TaSPDT protein, gene encoding same and use thereof

Abstract

The application discloses TaSPDT protein and a coding gene and application thereof, and belongs to the technical field of biotechnology, and particularly relates to TaSPDT protein and a coding gene and application thereof. The protein is any one of the following: a protein with an amino acid sequence as shown in SEQ ID No. 4, SEQ ID No. 5 or SEQ ID No. 6; or a protein obtained by substitution, deletion and / or addition of amino acid residues of the above-mentioned protein, the protein having more than 80% identity and having the property of regulating wheat growth and development and seed phosphorus accumulation; or a fusion protein obtained by connecting the N terminal or / and C terminal of the above-mentioned protein with a protein tag. By regulating the activity and / or content of the protein TaSPDT in a target plant, or / and the expression amount of the coding gene of the protein, the property of regulating wheat growth and development and seed phosphorus accumulation can be realized.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to the TaSPDT protein, its encoding gene, and its applications. Background Technology

[0002] Phosphorus is one of the three essential nutrients for plant growth and development, but it is also a major limiting factor for plant growth because phosphorus in the soil readily chelates with metal ions such as iron, aluminum, and calcium to form insoluble compounds that are difficult for plants to absorb. Phosphate fertilizers are typically applied to increase crop yields. However, due to the high fixation rate and low diffusion rate of phosphorus in the soil, excessive phosphorus application is needed to meet the needs of crop growth, which conflicts with the limited reserves of phosphate rock and also causes environmental pollution.

[0003] Wheat is one of the world's three major food crops, requiring a large amount of phosphate fertilizer. Phosphate fertilizer used for wheat accounts for 16.1% of the total phosphorus applied. However, the utilization rate of phosphate fertilizer in the current season is very low, only reaching 15-30%, with more than half being fixed by the soil as slow-release or unavailable phosphorus. This leads to a situation where large areas of arable land have high total phosphorus content but low available phosphorus content. The excessive application of phosphate fertilizer accelerates the depletion of phosphate rock, causes eutrophication of water bodies, and threatens the sustainable development of modern agriculture.

[0004] Phosphorus absorbed by plants is partially accumulated in organs such as roots and stems, and partially stored in seeds during reproductive growth. In crops such as wheat, approximately 30-60% of phosphorus accumulates as phytic acid in the aleurone layer and germ of the seeds. Non-ruminant animals such as humans, pigs, poultry, and fish cannot synthesize phytase, and phytates cannot be digested and absorbed. Large amounts of undigested phytates are excreted into the environment through livestock and poultry manure, causing agricultural phosphorus pollution and eutrophication of water bodies. Akond et al. found a negative correlation between phytic acid concentration in soybeans and the bioavailability of minerals such as Fe, Mg, and Ca. Excessive accumulation of phytic acid in seeds makes people whose staple foods are grains and legumes highly susceptible to mineral deficiencies. Furthermore, phytates can form complexes with basic amino acids, proteins stored in seeds, and enzymes in the digestive tract, reducing amino acid availability, protein digestibility, and digestive enzyme activity.

[0005] Therefore, from the perspective of optimizing phosphorus allocation in plants, research on the translocation and distribution mechanisms of phosphorus in the aboveground parts of crops such as wheat is expected to provide some theoretical guidance for improving phosphorus utilization efficiency, reducing resource input, and reducing the accumulation of "anti-nutritional substances" such as phytic acid. Summary of the Invention

[0006] The technical problem to be solved by this invention is how to regulate the growth and development performance of wheat and the amount of phosphorus accumulation in seeds.

[0007] To address the problems existing in the prior art, the present invention provides a protein.

[0008] The protein provided by this invention may be any of the following:

[0009] A1) A protein with the amino acid sequence shown in SEQ ID No. 4;

[0010] A2) Proteins with amino acid sequences as shown in SEQ ID No. 5;

[0011] A3) Proteins with the amino acid sequence shown in SEQ ID No. 6;

[0012] A4) Proteins obtained by substituting and / or deleting and / or adding amino acid residues of proteins A1), A2), or A3) possessing more than 80% identity with the proteins shown in A1), A2), or A3) and having the function of regulating wheat growth and development performance and seed phosphorus accumulation; for example, those skilled in the art can, based on the amino acid sequences shown in SEQ ID No. 4, SEQ ID No. 5, and SEQ ID No. 6 and other conventional techniques such as the conserved substitution of amino acids, obtain protein mutants with the same function as the amino acid sequences shown in SEQ ID No. 4, SEQ ID No. 5, and SEQ ID No. 6 by substituting, deleting, and / or adding one or more amino acids without affecting their activity;

[0013] A5) is a fusion protein obtained by attaching a protein tag to the N-terminus and / or C-terminus of A1), A2), A3) or A4).

[0014] The protein described in A1 above is named TaSPDT.

[0015] To facilitate the purification or detection of the protein in A1), a tag protein can be attached to the amino or carboxyl terminus of the protein, which consists of the amino acid sequences shown in SEQ ID No. 4, SEQ ID No. 5, and SEQ ID No. 6 in the sequence listing.

[0016] The proteins mentioned above can be synthesized artificially, or their encoding genes can be synthesized first and then expressed biologically.

[0017] The tagged proteins include, but are not limited to: GST (glutathione thiotransferase) tagged protein, His6 tagged protein (His-tag), MBP (maltose-binding protein) tagged protein, Flag tagged protein, SUMO tagged protein, HA tagged protein, Myc tagged protein, eGFP (enhanced green fluorescent protein), eCFP (enhanced cyan fluorescent protein), eYFP (enhanced yellow-green fluorescent protein), mCherry (monomer red fluorescent protein), or AviTag tagged protein.

[0018] Those skilled in the art can readily mutate the nucleotide sequence encoding the TaSPDT protein of this invention using known methods, such as directed evolution or point mutation. Any artificially modified nucleotides that have 75% or more identity with the nucleotide sequence of the TaSPDT protein isolated in this invention, as long as they encode and function the TaSPDT protein, are derived from and equivalent to the nucleotide sequence of this invention.

[0019] The aforementioned 75% or higher degree of identity can be 80%, 85%, 90%, or 95% or higher degree of identity.

[0020] In this article, identity refers to the similarity of amino acid or nucleotide sequences. The identity of amino acid sequences can be determined using homology search sites on the internet, such as the BLAST page on the NCBI homepage. For example, in Advanced BLAST 2.1, using blastp as the procedure, setting the Expect value to 10, setting all filters to OFF, using BLOSUM62 as the matrix, setting the Gap existence cost, Per residue gap cost, and Lambda ratio to 11, 1, and 0.85 (default values) respectively, and performing an identity search on a pair of amino acid sequences, the identity value (%) can then be obtained.

[0021] In this document, the 80% or more identity 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% identity.

[0022] In this document, the 90% or more identity can be at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity.

[0023] The protein mentioned above is derived from wheat (Triticum aestivum L.).

[0024] The present invention also provides biomaterials related to the above-mentioned proteins, said biomaterials may be any of the following:

[0025] B1) Nucleic acid molecules that encode the proteins described above;

[0026] B2) An expression cassette containing the nucleic acid molecule described in B1);

[0027] B3) A recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2);

[0028] B4) Recombinant microorganisms containing the nucleic acid molecules described in B1), or recombinant microorganisms containing the expression cassette described in B2), or recombinant microorganisms containing the recombinant vector described in B3);

[0029] B5) A transgenic plant cell line containing the nucleic acid molecule described in B1), or a transgenic plant cell line containing the expression cassette described in B2);

[0030] B6) Transgenic plant tissue containing the nucleic acid molecules described in B1), or transgenic plant tissue containing the expression cassette described in B2);

[0031] B7) Transgenic plant organs containing the nucleic acid molecules described in B1), or transgenic plant organs containing the expression cassette described in B2);

[0032] C1) Nucleic acid molecules that inhibit, reduce, or silence the expression of the genes encoding the proteins described above;

[0033] C2) expresses the gene encoding the nucleic acid molecule described in C1);

[0034] C3) contains an expression cassette encoding the gene described in C2);

[0035] C4) A recombinant vector containing the encoding gene described in C2), or a recombinant vector containing the expression cassette described in C3);

[0036] C5) A recombinant microorganism containing the encoding gene described in C2), or a recombinant microorganism containing the expression cassette described in C3), or a recombinant microorganism containing the recombinant vector described in C4);

[0037] C6) A transgenic plant cell line containing the encoding gene described in C2), or a transgenic plant cell line containing the expression cassette described in C3), or a transgenic plant cell line containing the recombinant vector described in C4);

[0038] C7) Transgenic plant tissue containing the encoding gene described in C2), or transgenic plant tissue containing the expression cassette described in C3), or transgenic plant tissue containing the recombinant vector described in C4);

[0039] C8) A transgenic plant organ containing the encoding gene described in C2), or a transgenic plant organ containing the expression cassette described in C3), or a transgenic plant organ containing the recombinant vector described in C4).

