Wheat salt tolerance genes and their applications

Abstract

This invention discloses a wheat salt-tolerance gene and its applications. Specifically, it discloses the application of the protein TaSGR (SEQ ID NO: 3, 6, and / or 9) and its encoding gene in regulating plant salt tolerance and / or plant height. This invention successfully created a [gene / gene / technology] using CRISPR / Cas9 technology. TaSGR Homozygous gene-edited plants with gene knockout. The results showed that knockout... TaSGR Following gene modification, wheat plant height was significantly reduced, and salt tolerance was significantly enhanced. This invention reveals for the first time the application of the protein TaSGR and its encoding gene, through downregulating the content and / or activity of the protein TaSGR (e.g., knockout or silencing). TaSGR This invention provides a valuable gene resource for breeding salt-tolerant wheat, enabling the use of genes to increase salt tolerance and / or reduce plant height. TaSGR The application of genes has opened up new fields and is of great significance for breeding new salt-tolerant wheat.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to wheat salt tolerance genes and their applications. Background Technology

[0002] Salt stress refers to the environmental pressure caused by excessively high soil salinity, which adversely affects plant growth and development. As a common abiotic stress, salt stress and saline-alkali stress severely impact crop yields. Saline-alkali land is an important reserve of arable land in my country, with approximately 1.5 billion mu (100 million hectares) of such land, of which about 500 million mu (33 million hectares) have development and utilization potential. Therefore, identifying important salt-alkali tolerance genes and applying them to molecular design breeding and cultivating salt-alkali tolerant crop varieties will help to fully utilize saline-alkali land and other sub-grade land, which is of great significance for ensuring food security.

[0003] wheat( Triticum aestivum Wheat (L.) is one of the world's most important food crops, ranking among the top in terms of planting area, total output, and trade volume. However, wheat struggles to survive in high-salt environments, and its production is frequently and severely impacted by soil salinization. Soil salinization is a major factor limiting crop production, especially exacerbated by climate change and seawater intrusion. Developing salt-tolerant wheat varieties is the best way to effectively utilize saline-alkali land and improve economic efficiency. Discovering and utilizing salt-tolerant genes is of significant practical importance not only for wheat but also for other crops. Because common wheat is an allohexaploid, comprising three genomes (A, B, and D), its genome is extremely large and complex (approximately 40 times the size of the rice genome), thus hindering the discovery and mechanistic analysis of salt-tolerant genes. Screening and identifying new salt-tolerant wheat germplasm and discovering new salt-tolerant wheat genes are crucial for increasing wheat yield, developing high-yield and high-quality wheat varieties, ensuring food security, and promoting sustainable development. Summary of the Invention

[0004] The technical problem to be solved by this invention is how to regulate plant salt tolerance and / or plant height. 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 an application of proteins, wherein the application may be any of the following:

[0006] A1) Application in regulating plant salt tolerance;

[0007] A2) Application in regulating plant height;

[0008] A3) Application in cultivating salt-tolerant plants or plants with altered plant height;

[0009] A4) Applications in molecular breeding for improving plant salt tolerance and / or plant height, or in the improvement of germplasm resources related to salt tolerance and / or plant height;

[0010] The protein (named TaSGR) may be protein 1, protein 2, and / or protein 3, wherein:

[0011] The name of protein 1 may be TaSGR-5A, and may be any of the following:

[0012] B1) The amino acid sequence of this protein is 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 obtained by attaching a tag to the N-terminus and / or C-terminus of B1) or B2);

[0015] The name of protein 2 may be TaSGR-5B, and may be any of the following:

[0016] C1) The amino acid sequence is that of the protein SEQ ID NO:6;

[0017] C2) A protein that has more than 80% identity with and has the same function as the protein shown in C1) obtained by substituting, deleting and / or adding amino acid residues of the amino acid sequence shown in SEQ ID NO:6.

[0018] C3) A fusion protein with the same function is obtained by attaching a tag to the N-terminus and / or C-terminus of C1) or C2);

[0019] The name of protein 3 may be TaSGR-5D, and may be any of the following:

[0020] D1) The amino acid sequence is that of the protein in SEQ ID NO:9;

[0021] D2) A protein that has more than 80% identity with and has the same function as the protein shown in D1) obtained by substituting, deleting and / or adding amino acid residues of the amino acid sequence shown in SEQ ID NO:9.

