Application of TaENO1-5D gene and protein coded by TaENO1-5D gene in regulation and control of drought resistance of wheat

Abstract

The invention discloses application of a TaENO1-5D gene and a protein coded by the TaENO1-5D gene in regulation and control of drought resistance of wheat. The cDNA (complementary deoxyribonucleic acid) of the TaENO1-5D gene disclosed by the invention is as shown in SEQ ID No.1, the genome sequence of the TaENO1-5D gene is as shown in SEQ ID No.3, and a TaENO1-5D protein as shown in SEQ ID No.2 is encoded. Experiments prove that the drought resistance of plants can be remarkably inhibited by overexpressing the TaENO1-5D gene in the plants, and the drought resistance of the plants can be remarkably improved by knocking out the TaENO1-5D gene. Therefore, the TaENO1-5D gene and the protein coded by the TaENO1-5D gene can regulate the drought resistance of the plant and have great production and application potential.

Description

Technical Field

[0001] The invention relates to the field of biotechnology and application of TaENO1-5D gene and protein encoded by the gene in regulating drought resistance of wheat. Background Art

[0002] Drought seriously affects the growth and development of plants, directly leading to a decrease in the yield and quality of crops. Wheat (Triticum aestivum L.) is one of the most widely distributed food crops in the world with the largest planting area and yield. It is often subjected to adverse stresses such as drought throughout its growth process, which seriously affects wheat growth and yield. Plants have developed complex mechanisms to perceive and respond to external stresses under ever-changing environmental conditions. Therefore, exploring and utilizing wheat drought resistance-related gene resources is of great significance for cultivating drought-resistant, high-yield and stable-yield wheat varieties and improving the level of wheat breeding. Summary of the invention

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

[0004] In order to solve the above technical problems, the present invention first provides any of the following applications of a protein or a substance for regulating the content or activity of the protein:

[0005] M1) regulates plant drought resistance;

[0006] M2) preparing products for regulating drought resistance of plants;

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

[0008] M4) preparing products for breeding plants with altered drought resistance;

[0009] The protein is derived from wheat (Triticum aestivum L.), and its name is TaENO1-5D. TaENO1-5D is as follows A1), A2) or A3):

[0010] A1) a protein whose amino acid sequence 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 list, wherein one or more amino acid residues are substituted and / or deleted and / or added;

[0012] A3) A fusion protein obtained by connecting 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 having an amino acid sequence identity of 75% or more to the protein shown in SEQ ID No. 2 and having the same function.

[0014] Identity refers to the identity of amino acid sequences. The identity of amino acid sequences can be determined using homology search sites on the Internet, such as the BLAST webpage on the NCBI homepage website. For example, in Advanced BLAST2.1, by using blastp as a program, setting the Expect value to 10, setting all Filters to OFF, using BLOSUM62 as a Matrix, setting the Gap existence cost, Per residue gap cost and Lambda ratio to 11, 1 and 0.85 (default values) respectively, and searching for a pair of amino acid sequence identities to calculate, then the identity value (%) can be obtained. The identity of 75% or more is 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%.

[0015] The tag in A3) can be a polypeptide or protein fused and expressed with the target protein using DNA in vitro recombination technology to facilitate the expression, detection, tracing and / or purification of the target protein. The tag 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 application, the substance for regulating the content or activity of TaENO1-5D protein may be any one of the following B1) to B9):

[0017] B1) Nucleic acid molecule encoding 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) a recombinant microorganism containing the nucleic acid molecule described in B1), or a recombinant microorganism containing the expression cassette described in B2), or a recombinant microorganism 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 molecule described in B1), or transgenic plant tissue containing the expression cassette described in B2);

[0023] B7) a transgenic plant organ containing the nucleic acid molecule described in B1) or a transgenic plant organ containing the expression cassette described in B2);

[0024] B8) a nucleic acid molecule that reduces the content of TaENO1-5D protein;

[0025] B9) An expression cassette, a recombinant vector, a recombinant microorganism, a transgenic plant cell line, a transgenic plant tissue or a transgenic plant organ containing the nucleic acid molecule described in B8).