[0040] In the above-mentioned biological materials, the nucleic acid molecule described in B1) is a gene as shown below:

[0041] E1) The coding sequence of the coding strand is the cDNA molecule or DNA molecule of SEQ ID No. 1;

[0042] The coding sequence of the E2) coding strand is the cDNA molecule or DNA molecule of SEQ ID No. 2;

[0043] The coding sequence of the E3 coding strand is the cDNA molecule or DNA molecule of SEQ ID No. 3.

[0044] The DNA molecule shown in SEQ ID No. 1, SEQ ID No. 2 or SEQ ID No. 3 (the gene that regulates wheat growth and development performance and seed phosphorus accumulation) encodes the protein TaSPDT with the amino acid sequence of SEQ ID No. 4, SEQ ID No. 5 or SEQ ID No. 6.

[0045] The nucleotide sequences shown in SEQ ID No. 1, SEQ ID No. 2 or SEQ ID No. 3 are the nucleotide sequences of the protein TaSPDT encoding gene (CDS).

[0046] The TaSPDT gene described in this invention can be any nucleotide sequence capable of encoding the protein TaSPDT. Considering codon degeneracy and codon preferences among different species, those skilled in the art can use codons suitable for expression in a specific species as needed.

[0047] B1) The nucleic acid molecule may also include nucleic acid molecules obtained by codon preference modification based on the nucleotide sequence shown in SEQ ID No. 1, SEQ ID No. 2 or SEQ ID No. 3.

[0048] B1) The nucleic acid molecule may also include nucleic acid molecules that have a nucleotide sequence identity of more than 95% with the nucleotide sequence shown in SEQ ID No. 1, SEQ ID No. 2 or SEQ ID No. 3 and originate from the same species.

[0049] 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 gRNA, mRNA, siRNA, shRNA, sgRNA, miRNA, or antisense RNA.

[0050] The vectors described herein are well-known to those skilled in the art and include, but are not limited to: plasmids, bacteriophages (such as λ phage or M13 filamentous phage), granules (i.e., Cosmids), Ti plasmids, or viral vectors. Specifically, they may be the vectors pYPQ131D-TaU6 and pYLCRISPR / Cas9.

[0051] Recombinant expression vectors containing the TaSPDT gene can be constructed using existing plant expression vectors. These plant expression vectors include, but are not limited to, binary Agrobacterium vectors and vectors suitable for plant microbombardment. The plant expression vectors may also contain the 3' untranslated region of the exogenous gene, i.e., containing a polyadenylate signal and any other DNA fragment involved in mRNA processing or gene expression. The polyadenylate signal can guide the addition of polyadenylate to the 3' end of the mRNA precursor; similar functions exist for the untranslated regions transcribed at the 3' end of genes including, but not limited to, Agrobacterium crown gall-inducing (Ti) plasmid genes (such as the Nos gene for lipase synthesis) and plant genes (such as the soybean storage protein gene).

[0052] When constructing recombinant plant expression vectors using the TaSPDT gene, any enhancing or constitutive promoter can be added before its transcription initiation nucleotide, including but not limited to the cauliflower mosaic virus (CAMV) 35S promoter and the maize ubiquitin promoter. These can be used alone or in combination with other plant promoters. Furthermore, when constructing plant expression vectors using the gene of this invention, enhancers, including translational enhancers or transcriptional enhancers, can also be used. These enhancer regions can be ATG start codons or adjacent region start codons, but they must be identical to the reading frame of the coding sequence to ensure correct translation of the entire sequence. The sources of the translation control signals and start codons are wide-ranging; they can be natural or synthetic. The translation initiation region can originate from the transcription initiation region or structural genes.

[0053] To facilitate the identification and screening of transgenic plant cells or plants, the plant expression vectors used can be processed, such as by adding genes that can be expressed in plants, encoding enzymes or luminescent compounds that produce color changes (GUS genes, luciferase genes, etc.), antibiotic resistance markers (gentamicin markers, kanamycin markers, etc.), or chemical reagent resistance marker genes (such as herbicide resistance genes). From a safety perspective, transgenic plants can be screened directly under stress without adding any selective marker genes.

[0054] By using any vector capable of guiding the expression of exogenous genes in plants, the TaSPDT gene or gene fragments provided by this invention can be introduced into plant cells or recipient plants, resulting in transgenic cell lines and transgenic plants with altered plant growth and development performance and seed phosphorus accumulation. The expression vector carrying the TaSPDT gene can be used to transform plant cells or tissues using conventional biological methods such as Ti plasmids, Ri plasmids, plant virus vectors, direct DNA transformation, microinjection, electrocoagulation, and Agrobacterium-mediated transformation, and the transformed plant tissues can be cultured into plants.

[0055] The intermediate carrier may specifically be pYPQ131D-gR1.

[0056] The intermediate vector pYPQ131D-gR1 is obtained by replacing the fragment between the BglII and BsmBⅠ recognition sites of the pYPQ131D-TaU6 vector with Target1 while keeping the other sequences of the vector unchanged. Specifically, the intermediate vector can be YPQ131D-gR1.

[0057] The primer sequences for synthesizing Target1 are as follows: 5'-CTTGACTACAGCCTCCGCCTCCTC--3', 5'-AAACGAGGAGGCGGAGGCTGTAGT-3'.

[0058] The intermediate vector pYPQ131D-gR2 is obtained by replacing the fragment between the BglII and BsmBⅠ recognition sites of the pYPQ131D-TaU6 vector with Target2, while keeping the other sequences of the vector unchanged. Specifically, the intermediate vector can be pYPQ131D-gR2.

[0059] The primer sequences for synthesizing Target2 are as follows: 5'-CTTGGCCAACCTGCCGCCCATCAT-3', 5'-AAACATGATGGGCGGCAGGTTGGC-3'.

[0060] The recombinant vector can specifically be a CRISPR recombinant expression vector, which is obtained by inserting the BsaI recognition site of the pYLCRISPR / Cas9 vector into the two expression cassettes TaU6-gR1 and TaU6-gR2 fragments on pYPQ131D-gR1 and pYPQ131D-gR2, while keeping the other sequences of the pYLCRISPR / Cas9 vector unchanged.

[0061] The TaU6-gR1 fragment is the nucleotide sequence shown in SEQ ID No. 12. The TaU6-gR2 fragment is the same as TaU6-gR1, except that positions 363 to 383 are replaced with 5'-GGCCAACCTGCCGCCCATCAT-3'.

[0062] The recombinant microorganism may specifically be recombinant Agrobacterium EHA105.

[0063] The recombinant Agrobacterium EHA105 is a recombinant bacterium obtained by introducing the CRISPR recombinant expression vector into Agrobacterium tumefaciens EHA105.

[0064] The microorganisms described in this article can be yeast, bacteria, algae, or fungi. Among them, bacteria can originate from genera such as *Escherichia*, *Erwinia*, *Agrobacterium*, *Flavobacterium*, *Alcaligenes*, *Pseudomonas*, and *Bacillus*. Specifically, *Agrobacterium tumefaciens* EHA105 is an example.

[0065] The present invention also provides the use of the protein TaSPDT described above, or the expression substance regulating the gene, or the substance regulating the activity or content of the protein, in any of the following:

[0066] The application of the protein or gene expression substance or substance that regulates the activity or content of the protein described in U1) in regulating plant growth and development performance and seed phosphorus accumulation.

[0067] The application of the protein or gene expression substance or substance regulating the activity or content of the protein described in U2) in the preparation of products that regulate plant growth and development performance and seed phosphorus accumulation.

[0068] The application of the protein or gene-regulating substance described in U3) or the substance regulating the activity or content of the protein in the cultivation of plants with altered growth and development performance and seed phosphorus accumulation.

[0069] The application of the protein or gene-regulating substance or substance regulating the activity or content of the protein described in U4) in the preparation of products that cultivate plants with altered growth and development performance and seed phosphorus accumulation.

[0070] The application of the protein or gene expression substance or substance that regulates the activity or content of the protein described in U5) in plant breeding.

[0071] In this article, the substance that regulates the activity and / or content of the protein may be a substance that regulates gene expression, wherein the gene encodes the protein TaSPDT.

