Rice osvwa2 gene and its encoded protein in regulating plant height and yield

Abstract

The application discloses rice OsVWA2 gene and application of a coded protein thereof in regulating plant plant height and yield. Specifically disclosed is application of a protein OsVWA2 with an amino acid sequence of SEQ ID NO: 3 in regulating plant plant height, yield and / or tiller number. The application uses CRISPR / Cas9 gene editing technology to knockout the OsVWA2 gene, successfully creates an OsVWA2 knockout mutant rice, and experiments prove that the plant height, yield and / or tiller number can be significantly reduced by knocking out the OsVWA2 gene. The application provides a good gene resource for rice breeding, opens up a new field for the application of the gene, enriches the genetic background of the current rice varieties, and has urgent and important significance for cultivating new rice, reducing potential biological invasion risks, effectively improving the yield and quality of rice, and promoting the commercial breeding process of rice.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to the application of the rice OsVWA2 gene and its encoded protein in regulating plant height and yield. Background Technology

[0002] Rice, as an important model plant and the world's most important food crop, has made significant contributions to elucidating plant growth and development mechanisms, increasing crop yield, and ensuring food security. Plant height and tillering are two key factors determining rice plant type and yield. Plant height is an important trait determining rice plant type and yield, influencing yield by directly affecting lodging resistance. Although plant height does not directly constitute rice yield, it is a fundamental prerequisite for high and stable rice yields. Excessive plant height leads to poor lodging resistance, resulting in decreased rice yield. The discovery and widespread use of dwarf genes have significantly improved rice's fertilizer tolerance and lodging resistance, increasing rice biomass and triggering the first "Green Revolution." However, current dwarf gene resources are still very limited. Rice plant height is mainly reflected by the length of the upper 4-5 internodes and panicle length. In normal rice plants, the completion of flowering induction is accompanied by the elongation of the upper 4-5 internodes, while the lower internodes do not elongate. Internode elongation begins with cell division in the intercalary meristem and is subsequently achieved by cell elongation in the elongation zone. Recent molecular genetic studies have shown that plant height variations are primarily regulated by plant hormones such as gibberellins, brassinolide, and strigolactones. While the development of modern biotechnology has led to the discovery of numerous genes related to rice plant height, this field still requires further exploration. In-depth research into genes related to plant height and yield, and the application of genetic transformation techniques for functional gene research and utilization, can not only accelerate the breeding of new rice varieties and provide a scientific basis for their development, offering significant theoretical guidance and practical application value for high-yield rice production, but also provide valuable genetic resources for crop germplasm innovation and genetic improvement, demonstrating broad application prospects in agricultural production.

[0003] Therefore, discovering and identifying genes that regulate rice plant height and yield in order to achieve targeted improvement of rice is an effective way to reduce the negative impact of lodging on rice yield and quality, and has important practical application value for improving rice production potential. Summary of the Invention

[0004] The technical problem to be solved by this invention is how to regulate plant height, yield, and / or tiller number, and how to cultivate plants with altered plant height, yield, and / or tiller number. The technical problem to be solved is not limited to the described technical subject matter; other technical subject matter not mentioned herein will be clearly understood by those skilled in the art through the following description.

[0005] To address the aforementioned technical problems, the present invention first provides the application of proteins in any of the following:

[0006] A1) Application in regulating plant height;

[0007] A2) Application in regulating plant yield;

[0008] A3) Application in regulating the number of plant tillers;

[0009] A4) Application in cultivating plants with altered plant height, yield, and / or tiller number;

[0010] A5) Applications in molecular breeding for improving plant height, yield and / or tiller number, or in germplasm resource improvement related to plant height, yield and / or tiller number;

[0011] The protein may be named OsVWA2, and may be any of the following:

[0012] B1) The amino acid sequence is that of the protein SEQ ID NO:3;

[0013] B2) A protein that has more than 80% identity with and has the same function as the protein shown in B1) obtained by substituting, deleting and / or adding amino acid residues of the amino acid sequence shown in SEQ ID NO:3.

[0014] B3) A fusion protein with the same function is obtained by attaching a tag to the N-terminus and / or C-terminus of B1) or B2).

[0015] In the above applications, the protein OsVWA2 can be derived from rice (Oryza sativa).

[0016] The substitution of amino acid residues described in B2) can be a conservative substitution of amino acid residues.

[0017] The connection described in B3) can be a direct connection via peptide bonds or a connection via a linker.

[0018] To facilitate the isolation, purification, detection, and / or localization of the protein described in B1), a tag protein may be attached to the amino or carboxyl terminus of the protein shown in SEQ ID NO:3. Such tags include, but are not limited to: GST (glutathione thioredoxin) tag protein, Trx (thioredoxin) tag protein, nitrogen utilization substrate A (NusA) tag protein, His tag protein (His-tag), MBP (maltose-binding protein) tag protein, Flag tag protein, SUMO tag protein, HA (influenza hemagglutinin) tag protein, Myc tag protein, LacZ tag protein, CBD (cellulose-binding domain) tag protein, phage T7 protein kinase (T7PK) tag protein, GFP (green fluorescent protein), CFP (cyan fluorescent protein), YFP (yellow-green fluorescent protein), mCherry (monomer red fluorescent protein), or AviTag tag protein. The use of tags does not alter the function of the target protein, and those skilled in the art know how to select appropriate tag proteins according to the desired purpose.

[0019] The application can be achieved by upregulating or downregulating the content and / or activity of the protein OsVWA2.

[0020] Furthermore, the application may include reducing plant height, yield, and / or tiller number by downregulating the content and / or activity of the protein OsVWA2 (e.g., knocking out or silencing the OsVWA2 gene).

[0021] Furthermore, the application may include increasing plant height, yield, and / or tiller number by upregulating the content and / or activity of the protein OsVWA2 (e.g., overexpressing the OsVWA2 gene).

[0022] The present invention also provides the use of biomaterials associated with the protein OsVWA2 in any of the following:

[0023] Application of C1 in regulating plant height;

[0024] Application of C2 in regulating plant yield;

[0025] Application of C3 in regulating the number of plant tillers;

[0026] C4) Application in cultivating plants with altered plant height, yield, and / or tiller number;

[0027] C5) Applications in molecular breeding for improving plant height, yield and / or tiller number, or in germplasm resource improvement related to plant height, yield and / or tiller number;

[0028] The biomaterial may be any one of the following D1) to D7):

[0029] D1) A nucleic acid molecule encoding the protein described in claim 1;

[0030] D2) An expression cassette containing the nucleic acid molecules described in D1);

[0031] D3) A recombinant vector containing the nucleic acid molecule described in D1), or a recombinant vector containing the expression cassette described in D2);

[0032] D4) Recombinant microorganisms containing the nucleic acid molecules described in D1), or recombinant microorganisms containing the expression cassette described in D2), or recombinant microorganisms containing the recombinant vector described in D3);

[0033] D5) Recombinant host cells containing the nucleic acid molecules described in D1), or recombinant host cells containing the expression cassette described in D2), or recombinant host cells containing the recombinant vector described in D3);

[0034] D6) Transgenic plant tissue containing the nucleic acid molecules described in D1), or transgenic plant tissue containing the expression cassette described in D2);

[0035] D7) Transgenic plant organs containing the nucleic acid molecules described in D1) or transgenic plant organs containing the expression cassette described in D2).

