TaENO1-5D gene and the protein encoded by the gene are applied to regulating drought resistance of wheat

Abstract

The application discloses TaENO1-5D gene and application of the protein coded by the gene in regulating drought resistance of wheat. The cDNA of the TaENO1-5D gene disclosed by the application is shown as SEQ ID No. 1, the genomic sequence is shown as SEQ ID No. 3, and the TaENO1-5D protein coded by SEQ ID No. 2 is shown. Experiments prove that overexpression of the TaENO1-5D gene in plants can significantly inhibit the drought resistance of the plants, and knockout of the TaENO1-5D gene can significantly improve the drought resistance of the plants. It is proved that the TaENO1-5D gene and the protein coded thereby can regulate the drought resistance of the plants, and have great production and application potential.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically the application of the TaENO1-5D gene and its encoded protein in regulating wheat drought resistance. Background Technology

[0002] Drought severely impacts plant growth and development, directly leading to a decline in crop yield and quality. Wheat (Triticum aestivum L.) is one of the world's most widely distributed, cultivated, and highest-yielding food crops, and it is frequently subjected to abiotic stresses such as drought throughout its growth cycle, severely affecting its growth and yield. Plants have developed complex mechanisms to sense and respond to external stresses under constantly changing environmental conditions. Therefore, exploring and utilizing wheat drought-resistance-related gene resources is of great significance for breeding drought-resistant, high-yielding, and stable-yielding wheat varieties and improving wheat breeding standards. Summary of the Invention

[0003] The technical problem to be solved by this invention is how to regulate the drought resistance of plants.

[0004] To address the aforementioned technical problems, the present invention first provides any of the following applications of proteins or substances that regulate the content or activity of said proteins:

[0005] M1) regulates plant drought resistance;

[0006] M2) to prepare products that regulate plant drought resistance;

[0007] M3) Cultivating plants with altered drought resistance;

[0008] M4) Products prepared by cultivating plants with altered drought resistance;

[0009] The protein is derived from wheat (Triticumaestivum L.) and is named TaENO1-5D, which is A1), A2), or A3):

[0010] A1) The amino acid sequence of this protein is SEQ ID No. 2;

[0011] A2) A protein having the same function as the amino acid sequence shown in SEQ ID No. 2 in the sequence listing, with one or more amino acid residues substituted and / or deleted and / or added;

[0012] A3) is a fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of A1) or A2).

[0013] The TaENO1-5D protein in A2) above is a protein that has 75% or more of the same amino acid sequence as the protein shown in SEQ ID No.2 and has the same function.

[0014] Identity refers to the similarity of 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, the identity value (%) can then be obtained. A 75% or higher identity is defined as 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity.

[0015] The tag described in A3) can be a polypeptide or protein fused 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 Poly-Arg, Poly-His, FLAG, Strep-tag II, c-myc, MBP tag, HA tag, GST tag, and / or SUMO tag, etc.

[0016] In the above applications, the substance that regulates the content or activity of TaENO1-5D protein can be any one of the following B1) to B9):

[0017] B1) Nucleic acid molecules encoding the TaENO1-5D protein;

[0018] B2) An expression cassette containing the nucleic acid molecule described in B1);

[0019] B3) A recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2);

[0020] B4) Recombinant microorganisms containing the nucleic acid molecules described in B1), or recombinant microorganisms containing the expression cassette described in B2), or recombinant microorganisms containing the recombinant vector described in B3);

[0021] B5) A transgenic plant cell line containing the nucleic acid molecule described in B1), or a transgenic plant cell line containing the expression cassette described in B2);

[0022] B6) Transgenic plant tissue containing the nucleic acid molecules described in B1), or transgenic plant tissue containing the expression cassette described in B2);

[0023] B7) Transgenic plant organs containing the nucleic acid molecules described in B1), or transgenic plant organs containing the expression cassette described in B2);

[0024] B8) Nucleic acid molecules that reduce the content of TaENO1-5D protein;

[0025] B9) Expression cassettes, recombinant vectors, recombinant microorganisms, transgenic plant cell lines, transgenic plant tissues, or transgenic plant organs containing the nucleic acid molecules described in B8).

