Wheat drought-enduring gene TaWAK5 as well as encoding protein and application thereof

Abstract

The invention discloses a wheat drought-enduring gene TaWAK5 as well as an encoding protein and application thereof. The invention provides application of protein TaWAK5 or related biological materials thereof in the following A1) or A2): A1) improving the drought resistance of plants; a2) cultivating a drought-resistant plant; experiments show that overexpression of the TaWAK5 gene in the plant can significantly improve the drought resistance of the plant, and knockout of the TaWAK5 gene can significantly reduce the drought resistance of the plant. The TaWAK5 protein and the coding gene thereof can regulate and control the drought resistance of plants, and have great production and application potential.

Description

Technical Field

[0001] The invention belongs to the technical field of plant genetic engineering and relates to a wheat drought-resistant gene TaWAK5 and a coded protein and application thereof. Background Art

[0002] Drought is one of the key limiting factors for global food production. Studies have shown that the reduction in food production due to drought accounts for about half of the world's total food production reduction. Wheat, as one of the three major staple crops for humans and also the main food crop in arid and semi-arid areas, is deeply affected by drought (Gale 2002, Interim Science Council SECRETARIAT FOOD AND AGRICULTURE ORGANIZATION OF THE UNITED NATIONS, p1-27). Therefore, exploring and utilizing wheat drought-resistant genes and cultivating new drought-resistant varieties are major needs for responding to climate change, ensuring food security and water security, and are also effective ways to ensure the sustainable development of wheat production. Summary of the invention

[0003] The technical problem solved by the invention is to provide application of gene TaWAK5 in improving drought resistance and breeding of wheat.

[0004] In order to solve the above technical problems, the first aspect of the present invention is the use of protein TaWAK5 or its related biological materials in the following A1) or A2):

[0005] A1) Improve plant drought resistance;

[0006] A2) Cultivating drought-resistant plants;

[0007] The protein TaWAK5, derived from wheat (Triticum aestivum L.), is B1) or B2) or B3) or B4):

[0008] B1) the amino acid sequence is the protein shown in SEQ ID No. 2;

[0009] B2) a fusion protein obtained by connecting a tag to the N-terminus and / or the C-terminus of the protein shown in SEQ ID No. 2;

[0010] B3) a protein having the same biological function obtained by replacing and / or deleting and / or adding one or more amino acid residues of the amino acid sequence shown in SEQ ID No. 2;

[0011] B4) A protein having 80% or more identity with the amino acid sequence defined by SEQ ID No. 2, derived from a plant and having the same biological function.

[0012] In the above, identity refers to the identity of the amino acid sequence. The identity of the amino acid sequence can be determined with the help of homology search sites on the Internet, such as the BLAST page on the NCBI homepage website. Specifically, in Advanced BLAST2.1, the program is set to blastp, the Expect value is set to 10, all Filters are set to OFF, BLOSUM62 is selected as the Matrix, and the Gap existence cost, Per residue gap cost and Lambda ratio are set to 11, 1 and 0.85 (default value) respectively, in order to retrieve and calculate the identity of a pair of amino acid sequences, and then obtain the value of identity (%). The 75% and above identity here covers 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity.

[0013] In the above, the tag is a polypeptide or protein that is fused and expressed with the target protein using DNA in vitro recombination technology, and its function is to facilitate the expression, detection, tracing and / or purification of the target protein. Common tags include Poly-Arg, Poly-His, FLAG, Strep-tagII, c-myc, MBP tag, HA tag, GST tag and / or SUMO tag, etc.

[0014] The above-mentioned improvement of plant drought resistance may be reflected in an increase in survival rate under drought conditions and / or an increase in thousand-grain weight under drought conditions.

