Application of the wheat TaSP1 gene in improving wheat drought resistance

Abstract

This invention discloses wheat TaSP1 Application of genes in improving drought resistance in wheat. This invention belongs to the field of biotechnology, specifically relating to wheat. TaSP1 Application of genes in improving the drought resistance of wheat. The gene encoding the protein TaSP1 or its regulatory proteins in this invention. TaSP1 The expressed substance or the substance that regulates the activity or content of the protein may be used in any of the following: 1) regulating plant drought resistance; 2) preparing products that regulate plant drought resistance; 3) cultivating plants with altered drought resistance; 4) preparing products that cultivate plants with altered drought resistance; 5) plant breeding. TaSP1 Genes can positively regulate the drought resistance of wheat.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to wheat. TaSP1 Application of genes in improving the drought resistance of wheat. Background Technology

[0002] Wheat is one of the most widely cultivated food crops in the world, providing humans with essential amino acids, minerals, vitamins, as well as beneficial phytochemicals and dietary fiber. High and stable wheat yields are crucial to ensuring global food security.

[0003] Plant growth faces numerous stresses, including biotic stress induced by organisms and abiotic stress induced by the environment. Drought is a significant abiotic stress, and its impact on food yield is increasingly severe due to global warming, population growth, increased agricultural water demand, and decreasing available freshwater. Wheat, as an important dryland food crop, is highly susceptible to water-related factors, and drought stress is considered the leading abiotic stress threatening wheat yield. Therefore, improving wheat's drought resistance is particularly important. However, increasing crop tolerance to abiotic stress often inhibits growth and yield. This growth-defense balance is prevalent in plants, making the coordination of drought resistance and yield a major challenge in the genetic improvement of crop drought resistance. Nitrogen is one of the most important macronutrients for plants, promoting growth. Studies have shown that drought stress limits plant growth by inhibiting nitrogen use, while adequate nitrogen supply can increase drought resistance. Therefore, regulating the expression of key nitrogen-use genes may offer a potential way to synergistically improve wheat drought resistance and yield. Summary of the Invention

[0004] The technical problem solved by this invention is how to regulate plant drought resistance, especially wheat.

[0005] To address the aforementioned problems, the present invention provides applications related to proteins, substances that regulate the expression of genes encoding said proteins, or substances that regulate the activity or content of said proteins.

[0006] The use of the protein, the substance regulating the expression of the gene encoding the protein, or the substance regulating the activity or content of the protein provided by this invention in any of the following: 1) Application in regulating plant drought resistance; 2) Application in the preparation of products that regulate plant drought resistance; 3) Application in cultivating plants with altered drought resistance; 4) Application in the preparation of products using plants with altered drought resistance; 5) Application in plant breeding.

[0007] The protein is any of the following proteins: a1) Proteins with the amino acid sequence SEQ ID No:1, SEQ ID No:4 or SEQ ID No:7; a2) A protein having the same function as the amino acid sequence shown in SEQ ID No:1, SEQ ID No:4 or SEQ ID No:7, by substitution and / or deletion and / or addition of one or more amino acid residues. Proteins that have more than 80% identity with the amino acid sequences defined in (a3), (a1), or (a2) and have the same function; The fusion protein is obtained by attaching a tag to the end of any of the proteins defined in a4), a1)-a3).

[0008] In the aforementioned proteins, the protein tag refers to a polypeptide or protein fused with the target protein using in vitro DNA recombination technology for expression, to facilitate the expression, detection, tracing, and / or purification of the target protein. The protein tag may be a Flag tag, His tag, MBP tag, HA tag, myc tag, GST tag, and / or SUMO tag, etc.

[0009] In the above-mentioned proteins, identity refers to the identity of the amino acid sequences. The identity of amino acid sequences can be determined using homology search sites on the Internet, such as the BLAST page on the NCBI homepage. For example, in Advanced BLAST 2.1, using blastp as the program, setting the Expect value to 10, setting all filters to OFF, using BLOSUM62 as the matrix, setting the Gap existence cost, Per residue gap cost, and Lambda ratio to 11, 1, and 0.85 (default values) respectively, and performing an identity search on a pair of amino acid sequences to calculate the identity value (%), then the identity value can be obtained.

[0010] In the aforementioned proteins, the 80% or more identity can be at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 95%, 96%, 98%, 99%, or 100% identity.

[0011] Of the proteins mentioned above, SEQ ID No: 1 is the TaSP1D protein, composed of 605 amino acid residues, whose encoding gene is: TaSP1D Gene.

[0012] SEQ ID No: 4 is the TaSP1A protein composed of 606 amino acid residues, and its encoding gene is: TaSP1A Gene.

[0013] SEQ ID No: 7 is the TaSP1B protein, composed of 604 amino acid residues, whose encoding gene is TaSP1B Gene.

[0014] In the above applications, the protein is derived from wheat ( Triticum aestivum L.).

[0015] In this article, protein TaSP1 may specifically refer to protein TaSP1A, TaSP1B, or TaSP1D.

[0016] In this article, the substances that regulate the activity and / or content of the protein may be substances that regulate gene expression, wherein the gene TaSP1A , TaSP1B or TaSP1D The protein encodes TaSP1A, TaSP1B, or TaSP1D.

[0017] In the above text, the substance regulating gene expression can be a substance that performs at least one of the following six types of regulation: 1) regulation at the transcriptional level of the gene; 2) post-transcriptional regulation of the gene (i.e., regulation of splicing or processing of the primary transcript of the gene); 3) regulation of RNA transport of the gene (i.e., regulation of mRNA transport of the gene from the nucleus to the cytoplasm); 4) regulation of translation of the gene; 5) regulation of mRNA degradation of the gene; and 6) post-translational regulation of the gene (i.e., regulation of the activity of the protein translated from the gene).

[0018] In this invention, the regulation can be increased, enhanced, or improved; the regulation can also be decreased, weakened, or reduced.

[0019] In this article, the enhancement, increase or upregulation of the expression level of the coding gene of the aforementioned protein in the recipient plant, and / or the enhancement, increase or upregulation of the activity and / or content of the coding gene of the aforementioned protein, is achieved by introducing the coding gene of the aforementioned protein into the recipient plant.

