Application of TaWDR3 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 TaWDR3 protein and a coding gene thereof in regulating drought resistance of wheat. According to the application, a recombinant vector p110UBI-R of a TaWDR3 gene CDS sequence is constructed and a wheat plant is transformed, so that the survival rate of a T3-generation wheat transgenic line obtained through TaWDR3 overexpression under a drought condition is obviously higher than that of wild type wheat; the yield of a single plant is averagely increased by about 18%, and statistically, the TaWDR3 protein and the coding gene thereof have the function of positively regulating the drought resistance of the wheat, the TaWDR3 gene is over-expressed in the wheat, the growth state of the wheat under the drought stress condition can be improved, and the survival rate can be increased; the method has important practical significance for culturing new varieties of drought-resistant crops.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a TaWDR3 protein for regulating wheat drought resistance. Background Art

[0002] In recent years, the development of molecular breeding technology has provided a new approach for improving wheat stress resistance. Genetic engineering breeding can overcome the disadvantages of traditional breeding, with a short cycle, and can break the boundaries of species, making limited genetic resources tend to be infinite. By introducing or regulating drought-related genes through genetic engineering means, the adaptability of crops to drought can be significantly improved. The drought resistance of plants is a quantitative trait controlled by multiple genes. Compared with other important crops such as rice and corn, wheat has a late start and a slow speed in gene breeding due to many problems such as a large genome and poor regeneration ability. Drought tolerance is a complex trait regulated by multiple genes, involving the synergistic effects of morphological structure, physiological metabolism, and molecular regulatory networks. Among them, the excavation and functional analysis of drought-related proteins and their encoding genes are the core links to reveal the drought resistance mechanism of wheat and are also important targets for molecular breeding. Patent 202411461093.8 discloses the application of an Aegilops tauschii AetDR1 protein and its encoding gene in regulating wheat drought resistance. The Aegilops tauschii AetDR1 protein plays a significant role in regulating plant drought resistance, plant seed size, plant yield, and cultivating transgenic plants with improved drought resistance, providing an effective method for cultivating high-yield and stress-resistant wheat varieties. Currently, the genes discovered related to drought response far from meet the requirements for systematically analyzing the drought resistance mechanism of wheat. There is an urgent need to find drought-resistant genes through various new technologies and means, and conduct functional verification and genetic analysis. The excavation and screening of effective stress-resistant genes have important practical significance for cultivating new varieties of drought-resistant crops. Summary of the Invention

[0003] To solve the above problems, the present invention proposes the application of a TaWDR3 protein and its encoding gene in regulating wheat drought resistance.

[0004] The technical solution of the present invention is realized as follows:

[0005] On the one hand, the present application provides a TaWDR3 protein, and its amino acid sequence is shown as any one of the following:

[0006] (1) The amino acid sequence is shown as SEQ ID No.1;

[0007] (2) A sequence in which one or several amino acids are inserted, deleted, or mutated in the amino acid sequence shown as SEQ ID No.1 and still has the function of the amino acids in (1);

[0008] (3) having an identity of more than 99%, more than 95%, more than 90%, more than 85% or more than 80% with the amino acid sequence shown in SEQ ID No. 1;

[0009] (4) a fusion protein obtained by linking a tag to the N-terminus and / or C-terminus of the protein of the amino acid sequence shown in SEQ ID No. 1.

[0010] Preferably, the sequence shown in SEQ ID No. 1 above consists of 70 amino acid residues.

[0011] Preferably, the above-mentioned tag refers to a polypeptide or protein that is fused and expressed together with the target protein by using in vitro DNA recombination technology for the purpose of facilitating 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.

[0012] Preferably, 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.

[0013] In a second aspect, a nucleic acid molecule encoding the TaWDR3 protein according to claim 1.