[0036] In the above applications, the nucleic acid molecule described in D1) can be any of the following:

[0037] E1) The coding sequence is a DNA molecule of SEQ ID NO:2;

[0038] E2) The nucleotide sequence is a DNA molecule of SEQ ID NO:1 or SEQ ID NO:2.

[0039] The nucleic acid molecules mentioned in this article can be DNA, such as cDNA, genomic DNA, or recombinant DNA; the nucleic acid molecules can also be RNA, such as mRNA or hnRNA.

[0040] D1) The nucleic acid molecule may also include a nucleic acid molecule obtained by codon preference modification based on the nucleotide sequence shown in SEQ ID NO:2.

[0041] Those skilled in the art can readily mutate the nucleotide sequence encoding the protein OsVWA2 using known methods, such as site-directed mutagenesis (including oligonucleotide primer-mediated site-directed mutagenesis, PCR-mediated site-directed mutagenesis, and cassette mutagenesis) or directed evolution (including error-prone PCR, DNA shuffling, and in vitro random recombination). Artificially modified nucleotide sequences that possess 75% or more identity with the nucleotide sequence encoding the protein OsVWA2, provided they encode the protein OsVWA2 and have the same function as the protein OsVWA2, are nucleotide sequences derived from and equivalent to those of the present invention.

[0042] The present invention also provides the use of a substance for reducing the activity and / or content of said protein OsVWA2 in any of the following:

[0043] Application of F1 in regulating plant height;

[0044] Application of F2 in regulating plant yield;

[0045] Application of F3 in regulating the number of plant tillers;

[0046] F4) Application in cultivating plants with altered plant height, yield and / or tiller number;

[0047] F5) Applications in molecular breeding for improving plant height, yield, and / or tiller number, or in the improvement of germplasm resources related to plant height, yield, and / or tiller number.

[0048] The substance may be any substance that reduces the activity and / or content of the protein OsVWA2 through gene-level expression regulation or protein-level regulation.

[0049] The gene-level expression regulation can include expression regulation at the chromatin level (such as histone modification and chromatin remodeling), transcriptional level (such as promoter, transcription factor, and co-regulatory factor regulation), post-transcriptional level (such as RNA splicing and microRNA regulation), and post-translational level (such as ubiquitination, SUMOylation, acetylation, glycosylation, phosphorylation, methylation, NEDD8 modification, etc.).

[0050] The regulation of protein levels may include regulating protein activity and / or content through protein degradation, protein interaction, or other methods that can modulate protein activity.

[0051] Furthermore, the substance may include a substance that causes the coding gene of the protein OsVWA2 to be deleted or inactivated by site-directed mutagenesis, gene knockdown, gene editing and / or gene knockout, or a substance that targets and binds to the protein OsVWA2 to reduce its content or inactivate its function.

[0052] It is well known to those skilled in the art to use site-directed mutagenesis (including oligonucleotide primer-mediated site-directed mutagenesis, PCR-mediated site-directed mutagenesis, and cassette mutagenesis), gene knockout techniques (including RNA interference, Morpholino interference, antisense nucleic acid techniques, and ribozyme techniques), gene editing techniques (including zinc finger ribozyme gene editing, TALEN gene editing, and CRISPR gene editing), or gene knockout techniques (including complete gene knockout and conditional gene knockout) to inhibit gene expression, silence, or knock out genes. For example, shRNA, siRNA, or miRNA targeting the OsVWA2 gene encoding the protein can be used to inactivate or silence gene expression at the post-transcriptional or translational level. The target gene can also be knocked out using a CRISPR-Cas system containing sgRNA and Cas protein. Alternatively, site-directed mutagenesis can be used to mutate the OsVWA2 gene to induce frameshift mutations or premature translation termination, thereby inactivating or weakening the function of the OsVWA2 gene. In some embodiments of the present invention, CRISPR / Cas9 gene editing technology is used to knock out the OsVWA2 gene in rice.

[0053] Furthermore, the substances may include nucleic acid molecules, carbohydrates, lipids, small molecule compounds, antibodies, peptides, proteins, recombinant vectors (such as gene editing vectors), recombinant cells, and viral vectors (such as lentiviruses and adeno-associated viruses).

[0054] Furthermore, the nucleic acid molecules may include (1) double-stranded RNA (dsRNA), small interfering RNA (siRNA), microRNA (miRNA), and short hairpin RNA (shRNA) used in RNA interference technology; (2) antisense RNA (asRNA) and antisense oligonucleotides (AON) used in antisense nucleic acid technology; (3) gRNA and sgRNA used in gene editing technology; and (4) aptamers and ribozymes.

[0055] Those skilled in the art will understand that nucleic acid molecules such as sgRNA, siRNA, miRNA, shRNA, or dsRNA can be designed based on the sequence of the OsVWA2 gene or its transcribed mRNA. These nucleic acid molecules can inhibit or interfere with gene transcription, translation, or post-transcriptional and post-translational modifications, thereby affecting protein expression.

[0056] In the above applications, the substance includes nucleic acid molecules that inhibit the replication, transcription, translation, post-transcriptional modification, and / or post-translational modification of nucleic acid molecules encoding the protein OsVWA2.

[0057] In the above applications, the nucleic acid molecule may be sgRNA or a CRISPR / Cas9 system containing the sgRNA, and the sgRNA may target the gene encoding the protein OsVWA2.

[0058] In the above applications, the target sequence of the sgRNA may be as shown in SEQ ID NO:4 and / or SEQ ID NO:5.

[0059] The present invention also provides a method for cultivating transgenic plants, the method comprising increasing or decreasing the content and / or activity of the protein OsVWA2 in the target plant to obtain plants with altered plant height, yield and / or tiller number.

[0060] By increasing the content and / or activity of the protein OsVWA2 in the target plant, plants with increased plant height, yield, and / or tiller number can be obtained.

[0061] By reducing the content and / or activity of the protein OsVWA2 in the target plant, plants with reduced plant height, yield, and / or tiller number can be obtained.

[0062] In the above method, the reduction of the content and / or activity of the protein OsVWA2 in the target plant is achieved by reducing the expression level of the gene encoding the protein OsVWA2 in the target plant.

[0063] In the above method, the reduction of the expression level of the gene encoding the protein OsVWA2 in the target plant is performed using a CRISPR / Cas9 system, which includes sgRNA targeting the gene encoding the protein OsVWA2.

[0064] Furthermore, the sgRNA may be sgRNA1 (target sequence is SEQ ID NO:4) and / or sgRNA2 (target sequence is SEQ ID NO:5).

[0065] Furthermore, the CRISPR / Cas9 system also includes the Cas9 protein.

[0066] The Cas9 protein is not limited to any specific protein, as long as it can be used in conjunction with the sgRNA of this invention.