[0022] D3) A fusion protein with the same function is obtained by attaching a tag to the N-terminus and / or D-terminus of D1) or D2).

[0023] In the above applications, the proteins TaSGR-5A, TaSGR-5B, and TaSGR-5D can be derived from wheat (Triticum aestivum ).

[0024] The connections described in B3), C3) and D3) can be directly connected via peptide bonds or via adapters.

[0025] The substitution of amino acid residues described in B2), C2) and D2) can be a conservative substitution of amino acid residues.

[0026] To facilitate the isolation, purification, detection, and / or localization of the proteins described in B1), C1), or D1), a tag protein may be attached to its amino or carboxyl terminus. 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.

[0027] The application can be achieved by upregulating or downregulating the content and / or activity of the protein TaSGR.

[0028] Furthermore, the application may include increasing plant salt tolerance and / or reducing plant height by downregulating the content and / or activity of the protein TaSGR (e.g., knocking out or silencing the gene encoding the protein TaSGR).

[0029] This invention also provides applications of biomaterials, which may be any of the following:

[0030] E1) Application in regulating plant salt tolerance;

[0031] E2) Application in regulating plant height;

[0032] E3) Application in cultivating salt-tolerant plants or plants with altered plant height;

[0033] E4) Applications in molecular breeding for improving plant salt tolerance and / or plant height, or in the improvement of germplasm resources related to salt tolerance and / or plant height;

[0034] The biomaterial may be any of the following:

[0035] F1) The nucleic acid molecule encoding the protein TaSGR;

[0036] F2) An expression cassette containing the nucleic acid molecule described in F1);

[0037] F3) A recombinant vector containing the nucleic acid molecule described in F1), or a recombinant vector containing the expression cassette described in F2);

[0038] F4) Recombinant microorganisms containing the nucleic acid molecules described in F1), or recombinant microorganisms containing the expression cassette described in F2), or recombinant microorganisms containing the recombinant vector described in F3);

[0039] F5) A recombinant host cell containing the nucleic acid molecule described in F1), or a recombinant host cell containing the expression cassette described in F2), or a recombinant host cell containing the recombinant vector described in F3);

[0040] F6) Transgenic plant tissue containing the nucleic acid molecules described in F1), or transgenic plant tissue containing the expression cassette described in F2);

[0041] F7) A transgenic plant organ containing the nucleic acid molecule described in F1) or a transgenic plant organ containing the expression cassette described in F2).

[0042] In the above applications, the nucleic acid molecule described in F1) may be a nucleic acid molecule encoding protein 1, a nucleic acid molecule encoding protein 2, and / or a nucleic acid molecule encoding protein 3, wherein:

[0043] The nucleic acid molecule encoding protein 1 may be any of the following:

[0044] G1) The coding sequence is a DNA molecule of SEQ ID NO:2;

[0045] G2) The nucleotide sequence is a DNA molecule of SEQ ID NO:1 or SEQ ID NO:2;

[0046] The nucleic acid molecule encoding protein 2 may be any of the following:

[0047] H1) The coding sequence is a DNA molecule of SEQ ID NO:5;

[0048] H2) The nucleotide sequence is a DNA molecule of SEQ ID NO:4 or SEQ ID NO:5;

[0049] The nucleic acid molecule encoding protein 3 may be any of the following:

[0050] J1) The coding sequence is a DNA molecule of SEQ ID NO:8;

[0051] J2) The nucleotide sequence is a DNA molecule of SEQ ID NO:7 or SEQ ID NO:8.

[0052] 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.

[0053] The nucleotide sequence shown in SEQ ID NO:2 can be TaSGR-5A The coding sequence (CDS) of the gene encodes the protein TaSGR-5A, as shown in SEQ ID NO:3. The nucleotide sequence shown in SEQ ID NO:1 may be... TaSGR- 5A The genome sequence of a gene.

