Application of wheat-derived protein and related biological materials thereof in dwarf wheat breeding

Abstract

The invention discloses application of protein derived from wheat and related biological materials thereof in dwarf wheat breeding. The TaWAK3-B gene in a wheat genome is knocked out by utilizing a gene editing technology, so that a dwarf wheat variety is cultivated, and a novel molecular breeding method is provided for improving the yield and quality of wheat. Experimental results show that compared with wild type wheat, after TaWAK3-B is knocked out, the plant height of the knocked-out line wheat is remarkably reduced, and the heading period is remarkably delayed. Therefore, the TaWAK3-B protein has the potential of regulating the plant height and the growth and development period of wheat, and can be applied to lodging-resistant breeding and plant type improvement of plants.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and particularly relates to application of a protein derived from wheat and related biological materials in breeding of short-stalk wheat. BACKGROUND

[0002] Plant height is one of the most important agronomic traits of wheat, and directly affects the resistance to lodging and yield. According to statistics, the yield loss rate caused by lodging is 0.25-0.5, so reducing plant height is an important goal to enhance the resistance to lodging of wheat and thus to improve the yield of wheat. The plant height of wheat is composed of different internode lengths, and each internode lengthens at different stages of wheat development, and finally each internode length determines the plant height of wheat. The second node below the ear and the second node from the bottom mainly determine the resistance to lodging of wheat. During the evolution from wild wheat to cultivated wheat, high-stalk and large-head are the main direction of evolution, but when the plant height reaches a certain height, it will affect the resistance to lodging of wheat, so appropriately reducing the plant height can enhance the resistance to lodging of wheat. However, too low plant height will affect the biological yield of wheat, and too short plant height will cause the leaves of the plant to be too close, resulting in poor light transmittance between plants and affecting the yield of wheat, so the plant of wheat should have a moderate height to maximize the yield. At present, the most commonly used short-stalk genes in wheat are Rht-B1b, Rht-D1b and Rht8, which are relatively single and have a narrow genetic background, and cannot meet the genetic diversity of wheat. It is found that Rht-B1b, Rht-D1b and Rht8 will reduce the thousand-grain weight and the number of grains per ear while reducing the plant height, so it is an important target of current short-stalk breeding to excavate new short-stalk genes. SUMMARY

[0003] The technical problem to be solved by the present application is how to regulate the plant height and / or how to reduce the plant height and / or how to breed short-stalk wheat and / or how to regulate the heading stage of wheat and / or how to delay the heading stage of plants.

[0004] In order to solve the above technical problem, the present application first provides the application of a protein or a substance for regulating the expression of a gene encoding the protein or a substance for regulating the activity or content of the protein, and the application can be any one of the following:

[0005] P1. The application of the protein or the substance for regulating the expression of a gene encoding the protein or the substance for regulating the activity or content of the protein in regulating the plant height;

[0006] P2. The application of the protein or the substance for inhibiting the expression of a gene encoding the protein or the substance for reducing the activity or content of the protein in reducing the plant height;

[0007] P3, use of the protein or a substance for regulating expression of a gene encoding the protein or a substance for regulating activity or content of the protein in regulating heading date of a plant;

[0008] P4, use of the protein or a substance for inhibiting expression of a gene encoding the protein or a substance for reducing activity or content of the protein in delaying heading date of a plant;

[0009] P5, use of the protein or a substance for regulating expression of a gene encoding the protein or a substance for regulating activity or content of the protein in breeding a dwarf plant;

[0010] The protein can be a protein as follows:

[0011] A1) a protein with an amino acid sequence of SEQ ID NO: 2 in the sequence listing;

[0012] A2) a protein with an amino acid sequence of SEQ ID NO: 4 in the sequence listing;

[0013] A3) a protein with an amino acid sequence of SEQ ID NO: 6 in the sequence listing;

[0014] A4) a protein derived from A1), A2) or A3) by substitution, deletion and / or addition of amino acid residues and having the same function, or a protein having more than 80% identity with the protein represented by A1), A2) or A3) and having the same function;

[0015] A5) a fusion protein obtained by linking a protein tag to the N terminus or / and C terminus of A1), A2), A3) or A4).

[0016] The protein described above can be derived from wheat.

[0017] In the above uses, the substance for regulating activity or content of the protein can be a substance for knocking out a gene encoding the protein and / or a substance for regulating expression of a gene encoding the protein.

[0018] In the above uses, the substance for regulating expression of a gene can be a substance for regulating at least one of the following six regulations: 1) regulation at the transcription level of the gene; 2) regulation after transcription of the gene (i.e. regulation of splicing or processing of the primary transcript of the gene); 3) regulation of RNA transport of the gene (i.e. regulation of mRNA transport from the nucleus to the cytoplasm of the gene); 4) regulation of translation of the gene; 5) regulation of mRNA degradation of the gene; and 6) post-translational regulation of the gene (i.e. regulation of activity of the protein translated from the gene).