[0067] Furthermore, the Cas9 proteins described herein include Streptococcus pyogenes Cas9 (spCas9, subtype II-A), spCas9 HF (high fidelity), nickase Cas9 (nCas9), Staphylococcus aureus Cas9 (saCas9, subtype II-A), Neisseria meningitidis Cas9 (NmCas9, subtype II-C), Francisella novicida Cas9 (FnCas9, subtype II-B), Streptococcus thermophilus Cas9 (St1Cas9, St3Cas9), Campylobacter jejuni Cas9 (CjCas9), and Treponema pallidum Cas9, as well as orthologs of Cas9 from other organisms, but not limited to these. The Cas9 protein may also include high-fidelity Cas9 mutants (such as SpCas9-HF1, eSpCas9-1.1, and TrueCut). TM HiFi Cas9 protein, etc.

[0068] The method of this invention can be implemented with any Cas9 protein known in the art. Those skilled in the art can make appropriate selections of the coding sequence of the Cas9 protein without departing from the principles of the embodiments of this invention.

[0069] Furthermore, reducing the expression level of the gene encoding the protein OsVWA2 in the target plant using the CRISPR / Cas9 system can be achieved by contacting the OsVWA2 gene in the target plant cells with any of the sgRNAs and Cas9 proteins described herein.

[0070] Furthermore, the contact step can be performed as follows (1) and (2):

[0071] (1) Directly introduce any of the sgRNAs described herein into the target plant cells, or first construct the DNA molecule encoding any of the sgRNAs described herein into an expression vector and then introduce it into the target plant cells;

[0072] (2) Directly introduce the Cas9 protein or the mRNA of the Cas9 protein into the target plant cell, or first construct the DNA molecule encoding the Cas9 protein into the expression vector and then introduce it into the target plant cell, or fuse the Cas9 protein with the membrane-penetrating peptide and then introduce it into the target plant cell through the membrane-penetrating peptide.

[0073] The membrane-penetrating peptide is used to promote the uptake and absorption of the Cas9 protein fused to it by the cell, and to enable it to perform its biological functions within the cell. Suitable membrane-penetrating peptides are not limited to specific types; any peptide capable of carrying the Cas9 protein across the membrane and internalizing it is acceptable. For example, the membrane-penetrating peptide could be Tat (Tat peptide), a transcriptional transactivator of human immunodeficiency virus (HIV).

[0074] Those skilled in the art know that Cas9 protein, Cas9 protein mRNA, Cas9 expression vectors (vectors containing and expressing DNA molecules encoding Cas9 protein), sgRNA, and sgRNA expression vectors (vectors containing and expressing DNA molecules encoding sgRNA) can be transferred into plant cells by various methods known in the art, such as chemical stimulation methods (including PEG, calcium phosphate, calcium chloride treatment, etc.), electroporation, liposome-mediated methods, microinjection, gene gun methods (also known as microparticle bombardment), laser microbeam methods, pollen tube pathway methods, ultrasonic methods, air gun methods, and eddy current methods. Furthermore, the target gene can be transferred into plant recipient cells using a vector as a medium, such as Agrobacterium Ti plasmid vector (including Ti plasmid-derived vectors such as co-integration vector systems and binary vector systems) mediated methods.

[0075] When using expression vectors to deliver sgRNA and Cas9 protein, the sgRNA and Cas9 protein can be expressed in different expression vectors or in the same expression vector.

[0076] Furthermore, the method for cultivating transgenic plants described herein can be a method for cultivating plants with reduced plant height, decreased yield, and / or reduced tiller number, and may include the following steps:

[0077] (1) Construct sgRNAs (such as sgRNA1 and / or sgRNA2) targeting the OsVWA2 gene into a Cas9 expression vector to obtain a CRISPR / Cas9 gene editing vector;

[0078] (2) The CRISPR / Cas9 gene editing vector was introduced into the target plant;

[0079] (3) Transgenic plants with OsVWA2 gene knockout obtained through screening and identification are plants with reduced plant height, reduced yield and / or reduced tiller number.

[0080] Further, the Cas9 expression vector described in step (1) contains the Cas9 gene and is capable of expressing the Cas9 protein. The Cas9 expression vector may also contain one or more of the following elements: origin of replication (ori), promoter (such as the U6 promoter, the U6-2 promoter of the present invention), enhancer (such as the CAG enhancer), tag (such as the FLAG tag), terminator (such as the bGH poly(A)terminator), resistance gene (such as the Kana antibiotic resistance gene, the ampicillin resistance gene), promoter of the resistance gene, selection gene (such as the bar gene), promoter of the selection gene, and promoter of the Cas9 gene (such as the Ubi promoter).

[0081] The Cas9 expression vector is commercially available. After designing the sgRNA targeting the gene, the DNA molecule encoding the sgRNA can be easily inserted into a commercial Cas9 expression vector, simultaneously expressing both the Cas9 protein and the sgRNA, thereby editing the target gene. Alternatively, conventional methods in the art can be used to construct the Cas9 expression vector. For example, the Cas9 gene can be amplified using the *Streptococcus pyogenes* genome as a template, and then cloned into a backbone expression vector (such as pET28a, pET32a, etc.) to obtain the Cas9 expression vector.

[0082] Further, the introduction in step (2) can be carried out by Agrobacterium-mediated transformation, which may include the following steps: introducing the CRISPR / Cas9 gene editing vector constructed in step (1) into Agrobacterium (such as Ca ion-induced transformation, polyethylene glycol-mediated transformation, metal cation-mediated transformation, electroporation transformation, phage transduction, etc.) to obtain recombinant Agrobacterium; infecting the callus or explant of the target plant with the recombinant Agrobacterium; and inducing and culturing the obtained positive callus or explant to obtain regenerated plants after identification.

[0083] The explants include, but are not limited to, seeds, roots, leaves, petioles, cotyledons, cotyledonary petioles, hypocotyls, stem segments, shoot apical meristems, epidermal parenchyma cells, tubers, stolons, embryogenic suspension cells, and protoplasts.

[0084] The screening and identification methods are known to those skilled in the art. For example, gene-edited plants (including progeny materials of gene-edited plants) can be identified by methods such as PCR detection, Sanger sequencing, high-throughput sequencing, Western blotting, and Southern blot.

[0085] While the OsVWA2 gene is knocked out using CRISPR / Cas9 technology in one or more embodiments provided in this invention, the invention is not limited to this specific method. Those skilled in the art will know that other gene knockout, gene editing, gene mutation, gene knockdown, homologous recombination, and other techniques known in the art can be used to delete or inactivate the OsVWA2 gene in the plant genome. These methods can also be used in this invention. These alternative methods do not depart from the scope of this invention, and this invention should include these alternative methods.

[0086] The present invention describes that increasing the content and / or activity of the protein OsVWA2 in the target plant can be achieved by increasing the expression level of the gene encoding the protein OsVWA2 in the target plant.

[0087] Increasing the expression level of the gene encoding the protein OsVWA2 in the target plant can be achieved through at least one of the following methods:

[0088] M1) increases the copy number of the gene encoding the protein OsVWA2;

[0089] M2) The gene encoding the protein OsVWA2 is expressed under the drive of a strong promoter;

[0090] M3) Increases the regulatory elements of the gene encoding the protein OsVWA2 to cause it to be overexpressed, said regulatory elements including enhancer elements, elements that improve mRNA stability, elements that improve translation efficiency, and / or elements that improve protein secretion;

[0091] M4) increases the ribosome binding site of the gene encoding the protein OsVWA2;

[0092] M5) Codon optimization was performed on the gene encoding the protein OsVWA2;

[0093] M6 upregulates the expression of the gene (encoding the protein OsVWA2) by altering epigenetic modifications such as DNA methylation or histone acetylation.