[0020] In this article, regulating the expression of the gene encoding the protein can be achieved by inhibiting, reducing, or downregulating the expression of the gene. Inhibition, reduction, or downregulation of the gene expression can be achieved through gene knockout or gene silencing.

[0021] In the above applications, the substance that regulates the expression of the gene encoding the protein or the substance that regulates the activity or content of the protein can be a biological material related to the protein described above, and the biological material can be any of the following: c1) The nucleic acid molecule that encodes the protein described above; c2) An expression cassette containing the nucleic acid molecule described in c1); c3) A recombinant vector containing the nucleic acid molecule described in c1), or a recombinant vector containing the expression cassette described in c2); c4) Recombinant microorganisms containing the nucleic acid molecules described in c1), or recombinant microorganisms containing the expression cassette described in c2), or recombinant microorganisms containing the recombinant vector described in c3); c5) A transgenic plant cell line containing the nucleic acid molecule described in c1), or a transgenic plant cell line containing the expression cassette described in c2); c6) Transgenic plant tissue containing the nucleic acid molecules described in c1), or transgenic plant tissue containing the expression cassette described in c2); c7) A transgenic plant organ containing the nucleic acid molecule described in c1), or a transgenic plant organ containing the expression cassette described in c2); e1) Nucleic acid molecules that inhibit, reduce, or silence the expression of the protein-coding genes mentioned above; e2) An expression cassette containing the nucleic acid molecule described in e1); e3) A recombinant vector containing the nucleic acid molecule described in e1), or a recombinant vector containing the expression cassette described in e2); e4) Recombinant microorganisms containing the nucleic acid molecules described in e1), or recombinant microorganisms containing the expression cassette described in e2), or recombinant microorganisms containing the recombinant vector described in e3); e5) A transgenic plant cell line containing the nucleic acid molecule described in e1), or a transgenic plant cell line containing the expression cassette described in e2); e6) Transgenic plant tissue containing the nucleic acid molecules described in e1), or transgenic plant tissue containing the expression cassette described in e2); e7) A transgenic plant organ containing the nucleic acid molecule described in e1) or a transgenic plant organ containing the expression cassette described in e2).

[0022] In the above applications, c1) the nucleic acid molecule can be any of the following DNA molecules: d1) The nucleotide sequence is the DNA molecule shown in SEQ ID No: 3, SEQ ID No: 6 or SEQ ID No: 9; d2) The coding sequence is a DNA molecule shown in SEQ ID No: 2, SEQ ID No: 5 or SEQ ID No: 8; d3) has 90% or more identity with the nucleotide sequence defined by d1) or d2) and is a DNA molecule encoding the protein described above; d4) A DNA molecule that hybridizes under strict conditions with a nucleotide sequence defined by d1) or d2) and encodes the protein described above.

[0023] The nucleic acid molecules mentioned in this article can be DNA, such as cDNA, genomic DNA, or recombinant DNA; the nucleic acid molecules can also be RNA, such as gRNA, mRNA, siRNA, shRNA, sgRNA, miRNA, or antisense RNA.

[0024] The vectors described herein are well-known to those skilled in the art and include, but are not limited to: plasmids, bacteriophages (such as λ phage or M13 filamentous phage), granules (i.e., Cosmids), Ti plasmids, or viral vectors. Specifically, they may be vectors PC186 and pBUE413.

[0025] Existing plant expression vectors can be used to construct structures containing... TaSP1 Recombinant gene expression vectors. These plant expression vectors include, but are not limited to, binary Agrobacterium vectors and vectors suitable for plant microbombardment. The plant expression vectors may also contain the 3' untranslated region of the exogenous gene, i.e., containing a polyadenylate signal and any other DNA fragment involved in mRNA processing or gene expression. The polyadenylate signal can guide the addition of polyadenylate to the 3' end of the mRNA precursor; similar functions exist for the untranslated regions transcribed at the 3' end of genes including, but not limited to, Agrobacterium crown gall-inducing (Ti) plasmid genes (such as the Nos gene for lipase synthesis) and plant genes (such as the soybean storage protein gene).

[0026] use TaSP1 When constructing recombinant plant expression vectors, any type of enhancing promoter or constitutive promoter can be added before the transcription initiation nucleotide, including but not limited to the cauliflower mosaic virus (CAMV) 35S promoter and the maize ubiquitin promoter. These can be used alone or in combination with other plant promoters. Furthermore, when constructing plant expression vectors using the genes of this invention, enhancers, including translational enhancers or transcriptional enhancers, can also be used. These enhancer regions can be ATG start codons or adjacent region start codons, but they must be identical to the reading frame of the coding sequence to ensure correct translation of the entire sequence. The sources of the translation control signals and start codons are wide-ranging; they can be natural or synthetic. The translation initiation region can originate from the transcription initiation region or structural genes.

[0027] To facilitate the identification and screening of transgenic plant cells or plants, the plant expression vectors used can be processed, such as by adding genes that can be expressed in plants, encoding enzymes or luminescent compounds that produce color changes (GUS genes, luciferase genes, etc.), antibiotic resistance markers (gentamicin markers, kanamycin markers, etc.), or chemical reagent resistance marker genes (such as herbicide resistance genes). From a safety perspective, transgenic plants can be screened directly under stress without adding any selective marker genes.

[0028] The present invention also provides a method for altering the drought resistance of plants, the method comprising the following steps M or P: Step M is to enhance, increase or upregulate the activity and / or content of the proteins mentioned above in the target plant, or / and enhance, increase or upregulate the expression level of the encoding genes of the proteins mentioned above, so as to improve the drought resistance of the plant. The method includes step P, which is to inhibit or reduce or silence the activity and / or content of the aforementioned protein in the target plant, or / and, inhibit or reduce or silence the expression level of the gene encoding the aforementioned protein, in order to reduce the plant's drought resistance.

[0029] In the above method, reducing the expression level and / or activity of the gene encoding the protein TaSP1 in the target plant can be achieved by using gene mutation, gene knockout, gene editing or gene knockdown techniques to reduce or inactivate the gene encoding the protein TaSP1 in the genome of the target plant.

