Application of wheat b1 protein and related biological materials in regulating wheat kernel size, kernel weight and protein content

Abstract

The application discloses application of wheat B1 protein and related biological materials thereof in regulating wheat kernel size, kernel weight and protein content. The application causes B1 protein not to be expressed or to be down-regulated by knocking out a coding gene of B1 protein which inhibits awn elongation in a wheat genome, and experimental results show that the absence of B1 protein expression in the wheat genome can improve the kernel size, kernel weight and protein content of awnless wheat. The application can be applied to high-yield and high-quality breeding and variety improvement of wheat.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to the application of wheat B1 protein and related biomaterials in regulating wheat grain size, grain weight, and protein content. Background Technology

[0002] Wheat, a widely cultivated food crop worldwide, is not only a major source of starch and energy for humans but also provides nutrients such as protein, vitamins, and dietary fiber. Yield and grain protein content are two key target traits in wheat breeding and production; however, these two traits are typically negatively correlated. Therefore, identifying the key genes in wheat that synergistically regulate grain weight and protein content is of great significance for high-yield and high-quality wheat breeding and variety improvement.

[0003] wheat B1 The (TraesCS5A02G542800) gene encodes the C2H2 zinc finger transcription factor, which has been reported to inhibit awn elongation. Summary of the Invention

[0004] The technical problem to be solved by the present invention is how to coordinate wheat grain size, grain weight and protein content and / or how to increase wheat grain size and grain weight while increasing grain protein content.

[0005] To address the aforementioned technical problems, the present invention first provides the application of a protein, a substance regulating the expression of the protein-encoding gene, or a substance regulating the activity or content of the protein, wherein the application may be any of the following:

[0006] P1. The application of the protein or the substance that regulates the expression of the protein-encoding gene or the substance that regulates the activity or content of the protein in increasing the size of plant seeds; P2. The application of the protein or a substance that inhibits the expression of the protein-coding gene or a substance that reduces the activity or content of the protein in increasing the grain weight of plant seeds; P3. The application of the protein or the substance that regulates the expression of the protein-encoding gene or the substance that regulates the activity or content of the protein in increasing the grain weight and grain protein content of plant seeds. P4. The application of the protein or the substance that inhibits the expression of the protein-coding gene or the substance that reduces the activity or content of the protein in improving plant seed size and weight; P5. The application of the protein or the substance that inhibits the expression of the protein-coding gene or the substance that reduces the activity or content of the protein in improving the size, weight and protein content of plant seeds. P6. Application of the protein or substances that regulate the expression of the protein-encoding gene or substances that regulate the activity or content of the protein in high-yield and high-quality plant breeding. The protein in question is the following: A1) The amino acid sequence is that of the protein in sequence 1 of the sequence listing; A2) A protein derived from A1) or having more than 80% identity with and the same function as the protein shown in A1) obtained by substituting and / or deleting and / or adding amino acid residues of the amino acid sequence shown in A1). A3) is a fusion protein obtained by attaching a protein tag to the N-terminus and / or C-terminus of A1) or A2).

[0007] In the above applications, the protein may be derived from wheat.

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

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

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

[0011] The inhibition or reduction of gene expression described above can be achieved through gene knockout or gene silencing.

[0012] Gene knockout refers to the process of inactivating a specific target gene by altering its DNA sequence.

[0013] Gene silencing refers to the phenomenon of preventing or reducing gene expression without damaging the original DNA. Gene silencing presupposes no change in the DNA sequence, resulting in the absence or reduction of gene expression. Gene silencing can occur at two levels: transcriptional silencing due to DNA methylation, heterochromatinization, and position effects; and post-transcriptional gene silencing, which inactivates the gene at the post-transcriptional level through specific inhibition of target RNA. This includes antisense RNA, co-suppression, quelling, RNA interference (RNAi), and microRNA (miRNA)-mediated translational repression.

[0014] In the above applications, the substance regulating gene expression can be a reagent that inhibits or reduces the expression of the gene. The reagent that inhibits or reduces the expression of the gene can be a gene knockout reagent, such as a reagent that knocks out the gene through homologous recombination or a reagent that knocks out the gene through CRISPR-Cas9. The reagent that inhibits or reduces the expression of the gene can contain a polynucleotide that targets the gene, such as siRNA, shRNA, sgRNA, miRNA, or antisense RNA.

[0015] The above-mentioned inhibition or reduction of the expression level of the protein-coding genes mentioned above in plants can be achieved by any means in the existing technology, so as to induce deletion mutations, insertion mutations or base change mutations in the genes, thereby reducing or losing gene function. Specifically, this can be achieved by chemical mutagenesis, physical mutagenesis, RNAi, site-directed genome editing or homologous recombination, etc.

[0016] Among the aforementioned site-specific genome editing methods, zinc finger nuclease (ZFN) technology, transcription activator-like effector nuclease (TALEN) technology, clustered regularly interspaced short palindromic repeats / CRISPR-associated (CRISPR / Cas9 system) technology, and other technologies capable of site-specific genome editing can be employed. Regardless of the method used, the entire coding gene of the aforementioned proteins can be targeted, or individual elements regulating the expression of the coding gene of the aforementioned proteins can be targeted, as long as gene function loss or reduction can be achieved.

