Use of gal-5b protein and coding gene therefor in regulating accumulation of raffinose in wheat grains

Abstract

The present invention provides a use of a GAL-5B protein and a coding gene therefor in plant breeding, wherein the loss of function of the GAL-5B protein can significantly promote the accumulation of raffinose in plant seeds, while simultaneously increasing the protein content and reducing the starch content in the seeds; moreover, overexpression of GAL-5B reduces the raffinose content in the plant seeds.

Description

Application of GAL-5B protein and its coding gene in regulating accumulation of raffinose in wheat grains

[0001] Cross-reference to Related Applications

[0002] This application claims priority to Chinese Patent Application No. 202411157999.0, filed on August 22, 2024, the entire contents of which are hereby incorporated by reference. TECHNICAL FIELD

[0003] The present application relates to the field of biotechnology, in particular to the application of GAL-5B protein and its coding gene in regulating accumulation of raffinose in wheat grains. BACKGROUND

[0004] Raffinose family oligosaccharides (RFOs) are a class of oligosaccharides with different numbers of galactosyl groups attached to the glucose residue end of sucrose. Previous studies have shown that RFOs are widely involved in plant responses to abiotic stress and also accumulate during grain maturation. When ingested by humans, RFOs cannot be digested and absorbed due to their special glycosidic bonds, but are instead absorbed and utilized by intestinal probiotics, which increases the biomass of intestinal probiotics and thus reduces the health risks associated with obesity by producing short-chain fatty acids or promoting bile acid metabolism.

[0005] Studies have found that RFOs synthesized and accumulated in monocotyledonous plants are almost raffinose, while in dicotyledonous plants, stachyose and verbascose with higher polymerization degree are mainly synthesized and accumulated. In catabolism, RFOs can be hydrolyzed by β-fructofuranosidase (β-FFase) to hydrolyze the fructose at one end, or by α-galactosidase (α-GAL) to sequentially hydrolyze the multiple galactosyl groups at the glucose residue end, ultimately becoming monosaccharides and sucrose to enter other metabolic pathways.

[0006] Humans lack endogenous digestive enzymes to break down α-(1,6) glycosidic bonds, so raffinose family oligosaccharides in food cannot be directly digested and absorbed. However, human gut probiotics can absorb and utilize these oligosaccharides. Raffinose family oligosaccharides with this characteristic belong to the nondigestible oligosaccharide (NDO) dietary fiber class and are considered a type of prebiotic with important functions. Related studies have shown that after humans ingest a certain amount of RFOs, the biomass of Bifidobacteria in the gut microbiota that can efficiently utilize this substrate significantly increases. Adding RFOs or galacttooligosaccharides (GOS) to the diet of mice fed a high-fat diet can significantly improve serum total cholesterol, low-density lipoprotein cholesterol, lipopolysaccharide, and serum total bile acid levels, reduce steatosis in liver tissue, and lower obesity-related health risks.

[0007] Invention Overview

[0008] The technical problem to be solved by this invention is how to regulate raffinose accumulation in wheat grains. The technical problem to be solved is not limited to the described technical subject matter; other technical subjects not mentioned herein will be clearly understood by those skilled in the art through the following description.

[0009] To address the aforementioned technical problems, the present invention first provides a novel use for GAL-5B protein or substances that regulate the content and / or activity of said GAL-5B protein.

[0010] This invention provides the use of GAL-5B protein or substances regulating the content and / or activity of said GAL-5B protein in any of the following A1)-A5):

[0011] A1) Regulates the raffinose content in plant seeds;

[0012] A2) Regulates the protein content in plant seeds;

[0013] A3) Regulates the starch content in plant seeds;

[0014] A4) Cultivate plants with altered raffinose and / or protein and / or starch content in their grains;

[0015] A5) Plant breeding or plant variety improvement;

[0016] The GAL-5B protein is any one of the following B1)-B4):

[0017] B1) The amino acid sequence is that of the protein SEQ ID NO: 2;

[0018] B2) a fusion protein having the same function as the amino acid sequence of B1) obtained by connecting a tag at the N-terminus and / or C-terminus of the amino acid sequence of B1);

[0019] B3) a protein having the same function as the amino acid sequence of B1) obtained by substitution and / or deletion and / or addition of one or several amino acid residues of the amino acid sequence of B1);

[0020] B4) a protein having 80% or more identity to the amino acid sequence of B1) and having the same function.

[0021] In the protein of B2) above, the tag refers to a polypeptide or protein that is expressed in fusion with the protein of interest by using DNA in vitro recombination technology, so as to facilitate the expression, detection, tracing and / or purification of the protein of interest. The tag includes but is not limited to: GST (glutathione S-transferase) tag protein, His6 tag protein (His-tag), MBP (maltose binding protein) tag protein, Flag tag protein, SUMO tag protein, HA tag protein, Myc tag protein, eGFP (enhanced green fluorescent protein), eCFP (enhanced cyan fluorescent protein), eYFP (enhanced yellow green fluorescent protein), mCherry (monomeric red fluorescent protein) or AviTag tag protein.

[0022] In the protein of B3) above, the substitution and / or deletion and / or addition of one or several amino acid residues is substitution and / or deletion and / or addition of no more than 10 amino acid residues or no more than 9 amino acid residues or no more than 8 amino acid residues or no more than 7 amino acid residues or no more than 6 amino acid residues or no more than 5 amino acid residues or no more than 4 amino acid residues or no more than 3 amino acid residues or no more than 2 amino acid residues or no more than 1 amino acid residue.