[0015] In the above application, the protein TaWAK5-related biological material is any one of C1) to C7):

[0016] C1) a nucleic acid molecule encoding the protein TaWAK5;

[0017] C2) an expression cassette comprising the nucleic acid molecule described in C1);

[0018] C3) a recombinant vector containing the nucleic acid molecule described in C1), or a recombinant vector containing the expression cassette described in C2);

[0019] C4) a recombinant microorganism containing the nucleic acid molecule described in C1), or a recombinant microorganism containing the expression cassette described in C2), or a recombinant microorganism containing the recombinant vector described in C3);

[0020] 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);

[0021] C6) transgenic plant tissue containing the nucleic acid molecule described in C1), or transgenic plant tissue containing the expression cassette described in C2);

[0022] 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).

[0023] In the above, the nucleic acid molecule can be DNA, such as cDNA, genomic DNA or recombinant DNA; or it can be RNA, such as mRNA or hnRNA.

[0024] A person skilled in the art can easily mutate the nucleotide sequence encoding the TaWAK5 protein of the present invention by using known methods, such as directed evolution and point mutation. As long as the nucleotide sequence has 75% or higher identity with the nucleotide sequence encoding the TaWAK5 protein of the present invention after artificial modification, and the nucleotide sequence encoding the TaWAK5 protein and having its function, it is derived from the nucleotide sequence of the present invention and is equivalent to the sequence of the present invention.

[0025] The term "identity" as used herein refers to sequence similarity with a natural nucleic acid sequence. "Identity" encompasses nucleotide sequences that are 75% or higher, or 85% or higher, or 90% or higher, or 95% or higher identical 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 determined by the naked eye or assessed with the aid of computer software. When using computer software, the identity between two or more sequences is expressed as a percentage (%) to measure the identity between related sequences.

[0026] In the above application, the stringent conditions are as follows: hybridization at 50°C in a mixed solution of 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO4 and 1 mM EDTA, and washing at 50°C in 2×SSC, 0.1% SDS.

[0027] The above-mentioned 75% and above identity may specifically be 80%, 85%, 90% or 95% or above identity.

[0028] The expression cassette mentioned above refers to a DNA that can express TaWAK5 protein in a host cell. The DNA contains not only a promoter for initiating transcription of the TaWAK5 gene, but also a terminator for terminating transcription of the TaWAK5 gene. Furthermore, the expression cassette may also contain an enhancer sequence.

[0029] The above vector can be a plasmid, cosmid, phage or virus vector. Among them, the plasmid can be a pWMB110 vector. Specifically, the recombinant vector can be pWMB110-TaWAK5, which is a wheat vector overexpressing TaWAK5 obtained by inserting the DNA molecule shown by nucleotides 1 to 2145 from the 5′ end of SEQ ID No.1 in the sequence table into the binary vector pWMB110, while keeping the other nucleotides of the binary vector pWMB110 unchanged.

[0030] In the above application, the nucleic acid molecule encoding the protein TaWAK5 is as follows D1) or D2) or D3) or D4):

[0031] D1) The nucleotide sequence is the DNA molecule shown in SEQ ID No.1;

[0032] D2) the nucleotide sequence is the DNA molecule shown in SEQ ID No.3;

[0033] D3) a DNA molecule having 90% or more identity with the nucleotide sequence defined in D1) or D2), derived from a plant and encoding the protein TaWAK5 described in claim 1;

[0034] D4) A DNA molecule that hybridizes with the nucleotide sequence defined in D1) or D2) under stringent conditions and encodes the protein TaWAK5 described in claim 1.

[0035] In a second aspect, the present invention provides a substance for inhibiting the activity of the protein TaWAK5 described in the first aspect or inhibiting the expression of a nucleic acid molecule encoding the protein TaWAK5 described in the first aspect, in the following E1) or E2):

[0036] E1) Reduce plant drought resistance;

[0037] E2) Cultivate plants with low drought resistance.

[0038] The above-mentioned reduction in plant drought resistance may be reflected in a reduction in survival rate under drought conditions and / or a reduction in thousand-grain weight under drought conditions.

