg; KI, 75 mg; NaMoO4·2H2O, 25 mg; CuSO4·5H2O, 2.5 mg; CoCl2·6H2O, 2.5 mg.

[0060] AA-2 100 mL: CaCl2·2H2O, 15 g.

[0061] AA-3 100 mL: MgSO4·7H2O, 25 g.

[0062] AA-4 100 mL: FeSO4·7H2O, 278 mg; Na2EDTA, 373 mg.

[0063] AA-5 100 mL: NaH2PO4·2H2O, 15 g.

[0064] AA-6 100 mL: Niacin, 20 mg; Vitamin B1, 20 mg; Vitamin B6, 20 mg; Inositol, 2 g.

[0065] AA-sol 100 mL: arginine, 176.67 mg; glycine, 75 mg.

[0066] N6D screening medium 1L: sucrose, 30g; NB Basal Medium, 4.1g; Casein, 0.3g; L-Proline, 2.875g; 2,4-D, 0.2g; Gelrite, 4g; pH=5.8; hygromycin 50mg or bifenthionine 2mg; carbobenzyl 200mg; cephalexin 250mg.

[0067] 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.

[0068] 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; ceftriaxone, 250 mg; NAA, 0.3 mg; 6-BA, 3 mg.

[0069] Rooting medium 1L: sucrose, 10g; MS Medium, 2.215g; Gelrite, 4g; pH = 5.8.

[0070] The above-mentioned RT-qPCR method for detecting transgenic wheat is as follows: wheat seedlings of the T2 transgenic line 14 days after germination and wild-type wheat seedlings under the same germination conditions were taken, and total RNA from the wheat leaves was extracted using the Tiangen Plant RNA Miniprep Kit, and DNA was digested with DNase I. After the concentration was measured using a NanoDrop2000 (Thermo Fisher, USA), 2 μg of RNA was taken and cDNA was synthesized using the Invitrogen reverse transcription kit with Oligo d(T) as the primer. TaCSP-H3 Gene-specific quantitative detection primer pair QF1 and QR1 TaCSP-H3 The cDNA of the gene was amplified using Actin The gene was used as an internal reference, and its primers were AF and AR. TaCSP-H3 Gene expression levels such as Figure 1 As shown in A; TaCSP-H4 Gene-specific quantitative detection primer pair QF2 and QR2 TaCSP-H4 The cDNA of the gene was amplified using Actin The gene was used as an internal reference, and its primers were AF and AR. TaCSP-H4 Gene expression levels such as Figure 1 As shown in B.

[0071] The sequences of the above primers are as follows: QF1:5'-GAGGCAGGTAGGGATAGTGA-3'; QR1:5'-TCCTTGAGGGATTTGAAGC-3'; QF2:5'-GGTAGGGATAGTGAAGTCGTTC-3'; QR2:5'-TCCTTGAGGGATTTGAAGC-3'; AF:5'- GACCGTATGAGCAAGGAGAT-3'; AR:5'- CAATCGCTGGACCTGACTC-3'.

[0072] The results showed that compared with the wild type, the overexpression TaCSP-H3 In two transgenic lines (OE-1 and OE-2), TaCSP-H3 The expression of genes was significantly increased ( Figure 1 A). Compared with wild type, overexpression TaCSP-H4 In the two transgenic lines (OE-1' and OE-2') TaCSP-H4 The expression of genes was significantly increased ( Figure 1 B).

[0073] 3. Phenotypic analysis of drought resistance in transgenic wheat Wild-type (Fielder) wheat, T2 generation seeds of OE-1 and OE-2, and T2 generation seeds of OE-1' and OE-2' were germinated and subsequently planted and harvested. TaCSP-H3 Overexpression wheat transgenic lines (OE-1 and OE-2), T3 generation homozygous TaCSP-H4 Seeds of overexpressing wheat transgenic lines (OE-1' and OE-2') and wild-type wheat (Fielder) were germinated and cultured in a greenhouse. After 20 days of growth under normal conditions, watering was stopped and phenotypes and plant survival were analyzed after an additional two weeks of growth.

[0074] The results showed that TaCSP-H3 The growth of the overexpressing wheat transgenic lines (OE-1 and OE-2) under drought conditions was significantly better than that of the wild type (Fielder), as shown by more stretched leaves and less wilting ( Figure 2 A), and the overall survival rate of the plants after drought treatment was significantly higher than that of the wild type ( Figure 3 A). TaCSP-H4 The growth of the overexpressing wheat transgenic lines (OE-1' and OE-2') under drought conditions was significantly better than that of the wild type (Fielder), as shown by more stretched leaves and less wilting ( Figure 2 B), and the overall survival rate of the plants after drought treatment was significantly higher than that of the wild type ( Figure 3 B).

[0075] The above results show that TaCSP-H3 Gene, TaCSP-H4 The gene and its encoded protein have the function of positively regulating wheat drought resistance and are overexpressed in wheat. TaCSP-H3 Gene or TaCSP-H4 Genes can improve the growth status and survival rate of wheat under drought stress conditions.

[0076] 4. Phenotypic analysis of transgenic wheat yield T3 generation homozygous TaCSP-H3 Overexpression wheat transgenic lines (OE-1 and OE-2), T3 generation homozygous TaCSP- H4 Seeds of overexpressing wheat transgenic lines (OE-1' and OE-2') and wild-type wheat (Fielder) were germinated and cultured in a greenhouse. After 30 days of growth under normal conditions, watering was restricted to maintain soil moisture content at around 8%. The seeds were grown to maturity, and mature seeds were selected for seed phenotype observation ( Figure 4 A and Figure 4 B) and statistical analysis of single plant yield.