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

[0027] b11) the coding sequence is the cDNA molecule or DNA molecule at positions 148 to 1488 of SEQ ID No. 1 in the sequence list;

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

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

[0030] b14) a DNA molecule having 75% or more identity with the nucleotide sequence defined in any one of b11) to b13) and encoding TaENO1-5D protein;

[0031] b15) A DNA molecule which hybridizes with any one of the nucleotide sequences specified in b11) to b14) under stringent conditions and encodes TaENO1-5D protein.

[0032] The nucleic acid molecule may be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule may also be RNA, such as mRNA or hnRNA.

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

[0034] The term "identity" as used herein refers to sequence similarity to a natural nucleic acid sequence. "Identity" includes nucleotide sequences that have 75% or more, or 85% or more, or 90% or more, or 95% or more identity to the nucleotide sequence of the present invention encoding the protein consisting of the amino acid sequence shown in SEQ ID No.2. Identity can be evaluated by the naked eye or by computer software. Using computer software, the identity between two or more sequences can be expressed as a percentage (%), which can be used to evaluate the identity between related sequences.

[0035] In the above application, the stringent conditions may be as follows: 50°C, in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO 4 The hybridization was carried out in a mixed solution of 1 mM EDTA and 1 mM EDTA, and the washing was carried out at 50°C in 2×SSC, 0.1% SDS.

[0036] The aforementioned 75% or more identity may be 80%, 85%, 90% or 95% or more identity.

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

[0038] B3) The vector may be a plasmid, cosmid, phage or virus vector. The plasmid may specifically be a pWMB110 vector.

[0039] B3) The recombinant vector may specifically be pWMB110-TaENO1-5D, which is a wheat vector overexpressing TaENO1-5D obtained by inserting the DNA molecule shown by nucleotides 148-1485 from the 5′ end of SEQ ID No.1 in the sequence list 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 a sgRNA targeting 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, cosmid, phage or virus vector. The plasmid may specifically be pCBC-MT1T2 or pBUE414.

[0043] B9) The recombinant vector can be a recombinant vector prepared by using the CRISPR-Cas9 system that can edit the TaENO1-5D gene. The recombinant vector can transcribe and express the sgRNA, that is, the sgRNA targeting the nucleic acid molecule in B1).

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

[0045] In the above application, the microorganism can be yeast, bacteria, algae or fungi. Among them, the 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 materials.

[0047] In the above application, 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 regulating plant drought resistance is to improve the drought resistance of the plant, and the cultivating plants with altered drought resistance is to cultivate plants with improved drought resistance.

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

[0049] X1) A method for improving drought resistance of a plant, comprising: reducing the content or activity of TaENO1-5D protein in a recipient plant, or knocking out a gene encoding TaENO1-5D protein in a recipient plant, or reducing the expression level of a gene encoding TaENO1-5D protein in a recipient plant, to obtain a target plant having improved drought resistance compared with the recipient plant;

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

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

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

[0053] Gene editing of the coding gene by the CRISPR-Cas9 system can be performed by introducing a recombinant vector (such as the recombinant vector B9) encoding Cas9 and capable of transcribing sgRNA targeting the coding gene) into the recipient plant to screen for the target plant in which the coding gene is edited.

[0054] In one embodiment of the present invention, the TaENO1-5D gene of the target plant is missing 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 missing positions 2498-2499 of SEQ ID No.3.

[0056] The recombinant vector can be introduced into plant cells by conventional biotechnology methods such as Ti plasmid, plant virus vector, direct DNA transformation, microinjection, electroporation, etc. (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 the first generation plant in which the coding gene of the TaENO1-5D protein is changed, but also its progeny. For the target plant, the gene can be propagated in the species, and the gene can also be transferred into other varieties of the same species using conventional breeding techniques, especially including commercial varieties. The target plant includes seeds, callus, complete plants and cells.

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

[0059] N1) monocots or dicots;

[0060] N2) Gramineae;

[0061] N3) Wheat.