[0014] Preferably, for the nucleic acid molecule encoding the TaWDR3 protein, the nucleic acid molecule is as shown in any of the following:

[0015] 1) a DNA molecule as shown in SEQ ID No. 2;

[0016] 2) a DNA molecule as shown in SEQ ID No. 3;

[0017] 3) a DNA molecule that hybridizes with the DNA molecule in 1) or 2) and encodes the TaWDR3 protein according to claim 1;

[0018] 4) a DNA molecule having an identity of more than 99%, more than 95%, more than 90%, more than 85% or more than 80% with the DNA sequence in 1) or 2) or 3) and encoding the TaWDR3 protein according to claim 1.

[0019] Among them, the nucleic acid molecule can be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule can also be RNA, such as mRNA or hnRNA, etc.

[0020] In a third aspect, a biological material, comprising an expression cassette, a recombinant vector, a recombinant microorganism or a transgenic plant cell line containing the above-mentioned nucleic acid molecule.

[0021] Specifically include:

[0022] A1) A nucleic acid molecule encoding the TaWDR3 protein;

[0023] A2) An expression cassette containing the nucleic acid molecule described in A1);

[0024] A3) A recombinant vector containing the nucleic acid molecule described in A1);

[0025] A4) A recombinant vector containing the expression cassette described in A2);

[0026] A5) A recombinant microorganism containing the nucleic acid molecule described in A1);

[0027] A6) A recombinant microorganism containing the expression cassette described in A2);

[0028] A7) A recombinant microorganism containing the recombinant vector described in A3);

[0029] A8) A recombinant microorganism containing the recombinant vector described in A4);

[0030] A9) A transgenic plant cell line containing the nucleic acid molecule described in A1);

[0031] A10) A transgenic plant cell line containing the expression cassette described in A2);

[0032] A11) A transgenic plant cell line containing the recombinant vector described in A3);

[0033] A12) A transgenic plant cell line containing the recombinant vector described in A4).

[0034] Those skilled in the art can easily mutate the nucleotide sequence encoding the TaWDR3 protein of the present invention by using known methods, such as directed evolution and point mutation methods. Those nucleotides that have been artificially modified and have 80% or higher identity with the nucleotide sequence encoding the TaWDR3 protein, as long as they encode the TaWDR3 protein and have the same function, are all derived from the nucleotide sequence of the present invention and are equivalent to the sequence of the present invention.

[0035] Preferably, the above "identity" refers to the sequence similarity with the natural nucleic acid sequence. "Identity" includes nucleotide sequences having 80% or higher, or 85% or higher, or 90% or higher, or 95% or higher identity with the nucleotide sequence encoding the protein consisting of the amino acid sequence shown in SEQ ID No.1 of the present invention. Identity can be evaluated by the naked eye or 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;

[0036] The above-mentioned identity of 80% or more can be an identity of 80%, 85%, 90% or 95% or more.

[0037] Preferably, the above-mentioned stringent conditions are hybridization and membrane washing twice in a solution of 2×SSC and 0.1% SDS at 68°C for 5 minutes each time, and then hybridization and membrane washing twice in a solution of 0.5×SSC and 0.1% SDS at 68°C for 15 minutes each time; or hybridization and membrane washing under the conditions of 65°C in a solution of 0.1×SSPE (or 0.1×SSC) and 0.1% SDS.

[0038] The expression cassette containing the nucleic acid molecule encoding the TaWDR3 protein (TaWDR3 gene expression cassette) in the above A2) refers to DNA that can express the TaWDR3 gene in a host cell. This DNA not only includes a promoter that initiates the transcription of the TaWDR3 gene, but also includes a terminator that terminates the transcription of the TaWDR3 gene. 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; the wound-inducible promoter, leucine aminopeptidase, from tomatoes; the chemically inducible promoter, pathogenesis-related 1 (PR1) (induced by salicylic acid and BTH (benzothiadiazole-7-thiocarboxylic acid S-methyl ester)), from tobacco; the tomato protease inhibitor II promoter (PIN2) or the LAP promoter (both can be induced by methyl jasmonate); the heat shock promoter; the tetracycline-inducible promoter; seed-specific promoters, such as the millet seed-specific promoter pF128, the promoters specific for seed storage proteins (for example: the promoters of vicilin, napin, oleosin, and soybean beta conglycin). They can be used alone or in combination with other plant promoters. Suitable transcription terminators include, but are not limited to: the Agrobacterium tumefaciens nopaline synthase terminator (NOS terminator), the cauliflower mosaic virus CaMV 35S terminator, the tml terminator, the pea rbcS E9 terminator, and the terminators of nopaline and octopine synthases.