[0019] In the above-mentioned applications, the regulation of gene expression can be inhibition or reduction of the gene expression, which can be achieved by gene knockout or by gene silencing.

[0020] The gene knockout refers to the inactivation of a specific target gene by homologous recombination. Gene knockout is the inactivation of a specific target gene by changes in DNA sequence.

[0021] The gene silencing refers to the phenomenon of non-expression or low expression of a gene without damaging the original DNA. Gene silencing is premised on not changing the DNA sequence to make the gene non-expressed or low-expressed. Gene silencing can occur at two levels. One is the transcriptional level of gene silencing caused by DNA methylation, heterochromatinization, and position effect, etc. The other is the post-transcriptional gene silencing, i.e. the inactivation of a gene by specific inhibition of target RNA after gene transcription, including antisense RNA, co-suppression, quelling, RNA interference (RNAi) and microRNA (miRNA) mediated translation inhibition, etc.

[0022] In the above-mentioned applications, the substance for regulating gene expression can be an agent for inhibiting or reducing the gene expression. The agent for inhibiting or reducing the gene expression can be an agent for knocking out the gene, such as an agent for knocking out the gene by homologous recombination or an agent for knocking out the gene by CRISPR-Cas9. The agent for inhibiting or reducing the gene expression can comprise a polynucleotide targeting the gene, such as siRNA, shRNA, sgRNA, miRNA or antisense RNA.

[0023] In the above-mentioned applications, the above 80% identity can be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 98% or 99% identity.

[0024] To solve the above technical problems, the present application also provides the following any one of the applications of the biological material related to the above-mentioned protein:

[0025] Q1. The application of the biological material in regulating plant height;

[0026] Q2. The application of the biological material in reducing plant height;

[0027] Q3. The application of the biological material in regulating the heading date of plants;

[0028] Q4. The application of the biological material in delaying the heading date of plants;

[0029] Q5, use of the biological material in plant breeding or quality improvement;

[0030] The biological material can be any one of the following:

[0031] B1) a nucleic acid molecule encoding the above-mentioned protein;

[0032] B2) an expression cassette containing the nucleic acid molecule of B1);

[0033] B3) a recombinant vector containing the nucleic acid molecule of B1), or a recombinant vector containing the expression cassette of B2);

[0034] B4) a recombinant microorganism containing the nucleic acid molecule of B1), or a recombinant microorganism containing the expression cassette of B2), or a recombinant microorganism containing the recombinant vector of B3);

[0035] B5) a transgenic plant cell line containing the nucleic acid molecule of B1), or a transgenic plant cell line containing the expression cassette of B2);

[0036] B6) a transgenic plant tissue containing the nucleic acid molecule of B1), or a transgenic plant tissue containing the expression cassette of B2);

[0037] B7) a transgenic plant organ containing the nucleic acid molecule of B1), or a transgenic plant organ containing the expression cassette of B2);

[0038] B8) a nucleic acid molecule inhibiting or reducing the expression of the gene encoding the above-mentioned protein or the activity of the above-mentioned protein;

[0039] B9) an expression cassette, a recombinant vector, a recombinant microorganism or a transgenic plant cell line containing the nucleic acid molecule of B8).

[0040] In the above-mentioned applications, the nucleic acid molecule can be a DNA molecule as shown below:

[0041] b1) a DNA molecule whose coding sequence is shown in SEQ ID NO: 1 of the sequence listing;

[0042] b2) a DNA molecule whose coding sequence is shown in SEQ ID NO: 3 of the sequence listing;

[0043] b3) a DNA molecule whose coding sequence is shown in SEQ ID NO: 5 of the sequence listing;

[0044] b4) a DNA molecule having 90% or more identity with the nucleotide sequence defined in b1), b2) or b3), and encoding the above-mentioned protein;

[0045] b5) hybridizes under stringent conditions to a nucleotide sequence defined in b1), b2) or b3) and encodes a DNA molecule of a protein as described above;

[0046] B8) the nucleic acid molecule is a DNA molecule expressing a gRNA targeting a gene encoding the protein of A1) above or a gRNA targeting a gene encoding the protein of A1) above;

[0047] The target sequence of the gRNA1 targeting a gene encoding the protein of A1) above is shown in SEQ ID NO: 1 at positions 950-969, and the target sequence of the gRNA2 targeting a gene encoding the protein of A1) above is shown in SEQ ID NO: 1 at positions 2995-3013.

[0048] The term "identity" refers to sequence similarity with a native nucleic acid sequence. Identity can be assessed by eye or by computer software. Using computer software, identity between two or more sequences can be expressed as a percentage (%) which can be used to assess identity between related sequences. The having 90% or more identity can be at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity.