[0030] The present invention also provides a method for cultivating highly drought-resistant plants, comprising upregulating or enhancing or increasing the expression level of the coding gene of the protein described above in the target plant, and / or, the activity and / or content of the protein to obtain highly drought-resistant plants, wherein the drought resistance of the highly drought-resistant plants is higher than that of the target plant.

[0031] In one specific embodiment, the upregulation, enhancement, or increase of the expression of the gene encoding the protein described above in the plant includes introducing the nucleic acid molecule, expression cassette, or recombinant vector described above into the target plant to obtain a plant with high drought resistance.

[0032] The present invention also provides a method for cultivating plants with low drought resistance, comprising inhibiting, reducing or silencing the expression level of the gene encoding the protein described above in the target plant, and / or, the activity and / or content of the protein to obtain a plant with low drought resistance, wherein the plant with low drought resistance is weaker than the target plant.

[0033] In one specific embodiment, the inhibition, reduction, or silencing of the expression of the gene encoding the protein described above in the plant includes introducing the nucleic acid molecule, expression cassette, or recombinant vector described above into the target plant to obtain a plant with low drought resistance.

[0034] In this article, the purpose of breeding includes cultivating plants with high drought resistance; the purpose of breeding also includes cultivating plants with low drought resistance.

[0035] The drought-resistant plants exhibited improved drought resistance compared to the target plants. The drought-resistant plants exhibited decreased drought resistance compared to the target plants.

[0036] The proteins and / or the biological materials mentioned above are also within the scope of protection claimed in this invention.

[0037] In the above applications or methods, the plant may be any of the following: C1) Monocotyledons; C2) Plants of the order Poales; C3) Gramineae plants; C4) Plants of the genus Triticum; C5) Wheat.

[0038] The wheat variety in question is Fielder.

[0039] Wheat is an important dryland food crop, and drought stress is the leading abiotic stress threatening wheat yield. Previous studies have shown that increasing drought resistance through genetic modification usually inhibits crop growth and yield. This gene, while improving wheat drought resistance, can ensure normal growth, development, and nutrient utilization, providing a new perspective and method for breaking the coupling between drought resistance and growth defects and breeding new drought-resistant wheat varieties. Attached Figure Description

[0040] Figure 1 For Fielder (WT) and Tasp1-aabbdd In mutants TaSP1-A / B / D sequence.

[0041] Figure 2 For Fielder (WT) and TaSP1-D Detection of expression levels in overexpressing plants.

[0042] Figure 3 For Fielder, Tasp1-aabbdd mutants and TaSP1-D Overexpressing plants were subjected to drought treatment during the booting stage. Where 'a' represents Fielder's expression during the booting stage under normal irrigation. Tasp1-aabbdd2 as well as p35S::TaSP1D-1 wheat plants; b represents Fielder wheat plants in the booting stage under drought stress. Tasp1-aabbdd2 as well asp35S::TaSP1D-1 Wheat plants; c represents Fielder plants under normal irrigation conditions and drought stress. Tasp1-aabbdd2 as well as p35S::TaSP1D-1 The results of soil moisture content measurement.

[0043] Figure 4 For Fielder (WT), [[ID= mutants and ​ The yield per plant of overexpressing plants under normal and drought conditions. Where 'a' represents the Fielder yield in a normally irrigated field. ​ ​ as well as ​ Single plant grains; b represents field samples under drought stress. ​ ​ as well as ​ Single plant grains; c and d represent Fielder's grains in normally irrigated fields and drought-stressed fields, respectively. ​ as well as ​ Statistics on yield data per plant.

[0044] ​ For Fielder, ​ mutants and ​ The yield ratio of overexpressing plants under drought and normal conditions. Detailed Implementation

[0045] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0046] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0047] Unless otherwise specified, the quantitative experiments in the following examples are all repeated three times, and the results are averaged.

[0048] The DH5α competent cells in the following examples are: MAX DH5α (Weidi Biotechnology, WD0462947); EHA105 competent cells are: EHA105 (Weidi Biotechnology, WD0453243).

[0049] The pBM27 / 10×Topo smart in the following examples: pBM27 Vector was purchased from Biomed Biotechnology, catalog number 775969AH.

[0050] The Green Taq Mix used in the following examples: Green Taq Mix was purchased from Vazyme, catalog number 037E3210CA.

[0051] In the following examples, KOD FX / KOD FX Buffer / dNTP: KOD FX was purchased from TOYOBO, part number 2406054. KOD Plus / KOD Plus Buffer / dNTP / MgSO4: KOD Plus was purchased from TOYOBO, part number 7653005.

[0052] The LR Enzyme Mix used in the following examples: Gateway™ LR Clonase™ II was purchased from Invitrogen, catalog number 2755278; ArtiCan in the following examples ATM SYBR qPCR Mix was purchased from Qingke Biotechnology, catalog number 1F124301.

[0053] The PC186 vector used in the following examples was kindly provided by Professor Daolin Fu of Shandong Agricultural University and is described in: Hao, Q., Wang, W., Han, X., et al. (2018). Isochorismate‐based salicylic acidbiosynthesis confers basal resistance to Fusarium graminearum inbarley. ​ , 19 (8), 1995-2010, the public can obtain relevant biological materials from the applicant, and the obtained biological materials can only be used for the verification of the embodiments of this application and cannot be used for other purposes.

[0054] The pBUE413 and pCBC-MT1T2 vectors used in the following examples were kindly provided by Professor Daolin Fu of Shandong Agricultural University and are described in: Xing, H., Dong, L., Wang, Z., et al. (2014). A CRISPR / Cas9toolkit for multiplex genome editing in plants. ​ , 14 1-12. The public may obtain relevant biological materials from the applicant. The obtained biological materials may only be used for the verification of the embodiments of this application and may not be used for other purposes.

[0055] The Fielder wheat material used in the following examples was kindly provided by Professor Daolin Fu of Shandong Agricultural University and is described in: Ni, F., Zheng, Y., Liu, X., et al. (2023). Sequencing trait-associated mutations to clone wheat rust-resistance gene ​ . ​ , 14 (1), 4353, The public can obtain relevant biological materials from the applicant. The obtained biological materials can only be used for the verification of the embodiments of this application and cannot be used for other purposes.