[0039] A recombinant vector containing the TaWDR3 gene expression cassette can be constructed using existing expression vectors. The plant expression vectors include binary Agrobacterium vectors and vectors that can be used for plant microprojectile bombardment, etc., such as pAHC25, pBin438, pCAMBIA1302, pCAMBIA2301, pCAMBIA1301, pCAMBIA1300, pBI121, pCAMBIA1391-Xa or pCAMBIA1391-Xb (from CAMBIA), etc. The plant expression vector may further contain the 3'-untranslated region of the foreign gene, that is, it contains a polyadenylation signal and any other DNA fragments involved in mRNA processing or gene expression. The polyadenylation signal can direct the addition of polyadenylate to the 3' end of the mRNA precursor. For example, the nopaline synthase gene Nos of the Agrobacterium tumefaciens Ti plasmid gene and the 3'-untranslated region transcribed by plant genes (such as the soybean storage protein gene) have similar functions. When constructing a plant expression vector using the gene of the present invention, enhancers can also be used, including translation enhancers or transcription enhancers. These enhancer regions can be the ATG start codon or the start codon in the adjacent region, etc., but must be in the same reading frame as the coding sequence to ensure the correct translation of the entire sequence. The sources of the translation control signal and the start codon are extensive and can be natural or synthetic. The translation initiation region can be from the transcription initiation region or the structural gene. For the convenience of identifying and screening transgenic plant cells or plants, the used plant expression vector can be processed, such as adding a gene encoding an enzyme or a luminescent compound that can produce a color change and can be expressed in plants (GUS gene, luciferase gene, etc.), a marker gene for antibiotics (such as the nptII gene conferring resistance to kanamycin and related antibiotics, the bar gene conferring resistance to the herbicide phosphinothricin, the hph gene conferring resistance to the antibiotic hygromycin, and the dhfr gene conferring resistance to methotrexate, the EPSPS gene conferring resistance to glyphosate) or a marker gene for anti-chemical reagents (such as an anti-herbicide gene), a mannose-6-phosphate isomerase gene providing the ability to metabolize mannose. Considering the safety of transgenic plants, no selective marker gene can be added, and the transformed plants can be directly screened under stress conditions.

[0040] Furthermore, the above vector can be a plasmid, cosmid, phage or viral vector.

[0041] Furthermore, the above microorganism can be yeast, bacteria, algae or fungi; such as Agrobacterium.

[0042] Fourthly, the application of the above TaWDR3 protein, or the above nucleic acid molecule, or the above biological material in regulating plant drought resistance and / or yield.

[0043] Fifth aspect, application of the above TaWDR3 protein, or the above nucleic acid molecule, or the above biological material in cultivating transgenic plants with improved drought resistance and / or increased yield.

[0044] Sixth aspect, a method for cultivating transgenic plants with improved drought resistance and / or increased yield, the steps are: increasing the content and / or activity of the above nucleic acid molecule in the target plant to obtain transgenic plants with improved drought resistance and / or increased yield.

[0045] Furthermore, the method for increasing the content and / or activity of TaWDR3 protein in the recipient plant is overexpressing TaWDR3 protein in the recipient plant; the method for overexpression is introducing the coding gene of TaWDR3 protein into the recipient plant.

[0046] The yield of the transgenic plant being greater than that of the recipient plant is specifically manifested as the yield per plant of the transgenic plant being greater than that of the recipient plant.