[0017] To address the aforementioned technical problems, the present invention also provides any of the following applications of biomaterials related to the proteins described above: P1. The application of the biomaterial in improving plant seed size; P2. Application of the aforementioned biomaterials in increasing the grain weight of plant seeds; P3. Application of the biomaterial in increasing the grain weight and protein content of plant seeds; P4. Application of the biomaterial in improving plant seed size and weight; P5. Application of the biomaterial in improving plant seed size, seed weight and seed protein content; P6. Application of the aforementioned biomaterials in high-yield and high-quality plant breeding; The biomaterial may be any of the following: B1) Nucleic acid molecules that encode the proteins described above; 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) Recombinant microorganisms containing the nucleic acid molecules described in B1), or recombinant microorganisms containing the expression cassette described in B2), or recombinant microorganisms containing the recombinant vector described in B3); 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 molecules described in B1), or transgenic plant tissue containing the expression cassette described in B2); B7) Transgenic plant organs containing the nucleic acid molecules described in B1), or transgenic plant organs containing the expression cassette described in B2); B8) Nucleic acid molecules that inhibit or reduce the expression of the genes encoding the proteins described above or the activity of the proteins described above; B9) Expression cassettes, recombinant vectors, recombinant microorganisms, or transgenic plant cell lines containing the nucleic acid molecules described in B8); B10) A transgenic plant containing the transgenic plant cell line described in B9); B11) Gene-edited wheat obtained by any of the methods described in claims 6-9.

[0018] In the above applications, the nucleic acid molecule can be a DNA molecule as shown below: b1) The coding sequence is the DNA molecule shown in sequence 2 of the sequence listing; b2) The coding sequence is the DNA molecule shown in sequence 7 of the sequence listing; b3) has 90% or more identity with the nucleotide sequence defined by b1) or b2) and is a DNA molecule encoding the protein of claim 1; b4) hybridizes under stringent conditions with the nucleotide sequence defined in b1), b2), or b3) and encodes a DNA molecule that encodes the protein of claim 1; B8) The nucleic acid molecule is a DNA molecule that expresses the gRNA of the protein-coding gene in claim 1(A1) or the gRNA that targets the protein-coding gene in claim 1(A1). The target sequence for sgRNA1, which targets the protein encoding gene A1 in claim 1, is shown at positions 60-81 of sequence listing sequence 3, and the target sequence for sgRNA2 is shown at positions 96-107 of sequence listing sequence 3.

[0019] In the above applications, the plant may be any of the following: D1) Dicotyledons; E1) Monocotyledons, E2) Plants of the order Poales, E3) Gramineae plants, E4) Wheat species, E5) Wheat.

[0020] To solve the above-mentioned technical problems, the present invention also provides a method for improving the size, weight and / or protein content of plant seeds, wherein the method may be M1 or M2; M1 includes improving the grain size, grain weight, and / or grain protein content of the wheat to be improved by inhibiting or reducing the expression level of the gene encoding the protein described in A1) above or the activity of the protein described in A1) above. M2 comprises gene-edited wheat obtained by knocking out the gene encoding the protein described in A1) above in the wheat to be improved, thereby increasing the grain size, grain weight, and / or grain protein content of the wheat to be improved; the grain size, grain weight, and / or grain protein content of the gene-edited wheat are higher than those of the target improved wheat; the wheat to be improved contains the gene encoding the protein described in A1) above.

[0021] The target improved wheat described above is a plant of the Triticum genus, more preferably hexaploid wheat, and even more preferably common wheat.

[0022] In one specific embodiment of the present invention, the target improved wheat is awnless common wheat and / or non-GMO common wheat.

[0023] In some embodiments, the wheat to be improved is awnless wheat. In some embodiments, knocking out the gene encoding the protein in the wheat to be improved to obtain gene-edited wheat includes: 1) Provide a CRISPR / Cas genome editing vector, wherein the CRISPR / Cas genome editing vector comprises a gRNA gene expression cassette and an RNA-directed nuclease gene expression cassette; the gRNA gene expression cassette is a DNA molecule introduced into wheat that can transcribe gRNA, wherein the gRNA targets the gene of the protein; the RNA-directed nuclease gene expression cassette is a DNA molecule introduced into wheat that can express the RNA-directed nuclease; 2) The CRISPR / Cas genome editing vector was introduced into awned wheat to obtain wheat containing gRNA gene expression cassettes and RNA-directed nuclease gene expression cassettes. This wheat was called the hybrid parent wheat. 3) The hybrid parent wheat is hybridized with the wheat to be improved to obtain gene-edited wheat in which the coding gene of the protein is knocked out.

[0024] The hybridization includes backcrossing. The transgenic plant described in B10 above may be wheat, the parent of the hybridization.

[0025] "Hybridization" refers to the production of offspring (e.g., cells, seeds, or plants) through fertilization, and includes hybridization (sexual) between plants and self-fertilization (self-pollination).

[0026] In this application, "RNA-directed nuclease" refers to RNA-directed DNA endonucleases associated with the CRISPR system. Unrestricted examples of RNA-directed nucleases include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, Cpf1, their homologs or modified forms thereof. In one implementation, the RNA-directed nuclease is Cas9.