[0049] In the above biological material, the expression cassette containing a nucleic acid molecule of B2) refers to a DNA capable of expressing the protein described above in the above applications in a host cell. The DNA can include not only a promoter that initiates transcription of the protein-encoding gene, but also a terminator that terminates transcription of the protein-encoding gene. Further, the expression cassette can include an enhancer sequence. Promoters that can be used in the present application include, but are not limited to, constitutive promoters, tissue-, organ-, and development-specific promoters, and inducible promoters.

[0050] A recombinant expression vector containing the expression cassette of the protein-encoding gene can be constructed using an existing plant expression vector.

[0051] In the above biological material, the recombinant microorganism can be specifically yeast, bacteria, algae, and fungi.

[0052] In the above applications, the plant can be any one of the following:

[0053] D1) a dicotyledonous plant;

[0054] E1) a monocotyledonous plant class,

[0055] E2) a plant of the order Poales,

[0056] E3) a plant of the family Poaceae,

[0057] E4) a plant of the genus Triticum,

[0058] E5) wheat.

[0059] To solve the above technical problems, the present application also provides a method for reducing plant height and / or delaying the heading stage of a plant, comprising reducing the plant height and / or delaying the heading stage of a plant by inhibiting or reducing the expression amount of a gene encoding the protein described in A1) above or the activity of the protein described in A1) above.

[0060] The above-mentioned reduction or inhibition of the expression amount of the gene encoding the protein described above in a plant can be achieved in any way known in the art, so as to cause a deletion mutation, an insertion mutation or a base substitution mutation of the gene, thereby reducing or losing the function of the gene. Specifically, it can be chemical mutagenesis, physical mutagenesis, RNAi, genome site-directed editing or homologous recombination, etc.

[0061] In the above-mentioned genome site-directed editing method, Zinc finger nuclease (ZFN) technology, Transcription activator-like effector nuclease (TALEN) technology or Clustered regularly interspaced short palindromic repeats / CRISPR associated (CRISPR / Cas9) system technology, and other technologies capable of achieving genome site-directed editing can be used. Regardless of which method is used, either the entire coding gene of the protein described above can be used as a target, or each element regulating the expression of the gene encoding the protein described above can be used as a target, as long as the function of the gene is lost or reduced. For example, the exons or 5'UTR of the coding gene of the protein described above can be used as a target.

[0062] The method can comprise introducing into the plant a substance that reduces or inhibits the expression of the gene encoding the protein described above; the substance that reduces or inhibits the expression of the gene encoding the protein described above can be any one of the following c1) to c4):

[0063] c1) a nucleic acid molecule that inhibits or reduces the expression of the gene encoding the protein described in A1) above;

[0064] c2) an expression cassette containing the nucleic acid molecule of c1);

[0065] c3) a recombinant vector containing the nucleic acid molecule of c1), or a recombinant vector containing the expression cassette of c2);

[0066] c4) a recombinant microorganism containing the nucleic acid molecule of c1), or a recombinant microorganism containing the expression cassette of c2), or a recombinant microorganism containing the recombinant vector of c3).

[0067] In the above method, the nucleic acid molecule of c1) can be a DNA molecule expressing a gRNA targeting the protein-coding gene of A1) above or a gRNA targeting the protein-coding gene of A1) above;

[0068] The target sequence of the gRNA1 targeting the protein-coding gene of A1) is shown in SEQ ID NO: 1, 950-969, and the target sequence of the gRNA2 is shown in SEQ ID NO: 1, 2995-3013.

[0069] The above method can be specifically a site-directed editing of the protein-coding gene of A1) above by a CRISPR / Cas9 method to reduce or inhibit the expression of the protein-coding gene of A1) above in plants, for example, a recombinant plasmid vector expressing Cas9 protein and gRNA is used to realize the transfer and integration of exogenous genes into plant cells through the infection of Agrobacterium.

[0070] In the above method, the inhibition or reduction of the expression of the protein-coding gene of A1) above in plants can be at least one mutation of the protein-coding gene shown in SEQ ID NO: 1 in plants:

[0071] 1) deletion of 1 nucleotide A at position 966 of SEQ ID NO: 1 in the plant sequence table;

[0072] 2) deletion of 3 nucleotides CAC at positions 963-965 of SEQ ID NO: 1 in the plant sequence table;

[0073] The plant can be wheat.

[0074] The protein described above and / or the biological material described above also belong to the protection scope of the present application. The beneficial effects of the present application are that:

[0075] The present application cultivates a dwarf wheat variety by knocking out TaWAK3-B gene through the use of gene editing technology, thereby providing a new molecular breeding method for improving the yield and quality of wheat. The experimental results show that, compared with the wild type, after knocking out TaWAK3-B, the plant height of the knockout strain is significantly reduced, and the heading period is significantly delayed. This shows that the TaWAK3-B protein has the potential to regulate plant height and wheat growth and development period, and can be applied to the research of plant lodging resistance and plant type improvement. The present application aims to cultivate a smaller wheat variety, which is expected to play a role in improving the yield and quality of wheat and has potential value in improving the lodging resistance of wheat and improving the excellent plant type.