[0047] Preferably, the above increase is to construct an overexpression vector p110UBI-R containing the above nucleic acid molecule, and then transfer it into the plant to be improved by the Agrobacterium-mediated transformation method, thus obtaining a plant with improved drought resistance and / or increased yield.

[0048] Preferably, the forward primer sequence for constructing the overexpression vector p110UBI-R is as shown in SEQ ID No.4, and the reverse primer sequence is as shown in SEQ ID No.5.

[0049] Preferably, the above plant is a dicotyledonous plant or a monocotyledonous plant.

[0050] Preferably, the above plant is a gramineous plant; the gramineous plant is wheat; the wheat is wild-type wheat Fielder.

[0051] The present invention has the following beneficial effects:

[0052] This application discloses a wheat drought tolerance-related protein TaWDR3 and its coding gene. By constructing a recombinant vector p110UBI-R of the CDS sequence of the TaWDR3 gene and transforming wheat plants, it is found that the survival rate of the T3 generation wheat transgenic lines obtained by overexpressing TaWDR3 is significantly higher than that of wild-type wheat under drought conditions; the yield per plant increases by about 18% on average, which is extremely significantly higher than that of wild-type wheat statistically, indicating that the TaWDR3 protein and its coding gene have the function of positively regulating wheat drought resistance. Overexpressing the TaWDR3 gene in wheat can improve the growth state of wheat under drought stress conditions and increase the survival rate, which has important practical significance for cultivating new varieties of drought-resistant crops. Description of the Drawings

[0053] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0054] Figure 1 This is the expression level of the TaWDR3 gene in different strains of wheat after introducing the p110UBI-R vector in the present invention.

[0055] Figure 2 This is the phenotype of different strains of wheat overexpressing the TaWDR3 gene before and after drought treatment in the present invention.

[0056] Figure 3 This is the statistical data of the survival rate of different strains of wheat overexpressing the TaWDR3 gene after drought treatment in the present invention.

[0057] Figure 4 This is the yield phenotype of different strains of wheat overexpressing the TaWDR3 gene under normal and drought conditions in the present invention.

[0058] Figure 5 This is the statistical data of the yield of different strains of wheat overexpressing the TaWDR3 gene under normal and drought conditions in the present invention. Detailed implementation manners

[0059] The following will clearly and completely describe the technical solutions of the present invention in combination with the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, rather than all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the scope of protection of the present invention.

[0060] Unless otherwise specified, the test methods used in the following experimental examples are all conventional methods; the materials, reagents, etc. used, unless otherwise specified, are reagents and materials that can be obtained from commercial channels.

[0061] In the early stage of this application, a drought-related protein (Wheat Drought Related 3, TaWDR3) was identified in wheat by genome-wide association analysis. Bioinformatics analysis shows that this protein belongs to the RNA-binding protein (RBP) family, and it may maintain the transcription process by recognizing and binding to precursor RNA and regulating the RNA secondary structure.

[0062] The p110UBI-GFP / Flag vector used in this application was independently modified by the applicant. This vector is based on the commercial vector pCAMBIA and contains the UBI gene promoter conserved in monocotyledonous plants, a multiple cloning site, and the GFP / Flag protein tag. It was ligated between the left and right borders of the vector through two restriction endonucleases, Pst I and EcoR I, and contains the kanamycin and rifamycin resistance genes for resistance screening. The public can obtain it from the College of Life Sciences, Henan University.

[0063] The Agrobacterium tumefaciens strain EHA105 used in this application can be obtained by the public from the College of Life Sciences, Henan University.

[0064] The wild-type wheat material used in this application is the variety "Fielder" (Triticum aestivum L. cv. Fielder), which can be obtained by the public from the College of Life Sciences, Henan University.

[0065] The medium formula used in the Agrobacterium-mediated wheat callus transformation process:

[0066] (1) 1L of N6D solid medium: 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.