[0062] The present invention also provides a product for regulating plant drought resistance, wherein the product contains (or its active ingredient is) TaENO1-5D protein, or the 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 the drought resistance of plants, while knocking out the TaENO1-5D gene can significantly improve the drought resistance of plants. Further testing of drought resistance-related indicators found that the TaENO1-5D overexpression strain had lower cell membrane stability and leaf water content, faster leaf water loss, and increased malondialdehyde content compared with the wild type after drought treatment; while the gene knockout strain had increased cell membrane stability and leaf water content, reduced leaf water loss, and reduced malondialdehyde content compared with the wild type. This indicates that the TaENO1-5D protein and its encoding gene can regulate the drought resistance of plants and have great potential for production and application.

[0064] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way. BRIEF DESCRIPTION OF THE DRAWINGS

[0065] Figure 1 This is the detection result of gene expression of overexpression strain TaENO1-5D.

[0066] Figure 2 Sequence changes in gene-edited strains.

[0067] Figure 3 The results of drought resistance test. A is the phenotype of TaENO1-5D overexpression strain and wild type under drought stress, B is the phenotype of gene-edited strain and wild type under drought stress, C is the seedling survival rate of TaENO1-5D overexpression strain and wild type under drought stress, and D is the seedling survival rate of gene-edited strain and wild type under drought stress.

[0068] Figure 4 Comparison of physiological indicators of wheat before and after drought stress. Comparison of detached leaf water loss rate (A, B), leaf water content (C, D), cell membrane stability (E, F) and malondialdehyde content (G, H) of TaENO1-5D overexpression line, gene-edited line and wild type under normal (CK) and drought stress conditions. DETAILED DESCRIPTION

[0069] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are carried out according to the techniques or conditions described in the literature in the art or according to the product instructions. The materials, reagents, instruments, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial sources. The quantitative tests in the following examples were all repeated at least three times, and the results were averaged. In the following examples, unless otherwise specified, the first position of each nucleotide sequence in the sequence table 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: Hexaploid common wheat, bred in the United States in 1974, commonly used for Agrobacterium-mediated transformation and gene editing receptors. References: 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. 2021Jun 25; 28(3): dsab008. doi: 10.1093 / dnares / dsab008. PMID: 34254113; PMCID: PMC8320877.

[0071] Drought-selected 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.; available to the public from the Institute of Crop Sciences, Chinese Academy of Agricultural Sciences.

[0073] Example 1: TaENO1-5D gene can regulate drought resistance in wheat

[0074] 1. Cloning of TaENO1-5D gene

[0075] 1. According to 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 Genetics Sinica, 32(8): 811-817), a full-length cDNA library of wheat was constructed using dry-selected wheat No. 10.

[0076] 2. Perform sequence analysis, segment extraction and functional verification on the wheat full-length cDNA to obtain candidate clones, and sequence to obtain the full-length sequence of the target clone, as shown in SEQ ID No. 1 in the sequence table, encoding the protein shown in SEQ ID No. 2 in the sequence table.

[0077] The protein shown in SEQ ID No.2 of the sequence table is named protein TaENO1-5D, which consists of 446 amino acid residues. The coding gene of protein TaENO1-5D is named TaENO1-5D gene. The full-length cDNA of TaENO1-5D gene is shown in SEQ ID No.1 of the sequence table (1680bp), wherein the nucleotides 1-147 from the 5' end are 5'UTR (147bp), the nucleotides 148-1488 are open reading frame (1341bp), and the nucleotides 1489-1680 are 3'UTR (192bp). The genomic sequence of TaENO1-5D gene is shown in SEQ ID No.3.

[0078] 2. Preparation of genetically modified wheat

[0079] 1) Construction of recombinant overexpression vector

[0080] 1. The pWMB110 vector was used as the original vector, wherein the pWMB110 vector was recorded in the following document: Liu H, Wang K, Jia Z, Gong Q, Lin Z, Du L, Pei X, Ye X. Efficient induction of haploid plants in wheat 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, and the name in the document is The plasmid pWMB110 or pWMB110.

[0081] 2. Extract total RNA from mixed tissue samples of dry selected wheat No. 10, and obtain cDNA through reverse transcription. Using cDNA as a template, use primers consisting of F and R to perform PCR amplification to obtain PCR products (sequencing shows that the DNA molecule is the 96th to 1666th nucleotides from the 5′ end of SEQ ID No. 1 in the sequence table). F: 5′-CCTCCGCTCCATTCGTTT-3′; R: 5′-ATTGGAGCAAGTTTGGAG-3′; Then PCR products are purified and recovered and connected to the intermediate vector T vector. Extract plasmids, and set aside after sequencing is correct.