[0039] In the application described above, the substance is a CRISPR system that inhibits the expression of a nucleic acid molecule encoding the protein TaWAK5;

[0040] The CRISPR system includes an sgRNA targeting a nucleic acid molecule encoding the protein TaWAK5,

[0041] The targets of the sgRNA are target 1 and target 2;

[0042] The nucleotide sequence of the target 1 is positions 557-576 of SEQ ID No. 3;

[0043] The nucleotide sequence of the target 2 is positions 740-759 of SEQ ID No.3.

[0044] The above CRISPR system includes a vector expressing sgRNA, and the backbone of the vector expressing sgRNA can be a plasmid, a cosmid, a phage or a viral vector. Among them, the plasmid vector backbone can be pCBC-MT1T2 or pBUE414. The vector expressing sgRNA can be the TaWAK5 gene editing wheat vector in Example 1.

[0045] In the above application, the microorganism may be yeast, bacteria, algae or fungi. Among them, the bacteria may 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 contain any propagation material.

[0047] In a third aspect, the present invention provides a method for improving drought resistance of plants, which is as follows F1) or F2):

[0048] F1) The method comprises the following steps: increasing the content and / or activity of the protein TaWAK5 in the target plant to improve the drought resistance of the plant;

[0049] F2) The method comprises the following steps: increasing the expression of a nucleic acid molecule encoding a protein TaWAK5 in a target plant, thereby increasing the drought resistance of the plant;

[0050] The target plant contains a nucleic acid molecule encoding protein TaWAK5;

[0051] The protein TaWAK5 is B1) or B2) or B3) or B4):

[0052] B1) the amino acid sequence is the protein shown in SEQ ID No. 2;

[0053] B2) a fusion protein obtained by connecting a tag to the N-terminus and / or the C-terminus of the protein shown in SEQ ID No. 2;

[0054] B3) a protein having the same biological function obtained by replacing and / or deleting and / or adding one or more amino acid residues of the amino acid sequence shown in SEQ ID No. 2;

[0055] B4) A protein having 80% or more identity with the amino acid sequence defined by SEQ ID No. 2, derived from a plant and having the same biological function.

[0056] The above-mentioned improvement of plant drought resistance may be reflected in an increase in survival rate under drought conditions and / or an increase in thousand-grain weight under drought conditions.

[0057] In a fourth aspect, the present invention provides a method for cultivating a highly drought-resistant plant, comprising the following steps: increasing the content and / or activity of the protein TaWAK5 described in the first aspect in a starting plant to obtain a transgenic plant, namely, a target plant;

[0058] The transgenic plants have higher drought resistance than the starting plants.

[0059] In a fifth aspect, the present invention provides a method for cultivating a highly drought-resistant plant, comprising the following steps: increasing the expression of the nucleic acid molecule encoding the protein TaWAK5 in the first aspect in a starting plant to obtain a transgenic plant, namely, a target plant;

[0060] The transgenic plants have higher drought resistance than the starting plants.

[0061] In the above, the plant is the following N1) or N2) or N3):

[0062] N1) monocots or dicots;

[0063] N2) Gramineae;

[0064] N3) Wheat.

[0065] The drought resistance of the transgenic plants described above is higher than that of the starting plants, which can be reflected in that the survival rate of the transgenic plants under drought conditions is higher than that of the starting plants, and / or the thousand-grain weight of the transgenic plants under drought conditions is higher than that of the starting plants.

[0066] The above recombinant vector can be introduced into plant cells through 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).).

[0067] The target plants mentioned above include not only the first generation plants in which the coding gene of TaWAK5 protein is changed, but also its offspring. For the target plants, the gene can be propagated in the species, and the gene can be transferred to other varieties of the same species, especially commercial varieties, using conventional breeding techniques. The target plants include seeds, callus tissues, complete plants and cells.

[0068] The experiments of the present invention show that overexpression of the TaWAK5 gene in plants can significantly improve the drought resistance of plants, while knocking out the TaWAK5 gene can significantly reduce the drought resistance of plants. This fully demonstrates that the TaWAK5 protein and its encoding gene can regulate the drought resistance of plants and have great production and application potential.