[0076] The present application knocks out the TaWAK3-B gene of wheat by CRISPR gene editing technology to breed short-stalk wheat varieties. Specifically, a CRISPR / Cas9 knockout expression vector of TaWAK3-B is constructed using a CRISPR / Cas9 knockout target primer pair, a wheat endogenous U6a promoter, and a pBUE414 plasmid. The construction of this vector includes steps such as restriction enzyme digestion, ligation, transformation, colony PCR detection, and sequencing analysis.

[0077] The present application introduces the constructed CRISPR / Cas9 knockout expression vector of TaWAK3-B into the wheat variety Fielder by Agrobacterium-mediated method. This process involves transforming the vector into wheat plants so that they carry the CRISPR / Cas9 system. In the subsequent generation segregation process, various molecular biology techniques including PCR, sequencing analysis, etc. are used to identify and analyze the transgenic wheat plants. Through these techniques, it is determined whether short-stalk wheat plants with TaWAK3-B homozygous mutations are obtained. These plants will not contain T-DNA insertion and will not have off-target effects. BRIEF DESCRIPTION OF DRAWINGS

[0078] Figure 1 Sequencing identification of CRISPR / Cas9 knockout target sites and gene editing materials for TaWAK3-B gene DNA sequence.

[0079] Figure 2 Differences in plant height, ear length, and internode length phenotypes of wild-type Fielder and three TaWAK3-B homozygous knockout mutants. The left graph shows the comparison of plant height phenotypes of wild-type Fielder and three TaWAK3-B homozygous knockout mutants; the right graph shows the comparison of ear length and internode length of wild-type Fielder and three TaWAK3-B homozygous knockout mutants.

[0080] Figure 3 Difference analysis of heading date, plant height, ear length, and spikelet number of wild-type Fielder and TaWAK3-B homozygous knockout mutant wheat. ns represents no significant difference.

[0081] Figure 4 pCBC-MT1T2 plasmid map. DETAILED DESCRIPTION

[0082] The present application will be further described in detail below in conjunction with specific embodiments, and the examples given are only to illustrate the present application, not to limit the scope of the present application. The examples provided below can serve as a guide for further improvement by those of ordinary skill in the art, and do not in any way constitute a limitation on the present application.

[0083] The experimental methods in the following examples 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, unless otherwise specified. The materials, reagents, etc. used in the following examples can be obtained commercially, unless otherwise specified.

[0084] The following examples use Excel 2016 statistical software to process data, and the experimental results are expressed as mean ± standard deviation. The t test is used, P<0.05 (*) indicates a significant difference, P<0.01 (**) indicates a highly significant difference, and P<0.001 (***) indicates a highly significant difference.

[0085] The test materials and sources in the embodiments of the present application are as follows:

[0086] Transgenic wheat material: Fielder.

[0087] The expression vector pBUE414 was donated by Professor Chen Qijun of China Agricultural University and was preserved in the laboratory. It is available to the public from the applicant and is only used to repeat the present application and cannot be used for other purposes. Related literature: Ke W, Xing J, Chen Z, Zhao Y, Xu W, Tian L, Guo J, Xie X, Du D, Wang Z, Li Y, Xu J, Xin M, Guo W, Hu Z, Su Z, Liu J, Peng H, Yao Y, Sun Q, Ni Z. The TaTCP4 / 10-B1 cascade regulates awn elongation in wheat (Triticum aestivum L.). Plant Commun. 2023 Jul 10;4(4):100590.

[0088] The pCBC-MT1T2 plasmid is preserved in the laboratory and is available to the public from the applicant and is only used to repeat the present application and cannot be used for other purposes. The vector map is shown in Figure 4 Related literature: Ke W, Xing J, Chen Z, Zhao Y, Xu W, Tian L, Guo J, Xie X, Du D, Wang Z, Li Y, Xu J, Xin M, Guo W, Hu Z, Su Z, Liu J, Peng H, Yao Y, Sun Q, Ni Z. The TaTCP4 / 10-B1 cascade regulates awn elongation in wheat (Triticum aestivum L.). Plant Commun. 2023 Jul 10;4(4):100590.

[0089] Strains: Escherichia coli competent cells were T1, and Agrobacterium competent cells for wheat infection transformation were EHA105.

[0090] Enzymes and reagents: DNA polymerase, T4 DNA ligase, restriction enzymes, Marker from Thermo, and gel recovery kit from Kangwei Century.