[0094] Furthermore, increasing the expression level of the gene encoding the protein OsVWA2 in the target plant can be achieved by introducing the gene encoding the protein OsVWA2 (such as SEQ ID NO:2) into the target plant.

[0095] In this article, the plant may be any of the following:

[0096] G1) Monocotyledons;

[0097] G2) Gramineae plants;

[0098] G3) Plants of the genus Oryza.

[0099] In this article, the yield mentioned may refer to the yield per plant.

[0100] In this article, the target plant may be a target plant containing the gene encoding the OsVWA2 protein.

[0101] In this document, the term "transgenic plant" is understood to include not only first-generation transgenic plants obtained by knocking out or overexpressing the OsVWA2 gene in the target plant, but also their progeny. The transgenic plant includes seeds, callus tissue, intact plants, and cells.

[0102] The method for cultivating transgenic plants according to the present invention may further include the step of hybridizing the transgenic plant obtained by any of the methods described above with the plant to be improved to obtain offspring transgenic plants; the offspring transgenic plants are substantially identical in phenotype to the transgenic plants.

[0103] This invention utilizes CRISPR / Cas9 technology with Nipponbare rice as the recipient material to create the OsVWA2 transgenic material and analyze its phenotype. The results showed that knocking out OsVWA2 significantly reduced plant height, tiller number, and yield per plant in rice, indicating that this gene holds promise for improving crop phenotypes (e.g., increasing plant height to improve yield, or decreasing plant height to improve lodging resistance and harvest index). Plant type is a crucial factor determining yield, and increased crop yield largely depends on improving plant type. Rice plant type mainly includes plant height, tiller number, tiller angle, leaf type, and panicle type, among which plant height and tiller number are key factors affecting yield. The discovery and application of dwarf genes was central to the first Green Revolution, and the effective tiller number directly determines the effective panicle number, thus affecting yield. Research on this gene not only addresses fundamental biological issues but also holds promise for improving crop phenotypes, increasing crop yield, or improving lodging resistance, making it of great significance to crop production.

[0104] OsVWA2 is crucial for the growth and development of rice. Rice stem length and grain size are closely related to yield, making research on the size of these two organs of paramount importance. Before the 1940s and 50s, rice cultivation primarily focused on tall varieties. However, with the rapid development of the chemical industry and the widespread use of chemical fertilizers in agricultural production, the lodging susceptibility and intolerance to fertilizer in tall varieties became apparent, leading to yield reductions due to lodging. Therefore, dwarf varieties began to attract the attention of rice breeders, with lodging resistance becoming a primary breeding objective. Dwarf rice is less prone to lodging, has greater adaptability, and can better withstand strong winds, reducing losses caused by natural disasters such as typhoons and tornadoes, thus ensuring harvest rate and quality.

[0105] Most existing research on rice dwarfing genes is based on the molecular mechanisms by which plant hormones, such as gibberellins, brassinolide, and strigolactone, regulate rice plant height. However, the OsVWA2 gene contains a vWA (von Willebrand factor A) domain. The von Willebrand factor A (vWA) domain was initially discovered in the blood clotting protein von Willebrand factor. While the vWA gene has been well-studied in humans, its research in plants is limited.

[0106] This invention reveals for the first time the role of the OsVWA2 gene and its encoded protein OsVWA2 in regulating plant height, yield, and / or tiller number. Reducing the content and / or activity of the OsVWA2 protein in the target plant (e.g., knocking out or silencing the OsVWA2 gene) can significantly reduce plant height, yield, and / or tiller number; conversely, increasing the content and / or activity of the OsVWA2 protein in the target plant (e.g., overexpressing the OsVWA2 gene) can significantly increase plant height, yield, and / or tiller number. This invention successfully created a dwarf rice variety with significantly reduced plant height, providing material accumulation and an efficient and safe technical method for rice germplasm resource improvement and breeding. This invention provides a valuable gene resource for rice breeding by deeply understanding and exploring the molecular mechanism by which the OsVWA2 gene regulates important agronomic traits in rice. It opens up new fields for the application of the OsVWA2 gene, enriches the genetic background of current rice varieties, and is of urgent and important significance for breeding new types of rice, reducing the risk of potential biological damage, effectively improving rice yield and quality, and promoting the commercialization of rice breeding.

[0107] Terminology Definition

[0108] In this invention, unless otherwise stated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, to better understand this invention, definitions and explanations of relevant terms are provided below.

[0109] The term "expression cassette" generally refers to a nucleic acid construct containing sufficient nucleic acid elements to express a target gene. A typical expression cassette includes a promoter, a multiple cloning site (MCS), and a terminator. Expression cassettes may also include the target gene, marker genes (such as TK, DHFR, CAT, and NEO genes), ribosome recognition and binding sites (SDs), transcription factor binding sites (TFBSs), enhancers, silencers, repressors, introns, poly(A) signal sequences, and / or mRNA splicing signal sequences. Elements within an expression cassette can be directly linked or indirectly linked through adapters.

[0110] The term "vector" generally refers to a vector capable of delivering exogenous DNA or a target gene into host cells for amplification and / or expression. This vector can be a cloning vector or an expression vector. Vectors can be introduced into host cells through transformation, transduction, or transfection, allowing the genetic material they carry to be amplified and / or expressed within the host cells. Those skilled in the art can select appropriate vectors based on the purpose of genetic engineering and the properties of the recipient cells. The vectors include, but are not limited to: plasmids, phages (such as λ phage or M13 phage), cosmids (i.e., Cosmids), phagemids, shuttle vectors (such as yeast expression vectors), Ti plasmids, artificial chromosomes (such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), P1 artificial chromosomes (PAC), or Ti plasmid artificial chromosomes (TAC)), and viral vectors (such as baculovirus vectors, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, poxviruses, papillomaviruses, papillomaviruses (such as SV40), and herpesviruses (such as herpes simplex virus)). A vector may contain multiple elements controlling expression, including but not limited to promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, the vector may also contain a replication initiation site.

[0111] The term "microorganism" typically includes bacteria, viruses, fungi, actinomycetes, rickettsiae, mycoplasma, chlamydia, spirochetes, algae, etc. For example, the bacteria mentioned can be from genera such as *Escherichia* sp. (e.g., *Escherichia coli*), *Erwinia* sp., *Agrobacterium* sp. (e.g., *Agrobacterium tumefaciens*), *Flavobacterium* sp., *Alcaligenes* sp., *Pseudomonas* sp., and *Bacillus* sp. (e.g., *Bacillus*). The viruses mentioned can include rotaviruses, baculoviruses, retroviruses (e.g., lentiviruses), adenoviruses, adeno-associated viruses, poxviruses, papillomaviruses, influenza viruses, papillomaviruses (e.g., SV40), and herpesviruses (e.g., herpes simplex virus). The fungi may originate from genera such as *Saccharomyces* sp. (e.g., *Saccharomyces cerevisiae*, *Methanolac*, *Pichia pastoris*), *Fusarium* sp., *Rhizoctonia* sp., *Verticillium* sp., *Penicillium* sp., *Aspergillus* sp., and *Cephalosporium* sp. The actinomycetes may originate from genera such as *Streptomyces* sp. The algae may originate from phyla such as *Cyanophyta* (e.g., cyanobacteria), genera such as *Fucus* sp., *Achnanthes* sp., *Amphiprora* sp., *Amphora* sp., *Ankistrodesmus* sp., *Asteromonas* sp., and *Boekelovia* sp. .