[0026] In the above applications, the nucleic acid molecule described in B1) can be any one of the following: (b11)-(b15)

[0027] b11) The coding sequence is a cDNA molecule or DNA molecule located at positions 148-1488 of SEQ ID No. 1 in the sequence listing;

[0028] b12) The cDNA molecule or DNA molecule shown in SEQ ID No. 1 of the sequence listing;

[0029] b13) The DNA molecule shown in SEQ ID No. 3 of the sequence listing;

[0030] b14) has 75% or more identity with any of the defined nucleotide sequences in b11)-b13) and is a DNA molecule encoding the TaENO1-5D protein.

[0031] b15) hybridizes under strict conditions with any of the defined nucleotide sequences in b11)-b14) and is a DNA molecule encoding the TaENO1-5D protein.

[0032] 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.

[0033] Those skilled in the art can readily mutate the nucleotide sequence encoding the TaENO1-5D protein of this invention using known methods, such as directed evolution and point mutation. Artificially modified nucleotides having 75% or higher identity with the nucleotide sequence of the TaENO1-5D protein of this invention, provided they encode and function the TaENO1-5D protein, are derived from and equivalent to the nucleotide sequence of this invention.

[0034] As used herein, the term "identity" refers to sequence similarity to a natural nucleic acid sequence. "Identity" includes nucleotide sequences that have 75% or higher, 85% or higher, 90% or higher, or 95% or higher identity with the nucleotide sequence encoding the amino acid sequence shown in SEQ ID No. 2 of this invention. Identity can be evaluated 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 evaluate the identity between related sequences.

[0035] In the above applications, the stringent conditions may be as follows: hybridization at 50°C in a mixed solution of 7% sodium dodecyl sulfate (SDS), 0.5M NaPO4 and 1mM EDTA, followed by rinsing at 50°C in 2×SSC and 0.1% SDS.

[0036] The aforementioned 75% or higher degree of identity can be 80%, 85%, 90%, or 95% or higher degree of identity.

[0037] In the above application, the expression cassette (TaENO1-5D gene expression cassette) containing a nucleic acid molecule encoding the TaENO1-5D protein described in B2) refers to DNA capable of expressing the TaENO1-5D protein in host cells. This DNA may include not only a promoter to initiate TaENO1-5D gene transcription but also a terminator to terminate TaENO1-5D gene transcription. Furthermore, the expression cassette may also include an enhancer sequence.

[0038] B3) The vector may be a plasmid, granule, bacteriophage, or viral vector. Specifically, the plasmid may be the pWMB110 vector.

[0039] B3) The recombinant vector may specifically be pWMB110-TaENO1-5D, which is a wheat vector that overexpresses TaENO1-5D by inserting the DNA molecule shown in SEQ ID No. 1 from the 5′ end of the sequence listing into the binary vector pWMB110 using BamHI, while keeping the other nucleotides of the binary vector pWMB110 unchanged.

[0040] B8) The nucleic acid molecule that reduces the content of TaENO1-5D protein may be an sgRNA that targets the TaENO1-5D gene.

[0041] The target sequence of the sgRNA may be positions 1085-1103 or 2484-2502 of SEQ ID No. 3.

[0042] B9) The vector may be a plasmid, granule, bacteriophage, or viral vector. Specifically, the plasmid may be pCBC-MT1T2 or pBUE414.

[0043] B9) The recombinant vector may be a recombinant vector prepared using the CRISPR-Cas9 system capable of editing the TaENO1-5D gene. The recombinant vector can transcribe and express the sgRNA, i.e., the sgRNA targeting the nucleic acid molecule described in B1).

[0044] In one embodiment of the present invention, the recombinant vector described in B9) may be the TaENO1 gene-editing wheat vector in Example 1.

[0045] In the above applications, the microorganisms can be yeast, bacteria, algae, or fungi. Among them, bacteria can be Agrobacterium, such as Agrobacterium EHA105.

[0046] In the above applications, the transgenic plant cell lines, transgenic plant tissues, and transgenic plant organs do not include propagation material.

[0047] In the above applications, the substance that regulates the content or activity of TaENO1-5D protein is a substance that reduces the content or activity of TaENO1-5D protein; the substance that regulates plant drought resistance is a substance that increases the drought resistance of the plant; and the substance that cultivates plants with altered drought resistance is a substance that cultivates plants with improved drought resistance.

[0048] The present invention also provides any of the following methods:

[0049] X1) Methods to improve plant drought resistance include: reducing the content or activity of TaENO1-5D protein in the recipient plant, or knocking out the gene encoding TaENO1-5D protein in the recipient plant, or reducing the expression level of the gene encoding TaENO1-5D protein in the recipient plant, to obtain a target plant with improved drought resistance compared to the recipient plant.