[0023] The identity in the above-mentioned B4) refers to the identity of the amino acid sequence. The identity of the amino acid sequence can be determined using a homology search site on the Internet, such as the BLAST page of the NCBI home page website. For example, the identity (%) can be obtained by calculating the identity of a pair of amino acid sequences using blastp as the program, setting the Expect value to 10, setting all Filters to OFF, using BLOSUM62 as the Matrix, setting Gap existence cost, Per residue gap cost and Lambda ratio to 11, 1 and 0.85 (default values), respectively, and performing a search in the advanced BLAST 2.1. The identity includes an amino acid sequence having 80% or more, or having 81% or more, or having 82% or more, or having 83% or more, or having 84% or more, or having 85% or more, or having 86% or more, or having 87% or more, or having 88% or more, or having 89% or more, or having 90% or more, or 91% or more, or 92% or more, or 93% or more, or 94% or more, or 95% or more, or 96% or more, or 97% or more, or 98% or more, or 99% or more identity to the amino acid sequence shown in SEQ ID NO: 2 of the present application.

[0024] The above-mentioned proteins of B1) to B4) can be artificially synthesized, or the encoding genes thereof can be synthesized first and then expressed biologically.

[0025] In the above-mentioned applications, the substance for regulating the content and / or activity of GAL-5B protein includes a substance for increasing the content and / or activity of GAL-5B protein or a substance for decreasing the content and / or activity of GAL-5B protein.

[0026] Further, the substance for increasing the activity of GAL-5B protein can be a protein, a polypeptide or a small molecule compound that enhances or promotes the function of GAL-5B protein.

[0027] The substance for increasing the content of GAL-5B protein can be a substance that promotes the synthesis of GAL-5B protein or inhibits the degradation of GAL-5B protein or overexpresses GAL-5B protein.

[0028] The substance for decreasing the activity of GAL-5B protein can be a protein, a polypeptide or a small molecule compound that inhibits the function of GAL-5B protein.

[0029] The substance for decreasing the content of GAL-5B protein can be a substance that inhibits the synthesis of GAL-5B protein or promotes the degradation of GAL-5B protein or knocks down (knocks down) or knocks out the GAL-5B protein encoding gene.

[0030] Further, the substance for knocking down (knocking out) the GAL-5B protein-encoding gene can be any nucleic acid molecule capable of inhibiting or interfering with the expression of the GAL-5B protein-encoding gene, such as gRNA (e.g., sgRNA), mRNA, siRNA, dsRNA, shRNA, miRNA, antisense RNA, etc.

[0031] The substance for knocking out the GAL-5B protein-encoding gene can be any substance that achieves that the host cell does not produce a functional protein product of the GAL-5B gene in any way, such as removing all or part of the coding gene sequence, introducing mutations so that no functional protein is produced, removing or changing regulatory components (e.g., promoter editing) so that the coding gene sequence is not transcribed, preventing translation by binding to mRNA, etc. Generally, the knockout is carried out at the genomic DNA level, so that the offspring of the cell also permanently carry the knockout.

[0032] Further, the substance for knocking out the GAL-5B protein-encoding gene can be a substance that mutates the GAL-5B gene in the plant (the mutated form can be a deletion mutation and / or an insertion mutation and / or a base substitution) so as to lose activity, and the mutation can be achieved in any way known in the art, such as zinc finger protein ZFN gene editing system, TALENs gene editing system, CRISPR / Cas9 gene editing system, T-DNA insertion, etc.

[0033] In some embodiments, the substance for knocking out the GAL-5B protein-encoding gene is a substance that mutates a mutation site in the wheat genome from base G to base A. The mutation site is at position 830 of SEQ ID NO: 1 or position 2938 of SEQ ID NO: 9.

[0034] In some specific embodiments, the substance that mutates a mutation site in the wheat genome from base G to base A is a cytosine base editor (CBE) that can achieve C·G to T·A conversion. The cytosine base editor includes necessary elements that can achieve C·G to T·A conversion, such as sgRNA, Cas9 protein, and cytosine deaminase.

[0035] To solve the above problems, the present application further provides a new use of biological materials related to GAL-5B protein.

[0036] The present application provides the use of biological materials related to GAL-5B protein in any one of the following A1)-A5):

[0037] A1) regulating the content of raffinose in plant seeds;

[0038] A2) modulating the protein content in the grain of a plant;

[0039] A3) modulating the starch content in the grain of a plant;

[0040] A4) breeding plants with altered content of raffinose and / or protein and / or starch in the grain;

[0041] A5) breeding or improving a plant variety;

[0042] The biological material is any one of the following E1) to E5):

[0043] E1) a nucleic acid molecule encoding the GAL-5B protein described above;

[0044] E2) a nucleic acid molecule that inhibits or interferes with the expression of the GAL-5B protein-encoding gene or that knocks out or knocks down the GAL-5B protein-encoding gene;

[0045] E3) an expression cassette containing the nucleic acid molecule of E1) or E2);

[0046] E4) a recombinant vector containing the nucleic acid molecule of E1) or E2);

[0047] E5) a recombinant microorganism containing the nucleic acid molecule of E1) or E2).

[0048] In the above applications, the nucleic acid molecule of E1) can be a DNA, such as a cDNA, a genomic DNA or a recombinant DNA.