[0072] In the above applications, the substance regulating gene expression or the substance regulating the activity or content of the protein is a biological material related to the protein, and the biological material may be any of the following:

[0073] B1) Nucleic acid molecules that encode the proteins described above;

[0074] B2) An expression cassette containing the nucleic acid molecule described in B1);

[0075] B3) A recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2);

[0076] B4) Recombinant microorganisms containing the nucleic acid molecules described in B1), or recombinant microorganisms containing the expression cassette described in B2), or recombinant microorganisms containing the recombinant vector described in B3);

[0077] B5) A transgenic plant cell line containing the nucleic acid molecule described in B1), or a transgenic plant cell line containing the expression cassette described in B2);

[0078] B6) Transgenic plant tissue containing the nucleic acid molecules described in B1), or transgenic plant tissue containing the expression cassette described in B2);

[0079] B7) Transgenic plant organs containing the nucleic acid molecules described in B1), or transgenic plant organs containing the expression cassette described in B2);

[0080] C1) Nucleic acid molecules that inhibit, reduce, or silence the expression of the genes encoding the proteins described above;

[0081] C2) expresses the gene encoding the nucleic acid molecule described in C1);

[0082] C3) contains an expression cassette encoding the gene described in C2);

[0083] C4) A recombinant vector containing the encoding gene described in C2), or a recombinant vector containing the expression cassette described in C3);

[0084] C5) A recombinant microorganism containing the encoding gene described in C2), or a recombinant microorganism containing the expression cassette described in C3), or a recombinant microorganism containing the recombinant vector described in C4);

[0085] C6) A transgenic plant cell line containing the encoding gene described in C2), or a transgenic plant cell line containing the expression cassette described in C3), or a transgenic plant cell line containing the recombinant vector described in C4);

[0086] C7) Transgenic plant tissue containing the encoding gene described in C2), or transgenic plant tissue containing the expression cassette described in C3), or transgenic plant tissue containing the recombinant vector described in C4);

[0087] C8) A transgenic plant organ containing the encoding gene described in C2), or a transgenic plant organ containing the expression cassette described in C3), or a transgenic plant organ containing the recombinant vector described in C4).

[0088] In the above text, the substance regulating gene expression can be a substance that performs at least one of the following six types of regulation: 1) regulation at the transcriptional level of the gene; 2) post-transcriptional regulation of the gene (i.e., regulation of splicing or processing of the primary transcript of the gene); 3) regulation of RNA transport of the gene (i.e., regulation of mRNA transport of the gene from the nucleus to the cytoplasm); 4) regulation of translation of the gene; 5) regulation of mRNA degradation of the gene; and 6) post-translational regulation of the gene (i.e., regulation of the activity of the protein translated from the gene).

[0089] The present invention also provides a method for regulating plant growth and development performance and seed phosphorus accumulation, including regulating the activity and / or content of the proteins described above in the target plant, and / or the expression level of the genes encoding the proteins, to regulate plant growth and development performance and seed phosphorus accumulation.

[0090] In the above method, regulating the activity and / or content of the protein TaSPDT in the target plant, or / and the expression level of the gene encoding the protein, includes introducing the gene encoding the protein TaSPDT into the recipient plant to change the plant's growth and development performance and seed phosphorus accumulation; the TaSPDT encoding gene encodes the protein TaSPDT.

[0091] In the above method, the regulation of the activity and / or content of the protein TaSPDT in the target plant, or / and the expression level of the gene encoding the protein, includes inhibiting, reducing or silencing the expression level of the gene encoding the protein mentioned above in the target plant, or / and inhibiting, reducing or silencing the activity and / or content of the gene encoding the protein mentioned above, thereby reducing plant growth and development performance and seed phosphorus accumulation.

[0092] The importation refers to the importation through recombination methods, including but not limited to Agrobacterium-mediated transformation, bio-projectile methods, electroporation, in-planta technology, and so on.

[0093] In the above applications and methods, the regulation can be to increase, enhance, or upregulate.

[0094] In the above applications and methods, the regulation can be suppression, reduction, or silencing.

[0095] To facilitate the identification and screening of transgenic cells or plants, the recombinant expression vectors used can be processed, such as by adding genes encoding enzymes or luminescent compounds that can be expressed in plants, antibiotic resistance markers, or chemical reagent resistance marker genes. Alternatively, without adding any selective marker genes, transformed plants can be directly screened for resistance under stress. Plants obtained by the above methods can be transgenic plants or plants obtained through conventional breeding techniques such as hybridization. In the above methods, the transgenic plants are understood to include not only first- and second-generation transgenic plants but also their progeny. For transgenic plants, the gene can be propagated within the species or transferred into other varieties of the same species using conventional breeding techniques, especially commercial varieties. The transgenic plants include seeds, callus tissue, intact plants, and cells.

[0096] The present invention also provides a method for cultivating plants with altered growth and development performance and seed phosphorus accumulation, comprising: 1) increasing, enhancing and / or upregulating the expression level of the coding gene of the protein described above in the target plant, or / and increasing, enhancing and / or upregulating the activity and / or content of the coding gene of the protein described above, to obtain plants with improved growth and development performance and seed phosphorus accumulation.

[0097] 2) Inhibit, reduce, or silence the expression level of the coding genes of the proteins mentioned above in the target plant, or / and inhibit, reduce, or silence the activity and / or content of the coding genes of the proteins mentioned above, to obtain plants with reduced growth and development performance and seed phosphorus accumulation.

[0098] As one embodiment of the present invention, the method for cultivating plants with altered growth and development performance and seed phosphorus accumulation includes the following steps:

[0099] (1) Construct expression vectors containing the coding sequences of sgRNA1 and sgRNA2 as shown in SEQ ID NO.10 and SEQ ID NO.11;

[0100] (2) Introduce the expression vector constructed in step (1) into plants;

[0101] (3) Plants with reduced growth and development performance and seed phosphorus accumulation were obtained through screening and identification.

[0102] In one specific embodiment, a method for cultivating plants with reduced growth and development performance and reduced seed phosphorus accumulation includes the following steps: inhibiting the expression of nucleic acid molecules encoding TaSPDT protein in the target plant to obtain transgenic plants with reduced growth and development performance and reduced seed phosphorus accumulation.

[0103] The inhibition of TaSPDT protein expression in the target plant can be achieved by introducing a knockout vector targeting the TaSPDT protein into the target plant. Specifically, the gene editing vector is a Cas9 gene editing technology-based vector. Specifically, the gene editing vector expresses sgRNA and Cas9 protein. The sgRNA targets the TaSPDT protein. Specifically, the target of the sgRNA is:

[0104] Target1:5'-ACTACAGCCTCCGCCTCCTC-3'

[0105] Target2:5'-GCCAACCTGCCGCCCATCAT-3'

[0106] The present invention also provides a method for cultivating plants with improved growth and development performance and seed phosphorus accumulation, comprising the following steps: increasing the content of TaSPDT protein in the plant to obtain plants with improved growth and development performance and seed phosphorus accumulation.

[0107] In this invention, the purpose of plant breeding includes cultivating plants with improved growth and development performance and increased seed phosphorus accumulation.

[0108] In this invention, the growth and development performance can be any of the following:

[0109] M1) Wheat seedlings have long roots;

[0110] M2) Diameter of wheat roots during the seedling stage;

[0111] M3) Dry weight of wheat straw at maturity;

[0112] M4) Dry weight of mature wheat seeds;

[0113] M5) Number of wheat ears at maturity.

[0114] In the above applications or methods, the plant is any one of the following:

[0115] C1) Monocotyledons;

[0116] C2) Plants of the order Poales;

[0117] C3) Gramineae plants;

[0118] C4) Plants of the genus Triticum;

[0119] C5) Wheat.

[0120] The TaSPDT protein of this invention can mediate the distribution of phosphorus to seeds. By providing a method for regulating phosphorus accumulation in wheat seeds, it is expected to provide some theoretical guidance for improving phosphorus utilization efficiency, reducing resource input, and reducing the accumulation of "anti-nutritional substances" such as phytic acid. Attached Figure Description

[0121] Figure 1 The values ​​represent the Pi transport activity of TaSPDT at different pH values. The values ​​are the biological replicate mean ± standard deviation (n = 4).

[0122] Figure 2 This illustrates the spatiotemporal expression pattern of TaSPDT. The relative expression levels of the TaSPDT gene in various organs of wheat at the seedling, flowering, and grain-filling stages were determined when cultured at concentrations of 0.2 mM and 0.02 mMPi, respectively.