[0082] 3. Using the plasmid obtained in step 2 as a template, use the primer pair consisting of F1 and R1 for PCR amplification, select the high-fidelity enzyme Pfu amplification system, and 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 homology arm (a fragment with the same sequence as the insertion position of the vector pWMB110).

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

[0084] 5. The PCR product containing homology arms obtained in step 3 and the vector backbone (10 kb vector backbone) in step 4 were homologously recombined, that is, the seamless ligase and the above two were mixed and incubated at 37°C for 30 minutes, and then transformed conventionally to obtain the recombinant expression vector pWMB110-TaENO1-5D. Seamless ligase In-fusion is a product of TaKaRa (Cat. No. 638947).

[0085] The sequencing results of pWMB110-TaENO1-5D showed that it was a wheat vector overexpressing TaENO1-5D obtained by inserting the DNA molecule shown by nucleotides 148-1485 from the 5′ end of SEQ ID No.1 in the sequence list into the binary vector pWMB110 using BamHI, while keeping the other nucleotides of the binary vector pWMB110 unchanged.

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

[0087] After pWMB110-TaENO1-5D was introduced into Agrobacterium tumefaciens EHA105, it was transferred into the wheat variety Fielder by Agrobacterium-mediated genetic transformation. The homozygous strains were obtained by selfing. The relative expression level of TaENO1-5D gene in T3 plants (OE-2, OE-5, and OE-10) of each tested strain was detected by qRT-PCR (with Actin gene as the internal reference gene). The primer pair consisting of F2 (5′-CTCCCTCACAACAACAACCGC-3′) and R2 (5′-TACCAGGAACTTCCATACCAAC-3′) was used to detect the expression of the internal reference gene Actin, and the primer pair consisting of primer F3 (5′-TGAGGACCCATTTGACCAGG-3′) and primer R3 (5′-GCACCAACCCCACTCATTCT-3′) was used to detect the expression of TaENO1-5D gene. The results are shown in Figure 2. Figure 1 As shown, there is a small amount of TaENO1-5D gene expression in the receptor material Fielder (WT), while the expression of TaENO1-5D gene in the overexpression strains OE-2, OE-5 and OE-10 is significantly higher than that in the Fielder material. The next step is to use OE-2, OE-5 and OE-10 for stress resistance testing.

[0088] II) Construction of recombinant gene editing vector

[0089] 1. Use the online website (http: / / www.e-crisp.org / E-CRISP / ) to predict the editing sites of the TaENO1 gene.

[0090] 2. The target site of TaENO1-5D gene (i.e., positions 1085-1103 and 2484-2502 of SEQ ID No. 3) was designed according to SEQ ID No. 3 of TaENO1-5D genome, and then four primers were designed according to the instructions for use of the vector, 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), use pCBC-MT1T2 vector as template and TaENO1-MT1T2 F / F0 / R0 / R as primers to obtain PCR products; use T4 DNA Ligase and endonuclease BsaI (both products of NEB) to digest and connect the obtained PCR products with the pBUE414 vector to obtain a recombinant vector with the correct sequence, namely the TaENO1 gene-edited 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 Cas protein.

[0092] 4. After the TaENO1 gene-edited wheat vector was introduced into Agrobacterium tumefaciens EHA105, it was transferred into the wheat variety Fielder through Agrobacterium-mediated genetic transformation. After obtaining the gene-edited plants, specific primers were synthesized for the editing sites, and the target genes were amplified respectively. Then, sequencing was performed to identify the editing situation to obtain gene-edited wheat of different combinations.

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

[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. After sequencing, two gene-edited strains (eno1-7 and eno1-8) with TaENO1-5D knocked out were obtained. The results are as follows Figure 2 As shown, the eno1-7 editing material lacks 6 nucleotides (i.e., lacks positions 2498-2503 of SEQ ID No.3), resulting in two amino acid reductions, and the eno1-8 editing material lacks 2 nucleotides (i.e., lacks positions 2498-2499 of SEQ ID No.3), resulting in frameshift mutations and premature termination of translation. Next, eno1-7 and eno1-8 were used for stress resistance testing.

