Application of TaMYB46L-5A protein or gene for coding TaMYB46L-5A protein in regulation and control of protein content of plant seeds

Abstract

The invention provides an application of a TaMYB46L-5A protein or a gene for coding the TaMYB46L-5A protein in regulating and controlling the protein content of plant seeds, and belongs to the technical field of molecular biology. The invention provides an application of a TaMYB46L-5A protein or a gene for coding the TaMYB46L-5A protein in at least one of regulation and control of protein content of plant seeds, creation of transgenic plants with dominant protein content and breeding of plant varieties with dominant protein content. The result of the embodiment of the invention shows that the seed protein content of a TaMYB46L-5A overexpression plant is obviously reduced, the seed protein content of a TaMYB46L-5A mutant plant is increased, and the contents of prolamin and glutenin are obviously increased. Therefore, the TaMYB46L-5A can negatively regulate and control the protein content of the plant seeds, and plays an important role in plant breeding.

Description

Technical Field

[0001] The present invention belongs to the field of molecular biotechnology, and particularly relates to the application of TaMYB46L-5A protein or a gene encoding TaMYB46L-5A protein in regulating the protein content of plant seeds. Background Art

[0002] Plant seed protein contains a variety of amino acids and is an important source of human nutrition. It can also enhance immunity, control weight, promote cardiovascular health, increase metabolic rate, and prevent chronic diseases. The content and composition of plant seed protein are important indicators for evaluating plant seed quality, and cultivating plant varieties with high protein content is a key goal of plant breeding today and in the future.

[0003] Traditional breeding processes are long, difficult, and require significant human and material resources. In recent years, molecular biology techniques have been widely used in biological breeding. Transgenic breeding allows for targeted plant improvement and shortens the breeding cycle. However, limited research has focused on genes related to seed protein content, severely hindering the development of molecular breeding. Summary of the Invention

[0004] The purpose of the present invention is to provide an application of a TaMYB46L-5A protein or a gene encoding the TaMYB46L-5A protein in regulating the protein content of plant seeds, thereby providing genetic resources for transgenic breeding of plant varieties with high protein content.

[0005] In order to achieve the above object, the present invention provides the following technical solutions:

[0006] The present invention provides an application of a TaMYB46L-5A protein or a gene encoding the TaMYB46L-5A protein in at least one of regulating the protein content of plant seeds, creating transgenic plants with superior protein content, and breeding plant varieties with superior protein content.

[0007] Preferably, the TaMYB46L-5A protein or the gene negatively regulates the protein content of plant seeds.

[0008] The present invention provides the use of TaMYB46L-5A protein or a derivative containing a gene encoding TaMYB46L-5A protein in reducing the protein content of plant seeds;

[0009] The derivative containing the gene encoding TaMYB46L-5A protein includes at least one of the following: a recombinant expression vector containing the gene, a recombinant bacterium containing the gene, and a gene expression cassette containing the gene.

[0010] Preferably, the plant seed protein comprises glutenin and / or prolamin.

[0011] Preferably, the plants include plants of the family Poaceae.

[0012] The present invention provides a method for regulating the protein content of plant seeds, wherein the gene encoding the TaMYB46L-5A protein is overexpressed in plants to reduce the protein content of plant seeds;

[0013] Alternatively, the biological function of the TaMYB46L-5A protein or the expression level of the gene encoding the TaMYB46L-5A protein can be inhibited to increase the protein content of plant seeds.

[0014] Preferably, the method for inhibiting the biological function of the TaMYB46L-5A protein or inhibiting the expression level of the gene encoding the TaMYB46L-5A protein comprises gene editing the gene encoding the TaMYB46L-5A protein.

[0015] The present invention provides a method for breeding plant varieties with superior seed protein content, which comprises detecting the expression level of TaMYB46L-5A protein or a gene encoding TaMYB46L-5A protein in the genome of the plant;

[0016] Plants with inferior expression levels of the TaMYB46L-5A protein or the gene encoding the TaMYB46L-5A protein are selected as breeding materials for breeding.