[0027] The term "sgRNA (single-guide RNA)" is a component of the CRISPR-Cas system, responsible for guiding the Cas protein to recognize and cleave target nucleic acid molecules. In practical gene editing applications, sgRNA can be synthesized directly or obtained through plasmid expression or in vitro transcription. In this field, "gRNA" and "sgRNA" are often used interchangeably. In this document, "gRNA" and "sgRNA" are also used interchangeably. sgRNA generally refers to a single RNA structure formed by artificially modifying the crRNA / tracrRNA complex (gRNA) with a dual RNA structure, directly (or through a linker) linking the crRNA and tracrRNA. sgRNA is a short RNA containing a recognition region and a framework region.

[0028] The term "recognition region," also known as a guide sequence, is typically an RNA sequence (referred to herein as the "guide sequence") that is identical to or complementary to the target sequence or target site within the target RNA (sgRNA or gRNA). The guide sequence is generally sufficiently complementary to the target sequence to hybridize with it and guide the CRISPR / Cas complex to specifically bind to the target sequence. Perfect complementarity between the guide sequence and the target sequence is preferred, but some mismatch (e.g., a mismatch of 1-6 nucleotides) is permissible, as long as it still results in gene knockout. The complementarity between the guide sequence and its corresponding target sequence is at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99%. Methods for determining the complementarity of two nucleic acid sequences are within the capabilities of those skilled in the art.

[0029] The term "scaffold" generally refers to the structural or scaffold RNA sequence that guides the binding or interaction of RNA with RNA-directed nucleases and / or other RNA molecules (e.g., tracrRNA) into RNA (sgRNA or gRNA), and can also be called the backbone sequence of sgRNA. The scaffold can be conventionally selected by those skilled in the art; for example, it can be the backbone sequence of the sgRNA corresponding to Cas9, or it can be a mutant constructed based on this sequence that still retains the function of binding the corresponding Cas9.

[0030] The method described above may include introducing a substance into the target improved wheat that reduces or inhibits the expression of the protein-coding genes described above; the substance that reduces or inhibits the expression of the protein-coding genes described above is any one of the following c1)-c4): c1) Inhibit or reduce the expression of nucleic acid molecules of the protein-coding genes described in A1) above; c2) An expression cassette containing the nucleic acid molecule described in c1); c3) A recombinant vector containing the nucleic acid molecule described in c1), or a recombinant vector containing the expression cassette described in c2); c4) Recombinant microorganisms containing the nucleic acid molecules described in c1), or recombinant microorganisms containing the expression cassette described in c2), or recombinant microorganisms containing the recombinant vector described in c3).

[0031] In the above method, the nucleic acid molecule described in c1) is a DNA molecule that expresses sgRNA targeting the protein-coding gene described in A1) above, or sgRNA targeting the protein-coding gene described in A1) above; The target sequence of sgRNA1, which targets the gene encoding the A1 protein, is shown at positions 46-68 of Sequence Listing 3, and the target sequence of sgRNA2 is shown at positions 94-116 of Sequence Listing 3.

[0032] In various specific embodiments of the present invention, the nucleic acid molecule containing c1) described above is sequence 7 in the sequence listing.

[0033] In the above method, inhibiting or reducing the activity of the protein described above and / or the expression of the gene encoding the protein in the target improved wheat constitutes at least one of the following mutations in the protein-encoding gene shown in sequence 1 in the target improved wheat: 1) The 22 nucleotides TGGGCATGGAGATGGAAGAGAGGG in sequence 3 of the plant sequence listing are missing. 2) The 12 nucleotides TGTCGCTGCGCC at positions 96-107 of sequence 3 in the plant sequence listing are missing.

[0034] The target improved wheat is a plant of the genus Triticum, more preferably hexaploid wheat, and even more preferably common wheat.

[0035] In one specific embodiment of the present invention, the target improved wheat is awnless common wheat and / or non-GMO common wheat.

[0036] In the above applications and / or methods, the breeding indicators include grain size, grain weight, and / or grain protein content.

[0037] The breeding objectives include developing wheat with large grains, high grain weight, and / or high grain protein content (wheat with larger grains than the parents, wheat with higher grain weight than the parents, and / or wheat with higher grain protein content than the parents).

[0038] The proteins and / or biological materials described above are also within the scope of protection of this invention.

[0039] The wheat receptor mentioned above is a plant of the genus *Triticum*, more preferably hexaploid wheat, even more preferably common wheat, and even more preferably awnless common wheat. The beneficial effects of this invention are as follows: This invention provides the B1 protein, its encoding gene, and its application in synergistically improving wheat yield and quality. The B1 protein is derived from common wheat (…). Triticum aestivum L.). The gene encoding the B1 protein also falls within the scope of protection of this invention. The gene encoding the B1 protein is named... B1 Genes. This invention also protects the use of B1 protein in regulating wheat grain weight and / or protein content. This invention is of great significance for the synergistic improvement of high-yield and high-quality wheat.