[0056] The WLS-AS medium formulation in the following examples is as follows: 1 / 100 volume 10× LS major salt, 1 / 1000 volume 100× FeEDTA, 1 / 1000 volume 100× LS trace salt, 1 / 1000 volume 100× MS vitamin, 8 g / L agarose, 10 mg / L glucose, 0.5 g / L MES, 0.85 mg / L AgNO3, 100 μM AS, 1.25 mg / L CuSO4·5H2O, and the remainder is water.

[0057] The WLS-Res medium formulation in the following examples is as follows: 1 / 10 volume 10× LS major salt, 1 / 100 volume 100× FeEDTA, 1 / 10 volume 100× LS trace salt, 0.5 mg / L 2,4-D, 1 / 100 volume 100× MS vitamins, 2.2 mg / L chlorhexidine, 0.75 g / L MgCl2·6H2O, 0.5 g / L glutamine, 0.1 g / L casein hydrolysate, 40 g / L maltose, 1.95 g / L MES, 5 g / L agarose, 250 mg / L carbenicillin, 100 mg / L ascorbic acid, 0.85 mg / L AgNO3, 100 mg / L cefotaxime, and the remainder is water.

[0058] The WLS-P5 culture medium formulation in the following examples is as follows: 1 / 10 volume 10× LS major salt, 1 / 100 volume 100× FeEDTA, 0.5 mg / L 2,4-D, 1 / 10 volume 100× LS trace salt, 1 / 100 volume 100× MS vitamins, 2.2 mg / L chlorhexidine, 0.1 g / L casein hydrolysate, 0.5 g / L glutamine, 0.75 g / L MgCl2·6H2O, 5 g / L agarose, 40 g / L maltose, 1.95 g / L MES, 100 mg / L ascorbic acid, 250 mg / L carbenicillin, 5 mg / L glufosinate, 0.85 mg / L AgNO3, with the remainder being water.

[0059] The LSZ-P5 culture medium formulation in the following examples is as follows: 1 / 10 volume of 10× LS major salt, 1 / 100 volume of 100× FeEDTA, 1 / 100 volume of 100× LS trace salt, 20 g / L sucrose, 1 / 100 volume of 100× modified LS vitamins, 5 mg / L zeatin, 0.5 g / L MES, 2.5 mg / L CuSO4·5H2O, 250 mg / L carbenicillin, 8 g / L agar, 100 mg / L cefotaxime, 5 mg / L glufosinate, and the remainder is water.

[0060] The LSF-P5 culture medium formulation in the following examples is as follows: 1 / 10 volume of 10× LS major salt, 1 / 100 volume of 100× FeEDTA, 1 / 100 volume of 100× LS trace salt, 0.5 g / L MES, 1 / 100 volume of 100× modified LS vitamins, 15 g / L sucrose, 3 g / L Gelrite, 0.2 mg / L IBA, 250 mg / L carbenicillin, 5 mg / L glufosinate, and the remainder is water.

[0061] Unless otherwise specified, the antibiotics used in the following examples are at commonly used working concentrations: kanamycin 50 μg / mL; rifampin 40 μg / mL; and spectinomycin 100 μg / mL.

[0062] Unless otherwise specified, the quantitative experiments in the following examples were performed in triplicate, and the results were averaged.

[0063] The following examples use GraphPad Prism statistical software to process the data. The experimental results are expressed as mean ± standard deviation. The t-test is used, and P < 0.05 (*) indicates a significant difference, and P < 0.01 (**) indicates a highly significant difference.

[0064] Example 1 ​ Construction of gene overexpression vectors ​ The coding sequence (CDS) of the gene in the wheat variety Chinese Spring is SEQ ID No:2, encoding the TaSP1-D protein with the amino acid sequence SEQ ID No:1. The genomic gene encoding the TaSP1-D protein in the genomic DNA of the wheat variety Chinese Spring is shown in SEQ ID No:3 of the sequence listing.

[0065] 1. ​ Obtaining the gene CDS Using Chinese spring wheat spikelet cDNA as a template, primers TaSP1-DF and TaSP1-DR were used to... ​ The CDS sequence was amplified.

[0066] TaSP1-DF: 5'-ACGACGCCACAGCAAACTCTCTC-3'; TaSP1-DR: 5'-TTAGTTGATGAGACCCATACACACA-3'.

[0067] PCR amplification reaction system: KOD FX 1 μL, KOD FX Buffer 25 μL, dNTPs 10 μL, cDNA 1 μL, TaSP1-DF 1.5 μL, TaSP1-DR 1.5 μL, H2O 10 μL PCR amplification reaction conditions: 94 ℃ pre-denaturation for 2 min, 98 ℃ denaturation for 10 s, 55 ℃ annealing for 30 s, 68 ℃ extension for 2 min, for a total of 34 cycles, with a final extension of 68 ℃ for 5 min.

[0068] 2. Add a connector The PCR product was diluted 200-fold and used as a template. Primers TaSP1D-pBM27-F and TaSP1D-pBM27-R were used to... ​ The CDS sequence was amplified and adapters were added.

[0069] TaSP1D-pBM27-F: 5'-CACCATGGATGTCGAGTCGTCGAGGCA-3'; TaSP1D-pBM27-R: 5'-TCACGCAGAACCCTTCTCATCCTCC-3'.

[0070] PCR amplification reaction system: 1 μL KOD FX, 25 μL KOD FX Buffer, 10 μL dNTPs, 1 μL PCR product (diluted 200 times), 1.5 μL TaSP1D-pBM27-F, 1.5 μL TaSP1D-pBM27-R, 10 μL H2O.

[0071] PCR amplification reaction conditions: Pre-denaturation at 94 ℃ for 2 min, denaturation at 98 ℃ for 10 s, annealing at 59 ℃ for 30 s, extension at 68 ℃ for 2 min, for a total of 34 cycles, followed by extension at 68 ℃ for 5 min.

[0072] 3. Connection The PCR products were recovered using a gel extraction kit, and the recovered products were ligated into the pBM27 vector to obtain the recombinant vector pBM27-TaSP1-D.

[0073] Enzyme ligation reaction system: pBM27 Vector 1 μL, 10×Topo smart 1 μL, gel recovery product (200 ng / μL) 1 μL, H2O 7 μL.