[0123] Figure 3 Tissue specificity of TaSPDT localization in nodes. Wheat node I (AE) and basal node (FH) were subjected to co-immunostained staining with TaSPDT antibody (red) and DAPI (blue). A is a cross-section immunostaining result of node I; B is an enlarged view of the dashed box in Figure A; C and D are enlarged views of the dashed boxes in Figure B; E is a partial negative control image of node I without primary antibody; F is a cross-section of the basal node; G is an enlarged view of the dashed box in Figure F; H is a partial negative control image of the basal node without primary antibody. DVB: Dispersed vascular bundle, PDV: Dispersed vascular bundle phloem, XDV: Dispersed vascular bundle xylem; EVB: Enlarged vascular bundle, PEV: Enlarged vascular bundle phloem, XEV: Enlarged vascular bundle xylem; RVB: Conventional vascular bundle, PCB: Thin-walled cell bridge, BS: Vascular bundle sheath cells, XPC: Xylem parenchyma cells, XTC: Xylem transfer cells. Scale bar = 100m.

[0124] Figure 4 This diagram illustrates the construction of the TaSPDT:GFP vector and shows the subcellular localization results of TaSPDT. A is a schematic diagram of the TaSPDT:GFP vector construction; B is a partial view of the cross-section immunostaining results at node I; C shows the localization of TaSPDT in wheat protoplasts. GFP emits green fluorescence; autofluorescence of chloroplasts is indicated by purple-red. Scale bar = 5 μm.

[0125] Figure 5 This is a diagram showing the structure of the TaSPDT gene and the location of its target sites.

[0126] Figure 6 For testing genetically modified wheat using Bar test strips.

[0127] Figure 7TaSPDT gene-edited wheat germination and seedling root morphology. Scale bar in A = 1 cm; B represents wheat germination rate at day 5, values ​​are the mean ± standard deviation of biological replicates (n = 10); CF represents root phenotypic data of 10-day-old wheat seedlings, values ​​are the mean ± standard deviation of biological replicates (n = 4).

[0128] Figure 8 The TaSPDT gene-edited wheat maturity phenotype is shown. A is a photograph of the wheat spike and aboveground parts at maturity, scale bar = 2cm; BF are the wheat plant height, straw dry weight, seed dry weight, number of spikes per plant, and average number of grains per spike at maturity, respectively, with values ​​representing the biological replicate mean ± standard deviation (n = 4).

[0129] Figure 9 Phosphorus concentration and distribution in TaSPDT gene-edited wheat. A represents the total phosphorus concentration in different aboveground organs of wheat; B represents the dry weight of different aboveground organs of wheat; C represents the proportion of phosphorus distribution in different aboveground organs of wheat; D represents the phytic acid concentration in seeds. The values ​​in the figure are the biological replicate mean ± standard deviation (n=4). Detailed Implementation

[0130] 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.

[0131] 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.

[0132] Unless otherwise specified, all quantitative experiments in the following examples are performed in triplicate.

[0133] The pxβg-ev1 plasmid used in the following examples was provided by Professor Maki Katsuhara of Okayama University, Japan, and is described in: Ma JF, Tamai K, Yamaji N, et al. A silicon transporter in rice. Nature. 2006; 440(7084):688-691. This biological material is available to the public from the applicant and is intended solely for the purpose of replicating the experiments of this invention and shall not be used for any other purpose.

[0134] The pYPQ131D-TaU6 in the following examples is described in: Zhang S, Zhang R, Song G, et al. Targeted mutagenesis using the Agrobacterium tumefaciens-mediated CRISPR-Cas9 system in common wheat. BMC Plant Biol. 2018; 18(1):302. Published 2018 Nov 26. The biological material is available to the public from the applicant and is intended solely for the purpose of repeating experiments of the present invention and shall not be used for any other purpose.

[0135] The pTF486 carrier in the following examples was provided by Professor Gong Haijun of Northwest A&F University and is described in Sun H, Duan Y, Mitani-Ueno N, et al. Tomato roots have a functional silicon influxtransporter but not a functional silicon efflux transporter. Plant Cell Environ. 2020; 43(3):732-744. The public can obtain this biological material from the applicant. This biological material is only used for repeating the experiments of this invention and cannot be used for other purposes.

[0136] The wild-type wheat cultivar Fielder in the following examples is described in: Sato K, Abe F, Mascher M, et al. Chromosome-scale genome assembly of the transformation-amenable commonwheat cultivar 'Fielder'. DNA Res. 2021; 28(3):dsab008. The biological material is available to the public from the applicant and is intended solely for the purpose of repeating experiments of the present invention and shall not be used for any other purpose.

[0137] The following examples use IBM SPSS Statistics 26 statistical software to process the data. The experimental results are expressed as mean ± standard deviation. The LSD method (α = 0.05) is used for significance analysis. P < 0.05 (*) indicates a significant difference, P < 0.01 (**) indicates a highly significant difference, and P < 0.001 (***) indicates a highly significant difference.

[0138] Example 1: Obtaining the TaSPDT protein and its encoding gene

[0139] A gene encoding the TaSPDT protein (a protein in wheat responsible for phosphate transport via a sulfur transporter) was isolated and cloned from wheat. The specific steps are as follows: Using cDNA from the wheat variety Zhengmai 7698 (Triticum aestivum L.cv.Zhengmai7698) as a template, the TaSPDT coding region was amplified using PrimeSTAR Max DNA polymerase (Takara, Japan). The primers were TaSPDT-F: 5'-ACTCTCTGCTTTAGCCGTCG-3'; TaSPDT-R: 5'-AGATCAGCACCGCCGTTATT-3'. The PCR amplification program was as follows: the pre-denaturation temperature was set to 95℃ for 3 min; the denaturation temperature was set to 95℃ for 15 sec; the annealing temperature was set to 60℃ for 15 sec; the extension temperature was set to 72℃ for 30 sec, for a total of 30 cycles, with a final extension at 72℃ for 5 min. PCR products were separated by electrophoresis, and the target band was excised and recovered by gel extraction. The PCR products were then extracted and A was added to the 3' end of the PCR products using 2×Rapid Taq Master Mix (vazyme, China) to recover the TaSPDT fragment.

[0140] The TaSPDT-4A gene in wheat genomic DNA is shown in SEQ ID No. 7 of the sequence listing. The coding sequence of the TaSPDT-4A gene is shown in SEQ ID No. 1 of the sequence listing, and it encodes the protein TaSPDT-4A with the amino acid sequence shown in SEQ ID No. 4 of the sequence listing. The TaSPDT-7A gene in wheat genomic DNA is shown in SEQ ID No. 8 of the sequence listing. The coding sequence of the TaSPDT-7A gene is shown in SEQ ID No. 2 of the sequence listing, and it encodes the protein TaSPDT-7A with the amino acid sequence shown in SEQ ID No. 5 of the sequence listing. The TaSPDT-7D gene in wheat genomic DNA is shown in SEQ ID No. 9 of the sequence listing. The coding sequence of the TaSPDT-7D gene is shown in SEQ ID No. 3 of the sequence listing, and it encodes the protein TaSPDT-7D with the amino acid sequence shown in SEQ ID No. 6 of the sequence listing.

[0141] 2. Functional validation of TaSPDT protein

[0142] 1) TaSPDT protein transport activity analysis

[0143] To investigate the phosphorus transport activity and characteristics of the SPDT protein in wheat, this study used Xenopus oocytes (purchased from the Xenopus Resource Center (Hangzhou)) as a heterologous expression system. TaSPDT cRNA was injected into the oocytes, and the phosphorus transport activity of this protein under different pH conditions was detected. The specific experimental steps are as follows:

[0144] The TaSPDT-7A fragment obtained in step 1 was ligated into a linearized pGEM-T Easy vector system (Promega, USA) using T4 ligase. The ligation product was transformed into *E. coli* DH5α competent cells using a freeze-thaw method. Colonies that appeared after overnight culture were verified by PCR. Single clones that amplified the correct target band were selected and cultured overnight. Plasmids were extracted and digested with the restriction enzyme BamHI (New England Biolab, USA) for verification. Plasmids with correct digestion results were sequenced. Correctly ligated plasmids were digested with restriction endonucleases, and the digestion products were separated by gel electrophoresis. The target fragment TaSPDT was recovered from the gel.

[0145] The vector pxβg-ev1 was linearized with the restriction endonuclease Bgl II (New England Biolab, USA) and recovered by alcohol precipitation.

[0146] After detecting the concentration of the recovered TaSPDT fragment and the linearized pxβg-ev1 vector, the fragment was ligated and transformed using T4 ligase (NEB, USA). The plasmid was extracted and digested to verify the results, yielding a single clone containing the correct recombinant plasmid pxβg-ev1-TaSPDT. The plasmid was then extracted by shaking for later use.

[0147] The structure of the recombinant plasmid pxβg-ev1-TaSPDT is described as follows: The recombinant expression vector is obtained by replacing the small fragment between the Bgl II restriction sites of the pxβg-ev1 vector with a nucleotide sequence containing the coding sequence of TaSPDT, while keeping the other sequences of the pxβg-ev1 vector unchanged.