[0091] Example 1. Application of TaWAK3-B gene in regulating wheat plant height and heading date

[0092] 1. Obtaining of wheat TaWAK3-B gene edited plants

[0093] 1.1 Target design

[0094] After downloading the CDS sequence of TaWAK3-B (Sequence 1 in the sequence listing), a segment was selected. The online target design website http: / / www.e-crisp.org / E-CRISP / was opened, and appropriate target sequences were designed and selected. The gene sequence was copied into the text box, the PAM requirement of NGG was selected, and the wheat database was selected. The designed sgRNA target sequence was subjected to BLAST detection in the wheat gene library. The target primer specific to TaWAK3-B gene was designed for knocking out TaWAK3-B gene; the nucleotide sequence of sgRNA1 target was 5'-GCAATCCTTACCTCACAGAC-3', which was targeted to the 950-969th nucleotide of TaWAK3-B gene (Sequence 1 in the sequence listing); for knocking out TaWAK3-B gene; the nucleotide sequence of sgRNA2 target was 5'-GATGAAATCGCTGCAATTG-3', which was targeted to the 2995-3013th nucleotide of TaWAK3-B gene (Sequence 1 in the sequence listing). The amino acid sequence of TaWAK3-B protein is shown in Sequence 2 in the sequence listing.

[0095] 1.2 Construction of CRISPR / Cas9 knockout expression vector of TaWAK3-B gene

[0096] 1.2.1 First PCR amplification

[0097] The primer specific to the target of TaWAK3-B gene was designed for knocking out TaWAK3-B gene. pCBC-MT1T2 plasmid was used as the template, and MT1T2-F0 / MT1T2-R0 was used as the primer for first PCR amplification. 60ul of PCR product was subjected to gel recovery purification to obtain the first purified product.

[0098] MT1T2-F0: 5'-GCAATCCTTACCTCACAGAC GTTTTAGAGCTAGAAATAGC-3';

[0099] MT1T2-R0: 5'-CAATTGCAGCGATTTCATCCGCTTCTTGGTGCC-3';

[0100] Amplification system:

[0101]

[0102] Amplification procedure:

[0103]

[0104] 1.2.2 Second PCR amplification

[0105] The first purified product obtained by the above steps was used as a template for second PCR amplification with the primer pair MT1T2-F / MT1T2-R. The 60ul PCR product obtained was purified by gel recovery to obtain the second purified product.

[0106] MT1T2-F: 5'-AATAATGGTCTCAAGCGCAATCCTTACCTCACAGAC-3';

[0107] MT1T2-R: 5'-ATTATTGGTCTCTAAACCAATTGCAGCGATTTCATC-3';

[0108] Amplification system:

[0109]

[0110] The PCR amplification step was the same as the first PCR amplification.

[0111] 1.2.3 Enzyme digestion and ligation system

[0112] The pBUE414 vector was digested with BsaI and simultaneously ligated with T4 Ligase and the second purified product. The enzyme digestion and ligation were performed simultaneously. The enzyme digestion and ligation system was as follows:

[0113]

[0114] Enzyme digestion and ligation conditions:

[0115]

[0116] 1.2.4 Transformation of ligation product

[0117] The ligation product is transferred into E. coli, and positive E. coli is obtained by PCR detection, ensuring that the target plant has been successfully transformed and contains the CRISPR / Cas9 system. Further sequencing analysis of the sgRNA sequence in the CRISPR / Cas9 system is performed to confirm its correctness and pairing with the TaWAK3-B gene (Qingke Biology), and positive plasmid is obtained by extracting the plasmid of the positive E. coli.

[0118] Sequencing primers:

[0119] 414-F: 5'-TTTCCCAGTCACGACGTTGT-3';

[0120] 414-R: 5'-ATCTCTAGAGAGGGGCACGA-3';

[0121] 1.2.5 Transformation of plasmid into Agrobacterium

[0122] The sequence-corrected positive plasmid obtained in step 1.2.5 is transferred into EHA105 Agrobacterium competent cells using the heat shock method, and recombinant Agrobacterium EHA105 / TaWAK3-B-gRNA containing the CRISPR / Cas9 knockout expression vector of TaWAK3-B is obtained, which is used for knocking out the expression of the TaWAK3-B gene.

[0123] 1.3 Obtaining of TaWAK3-B gene edited plant

[0124] The method used for transformation in the present application is Agrobacterium-mediated method, and the operation procedure is as follows:

[0125] Preparation of Agrobacterium suspension: the recombinant Agrobacterium EHA105 / TaWAK3-B-gRNA obtained in step 1.2 is shaken at a speed of 160 rpm and 28°C the day before the test. After the wheat Fielder ear is ready, the preparation of the Agrobacterium suspension is started. 1 ml of bacterial solution is taken in a 1.5 ml centrifuge tube, 1.4 ul of acetyl-syringone (0.1M) is added and mixed to obtain the Agrobacterium suspension.