[0054] The nucleotide sequence shown in SEQ ID NO:5 can be TaSGR-5B The coding sequence (CDS) of the gene encodes the protein TaSGR-5B, as shown in SEQ ID NO:6. The nucleotide sequence shown in SEQ ID NO:4 may be... TaSGR- 5B The genome sequence of a gene.

[0055] The nucleotide sequence shown in SEQ ID NO:8 can be TaSGR-5D The coding sequence (CDS) of the gene encodes the protein TaSGR-5D, as shown in SEQ ID NO:9. The nucleotide sequence shown in SEQ ID NO:7 may be... TaSGR- 5D The genome sequence of a gene.

[0056] The nucleic acid molecules described herein may also include nucleic acid molecules obtained by codon preference modification based on the nucleotide sequences shown in SEQ ID NO:2, SEQ ID NO:5 and / or SEQ ID NO:8.

[0057] Those skilled in the art can readily mutate the nucleotide sequence encoding the protein TaSGR 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 TaSGR, provided they encode the protein TaSGR and have the same function as the protein TaSGR, are nucleotide sequences derived from and equivalent to those of the present invention.

[0058] The present invention also provides the use of a substance for reducing the activity and / or content of said protein TaSGR in any of the following:

[0059] Application of K1 in regulating plant salt tolerance;

[0060] Application of K2 in regulating plant height;

[0061] Application of K3 in cultivating salt-tolerant plants or plants with altered plant height;

[0062] Application of K4 in molecular breeding for improving plant salt tolerance and / or plant height, or in the improvement of germplasm resources related to salt tolerance and / or plant height.

[0063] The substance may be any substance that reduces the activity and / or content of the protein TaSGR through gene-level expression regulation or protein-level regulation.

[0064] The gene-level expression regulation may include expression regulation at the chromatin level (such as histone modification and chromatin remodeling), transcriptional level (such as regulation by promoters, transcription factors, and co-regulatory factors), 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.).

[0065] 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.

[0066] In the above applications, the substance includes substances that inhibit the replication, transcription, translation, post-transcriptional modification, and / or post-translational modification of nucleic acid molecules encoding the protein TaSGR.

[0067] The nucleic acid molecule encoding the protein TaSGR can be TaSGR-5A , TaSGR-5B and / or TaSGR-5D Gene.

[0068] Furthermore, the substance may include a substance that causes the coding gene of the protein TaSGR 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 TaSGR to reduce its content or inactivate its function.

[0069] 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 gene encoding the protein TaSGR 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 gRNA (sgRNA) and Cas protein. Alternatively, site-directed mutagenesis can be used to mutate the gene encoding the protein TaSGR to induce a frameshift mutation or premature translation termination, thereby inactivating or weakening the gene. In some embodiments of the present invention, CRISPR / Cas9 gene editing technology is used to knock out genes in wheat. TaSGR-5A , TaSGR-5B and / or TaSGR-5D Gene.

[0070] Those skilled in the art will know that, according to TaSGR-5A , TaSGR-5B and / or TaSGR-5D Nucleic acid molecules such as siRNA, miRNA, shRNA, or dsRNA are designed by selecting target sequences from gene sequences or the sequences of mRNA transcribed from them. These nucleic acid molecules can inhibit or interfere with gene transcription, translation, or post-transcriptional and post-translational modifications, thereby affecting protein expression.

[0071] 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).

[0072] 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.

[0073] In the above applications, the substance may be sgRNA or a CRISPR / Cas9 system containing the sgRNA, wherein the sgRNA targets the gene encoding the protein TaSGR.

[0074] Further, the target sequence of the sgRNA may be as shown in SEQ ID NO:10 (target sequence of sgRNA1) and / or SEQ ID NO:11 (target sequence of sgRNA2). Wherein: sgRNA1 can target... TaSGR-5A , TaSGR-5B and TaSGR-5D Genes, sgRNA2 can also be targeted TaSGR-5A , TaSGR-5B and TaSGR-5D Genes, in conjunction with the Cas9 protein, sgRNA1 and / or sgRNA2 can be used for knockout. TaSGR-5A , TaSGR-5B and TaSGR-5D Gene.

[0075] The present invention also provides a method for cultivating salt-tolerant plants or plants with altered plant height, the method comprising reducing the content and / or activity of the protein TaSGR in the target plant to obtain plants with increased salt tolerance and / or reduced plant height.