[0067] (2) 1L of AAM Agrobacterium activation medium: 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.

[0068] Among them, 100 mL of AA-1: 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.

[0069] 100 mL of AA-2: CaCl2·2H2O, 15 g.

[0070] 100 mL of AA-3: MgSO4·7H2O, 25 g.

[0071] AA-4 100 mL: FeSO4·7H2O, 278 mg; Na2EDTA, 373 mg.

[0072] AA-5 100 mL: NaH2PO4·2H2O, 15 g.

[0073] AA-6 100 mL: Nicotinic acid, 20 mg; Vitamin B1, 20 mg; Vitamin B6, 20 mg; Inositol, 2 g.

[0074] AA-sol 100 mL: Arginine, 176.67 mg; Glycine, 75 mg.

[0075] (3) N6D Screening Medium 1 L: 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; Hygromycin 50 mg or Bialaphos 2 mg; Carbenicillin 200 mg; Cefotaxime 250 mg.

[0076] (4) 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; Carbenicillin, 200 mg; Cefotaxime, 250 mg; NAA, 0.3 mg; 6-BA, 3 mg.

[0077] (5) 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; Carbenicillin, 200 mg; Cefotaxime, 250 mg; NAA, 0.3 mg; 6-BA, 3 mg.

[0078] (6) Rooting Medium 1 L: Sucrose, 10 g; MS Medium, 2.215 g; Gelrite, 4 g; pH = 5.8. Example: Obtaining Protein TaWDR3 and Its Encoding Gene

[0079] 1. Take the seeds of wheat Fielder, soak them in water at room temperature for 48 hours to absorb moisture and then place them in a 4°C refrigerator for 24 hours to germinate. Transfer the germinated wheat seeds to nutrient soil and cultivate for two weeks. Take the whole plant, quickly freeze it in liquid nitrogen, grind it to extract total RNA, perform reverse transcription, and obtain cDNA.

[0080] 2. Using the cDNA obtained in step 1 as a template, perform PCR amplification with 5'-ATGGCGCCGAGGCAGGTAGGG-3' (as shown in SEQ ID No. 4) as the forward primer and 5'-TTACTCAGTGATGACTTCATTGGCTTG-3' (as shown in SEQ ID No. 5) as the reverse primer to obtain a PCR product. The PCR reaction system and reaction procedure are as follows:

[0081] The reaction system is shown in Table 1:

[0082] Table 1 PCR reaction system

[0083]

[0084] The reaction procedure is shown in Table 2:

[0085] Table 2 PCR reaction procedure

[0086]

[0087] 3. Perform agarose gel electrophoresis on the amplified PCR product, separate and purify the DNA fragment approximately 213 bp in length and perform sequencing. The sequencing results show that the nucleotide sequence of this DNA fragment is as shown in positions 1-213 of SEQ ID No. 2 in the sequence listing.

[0088] The sequence shown in SEQ ID No. 2 in the sequence listing is the full-length coding region sequence of the protein TaWDR3 encoded by SEQ ID No. 1 in the sequence listing in wheat Fielder. The gene encoding the protein TaWDR3 is named gene TaWDR3.

[0089] Application example: Application of TaWDR3 in regulating wheat drought resistance

[0090] I. Construction of recombinant overexpression vector p110UBI-R and recombinant Agrobacterium R1

[0091] 1. Construction of recombinant overexpression vector p110UBI-R

[0092] Clone the DNA fragment shown in positions 1-213 of SEQ ID No. 2 in the sequence listing between the BamH I restriction enzyme sites of vector p110UBI-GFP / Flag. After sequencing and alignment, obtain the recombinant overexpression vector p110UBI-R.

[0093] The recombinant overexpression vector p110UBI-R was obtained by inserting the DNA fragment shown in positions 1-213 of SEQ ID No.2 in the sequence listing between the BamH I restriction enzyme sites of the vector p110UBI-GFP / Flag, while keeping the other sequences of the vector p110UBI-GFP / Flag unchanged.