[0017] Preferably, the reagent for detecting the expression level of the gene encoding the TaMYB46L-5A protein comprises a forward primer having a nucleotide sequence as shown in SEQ ID NO: 3 and a reverse primer having a nucleotide sequence as shown in SEQ ID NO: 4.

[0018] Preferably, the plants include plants of the family Poaceae.

[0019] Beneficial effects:

[0020] The present invention provides an application of a TaMYB46L-5A protein or a gene encoding the TaMYB46L-5A protein in at least one of regulating the protein content of plant seeds, creating transgenic plants with superior protein content, and breeding plant varieties with superior protein content. The TaMYB46L-5A protein or the gene negatively regulates the protein content of plant seeds. The present invention uses TaMYB46L-5A gene-edited mutant wheat plants and TaMYB46L-5A gene-overexpressing wheat plants as research objects, respectively, and detects the protein content of grains after harvest. The results showed that the protein content of the grains of the TaMYB46L-5A gene-edited mutant plants was significantly increased compared with the control, that is, knocking out the TaMYB46L-5A gene can increase the protein content of wheat grains. The protein content of the grains of the TaMYB46L-5A overexpressing transgenic lines was significantly decreased compared with the wild-type control. Therefore, the TaMYB46L-5A gene can regulate the protein content of wheat grains, and the TaMYB46L-5A protein and its encoding gene will play an important role in breeding high-quality specialized wheat varieties. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Figure 2 shows the mutation site detection results of TaMYB46L-5A gene-edited mutants. TaMYB46L-5A-KO1 has a 3-base deletion, and TaMYB46L-5A-KO2 has a 1-base insertion.

[0022] Figure 2 The figure shows the expression level detection results of TaMYB46L-5A gene in TaMYB46L-5A overexpressing transgenic lines compared with wild-type control plants;

[0023] Figure 3 The graph shows the results of the grain protein content test of the TaMYB46L-5A overexpressing transgenic line and the wild-type control plant;

[0024] Figure 4 These are the results of grain protein detection of the TaMYB46L-5A gene-edited mutant and the wild-type control plant, where A represents the glutenin detection result and B represents the alcohol-soluble protein detection result. DETAILED DESCRIPTION

[0025] The present invention provides an application of a TaMYB46L-5A protein or a gene encoding the TaMYB46L-5A protein in at least one of regulating the protein content of plant seeds, creating transgenic plants with superior protein content, and breeding plant varieties with superior protein content.

[0026] In the present invention, the TaMYB46L-5A protein includes at least one of the following proteins;

[0027] 1) Amino acid sequence such as SEQ ID NO:1(MRKPVECPATKCSGGAAPGNSNVAAAAAKLRKGLWSPEEDERLVAYMLRSGQGSWSDVARNAGLQRCGKSCRLRWINYLRPDLKRGAFSPQEEDLIVNLHAILGNRWSQIAARLPGRTDNEIKNFWNSTIKKRLKMNSAASSPATTECASPREPKLDGGSASCLDLTSLEDGSHHGMKSMWRMDSSSSSSSSSSIQ QSRPSTMAPAAANRGYGGLLLPLPDQFCGVAPSTHTSVPPFFHDHSSFKQVSPLRAGGYYPHGMAMEGGGGSCFTGEEAVGGGGEHSVLFSVPPLLEPMAVALQDQTLMASTGNSDNNHRNTNSTAEGTTLSSKNGCNINDDNNSKNNINSVVSYWEQHGHQQHMSRNVVMGEWDLEELMKDVSCLPFLDFQVE);

[0028] 2) A fusion protein formed by the protein described in item 1) and a tag sequence;

[0029] 3) A protein with unchanged biological function obtained by replacing, deleting or inserting one or more amino acid residues based on the protein described in item 1).

[0030] In the present invention, the TaMYB46L-5A protein preferably includes a protein that has 98% or more identity with the protein described in item 1) and is associated with wheat disease resistance. The identity 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 on the NCBI homepage. For example, in Advanced BLAST 2.1, by using BLASTP as a program, setting the Expect value to 10, setting all filters to OFF, using BLOSUM62 as a matrix, setting the Gap existence cost, Perresidue gap cost, and Lambdaratio to 11, 1, and 0.85 (default values), respectively, and searching for the identity of a pair of amino acid sequences for calculation, the identity value (%) can be obtained.