[0076] In the above method, reducing the content and / or activity of the protein TaSGR in the target plant can be achieved by reducing the expression level of the gene encoding the protein TaSGR (TaSGR-5A, TaSGR-5B and / or TaSGR-5D) in the target plant.

[0077] The nucleotide sequence of the gene encoding the protein TaSGR-5A is as shown in SEQ ID NO:2; the nucleotide sequence of the gene encoding the protein TaSGR-5B is as shown in SEQ ID NO:5; and the nucleotide sequence of the gene encoding the protein TaSGR-5D is as shown in SEQ ID NO:8.

[0078] In the above method, the reduction of the expression level of the gene encoding the protein in the target plant can be performed using a CRISPR / Cas9 system, wherein the CRISPR / Cas9 system includes sgRNA targeting the TaSGR gene encoding the protein.

[0079] In the above method, the target sequence of sgRNA may be as shown in SEQ ID NO:10 and / or SEQ ID NO:11.

[0080] Furthermore, the CRISPR / Cas9 system also includes the Cas9 protein. Furthermore, the Cas9 protein described herein is not limited to any specific protein, as long as it can be used in conjunction with the sgRNA of this invention.

[0081] Furthermore, the Cas9 protein described herein includes Streptococcus pyogenes (Streptococcus pyogenes) Streptococcus pyogenes Cas9 (spCas9, subtype II-A), spCas9 HF (high fidelity), nicked Cas9 (nCas9), Staphylococcus aureus ( Staphylococcus aureus Cas9 (saCas9, subtype II-A), Neisseria meningitidis ( Neisseria meningitidis Cas9 (NmCas9, subtype II-C), the new culprit Francisella ( Francisella novicida Cas9 (FnCas9, subtype II-B), Streptococcus thermophilus ( Streptococcus thermophilus Cas9 (St1Cas9, St3Cas9), Campylobacter jejuni ( Campylobacter jejuni Cas9 (CjCas9) and spirochetes ( Treponema sp.) Cas9, and other Cas9 orthologs 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™ HiFi Cas9 protein).

[0082] 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.

[0083] Furthermore, reducing the expression level of the gene encoding the protein TaSGR in the target plant using the CRISPR / Cas9 system can be achieved by contacting the gene encoding the protein TaSGR in the target plant cells with any of the sgRNAs described herein (such as sgRNA1 and / or sgRNA2) and the Cas9 protein.

[0084] Furthermore, the contact step can be performed as follows (1) and (2):

[0085] (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;

[0086] (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.

[0087] 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, as long as they can achieve the purpose of carrying the Cas9 protein across the membrane and internalizing it. For example, the membrane-penetrating peptide can be Tat (Tat peptide), a transcriptional transactivator of human immunodeficiency virus (HIV).

[0088] 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.

[0089] 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.

[0090] Furthermore, the methods for cultivating salt-tolerant plants or plants with altered plant height described herein may include the following steps:

[0091] (1) Construct sgRNAs (such as sgRNA1 and / or sgRNA2) targeting the TaSGR protein encoding gene into a Cas9 expression vector to obtain a CRISPR / Cas9 gene editing vector;

[0092] (2) The CRISPR / Cas9 gene editing vector was introduced into the target plant;

[0093] (3) Salt-tolerant plants or plants with reduced plant height whose coding gene for the protein TaSGR has been knocked out after screening and identification.

[0094] 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).

[0095] 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.

[0096] 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.

[0097] 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.

[0098] 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 blot, and Southern blot.

[0099] Although in one or more embodiments provided by the present invention, CRISPR / Cas9 technology is utilized. TaSGR-5A , TaSGR-5B and TaSGR-5D Gene knockout is an important technique, but this 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 knock out genes in the plant genome. TaSGR-5A , TaSGR-5B and / or TaSGR-5D Gene deletion or inactivation. 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.

[0100] In this article, the plant may be any one of the following (1)-(3):

[0101] (1) Monocotyledons; (2) Gramineae; (3) Triticum.

[0102] In this article, the target plant may be a target plant containing the gene encoding the protein TaSGR.