[0094] 2. Obtaining of Agrobacterium tumefaciens EHA105 harboring p110UBI-R

[0095] The recombinant overexpression vector p110UBI-R was transformed into Agrobacterium tumefaciens strain EHA105. After PCR detection, the recombinant Agrobacterium R1 containing the recombinant vector p110UBI-R was obtained.

[0096] II. Obtaining of transgenic wheat lines overexpressing TaWDR3 gene

[0097] The wild-type wheat Fielder was transformed with the recombinant Agrobacterium R1 by the method of infecting embryogenic callus. The T0 generation of wheat lines was harvested and the T1 generation of seeds was obtained; after the T1 generation of seeds germinated, resistant seedlings were obtained for subsequent planting and seed harvesting to obtain the T2 generation of seeds; after the T2 generation of seeds germinated, different transgenic lines were randomly selected for RT-qPCR detection to determine the wheat lines overexpressing TaWDR3. Two transgenic lines with relatively high expression levels of the TaWDR3 gene were selected for drought resistance and yield determination, and were named OE-1 and OE-2 respectively.

[0098] The T0 generation represents the plants of the transformation generation, the T1 generation represents the seeds produced by self-crossing of the T0 generation and the plants grown from them, and the T2 generation represents the seeds produced by self-crossing of the T1 generation and the plants grown from them.

[0099] The specific operation steps of the above Agrobacterium-mediated wheat transgenic process are as follows:

[0100] (1) Obtaining callus induced from mature wheat embryos: Take the dried seeds after wheat maturity, remove the hulls and place them in a sterile 100 mL Erlenmeyer flask. Add 70% alcohol in the laminar flow hood for surface disinfection for 45 s, shaking the Erlenmeyer flask constantly during this period. Then transfer the seeds to a 2.5% sodium hypochlorite solution, add a drop of Triton X-100, place the seeds in a shaker at 28 °C, shake and disinfect at 200 rpm for 15 min. Then transfer the seeds to a 2.5% sodium hypochlorite solution again, place them in a shaker at 28 °C, shake and disinfect at 200 rpm for 15 min. Discard the disinfectant solution, and rinse repeatedly with sterile distilled water until the rinsed solution is clear and free of foreign objects. Pour out the seeds and place them on a Petri dish lined with sterile filter paper, and dry them in the laminar flow hood for 45 min. Arrange the seeds on the N6D solid medium in sequence, place them in an incubator at 28 °C, and culture for 4 weeks. Pick out the light yellow and smooth embryogenic callus and subculture it on a new N6D solid medium for 5 days, which can be used for Agrobacterium transformation.

[0101] (2) Culturing Agrobacterium: Take the transformed Agrobacterium stored at -80 °C, coat it on the YEB solid medium containing the corresponding antibiotics for streak activation, and then place the Agrobacterium in an incubator at 28 °C for 3 days. Pick out the monoclonal strains with good growth and streak-activate them again on the YEB solid medium containing the corresponding antibiotics. After culturing at 28 °C for 1 day, scrape off a solid strain the size of a match head with a sterile medicine spoon and suspend it in 30 mL of AAM (the final concentration of acetosyringone is 40 mg / L) liquid medium for transformation.

[0102] (3) Transformation and co-cultivation of Agrobacterium: Scrape off the vigorously growing small granular callus after subculture from the N6D medium and place it in a 100 mL Erlenmeyer flask. Suspend the callus with the AAM medium containing the Agrobacterium strain, soak for 20 min, and then discard the AAM medium. Place the callus on a Petri dish lined with sterile filter paper, blow-dry it in the laminar flow hood for 30 min, and then place it on the N6D + AAM co-culture medium lined with a layer of filter paper. Place the callus in an incubator at 23 °C and perform dark culture for 4 days.