[0112] The term "host cell," also known as the recipient cell, generally refers to any type of cell that can be used to introduce a vector, such as plant and animal cells. The term "host cell" can be understood not only to the specific recipient cell but also to its offspring, which, due to natural, accidental, or intentional mutations and / or alterations, may not necessarily be identical to the original parent cell but are still included within the scope of the host cell. Suitable host cells are those known in the art, including: plant cells such as Arabidopsis thaliana, tobacco (Nicotiana tabacum), maize (Zea mays), rice (Oryza sativa), wheat (Triticum aestivum), etc., but not limited to these; animal cells such as mammalian cells (e.g., Chinese hamster ovary cells (CHO cells), Chinese hamster ovary cell subline (CHO-K1 cells), African green monkey kidney cells (Vero cells), SV40-transformed African green monkey kidney cells (COS cells), young hamster kidney cells (BHK cells), mouse breast cancer cells (C127 cells), human embryonic kidney cells (HEK293 cells), human HeLa cells, fibroblasts, bone marrow cell lines, T cells or NK cells, etc.), avian cells (e.g., chicken or duck cells), and amphibian cells (e.g., Xenopus laevis cells or Andrias davidianus cells). These include, but are not limited to, davidianus cells, fish cells (e.g., grass carp, carp, rainbow trout, or catfish cells), insect cells (e.g., Sf21 cells, Sf-9 cells, or Hi-5 cells).

[0113] The term "recombinant vector" generally refers to a recombinant DNA molecule constructed by linking a foreign target gene to a vector in vitro. It can be constructed in any suitable way, as long as the constructed recombinant vector can carry the foreign target gene into the recipient cell and provide the foreign target gene with the ability to replicate, integrate, amplify and / or express in the recipient cell.

[0114] The term "recombinant microorganism" generally refers to a recombinant microorganism whose genes have been manipulated and modified to obtain a functionally altered microorganism. This can be achieved by introducing a foreign target gene or recombinant vector into the target microorganism, or by directly editing the endogenous genes of the target microorganism.

[0115] The term "recombinant host cell" generally refers to a recombinant host cell whose genes have been manipulated and modified to obtain a recombinant host cell with altered function. This can include introducing a foreign target gene or recombinant vector into the host cell, or directly editing the host cell's endogenous genes.

[0116] The term "linkage" generally refers to the association of two or more molecules. Linkages can be covalent or non-covalent. The linkages described herein can be direct peptide bonds or linkages via linkers (connectors).

[0117] The term "identity" generally refers to the degree to which two (nucleotide or amino acid) sequences have identical residues at the same position in an alignment, and is usually expressed as a percentage. The identity described herein can refer to the identity of an amino acid sequence or a nucleotide sequence. Two copies having completely identical sequences have 100% identity. Those skilled in the art will recognize that the identity of an amino acid sequence or nucleotide sequence can be determined using identity search sites on the Internet, such as the BLAST page on the NCBI homepage. For example, in Advanced BLAST 2.1, the identity of an amino acid sequence can be calculated by 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, Perresidue gap cost, and Lambda ratio to 11, 1, and 0.85 (default values), and performing a search, thus obtaining the identity value (%). Alternatively, sequence analysis software such as CLC Main Workbench and MegAlign can be used. TM The determination can be performed, for example, using a computer program BLAST with default parameters, especially BLASTP or TBLASTN. The 75% or higher identity mentioned herein can mean at least 75%, 80%, 85%, 90%, or 95% or higher. The 80% or higher identity mentioned herein can mean at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or higher.

[0118] The term "conservative substitution" generally refers to the replacement of one amino acid residue with another amino acid residue in a side chain that has similar physicochemical properties. For example, conservative substitutions can occur between hydrophobic side chain amino acid residues (e.g., Met, Ala, Val, Leu, and Ile), between neutral hydrophilic side chain residues (e.g., Cys, Ser, Thr, Asn, and Gln), between acidic side chain residues (e.g., Asp, Glu), between basic side chain amino acids (e.g., His, Lys, and Arg), or between aromatic side chain residues (e.g., Trp, Tyr, and Phe). It is known in the art that conserved substitutions generally do not cause significant changes in protein conformation and structure, and essentially do not alter the protein's biological activity. Conservative substitutions in the protein sequence that are expected to have only a minimal or no effect on protein structure or function can be readily designed by those skilled in the art.

[0119] The term "introduction" generally refers to the transfer of a foreign gene into a recipient cell, such as a eukaryotic or prokaryotic recipient cell. There are no particular limitations on the method of introduction; any known transformation method that can transfer the target gene (such as the DNA molecule of this invention) into the recipient cell is acceptable. The methods of introduction may include any of the following: (1) introducing the target gene or a recombinant vector containing the target gene into the host bacteria via chemical transformation (such as Ca ion-induced transformation, polyethylene glycol-mediated transformation, or metal cation-mediated transformation, etc.) or physical transformation (such as electroporation transformation). (2) transducing the target gene into the host bacteria via bacteriophage transduction. (3) transferring the target gene into plant recipient cells via physical or chemical methods, such as gene gun method (also known as microparticle bombardment method or biological missile method), chemical stimulation method, electroshock method, liposome-mediated method, microinjection method, laser microbeam method, pollen tube channel method, ultrasound method, air gun method, and eddy current method, etc. (4) Transformation of the target gene into plant recipient cells using vectors, such as Agrobacterium Ti plasmid vector (including Ti plasmid-derived vectors such as co-integration vector systems and binary vector systems) mediated by Agrobacterium, transformation mediated by plant virus vectors, etc. (5) Transformation of the target gene into isolated animal cells (transfection) through calcium phosphate coprecipitation, cationic polymer methods (such as DEAE-dextran transfection), cationic liposome methods, electroporation (i.e., electrotransfection), microinjection, gene gun methods, or virus-mediated methods (such as retrovirus infection, adenovirus infection, lentivirus infection), etc. (6) Transformation of the target gene into in vivo animal cells through microinjection, retroviral vector methods, somatic cell nuclear transfer methods, sperm vector methods, or embryonic stem cell methods, etc., to further prepare transgenic animals.

[0120] The term "gene editing" generally refers to the ability to alter specific gene sequences within any cell, including somatic cells, resulting in base deletions, duplications, insertions, frameshift mutations, and replacements or knockouts of target genes. This allows for the substitution, deletion, splicing, and single-base changes of the genome sequence—essentially, the technology to arbitrarily "edit" the genome or the sequence of a specific gene. Gene editing includes zinc finger ribozyme knockout technology, TALEN gene editing technology, and CRISPR gene editing technology.