[0103] The method for cultivating salt-tolerant plants or plants with altered plant height described in this invention may further include the step of hybridizing the salt-tolerant plants or plants with altered plant height obtained by any of the methods described above with the plant to be improved to obtain offspring transgenic plants; the offspring transgenic plants have a phenotype substantially consistent with the transgenic plants.

[0104] In this article, the salt-tolerant plants or plants with altered plant height are understood to include not only the first-generation gene-edited plants obtained by knocking out the TaSGR protein encoding gene in the target plant, but also their progeny. The gene-edited plants include seeds, callus tissue, intact plants, and cells.

[0105] In this article, " TaSGR "Gene" means TaSGR-5A , TaSGR-5B and / or TaSGR-5D Gene.

[0106] In this article, “protein TaSGR” refers to TaSGR-5A, TaSGR-5B and / or TaSGR-5D proteins.

[0107] This invention utilizes CRISPR / Cas9 technology to knock out three alleles in wheat, namely... TaSGR-5A , TaSGR-5B and TaSGR-5D Genes have successfully created TaSGR Homozygous gene-edited plants with gene knockout. Experimental results show that knockout... TaSGR After gene modification, wheat plant height was significantly reduced and salt tolerance was significantly enhanced.

[0108] This invention reveals for the first time the application of the protein TaSGR and its encoding gene in regulating plant salt tolerance and / or plant height. This is achieved by downregulating the content and / or activity of the protein TaSGR (e.g., knockout or silencing). TaSGR This invention provides a valuable genetic resource for breeding salt-tolerant wheat, enabling the development of genes to increase salt tolerance and / or reduce plant height (for better lodging resistance). TaSGR The application of genes has opened up new fields and is of great significance for breeding new types of wheat and promoting the commercialization of wheat breeding.

[0109] Terminology Definition

[0110] 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.

[0111] 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.

[0112] 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 contain a replication origin site.

[0113] The term "microorganism" generally includes bacteria, viruses, fungi, actinomycetes, rickettsiae, mycoplasmas, chlamydiae, spirochetes, algae, etc. For example, the bacteria mentioned may originate from the genus *Escherichia* (…). Escherichia sp. (such as Escherichia coli), Erwinia spp. Erwinia sp. ), Agrobacterium ( Agrobacterium sp. (such as Agrobacterium tumefaciens), Flavobacterium ( Flavobacterium sp. ), Alcaligenes ( Alcaligenes sp. ), Pseudomonas spp. Pseudomonas sp. ) and Bacillus spp. ( Bacillus sp. (e.g., Bacillus subtilis). The viruses may include rotavirus, baculovirus, retrovirus (e.g., lentivirus), adenovirus, adeno-associated virus, poxvirus, papillomavirus, influenza virus, papillomavirus (e.g., SV40), and herpesvirus (e.g., herpes simplex virus). The fungi may be derived from yeasts (e.g., Bacillus subtilis). Saccharomyces sp. (such as Saccharomyces cerevisiae, Saccharomyces methylbenzene, Pichia pastoris), Fusarium genus ( Fusarium sp. ), Rhizoctonia spp. Rhizoctonia sp. Verticillium ( Verticillium sp. ), Penicillium ( Penicillium sp. Aspergillus ( ) Aspergillus sp. ) and Cephalosporin ( Cephalosporium sp. The actinomycetes may originate from the genus Streptomyces (…).Streptomyces sp. (e.g., Streptomyces). The algae may originate from the phylum Cyanophyta (e.g., cyanobacteria), genus Fucus (e.g., fucus vesiculosus). Fucus sp. ), genus *Cyclocarya* ( Achnanthes sp. ), genus *Codonopsis* ( Amphiprora sp. ), genus Dipterocarpa ( Amphora sp. ), Fiber Algae ( Ankistrodesmus sp. ), genus *Stellaria* ( Asteromonas sp. ) and the genus *Golden Color Algae* ( Boekelovia sp. )wait.