[0049] In some embodiments, the nucleic acid molecule of E1) is any one of the following:

[0050] F1) a DNA molecule whose nucleotide sequence is SEQ ID NO: 1 or SEQ ID NO: 9;

[0051] F2) a DNA molecule having 75% or more identity with the nucleotide sequence of F1) and encoding the GAL-5B protein described above.

[0052] The nucleotide sequence encoding the GAL-5B protein of the present application can be easily mutated by those of ordinary skill in the art using known methods, such as directed evolution and point mutation. Those nucleotides that have been artificially modified and have 75% or more identity to the GAL-5B nucleotide sequence isolated from the present application, as long as they encode the GAL-5B protein and have the same function, are derived from the nucleotide sequence of the present application and are equivalent to the sequence of the present application. The identity refers to the sequence similarity to the natural nucleic acid sequence, including the nucleotide sequence having 75% or more, or 80% or more, or 85% or more, or 90% or more, or 95% or more identity to the nucleotide sequence encoding the protein consisting of the amino acid sequence shown in SEQ ID NO: 2 of the present application. The identity can be evaluated by naked eyes or computer software. Using computer software, the identity between two or more sequences can be expressed in percentage (%), which can be used to evaluate the identity between related sequences.

[0053] E2) The nucleic acid molecule can be a gRNA (such as sgRNA), mRNA, siRNA, dsRNA, shRNA, miRNA or antisense RNA.

[0054] Any of the above expression cassettes can include a promoter, the nucleic acid molecule of E1) or E2) above, and a terminator. Promoters useful in the present application include, but are not limited to, constitutive promoters, tissue-, organ-, and development-specific promoters, and inducible promoters. Further, the expression cassette can also include an enhancer sequence. Promoters useful in the present application include, but are not limited to, constitutive promoters; tissue-, organ-, and development-specific promoters, and inducible promoters. Suitable transcription terminators include, but are not limited to, the Agrobacterium nopaline synthase terminator (NOS terminator), the CaMV 35S terminator, the tml terminator, the pea rbcS E9 terminator, and the nopaline synthase and octopine synthase terminators.

[0055] Any of the above vectors refers to a vector that can carry the nucleic acid molecule of E1) or E2) above into a host cell for amplification and expression. The vector can be a cloning vector or an expression vector, including but not limited to, plasmids, bacteriophages (such as lambda phage or M13 filamentous phage, etc.), cosmids (i.e., cosmids), Ti plasmids, viral vectors (such as retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, etc.).

[0056] The recombinant vector of any of the above is a recombinant DNA molecule constructed by in vitro ligation of the nucleic acid molecule of E1) or E2) above with the vector. The recombinant vector containing the nucleic acid molecule of E1) or E2) above can be constructed using an existing plant expression vector. The plant expression vector includes a binary Agrobacterium vector and a vector useful for plant microprojectile bombardment, etc. Examples of the plant expression vector include pAHC25, pBin438, pCAMBIA1302, pCAMBIA2300, pCAMBIA2301, pCAMBIA1301, pCAMBIA1300, pBI121, pCAMBIA1391-Xa, or pCAMBIA1391-Xb (CAMBIA), etc. The plant expression vector can further comprise a 3' untranslated region of a foreign gene, i.e., a DNA fragment comprising a polyadenylation signal and any other DNA fragment involved in mRNA processing or gene expression. The polyadenylation signal can direct the addition of polyadenosine to the 3' end of the mRNA precursor, and the untranslated region of the 3' end of the transcription of the Agrobacterium Ti plasmid gene (e.g., the nopaline synthase gene Nos), the plant gene (e.g., the soybean storage protein gene), etc. have similar functions. When the recombinant vector is constructed using the gene of the present application, an enhancer, including a translational enhancer or a transcriptional enhancer, can also be used. The enhancer region can be the ATG initiation codon or the adjacent region of the initiation codon, but must be in the same reading frame as the coding sequence to ensure correct translation of the entire sequence. The source of the translational control signal and the initiation codon is broad, and can be natural or synthetic. The translational initiation region can be derived from a transcriptional initiation region or a structural gene. In order to facilitate the identification and selection of transgenic plant cells or plants, the plant expression vector can be processed, such as by adding a gene that can be expressed in plants to produce a color change or a luminescent compound (GUS gene, luciferase gene, etc.), a marker gene for an antibiotic (e.g., the nptll gene conferring resistance to kanamycin and related antibiotics, the bar gene conferring resistance to the herbicide phosphinothricin, the hph gene conferring resistance to the antibiotic hygromycin, the dhfr gene conferring resistance to methotrexate, and the EPSPS gene conferring resistance to glyphosate), or a marker gene for a chemical agent (e.g., a herbicide resistance gene), a mannose-6-phosphate isomerase gene providing the ability to metabolize mannose. For safety considerations regarding transgenic plants, no selective marker gene can be added, and the transformed plants can be directly screened under stress conditions.