[0069] The present invention will be described in more detail below in conjunction with specific embodiments. The examples provided are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The examples provided below may be used as a reference for further improvement by those of ordinary skill in the art and are not intended to limit the present invention in any form. BRIEF DESCRIPTION OF THE DRAWINGS

[0070] Figure 1 These are the detection results of the transgenic lines overexpressing the TaWAK5 gene.

[0071] Figure 2 Sequence changes in gene-edited strains.

[0072] Figure 3 This is the result of drought resistance test under repeated drought.

[0073] Figure 4 This is the result of thousand-grain weight test. DETAILED DESCRIPTION

[0074] 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.

[0075] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0076] Unless otherwise specified, the quantitative tests in the following examples were performed three times and the results were averaged.

[0077] Fielder wheat: gifted by Xin Mingming's laboratory at China Agricultural University. Hexaploid common wheat, bred in the United States in 1974, is 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. 2021 Jun 25; 28(3): dsab008. doi: 10.1093 / dnares / dsab008. PMID: 34254113; PMCID: PMC8320877.

[0078] 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.

[0079] Related information about the pWMB110 vector is recorded in the literature: 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. In the literature, its name is The plasmid pWMB110 or pWMB110.

[0080] Plasmids pCBC-MT1T2 and pBUE414 are recorded in the following documents: Plant Biotechnol J.2021Dec24;20(5):862–875.doi:10.1111 / pbi.13765; TaMOR is essential for root initiation and improvement of root system architecture in wheat; Chaonan Li, Jingyi Wang, Long Li, Jialu Li, Mengjia Zhuang,Bo Li,Qiaoru Li,Junfang Huang,Yan Du,JinpingWang,Zipei Fan,Xinguo Mao,Ruilian Jing.

[0081] Example 1. TaWAK5 gene

[0082] The nucleotide sequence of the genomic DNA of the TaWAK5 gene is SEQ ID No.3; the nucleotide sequence of the cDNA of the TaWAK5 gene is SEQ ID No.1; the protein encoded by the TaWAK5 gene is TaWAK5, and its amino acid sequence is SEQ ID No.2.

[0083] Example 2: Functional study of TaWAK5 gene

[0084] 1. Construction of transgenic wheat overexpressing TaWAK5

[0085] 1. Construction of recombinant overexpression vector

[0086] In this experiment, pWMB110 vector was selected as the original vector.

[0087] Total RNA was extracted from mixed tissue samples of wheat Hanxuan No. 10, and cDNA was successfully obtained through reverse transcription. Using the cDNA as a template, PCR amplification was performed using a primer pair consisting of primer F (5′-ATGTCGCCGATGGCATGGATG-3′) and primer R (5′-TCGAGGGTAGCTCGCGGACAA-3′) to obtain a PCR product.

[0088] The PCR product was verified by sequencing to be a DNA molecule shown in nucleotides 1 to 2145 of the 5′ end of SEQ ID No. 1 in the sequence table. Subsequently, the PCR product was purified and recovered, and connected to the intermediate vector T vector to obtain a recombinant intermediate plasmid. The recombinant intermediate plasmid was extracted, and after the sequence was confirmed to be correct by sequencing, it was set aside.

[0089] The recombinant intermediate plasmid was used as a template and the primer F1 (5′-CGACTCTAGA GGATCC ATGTCGCCGATGGCATGGATG-3′) and primer R1 (5′-CGGTACCCGG GGATCC TCGAGGGTAGCTCGCGGACAA-3′) was used for PCR amplification. The high-fidelity enzyme Pfu amplification system was used in the amplification process, and finally a PCR amplification product was obtained, and a PCR product containing homology arms was obtained.

[0090] In the F1 and R1 primers, the underlined portion is the Bam HI restriction site; the sequence in front of the underline (5' side) is the homology arm, which is a fragment identical to the sequence near the position to be inserted in the vector pWMB110.