[0126] Infection: take the wheat ear that has been pollinated for about 15 days, remove the embryo, and add the prepared Agrobacterium suspension for infection for 5 minutes, and then place it on the co-culture medium at 23°C for 3 days of co-culture.

[0127] Rest: after co-culture, place it on the rest medium and incubate it in the dark at 25°C for 5 days.

[0128] Screening 1: transfer the callus after rest culture to screening medium 1 (containing hygromycin). Seal the culture dish with sealing film and incubate it in the dark at 25.5°C for 2 weeks for the first screening.

[0129] Screening 2: After the first screening, the resistant calli were cut and transferred to screening medium 2 (containing hygromycin). The culture dish was sealed with sealing film and cultured in a dark incubator at 25.5 degrees for 2 weeks before the second screening.

[0130] Regeneration 1: Transfer the calli that showed resistance in the second screening to the regeneration medium. Resistant calli generally have green buds or green spots, or beautiful beige spherical structures. Do not transfer mushy or brown calli. Calli with proliferation can be cut into smaller calli. Small pieces from the same callus (one line) should be placed on the same line. Pay attention to the orientation of the callus, for example, the green buds and green spots are facing upwards. Seal the culture dish and place it in an incubator at 25 degrees Celsius with light (16 hours) for 2 weeks for regeneration culture.

[0131] Regeneration 2: After 2 weeks of regeneration culture, transfer healthy seedlings to new resistant regeneration boxes. Once the seedlings reach a certain size, they can be sampled and transplanted into soil for growth to obtain T0 generation transgenic seedlings.

[0132] 1.4 Identification of transgenic seedlings

[0133] 1.4.1 Extraction of genomic DNA from these transgenic wheat plants using the CTAB method

[0134] Place an appropriate amount of fresh T0 transgenic wheat seedling leaves obtained in step 1.3 into a 2.0ml centrifuge tube and freeze in liquid nitrogen. Grind the liquid nitrogen-frozen leaves into a powder using a grinding rod. Add 700μl of CTAB preheated at 65°C, shake vigorously, and immediately return to a 65°C water bath for 30 minutes, mixing every 10 minutes. Add an equal volume (700μl) of chloroform and isoamyl alcohol (24:1) to the 2.0ml centrifuge tube, mix thoroughly, balance, and centrifuge at 12,000 rpm for 10-15 minutes. Transfer the supernatant to a 1.5ml centrifuge tube, add 1 volume of cold isopropanol (600μl + 600μl isopropanol), centrifuge at 12,000 rpm for 10 minutes, and discard the supernatant. Wash with 75% ethanol, discard the ethanol, air-dry, and add 200μl of sterile water to dissolve the DNA.

[0135] 1.4.2 PCR detection

[0136] The extracted DNA of the T0 generation transgenic plants was used as a template for identification using the following PCR system to obtain PCR products. The T0 generation transgenic plants containing a 383 bp PCR product in the PCR amplification product were positive T0 generation transgenic plants.

[0137] Cas9-F:5'-CTAAGCGGAACAGCGACAAG-3';

[0138] Cas9-R: 5'-GGCCAGGTAGAGGAAGTTCAC-3';

[0139]

[0140] PCR reaction program:

[0141]

[0142] 1.4.3 Sequencing identification results of three lines of editing sites:

[0143] The following primer pairs will be used to amplify the PCR products of the T0 generation plants:

[0144] 1.4.2 The PCR products obtained are subjected to Sanger sequencing: 6B-F: 5'-TTGGGTCGTTGGAACCGTAG-3' and 6B-R: 5'-GCACCCACACATGAAGGATC-3'. The sequencing results are compared with the target sequence of the TaWAK3-B gene (nucleotide molecule shown in SEQ ID NO: 1 in the sequence listing) to identify whether an insertion or deletion occurs at the editing site, and the mutation type is counted.

[0145] It is detected that among the positive T0 generation transgenic wheat plants, 3 plants have undergone gene editing.

[0146] The T0 generation gene edited plants are selfed for 2 generations to obtain T2 generation gene edited plants. Then the mutation types of the T2 generation gene edited plants are detected according to the above method. A total of 3 homozygous (the same mutation occurs on both homologous chromosomes in the wheat genome) mutants are obtained. The 3 homozygous mutants are named Tawak3-b#1, Tawak3-b#2 and Tawak3-b#3, respectively.

[0147] The detection results of the 3 homozygous mutants are shown in Table 1. Figure 1 .

[0148] Tawak3-b#1 has a deletion of 1 nucleotide "A" (i.e., the 966th nucleotide of SEQ ID NO: 1 in the sequence listing) in the TaWAK3-B gene, which causes a frame shift and premature termination, resulting in loss of function of the TaWAK3-B protein.