[0050] X2) Methods for cultivating plants with improved drought resistance include: reducing the content or activity of TaENO1-5D protein in recipient plants, or knocking out the gene encoding TaENO1-5D protein in recipient plants, or reducing the expression level of the gene encoding TaENO1-5D protein in recipient plants, to obtain target plants with improved drought resistance compared with the recipient plants, thereby improving the drought resistance of plants.

[0051] In the above method, the encoding gene can be the nucleic acid molecule described in B1).

[0052] In the above method, the gene encoding the TaENO1-5D protein can be knocked out using the CRISPR-Cas9 system.

[0053] Gene editing of the coding gene using the CRISPR-Cas9 system can be achieved by introducing a recombinant vector (such as B9) encoding Cas9 and capable of transcribing sgRNA that targets the coding gene into the recipient plant and screening to obtain the target plant in which the coding gene has been edited.

[0054] In one embodiment of the present invention, the TaENO1-5D gene of the target plant is deleted at positions 2498-2503 of SEQ ID No. 3.

[0055] In one embodiment of the present invention, the TaENO1-5D gene of the target plant is deleted at positions 2498-2499 of SEQ ID No. 3.

[0056] The recombinant vector can be introduced into plant cells using conventional biotechnological methods such as Ti plasmids, plant virus vectors, direct DNA transformation, microinjection, and electroporation (Weissbach, 1998, Method for Plant Molecular Biology VIII, Academy Press, New York, pp. 411-463; Geiserson and Corey, 1998, Plant Molecular Biology (2nd Edition)).

[0057] The target plant is understood to include not only first-generation plants containing the altered gene encoding the TaENO1-5D protein, but also their progeny. For the target plant, the gene can be propagated within the species, or it can be transferred into other varieties of the same species, particularly commercial varieties, using conventional breeding techniques. The target plant includes seeds, callus tissue, intact plants, and cells.

[0058] In this invention, the plant may be N1, N2, or N3:

[0059] N1) Monocotyledonous or dicotyledonous plants;

[0060] N2) Gramineae plants;

[0061] N3) Wheat.

[0062] The present invention also provides a product for regulating plant drought resistance, the product containing (or having its active ingredient as) TaENO1-5D protein, or a substance for regulating the content or activity of TaENO1-5D protein.

[0063] Experiments have shown that overexpression of the TaENO1-5D gene in plants can significantly inhibit drought resistance, while knockout of the TaENO1-5D gene can significantly improve drought resistance. Further analysis of drought-related indicators revealed that, compared with the wild type, TaENO1-5D overexpressing lines showed decreased cell membrane stability and leaf water content after drought treatment, with faster leaf water loss and increased malondialdehyde (MDA) content. Conversely, compared with the wild type, gene knockout lines showed increased cell membrane stability and leaf water content, decreased leaf water loss, and decreased MDA content. This indicates that the TaENO1-5D protein and its encoding gene can regulate plant drought resistance and have great potential for industrial application.

[0064] 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. Attached Figure Description

[0065] Figure 1 The results show the detection of TaENO1-5D gene expression in the overexpression line.

[0066] Figure 2 This refers to sequence changes in gene-edited strains.

[0067] Figure 3 The results are for drought resistance testing. A shows the phenotypes of TaENO1-5D overexpressing lines and wild-type under drought stress; B shows the phenotypes of gene-edited lines and wild-type under drought stress; C shows the seedling survival rate of TaENO1-5D overexpressing lines and wild-type under drought stress; and D shows the seedling survival rate of gene-edited lines and wild-type under drought stress.

[0068] Figure 4 To compare the physiological indicators of wheat before and after drought stress, the following comparisons were made between TaENO1-5D overexpressing lines, gene-edited lines, and wild-type detached leaf water loss rate (A, B), leaf water content (C, D), cell membrane stability (E, F), and malondialdehyde content (G, H) under normal (CK) and drought stress conditions. Detailed Implementation

[0069] 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, reagents, instruments, etc., used in the following examples are all commercially available. All quantitative experiments in the following examples were performed in at least three replicates, and the results were averaged. Unless otherwise specified, in the following examples, the first position of each nucleotide sequence in the sequence listing is the 5′ terminal nucleotide of the corresponding DNA / RNA, and the last position is the 3′ terminal nucleotide of the corresponding DNA / RNA.