[0074] Enzyme ligation reaction conditions: 25 ℃ for 15 min.

[0075] The structure of the recombinant vector pBM27-TaSP1-D is described as follows: It is a recombinant vector obtained by inserting a DNA fragment with sequence SEQ ID No:2 from position 182 to 1999 between the 5'-CTTGTGTCGCCCTTCACC-3' and 5'-AAGGGCGACACCGGATC-3' fragments of the starting vector pBM27, while keeping the other sequences of the starting vector pBM27 unchanged.

[0076] 4. Transformation The ligation product was transformed into *E. coli* DH5α and cultured at 37 °C for 10 h. PCR amplification was performed, and single colonies were screened using primers M13-F and M13-R: M13-F: 5'-GTTGTAAAACGACGGCCAG-3'; M13-R: 5'-CAGGAAACAGCTATGAC-3'. Positive single colonies were selected for sequencing.

[0077] Add 10 μL of the ligation product to E. coli DH5α competent cells and gently mix. After incubating on ice for 30 min, heat shock in a 42 ℃ water bath for 60 s. Quickly transfer to ice and incubate on ice for 2 min. Add 1 ml of antibiotic-free LB liquid medium and incubate in a shaker at 37 ℃ for 45 min. Then spread the bacterial culture onto LB solid medium supplemented with spectinomycin.

[0078] Colony PCR amplification reaction system: 7.5 μL Green Taq Mix, 0.3 μL M13-F, 0.3 μL M13-R, 6.9 μL H2O Colony PCR amplification reaction conditions: 95 ℃ pre-denaturation for 2 min, 95 ℃ denaturation for 30 s, 55 ℃ annealing for 30 s, 72 ℃ extension for 2 min, for 28 cycles, followed by a 72 ℃ extension for 5 min. 5. ​ Cloning to PC186 vector Single colonies matching the reference sequence were selected through sequence alignment. Plasmids were then extracted and processed using the Gateway system. ​ Cloned into the PC186 vector.

[0079] Reaction system: LR Enzyme Mix 2 μL, pBM27-TaSP1-D 2 μL, PC186 Vector 2 μL, H2O4 μL.

[0080] Reaction conditions: 25 °C for 1 hour.

[0081] 6. Transformation The ligation product was transformed into *E. coli* DH5α and cultured at 37 °C for 10 h. PCR amplification was performed, and single-clone colonies were screened using primers PC186-F and TaSP1D-PC186-R. Positive single-clone colonies were selected for sequencing.

[0082] Transformation method: 10 μL of ligation product was added to E. coli DH5α competent cells and gently mixed. After incubating on ice for 30 min, the cells were heat-shocked in a 42 ℃ water bath for 60 s. The cells were then quickly transferred to ice and incubated on ice for 2 min. 1 ml of antibiotic-free LB liquid medium was added and cultured in a shaker at 37 ℃ for 45 min. The bacterial culture was then spread on LB solid medium supplemented with kanamycin.

[0083] Colony PCR amplification reaction system: Green Taq Mix 7.5 μL, PC186-F 0.3 μL, TaSP1D-pC186-R 0.3 μL, H2O 6.9 μL.

[0084] The colony PCR amplification reaction conditions are the same as in step 4.

[0085] PC186-F: 5'-CTGCCTTCATACGCTATTTATTTGC-3'; TaSP1D-PC186-R: 5'-CCGCGTTGAAGTAGCTGGAGAGCC-3'.

[0086] Positive monoclonal colonies were selected for sequencing verification. Plasmids were extracted from the correctly sequenced monoclonal colonies for later use and named PC186-TaSP1-D. The nucleotide sequence of the recombinant vector PC186-TaSP1-D is SEQ ID No:10.

[0087] The structure of the recombinant vector PC186-TaSP1-D is described as follows: It is a recombinant vector obtained by inserting a DNA fragment with sequence SEQ ID No:2 from position 182 to 1999 between the 5'-CTCCAAGCTTGTGTCGCCCTTCACC-3' and 5'-CAGCTTTCTTGTACAAAGTGGTGATC-3' fragments of the starting vector PC186, while keeping the other sequences of the starting vector PC186 unchanged.

[0088] Example 2: Construction of the knockout vector 1. Target sequence selection ​ The coding sequence (CDS) of the wheat variety Chinese Spring is SEQ ID No:5, encoding the TaSP1-A protein with the amino acid sequence SEQ ID No:4. The genomic gene encoding the TaSP1-A protein in the genomic DNA of the wheat variety Chinese Spring is shown in SEQ ID No:6 of the sequence listing.

[0089] ​ The coding sequence (CDS) of the gene in the wheat variety Chinese Spring is SEQ ID No:8, encoding the TaSP1-B protein with the amino acid sequence SEQ ID No:7. The genomic gene encoding the TaSP1-B protein in the genomic DNA of the wheat variety Chinese Spring is shown in SEQ ID No:9 of the sequence listing.

[0090] ​ The coding sequence (CDS) of the gene in the wheat variety Chinese Spring is SEQ ID No:2, encoding the TaSP1-D protein with the amino acid sequence SEQ ID No:1. The genomic gene encoding the TaSP1-D protein in the genomic DNA of the wheat variety Chinese Spring is shown in SEQ ID No:3 of the sequence listing.

[0091] exist ​ Two CRISPR / Cas9 target sites were selected from the conserved sequences to design two sgRNAs, sgRNA1 and sgRNA2: 1) The target sequence of sgRNA1 is: 5'-GCACTTCCCGCTGTCCAAGTCGG-3', located at... ​The target sequence of sgRNA1 in exon 2 of the gene is positions 2076-2098 of SEQ ID No:3 (corresponding to positions 406-428 of SEQ ID No:2); located in ​ The target sequence of sgRNA1 in exon 2 of the gene is positions 2205-2227 of SEQ ID No:6 (corresponding to positions 399-421 of SEQ ID No:5); located at ​ The target sequence of exon 2 of the gene, sgRNA1, is positions 2083-2105 of SEQ ID No:9 (corresponding to positions 399-421 of SEQ ID No:8).