[0057] The microorganism can be a bacterium, a fungus, an actinomycete, an alga, or a virus. The bacterium can be from the genus Escherichia sp., Erwinia sp., Agrobacterium sp., Flavobacterium sp., Alcaligenes sp., Pseudomonas sp., Bacillus sp., etc., but is not limited thereto, and can be Escherichia coli, Bacillus subtilis, or Bacillus pumilus. The fungus can be a yeast from the genus Saccharomyces sp. (e.g., Saccharomyces cerevisiae), Kluyveromyces sp. (e.g., Kluyveromyces lactis), Pichia sp. (e.g., Pichia pastoris), Schizosaccharomyces sp. (e.g., Schizosaccharomyces pombe), Hansenula sp. (e.g., Hansenula polymorpha), etc., but is not limited thereto. The fungus can also be from the genus Fusarium sp., Rhizoctonia sp., Verticillium sp., Penicillium sp., Aspergillus sp., Cephalosporium sp., etc., but is not limited thereto. The actinomycete can be from the genus Streptomyces sp., Nocardia sp., Micromonospora sp., Streptosporangium sp., Actinoplanes sp., Thermoactinomyces sp., etc., but is not limited thereto. The alga can be from the genus Fucus sp., Achnanthes sp., Amphiprora sp., Amphora sp., Ankistrodesmus sp., Asteromonas sp., Boekelovia sp., etc., but is not limited thereto. The virus can be a rotavirus, a herpes virus, an influenza virus, an adenovirus, etc., but is not limited thereto.

[0058] The recombinant microorganism as described above refers to a recombinant microorganism obtained by operating and modifying the gene of the microorganism of interest. For example, the recombinant microorganism obtained after introducing the recombinant vector as described above into the microorganism of interest. The recombinant microorganism can be understood as not only referring to a specific recombinant microorganism, but also referring to the offspring of such cells, and due to natural, accidental or intentional mutations and / or changes, the offspring can not necessarily be completely consistent with the original parent cell, but is still included in the scope of the recombinant microorganism.

[0059] In any of the above applications, the regulation of the raffinose content in the plant seed is to increase the raffinose content in the plant seed or to decrease the raffinose content in the plant seed. The regulation mode is negative regulation, that is, when the GAL-5B protein content and / or activity in the plant is increased, the raffinose content in the plant seed is decreased, and when the GAL-5B protein content and / or activity in the plant is decreased or deleted, the raffinose content in the plant seed is increased.

[0060] In some embodiments, when the expression amount of the GAL-5B gene in the plant is increased, the raffinose content in the plant seed is decreased.

[0061] In some embodiments, when the GAL-5B gene mutation in the plant results in the loss of function of the GAL protein, the raffinose content in the plant seed is increased.

[0062] In any of the above applications, the regulation of the protein content in the plant seed is to increase the protein content in the plant seed, which is specifically embodied in that when the GAL-5B protein content and / or activity in the plant is decreased or deleted, the protein content in the plant seed is increased.

[0063] In some embodiments, when the GAL-5B gene mutation in the plant results in the loss of function of the GAL-5B protein, the protein content in the plant seed is increased.

[0064] In any of the above applications, the regulation of the starch content in the plant seed is to decrease the starch content in the plant seed, which is specifically embodied in that when the GAL-5B protein content and / or activity in the plant is decreased or deleted, the starch content in the plant seed is decreased.

[0065] In some embodiments, when the GAL-5B gene mutation in the plant results in the loss of function of the GAL-5B protein, the starch content in the plant seed is decreased.

[0066] In any of the above applications, the regulation of the starch content in the plant seed is to decrease the starch content in the plant seed, which is specifically embodied in that when the GAL-5B protein content and / or activity in the plant is decreased or deleted, the starch content in the plant seed is decreased.

[0067] The breeding of the plant or the improvement of the plant variety aims to breed a plant variety with high raffinose content and / or high protein content and / or low starch content.

[0068] To solve the above technical problems, the present application further provides a method for increasing the raffinose content and / or the protein content or decreasing the starch content in the grain of a plant.

[0069] The method for increasing the raffinose content and / or the protein content or decreasing the starch content in the grain of a plant provided by the present application is method one or method two.

[0070] The method one comprises the following steps: reducing the content and / or activity of the GAL-5B protein in the target plant, so as to increase the raffinose content and / or the protein content or decrease the starch content in the grain of the target plant.

[0071] The method two comprises the following steps: replacing the base G at the 830th position of the GAL-5B gene in the genome of the target plant with a base A, so as to increase the raffinose content and / or the protein content or decrease the starch content in the grain of the plant.

[0072] To solve the above technical problems, the present application further provides a method for breeding a transgenic plant with increased raffinose content and / or increased protein content and / or decreased starch content in the grain.

[0073] The method for breeding a transgenic plant with increased raffinose content and / or increased protein content and / or decreased starch content in the grain provided by the present application is method three or method four.

[0074] The method three comprises the following steps: reducing the content and / or activity of the GAL-5B protein in the target plant, so as to obtain a transgenic plant; the raffinose content and / or the protein content in the grain of the transgenic plant is higher than that of the target plant, and the starch content is lower than that of the target plant.

[0075] The method four comprises the following steps: replacing the base G at the 830th position of the GAL-5B gene in the genome of the target plant with a base A, so as to obtain a transgenic plant; the raffinose content and / or the protein content in the grain of the transgenic plant is higher than that of the target plant, and the starch content is lower than that of the target plant.

[0076] The method four comprises the following steps: replacing the base G at the 830th position of the GAL-5B gene in the genome of the target plant with a base A, so as to obtain a transgenic plant; the raffinose content and / or the protein content in the grain of the transgenic plant is higher than that of the target plant, and the starch content is lower than that of the target plant.

[0077] In any of the above-mentioned methods, the plant of interest contains a GAL-5B protein or a gene (genomic gene and / or cDNA gene) encoding the GAL-5B protein.

[0078] In any of the above-mentioned methods, the GAL-5B gene encodes the above-mentioned GAL-5B protein.