[0091] The vector pWMB110 was digested with restriction endonuclease Bam HI, and then the vector backbone of about 10 kb was recovered.

[0092] The above-mentioned PCR product containing homology arms was connected to the recovered 10kb vector backbone by homologous recombination method; the specific operation was to mix the seamless ligase (TaKaRa product, item number: 638947) with the above-mentioned two, incubate at 37°C for 30 minutes, and then undergo conventional transformation steps to successfully obtain the recombinant vector pWMB110-TaWAK5.

[0093] The sequencing results of pWMB110-TaWAK5 showed that the vector was a wheat vector overexpressing TaWAK5 obtained by inserting the DNA molecule represented by nucleotides 1-2145 of SEQ ID No.1 from the 5′ end into the BamHI restriction site of the pWMB110 vector while keeping the other nucleotide sequences of the binary vector pWMB110 unchanged.

[0094] 2. Construction of transgenic wheat overexpressing TaWAK5 gene

[0095] The above-mentioned recombinant vector pWMB110-TaWAK5 was introduced into Agrobacterium tumefaciens EHA105 to obtain recombinant bacteria; then it was transferred into the wheat variety Fielder (hereinafter also referred to as wild-type wheat) through Agrobacterium-mediated genetic transformation to obtain T0 generation TaWAK5 transgenic wheat, and after self-pollination operation, the homozygous strain T3 generation TaWAK5 transgenic wheat was obtained, that is, transgenic wheat overexpressing the TaWAK5 gene.

[0096] RNA was extracted from the leaves of homozygous TaWAK5 transgenic wheat OE3 and OE6, and reverse transcribed to obtain cDNA. Primers F3 and R3 were used as primers for qRT-PCR amplification to detect the relative expression level of TaWAK5 gene in each tested line. Tubulin gene was used as the internal reference gene in the detection process, and the primers for amplifying the internal reference gene were F2 and R2.

[0097] The above primer sequences are as follows:

[0098] Primer F2 (5′-CGTGCTGTCTTTGTAGATCTCG-3′);

[0099] Primer R2 (5′-GACCAGTGCAGTTGTCTGAAAG-3′);

[0100] Primer F3 (5′-GAGACTGCCCATCTGAAGAGGAAACC-3′);

[0101] Primer R3 (5′-GTTACCTCTGTGCATCCATCGATAAGGTATG-3′).

[0102] The results are as follows Figure 1 As shown, it can be seen that compared with the recipient material Fielder (WT), the expression level of the TaWAK5 gene in the homozygous T3 generation TaWAK5 transgenic wheat OE3 and OE6 was significantly increased, indicating that OE3 and OE6 are transgenic wheat overexpressing the TaWAK5 gene.

[0103] 2. Construction of transgenic wheat with TaWAK5 knockout gene

[0104] 1. Construction of recombinant gene editing vector

[0105] In order to edit the TaWAK5 gene to obtain gene-edited transgenic wheat, the following series of experimental operations were carried out:

[0106] 1) Editing site prediction

[0107] The editing site of the TaWAK5 gene was predicted with the help of an online website (http: / / www.e-crisp.org / E-CRISP / ) to provide accurate target locations for subsequent gene editing work.

[0108] 2) Primer design

[0109] According to the sequence characteristics of the TaWAK5 genome, the target sites of the TaWAK5 gene were carefully designed (T1 target site: positions 557-576 of SEQ ID No. 3, T2 target site: positions 740-759 of SEQ ID No. 3). Subsequently, four primers were designed according to the instructions for use of the vector.

[0110] The specific primer sequences are as follows: upstream primer F (5'-aataatggtctcTGGCgCTGCCACGTCAACATCTCG-3'), F0 (5'-gCTGCCACGTCAACATCTCGgttttagagctagaaatagc-3'); downstream primer R (5'-GTCCAGCCAAAGCGGCATGTGCTTCTTGGTGCCGC-3'), R0 (5'-ATTATTGGTCTCTAAACGTCCAGCCAAAGCGGCATG-3'). The design of these primers is the key basis for the subsequent construction of gene editing vectors.