[0031] In the present invention, the tag sequence described in item 2) refers to a polypeptide or protein that is fused with the target protein to facilitate expression, detection, tracing, and / or purification of the target protein during in vitro DNA recombination techniques. The tag sequence preferably includes at least one of the following: a FLAG tag, a polyhistidine tag, an MBP tag, a hemagglutinin tag, a Myc tag, a GST tag, and a SUMO tag. The tag sequence is preferably linked to the 5' or 3' end of the gene encoding the TaMYB46L-5A protein.

[0032]

[0033] In the present invention, the plant preferably comprises a grass plant, more preferably at least one of the following: corn, rice, oats, sugarcane, and wheat, and most preferably wheat. The present invention does not impose any particular limitation on the wheat variety. In the present embodiment, the wheat variety is the wild-type hexaploid wheat Kenong 199.

[0034] In the present invention, the TaMYB46L-5A protein or the gene preferably negatively regulates the protein content of plant seeds.

[0035] In the present invention, the plant seed protein preferably includes glutenin and / or prolamin.

[0036] In the present examples, the grain protein content of wheat plants with TaMYB46L-5A gene editing and wheat plants with TaMYB46L-5A gene overexpression was measured. The results showed that compared with wild-type control plants, the grain protein content of TaMYB46L-5A gene editing mutants was significantly increased, while the grain protein content of TaMYB46L-5A gene overexpression plants was significantly decreased. Therefore, the TaMYB46L-5A gene can regulate the protein content of plant seeds.

[0037] The present invention provides a use of a TaMYB46L-5A protein or a derivative containing a gene encoding the TaMYB46L-5A protein in reducing the protein content of plant seeds; the derivative containing the gene encoding the TaMYB46L-5A protein comprises at least one of the following: a recombinant expression vector containing the gene, a recombinant bacterium containing the gene, and a gene expression cassette containing the gene.

[0038] In the present invention, the backbone vector type of the recombinant expression vector preferably includes at least one of the following: plasmid, cosmid and phage. The host bacteria type of the recombinant bacteria preferably includes bacteria and / or fungi. The bacteria preferably include Agrobacterium or Escherichia coli. The Agrobacterium strain is preferably Agrobacterium EHA105 and / or Agrobacterium C58C1. The gene expression cassette preferably includes DNA capable of expressing TaMYB46L-5A protein in host cells. The DNA may include not only a promoter for initiating TaMYB46L-5A transcription, but also a terminator for terminating TaMYB46L-5A transcription. The expression cassette preferably also includes an enhancer sequence. In an embodiment of the present invention, the method for constructing the recombinant expression vector is preferably: seamlessly cloning the coding gene of the TaMYB46L-5A protein into the backbone vector, verifying, and obtaining the recombinant expression vector. The backbone vector of the recombinant expression vector is preferably a pUbi-FLAG vector, and the cloning site is BamHI. The pUbi-FLAG vector is preserved by the Wang Daowen research group of Henan Agricultural University (reference to the prior art: New Phytologist (2020) 225: 2526-2541 doi: 10.1111 / nph.16305). The recombinant vector is preferably verified by sequencing, and the forward primer pUbi-Seq-f and the reverse primer FLAG-Seq-r shown in the nucleotide sequence such as SEQ ID NO: 7 are preferably used for sequencing. The construction method of the recombinant bacteria is preferably to transfect the recombinant vector into the host bacteria, and the recombinant bacteria are obtained by screening and verification. The transfection method is preferably any one of the following: electric shock method, calcium phosphate method and freeze-thaw method, more preferably freeze-thaw method. The host bacteria is preferably Agrobacterium C58C1, purchased from Beijing Zhuangmeng International Biogene Technology Co., Ltd., and the product number is ZC1504. The screening method is preferably screened by antibiotics. The antibiotics are preferably rifampicin and kanamycin sulfate, and the concentration is preferably 50 μg / mL. After the antibiotic screening, it is preferably verified by sequencing. The primers preferably used in sequencing are preferably the same as those used for sequencing verification of the recombinant vector, and the primer pair used is pUbi-Seq-f (SEQ ID NO: 7) and FLAG-Seq-r (SEQ ID NO: 8).