[0092] 2) The target sequence of sgRNA2 is 5'-CCGGCGTGCGACATGAAGGCGGC-3', located at... ​ The target sequence of sgRNA2 in exon 3 of the gene is positions 2705-2727 of SEQ ID No:3 (corresponding to positions 599-621 of SEQ ID No:2); located at ​ The target sequence of sgRNA2 in exon 3 of the gene is positions 2833-2855 of SEQ ID No:6 (corresponding to positions 592-614 of SEQ ID No:5); located in ​ The target sequence of sgRNA2 in exon 3 of the gene is positions 2699-2721 of SEQ ID No:9 (corresponding to positions 592-614 of SEQ ID No:8).

[0093] Using plasmid pCBC-MT1T2 as a template, four-primer PCR amplification was performed using primers TaSP1-MT1T2-F, TaSP1-MT1T2-F0, TaSP1-MT1T2-R0, and TaSP1-MT1T2-R. The PCR products were then recovered from the gel.

[0094] Reaction system: KOD Plus 1 μL, KOD Plus Buffer 5 μL, dNTPs 4 μL, MgSO43 μL, pCBC-MT1T2 1 μL, TaSP1-MT1T2-F (10 μM) 2 μL, TaSP1-MT1T2-F0 (1 μM) 1 μL, TaSP1-MT1T2-R (10 μM) 2 μL, TaSP1-MT1T2-R0 (1μM) 1 μL, H2O 30 μL Reaction conditions: 94 °C pre-denaturation for 2 min, 94 °C denaturation for 15 s, 60 °C annealing for 30 s, 68 °C extension for 1 min, for 30 cycles, followed by 68 °C extension for 5 min.

[0095] Primer sequences: TaSP1-MT1T2-F: 5'-AATAATGGTCTCAAGCG ​ -3'; TaSP1-MT1T2-F0:5'-G ​ GTTTTAGAGCTAGAAATAGA-3'; TaSP1-MT1T2-R0:5'- ​ CGCTTCTTGGTGCC-3'; TaSP1-MT1T2-R: 5'-ATTATTGGTCTCTAAAC ​ -3'.

[0096] 2. The gel-recovered product was ligated into the pBUE413 vector (simultaneous cleavage and ligation reaction), and the ligation product was transformed into Escherichia coli DH5α and cultured at 37 ℃ for 10 h.

[0097] Simultaneous cleavage and ligation reaction system: gel recovery product (100 ng / μL) 2 μL, pBUE413 2 μL, 10x T4 DNALigase Buffer 1.5 μL, 10x BSA 1.5 μL, ​ 1 μL, T4 DNA Ligase 1 μL, H2O 6 μL.

[0098] The reaction conditions for simultaneous cutting and connecting were: 37 °C for 5 hours, 50 °C for 5 minutes, and 80 °C for 10 minutes.

[0099] 3. PCR amplification was performed, and single-clone colonies were screened using primers TaU3p-411-seq-F and TaU3p-411-seq-R. Positive single-clone colonies were selected for sequencing.

[0100] Colony PCR reaction system: Green Taq Mix 7.5 μL, TaU3p-411-seq-F 0.3 μL, TaU3p-411-seq-R 0.3 μL, H2O 6.9 μL.

[0101] The colony PCR reaction conditions are the same as step 4 in Example 1.

[0102] Primer sequences: TaU3p-411-seq-F: 5'-TTTCCCAGTCACGACGTTGT-3'; TaU3p-411-seq-R: 5'-ATCTCTAGAGAGGGGCACGA-3'.

[0103] Positive monoclonal colonies were selected for sequencing verification. Plasmids were extracted from the monoclonal colonies that were correctly sequenced and reserved for use. The plasmids were named pBUE413-TaSP1 and the nucleotide sequence of the recombinant vector pBUE413-TaSP1 is SEQ ID No:11.

[0104] Example 3: Creation of transgenic plants 1. PC186-TaSP1-D and pBUE413-TaSP1 were transformed into EHA105 Agrobacterium competent cells by electroporation to obtain EHA105 / PC186-TaSP1-D and EHA105 / pBUE413-TaSP1.

[0105] Electroconvulsive therapy: Agrobacterium EHA105 was thawed on ice, and 1 μL of plasmid (100 ng / μL) was added, followed by an ice bath for 10 min. The bacterial culture containing the plasmid was then transferred to an ice-cold electroporation cuvette, which was then transferred to an electroporator with the following parameters: C=25 μF, PC=200 ohm, V=2400 V. After electroporation, 1 mL of antibiotic-free LB broth was added and cultured at 28 °C with shaking for 2 h. Then, 50 μL of the bacterial culture was spread onto LB solid medium containing kanamycin and rifampin and incubated at 28 °C for 48 h.

[0106] 2. Agrobacterium infection Wheat genetic transformation methods: Fielder wheat was cultivated, and immature embryos were harvested 14 days after flowering for transformation. Agrobacterium strains EHA105 / PC186-TaSP1-D and EHA105 / pBUE413-TaSP1 containing PC186-TaSP1-D and pBUE413-TaSP1 were used to infect the immature embryos. The embryos were then cultured in WLS-AS medium for two days. After removing the hypocotyls and exophytes, the embryos were transferred to WLS-Res medium and cultured for another 5 days. The explants were then transferred to WLS-P5 medium and cultured for 14 days. Subsequently, the explants were divided into two parts and transferred to WLS-P10 medium for 21 days to induce callus formation. The callus was then transferred to LSZ-P5 medium to induce aboveground regeneration. The regenerated portion was transferred to LSF-P5 medium for rooting. The transformed wheat plants were then planted in a greenhouse.

[0107] 3. DNA was extracted from the obtained mutants, and the mutation status of the knockout mutants was identified by PCR amplification and sequencing. The obtained homozygous triple mutant was named... ​ and ​ .

[0108] PCR reaction system: Green Taq Mix 25 μL, TaSP1-A / B / D-target-F 1.5 μL, TaSP1-A / B / D-target-R1.5 μL, H2O 20 μL, DNA 2 μL.