[0149] The amino acid sequence of the mutated TaWAK3-b1 protein in the above Tawak3-b#1 mutant is shown in SEQ ID NO: 4, and its coding sequence is shown in SEQ ID NO: 3.

[0150] Tawak3-b#2 has a deletion of three nucleotides "CAC" in the TaWAK3-B gene (i.e., the 963rd to 965th nucleotides of sequence 1 in the sequence list are missing), which causes a frameshift and produces the mutated TaWAK3-b2 protein.

[0151] The amino acid sequence of the mutated TaWAK3-b2 protein in the above-mentioned Tawak3-b#2 mutant is shown in SEQ ID NO: 6, and its coding sequence is shown in SEQ ID NO: 5.

[0152] Tawak3-b#3 had a deletion of 1 nucleotide "A" in the TaWAK3-B gene (i.e., the 966th nucleotide of sequence 1 in the sequence list was missing), which caused a frameshift and premature termination, resulting in loss of TaWAK3-B protein function.

[0153] The amino acid sequence of the mutated TaWAK3-b3 protein in the above-mentioned Tawak3-b#3 mutant is shown in SEQ ID NO: 4, and its coding sequence is shown in SEQ ID NO: 3.

[0154] The seeds of Tawak3-b#1, Tawak3-b#2 and Tawak3-b#3 plants were harvested and their phenotypic characteristics were observed.

[0155] 2. Phenotypic Observation of TaWAK3-B Gene-Edited Plants

[0156] The wild-type Fielder and three homozygous mutants of TaWAK3-B (Tawak3-b#1, Tawak3-b#2, and Tawak3-b#3) were planted in the field. Figure 2 and Figure 3 , it was observed at the late heading stage that the plant height of TaWAK3-B gene-edited (homozygous mutant) plants was significantly shorter than that of the wild-type Fielder.

[0157] A comparative analysis of the heading date, plant height, spike length, and spikelet number of Fielder, Tawak3-b#1, Tawak3-b#2, and Tawak3-b#3 at maturity showed that compared with Fielder, Tawak3-b#1, Tawak3-b#2, and Tawak3-b#3 had significantly reduced plant height (P < 0.001) and a significantly delayed heading date (P < 0.001), while spike length and spikelet number remained unchanged. This suggests that TaWAK3-B deletion significantly reduces plant height and delays heading date. These results indicate that the homozygous TaWAK3-B mutation causes dwarfing in wheat, which has important potential for breeding dwarf wheat varieties to increase and stabilize yield.

[0158] Sequence 1:

[0159]

[0160] Sequence 2:

[0161]

[0162] Sequence 3:

[0163]

[0164] 序列4:

[0165] MAQITISAVAIAAAVLILVPVFTFTRGTVEAADPIGMPNCVISCGDVEVPYPFGIGANTSCYLPGFNLTCDNTTGSSPRLLLDADGTIQVLGIMNLDSGGYMYVQRNGDVKIKVDANGNGNGTFSQGLKHDGPYMLLTVLLPFRSELILMGCNVQATVKSGNATVASCTSLCDDTADYETSVIDRCSGGSTGCCRANIIKPGYDDDGRGEVYSSSSTAYDVQLRRLGRWNRSADRDRFPVRVFIAERGWFDNTSVSNDLLQTGRPPSKETLKVPVWLSWEVVDHPNRSSVCKSNHSQRGNGRKRGGYACACKNGYEGNPYLTTGAKISMSASGHQHSLDAMVNASTRKDPSCVGARQEPPATSPYPVGASTRKIFAGHPAGMWRCHTRLVLARPSATGQGSASSVTRAINPRVCYSVMVGSRSSVSPFTTPRCVLSGSTTLGPTQRYQLQWPLI。

[0166] 序列5:

[0167]

[0168] Sequence 6:

[0169]

[0170] The application has been described in detail. Those skilled in the art will understand that they can make modifications and alterations to this application without departing from the spirit and scope of the application. Although this application presents specific examples, it is to be understood that further modifications can be made. In general, the application is intended to cover any adaptations or variations of the present application including modifications based on the disclosure in the patent application as filed, which are apparent to those skilled in the art to which the present application pertains.