[0148] The recombinant plasmid was linearized using the restriction endonuclease Xba I (New England Biolab, USA) and a kit was used. The preparation, purification, and recovery of TaSPDT cRNA were performed using System-T3 and Ribo m7G Cap Analog (Promega, USA). TaSPDT cRNA was injected into oocytes using a micromanipulator (Eppendorf, Germany). Frog eggs injected with TaSPDT cRNA or RNase-free ddH2O were incubated at 18°C ​​for 24 hours, and then placed in a solution containing [unspecified substance] at pH 7.5 or 5.5. 32 P in MBS solution for 30 min. (By...)32 The phosphorus transport activity of the TaSPDT gene expressed in frog eggs was determined by a P isotope uptake assay.

[0149] The results showed that TaSPDT protein exhibited phosphorus transport activity at an external solution pH of 5.5, i.e., when a proton gradient existed inside and outside the oocyte, while this transport activity disappeared at pH 7.5. Figure 1 These results indicate that TaSPDT in wheat is an H... + / Pi cotransporter.

[0150] 2) TaSPDT gene expression pattern analysis

[0151] To investigate the expression pattern of TaSPDT, this study used qPCR to detect the expression level of TaSPDT in various tissues and organs of wheat at different growth stages.

[0152] Twelve-day-old wheat seedlings were transferred to hydroponic nutrient solutions containing 200 μM P (CT) and 20 μM P (LP), respectively. After 7 days of cultivation, samples were taken from leaves, stem base, and roots. Potted wheat seedlings with good growth and uniform growth at the heading stage were selected, their roots were rinsed clean, and they were transferred to 200 μM P nutrient solution for 3 days of cultivation. Then, they were cultivated in 200 μM P nutrient solution and 20 μM P nutrient solution, respectively. Samples were taken from different organs at the flowering and grain-filling stages.

[0153] RNA was extracted from plant samples using the Plant RNA Kit R6827 (Omega Biotek, USA), and cDNA was prepared using the HiScript II Q RT SuperMix for qPCR (Vazyme, China). The products were stored at -80°C. qRT-PCR was performed using the ChamQ Universal SYBR qPCR Master Mix (Vazyme, China). Wheat TEF-1 and cyclin-T1-3 were used as internal control genes. -ΔΔCt Calculate the relative expression level of TaSPDT.

[0154] The primers used are as follows:

[0155] SPDT-qF: 5'-GGGCATCTCGCTGTTCAAGA-3'; SPDT-qR: 5'-TCCACCAGGTAGGTGGAGTT-3'.

[0156] TEF-qF: 5'-CAGATTGGCAACGGCTACG-3'; TEF-qR: 5'-CGGACAGCAAAACGACCAAG-3'.

[0157] cyclin-qF: 5'-CGGACAGCAAAACGACCAAG-3'; cyclin-qR: 5'-CGGACAGCAAAACGACCAAG-3'.

[0158] The results showed that during the vegetative growth stage, the relative expression level of the TaSPDT gene was generally low, with the highest expression level at the stem base, more than six times that of the underground and aboveground parts. During the reproductive growth stage, the expression level of the TaSPDT gene at nodes was high, and the expression level was significantly upregulated under low phosphorus induction. Specifically, under low phosphorus conditions, the expression level of the wheat TaSPDT gene was highest at the basal nodes during the flowering stage, followed by the expression levels at the rachis and spikelets. During the grain-filling stage, the expression level of the wheat TaSPDT gene decreased at the basal nodes, but the expression level at node I was significantly increased, reaching the highest value of the entire growth period, and the expression level under low phosphorus conditions was 63.6% higher than that of the control. Figure 2 ).

[0159] As shown in step 2), the expression of TaSPDT in different growth stages of wheat is highest at the nodes. In this study, the basal nodes and node I were selected, and the tissue localization of TaSPDT was investigated by immunofluorescence staining with TaSPDT antibody.

[0160] The specific steps of immunofluorescence staining are as follows: The preparation of the antigenic peptide and the rabbit-derived antibody were completed by Genscript Biotech Co., Ltd. Wheat node I during the grain-filling stage was cut with a sharp blade and immersed in fixative [4% (w / v) paraformaldehyde, 60 mM sucrose, and 50 mM diarsonic acid (pH 7.4)] for 2 hours at room temperature. After washing three times with a solution containing 60 mM sucrose and 50 mM diarsonic acid (pH 7.4), the tissue was embedded in paraffin and then sectioned using a microtome.

[0161] Soak the slides in xylene for 15 minutes, then replace the xylene. Next, soak them sequentially in 100%, 100%, 85%, and 75% ethanol for 5 minutes each, and finally rinse with ddH2O. Circle the tissue location with an immunohistochemical pen, add pepsin antigen retrieval solution, and incubate the slides in a humidified chamber at 37°C for 30 minutes. Then, place the slides in PBS (pH 7.4) and wash them on a destaining shaker for 5 minutes, repeating this process three times.

[0162] Gently shake off the PBS solution, add 3% BSA, and block for 30 min. Carefully remove the blocking solution, add primary antibody to the slide, and then place the slide flat in a humidified chamber and incubate overnight at 4°C. Then place the slide in PBS (pH 7.4), shake on a decolorizing shaker for 5 min, and repeat 3 times. Gently shake off the PBS solution, add secondary antibody, and incubate at room temperature in the dark for 50 min. Wash 3 times with PBS as above, gently shake off the PBS solution, add DAPI staining solution, and incubate at room temperature in the dark for 10 min. Wash 3 times with PBS as above, gently shake off the PBS solution, add autofluorescence quencher, react for 5 min, and rinse with running water for 10 min. Gently shake off the PBS solution and mount with antifluorescence quenching mounting medium.

[0163] The slides were observed and images were acquired under a fluorescence microscope (NIKON, Japan). DAPI excitation / emission wavelengths were 330-380 / 420 nm, emitting blue light; CY3 excitation / emission wavelengths were 510-560 / 590 nm, emitting red light.

[0164] The results showed that TaSPDT was located on the enlarged vascular bundles and dispersed vascular bundles at node I, as well as on the thin-walled cell bridges between them. Figure 3 (AD). TaSPDT distribution at the basal nodes is similar to that at node I, located on the enlarged vascular bundles and regular vascular bundles and the thin-walled cell bridges between them. Figure 3 (E, F, and G).

[0165] 4) Subcellular localization of TaSPDT protein

[0166] TaSPDT protein exhibits typical transmembrane protein characteristics and is likely localized on the cell membrane. This study constructed a recombinant vector containing green fluorescent protein (GFP) fused to the C-terminus of TaSPDT protein. Figure 4 In study A), TaSPDT was introduced into wheat protoplasts using a PEG-mediated method. The subcellular localization distribution of TaSPDT was investigated by observing the green fluorescence signal in the protoplasts.

[0167] The specific experimental steps are as follows:

[0168] Primers were designed based on the obtained TaSPDT-7A sequence and pTF486 vector, introducing Sal I and BamHI restriction sites. Using wheat cDNA as a template, the TaSPDT-7A coding region was amplified using PrimeSTAR Max DNA polymerase (Takara, Japan), and the amplified product was recovered by gel extraction. pTF486 was simultaneously digested with restriction endonucleases Sal I and BamHI (New England Biolab, USA). The linearized pTF486 vector was recovered by alcohol precipitation. The recovered TaSPDT-7A fragment and the linearized pTF486 vector were then... Ligation was performed using Snap Assembly Master Mix (Takara, Japan). The ligation product was transformed into E. coli DH5α using the freeze-thaw method. The transformed single clones were detected by PCR, enzyme digestion, and sequencing, and the recombinant vector pTF486-TaSPDT-7A was successfully obtained. The plasmids of the correctly detected single clones were extracted for later use.

[0169] The recombinant vector pTF486-TaSPDT-7A is a recombinant expression vector obtained by replacing a small fragment between the Sal I and BamHI restriction sites of the pTF486 vector with the coding sequence of TaSPDT-7A SEQ ID No.2, while keeping the other sequences of the pTF486 vector unchanged.

[0170] Wheat leaf protoplasts were extracted using a wheat protoplast preparation and transformation kit (Coolaber, China), and recombinant vectors were transformed into wheat protoplasts via PEG-mediated transformation for protein expression. After incubation at 28°C for 16 h, observation and photography were performed using a confocal microscope (Zeiss, Germany). The excitation / emission wavelengths of the GFP fluorescence signal were 488 nm / 495-556 nm, and the excitation / emission wavelengths of the chloroplast autofluorescence signal were 488 nm / 637-758 nm, respectively. Subcellular localization of the protein was determined by fluorescence localization.