[0103] (4) Elimination and screening culture of Agrobacterium: Scrape the callus after co-cultivation into a sterile Erlenmeyer flask with a sterile medicine spoon, and rinse it repeatedly with sterile distilled water until the rinsed water is clear. Add a certain volume of sterile distilled water again, let it stand in the laminar flow hood for 15 min, and then soak it twice with sterile distilled water containing carbenicillin with a final concentration of 400 mg / L, 15 min each time. Pour the soaked callus onto a Petri dish lined with sterile filter paper, blow-dry it in the laminar flow hood for 3 h, and then spread the callus on the N+ medium containing the corresponding antibiotics. Place it in an incubator at 30 °C for dark culture, subculture once every two weeks, and subculture twice.

[0104] (5) Redifferentiation of resistant calli after infection: Transfer the subcultured and screened calli to hypertonic medium according to different transgenic lines in sequence, place them in a light incubator at 30 °C, and culture them under light for 2 weeks until the calli turn green. Transfer the green calli to hypotonic medium again until tender shoots grow. Transfer the tender shoots to rooting medium in Erlenmeyer flasks according to different clones in sequence, place them in a light incubator at 30 °C, and culture them under light for 2 weeks to obtain resistant seedlings.

[0105] (6) Hydroponics and transplantation of resistant seedlings: When the resistant seedlings grow to the top of the Erlenmeyer flask, remove the top filter membrane and place the seedlings in the air. At the same time, add sterile distilled water to the Erlenmeyer flask and acclimatize them in a light incubator. After the seedlings' leaves straighten, pull out the seedlings from the Erlenmeyer flask, wash the medium from the roots of the seedlings, and transplant them into a greenhouse for cultivation management. After the plants flower and pollinate under normal greenhouse conditions, harvest the seeds.

[0106] The method for detecting transgenic wheat by the above RT-qPCR is as follows: Take wheat seedlings at 14 days after germination of the T2 generation transgenic lines and wild-type wheat seedlings under the same germination conditions, extract the total RNA of wheat leaves using the Plant RNA Mini Kit from Tiangen Biotech (Beijing) Co., Ltd., and digest the DNA with DNase I. After measuring the concentration with NanoDrop2000 (Thermo Fisher, USA), take 2 μg and synthesize cDNA using the reverse transcription kit from Invitrogen Corporation with Oligo d(T) as the primer. Amplify the cDNA of the TaWDR3 gene using the specific quantitative detection primers QF and QR for the TaWDR3 gene, and use the Actin gene in wheat as an internal reference, with its primers being AF and AR. After analyzing the quantitative results, the expression levels of the TaWDR3 gene in wheat of different transgenic lines are as Figure 1 shown:

[0107] The sequences of the above primers are as follows:

[0108] QF: 5'-GGTAGGGATAGTGAAGTCGTTC-3';

[0109] QR: 5'-TCCTTGAGGGATTTGAAGC-3';

[0110] AF: 5'-GACCGTATGAGCAAGGAGAT-3';

[0111] AR: 5'-CAATCGCTGGACCTGACTC-3'.

[0112] The results show that compared with the wild type, the expression levels of the TaWDR3 gene in the two transgenic lines (OE-1 and OE-2) overexpressing the TaWDR3 gene are significantly increased (Figure 1 ).

[0113] 3. Phenotypic analysis of drought resistance in transgenic wheat

[0114] The seeds of the T3 generation homozygous TaWDR3 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 the phenotype and plant survival rate were counted after another 2 weeks of growth.

[0115] The results showed that the growth of TaWDR3 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 ), and the overall survival rate of the plants after drought treatment was increased by about 45% compared with the wild type ( Figure 3 The above results indicate that the TaWDR3 gene and its encoded protein have the function of positively regulating the drought resistance of wheat. Overexpression of the TaWDR3 gene in wheat can improve the growth state and survival rate of wheat under drought stress conditions.