[0070] Fielder wheat: a hexaploid common wheat, bred in the United States in 1974, commonly used for Agrobacterium-mediated transformation and gene editing receptors. Reference: Sato K, Abe F, Mascher M, Haberer G, Gundlach H, Spannagl M, Shirasawa K, Isobe S. Chromosome-scale genome assembly of the transformation-amenable common wheat cultivar 'Fielder'. DNA Res. 2021 Jun 25;28(3):dsab008.doi:10.1093 / dnares / dsab008.PMID:34254113;PMCID:PMC8320877.

[0071] Hanxuan No. 10 wheat: National Germplasm Bank (http: / / www.cgris.net / ), No.: ZM009279.

[0072] Agrobacterium tumefaciens EHA105: Reference: Torisky RS, Kovacs L, Avdiushko S, Newman JD, Hunt AG, Collins GB. Development of a binary vector system for plant transformation based on the supervirulent Agrobacterium tumefaciens strain Chry5. Plant Cell Reports, (1997) 17: 102-108.; It can be obtained from the Institute of Crop Science, Chinese Academy of Agricultural Sciences.

[0073] Example 1: The TaENO1-5D gene can regulate the drought resistance of wheat.

[0074] I. Cloning of the TaENO1-5D gene

[0075] 1. Following the method in the literature (Mao Xinguo et al., 2005, Construction of a full-length cDNA library of Aegilops speltii using an improved Cap-trapper method. Acta Genetica Sinica, 32(8): 811-817), a full-length cDNA library of wheat was constructed using Hanxuan No. 10 wheat.

[0076] 2. Sequence analysis, segment extraction, and functional verification were performed on the full-length wheat cDNA to obtain candidate clones. Sequencing yielded the full-length sequence of the target clone, as shown in SEQ ID No. 1 of the sequence listing, which encodes the protein shown in SEQ ID No. 2 of the sequence listing.

[0077] The protein shown in SEQ ID No. 2 of the sequence listing is named TaENO1-5D, consisting of 446 amino acid residues. The gene encoding TaENO1-5D is named the TaENO1-5D gene. The full-length cDNA of the TaENO1-5D gene is shown in SEQ ID No. 1 of the sequence listing (1680 bp), with nucleotides 1-147 from the 5′ end forming the 5′ UTR (147 bp), nucleotides 148-1488 forming the open reading frame (1341 bp), and nucleotides 1489-1680 forming the 3′ UTR (192 bp). The genomic sequence of the TaENO1-5D gene is shown in SEQ ID No. 3.

[0078] II. Preparation of Genetically Modified Wheat

[0079] 1) Construction of recombinant overexpression vectors

[0080] 1. The pWMB110 vector was used as the original vector. The pWMB110 vector is described in the following literature: Liu H, Wang K, Jia Z, Gong Q, Lin Z, Du L, Pei X, Ye X. Efficient induction of haploid plants inwheat by editing of TaMTL using an optimized Agrobacterium-mediated CRISPR system. J Exp Bot. 2020 Feb 19; 71(4):1337-1349. doi:10.1093 / jxb / erz529.PMID:31760434;PMCID:PMC7031065. In this literature, it is called The plasmid pWMB110 or pWMB110.

[0081] 2. Total RNA was extracted from mixed tissue samples of dry-selected wheat variety 10 and reverse transcribed to obtain cDNA. Using the cDNA as a template, PCR amplification was performed using primers F and R to obtain the PCR product (sequencing revealed as the DNA molecule represented by nucleotides 96-1666 from the 5′ end of SEQ ID No. 1). F: 5′-CCTCCGCTCCATTCGTTT-3′; R: 5′-ATTGGAGCAAGTTTGGAG-3′. The PCR product was then purified and recovered, and ligated into the intermediate vector T. The plasmid was extracted, sequenced correctly, and then used for later use.

[0082] 3. Using the plasmid obtained in step 2 as a template, PCR amplification was performed using the primer pair composed of F1 and R1. A high-fidelity Pfu amplification system was selected to obtain the PCR amplification product. F1: 5′-CGACTCTAGA GGATCC ATGGCGGCGACGATCC-3′; R1: 5′-AGCTCCGGTACCCGG GGATCC GTATGGCTCCACCGGTGC-3′; In F1 and R1, the underline marks the BamHI restriction site; the sequence in front of the underline (5′ side) is the homologous arm (the same fragment as the sequence near the insertion site of the vector pWMB110).