[0171] The results showed that obvious green fluorescence was observed on the cytoplasmic membrane of protoplasts transformed with the TaSPDT:GFP recombinant vector, while fluorescence was observed throughout the cytoplasm of protoplasts transformed with the empty vector. Figure 4 (C). Simultaneously, immunofluorescence results also revealed that red fluorescence was distributed in a ring around the inner side of the cell wall (C). Figure 4 These results collectively indicate that TaSPDT in wheat is located on the wheat cell membrane.

[0172] Example 2: Creation and Identification of Gene-Edited Wheat

[0173] TaSPDT gene-edited wheat was created using the Crisper-Cas9 system. The wheat variety used was Fileder. The specific experimental steps for gene editing vector construction and genetic transformation are as follows:

[0174] Based on the conserved sequences of the TaSPDT gene on chromosomes 4A, 7A, and 7D, two target sites, Target 1 and Target 2, were designed, located in the first and second exons, respectively. Figure 5 The target sequence is as follows:

[0175] Target1:5'-ACTACAGCCTCCGCCTCCTC-3'

[0176] Target2:5'-GCCAACCTGCCGCCCATCAT-3'

[0177] Target1 and Target2 were synthesized. The primer sequences for Target1 are as follows: 5'-CTTGACTACAGCCTCCGCCTCCTC-3', 5'-AAACGAGGAGGCGGAGGCTGTAGT-3'; the primer sequences for Target2 are as follows: 5'-CTTGGCCAACCTGCCGCCCATCAT-3', 5'-AAACATGATGGGCGGCAGGTTGGC-3'. The primers were phosphorylated and annealed to form double-stranded sgRNA1 and sgRNA2.

[0178] First, replace the fragment between the BglII and BsmBI recognition sites in the pYPQ131D-TaU6 vector with sgRNA1 and sgRNA2. Then, amplify the two expression cassettes TaU6-gR1 and TaU6-gR2 on pYQ131D-gR1 and pYPQ131D-gR2. The nucleotide sequence of the expression cassette TaU6-gR1 is sequence 12 in the sequence listing. The TaU6-gR2 fragment is the same as TaU6-gR1, except that positions 363 to 383 of sequence 12 in the sequence listing are replaced with 5'-GGCCAACCTGCCGCCCATCAT-3'.

[0179] Finally, the BsaI recognition site of the pYLCRISPR / Cas9 vector was inserted into two expression cassettes, TaU6-gR1 and TaU6-gR2, while keeping the other sequences of the pYLCRISPR / Cas9 vector unchanged, to obtain the CRISPR / Cas9 gene editing vector.

[0180] Identification of transgenic positive plants using Gene True TMThe BAR Test Kit (Artron, Canada) was used for testing. The transformed embryos underwent resistance selection and induced culture, resulting in 24 T0 generation transgenic plants. These 24 wheat plants were tested with Bar test strips (Artron, Canada), and 22 plants (excluding plants 10 and 13) showed positive results. Figure 6 ).

[0181] Genomic DNA was extracted from 22 positive plants of generation T0 using the CTAB method. 0.2 g of fresh wheat leaves at the two-leaf-one-heart stage were placed in a 2 mL sterile centrifuge tube and rapidly ground into powder using liquid nitrogen. 700 μL of preheated CTAB solution was added, and the mixture was inverted and mixed thoroughly. The tube was then incubated at 65°C for 1 hour, inverting and mixing every 10-14 minutes. 700 μL of chloroform-isoamyl alcohol (24:1) was added to each centrifuge tube, and the tube was shaken at 50 rpm for 20 minutes at room temperature. The tube was then centrifuged at 12000 rpm for 8 minutes at room temperature, and 500 μL of the supernatant was pipetted into a 1.5 mL centrifuge tube. 1000 μL of ice-cold anhydrous ethanol was added to the centrifuge tube, and the tube was incubated at -20°C for 30 minutes. The tube was then centrifuged at 8000 rpm for 10 minutes at room temperature, and the supernatant was discarded. 200 μL of 75% ethanol was added to the centrifuge tube, and the tube was rinsed twice and dried with an air dryer. Finally, add 50 μL of TE buffer containing RNase, incubate at 4°C for 24 h, and then store at -20°C.

[0182] Using extracted DNA as a template, the target site sequence was amplified using the high-fidelity enzyme PrimeSTAR Max DNA polymerase (Takara, Japan) with the following primers: Hi-Tom-F: 5'-GGAGTGAGTACGGTGTGCGCTGCAGTACTTCTTCCCCA-3'; Hi-Tom-R: 5'-GAGTTGGATGCTGGATGGTTATGGAGCAGAGGCGTG-3'. 4 μL of the PCR product was run on a gel to verify the amplification results, and the remaining reaction solution was sent to the Hi-Tom platform for sequencing.

[0183] The results showed that no editing occurred at Target 2, while 15 strains showed editing at Target 1, with a total of 15 mutation types, including single-base insertions and deletions of varying numbers of bases. Among them, 7 strains showed simultaneous biallelic mutations in homologous genes at 4A, 7A, and 7D (Table 1).

[0184] Compared to wild-type WT, taspdt-6 exhibits the following mutations in the TaSPDT gene on chromosome 4A: an insertion of a single base "A" (corresponding to positions 319-320 of SEQ ID No. 1 and 433-434 of SEQ ID No. 7) in one chromosome, and a deletion of two bases "5'-CA-3'" (corresponding to positions 321-322 of SEQ ID No. 1 and 435-436 of SEQ ID No. 7) in the other chromosome; and a deletion of two bases "5'-CA-3'" (corresponding to positions 321-322 of SEQ ID No. 1 and 435-436 of SEQ ID No. 7) in the TaSPDT gene on chromosome 7A. For the TaSPDT gene, the following mutations occur: a deletion of two bases "5'-CA-3'" (corresponding to positions 318-319 of SEQ ID No. 2 and 445-446 of SEQ ID No. 8) in the TaSPDT gene on one chromosome, and a deletion of three bases "5'-AGC-3'" (corresponding to positions 319-321 of SEQ ID No. 2 and 433-434 of SEQ ID No. 7) in the other chromosome. The deletion of positions 446-448 of SEQ ID No. 8; for the TaSPDT gene on chromosome 7D, the following mutations occurred: a deletion of 5 bases "5'-ACAGC-3'" (corresponding to positions 320-324 of SEQ ID No. 3 and positions 528-532 of SEQ ID No. 9) occurred in the TaSPDT gene on one chromosome, and a deletion of 2 bases "5'-CA-3'" (corresponding to positions 321-322 of SEQ ID No. 3 and positions 529-530 of SEQ ID No. 9) occurred in the TaSPDT gene on the other chromosome.

[0185] Compared to wild-type WT, taspdt-7 exhibits the following mutations in the TaSPDT gene on chromosome 4A: a deletion of 4 bases "5'-ACAG-3'" (corresponding to positions 320-323 of SEQ ID No. 1 and positions 434-437 of SEQ ID No. 7) in one chromosome and a deletion of 3 bases "5'-ACA-3'" (corresponding to positions 320-322 of SEQ ID No. 1 and positions 434-436 of SEQ ID No. 7) in the other chromosome; and a deletion of 2 bases "5'-CA-3'" (corresponding to positions 318-319 of SEQ ID No. 2 and positions 445-446 of SEQ ID No. 8) in the other chromosome and a deletion of 3 bases "5'-TAC-3'" (corresponding to positions 320-322 of SEQ ID No. 1 and positions 434-436 of SEQ ID No. 7) in the other chromosome. The deletion of positions 316-318 of SEQ ID No. 2 and positions 443-445 of SEQ ID No. 8; for the TaSPDT gene on chromosome 7D, the following mutations occurred: an insertion of a single base "A" (corresponding to positions 319-320 of SEQ ID No. 3 and positions 527-528 of SEQ ID No. 9) occurred in the TaSPDT gene on one chromosome, and a deletion of three bases "5'-ACA-3'" (corresponding to positions 320-322 of SEQ ID No. 3 and positions 528-530 of SEQ ID No. 9) occurred in the TaSPDT gene on the other chromosome.