[0121] The term "gene knock-down," also known as gene knockdown, generally refers to techniques that inactivate or silence gene expression at the post-transcriptional or translational level without altering the gene's DNA sequence. Gene knockdown includes techniques such as RNA interference, Morpholino interference, antisense nucleic acid techniques, and ribozyme techniques.

[0122] The term "gene knockout" generally refers to a technique that uses a foreign mutated gene to replace an endogenous normal homologous gene through homologous recombination, thereby inactivating the endogenous gene. This includes complete gene knockout (e.g., complete mutation of the target gene based on substitution or insertion targeting vectors) and conditional gene knockout (e.g., tissue-specific knockout based on the Cre-LoxP recombinase system or the FLP-FRT recombinase system).

[0123] The term "RNA interference (RNAi)" generally refers to the technique of using double-stranded RNA (dsRNA) to induce the degradation of mRNA of a target gene homologously complementary to it, thereby silencing gene expression and inducing post-transcriptional gene silencing (PTGS), thus preventing gene expression. Long dsRNA can be cleaved into smaller dsRNA fragments, known as small interfering RNA (siRNA), by the enzyme Dicer within the cell, and siRNA mediates mRNA cleavage. In a broader sense, RNA interference also includes transcriptional gene silencing (pre-transcriptional gene silencing) induced in gene regulatory regions. This silencing process involves DNA methylation rather than mRNA degradation, and the siRNA used to silence genes acts directly on the regulatory regions of the gene, not the coding regions. RNA interference can also include translational gene silencing (translational silencing), for example, microRNA (miRNA, a single-stranded RNA molecule) mainly silences gene expression by preventing mRNA translation and interfering with the accumulation of target mRNA protein products.

[0124] The term "Morpholino interference technology" typically refers to replacing the five-carbon sugar ring on a traditional nucleotide with morpholino, altering the original phosphate group. This results in a molecule that carries no charge, cannot be recognized or degraded by RNases and DNases, and is extremely stable. Its principle is similar to antisense nucleic acid technology; it binds to the mRNA molecule through complementarity with the homologous sequence of the target gene mRNA, thereby preventing the binding of other molecules and proteins to the specific mRNA nucleic acid sequence, ultimately preventing the target gene mRNA from being translated into protein.

[0125] The term "antisense nucleic acid technology" generally refers to the technology that utilizes the principle that antisense RNA can bind complementary to specific mRNA molecules with homologous sequences, thereby inhibiting the processing and translation of that mRNA. This involves artificially synthesizing antisense RNA or its gene and introducing it into cells to suppress the expression of specific genes. Antisense nucleic acid technology mainly includes antisense RNA (asRNA) and antisense oligonucleotides (AON).

[0126] The term "ribozyme technology" generally refers to the technique of using ribozymes to cleave and degrade target RNA molecules. Ribozymes are a class of RNA molecules with biocatalytic activity that can specifically bind to and cleave target RNA molecules, thereby inhibiting the expression of target genes. Ribozymes include hammerhead ribozymes, hairpin ribozymes, hepatitis D virus ribozymes, VS (Varkud satellite) ribozymes, and class I intron ribozymes, among others.

[0127] The term "Cas9 protein" generally refers to a Cas endonuclease of the type II CRISPR system that forms a complex with crRNA and tracrRNA or with guide RNA, used to specifically recognize and cleave all or part of a DNA target sequence. Cas9 proteins have two distinct domains: the HNH domain and the RuvC domain. The HNH domain is responsible for cleaving the DNA strand complementary to the crRNA (or gRNA) (the target strand), while the RuvC domain is responsible for cleaving the non-complementary strand (the non-target strand). The Cas9 protein is not limited to a specific type of protein, as long as it can interact with sgRNA (gRNA). The Cas9 protein can be derived from bacterial species.

[0128] The term "sgRNA (single-guide RNA)" generally refers to a single RNA structure created by artificially modifying a crRNA / tracrRNA complex (gRNA) with a dual RNA structure, linking the crRNA and tracrRNA directly (or through a linker). sgRNA is a component of the CRISPR-Cas9 system, responsible for guiding the Cas9 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. sgRNA includes a recognition region and a scaffold region. The scaffold region, as known to those skilled in the art, is responsible for binding to the Cas protein, while the recognition region is responsible for binding to the target site of the target gene, guiding the Cas protein to the target site. In this invention, sgRNA and gRNA are used interchangeably.

[0129] The term "callus" generally refers to the new tissue that forms on the surface of a wound after a localized injury to the original plant. It consists of living parenchyma cells and can originate from living cells in various tissues within any organ of the plant. In plant tissue culture, it can refer to a cluster of disordered, rapidly dividing parenchyma cells formed from an explant. Cultivating callus on a suitable culture medium can induce the formation of a whole plant.

[0130] The term "explant" generally refers to a part of a plant used as in vitro culture material in plant tissue culture, which, after appropriate treatment and under suitable conditions, can regenerate into a whole plant. In practice, those skilled in the art select suitable explants for transformation based on different plants. Explants include seeds, roots, leaves, petioles, cotyledons, cotyledonary petioles, hypocotyls, stem segments, shoot apical meristems, epidermal parenchyma cells, tubers, stolons, embryogenic suspension cells, and protoplasts, etc.

[0131] The term "comprising" is not intended to be restrictive, but rather inclusive and implies the presence of other elements besides those listed, and can be interpreted as "including but not limited to". The term "comprising" also encompasses the terms "consisting of" and "substantially consisting of". In this document, the terms "comprising" and "including" are used interchangeably. Attached Figure Description

[0132] Figure 1 This is a map of the CRISPR / Cas9 gene editing vector used to knock out the OsVWA2 gene in Example 2.

[0133] Figure 2 This is a schematic diagram of the rice CRISPR-Cas9 dual-target design and edited gene sequence in Example 2. Figure 2 A in the diagram represents the dual target sites of CRISPR-Cas9 in rice.Figure 2 B in the diagram represents the edited gene sequence.

[0134] Figure 3 Phenotypic identification of wild-type Nipponbare (WT) and knockout line OsVWA2 in Example 3. Figure 3 A represents the overall phenotype of OsVWA2 at maturity. The scale bar is 10 cm long. Figure 3 Figure B shows the plant height statistics. Figure 3 C represents the statistical chart of the number of ears (tillers) per plant; Figure 3 Figure D shows the yield statistics for a single plant. Data in the figure are mean ± SE (n = 10–20). The t-test was used to examine the significance of differences between the wild-type and knockout line OsVWA2. * indicates no significant difference (ns), and * and ** represent differences between the wild-type and knockout line OsVWA2 reaching the P < 0.05 and P < 0.01 levels, respectively. Detailed Implementation

[0135] 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.

[0136] 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.

[0137] Unless otherwise specified, the quantitative experiments in the following examples were performed in triplicate, and the results were averaged.