[0095] 3. Drought resistance identification

[0096] Plants to be tested: transgenic receptor variety wheat Fielder (negative control, WT), T3 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, drought treatment group and control group.

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

[0098] 2. Pick the seedlings with consistent germination and growth and plant them in the same box (separate boxes for overexpression strains and gene-edited strains to observe phenotypes), sow 30 seeds for each material. Fill the plastic box (35cm×20cm×15cm) with an equal volume of soil and vermiculite mixture, and set the culture conditions to 16h of light (23℃) and 8h of darkness (20℃).

[0099] 3. Water treatment was performed on seedlings that had grown to the two-leaf and one-heart stage. Water supply was stopped for the drought treatment group, while normal watering was given to the control group. When the phenotypes of wild-type and transgenic wheat plants in the drought treatment group showed obvious differences, a portion of the leaves were taken for testing physiological and biochemical traits, and the other portion of the material continued to be drought treated to calculate the survival rate. Rehydration began 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 was calculated according to the following formula: survival rate (%) = number of surviving plants / number of treated plants × 100%.

[0101] The results are as follows Figure 3 As shown, under normal conditions, there were no obvious morphological differences between transgenic plants and WT ( Figure 3 A and B). After one week of drought treatment, the gene overexpression lines OE-2, OE-5, and OE-10 showed high sensitivity to drought stress, and the leaves began to curl and wilt severely ( Figure 3 In contrast, the leaves of the gene-edited lines eno1-7 and eno1-8 curled slightly and wilted ( Figure 3 Middle B). After drought treatment and rehydration, the survival rate of the overexpression line was significantly reduced compared with the WT ( Figure 3 C), the survival rate of seedlings of the gene-edited lines was significantly increased ( Figure 3 (middle D).

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

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

[0104] The water loss rate of detached leaves is determined by the weighing method. The test site should be selected in a windless room at room temperature. First, use small scissors and tweezers to cut off the leaves at the same part of the plant and weigh them (W1). Each strain is weighed 10 times. Then the leaves are placed on filter paper to allow them to lose water naturally. After that, use tweezers to pick up the leaves and weigh them at 0.5h, 1h, 2h, 3h, 4h, 5h and 6h of water loss (W2). The water loss rate at each time point is calculated according to the formula: Water loss rate (%) = (W1-W2) / W1×100%.

[0105] Leaf water content (WC) determination:

[0106] Weigh 4 portions of wheat leaves after drought treatment, record the fresh weight, bake them in a 120°C oven for 1-1.5 h, then adjust to 80°C and dry them to constant weight, weigh the dry weight, and calculate the moisture content according to the formula: moisture content (%) = (fresh weight - dry weight) / fresh weight × 100%.

[0107] Cell membrane stability (CMS) assay:

[0108] Take the control and drought-treated leaf samples and immerse them in 45 mL of ddH 2O in a test tube, incubate at room temperature on a horizontal shaker at 60 rpm for 30 min, detect the initial conductivity (C1) of the solution, then boil in a water bath for 30 min, and detect the conductivity (C2). Calculate the cell membrane stability (CMS) according to the following formula: CMS (%) = (1-C1 / C2) × 100%.

[0109] Malondialdehyde (MDA) content detection:

[0110] Use the catalase (CAT) test kit (BC0025) provided by Beijing Solebow Biotechnology Co., Ltd. and operate according to the instructions to determine the malondialdehyde content of the sample to be tested. Weigh about 0.1g of sample (W), grind it into powder with a proofing machine, add 1mL of extract solution to shake and mix, centrifuge at 8000g, 4℃ for 10min, take the supernatant, and place it on ice for testing. Add 15mL of reagent 1 to reagent 2, dissolve and mix, and use it as the MDA detection working solution. The working solution is difficult to dissolve, so it can be heated at 70℃ and shaken vigorously to promote dissolution. Add 300 μL of working solution, 100 μL of sample and 100 μL of reagent three to the EP tube in order, mix well and keep warm in a 100°C water bath for 30 min (cover tightly to prevent water loss and tube burst), cool in an ice bath, centrifuge at 10000g for 10 min at room temperature, draw 200 μL of supernatant into the ELISA plate, measure the absorbance of each sample at 450 nm, 532 nm and 600 nm, and calculate the MDA content using the public. MDA (nmol / g fresh weight) = 5 × (6.45 × (A532-A600)-0.56 × A450) ÷ W.