[0114] The term "host cell," also known as 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 to refer not only to the specific recipient cell but also to its offspring, which, due to natural, accidental, or intentional mutations and / or alterations, need not be completely 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, wherein: the plant cell may be Arabidopsis thaliana (…). Arabidopsis thaliana ),tobacco( Nicotiana tabacum ),corn( Zea mays ), rice ( Oryza sativa ),wheat( Triticum aestivum Plant cells, but not limited to these; animal cells may be 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), amphibian cells (e.g., African clawed frog cells), etc. Xenopus laevis ) cells or giant salamander ( Andrias davidianus Cells include, but are not limited to, 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), etc.

[0115] 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.

[0116] 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.

[0117] 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.

[0118] 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).

[0119] 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, Per residuegap 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 MainWorkbench 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 be at least 75%, 80%, 85%, 90%, or 95% or higher. The 80% or higher identity mentioned herein can be 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.

[0120] 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.

[0121] 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) 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. (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 method (Agrobacterium-mediated method), plant virus vector mediated transformation method, etc. (5) Transformation of the target gene into isolated animal cells (transfection) through calcium phosphate coprecipitation method, cationic polymer method (such as DEAE-dextran transfection method), cationic liposome method, electroporation method (i.e., electrotransfection method), microinjection, gene gun method or virus-mediated method (such as retrovirus infection method, adenovirus infection method, lentivirus infection method), etc. (6) Transformation of the target gene into in vivo animal cells through microinjection method, retroviral vector method, somatic cell nuclear transfer method, sperm vector method or embryonic stem cell method, etc., to further prepare transgenic animals.

[0122] 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 protein, as long as it can interact with sgRNA (gRNA). The Cas9 protein can be derived from bacterial species.

[0123] 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.

[0124] The term "explant" generally refers to a portion 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 the specific plant species.

[0125] 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. Attached Figure Description

[0126] Figure 1 In Example 2 TaSGR Phenotypic and mutant sequences of gene-edited wheat materials.

[0127] Figure 2 In Example 3 TaSGR Results of salt tolerance phenotype identification of gene-edited mutants.

[0128] Figure 3 This is a structural diagram of the 964 bp PCR product fragment from Example 1. Detailed Implementation

[0129] 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.

[0130] 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.

[0131] The following examples use Excel statistical software to process the data. The experimental results are expressed as mean ± standard deviation. , s t The t-test method is used, where P < 0.05 (*) indicates a statistically significant difference, P < 0.01 (**) indicates a statistically significant difference, and P < 0.001 (***) indicates a highly statistically significant difference. Unless otherwise specified, the quantitative experiments in the following examples are performed in triplicate, and the results are averaged.

[0132] The pCBC-DT1T2 and pBUE411 vectors in the following examples are described in the following literature: He, G., Zhang, Y., Liu, P., Jing, Y., Zhang, L., Zhu, Y. et al. (2021) The transcription factor TaLAX1 interacts with Q to antagonistically regulate grainthreshability and spike morphogenesis in bread wheat. New phytologist, 230, 988-1002.

[0133] Example 1, Wheat TaSGR Construction of gene editing vectors

[0134] Through transcriptome analysis of salt-tolerant wheat varieties, we found that salt-tolerant wheat varieties... TraesCS5B03G0809200 The transcriptional expression level of the STAYGREEN (TaSGR) gene was significantly lower than that of the control variety.TaSGR The three alleles of a gene TaSGR-5A , TaSGR-5B and TaSGR-5D The sequence of the protein it encodes is as follows:

[0135] TaSGR-5A The genomic nucleotide sequence of the gene is shown in SEQ ID NO:1. [[ID= The coding region (CDS) nucleotide sequence of the gene is shown in SEQ ID NO:2, which encodes a protein with the amino acid sequence shown in SEQ ID NO:3, named TaSGR-5A.

[0136] ​ The genomic nucleotide sequence of the gene is shown in SEQ ID NO:4. TaSGR-5B The coding region (CDS) nucleotide sequence of the gene is shown in SEQ ID NO:5, which encodes a protein with the amino acid sequence shown in SEQ ID NO:6, named TaSGR-5B.

[0137] TaSGR-5D The genomic nucleotide sequence of the gene is shown in SEQ ID NO:7. TaSGR-5D The coding region (CDS) nucleotide sequence of the gene is shown in SEQ ID NO:8, which encodes a protein with the amino acid sequence shown in SEQ ID NO:9, named TaSGR-5D.