[0109] Primer sequences: TaSP1-A-target-F: 5'-TAAATAACCGGCCAAGTTGCCTAC-3'; TaSP1-A-target-R: 5'-ATTGAATGCCCATGGTAACTTTGGC-3'; TaSP1-B-target-F: 5'-AGTACCAGTAGTAAACAACAGAGA-3'; TaSP1-B-target-R: 5'-GGTGGCTTTGTTCCGGTAGAAGAAA-3'; TaSP1-D-target-F: 5'-CAAGTTGCCTAGAATTTACCAAGACG-3'; TaSP1-D-target-R: 5'-CGGTGGCTTGTTCCGGTAGAAGAC-3'.

[0110] Identified by sequencing ( ​ Compared to the genomic DNA of wild-type Fielder, the mutation type is: ​ ​ In the chromosome, the gene encoding the TaSP1-D protein underwent the following mutation: nucleotide "A" was deleted at position 2093 of SEQ ID No:3 (corresponding to position 242 of SEQ ID No:2), and a nucleotide "T" was inserted between nucleotides 2711 and 2712 (corresponding to positions 424 and 425 of SEQ ID No:2), resulting in an alteration in the amino acid sequence of the gene, thereby... ​ Gene knockout; the gene encoding the TaSP1-A protein underwent the following mutations: deletion of nucleotides "CA" at positions 2220 and 2221 of SEQ ID No:6 (corresponding to positions 240 and 241 of SEQ ID No:5), and deletion of nucleotides "GT" at positions 2838 and 2839 (corresponding to positions 423 and 424 of SEQ ID No:5), resulting in an amino acid sequence mutation in the gene, thereby... ​Gene knockout; the gene encoding the TaSP1-B protein underwent the following mutation: "nucleotide sequence 2098 to 2704 of SEQ ID No:9 was mutated to nucleotide "A" (corresponding to positions 240 to 423 of SEQ ID No:8), resulting in a mutation in the amino acid sequence of the gene, thereby..." ​ Gene knockout; sequencing results of the mutation site and surrounding nucleotides are shown in [link to sequencing results]. ​ .

[0111] Compared to the genomic DNA of wild-type Fielder, the mutation type is: ​ In the chromosome, the gene encoding the TaSP1-D protein underwent the following mutation: nucleotide "A" was deleted at position 2093 of SEQ ID No:3 (corresponding to position 242 of SEQ ID No:2), and a nucleotide "T" was inserted between nucleotides 2711 and 2712 (corresponding to positions 424 and 425 of SEQ ID No:2), resulting in an alteration in the amino acid sequence of the gene, thereby... ​ Gene knockout; the gene encoding the TaSP1-A protein underwent the following mutations: deletion of nucleotides "CA" at positions 2220 and 2221 of SEQ ID No:6 (corresponding to positions 240 and 241 of SEQ ID No:5), and deletion of nucleotides "GT" at positions 2838 and 2839 (corresponding to positions 423 and 424 of SEQ ID No:5), resulting in an amino acid sequence mutation in the gene, thereby... ​ Gene knockout; the gene encoding the TaSP1-B protein underwent the following mutation: "The deletion of nucleotides 2096 to 2099 'TCCA' in SEQ ID No:9 (corresponding to positions 238 to 241 in SEQ ID No:8) resulted in a mutation in the amino acid sequence of the gene, thereby..." ​ Gene knockout; sequencing results of the mutation site and surrounding nucleotides are shown in [link to sequencing results]. ​ .

[0112] The selected three-protrusion materials ​ and ​ Propagate the seeds and harvest homozygous seeds for subsequent experiments.

[0113] 4. RNA was extracted from the leaves of the obtained overexpression plants, and cDNA was obtained by reverse transcription. Real-time quantitative PCR was used to verify whether TaSP1-D was upregulated in the overexpression plants. The obtained overexpression plants were named. ​ and ​ .

[0114] The real-time PCR reaction system consisted of: ArtiCanATM SYBR qPCR Mix 5 μL, TaSP1-D-qF 0.5 μL, TaSP1-D-qR 0.5 μL, H2O 3 μL, and cDNA 3 μL.

[0115] The quantitative PCR reaction program was as follows: 95 °C for 1 min, 95 °C for 10 s, 60 °C for 20 s, for 40 cycles.

[0116] Primer sequences for quantitative fluorescence: TaSP1-D-qF: 5'-AGGCGTTTGAGATCATGGCGATT -3'; TaSP1-D-qR: 5'-AGAGGAGGAACATGGTGCCG-3'.

[0117] ​ For Fielder, ​ as well as ​ middle ​ Gene expression levels, by ​ It can be known ​ Genes in ​ as well as ​ The middle part was significantly increased. ​ ​ and ​ for ​ Overexpression of the plant.

[0118] Example 4: Drought-resistant phenotypes of TaSP1D mutants and overexpression strains The plant under test: a homozygous three-mutant. ​ 1 and ​ Overexpression plants ​ ​ and p35S::TaSP1D-2 Wild wheat Fielder (WT).

[0119] 1. Place Fielder, Tasp1-aabbdd2 ,as well as p35S::TaSP1D-1 During the greenhouse cultivation period, soil moisture content was measured (6 replicates for each line), and phenotypic observations and photographs were taken. After a drought treatment, soil moisture content was measured again 5 days later (6 replicates for each line), and phenotypic observations and photographs were taken again.

[0120] 2. Place Fielder, Tasp1-aabbdd1 , Tasp1-aabbdd2 , p35S::TaSP1D-1 as well as p35S:: TaSP1D-2Planting was carried out in normally irrigated fields (irrigated 3 times after sowing, during the seedling stage, and during the heading stage) and in dry fields (irrigated once after sowing). After maturity, the yield per plant was counted (each plant was counted as 8 or more plants), and the yield ratio between dry fields and normally irrigated fields was calculated.

[0121] The results are as follows: 1) Compared with normal irrigation conditions, the soil moisture content of wheat under drought stress was significantly reduced, indicating that the drought treatment was successful. Under normal irrigation conditions, Fielder... Tasp1-aabbdd2 as well as p35S::TaSP1D-1 There was no significant difference in wheat plants under drought stress. Tasp1-aabbdd2 The degree of leaf wilting was significantly increased compared to the wild-type Fielder. p35S::TaSP1D-1 The degree of leaf wilting was significantly reduced compared to the wild-type Fielder. Figure 3 (c).