[0111] 3) Construction of recombinant vector

[0112] Using plasmid pCBC-MT1T2 as a template, PCR amplification was performed using the four primers F, F0, R0 and R in 2 above to obtain a product of about 900 bp.

[0113] The PCR product was then connected to the pBUE414 vector by homologous recombination to obtain the TaWAK5 gene-edited wheat vector, which contained a T1 target site and a T2 target site.

[0114] 2. Genetic transformation and identification

[0115] The constructed TaWAK5 gene editing wheat vector was introduced into Agrobacterium tumefaciens EHA105, and then transformed into wheat Fielder (hereinafter referred to as wild-type wheat) by Agrobacterium-mediated genetic transformation to obtain T0 generation CRISPR knockout transgenic wheat.

[0116] The genomic DNA of leaves extracted from T0 generation CRISPR knockout transgenic wheat was used as a template, and PCR identification was performed using genome-specific primers F4 (5′-GATGTCACCTTCAAGGGCCACAC-3′), R4 (5′-GACCATCATGGCTTTTCCATAGCTACTCC-3′), F5 (5′-CTGACATTCAACAAGTGGCGCGTTTAC-3′) and R5 (5′-CAAGCGTATTCTCGCGTCTTGACC-3′). The PCR product was sent for sequencing. Compared with the sequence of the TaWAK5 gene (SEQ ID No. 3) in the wild-type wheat Fielder, a sense mutation occurred, which was recorded as a positive T0 generation CRISPR knockout transgenic wheat.

[0117] The positive T0 generation CRISPR knockout transgenic wheat was cultured continuously until homozygous T3 generation CRISPR knockout transgenic wheat plants were obtained.

[0118] The genomic DNA of leaves extracted from T3 generation CRISPR knockout transgenic wheat homozygous plants was used as a template, and PCR identification was performed using genome-specific primers F4 (5′-GATGTCACCTTCAAGGGCCACAC-3′), R4 ( 5′-GACCATCATGGCTTTTCCATAGCTACTCC-3′ ), F5 ( 5′-CTGACATTCAACAAGTGGCGCGTTTAC-3′ ) and R5 (5′-CAAGCGTATTCTCGCGTCTTGACC-3′), and the products were sent for sequencing.

[0119] The sequencing results are as follows Figure 2 As shown,

[0120] Compared with the sequence of TaWAK5 gene (SEQ ID No.3) in wild-type wheat Fielder, it was found that the regions corresponding to TaWAK5 gene on two homologous chromosomes in T3 generation CRISPR knockout transgenic wheat homozygous plant tawak5-1 had the following changes: 2 bp were deleted at target site 1 (SEQ ID No.3 573-574), and 1 bp base (g) was inserted at target site 2 (insertion between SEQ ID No.3 755-756), resulting in gene frameshift mutation and premature termination of translation;

[0121] Compared with the sequence of the TaWAK5 gene (SEQ ID No. 3) in the wild-type wheat Fielder, it was found that the regions corresponding to the TaWAK5 genes on the two homologous chromosomes in the T3 generation CRISPR knockout transgenic wheat homozygous plant tawak5-2 had the following changes: 2bp were deleted at target site 1 (SEQ ID No. 3 573-574), resulting in a gene frameshift mutation and premature termination of translation.

[0122] The above results indicated that tawak5-1 and tawak5-2 were positive CRISPR knockout transgenic wheat, named CRISPR knockout TaWAK5 transgenic wheat.

[0123] 3. Identification of drought resistance of transgenic wheat

[0124] In this experiment, the wheat to be tested was used as the research object, including the transgenic recipient variety wheat Fielder (recorded as wild-type wheat, as a negative control), T3 generation transgenic wheat OE3 and OE6 overexpressing the TaWAK5 gene, and T3 generation CRISPR knockout TaWAK5 transgenic wheat tawak5-1 and tawak5-2.