[0040] This invention utilizes genetic engineering technology to inhibit, reduce, or downregulate... B1 The expression of the gene and / or the activity or content of the B1 protein significantly improve wheat grain weight, yield, and protein content. This invention is of great significance for the synergistic improvement of high-yield and high-quality wheat. Attached Figure Description

[0041] Figure 1 Electrophoresis diagram of T0 generation transgenic seedlings using CRISPR / Cas9 vector detection. M is a DNA molecular weight indicator, B1-CR#1 to B1-CR#12 are regenerated seedlings of T0 generation transgenic plants, and WT is the recipient wheat Fielder.

[0042] Figure 2 for B1 Electrophoresis diagram of gene promoter genotype detection. The type of band A indicates the strain. B1 The gene promoter type is Fielder long awn strain B1 Gene promoter type; B-band type indicates the strain B1 The gene promoter type is CAU157 awnless strain B1 Gene promoter type; H band type indicates heterozygous genotype, and both A and B band types exist simultaneously.

[0043] Figure 3 This is a schematic diagram illustrating the editing types of transgenic pure-line plants.

[0044] Figure 4 The results of measurements and statistics on the improvement of wheat grain size and weight by knocking out the B1 gene. P A value <0.05 indicates statistical significance. P A value <0.01 indicates statistical significance. P A value > 0.05 indicates no statistical significance; therefore, the Student's score is used. t The test was performed using statistical analysis. A is... B1Comparison of grain size between gene-edited lines and control lines; the left image shows a comparison of grain length, and the right image shows a comparison of grain width; B represents... B1 A statistical comparison of the thousand-grain weight of gene-edited lines and control lines, with the vertical axis representing thousand-grain weight (TGW); C represents... B1 Comparison of grain length statistics between gene-edited lines and control lines, with the vertical axis representing grain length (GL) and the horizontal axis representing the vertical axis. B1 Comparison of grain width statistics between gene-edited lines and control lines, with the vertical axis representing grain width (GW); E represents... B1 A statistical comparison of awn length between gene-edited strains and control strains, with the vertical axis representing awn length.

[0045] Figure 5 To knock out B1 Measurement and statistical results of genes improving wheat protein content and yield. P A value <0.05 indicates statistical significance. P A value <0.01 indicates statistical significance. P A value > 0.05 indicates no statistical significance; therefore, the Student's score is used. t The test was performed using statistical analysis. A is... B1 A comparative chart of yield statistics per plant between gene-edited lines and control lines, with the vertical axis representing grain yield per plant; B represents... B1 A comparative chart of grain nitrogen content between gene-edited lines and control lines, with the vertical axis representing grain nitrogen content; C represents... B1 A statistical comparison of grain protein content between gene-edited lines and control lines, with the vertical axis representing the protein content of a single grain. Detailed Implementation

[0046] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0047] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0048] The following examples use Excel software to process the data. The experimental results are expressed as mean ± standard error, using Student's algorithm. t -Test verification, P <0.05 indicates a significant difference. P <0.01 indicates a highly significant difference. P <0.001 indicates a highly significant difference.

[0049] The pCBC-MT1T2 and pBUN411-ZT1 plasmids used in the following examples are described in the following literature: Xing, HL, Dong, L., Wang, ZP, Zhang, HY, Han, CY, Liu, B., Wang, XC, & Chen, QJ (2014). A CRISPR / Cas9 toolkit for multiplex genome editing inplants. BMC plant biology , 14 , 327. 3, which is available to the public from the applicant and is intended solely for the purpose of replicating the invention and shall not be used for any other purpose.

[0050] The wheat CAU157 used in the following examples is the CAU157 mentioned in the following literature, which is publicly available from the applicant and is used only for repeating the present invention. It should not be used for any other purpose. Related literature: Ke, W., Xing, J., Chen, Z., Zhao, Y., Xu, W., Tian, ​​L., Guo, J., Xie, X., Du, D., Wang, Z., et al., 2023. The TaTCP4 / 10-B1 cascade regulates awn elongation in wheat ( Triticum aestivum L.).Plant Commun. 4, 100590).

[0051] Example 1. Obtaining and phenotypic identification of target wheat with large grains, high grain weight, and high grain protein content. This embodiment uses wheat CAU157 (awnless wheat) as the wheat to be improved. The coding gene for the B1 protein in the wheat was knocked out using a CRISPR / Cas genome editing system to obtain the target wheat with large grains, high grain weight, and high grain protein content. Wheat CAU157 contains the B1 protein and its genomic gene. The amino acid sequence of the B1 protein is sequence 1 in the sequence listing, and the nucleotide sequence of the B1 protein genomic gene is sequence 3 in the sequence listing. The CDS (Coding Sequence) nucleotide sequence of the B1 protein coding gene is sequence 2 in the sequence listing.

[0052] The specific method is as follows: 1. Construction of recombinant plasmids Sequence 1: MEMEEGLDLSLSLRQYTPPWSQLVFACSYCSRSFKNSQALGGHQNAHKLARRATALSSAAAGELAIENHHSAPPRPGARAWRAGYRQQARRTGTGSGATSSGARRGDQELAEEAIDLSLKL.