[0138] Example 1: Cloning of the OsVWA2 gene and its related sequence

[0139] This invention screened and identified a gene related to plant height and yield, named the OsVWA2 gene. The clone and sequence of the OsVWA2 gene are as follows:

[0140] RNA was extracted from Nipponbare rice, and cDNA was prepared by reverse transcription. The OsVWA2 gene was cloned from the cDNA. The primer sequences used for cloning the OsVWA2 gene were: upstream primer VWA2F: 5'-ATGGCGTTTAACGACGATGA GAA-3', downstream primer VWA2R: 5'-CTAGGTGGAGGTGGAGGTGGAGG-3'. The OsVWA2 gene sequence is as follows:

[0141] The genomic nucleotide sequence of the OsVWA2 gene is shown in SEQ ID NO:1. The coding region (CDS) nucleotide sequence of the OsVWA2 gene is shown in SEQ ID NO:2, which encodes a protein with the amino acid sequence shown in SEQ ID NO:3, named OsVWA2.

[0142] Example 2: Creation of OsVWA2 knockout system materials

[0143] 1. Construction of CRISPR / Cas9 gene editing vector

[0144] Based on the OsVWA2 gene sequence, a CRISPR / Cas9 dual-target design was created. A schematic diagram of the dual-target design is shown below. Figure 2 As shown in Figure A, the target design principles are as follows: 1) The knockout site should be located in the coding sequence (CDS) region and preferably at the protein's front end or in an important functional domain; 2) It should cover a higher proportion of transcripts; 3) There should be no off-target effects or off-target effects should be located in intergenic regions; 4) Targets with higher editing efficiency should be preferred; 5) The sequence should have a relatively balanced GC content and be less prone to secondary structure formation. The successful application of this program in the design of whole-genome targets in rice has proven its feasibility. We used the Huazhong Agricultural University CRISPR-P website (http: / / crispr.hzau.edu.cn / CRISPR2 / ) for design, selecting targets with high target scores, low off-target rates, and suitable locations. The two designed sgRNA target sequences are:

[0145] sgRNA1 target sequence: 5'-TGCTCTTCGTCATCCGCAAG-3' (SEQ ID NO:4),

[0146] sgRNA2 target sequence: 5'-GTTCGGGACGCTGTACAGCG-3' (SEQ ID NO:5).

[0147] The primer sequences involved in constructing the gene editing vector are as follows:

[0148] Upstream primer y2381316bar-CZF-C026: 5'-CGCGCTGTCGCTTGTGTGCTTGCGGATGACGAAGAGCAGTTTTAGAGCTAGAAAT-3',

[0149] Downstream primer y2381316bar-CZR-C026: 5'-CTATTTCTAGCTCTAAAACCGCTGTACAGCGTCCCGAACGCCACGGATCATCTGCA-3'.

[0150] A map of the CRISPR / Cas9 gene editing vector used to knock out the OsVWA2 gene is shown below. Figure 1 As shown, the vector construction steps are as follows:

[0151] Annealing primers were used to synthesize an sgRNA fragment with homologous arms at both ends and the target sequence in the middle. This sgRNA fragment was then constructed into the vector pCBSG032 to obtain the gene editing vector pCBSG032-OsVWA2.

[0152] The linearization reaction system with the support is as follows:

[0153] Table 1. Linearization reaction system of the carrier

[0154] Component Volume pCBSG032 15 μl BsaI 1 μl Buffer 4 μl ddH2O 20 μl

[0155] Reaction conditions: 37℃, 2h.

[0156] Inactivate the endonuclease at 65℃ for 20 minutes, then set aside for later use.

[0157] The connection system is shown in Table 2 below:

[0158] Table 2. Connection System

[0159]

[0160]

[0161] Connection reaction conditions: 25℃, 5min.

[0162] The recombinant vector for knocking out the OsVWA2 gene, constructed through the above steps, was named pCBSG032-OsVWA2.

[0163] The recombinant vector pCBSG032-OsVWA2 contains two editing target sites (SEQ ID NO:4 and SEQ ID NO:5) and the gene encoding the Cas9 protein on the vector. After being introduced into the recipient, the two transcribed guide RNAs can target the target sequence near the PAM of the recipient genome through base complementarity, that is, the OsVWA2 gene. The Cas9 protein causes a double-strand break in the DNA at the target site of the OsVWA2 gene. Through the organism's own DNA damage repair response mechanism, gene mutations occur in the cleaved region during the repair process, resulting in frameshift mutations or premature termination of translation in the encoding gene, thereby knocking out the OsVWA2 gene.

[0164] 2. Genetic transformation of rice

[0165] The recombinant vector pCBSG032-OsVWA2 was transformed into rice using Agrobacterium-mediated genetic transformation. The specific steps are as follows:

[0166] (1) Callus induction and subculture: Select mature rice seeds (preferably newly harvested seeds of the current year), peel off the husks, pour into 50mL centrifuge tubes, add 75% ethanol for 1 min to sterilize, discard the ethanol, rinse once with sterile water, discard the ethanol, then add 30% sodium hypochlorite for 20 min to sterilize, discard the sodium hypochlorite, and rinse 5-6 times with sterile water. Use a pipette to remove excess water (or use sterile filter paper to dry), and transfer the seeds to induction medium, 20-25 seeds per dish. After callus growth, the proembryo can be directly used for transformation. Small particles growing next to the proembryo can be picked and transferred to a new induction medium for subculture. When they reach a suitable size, they can also be transformed.

[0167] (2) Obtaining and culturing recombinant Agrobacterium: Agrobacterium is a common soil bacterium with natural genetic transformation capabilities. Utilizing the characteristics of Agrobacterium, the recombinant vector pCBSG032-OsVWA2 was introduced into Agrobacterium EHA105 to obtain recombinant Agrobacterium EHA105 / pCBSG032-OsVWA2. The recombinant Agrobacterium EHA105 / pCBSG032-OsVWA2 was streaked on a plate containing 50 mg / mL kanamycin and 25 mg / mL rifampicin antibiotics, and incubated in the dark at 28°C for 2 days until single colonies appeared.

[0168] (3) Agrobacterium infection: Prepare the infection solution. Use a pipette to aspirate the infection solution and wash the recombinant Agrobacterium off the plate to obtain an Agrobacterium suspension for co-culture transformation of rice. Select a sufficient number of callus tissues (the callus should be in good condition, bright yellow in color, round and firm in texture, and the particle diameter should be about 3 mm) and place them in a 100 mL sterile Erlenmeyer flask. Add an appropriate amount of Agrobacterium suspension (ensure that there is enough bacterial solution in contact with the material). Incubate at room temperature for 20 min, shaking occasionally. Discard the bacterial solution, place the callus tissues on sterile filter paper to absorb excess bacterial solution, and then transfer them to a solid co-culture medium covered with a layer of sterile filter paper. Incubate in the dark at 26 °C for 3 days.

[0169] (4) Screening Culture: After 3 days of co-culture, the callus tissue needs to be cleaned. The steps are as follows: Use a 1mL blue pipette tip to transfer the callus tissue from the co-culture medium to a sterilized Erlenmeyer flask, add sterile water to rinse twice, and rinse a third time with sterile water containing 500μl / L carbenicillin. After pipetting away excess water, transfer the callus tissue to sterile filter paper, and use the air blower in the laminar flow hood to dry the water on the callus tissue. The blowing time should be controlled at about 30 minutes. After the callus tissue is dried, transfer it to the screening medium for screening culture. The culture conditions are 28-30℃, dark culture. The screening time is 3-4 weeks.