[0138] Furthermore, in order to verify TaSGR The function of genes, we have constructed TaSGR-5A / 5B / 5D ( TraesCS5A02G319900 / TraesCS5B02G320200 / TraesCS5D02G325900 The CRRISPR / Cas9 gene editing vector used was pBUE411.

[0139] Because the three genes are highly homologous, therefore, targeting TaSGR The CRISPR / Cas9 dual-target sequence was designed based on conserved gene sequences, and the designed sgRNA target sequence is shown below:

[0140] sgRNA1 target sequence: 5'-TCACGTCGCTGTGGGTGAG-3' (SEQ ID NO:10).

[0141] sgRNA2 target sequence: 5'-TGAGCTTGGACGCCTCGAA-3' (SEQ ID NO:11).

[0142] The target sequence of sgRNA1 is located at TaSGR-5A , TaSGR-5B and TaSGR-5D Genetically; the target sequence of sgRNA2 is also located in TaSGR-5A, TaSGR-5B and TaSGR-5D Genetically; utilizing sgRNA1 and sgRNA1 dual targets TaSGR-5A , TaSGR-5B and TaSGR-5D Genes (also known as genes) TaSGR-5A / 5B / 5D Gene ） Knockout.

[0143] The PCR amplification primers are shown in Table 1.

[0144]

[0145] The gene editing vector construction process is as follows:

[0146] 1. PCR amplification

[0147] Dissolve and mix the four primers, with the concentrations of TaSGR-F / R primers being 10 µM and the concentrations of TaSGR-F0 / R0 primers being 0.5 µM. The PCR reaction system is as follows:

[0148]

[0149] Table 2 shows the composition of the KOD Plus PCR reaction solution: KOD Plus buffer 5 µl, 2 mM dNTP 5 µl, 25 mM MgSO4 5 µl, and KOD-Plus 1 µl.

[0150] 2. The PCR product fragment (964 bp, nucleotide sequence SEQ ID NO:12, structure as shown) was recovered. Figure 3 As shown in Table 3, the enzyme was digested with BsaI and ligated into the pBUE411 vector via T4 Ligase.

[0151]

[0152] The above steps are used to construct a method for knockout. TaSGR-5A , TaSGR-5B and TaSGR-5D The recombinant vector for the gene was named pBUE411-TaSGR.

[0153] The recombinant vector pBUE411-TaSGR contains two editing target sites (SEQ ID NO:10 and SEQ ID NO:11) 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 in the recipient genome through base complementarity, i.e., targeting... TaSGR-5A , TaSGR-5B and TaSGR-5DGenes, Cas9 protein TaSGR-5A , TaSGR-5B and TaSGR-5D A double-strand break in DNA at a gene target site triggers a gene mutation in the cut region through the organism's own DNA damage repair response mechanism. This mutation leads to a frameshift mutation or premature termination of translation in the coding gene, thereby achieving the desired effect. TaSGR-5A , TaSGR-5B and TaSGR-5D Gene knockout.

[0154] Example 2 TaSGR Construction of gene-edited wheat materials

[0155] Will TaSGR The gene-editing vector (pBUE411-TaSGR) was transformed into the Fielder wheat variety using Agrobacterium-mediated transformation. Two gene editing vectors were obtained after PCR sequencing identification. TaSGR-5A / 5B / 5D Homozygous gene-edited lines (named respectively) Tasgr- aabbdd #1 and Tasgr-aabbdd #2, planted in spring in the wheat experimental field within the Chinese Academy of Agricultural Sciences. The gene-edited lines morphologically exhibited a reduced plant height phenotype. Figure 1 (b and c). TaSGR-5A / 5B / 5D Gene-edited mutant mutation sequences such as Figure 1 As shown in Figure a.