[0053] Sequence 2 (5'-3'): ATGGAGATGGAAGAGGGGCTCGATCTGAGCCTGTCGCTGCGCCAGTACACGCCGCCGTGGTCGCAGCTGGTGTTCGCCTGCTCCTACTGCTCACGCAGCTTCAAGAACTCGCAGGCGCTCGGCGGCCACCAGAACGCTCACAAGCTGGCCAGGCGGGCCACGGCGCTCTCTTCAGCGGCGGCG GGAGAGCTCGCCATCGAGAACCACCACAGCGCTCCGCCGCGCCCCGGCGCCCGCGCTTGGCGAGCAGGATATCGTCAGCAGGCGCGGCGCACGGGTACTGGTTCGGGGGCAACCTCGTCCGGGGCAAGAAGAGGCGACCAAGAGCTTGCGGAGGAGGCGATTGACCTGTCCCTCAAGCTATGA.

[0054] Sequence 3 (5'-3'): .

[0055] 1. The target sequence for the B1 sgRNA is designed as follows: sgRNA1 gene: 5'-GTTGTGACAAGGGCTGGGCATGG-3' (Sequence 4, targeting nucleotides 46-68 of sequence 3); sgRNA2 gene: 5'-CCTGTCGCTGCGCCAGTACACGC-3' (Sequence 5, targeting nucleotides 94-116 of sequence 3).

[0056] 2. Primer sequences for constructing the B1 sgRNA expression cassette: B1-MT1T2-F: 5'-aataatggtctcAAGCgTTGTGACAAGGGCTGGGCA-3'; B1-MT1T2-F0: 5'-gTTGTGACAAGGGCTGGGCAgttttagagctagaaatagc-3'; B1-MT1T2-R0: 5'-GTCGCTGCGCCAGTACACGcgcttcttggtgcc-3'; B1-MT1T2-R: 5'-attattggtctctaaacGTCGCTGCGCCAGTACACG-3'.

[0057] 3. PCR amplification: Using plasmid pCBC-MT1T2 as a template, the DNA fragment containing the vector adapter and target site was amplified using the above four primers, and the PCR product was obtained by gel extraction and purification. B1 The sgRNA of the gene is linked to the wheat U6 promoter to form an sgRNA expression cassette. Specifically: Using plasmid pCBC-MT1T2 as a template, the first PCR amplification was performed using primers B1-MT1T2-F0 and B1-MT1T2-R0. 60 μL of the PCR product was purified by gel extraction to obtain the first purified product.

[0058] Using the first purified product as a template, a second PCR amplification was performed using primers B1-MT1T2-F and B1-MT1T2-R. The resulting 60 μL of PCR product was then purified by gel extraction to obtain the second purified product, which is the final purified product. B1 The fragment formed by the sgRNA of the gene linked to the wheat U6 promoter.

[0059] 4. Enzyme digestion and vector ligation The vector pBUN411-ZT1 was digested with BsaI and the second purified product obtained in step 3 was ligated overnight using T4 ligase to ligate the sgRNA expression cassette with the pBUN411-ZT1 plasmid, thus obtaining the recombinant plasmid pBUN411- B1 , The enzyme digestion and ligation system (15 μL) was as follows: second purified product, 6 μL; pBUN411-ZT1 vector, 4 μL; BsaI, 1 μL; T4 ligase, 1 μL; 10× Cutsmart Buffer, 1.5 μL; 10× T4 Buffer, 1.5 μL. Enzyme digestion and ligation conditions: 37℃, 5 h; 50℃, 5 min; 80℃, 10 min.

[0060] 5. Conversion of Linkage Products The ligation product obtained in step 4 was transformed into *E. coli* Mach1-T1 competent cells. Positive *E. coli* samples were obtained by PCR detection, confirming successful transformation. B1The gene's target sequence contains the CRISPR / Cas9 system. Further sequencing analysis of the sgRNA sequence within the CRISPR / Cas9 system was performed to confirm its correctness and compatibility with the target sequence. B1 Gene target sequence pairing was performed, and plasmids were extracted from positive *E. coli* to obtain positive plasmids, resulting in the pBUN411-B1 knockout vector. The pBUN411-B1 knockout vector contains... B1 sgRNA expression cassette vector. The recombinant T-DNA of the pBUN411-B1 knockout vector contains an expression cassette for sgRNA targeting B1 and a Cas9 gene expression cassette for Cas9.

[0061] 6. Agrobacterium-mediated genetic transformation for the preparation of transgenic wheat The pBUN411-B1 knockout vector was transformed into Agrobacterium EHA105 to obtain recombinant Agrobacterium EHA105 / pBUN411-B1, which was then sent to a wheat transgenic platform for genetic transformation. The recipient plant was the wheat Fielder variety (considering that the recipient Fielder carries...). B1B1 The gene contains a naturally mutated allele (b1b1) and exhibits a long awn phenotype. Therefore, this invention first transforms the pBUN411-B1 knockout vector into Fielder, and then combines the transgenic line with an awnless material (CAU157, containing...). B1B1 Allele hybridization was performed, and B1 was knocked out in an awnless background. Recombinant Agrobacterium was used to infect embryogenic callus of wheat Fielder, followed by differentiation culture, rooting culture, and herbicide resistance screening (screening concentration of 250 mg / L) to obtain 12 T0 generation transgenic plants.