[0122] 2) Under normal watering conditions, Tasp1-aabbdd The yield per plant was significantly lower than that of the wild type. p35S:: TaSP1D-1 as well as p35S::TaSP1D-2 The yield per plant was not significantly different from that of the wild type; under drought stress conditions Tasp1-aabbdd The yield per plant was significantly lower than that of the wild type. p35S::TaSP1D The yield per plant was significantly increased compared to the wild type. Figure 4 (ad). Further calculations of the yield per plant under drought conditions versus normal conditions revealed... Tasp1-aabbdd The yield was significantly lower than that of the wild type. p35S::TaSP1D The yield ratio was significantly increased compared to the wild type, indicating that... TaSP1 Positive regulation of wheat drought resistance ( Figure 5 ).

[0123] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. The use of a protein or a substance that regulates the expression of a gene encoding the protein or a substance that regulates the activity or content of the protein in any of the following; 1) Application in regulating plant drought resistance; 2) Application in the preparation of products that regulate plant drought resistance; 3) Application in cultivating plants with altered drought resistance; 4) Application in the preparation of products using plants with altered drought resistance; 5) Applications in plant breeding; The protein is any of the following proteins: a1) Proteins with the amino acid sequence SEQ ID No:1, SEQ ID No:4 or SEQ ID No:7; a2) A protein having the same function as the amino acid sequence shown in SEQ ID No:1, SEQ ID No:4 or SEQ ID No:7, by substitution and / or deletion and / or addition of one or more amino acid residues. a3) Proteins that share more than 80% identity with the amino acid sequence defined by a1) or a2) and have the same function; a4) A fusion protein obtained by attaching a tag to the end of any of the proteins defined in a1)-a3).

2. The application according to claim 1, characterized in that, The protein is derived from wheat.

3. The application according to claim 1 or 2, characterized in that, The substance that regulates gene expression or the substance that regulates the activity or content of the protein is a biological material related to the protein in the application of claim 1 or 2, and the biological material is any one of the following: c1) The nucleic acid molecule encoding the protein; c2) An expression cassette containing the nucleic acid molecule described in c1); c3) A recombinant vector containing the nucleic acid molecule described in c1), or a recombinant vector containing the expression cassette described in c2); c4) Recombinant microorganisms containing the nucleic acid molecules described in c1), or recombinant microorganisms containing the expression cassette described in c2), or recombinant microorganisms containing the recombinant vector described in c3); c5) A transgenic plant cell line containing the nucleic acid molecule described in c1), or a transgenic plant cell line containing the expression cassette described in c2); c6) Transgenic plant tissue containing the nucleic acid molecules described in c1), or transgenic plant tissue containing the expression cassette described in c2); c7) A transgenic plant organ containing the nucleic acid molecule described in c1), or a transgenic plant organ containing the expression cassette described in c2); e1) Nucleic acid molecules that inhibit, reduce, or silence the expression of the protein-encoding gene; e2) An expression cassette containing the nucleic acid molecule described in e1); e3) A recombinant vector containing the nucleic acid molecule described in e1), or a recombinant vector containing the expression cassette described in e2); e4) Recombinant microorganisms containing the nucleic acid molecules described in e1), or recombinant microorganisms containing the expression cassette described in e2), or recombinant microorganisms containing the recombinant vector described in e3); e5) A transgenic plant cell line containing the nucleic acid molecule described in e1), or a transgenic plant cell line containing the expression cassette described in e2); e6) Transgenic plant tissue containing the nucleic acid molecules described in e1), or transgenic plant tissue containing the expression cassette described in e2); e7) A transgenic plant organ containing the nucleic acid molecule described in e1) or a transgenic plant organ containing the expression cassette described in e2).

4. The application according to claim 3, characterized in that, c1) The nucleic acid molecule is any of the following DNA molecules: d1) The nucleotide sequence is a DNA molecule shown in SEQ ID No:3, SEQ ID No:6 or SEQ ID No:9; d2) The coding sequence is a DNA molecule shown in SEQ ID No:2, SEQ ID No:5, or SEQ ID No:8; d3) has 90% or more identity with the nucleotide sequence defined by d1) or d2) and is a DNA molecule encoding the protein of claim 1; d4) Hybridizes under stringent conditions to a nucleotide sequence defined by d1) or d2) and encodes a DNA molecule that encodes the protein of claim 1.

5. A method for altering plant drought resistance, characterized in that: The method includes step M or P, wherein step M is to inhibit or reduce or silence the activity and / or content of the protein described in claim 1 or 2 in the target plant, or / and, inhibit or reduce or downregulate the expression level of the gene encoding the protein described in claim 1 or 2, so as to reduce the drought resistance of the plant. The method includes step P, which is to enhance, increase or upregulate the activity and / or content of the protein described in claim 1 or 2 in the target plant, or / and enhance, increase or upregulate the expression level of the gene encoding the protein described in claim 1 or 2, so as to improve the drought resistance of the plant.

6. A method for cultivating highly drought-resistant plants, characterized in that, This includes enhancing, increasing, or upregulating the expression level of the gene encoding the protein described in claim 1 or 2 in the target plant, and / or, the activity and / or content of the protein to obtain a highly drought-resistant plant, wherein the drought-resistant plant has higher drought resistance than the target plant.

7. The method according to claim 6, characterized in that, The enhancement, improvement, or upregulation of the expression of the gene encoding the protein of claim 1 or 2 in the plant comprises introducing the nucleic acid molecule of claim 4c1) into the target plant to obtain a highly drought-resistant plant.

8. A method for cultivating plants with low drought resistance, characterized in that, This includes inhibiting, reducing, or silencing the expression level of the gene encoding the protein described in claim 1 or 2 in the target plant, and / or, the activity and / or content of the protein, to obtain a low drought-resistant plant, wherein the low drought-resistant plant has lower drought resistance than the target plant.

9. The protein as described in claim 1 or 2 and / or the biomaterial as described in claim 3 or 4.

10. The method according to any one of claims 5-8, characterized in that, The plant is any one of the following: C1) Monocotyledons; C2) Plants of the order Poales; C3) Gramineae plants; C4) Plants of the genus Triticum; C5) Wheat.

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