[0125] 1. Identification of drought resistance in repeated droughts

[0126] 1) Seed pretreatment: The wheat seeds to be tested were treated with 1% hydrogen peroxide for 1 day to break the seed dormancy and prepare for subsequent germination.

[0127] 2) Seedling planting: Select seedlings with the same germination and growth conditions and plant them in plastic boxes of the same size of 56cm×38cm×11cm. Sow 30 seeds for each strain, then bury the plastic boxes in outdoor soil, ensuring that the height of the plastic boxes is flush with the ground to simulate the natural growth environment.

[0128] 3) Drought treatment and data statistics: When the seedlings grew to the three-leaf and one-heart stage, the drought treatment group was subjected to drought treatment, that is, watering was stopped. After 25 days of drought treatment (at this time, the phenotypes of wild-type and transgenic wheat plants had shown obvious differences), all plants were rehydrated. The survival rate was counted 5 days after rehydration. Three independent biological replicates were set to ensure the reliability of the experimental results.

[0129] The formula for calculating the seedling survival rate is: survival rate (%) = number of surviving plants / number of planted plants (30) × 100%.

[0130] The experimental results are as follows Figure 3As shown, after rehydration, the survival rate of wild-type wheat was significantly higher than that of transgenic wheat with CRISPR knockout of TaWAK5 (tawak5-1 and tawak5-2), but lower than that of transgenic wheat overexpressing the TaWAK5 gene (OE3 and OE6).

[0131] This result shows that the TaWAK5 gene can regulate the drought resistance of wheat at the seedling stage, which is reflected in: the TaWAK5 gene improves the drought resistance of wheat; knocking out or reducing the expression of the TaWAK5 gene reduces the drought resistance of wheat.

[0132] 2. Thousand-grain weight test

[0133] The experiment was conducted in the dry greenhouse of the Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, with two treatments: drought stress (DS) and well watered (WW). The drought stress crops were planted in the dry greenhouse without watering during the entire growth period; the well watered crops were irrigated before wintering, during the booting period, and during the flowering period (750m 3 ha -1 ). Each wheat strain to be tested was sown in 4 rows, with a row length of 2 m, a row spacing of 30 cm, and 40 seeds per row, with conventional field management. After harvest, the thousand-grain weight of each strain was measured.

[0134] The experimental results are as follows Figure 4 As shown in the figure, under drought stress (DS), the thousand-grain weight of wild-type wheat was significantly higher than that of transgenic wheat with CRISPR knockout of TaWAK5 (tawak5-1 and tawak5-2), but lower than that of transgenic wheat overexpressing the TaWAK5 gene (OE3 and OE6).

[0135] This result indicates that the TaWAK5 gene can regulate the thousand-grain weight of wheat under drought conditions, which is reflected in: the TaWAK5 gene increases the thousand-grain weight of wheat under drought conditions; knocking out or reducing the expression of the TaWAK5 gene reduces the thousand-grain weight of wheat under drought conditions.

[0136] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be implemented in a wide range under equivalent parameters, concentrations and conditions without departing from the spirit and scope of the present invention and without the need for unnecessary experimentation. Although the present invention provides specific embodiments, it should be understood that further improvements may be made to the present invention. In short, according to the principles of the present invention, this application intends to include any changes, uses or improvements to the present invention, including changes made by conventional techniques known in the art that depart from the scope disclosed in this application. Applications of some of the basic features may be made within the scope of the following appended claims.

Claims

1. Application of protein TaWAK5 or its related biomaterials in the following A1) or A2): A1) Improve plant drought resistance; A2) Cultivating drought-resistant plants; The protein TaWAK5 is B1) or B2) or B3) or B4): B1) the amino acid sequence is the protein shown in SEQ ID No. 2; B2) a fusion protein obtained by connecting a tag to the N-terminus and / or the C-terminus of the protein shown in SEQ ID No. 2; B3) a protein having the same biological function obtained by replacing and / or deleting and / or adding one or more amino acid residues of the amino acid sequence shown in SEQ ID No. 2; B4) A protein having 80% or more identity with the amino acid sequence defined by SEQ ID No. 2, derived from a plant and having the same biological function.