[0062] The specific operating procedures are as follows: Preparation of Agrobacterium suspension: Recombinant Agrobacterium EHA105 / pBUN411-B1 was cultured by shaking at 160 rpm and 28℃ one day before the experiment. After the wheat Fielder ears were prepared, the Agrobacterium suspension was prepared. 1 ml of bacterial culture was placed in a 1.5 ml centrifuge tube, and 1.4 μl of acetylsuccinone (0.1 M) was added and mixed well to obtain the Agrobacterium suspension.

[0063] Infection: Take wheat ears that have been pollinated for about 15 days, remove the kernels and remove the embryos, add the prepared Agrobacterium suspension for 5 minutes, and then place them on a co-culture medium and co-culture at 23°C for 3 days.

[0064] Resting: After co-culturing, place on resting medium and incubate in the dark at 25°C for 5 days.

[0065] Screening 1: After resting culture, the callus was transferred to screening medium 1 (containing the herbicide Basta). The culture dishes were sealed with sealing film and incubated in the dark at 25.5℃ for 2 weeks for the first screening.

[0066] Screening 2: The resistant callus from the first screening was cut and transferred to screening medium 2 (containing the herbicide Basta). The culture dishes were sealed with sealing film and incubated in the dark at 25.5℃ for 2 weeks for the second screening.

[0067] Regeneration 1: Transfer the calluses that showed resistance in the second screening to regeneration medium. Resistant calluses generally have green buds or green spots, or beautiful pale yellow spherical structures. Do not transfer pasty or brown calluses. Calluses with proliferation can be cut into smaller pieces. Small pieces from the same callus (along a line) should be placed back in the same line. Pay attention to the orientation of the callus, for example, with the green buds and green spots facing upwards. Seal the petri dishes and incubate at 25°C under light (16 hours) for 2 weeks for regeneration.

[0068] Regeneration 2: After two weeks of regeneration culture, the healthy seedlings are transferred to new resistance regeneration boxes. Once the seedlings have grown to a certain size, samples can be taken for testing and then transplanted into soil to obtain T0 generation transgenic seedlings.

[0069] 7. Identification of transgenic plants PCR identification was used: Genomic DNA was extracted from leaves of T0 generation transgenic plantlets and amplified by PCR using primers 411-F and Cas9-R. If an amplification product (approximately 1 kb, corresponding to nucleotides 210-1176 of sequence 7) was obtained, the identification result was positive, indicating that the regenerated plant was a positive transgenic plant carrying recombinant T-DNA pBUN411-B1. The electrophoresis image of the PCR identification results of the transgenic plant is shown below. Figure 1 . Figure 1 In the diagram, M represents the molecular weight marker, B1-CR#1 to #12 represent different transgenic plants, and WT represents the wheat Fielder plant (negative control). The primer sequences for 411-F and cas9-R are shown below: 411-F: 5'-TTTCCCAGTCACGACGTTGT-3'; Cas9-R: 5'-GGATTCATGAGCAGCAAGCA-3'.

[0070] 8. The genetically modified plants B1 The sgRNA expression cassette vector was transformed into awnless common wheat (in this example, the awnless common wheat variety CAU157 was used). T0 generation transgenic plants were self-crossed once to obtain T1 generation transgenic plants. Leaves of T1 generation transgenic plantlets were taken, genomic DNA was extracted, and PCR amplification was performed using primers consisting of 411-F and cas9-R. Plants that yielded amplification products (approximately 1 kb) (carrying recombinant T-DNA of pBUN411-B1) were selected as the male parent plants for hybridization.

[0071] The male parent plant (as the male parent) was crossed with CAU157 (as the female parent, to be improved wheat) to obtain F1 seeds. The F1 plants were backcrossed with the wheat line CAU157 for one generation to obtain BC1F1 seeds. Then, following the method in step 7, BC1F1 plants carrying recombinant T-DNA pBUN411-B1 were selected and backcrossed with the wheat line CAU157 for one generation to obtain BC2F1 seeds. The BC2F1 seeds were self-pollinated to obtain the BC2F2 population.

[0072] Using a primer pair consisting of ID-5A698F and ID-5A698R, PCR amplification was performed with genomic DNA as a template to detect individual plants in the BC2F2 population. B1 Genotype of gene promoter. PCR product bands were separated by 10% polyacrylamide gel electrophoresis. Figure 2 ), selecting individuals within the group B1 Promoter genotype (the B1 gene promoter genotype in CAU157 is...) Figure 2 In the B genotype, Fielder is the promoter genotype of the B1 gene. Figure 2 (A genotype) and wheat line CAU157 B1 Genotype of gene promoter ( Figure 2 Plants with the same B genotype were subjected to PCR amplification of their genomic DNA using primer pairs consisting of B1-CR-F and B1-CR-R. The amplification products (containing...) B1 Gene sequences were subjected to first-generation sequencing to detect... B1 Whether the sequence near the gene target has been edited or altered, and further selection from it. B1 Gene-edited heterozygous plants were self-crossed to produce BC2F. 2:3 The group continues to target BC2F 2:3 Genotyping and screening of individual plants in the population B1 Gene-edited heterozygous plants were self-crossed to produce BC2F. 2:4 Population seeds. The number of backcrosses with awnless common wheat mentioned in this invention is not limited to two times; it can be backcrossed more than two times. In this embodiment, it is backcrossed twice. The seeds mentioned in this invention... B1The number of self-crosses for the gene-edited heterozygous plants is not limited to two; they can be self-crossed more than two times. In this example, they are self-crossed twice. The primer sequences for ID-5A698F, ID-5A698R, B1-CR-F, and B1-CR-R are as follows: ID-5A698F: 5'-TAGAATGGAGGGCGTACAAA-3'; ID-5A698R: 5'-CACCCTACACATCTACTGCA-3'; B1-CR-F: 5'-TACAGGGTGACTCGAGCAGT-3'; B1-CR-R: 5'-GTCCTTAGTGCCTGCACCTG-3'.