[0156] Example 3 TaSGR Identification of salt tolerance phenotype in gene-edited mutants

[0157] The test plants were third-generation plants, initially tested on Fielder and... Tasgr-aabbdd #1 and Tasgr-aabbdd Seeds of material #2 were subjected to germination treatment. After the seeds sprouted, they were transplanted into 96-cell hydroponic boxes and cultured in Hoagland's solution. After 7-10 days of culture, when the materials reached the two-leaf-one-heart stage, photographs were taken of the materials before treatment. The Hoagland's solution was then replaced with a 150 mM NaCl salt solution (with 8.766 g of sodium chloride added). The materials were cultured in the salt solution for at least two weeks, after which the salt solution was replaced with Hoagland's solution again for rehydration treatment. Phenotypic data were collected 7 days after rehydration. The hydroponic nutrient solution was changed every 2-3 days. All materials were cultured in a wheat culture incubator under the following conditions: 22℃, 16 hours of light; 19℃, 8 hours of darkness.

[0158] The results are as follows Figure 2 As shown, compared with the control variety Fielder, TaSGR The gene-edited mutants exhibited significantly enhanced salt tolerance.

[0159] The results in summary indicate that gene editing can be used to knock out wheat germ cells. TaSGR-5A , TaSGR-5B and TaSGR-5D Following gene editing, the resulting gene-edited plants exhibited significantly reduced plant height and showed better growth under salt stress conditions than wild-type plants, indicating that... TaSGR Genes can be used to regulate wheat plant height and salt tolerance.

[0160] 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. An application for downregulating the content and / or activity of proteins in plants, characterized in that, The application is any one of the following: A1) Application in improving plant salt tolerance; A2) Application in reducing plant height; A3) Application in cultivating salt-tolerant plants or plants with reduced plant height; A4) Applications in molecular breeding for improving plant salt tolerance and / or reducing plant height, or in germplasm resources for improving plant salt tolerance and / or reducing plant height; The proteins are protein 1, protein 2, and protein 3, wherein: Protein 1 is a protein with the amino acid sequence SEQ ID NO:3; Protein 2 is a protein with the amino acid sequence SEQ ID NO:6; Protein 3 is a protein whose amino acid sequence is SEQ ID NO:9; The plant in question is wheat.

2. Knock out plants TaSGR-5A Gene, TaSGR-5B Genes and TaSGR-5D Applications of genes, wherein the application is any of the following: E1) Application in improving plant salt tolerance; E2) Application in reducing plant height; E3) Application in cultivating salt-tolerant plants or plants with reduced plant height; E4) Applications in molecular breeding for improving plant salt tolerance and / or reducing plant height, or in germplasm resources for improving plant salt tolerance and / or reducing plant height; The plant is wheat. TaSGR-5A The nucleotide sequence of the gene is shown in SEQ ID NO:2; TaSGR-5B The nucleotide sequence of the gene is shown in SEQ ID NO:5; TaSGR-5D The nucleotide sequence of the gene is shown in SEQ ID NO:

8.

3. 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 K1 in improving plant salt tolerance; Application of K2 in reducing plant height; Application of K3 in cultivating salt-tolerant plants or plants with reduced plant height; K4) Applications in molecular breeding for improving plant salt tolerance and / or reducing plant height, or in germplasm resources for improving plant salt tolerance and / or reducing plant height; The plant is wheat; the application is achieved by downregulating the content and / or activity of the protein in the plant, and the substance improves the plant's salt tolerance and / or reduces the plant's height by causing the coding gene of the protein in claim 1 to be deleted or inactivated; the substance is a nucleic acid molecule that causes the coding gene of the protein in claim 1 to be deleted or inactivated through site-directed mutagenesis, gene knockdown, gene editing, and / or gene knockout techniques.

4. The application according to claim 3, characterized in that, The substance is a CRISPR / Cas9 system containing sgRNA, wherein the sgRNA targets the gene encoding the protein described in claim 1.

5. A method for cultivating salt-tolerant plants or plants with reduced plant height, characterized in that, The method includes reducing the content and / or activity of the protein described in claim 1 in the target plant to obtain a plant with increased salt tolerance and / or reduced plant height, wherein the plant is wheat.

6. The method according to claim 5, 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.

7. The method according to claim 6, characterized in that, The reduction of the expression level of the protein-coding gene in the target plant is performed using a CRISPR / Cas9 system, which includes sgRNA targeting the protein-coding gene of claim 1.

8. The method according to claim 7, characterized in that, The target sequences of sgRNA are shown in SEQ ID NO:10 and SEQ ID NO:11.

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