2. The use according to claim 1, characterized in that: The protein TaWAK5-related biological material is any one of C1) to C7): C1) a nucleic acid molecule encoding the protein TaWAK5; C2) an expression cassette comprising 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) a recombinant microorganism containing the nucleic acid molecule described in C1), or a recombinant microorganism containing the expression cassette described in C2), or a recombinant microorganism 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 molecule 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).

3. The use according to claim 2, characterized in that: The nucleic acid molecule encoding the protein TaWAK5 is as follows D1) or D2) or D3) or D4): D1) The nucleotide sequence is the DNA molecule shown in SEQ ID No.1; D2) the nucleotide sequence is the DNA molecule shown in SEQ ID No.3; D3) a DNA molecule having 90% or more identity with the nucleotide sequence defined in D1) or D2), derived from a plant and encoding the protein TaWAK5 described in claim 1; D4) A DNA molecule that hybridizes with the nucleotide sequence defined in D1) or D2) under stringent conditions and encodes the protein TaWAK5 described in claim 1.

4. Use of a substance that inhibits the activity of the protein TaWAK5 described in claim 1 or inhibits the expression of a nucleic acid molecule encoding the protein TaWAK5 described in claim 1 in the following E1) or E2): E1) Reduce plant drought resistance; E2) Cultivating plants with low drought resistance.

5. The use according to claim 4, characterized in that: The substance is a CRISPR system that inhibits the expression of a nucleic acid molecule encoding the protein TaWAK5; The CRISPR system includes an sgRNA targeting a nucleic acid molecule encoding the protein TaWAK5, The targets of the sgRNA are target 1 and target 2; The nucleotide sequence of the target 1 is positions 557-576 of SEQ ID No. 3; The nucleotide sequence of the target 2 is positions 740-759 of SEQ ID No.

3.

6. A method for improving drought resistance of plants, comprising the following F1) or F2): F1) The method comprises the following steps: increasing the content and / or activity of the protein TaWAK5 in the target plant to improve the drought resistance of the plant; F2) The method comprises the following steps: increasing the expression of a nucleic acid molecule encoding a protein TaWAK5 in a target plant, thereby increasing the drought resistance of the plant; The target plant contains a nucleic acid molecule encoding protein TaWAK5; The protein TaWAK5 is B1) or B2) or B3) or B4): B1) the amino acid sequence is the protein shown in SEQ ID No. 2; B2) a fusion protein obtained by connecting a tag to the N-terminus and / or the C-terminus of the protein shown in SEQ ID No. 2; B3) a protein having the same biological function obtained by replacing and / or deleting and / or adding one or more amino acid residues of the amino acid sequence shown in SEQ ID No. 2; B4) A protein having 80% or more identity with the amino acid sequence defined by SEQ ID No. 2, derived from a plant and having the same biological function.

7. A method for cultivating a highly drought-resistant plant, comprising the following steps: increasing the content and / or activity of the protein TaWAK5 of claim 1 in a starting plant to obtain a transgenic plant, namely, the target plant; The transgenic plants have higher drought resistance than the starting plants.

8. A method for cultivating plants with high drought resistance, comprising the following steps: increasing the expression of the nucleic acid molecule encoding the protein TaWAK5 in claim 1 in a starting plant to obtain a transgenic plant, namely, the target plant; The drought resistance of the transgenic plants is higher than that of the starting plants.

9. The use according to any one of claims 1 to 5 or the method according to any one of claims 6 to 8, characterized in that: The plant is the following N1) or N2) or N3): N1) monocots or dicots; N2) Gramineae; N3) Wheat.

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