[0170] (5) Differentiation and regeneration: One month after screening, bright yellow positive callus tissues can be seen growing. At this time, the positive callus tissues can be picked and placed on differentiation medium for differentiation and regeneration. Place 16 positive callus tissues on each differentiation dish and place them in a greenhouse at 28-30℃ for light culture. Generally, green spots will appear on the callus tissues in about 10 days, and seedlings will differentiate in about 10 days.

[0171] (6) Seedling rooting: When the differentiated seedlings grow to about 2-3cm and have obvious roots, they can be transferred to the rooting medium to allow them to grow. The rooting medium should be poured into a relatively tall bottle or tube so that the seedlings have enough space to grow tall after rooting. The rooting culture conditions are 28-30℃ and sterile light culture.

[0172] 3. Identification of OsVWA2 knockout materials

[0173] Ten rice OsVWA2 knockout mutant strains were obtained through Agrobacterium-mediated genetic transformation. Sequencing primers were designed based on the editing target location of the OsVWA2 gene in the gene editing vector (recombinant vector pCBSG032-OsVWA2) as follows:

[0174] Upstream primer v2F1: 5'-CAACAATGGCGGGACGACG-3',

[0175] Downstream primer v2R1: 5'-CCCTGGAGGCTGTACCTGAACT-3'.

[0176] Following standard screening procedures, homozygous mutant lines were identified and sequenced. Two homozygous deletion mutant lines (named OsVWA2#1 and OsVWA2#2, respectively) were selected for subsequent experiments. The OsVWA2#1 mutant lacked a T base at the target site, specifically the T base deletion at position 281 of the CDS region (SEQ ID NO:2). Correspondingly, a stop codon appeared at amino acid position 170 of its encoded amino acid sequence, leading to premature translation termination. The OsVWA2#2 mutant lacked two CT bases at the target site, specifically the CT base deletion at positions 282-283 of the CDS region (SEQ ID NO:2). Correspondingly, a stop codon appeared at amino acid position 332 of its encoded amino acid sequence, also leading to premature translation termination. Figure 2 (B)

[0177] Example 3: Phenotypic Identification of OsVWA2 Knockout Line Materials

[0178] To further clarify the phenotypic characteristics of the knockout line OsVWA2, we conducted statistical analysis on its agronomic traits: plant height, spike number, and grain yield per plant. Figure 3 The results showed that the plant height of the knockout line OsVWA2 was significantly different from that of the wild-type Nipponbare. OsVWA2#1 was reduced by an average of 20%, to approximately 18.4 cm, decreasing from 91.2 cm to 72.8 cm; OsVWA2#2 was reduced by an average of 16%, to approximately 15 cm, decreasing from 91.2 cm to 76 cm. The number of tillers in the knockout line OsVWA2#1 decreased by an average of 7 compared to the wild-type Nipponbare, from 15 to 8; the number of tillers in OsVWA2#2 decreased by an average of 6.2 compared to the wild-type Nipponbare, from 15 to 8.8. The yield per plant in the knockout line OsVWA2#1 decreased by an average of approximately 69% compared to the wild-type Nipponbare, from 18.7 g to 5.7 g; the yield per plant in the knockout line OsVWA2#2 decreased by an average of approximately 70% compared to the wild-type Nipponbare, from 18.7 g to 5.6 g.

[0179] 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 proteins in any of the following: A1) Application in regulating plant height; A2) Application in regulating plant yield; A3) Application in regulating the number of plant tillers; A4) Application in cultivating plants with altered plant height, yield, and / or tiller number; A5) Applications in molecular breeding for improving plant height, yield and / or tiller number, or in germplasm resource improvement related to plant height, yield and / or tiller number; The protein is any one of the following: B1) The amino acid sequence is that of the protein SEQ ID NO:3; B2) A protein that has more than 80% identity with and has the same function as the protein shown in B1) obtained by substituting, deleting and / or adding amino acid residues of the amino acid sequence shown in SEQ ID NO:

3. B3) A fusion protein with the same function is obtained by attaching a tag to the N-terminus and / or C-terminus of B1) or B2).

2. Use of biomaterials related to the protein of claim 1 in any of the following: Application of C1 in regulating plant height; Application of C2 in regulating plant yield; Application of C3 in regulating the number of plant tillers; C4) Application in cultivating plants with altered plant height, yield, and / or tiller number; C5) Applications in molecular breeding for improving plant height, yield and / or tiller number, or in germplasm resource improvement related to plant height, yield and / or tiller number; The biomaterial is any one of the following D1) to D7): D1) A nucleic acid molecule encoding the protein described in claim 1; D2) An expression cassette containing the nucleic acid molecules described in D1); D3) A recombinant vector containing the nucleic acid molecule described in D1), or a recombinant vector containing the expression cassette described in D2); D4) Recombinant microorganisms containing the nucleic acid molecules described in D1), or recombinant microorganisms containing the expression cassette described in D2), or recombinant microorganisms containing the recombinant vector described in D3); D5) Recombinant host cells containing the nucleic acid molecules described in D1), or recombinant host cells containing the expression cassette described in D2), or recombinant host cells containing the recombinant vector described in D3); D6) Transgenic plant tissue containing the nucleic acid molecules described in D1), or transgenic plant tissue containing the expression cassette described in D2); D7) Transgenic plant organs containing the nucleic acid molecules described in D1) or transgenic plant organs containing the expression cassette described in D2).

3. The application according to claim 2, characterized in that, D1) The nucleic acid molecule is any one of the following: E1) The coding sequence is a DNA molecule of SEQ ID NO:2; E2) The nucleotide sequence is a DNA molecule of SEQ ID NO:1 or SEQ ID NO:

2.

4. The use of a substance for reducing the activity and / or content of the protein of claim 1 in any of the following: Application of F1 in regulating plant height; Application of F2 in regulating plant yield; Application of F3 in regulating the number of plant tillers; F4) Application in cultivating plants with altered plant height, yield and / or tiller number; F5) Applications in molecular breeding for improving plant height, yield, and / or tiller number, or in the improvement of germplasm resources related to plant height, yield, and / or tiller number.

5. The application according to claim 4, characterized in that, The substance includes nucleic acid molecules that inhibit the replication, transcription, translation, post-transcriptional modification, and / or post-translational modification of nucleic acid molecules encoding the protein of claim 1.

6. The application according to claim 5, characterized in that, The nucleic acid molecule is sgRNA or a CRISPR / Cas9 system containing the sgRNA, wherein the sgRNA targets the gene encoding the protein of claim 1.

7. The application according to claim 6, characterized in that, The target sequence of the sgRNA is shown in SEQ ID NO:4 and / or SEQ ID NO:

5.

8. A method for cultivating transgenic plants, characterized in that, The method includes increasing or decreasing the content and / or activity of the protein described in claim 1 in the target plant to obtain plants with altered plant height, yield, and / or tiller number.

9. The method according to claim 8, characterized in that, The reduction of the content and / or activity of the protein described in claim 1 in the target plant is achieved by reducing the expression level of the gene encoding the protein in the target plant.

10. The application according to any one of claims 1-7 or the method according to claim 8 or 9, characterized in that, The plant is any one of the following: G1) Monocotyledons; G2) Gramineae plants; G3) Plants of the genus Oryza.

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