[0186] Compared to wild-type WT, taspdt-11 exhibits the following mutations in the TaSPDT gene on chromosome 4A: a deletion of four bases "5'-ACAG-3'" (corresponding to positions 320-323 of SEQ ID No. 1 and positions 434-437 of SEQ ID No. 7) in one chromosome, and an insertion of one base "T" (corresponding to positions 319-320 of SEQ ID No. 1 and positions 433-434 of SEQ ID No. 7) in the other chromosome; and an insertion of three bases "5'-ACA-3'" (corresponding to positions 317-319 of SEQ ID No. 2 and positions 444-446 of SEQ ID No. 8) in the other chromosome, and an insertion of one base "A" (corresponding to positions 316-317 of SEQ ID No. 2 and positions 434-437 of SEQ ID No. 7) in the other chromosome. An insertion occurred between positions 443 and 444 of SEQ ID No. 8; for the TaSPDT gene on chromosome 7D, the following mutations occurred: a deletion of 3 bases "5'-ACA-3'" (corresponding to positions 320-322 of SEQ ID No. 3 and positions 528-530 of SEQ ID No. 9) occurred in the TaSPDT gene on one chromosome, and a deletion of 2 bases "5'-CA-3'" (corresponding to positions 321-322 of SEQ ID No. 3 and positions 529-530 of SEQ ID No. 9) occurred in the TaSPDT gene on the other chromosome.

[0187] Compared to wild-type WT, taspdt-14 exhibits the following mutations in the TaSPDT gene on chromosome 4A: an insertion of one base "A" (corresponding to positions 319-320 of SEQ ID No. 1 and 433-434 of SEQ ID No. 7) in one chromosome and an insertion of one base "T" (corresponding to positions 319-320 of SEQ ID No. 1 and 433-434 of SEQ ID No. 7) in the other chromosome; and an insertion of the following mutations in the TaSPDT gene on chromosome 7A: a deletion of three bases "5'-ACA-3'" (corresponding to positions 317-319 of SEQ ID No. 2 and 444-446 of SEQ ID No. 8) in one chromosome and a deletion of two bases "5'-CA-3'" (corresponding to positions 318-319 of SEQ ID No. 2 and 433-434 of SEQ ID No. 7) in the other chromosome. The deletion of position 445-446 of SEQ ID No. 8; and the insertion of one base "A" (corresponding to position 319-320 of SEQ ID No. 3 and position 527-528 of SEQ ID No. 9) in the TaSPDT gene on chromosome 7D.

[0188] Compared to wild-type WT, taspdt-16 exhibits the following mutations in the TaSPDT gene on chromosome 4A: a deletion of one base "A" (corresponding to position 320 of SEQ ID No. 1 and position 434 of SEQ ID No. 7) in one chromosome and a deletion of one base "C" (corresponding to position 321 of SEQ ID No. 1 and position 435 of SEQ ID No. 7) in the other chromosome; and a deletion of the following mutations in the TaSPDT gene on chromosome 7A: a deletion of three bases "5'-ACA-3'" (corresponding to positions 317-319 of SEQ ID No. 2 and positions 444-446 of SEQ ID No. 8) in one chromosome and a deletion of four bases "5'-ACAG-3'" (corresponding to positions 317-320 of SEQ ID No. 2 and position 435 of SEQ ID No. 7) in the other chromosome. The deletion of positions 444-447 of SEQ ID No. 8; for the TaSPDT gene on chromosome 7D, the following mutations occurred: a deletion of 4 bases "5'-ACAG-3'" (corresponding to positions 320-323 of SEQ ID No. 3 and positions 528-531 of SEQ ID No. 9) occurred in the TaSPDT gene on one chromosome, and a deletion of 3 bases "5'-ACA-3'" (corresponding to positions 320-322 of SEQ ID No. 3 and positions 528-530 of SEQ ID No. 9) occurred in the TaSPDT gene on the other chromosome.

[0189] Compared to wild-type WT, taspdt-21 exhibits the following mutations in the TaSPDT gene on chromosome 4A: an insertion of one base "A" (corresponding to positions 319-320 of SEQ ID No. 1 and 433-434 of SEQ ID No. 7) in one chromosome and an insertion of one base "T" (corresponding to positions 319-320 of SEQ ID No. 1 and 433-434 of SEQ ID No. 7) in the other chromosome; and an insertion of the following mutations in the TaSPDT gene on chromosome 7A: a deletion of 24 bases "5'-CTTCCAGTGGGGCTCCAACTACAG-3'" (corresponding to positions 296-320 of SEQ ID No. 2 and 423-447 of SEQ ID No. 8) in one chromosome and a deletion of 2 bases "5'-CA-3'" (corresponding to positions 296-320 of SEQ ID No. 2 and 423-447 of SEQ ID No. 8) in the other chromosome. The deletion of positions 318-319 of SEQ ID No. 2 and positions 445-446 of SEQ ID No. 8; for the TaSPDT gene on chromosome 7D, the following mutations occurred: a deletion of 3 bases "5'-AGC-3'" (corresponding to positions 322-324 of SEQ ID No. 3 and positions 530-522 of SEQ ID No. 9) occurred in the TaSPDT gene on one chromosome, and a deletion of 3 bases "5'-ACA-3'" (corresponding to positions 320-322 of SEQ ID No. 3 and positions 528-530 of SEQ ID No. 9) occurred in the TaSPDT gene on the other chromosome.

[0190] Compared to wild-type WT, taspdt-24 showed the following mutations: a deletion of two 5'-CA-3' bases in the TaSPDT gene on chromosome 4A (corresponding to positions 321-322 of SEQ ID No. 1 and positions 435-436 of SEQ ID No. 7); a deletion of four 5'-ACAG-3' bases in the TaSPDT gene on chromosome 7A (corresponding to positions 317-320 of SEQ ID No. 2 and positions 444-447 of SEQ ID No. 8), and an insertion of one 'A' base (corresponding to positions 316-317 of SEQ ID No. 2 and positions 443-444 of SEQ ID No. 8); and an insertion of three 5'-ACA-3' bases in the TaSPDT gene on chromosome 7D (corresponding to positions 321-322 of SEQ ID No. 1 and positions 435-436 of SEQ ID No. 7). The TaSPDT gene on another chromosome has a deletion at positions 320-322 of SEQ ID No. 3 and positions 528-530 of SEQ ID No. 9, and an insertion of a single base "T" (corresponding to positions 319-320 of SEQ ID No. 3 and positions 527-528 of SEQ ID No. 9).

[0191] Table 1 Gene editing status of homozygous mutant lines

[0192]

[0193]

[0194]

[0195]

[0196] PAM identification sites are marked with dashed underlines, target locations are marked with solid underlines, base insertions are marked with lowercase letters, and base deletions are replaced with red "·".

[0197] Example 3: Effects of TaSPDT gene editing on wheat germination rate and seedling root development.

[0198] Using the T1 generation plants of the mutant wheat taspdt-16 and taspdt-21 obtained in Example 2 as the research object, compared with wild-type WT, the taspdt-16 mutant wheat showed a deletion of base C in the TaSPDT gene on both chromosomes 4A (corresponding to position 321 of SEQ ID No. 1 and position 435 of SEQ ID No. 7), and a deletion of base ACA in the TaSPDT gene on chromosomes 7A and 7D (corresponding to positions 317-319 of SEQ ID No. 2 and positions 444-446 of SEQ ID No. 8). Compared with wild-type WT, the taspdt-21 mutant wheat showed an insertion of base A in the TaSPDT gene on both chromosomes 4A (corresponding to positions 319-320 of SEQ ID No. 1 and positions 433-434 of SEQ ID No. 7), and a deletion of base CA in the TaSPDT gene on chromosome 7AD (corresponding to positions 318-319 of SEQ ID No. 2 and positions 433-434 of SEQ ID No. 7). The TaSPDT gene on chromosome 7DD was deleted at positions 445-446 of SEQ ID No. 8, and at positions 320-322 of SEQ ID No. 3 and positions 528-530 of SEQ ID No. 9. The wild-type wheat variety Fielder was used as a control (WT) to investigate the effects of TaSPDT gene editing on wheat growth and development and phosphorus accumulation in seeds.

[0199] 1. Seed germination rate determination and seedling growth monitoring

[0200] Seeds of wheat plants taspdt-16, taspdt-21, and the wild-type wheat variety Fielder were rinsed three times with deionized water, sterilized in a 55°C water bath for 15 min, and then germinated on moistened gauze at 28°C in the dark. Germination rate was measured on day 5. Germinated seeds were then placed on a net floating with 0.5 mM CaCl2 (pH 5.6) for further cultivation. Root phenotypes were analyzed on day 10 using a RHIZO 2020a Operator root analysis system.

[0201] This study recorded the wheat germination rate on day 5 of TaSPDT gene-edited plants and observed the root morphology of 10-day-old wheat seedlings using a root scanner. Figure 7 (Associated with AF). The study found that TaSPDT knockout had no effect on wheat germination rate, but it affected root development during the seedling stage. Compared with the wild type, TaSPDT knockout resulted in a 14-31% reduction in wheat root length and a slight decrease in the number of lateral roots, although the difference was not significant. At the same time, the root diameter was significantly increased by 16-25%. There was no significant difference in surface area and root volume between TaSPDT-edited wheat and the wild type.