[0079] In any of the above-mentioned methods, the method for reducing the content and / or activity of the above-mentioned GAL-5B protein in the plant of interest can be knocking out the gene encoding the GAL-5B protein in the plant of interest.

[0080] The method for knocking out the gene encoding the GAL-5B protein in the plant of interest can be replacing the base G at position 830 of the GAL-5B gene in the genome of the plant of interest with a base A.

[0081] In any of the above-mentioned methods for replacing the base G at position 830 of the GAL-5B gene in the genome of the plant of interest with a base A, the method can be introducing a substance that replaces the base G at position 830 of the GAL-5B gene in the genome of the plant of interest with a base A into the plant of interest.

[0082] In some embodiments, the substance that replaces the base G at position 830 of the GAL-5B gene in the genome of the plant of interest with a base A can be a cytosine base editor that replaces the base G at position 830 of the GAL-5B gene in the genome of the plant of interest with a base A.

[0083] In any of the above-mentioned methods, the position 830 of the GAL-5B gene refers to position 830 in the cDNA sequence of the GAL-5B gene (SEQ ID NO: 1), which corresponds to position 2938 in the genomic sequence of the GAL-5B gene (SEQ ID NO: 9).

[0084] In any of the above-mentioned methods, the replacement is a homozygous replacement, i.e., the same replacement occurs in homologous chromosomes.

[0085] To solve the above technical problem, the present application further provides a method for reducing the content of raffinose in plant seeds.

[0086] The method for reducing the content of raffinose in plant seeds provided by the present application comprises the following steps: increasing the content and / or activity of the protein in the plant of interest as claimed in claim 1, thereby reducing the content of raffinose in the seeds of the plant of interest.

[0087] To solve the above technical problem, the present application finally provides a method for cultivating a transgenic plant with reduced raffinose content in seeds.

[0088] The method for cultivating the transgenic plant with reduced content of raffinose in the seed can be a method for producing a transgenic plant with reduced content of raffinose in the seed.

[0089] The method for cultivating the transgenic plant with reduced content of raffinose in the seed provided by the present application comprises the following steps: increasing the content and / or activity of the GAL-5B protein in the target plant to obtain a transgenic plant; and the content of raffinose in the seed of the transgenic plant is lower than that of the target plant.

[0090] Further, the method for increasing the content and / or activity of the GAL-5B protein in the target plant is overexpression of the GAL-5B protein in the target plant.

[0091] Further, the method for overexpression is introducing the coding gene of the GAL-5B protein into the target plant.

[0092] The nucleotide sequence of the coding gene of the GAL-5B protein is shown in SEQ ID NO: 1.

[0093] In some embodiments, the coding gene of the GAL-5B protein is introduced into the target plant by the recombinant vector pGWB18-Ubi::GAL-5B.

[0094] In any of the above-mentioned applications or methods, the transgenic plant not only includes the first generation transgenic plant obtained by transforming the target plant with the substance for overexpression or inhibition or knock-out of the GAL-5B gene, but also includes the offspring thereof. For the transgenic plant, the gene can be propagated in the species, or the gene can be transferred into other varieties of the same species by conventional breeding techniques, especially including commercial varieties. The transgenic plant includes seeds, callus, whole plants and cells.

[0095] In any of the above-mentioned applications or methods, the plant is a monocotyledon.

[0096] Further, the monocotyledon is a plant of the family Poaceae.

[0097] Further, the plant of the family Poaceae is a plant of the genus Triticum.

[0098] Further, the plant of the genus Triticum is wheat.

[0099] In some embodiments, the wheat is wild-type wheat Kronos.

[0100] The transgenic plant produced according to any of the above-mentioned methods also falls within the protection scope of the present application.

[0101] The application finds that the functional loss of GAL-5B protein can significantly promote the accumulation of raffinose in wheat kernels, increase the protein content in the kernels and reduce the starch content in the kernels; and overexpression of GAL-5B reduces the raffinose content in the kernels. The application first finds that the GAL-5B protein and related biological materials can be used to regulate the raffinose content, protein content and starch content in plant kernels, and will play an important role in the cultivation of functional wheat varieties. BRIEF DESCRIPTION OF DRAWINGS

[0102] Figure 1 is the identification of gal-5b mutant materials. A is the sequencing results of the target gene in wild-type wheat Kronos and gal-5b mutant, the sequence before mutation is TTTCAGCATCTGGGCACTT (SEQ ID NO: 5), and the sequence after mutation is TTTCAGCATCTAGGCACTT (SEQ ID NO: 6). B is the kernel phenotype of wild-type wheat Kronos, gal-5b mutant and transgenic GAL-5B wheat.

[0103] Figure 2 is the statistics of the agronomic traits of wild-type wheat Kronos, gal-5b mutant and transgenic GAL-5B wheat. A is the thousand-grain weight of wild-type wheat Kronos, gal-5b mutant and transgenic GAL-5B wheat. B is the grain number per plant of wild-type wheat Kronos, gal-5b mutant and transgenic GAL-5B wheat.

[0104] Figure 3 is the statistics of the quality traits of wild-type wheat Kronos, gal-5b mutant and transgenic GAL-5B wheat kernels. A is the protein content in the kernels of wild-type wheat Kronos, gal-5b mutant and transgenic GAL-5B wheat. B is the starch content in the kernels of wild-type wheat Kronos, gal-5b mutant and transgenic GAL-5B wheat. C is the raffinose content in the kernels of wild-type wheat Kronos, gal-5b mutant and transgenic GAL-5B wheat.