[0077] The results showed that wild-type wheat Fielder, TaCSP-H3 The yield per plant of two overexpressing wheat transgenic lines (OE-1 and OE-2) was 9.43 g, 9.60 g and 9.76 g respectively under normal conditions, and 4.74 g, 5.56 g and 5.42 g respectively under drought conditions. TaCSP-H3 The yield per plant of the overexpressing wheat transgenic lines (OE-1 and OE-2) increased by 17.3% and 14.3%, respectively, under drought conditions ( Figure 5 A); Description TaCSP-H3 Overexpression of the gene significantly increases the yield per plant of wheat under drought stress conditions. TaCSP-H4 The yield per plant of two overexpressing wheat transgenic lines (OE-1' and OE-2') was 9.43 g, 9.69 g and 10.10 g respectively under normal conditions, and 4.74 g, 5.80 g and 5.42 g respectively under drought conditions. TaCSP-H4 The yield per plant of the overexpressing wheat transgenic lines (OE-1' and OE-2') increased by 22.4% and 14.3%, respectively, under drought conditions ( Figure 5 B); Description TaCSP-H4 Overexpression of the gene can significantly increase the yield per plant of wheat under drought stress conditions.

[0078] The above results show that TaCSP-H3 Gene, TaCSP-H4 The gene and its encoded protein have the function of positively regulating wheat plant yield and are overexpressed in wheat. TaCSP-H3 Gene or TaCSP-H4 Gene that can improve wheat yield under drought conditions.

[0079] 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 various embodiments of the present invention.

Claims

1. Use of TaCSP-H protein or TaCSP-H protein-related biomaterials in one or more of the following applications, characterized in that: p1. Regulate wheat drought resistance; p2. Regulate wheat yield; p3. Cultivate transgenic wheat with improved drought resistance; p4. Cultivate transgenic wheat with increased yield under drought conditions; The TaCSP-H protein is a1 or a2: a1, a protein having an amino acid sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 4; a2. A fusion protein obtained by connecting a tag to the N-terminus and / or C-terminus of the protein shown in SEQ ID NO: 1 or SEQ ID NO: 4; The biological material is any one of the following A1 to A12: A1, nucleic acid molecule encoding TaCSP-H protein; A2, an expression cassette containing the nucleic acid molecule described in A1; A3, a recombinant vector containing the nucleic acid molecule described in A1; A4, a recombinant vector containing the expression cassette described in A2; A5, a recombinant microorganism containing the nucleic acid molecule described in A1; A6, a recombinant microorganism containing the expression cassette described in A2; A7, a recombinant microorganism containing the recombinant vector described in A3; A8, a recombinant microorganism containing the recombinant vector described in A4; A9, a transgenic plant cell line containing the nucleic acid molecule described in A1; A10, a transgenic plant cell line containing the expression cassette described in A2; A11, a transgenic plant cell line containing the recombinant vector described in A3; A12. A transgenic plant cell line containing the recombinant vector described in A4.

2. The use according to claim 1, characterized in that In a2, the tag is a Flag tag, a GFP tag, a His tag, an MBP tag, an HA tag, a myc tag, a GST tag and / or a SUMO tag.

3. The use according to claim 1, characterized in that In A1, the nucleic acid molecule is any one of the following B1, B2 or B3: B1, the cDNA molecule shown in SEQ ID NO: 2 or SEQ ID NO: 5; B2, a genomic DNA molecule represented by SEQ ID NO: 3 or SEQ ID NO: 6; RNA transcribed from B3, B1 or B2.

4. The use according to claim 1, characterized in that The vector is a plasmid, cosmid, phage or viral vector.

5. The use according to claim 1, characterized in that The microorganisms are bacteria, algae or fungi.

6. The use according to claim 5, characterized in that The bacteria is Agrobacterium.

7. A method for cultivating transgenic wheat with improved drought resistance and / or increased yield under drought conditions, characterized in that: The method comprises the following steps: increasing the content and / or activity of TaCSP-H protein in recipient wheat to obtain transgenic wheat; the drought resistance and / or yield of the transgenic wheat are greater than those of the recipient wheat; The TaCSP-H protein is a1 or a2: a1, a protein having an amino acid sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 4; a2. A fusion protein obtained by connecting a tag to the N-terminus and / or C-terminus of the protein shown in SEQ ID NO: 1 or SEQ ID NO:

4.

8. The method according to claim 7, characterized in that The method for increasing the content and / or activity of TaCSP-H protein in the recipient wheat is: overexpressing TaCSP-H protein in the recipient wheat; the overexpression method is to introduce a nucleic acid molecule encoding TaCSP-H protein into the recipient wheat.

9. The method according to claim 7, characterized in that The yield of the transgenic wheat is greater than that of the recipient wheat, which is reflected in that the yield per plant of the transgenic wheat is greater than the yield per plant of the recipient wheat.

10. The method according to claim 8, characterized in that The nucleic acid molecule is any one of the following B1, B2 or B3: B1, the cDNA molecule shown in SEQ ID NO: 2 or SEQ ID NO: 5; B2, a genomic DNA molecule represented by SEQ ID NO: 3 or SEQ ID NO: 6; RNA transcribed from B3, B1 or B2.

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