[0202] 2. Effects of TaSPDT gene editing on wheat maturity phenotype

[0203] Phenotypic indicators such as biomass at maturity in TaSPDT gene-edited wheat were investigated through pot experiments. Germinated wheat plants (taspdt-16, taspdt-21, and the wild-type wheat variety Fielder) were transferred to 22.5cm*18.5cm*20.5cm pots, with 5 plants per pot. The cultivation substrate was a 1:1 (v / v) mixture of Pindstrup substrate (Denmark) and vermiculite. After reaching maturity, the aboveground parts were harvested, and morphological characteristics were photographed and recorded. Plant height was measured using ImagJ 1.53t software. After counting the number of spikes and grains per spike, the seeds and straw were separated. The straw was placed in an oven at 105℃ for 30 min to kill the enzymes, then dried at 80℃ to constant weight. The seeds were dried at 40℃ for 3 days. The dry weight of straw and seeds per plant was weighed and recorded.

[0204] A study on the effects of TaSPDT gene editing on the phenotype of mature wheat found that TaSPDT gene editing affected wheat plant height (…). Figure 8 The effects on (A and B) and ear grain number were not significant, but compared to the wild type, straw dry weight decreased by about 30%, seed dry weight decreased by 33-50%, and ear number decreased by about 35%. Figure 8 (CF).

[0205] 3. Determination of total phosphorus content in each part

[0206] Total phosphorus was determined using the molybdenum-antimony colorimetric method. The above-ground parts of wheat plants (TASPDT-16, TASPDT-21, and the wild-type wheat variety Fielder) were divided into six parts: seeds, glumes, rachis and pedicels, flag leaf, node I, and other parts. Approximately 0.05 g of each part was ground into powder and placed in a digestion tube. 1 mL of H₂O was added, followed by 5 mL of concentrated H₂SO₄, and the mixture was gently shaken and allowed to stand for at least 8 hours. A small funnel was placed at the mouth of the digestion tube, and the tube was digested on an electric stove at 280℃ for 30 minutes. When a large amount of white smoke was observed in the digestion tube, it was removed, and approximately 10 drops of H₂O₂ were added and shaken well. The mixture was heated to a gentle boil for 5-10 minutes and then removed. After slightly cooling, 5-10 drops of H₂O₂ were added again, and the digestion process continued. This process was repeated 2-3 times, with the amount of H₂O₂ added decreasing each time. After digesting until the solution is colorless or clear, heat for another 5-10 minutes to remove any remaining H2O2. Turn off the furnace and allow it to cool naturally for 30 minutes. Remove the furnace and continue cooling. Rinse the funnel with a small amount of water, allowing the washings to flow into the digestion tube. Add water to bring the volume to 100 mL, shake well, and cool to room temperature for phosphorus determination. Pipette 4 mL of the digestion solution into a 50 mL volumetric flask (adjust the amount of reaction solution according to the sample concentration), and add water to approximately 30 mL. Add 2 drops of dinitrophenol indicator, and add 4N NaOH solution until the solution turns yellow. Add a few drops of 2N H2SO4 until the yellow color just fades. Then add 5 mL of molybdenum antimony reagent and add water to bring the volume to 50 mL. Shake well, and after 30 minutes, perform colorimetric determination using a microplate reader at 820 nm.

[0207] 4. Determination of phytic acid content in seeds

[0208] Phytic acid content in seeds was determined spectrophotometrically. 30 mg of whole wheat TASPDT-16, TASPDT-21, and wild-type wheat Fielder seed powder were placed in a 1.5 mL centrifuge tube, 1 mL of 0.4 mol / L HCl and 15% TCA extraction solution were added, and the mixture was shaken at room temperature for 3 h. Then, it was centrifuged at 2000 × g for 10 min. 50 μL of the supernatant was then placed in a 1.5 mL centrifuge tube (containing 550 μL of 36.3 mmol / L NaOH), and 200 μL of colorimetric reagent (containing 0.03% ferric chloride and 0.3% sulfosalicylic acid) was added. After the reaction, 200 μL of the solution was taken, and the phytic acid content was determined using a microplate reader at 500 nm.

[0209] By detecting the levels of total phosphorus in different parts of transgenic plants and phytic acid in seeds, it was found that ( Figure 9In TaSPDT-edited wheat (AC): the dry weight of the portion below node I did not change significantly. Compared with wild-type plants, phosphorus concentration increased by about 30%, and phosphorus distribution accounted for about 40% of the total phosphorus content of the wheat plant, which was 60% higher than the same part in the wild type. Compared with the wild type, although the dry weight of node I, rachis, peduncle, and glumes of TaSPDT-edited wheat decreased overall, the total phosphorus concentration and distribution did not change significantly. Compared with the wild type, the total phosphorus concentration in TaSPDT-edited wheat seeds decreased by more than 12%, and the proportion decreased by 22-36%, and the phytic acid concentration also decreased significantly. Figure 9 (D). This indicates that after TaSPDT gene editing, the level of phosphorus transported from the plant to the seed through node I was reduced, suggesting that TaSPDT may be involved in phosphorus allocation to the seed.

[0210] 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 use of a substance that regulates a protein-coding gene or reduces the content of said protein in any of the following: Application of U1 in regulating wheat growth and development performance and seed phosphorus accumulation; U2) is used in the preparation of products that regulate wheat growth and development performance and seed phosphorus accumulation. Application of U3 in the cultivation of wheat with altered growth and development performance and seed phosphorus accumulation; U4) Application in the preparation of products for wheat with altered growth and development performance and seed phosphorus accumulation; U5) is used in wheat breeding, the purpose of which is to select wheat varieties with reduced seed phosphorus concentration, phosphorus distribution ratio and phytic acid concentration. The amino acid sequence of the protein is shown in SEQ ID No. 4 and SEQ ID No. 5; The regulation or alteration of growth and development performance is to reduce wheat root length and straw dry weight, and increase root diameter; The regulation or alteration of seed phosphorus accumulation involves increasing the phosphorus concentration and phosphorus distribution ratio in wheat below node I, and decreasing the phosphorus concentration, phosphorus distribution ratio, and phytic acid concentration in wheat seeds. The substance is any one of the following: C1) Nucleic acid molecules that inhibit, reduce, or silence the expression of the genes encoding the proteins described above; C2) expresses the gene encoding the nucleic acid molecule described in C1); C3) contains an expression cassette containing the gene encoding described in C2); C4) A recombinant vector containing the encoding gene described in C2), or a recombinant vector containing the expression cassette described in C3); C5) A recombinant microorganism containing the encoding gene described in C2), or a recombinant microorganism containing the expression cassette described in C3), or a recombinant microorganism containing the recombinant vector described in C4).

2. A method for regulating wheat growth and seed phosphorus accumulation, characterized in that, This includes reducing the content of the protein described in claim 1 in the target plant, or reducing the expression level of the gene encoding the protein described in claim 1, to regulate wheat growth and development performance and seed phosphorus accumulation. The regulation of growth and development performance is to reduce wheat root length and straw dry weight, and increase root diameter; The regulation of seed phosphorus accumulation involves increasing the phosphorus concentration and phosphorus distribution ratio in wheat below node I, and decreasing the phosphorus concentration, phosphorus distribution ratio, and phytic acid concentration in wheat seeds.

3. The method according to claim 2, characterized in that, The method includes introducing a substance that inhibits the expression of the gene encoding the protein into recipient wheat, thereby reducing wheat root length and straw dry weight, and increasing root diameter; increasing phosphorus concentration and phosphorus distribution ratio in the wheat below node I, and reducing phosphorus concentration, phosphorus distribution ratio, and phytic acid concentration in wheat seeds. The encoding gene encodes the protein described in claim 1; the substance is the substance described in claim 1.

4. Methods for cultivating plants that alter growth, development, and seed phosphorus accumulation, including: Inhibiting or reducing the expression level of the gene encoding the protein described in claim 1 in the target plant, or inhibiting or reducing the content of the protein described in claim 1, results in a plant with altered growth, development, and seed phosphorus accumulation. The changes in growth and development performance and seed phosphorus accumulation are as follows: 1) Wheat roots are longer, straw dry weight is reduced, and root diameter is increased; 2) Phosphorus concentration and phosphorus distribution ratio increased in wheat below node I, while phosphorus concentration, phosphorus distribution ratio and phytic acid concentration decreased in wheat seeds.

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