[0105] Figure 4 is a sample chromatogram. The red peak is the target peak. The vertical coordinate is the normalized intensity, and the horizontal coordinate is the minute. EMBODIMENTS OF THE INVENTION

[0106] The application will be further described in detail below in conjunction with the specific embodiments, and the examples given are only for illustrating the application, but not for limiting the scope of the application. The examples provided below can serve as a guide for further improvement by those skilled in the art, and do not constitute any limitation on the application in any way.

[0107] The experimental methods in the following examples are all conventional methods, and are carried out according to the techniques or conditions described in the literature in the art or according to the product instructions, unless otherwise specified. The materials, reagents, etc. used in the following examples can be obtained commercially, unless otherwise specified.

[0108] The wild-type wheat Kronos in the following examples is described in the literature "Krasileva, K. V., et al. (2017). Uncovering hidden variation in polyploid wheat. Proceedings of the National Academy of Sciences 114(6): E913-E921."

[0109] The molecular formula of raffinose in the following examples is C 18 H 34 O 17 , CAS No. 512-69-6, and the structural formula is shown as Formula I.

[0110] Formula I.

[0111] The CDS sequence of the GAL-5B gene in the following examples is shown as SEQ ID NO: 1, the amino acid sequence of the GAL-5B protein encoded thereby is shown as SEQ ID NO: 2, and the genomic sequence of the GAL-5B gene is shown as SEQ ID NO: 9.

[0112] Example 1, obtaining and preparation of GAL-5B related genetic materials

[0113] I. gal-5b mutants and sequence analysis thereof

[0114] 1. Source and mutation information of gal-5b mutants

[0115] The seeds of the gal-5b mutant can be obtained by ordering from the British SeedStor or the laboratory of Dubcovsky in the United States, and the mutant number is Kronos3147. The relevant mutation information of the gal-5b mutant can be queried through the following website: http: / / plants.ensembl.org / Triticum_aestivum / Variation / Explore?db=core;r=5B:11515909-11516909;v=Kronos3147.chr5B.11516409;vdb=variation;vf=57902380.

[0116] 2. Sequencing of the gal-5b mutant

[0117] The genomic DNA of wild-type wheat Kronos and gal-5b mutant (homozygous) was extracted, PCR amplification was performed using GAL-5B gene specific primers, and the PCR product was sequenced.

[0118] The sequencing results are shown in Figure 1. By sequencing the GAL-5B gene in wild-type wheat Kronos and gal-5b mutant material, it was found that compared with wild-type wheat Kronos, the gal-5b mutant had a base mutation in the gene sequence (SEQ ID NO: 1) encoding the GAL-5B protein on wheat chromosome 5B, which was a mutation from base G to base A at position 830 of SEQ ID NO: 1 (corresponding to position 2938 of SEQ ID NO: 9), which resulted in a mutation from TGG to TAG at positions 829-831 of SEQ ID NO: 1, causing premature termination of protein translation.

[0119] II. Preparation of GAL-5B wheat

[0120] 1. Construction of recombinant vector pGWB18-Ubi::GAL-5B

[0121] The GAL-5B gene sequence shown in SEQ ID NO: 1 was connected to the pDONR207 vector (invitrogen, CAT#: 12535035) using the Gateway method to obtain the recombinant vector pDONR207-GAL-5B, which was sequenced and verified. Sequencing showed that the GAL-5B gene in the recombinant vector pDONR207-GAL-5B did not have any mutations.

[0122] The recombinant vector pDONR207-GAL-5B, the pGWB18 vector (NCBI: txid419562, the nucleotide sequence of the pGWB18 vector consists of the DNA molecule shown in SEQ ID NO: 3 and the DNA molecule shown in SEQ ID NO: 4 in order) and the recombinase (ThermoFisher Scientific, Cat. No. 11789100) were mixed and reacted at 22°C overnight to recombine the GAL-5B gene shown in SEQ ID NO: 1 between the attR1 site (CAAGTTTGTACAAAAAA, SEQ ID NO: 7) and the attR2 site (TTTCTTGTACAAAGTGG, SEQ ID NO: 8) of the pGWB18 vector to obtain the recombinant vector pGWB18-Ubi::GAL-5B. The recombinant vector pGWB18-Ubi::GAL-5B expresses the GAL-5B protein.

[0123] 2. Obtaining of recombinant bacteria

[0124] The recombinant vector pGWB18-Ubi::GAL-5B was transformed into Agrobacterium EHA105 (Eubio, CAT#: AC1010), and after identification, the recombinant bacteria pGWB18-Ubi::GAL-5B / EHA105 containing the recombinant vector pGWB18-Ubi::GAL-5B were obtained.

[0125] 3. Obtaining of GAL-5B transgenic wheat

[0126] Wild-type wheat Kronos in good growth state was selected as the receptor, and genetic transformation was performed by the method of embryo stripping. After infection and differentiation using the recombinant bacteria pGWB18-Ubi::GAL-5B / EHA105, screening was performed, and GAL-5B transgenic wheat lines were obtained. The GAL-5B transgenic wheat line with a significantly higher GAL-5B expression level than the wild-type wheat Kronos was recorded as GAL-5B OE.

[0127] Example 2. Analysis of agronomic traits and quality traits of GAL-5B related genetic materials

[0128] I. Analysis of agronomic traits of GAL-5B related genetic materials

[0129] Test materials: wild-type wheat Kronos, gal-5b mutant (homozygous), and GAL-5B transgenic wheat line GAL-5B OE.