[0083] 4. Digest the vector pWMB110 with the restriction endonuclease BamHI and recover approximately 10 kb of the vector backbone.

[0084] 5. The PCR product containing homologous arms obtained in step 3 and the vector backbone (10kb vector backbone) from step 4 were subjected to homologous recombination. This involved mixing the seamless ligase with both and incubating at 37°C for 30 min. Following standard transformation, the recombinant expression vector pWMB110-TaENO1-5D was obtained. The seamless ligase In-fusion was a product of TaKaRa (catalog number: 638947).

[0085] Sequencing results of pWMB110-TaENO1-5D showed that it was a DNA molecule obtained by inserting nucleotides 148-1485 from the 5' end of SEQ ID No. 1 into the binary vector pWMB110 using BamHI, while keeping the other nucleotides of the binary vector pWMB110 unchanged, thus obtaining a wheat vector overexpressing TaENO1-5D.

[0086] 6. Obtain transgenic wheat varieties OE-2, OE-5, and OE-10 overexpressing the TaENO1-5D gene.

[0087] After introducing pWMB110-TaENO1-5D into Agrobacterium tumefaciens EHA105, it was transformed into the wheat variety Fielder via Agrobacterium-mediated genetic transformation. Homozygous lines were obtained through self-pollination. The relative expression level of the TaENO1-5D gene in the T3 generation plants (OE-2, OE-5, and OE-10) of each test line was detected using qRT-PCR (with Actin as an internal reference gene). The expression of the internal reference gene Actin was detected using primer pairs consisting of F2 (5′-CTCCCTCACAACAACAACCGC-3′) and R2 (5′-TACCAGGAACTTCCATACCAAC-3′), and the expression of the TaENO1-5D gene was detected using primer pairs consisting of F3 (5′-TGAGGACCCATTTGACCAGG-3′) and R3 (5′-GCACCAACCCCACTCATTCT-3′). The results are as follows: Figure 1 As shown, the recipient material Fielder (WT) showed very low expression of the TaENO1-5D gene, while the overexpression lines OE-2, OE-5, and OE-10 showed significantly higher TaENO1-5D gene expression compared to the Fielder material. The next step will be to assess stress resistance using OE-2, OE-5, and OE-10.

[0088] II) Construction of Recombinant Gene Editing Vectors

[0089] 1. Predict the editing site of the TaENO1 gene using an online website (http: / / www.e-crisp.org / E-CRISP / ).

[0090] 2. Design the target sites of the TaENO1-5D gene (i.e., positions 1085-1103 and 2484-2502 of SEQ ID No. 3) based on the TaENO1-5D genome SEQ ID No. 3. Then, design four primers according to the vector usage instructions, specifically: upstream primer F (5′-aataatggtctcAGGCgTTGATGTGTGCTGCTCAGA-3′), F0 (5′-gTTGATGTGTGCTGCTCAGAgttttagagctagaaatagc-3′) and downstream primer R (5′-ATTATTGGTCTCTAAACGGATTGCATTAGCACCAAG-3′), R0 (5′-GGATTGCATTAGCACCAAGCGCTTCTTGGTGCC-3′).

[0091] 3. Dissolve and mix four primers (TaENO1-MT1T2 F / F0 / R0 / R), using the pCBC-MT1T2 vector as a template and TaENO1-MT1T2 F / F0 / R0 / R as primers to obtain PCR products; use T4 DNA Ligase and BsaI restriction enzyme (both products of NEB) to ligate the obtained PCR products with the pBUE414 vector while digesting the enzymes, to obtain a recombinant vector with the correct sequence, which is the TaENO1 gene editing wheat vector. The obtained vector can transcribe two sgRNAs targeting positions 1085-1103 and 2484-2502 of SEQ ID No. 3, respectively, and can express the Cas protein.

[0092] 4. After introducing the TaENO1 gene-editing wheat vector into Agrobacterium tumefaciens EHA105, it was transformed into the wheat variety Fielder through Agrobacterium-mediated genetic transformation. After obtaining gene-edited plants, specific primers were synthesized for the editing site to amplify the target gene. Then, sequencing was performed to identify the editing status in order to obtain gene-edited wheat with different combinations.

[0093] 5. Obtain transgenic wheat eno1-7 and eno1-8 with TaENO1-5D gene editing.