[0073] 9. B1 Screening of homozygous strains with edited genes The BC2F generation derived from the heterozygous single plants selected in step 8 was obtained through self-pollination. 2:4 The seed population was multiplied in a greenhouse, and genomic DNA was extracted from the multiplied plants. PCR amplification and sequencing were performed using B1-CR-F and B1-CR-R primer pairs to screen for... B1 The gene-edited homozygous lines (named 157-CR1-b1b1 and 157-CR2-b1b1, respectively) and B1 The unedited control lines (named 157-CR1-B1B1 and 157-CR2-B1B1, respectively), and the genotypes of the edited lines are as follows: Figure 3 As shown.

[0074] A homozygous mutant strain refers to a plant with two homologous chromosomes. B1 The same mutation occurred in the gene.

[0075] 157-CR1-b1b1 and 157-CR2-b1b1 in B1 The gene contains a 22-nucleotide deletion of “TGGGCATGGAGATGGAAGAGGG” (i.e., the deletion of nucleotides 60-81 of sequence 3 in the sequence listing) and a 12-nucleotide deletion of “TGTCGCTGCGCC” (i.e., the deletion of nucleotides 32-43 of sequence 2 in the sequence listing), which leads to the deletion of the start codon ATG of the B1 gene and the termination of protein translation, resulting in the loss of function of the B1 protein.

[0076] The above 157-CR1-b1b1 After mutation in the mutant B1-CR1 The gene is unable to initiate the translation process normally due to the absence of the start codon, thus failing to generate the amino acid sequence of the B1 protein. The gene coding sequence after the mutation is shown in Sequence 6.

[0077] Sequence 6 (5'-3'): .

[0078] 10. Phenotypic detection The filtered results from step 9 B1 Three hundred seeds each from the homozygous edited lines and control lines were propagated in a greenhouse and sown in the field between March 8th and June 26th, 2024, in Beijing (40°13'28''N, 116°33'37''E). A randomized block design with five replicates was used. Rows were 1.5m long, with 30 seeds per row and two rows per replicate. After harvest, grain shape, weight, and protein content at maturity were observed and recorded. Field management followed conventional wheat field production management.

[0079] Seed characteristics were measured using a Wanshen SC-G type seed quality analyzer after harvest, and protein content was determined using the Kjeldahl method. Compared with control lines (157-CR1-B1B1 and 157-CR2-B1B1), B1 The gene knockout lines (157-CR1-b1b1 and 157-CR2-b1b1) showed significantly increased awn length, grain length, and thousand-grain weight, indicating that gene knockout in awnless common wheat... B1Genes can increase wheat grain size and weight. Figure 4 The protein content determination results showed that... B1 The nitrogen and protein content of the grains in the gene knockout lines were significantly increased compared with the control lines, and the yield per plant in the knockout lines was also significantly increased. Figure 5 In summary, knocking out awnless common wheat... B1 The gene's function can achieve a synergistic increase in wheat yield and protein content, and can be applied in high-yield and high-quality wheat breeding or variety improvement.

[0080] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. The application of a protein, or a substance regulating the expression of the protein-encoding gene, or a substance regulating the activity or content of the protein, characterized in that: The application is any one of the following: P1. The application of the protein or the substance that regulates the expression of the protein-encoding gene or the substance that regulates the activity or content of the protein in increasing the size of plant seeds; P2. The application of the protein or a substance that inhibits the expression of the protein-coding gene or a substance that reduces the activity or content of the protein in increasing the grain weight of plant seeds; P3. The application of the protein or the substance that regulates the expression of the protein-encoding gene or the substance that regulates the activity or content of the protein in increasing the grain weight and grain protein content of plant seeds. P4. The application of the protein or the substance that inhibits the expression of the protein-coding gene or the substance that reduces the activity or content of the protein in improving plant seed size and weight; P5. The application of the protein or a substance that inhibits the expression of the protein-coding gene or a substance that reduces the activity or content of the protein in improving the size, weight and protein content of plant seeds. P6. Application of the protein or substances that regulate the expression of the protein-encoding gene or substances that regulate the activity or content of the protein in high-yield and high-quality plant breeding. The protein in question is the following: A1) The amino acid sequence is that of the protein in sequence 1 of the sequence listing; A2) A protein derived from A1) or having more than 80% identity with and the same function as the protein shown in A1) obtained by substituting and / or deleting and / or adding amino acid residues of the amino acid sequence shown in A1). A3) is a fusion protein obtained by attaching a protein tag to the N-terminus and / or C-terminus of A1) or A2).