[0130] 1. Field planting

[0131] Seeding was performed in the test field of Shangzhuang Experimental Station of China Agricultural University in March 2023 according to the conventional method, with a plant spacing of 5 cm and a row spacing of 20 cm. Regular weeding and fertilization were performed, and single plant harvesting and threshing were performed after the plants were fully mature in June.

[0132] 2. Statistical analysis of agronomic traits

[0133] Automatic seed analysis and non-destructive measurement of thousand seed weight and single plant grain number were performed using a thousand seed weight instrument. The brand of the automatic seed analysis and thousand seed weight instrument was Wanshen, and the model was SC-A.

[0134] The results are shown in Figure 2, which show that the average 1000-grain weight of wild-type wheat Kronos was 36.73±0.75 g, the average 1000-grain weight of the gal-5b mutant was 37.01±0.26 g, and the average 1000-grain weight of the GAL-5B wheat was 35.83±0.85 g, and there was no significant difference among the three. The average number of grains per plant of wild-type wheat Kronos was 164±5 grains, the average number of grains per plant of the gal-5b mutant was 168±7 grains, and the average number of grains per plant of the GAL-5B wheat was 159±3 grains, and there was no significant difference among the three.

[0135] II. Analysis of quality traits of GAL-5B-related genetic materials

[0136] Test materials: wild-type wheat Kronos, gal-5b mutant (homozygous), and GAL-5B wheat line GAL-5B OE.

[0137] 1. Field planting

[0138] Step 1 in Step One.

[0139] 2. Quality trait statistics

[0140] The raffinose content, protein content, and starch content in the grains of the test materials were detected.

[0141] The raffinose content in the grains was detected by HPLC method, and the raffinose content in the grains was calculated according to the following standard curve: y = 1.028e5x + 2.71e7 (R 2 = 0.9945). The instruments used in the HPLC method are as follows: analytical balance; 0.22 μm micro membrane filter; shaking mixer; ball mill; metal bath; vacuum dryer; Waters HPLC-QDa. The specific steps are as follows: the sample is wheat grain, and the experimental amount is 0.1 g. Grind the sample into powder on the ball mill, and weigh 100 mg of the sample. Add pre-frozen 75% methanol water 1.4 mL and vortex. After heating at 70°C for 10 min, centrifuge for 10 min, and transfer the supernatant to a new centrifuge tube. Add 0.75 mL of chloroform and 1.4 mL of high-purity water, vortex, and centrifuge. Transfer 0.8 mL of the supernatant to a 1.5 mL reaction tube and vacuum dry. Then add 0.15 mL of 80% methanol for redissolution, and detect on the instrument. The gradient elution conditions are as follows: chromatographic column: Waters Amide chromatographic column, sample amount: 10 μL; column temperature: 35°C; gradient elution settings (flow rate: 0.6 mL / min) as shown in Table 1; mass spectrometry monitoring ion: m / z = 503.1 (negative ion mode). The sample chromatogram is shown in Figure 4.

[0142] Table 1

[0143] The detection method of the protein content and starch content of the grain is as follows: the harvested wheat grains are placed in a dry environment for lossless placement to October, and the protein and starch content is detected using a multifunctional near-infrared analyzer (Perten, model DA7200). The Simplicity software is used for daily analysis, and the chemical dosage software is used to establish a calibration sample model.

[0144] The results are shown in Figure 3, which show that the average protein content in the wild-type wheat Kronos grain is 13.62 ± 0.23%, the average protein content in the GAL-5B transformed wheat grain is 13.22 ± 0.54%, but the average protein content in the gal-5b mutant grain is 15.88 ± 0.42%. Compared with the wild-type wheat Kronos, the protein content in the gal-5b mutant grain is significantly increased by more than 2%. The average starch content in the wild-type wheat Kronos grain is 76.99 ± 0.17%, the average starch content in the GAL-5B transformed wheat grain is 77.21 ± 0.28%, but the average starch content in the gal-5b mutant grain is 74.67 ± 0.28%. Compared with the wild-type wheat Kronos, the starch content in the gal-5b mutant grain is significantly reduced by nearly 2%. The average raffinose content in the wild-type wheat Kronos grain is 0.3488 ± 0.0012 micrograms / gram, the average raffinose content in the GAL-5B transformed wheat grain is 0.2879 ± 0.00028 micrograms / gram, but the average raffinose content in the gal-5b mutant grain is 1.0474 ± 1655 micrograms / gram. Compared with the wild-type wheat Kronos, the raffinose content in the gal-5b mutant grain is significantly increased by 200%.

[0145] The results of the above yield and nutrient determination show that, on the one hand, interfering with GAL-5B changes the grain sugar metabolic flow, which can significantly reduce the starch content in the wheat grain, reduces the sugar intake of consumers, and is expected to reduce blood sugar and promote health through a mechanism similar to "sugar starvation"; on the other hand, interfering with GAL-5B can significantly increase the raffinose content in the wheat grain, and raffinose as a prebiotic has the potential to increase the abundance of probiotics such as intestinal bifidobacteria, and is expected to achieve the reported phenomenon of enhancing the synthesis of short-chain fatty acids or human bile acid metabolism by intestinal microorganisms, promoting human heat production, regulating blood sugar levels, and reducing the risk of obesity. Industrial applicability

[0146] The application provides application of a GAL-5B protein in regulating contents of raffinose, protein and starch in wheat kernels. Experiments prove that interfering with the function of the GAL-5B gene can significantly promote the accumulation of raffinose in the wheat kernels, increase the protein content in the kernels and reduce the starch content in the kernels; and overexpression of the GAL-5B gene reduces the raffinose content in the wheat kernels. The GAL-5B protein and related biological materials are found to be used for regulating the raffinose content, the protein content and the starch content in the kernels of plants for the first time, and will play an important role in cultivating functional wheat varieties.