[0116] 4. Phenotypic analysis of transgenic wheat yield

[0117] The T3 generation homozygous TaWDR3 overexpressing wheat transgenic lines (OE-1 and OE-2) and wild-type wheat (Fielder) were taken and the seeds 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 phenotypes ( Figure 4 ).

[0118] The results showed that the yield per plant of the wild-type wheat Fielder and two transgenic lines of TaWDR3 overexpression wheat (OE-1 and OE-2) under normal conditions were 9.43g, 9.69g and 10.10g, respectively, and the yield per plant under drought conditions were 4.74g, 5.80g and 5.42g, respectively. Compared with the wild type, the yield per plant of the two transgenic lines of TaWDR3 overexpression wheat (OE-1 and OE-2) under drought conditions increased by 22.4% and 14.3%, respectively. Figure 5 ). This indicates that overexpression of the TaWDR3 gene will significantly increase the yield per plant of wheat under drought stress conditions. The above results indicate that the TaWDR3 gene and its encoded protein have the function of positively regulating the yield per plant of wheat. Overexpression of the TaWDR3 gene in wheat can increase the yield of wheat under drought conditions.

[0119] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included within the protection scope of the present invention.

Claims

1. A TaWDR3 protein, the amino acid sequence of which is shown as any one of the following: (1) The amino acid sequence is shown as SEQ ID No.1; (2) A sequence in which one or more amino acids are inserted, deleted or mutated in the amino acid sequence shown as SEQ ID No.1 and still has the function of the amino acids in (1); (3) Having an identity of more than 99%, more than 95%, more than 90%, more than 85% or more than 80% with the amino acid sequence shown as SEQ ID No.1; (4) A fusion protein obtained by connecting a tag to the N-terminus and / or C-terminus of the protein with the amino acid sequence shown as SEQ ID No.

1.

2. A nucleic acid molecule encoding the TaWDR3 protein according to claim 1.

3. The nucleic acid molecule according to claim 2, wherein The nucleic acid molecule is shown as any one of the following: 1) A DNA molecule shown as SEQ ID No.2; 2) A DNA molecule shown as SEQ ID No.3; 3) A DNA molecule that hybridizes with the DNA molecule in 1) or 2) and encodes the TaWDR3 protein according to claim 1; 4) A DNA molecule having an identity of more than 99%, more than 95%, more than 90%, more than 85% or more than 80% with the DNA sequence in 1) or 2) or 3) and encoding the TaWDR3 protein according to claim 1.

4. A biological material, characterized in that: An expression cassette, recombinant vector, recombinant microorganism or transgenic plant cell line containing the nucleic acid molecule according to claim 3.

5. Use of the TaWDR3 protein according to claim 1 or the nucleic acid molecule according to claim 3 or the biological material according to claim 4 in regulating plant drought resistance and / or yield.

6. Use of the TaWDR3 protein according to claim 1 or the nucleic acid molecule according to claim 3 or the biological material according to claim 4 in cultivating transgenic plants with improved drought resistance and / or increased yield.

7. A method for cultivating a transgenic plant with improved drought resistance and / or increased yield, characterized in that, The steps are: increasing the content and / or activity of the nucleic acid molecule according to claim 3 in the target plant to obtain a transgenic plant with improved drought resistance and / or increased yield.

8. The method for cultivating a transgenic plant with improved drought resistance and / or increased yield according to claim 7, characterized in that: The increase is to construct an overexpression vector p110UBI-R containing the nucleic acid molecule according to claim 3, and then transfer it into the plant to be improved by the Agrobacterium transformation method, and a plant with improved drought resistance and / or increased yield is obtained.

9. The method for cultivating a transgenic plant with enhanced drought resistance and / or enhanced yield according to claim 8, wherein: The forward primer sequence for constructing the overexpression vector p110UBI-R is shown as SEQ ID No.4, and the reverse primer sequence is shown as SEQ ID No.

5.

10. The method for cultivating a transgenic plant with improved drought resistance and / or increased yield according to claim 9, characterized in that: The plant is wheat.

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