[0094] Two pairs of TaENO1-5D genome-specific primers, F(5′-CTGCCAGGCGTCCATTGTAG-3′), R(5′-GCTCAAATGGATGTAAACCTTGG-3′) and F1(5′-GGTTGGTGCAAGCAAAAGGTATA-3′), R1(5′-TAGGCAATAGCAAACAAATGTAGAATA-3′), were used for amplification. Sequencing yielded two gene-edited lines (eno1-7 and eno1-8) with TaENO1-5D knocked out individually. The results are as follows: Figure 2 As shown, the eno1-7 edited material has a 6-nucleotide deletion (i.e., deletion of positions 2498-2503 of SEQ ID No. 3), resulting in a two-amino acid reduction. The eno1-8 edited material has a 2-nucleotide deletion (i.e., deletion of positions 2498-2499 of SEQ ID No. 3), resulting in a frameshift mutation and premature termination of translation. The next step is to use eno1-7 and eno1-8 for stress resistance testing.

[0095] III. Drought Resistance Assessment

[0096] Test plants: transgenic recipient wheat Fielder (negative control, WT), T3 generation plants of overexpression lines OE-2, OE-5 and OE-10, and gene-edited lines eno1-7 and eno1-8. The experiment was divided into two groups: a drought treatment group and a control group.

[0097] 1. Treat the wheat seeds to be treated (wheat Fielder (negative control), T3 generation seeds of overexpression lines OE-2, OE-5 and OE-10, and gene-edited lines eno1-7 and eno1-8) with 1% hydrogen peroxide for 2 days to break dormancy.

[0098] 2. Select seedlings with consistent germination and growth and plant them in the same box (overexpression lines and gene-edited lines are observed in separate boxes for phenotypic observation), with 30 seeds sown in each sample. Fill plastic boxes (35cm×20cm×15cm) with an equal volume of soil and vermiculite mixture, and set the culture conditions to 16h light (23℃) and 8h darkness (20℃).

[0099] 3. Water treatment was applied to seedlings that had grown to the two-leaf-one-heart stage. Watering was stopped for the drought treatment group, while the control group was watered normally. When there was a significant difference in phenotype between wild-type and transgenic wheat plants in the drought treatment group, a portion of the material's leaves were taken for physiological and biochemical trait testing, while the other portion of the material continued to be drought-treated to count the survival rate. Rehydration was started after about 20 days (the rehydration time depended on the drought status of the material). Seven days after rehydration, the plants were photographed and the number of surviving plants was counted. Three independent biological replicates were set up for each group.

[0100] The seedling survival rate is calculated using the following formula: Survival rate (%) = Number of surviving plants / Number of plants in each treatment × 100%.

[0101] The results are as follows Figure 3 As shown, under normal conditions, there are no significant morphological differences between transgenic plants and WT plants. Figure 3 (A, B). One week after drought treatment, the gene-overexpressing lines OE-2, OE-5, and OE-10 showed high sensitivity to drought stress, with leaves beginning to curl severely and wilt. Figure 3 In contrast, the gene-edited lines eno1-7 and eno1-8 showed slight leaf curling and wilting (in the middle A). Figure 3 (B). After rehydration following drought treatment, the survival rate of the overexpression lines was significantly lower than that of the WT lines. Figure 3 In the middle (C), the survival rate of seedlings of gene-edited lines was significantly increased ( Figure 3 (D).

[0102] Physiological and biochemical traits included water loss rate, leaf water content, cell membrane stability (CMS), and malondialdehyde (MDA) content in detached leaves. Results showed that TaENO1-5D overexpression lines (OE-2, OE-5, OE-10) lost water faster than WT detached leaves. Figure 4In the middle A), the water loss rate of gene-edited lines (eno1-7, eno1-8) was reduced ( Figure 4 (B). The water content of wheat leaves in TaENO1-5D overexpression lines was significantly lower than that in WT ( Figure 4 The C-type gene-edited lines were significantly higher than the WT-type lines. Figure 4 The results of cell membrane stability and malondialdehyde (MDA) content measurements showed that under drought stress, the cell membrane stability of the TaENO1-5D overexpressing lines was significantly lower than that of WT (wt). Figure 4 The content of malondialdehyde (MDA) in the middle (E) was significantly higher than that in the WT (Wt). Figure 4 The cell membrane stability of the gene-edited lines was significantly higher than that of the WT lines (G), while that of the gene-edited lines was significantly higher. Figure 4 The content of malondialdehyde (MDA) in the medium F was significantly lower than that in the WT (Wt). Figure 4 The result (H) indicates that drought treatment causes more severe cell damage to overexpression lines and less damage to gene knockout plants.