[0111] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that the present invention can be further improved. In a word, according to the principles of the present invention, the application is intended to include any changes, uses or improvements to the present invention, including departure from the disclosed scope in the application, and changes made with conventional techniques known in the art.

Claims

1. Any of the following uses of a protein or a substance for regulating the content or activity of the protein: M1) regulates plant drought resistance; M2) preparing products for regulating drought resistance of plants; M3) Breeding plants with altered drought resistance; M4) preparing products for breeding plants with altered drought resistance; The protein is the following A1), A2) or A3): A1) a protein whose amino acid sequence is SEQ ID No. 2; A2) a protein having the same function as the amino acid sequence shown in SEQ ID No. 2 in the sequence list, wherein one or more amino acid residues are substituted and / or deleted and / or added; A3) A fusion protein obtained by connecting a tag to the N-terminus and / or C-terminus of A1) or A2).

2. The use according to claim 1, characterized in that: The substance that regulates the protein content or activity is any one of the following B1) to B9): B1) a nucleic acid molecule encoding the protein according to claim 1; B2) an expression cassette containing the nucleic acid molecule described in B1); B3) a recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2); B4) a recombinant microorganism containing the nucleic acid molecule described in B1), or a recombinant microorganism containing the expression cassette described in B2), or a recombinant microorganism containing the recombinant vector described in B3); 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); B6) transgenic plant tissue containing the nucleic acid molecule described in B1), or transgenic plant tissue containing the expression cassette described in B2); B7) a transgenic plant organ containing the nucleic acid molecule described in B1) or a transgenic plant organ containing the expression cassette described in B2); B8) A nucleic acid molecule that reduces the protein content of claim 1; B9) An expression cassette, a recombinant vector, a recombinant microorganism, a transgenic plant cell line, a transgenic plant tissue or a transgenic plant organ containing the nucleic acid molecule described in B8).

3. The use according to claim 2, characterized in that: B1) The nucleic acid molecule is any one of the following b11)-b15): b11) the coding sequence is the cDNA molecule or DNA molecule at positions 148 to 1488 of SEQ ID No. 1 in the sequence list; b12) cDNA molecule or DNA molecule shown in SEQ ID No.1 in the sequence list; b13) the DNA molecule shown in SEQ ID No. 3 in the sequence list; b14) a DNA molecule having 75% or more identity with the nucleotide sequence defined in any one of b11) to b13) and encoding the protein described in claim 1; b15) A DNA molecule which hybridizes with the nucleotide sequence specified in any one of b11) to b14) under stringent conditions and encodes the protein described in claim 1.

4. The use according to any one of claims 1 to 3, characterized in that: The substance that regulates the protein content or activity is a substance that reduces the protein content or activity, the substance that regulates plant drought resistance is to improve the drought resistance of the plant, and the substance that cultivates plants with altered drought resistance is to cultivate plants with improved drought resistance.

5. The use according to any one of claims 1 to 4, characterized in that: The plant is N1) or N2) or N3): N1) monocots or dicots; N2) Gramineae; N3) Wheat.

6. Any of the following methods: X1) A method for improving drought resistance of plants, comprising: Reducing the content or activity of the protein described in claim 1 in a recipient plant, 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, to obtain a target plant with improved drought resistance compared with the recipient plant; X2) A method for cultivating plants with improved drought resistance, comprising: reducing the content or activity of the protein described in claim 1 in a recipient plant, 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, to obtain a target plant with improved drought resistance compared with the recipient plant, thereby achieving improved plant drought resistance.

7. The method according to claim 6, characterized in that: Knocking out the gene encoding the protein described in claim 1 is accomplished using the CRISPR-Cas9 system.

8. The method according to claim 6 or 7, characterized in that: The plant is N1) or N2) or N3): N1) monocots or dicots; N2) Gramineae; N3) Wheat.

9. A product for regulating plant drought resistance, comprising the protein of claim 1, or the substance for regulating protein content or activity of any one of claims 1 to 3.

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