Claims

1. Use of a protein or a substance that regulates the expression of a gene encoding the protein or a substance that regulates the activity or content of the protein, characterized in that: The application is any of the following: P1. Use of the protein or the substance regulating the expression of the gene encoding the protein or the substance regulating the activity or content of the protein in regulating plant growth; P2. Use of the protein or a substance that inhibits the expression of a gene encoding the protein or a substance that reduces the activity or content of the protein in reducing plant strain height; P3. Use of the protein or a substance that regulates the expression of a gene encoding the protein or a substance that regulates the activity or content of the protein in regulating the heading period of a plant; P4. Use of the protein or a substance that inhibits the expression of a gene encoding the protein or a substance that reduces the activity or content of the protein in delaying the heading period of a plant; P5. Use of the protein or a substance that regulates the expression of a gene encoding the protein or a substance that regulates the activity or content of the protein in dwarf plant breeding; The protein is the following protein: A1) The amino acid sequence is the protein of sequence 2 in the sequence listing; A2) The amino acid sequence is the protein of sequence 4 in the sequence listing; A3) The amino acid sequence is the protein of sequence 6 in the sequence listing; A4) a protein derived from the amino acid sequence of A1), A2) or A3) by substitution and / or deletion and / or addition of amino acid residues, or a protein having 80% or more identity with the protein of A1), A2) or A3) and having the same function; A5) A fusion protein obtained by connecting a protein tag to the N-terminus or / and C-terminus of A1), A2), A3) or A4).

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

3. Any of the following uses of a biomaterial related to the protein of claim 1 or 2: Q1. Application of the biomaterial in regulating plant strain growth; Q2. Application of the biomaterial in reducing plant strain height; Q3. Application of the biomaterial in regulating the heading period of plants; Q4. Use of the biological material in delaying the heading period of plants; Q5. Application of the biomaterial in plant breeding or quality improvement; The biological material is any one of the following: 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 inhibits or reduces the expression of a gene encoding the protein of claim 1 or the activity of the protein of claim 1; B9) An expression cassette, recombinant vector, recombinant microorganism or transgenic plant cell line containing the nucleic acid molecule described in B8).

4. The use according to claim 3, characterized in that: The nucleic acid molecule is a DNA molecule as shown below: b1) The coding sequence is the DNA molecule shown in Sequence 1 in the sequence listing; b2) The coding sequence is the DNA molecule shown in Sequence 3 in the sequence listing; b3) the coding sequence is the DNA molecule shown in Sequence 5 in the sequence listing; b4) a DNA molecule that has 90% or more identity with the nucleotide sequence defined in b1), b2) or b3) and encodes the protein of claim 1; b5) a DNA molecule that hybridizes under stringent conditions to the nucleotide sequence defined in b1), b2) or b3) and encodes the protein of claim 1; B8) the nucleic acid molecule is a DNA molecule that expresses a gRNA targeting the protein-coding gene described in A1) of claim 1 or a gRNA targeting the protein-coding gene described in A1) of claim 1; The target sequence of the gRNA1 targeting the protein encoding gene A1) in claim 1 is shown as positions 950-969 of sequence 1 in the sequence listing, and the target sequence of the gRNA2 is shown as positions 2995-3013 of sequence 1 in the sequence listing.

5. The use according to any one of claims 1 to 4, characterized in that: The plant is any one of the following: D1) Dicotyledons; E1) Monocotyledons, E2) plants of the order Poaceae, E3) Gramineae plants, E4) Triticum plants, E5) Wheat.

6. A method for reducing plant height and / or delaying plant heading period, comprising reducing plant height and / or delaying plant heading period by inhibiting or reducing the expression of a gene encoding the protein described in claim 1 A1) or the activity of the protein described in claim 1 A1) in a plant.

7. The method according to claim 6, characterized in that: The method comprises introducing into the plant a substance that reduces or inhibits the expression of the protein-encoding gene according to claim 1; the substance that reduces or inhibits the expression of the protein-encoding gene according to claim 1 is any one of the following substances c1) to c4): c1) a nucleic acid molecule that inhibits or reduces the expression of the gene encoding the protein described in A1) of claim 1; c2) an expression cassette containing the nucleic acid molecule described in c1); c3) a recombinant vector containing the nucleic acid molecule described in c1), or a recombinant vector containing the expression cassette described in c2); c4) 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).

8. The method according to claim 7, wherein: The nucleic acid molecule described in c1) is a DNA molecule that expresses a gRNA targeting the protein-coding gene described in A1) of claim 1 or a gRNA targeting the protein-coding gene described in A1) of claim 1; The target sequence of the gRNA1 targeting the A1) protein encoding gene is shown in positions 950-969 of Sequence 1 in the sequence listing, and the target sequence of the gRNA2 is shown in positions 2995-3013 of Sequence 1 in the sequence listing.

9. The method according to any one of claims 6 to 8, characterized in that: The method of inhibiting or reducing the expression of the gene encoding the protein of claim 1 in a plant is to perform at least one of the following mutations on the gene encoding the protein shown in Sequence 1 in the plant: 1) A nucleotide A at position 966 of sequence 1 in the plant sequence list is missing; 2) The three nucleotides CAC at positions 963-965 of sequence 1 in the plant sequence list are missing; The plant is wheat.

10. The protein according to claim 1 or 2 and / or the biomaterial according to claim 3 or 4.

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