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

3. Any of the following applications of biomaterials related to the protein described in claim 1 or 2: P1. The application of the biomaterial in improving plant seed size; P2. Application of the aforementioned biomaterials in increasing the grain weight of plant seeds; P3. Application of the biomaterial in increasing the grain weight and protein content of plant seeds; P4. Application of the biomaterial in improving plant seed size and weight; P5. Application of the biomaterial in improving plant seed size, seed weight and seed protein content; P6. Application of the aforementioned biomaterials in high-yield and high-quality plant breeding; The biomaterial is any one of the following: B1) A nucleic acid molecule encoding the protein described in 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) Recombinant microorganisms containing the nucleic acid molecules described in B1), or recombinant microorganisms containing the expression cassette described in B2), or recombinant microorganisms containing the recombinant vector described in B3); 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 molecules described in B1), or transgenic plant tissue containing the expression cassette described in B2); B7) Transgenic plant organs containing the nucleic acid molecules described in B1), or transgenic plant organs containing the expression cassette described in B2); B8) A nucleic acid molecule that inhibits or reduces the expression of the gene encoding the protein of claim 1 or the activity of the protein of claim 1; B9) Expression cassettes, recombinant vectors, recombinant microorganisms, or transgenic plant cell lines containing the nucleic acid molecules described in B8); B10) A transgenic plant containing the transgenic plant cell line described in B9); B11) Gene-edited wheat obtained by any of the methods described in claims 6-9.

4. The application 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 2 of the sequence listing; b2) The coding sequence is the DNA molecule shown in sequence 7 of the sequence listing; b3) has 90% or more identity with the nucleotide sequence defined by b1) or b2) and is a DNA molecule encoding the protein of claim 1; b4) hybridizes under stringent conditions with the nucleotide sequence defined in b1), b2), or b3) and encodes a DNA molecule that encodes the protein of claim 1; B8) The nucleic acid molecule is a DNA molecule that expresses the gRNA of the protein-coding gene in claim 1(A1) or the gRNA that targets the protein-coding gene in claim 1(A1). The target sequence for sgRNA1, which targets the protein encoding gene A1 in claim 1, is shown at positions 60-81 of sequence listing sequence 3, and the target sequence for sgRNA2 is shown at positions 96-107 of sequence listing sequence 3.

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

6. A method for improving wheat grain size, grain weight, and / or grain protein content, said method being M1 or M2; The M1 includes improving the grain size, grain weight, and / or grain protein content of the wheat to be improved by inhibiting or reducing the expression level of the gene encoding the protein described in claim 1(A1) or the activity of the protein described in claim 1(A1) in the wheat to be improved. The M2 comprises gene-edited wheat obtained by knocking out the gene encoding the protein described in claim 1(A1) of the wheat to be improved, thereby increasing the grain size, grain weight and / or grain protein content of the wheat to be improved; the grain size, grain weight and / or grain protein content of the gene-edited wheat is higher than that of the target improved wheat; the wheat to be improved contains the gene encoding the protein described in claim 1(A1).

7. The method according to claim 6, characterized in that: The method includes introducing a substance into the target improved wheat that reduces or inhibits the expression of the protein-coding gene as described in claim 1; the substance that reduces or inhibits the expression of the protein-coding gene as described in claim 1 is any one of the following c1)-c4): c1) Inhibit or reduce the expression of the protein-coding gene described in claim 1A1); c2) An expression cassette containing the nucleic acid molecule described in c1); c3) A recombinant vector containing the nucleic acid molecule described in c1), or a recombinant vector containing the expression cassette described in c2); c4) Recombinant microorganisms containing the nucleic acid molecules described in c1), or recombinant microorganisms containing the expression cassette described in c2), or recombinant microorganisms containing the recombinant vector described in c3).

8. The method according to claim 7, characterized in that: c1) The nucleic acid molecule described is a DNA molecule that expresses the sgRNA of the protein-coding gene described in claim 1(A1), or the sgRNA that targets the protein-coding gene described in claim 1(A1). The target sequence of sgRNA1, which targets the gene encoding the A1 protein, is shown at positions 46-68 of Sequence Listing 3, and the target sequence of sgRNA2 is shown at positions 94-116 of Sequence Listing 3.

9. The method according to any one of claims 6-8, characterized in that: The inhibition or reduction of the activity of the protein of claim 1 and / or the expression of the gene encoding the protein in the target improved wheat is achieved by performing at least one of the following mutations on the protein-encoding gene shown in sequence 1 in the target improved wheat: 1) The 22 nucleotides TGGGCATGGAGATGGAAGAGAGGG in sequence 3 of the plant sequence listing are missing. 2) The 12 nucleotides TGTCGCTGCGCC at positions 96-107 of sequence 3 in the plant sequence listing are missing.

10. The protein as described in claim 1 or 2 and / or the biological material as described in claim 3 or 4.

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