Claims

1. Use of a protein or a substance regulating the content and / or activity of the protein in any one of A1) to A5) below: A1) regulating the content of raffinose in the grain of a plant; A2) regulating the content of protein in the grain of a plant; A3) regulating the content of starch in the grain of a plant; A4) breeding a plant with altered content of raffinose and / or protein and / or starch in the grain; A5) plant breeding or plant variety improvement; the protein being any one of B1) to B4) below: B1) a protein with an amino acid sequence of SEQ ID NO: 2; B2) a fusion protein with the same function obtained by linking a tag to the N-terminus and / or C-terminus of the amino acid sequence of B1); B3) a protein with the same function obtained by substitution and / or deletion and / or addition of one or several amino acid residues of the amino acid sequence of B1); B4) a protein with 80% or more identity to the amino acid sequence of B1) and with the same function.

2. Use of a biological material related to the protein of claim 1 in any one of A1) to A5) below: A1) regulating the content of raffinose in the grain of a plant; A2) regulating the content of protein in the grain of a plant; A3) regulating the content of starch in the grain of a plant; A4) breeding a plant with altered content of raffinose and / or protein and / or starch in the grain; A5) plant breeding or plant variety improvement; the biological material being any one of E1) to E5) below: E1) a nucleic acid molecule encoding the protein of claim 1; E2) a nucleic acid molecule inhibiting or interfering with the expression of the gene encoding the protein of claim 1 or a nucleic acid molecule knocking out or knocking down the gene encoding the protein of claim 1; E3) an expression cassette comprising the nucleic acid molecule of E1) or E2); E4) a recombinant vector comprising the nucleic acid molecule of E1) or E2); E5) a recombinant microorganism comprising the nucleic acid molecule of E1) or E2). E1) the nucleic acid molecule being any one of F1) a DNA molecule with a nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 9; F2) a DNA molecule with 75% or more identity to the nucleotide sequence of F1) and encoding the protein. The plant is a monocotyledonous plant. The monocotyledonous plant is a plant of the family Poaceae. The plant of the family Poaceae is a plant of the genus Triticum. The plant of the genus Triticum is wheat.

8. A method for increasing the content of raffinose and / or protein or decreasing the content of starch in the grain of a plant, comprising the step of decreasing the content and / or activity of the protein of claim 1 in a plant of interest, thereby increasing the content of raffinose and / or protein or decreasing the content of starch in the grain of the plant of interest.

9. A method for increasing the content of raffinose and / or protein or decreasing the content of starch in the grain of a plant, comprising the step of replacing the base G at position 830 of the GAL-5B gene in the genome of a plant of interest with the base A, thereby increasing the content of raffinose and / or protein or decreasing the content of starch in the grain of the plant of interest. ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ 3. Use according to claim 2, characterized in that: ​ ​ ​ 4. Use according to any one of claims 1 to 3, characterized in that: ​ 5. Use according to claim 4, characterized in that: ​ 6. Use according to claim 5, characterized in that: ​ 7. Use according to claim 6, characterized in that: ​ ​ ​ 10. A method for breeding a transgenic plant with increased raffinose content and / or increased protein content and / or decreased starch content in its seeds, comprising the step of: reducing the content and / or activity of the protein as claimed in claim 1 in a plant of interest to obtain a transgenic plant; wherein the transgenic plant has a higher raffinose content and / or a higher protein content and a lower starch content in its seeds than the plant of interest.

11. A method for breeding a transgenic plant with increased raffinose content and / or increased protein content and / or decreased starch content in its seeds, comprising the step of: replacing the base G at position 830 of the GAL-5B gene in the genome of a plant of interest with base A to obtain a transgenic plant; wherein the transgenic plant has a higher raffinose content and / or a higher protein content and a lower starch content in its seeds than the plant of interest.

12. The method of any of claims 8-11, wherein: The plant is a monocotyledon.

13. The method of claim 12, wherein: The monocotyledon is a plant of the family Poaceae.

14. The method of claim 13, wherein: The plant of the family Poaceae is a plant of the genus Triticum.

15. The method of claim 14, wherein: The plant of the genus Triticum is wheat.

16. A method for decreasing the raffinose content in seeds of a plant, comprising the step of: increasing the content and / or activity of the protein as claimed in claim 1 in a plant of interest to decrease the raffinose content in seeds of the plant of interest.

17. A method for breeding a transgenic plant with decreased raffinose content in its seeds, comprising the step of: increasing the content and / or activity of the protein as claimed in claim 1 in a plant of interest to obtain a transgenic plant; wherein the transgenic plant has a lower raffinose content in its seeds than the plant of interest.

18. The method of claim 16 or 17, wherein: The plant is a monocotyledon.

19. The method of claim 18, wherein: The monocotyledon is a plant of the family Poaceae.

20. The method of claim 19, wherein: The plant of the family Poaceae is a plant of the genus Triticum.

21. The method of claim 20, wherein: The plant of the genus Triticum is wheat.

22. A transgenic plant produced according to any one of claims 8 to 21.

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