[0103] Detached leaf water loss rate (WLR) determination:

[0104] The water loss rate of detached leaves was determined by weighing. The test site should be a windless room with room temperature. First, leaves from the same part of the plant were cut off with small scissors and tweezers and weighed (W1). Ten replicates were made for each strain. Then, the leaves were placed on filter paper to lose water naturally. After that, the leaves were picked up with tweezers and weighed at 0.5h, 1h, 2h, 3h, 4h, 5h and 6h of water loss (W2). The water loss rate at each time point was calculated according to the formula: Water loss rate (%) = (W1-W2) / W1×100%.

[0105] Leaf water content (WC) measurement:

[0106] Four portions of wheat leaves after drought treatment were weighed, and their fresh weight was recorded. They were then placed in an oven at 120℃ for 1-1.5 hours and dried at 80℃ until constant weight was achieved. The dry weight was then recorded. The moisture content was calculated using the formula: Moisture content (%) = (fresh weight - dry weight) / fresh weight × 100%.

[0107] Cell membrane stability (CMS) assay:

[0108] Leaf samples from the control and drought-treated plants were immersed in test tubes containing 45 mL of ddH2O and incubated on a horizontal shaker at 60 rpm for 30 min at room temperature. The initial conductivity of the solution (C1) was measured, followed by boiling in a water bath for 30 min, and the conductivity (C2) was measured. Cell membrane stability (CMS) was calculated using the following formula: CMS (%) = (1 - C1 / C2) × 100%.

[0109] Malondialdehyde (MDA) content detection:

[0110] Using the catalase (CAT) test kit (BC0025) provided by Beijing Solarbio Biotechnology Co., Ltd., the malondialdehyde (MDA) content of the sample was determined according to the instructions. Approximately 0.1 g of sample (W) was weighed, ground into powder using a sample grinder, and 1 mL of extraction buffer was added. The mixture was shaken and vortexed, then centrifuged at 8000 g, 4°C for 10 min. The supernatant was collected and placed on ice for testing. 15 mL of reagent 1 was added to reagent 2 and dissolved and mixed to form the working solution for MDA detection. Since the working solution is difficult to dissolve, it can be heated at 70°C with vigorous shaking to promote dissolution. Add 300 μL of working solution, 100 μL of sample, and 100 μL of reagent 3 to the EP tube in sequence. Mix well and incubate in a 100℃ water bath for 30 min (tightly capped to prevent moisture loss and tube bursting). Cool in an ice bath and centrifuge at 10000g at room temperature for 10 min. Transfer 200 μL of supernatant to an ELISA plate and measure the absorbance of each sample at 450 nm, 532 nm, and 600 nm. Calculate the MDA content using the formula: MDA (nmol / g fresh weight) = 5 × (6.45 × (A532 - A600) - 0.56 × A450) ÷ W.

[0111] 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. Any of the following applications of substances that reduce protein content: M1) improves wheat's drought resistance; M2) Breed wheat with improved drought resistance; The protein is either A1 or A2 as follows: A1) The amino acid sequence of this protein is SEQ ID No. 2; A2) A fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of A1); The substance that reduces protein content is either B1 or B2 below. B1) Nucleic acid molecules that reduce the content of the aforementioned protein; B2) Expression cassettes, recombinant vectors, recombinant microorganisms, transgenic plant cell lines, transgenic plant tissues, or transgenic plant organs containing the nucleic acid molecules described in B1).

2. Any of the following methods: X1) A method for improving drought resistance in wheat comprising: By reducing the content of the protein described in claim 1 in the recipient wheat, or knocking out the gene encoding the protein described in claim 1, or reducing the expression level of the gene encoding the protein described in claim 1, a target wheat with improved drought resistance compared to the recipient wheat is obtained. X2) A method for cultivating drought-resistant wheat, comprising: reducing the content of the protein described in claim 1 in the recipient wheat, or knocking out the coding gene of the protein described in claim 1, or reducing the expression level of the coding gene of the protein described in claim 1, to obtain target wheat with improved drought resistance compared with the recipient wheat, thereby improving the drought resistance of wheat.

3. The method according to claim 2, characterized in that: The knockout of the gene encoding the protein described in claim 1 was accomplished using a CRISPR-Cas9 system.

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