[0039] The present invention provides a method for regulating the protein content of plant seeds, wherein the gene encoding the TaMYB46L-5A protein is overexpressed in plants to reduce the protein content of plant seeds;

[0040] Alternatively, the biological function of the TaMYB46L-5A protein or the expression level of the gene encoding the TaMYB46L-5A protein can be inhibited to increase the protein content of plant seeds.

[0041] In the present invention, the method for overexpressing the TaMYB46L-5A protein in plants preferably comprises transfecting a host bacterium with a recombinant expression vector containing the gene encoding the TaMYB46L-5A protein, and infecting a host plant with the resulting recombinant bacterium to obtain a plant overexpressing the TaMYB46L-5A gene or TaMYB46L-5A protein. The recombinant expression vector, host bacterium, and method for infecting a host plant with the recombinant bacterium are preferably the same as the method for using the derivative containing the gene encoding the TaMYB46L-5A protein to reduce the protein content of plant seeds, and will not be further described here.

[0042] In the present invention, the method for inhibiting the biological function of TaMYB46L-5A protein or inhibiting the expression level of the gene encoding TaMYB46L-5A protein preferably includes gene editing of the gene encoding TaMYB46L-5A protein. The gene editing preferably includes gene silencing, gene knockout or gene mutation. In the embodiment of the present invention, gene mutation is used as an example to illustrate the biological function of TaMYB46L-5A protein or the effect of inhibiting the gene encoding TaMYB46L-5A protein. The method for gene mutation refers to the prior art (Zhang Y, Li D, Zhang D, et al. Analysis of the functions of TaGW2 homoeologs in wheat grain weight and protein content traits. Plant J. 2018; 94 (5): 857-866. doi: 10.1111 / tpj.13903). The nucleotide sequence of the gRNA target region of the gene mutation is shown in SEQ ID NO: 9 (AAACAGCAATGTGGCTGCAG).

[0043] The present invention provides a method for breeding plant varieties with superior seed protein content, which comprises detecting the expression level of TaMYB46L-5A protein or a gene encoding TaMYB46L-5A protein in the genome of the plant;

[0044] Plants with inferior expression levels of the TaMYB46L-5A protein or the gene encoding the TaMYB46L-5A protein are selected as breeding materials for breeding.

[0045] In the present invention, the reagent for detecting the expression level of the gene encoding the TaMYB46L-5A protein includes a forward primer having a nucleotide sequence as shown in SEQ ID NO: 3 and a reverse primer having a nucleotide sequence as shown in SEQ ID NO: 4. The amplification system of the primers is preferably: 10.0 μL of 2×ChamQ Universal SYBR qPCR MasterMix, 0.4 μL of a 10 μM forward primer, 0.4 μL of a 10 μM reverse primer, 1 μL of template cDNA, and ddH2O to a volume of 20 μL. The amplification program of the primers is preferably: 95°C for 5 min; 95°C for 10 s, 60°C for 30 s, for 40 cycles. In the embodiment of the present invention, 2×ChamQ Universal SYBR qPCR MasterMix was purchased from Novozymes, catalog number Q711.

[0046] In the present invention, the plants are preferably the same as the plants in the above application, and will not be described in detail here.

[0047] In order to further illustrate the present invention, the solutions provided by the present invention are described in detail below with reference to the accompanying drawings and embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0048] Example 1

[0049] Obtaining and testing TaMYB46L-5A gene-edited mutant plants

[0050] 1. Obtaining TaMYB46L-5A gene-edited mutant plants

[0051] The TaMYB46L-5A gene-edited mutant materials (TaMYB46L-5A-KO1 and TaMYB46L-5A-KO2) were obtained by gene editing of the common wheat variety KN199. For specific steps, please refer to the existing technology (Zhang Y, Li D, Zhang D, et al. Analysis of the functions of TaGW2 homoeologs in wheat grain weight and protein content traits. Plant J. 2018; 94(5): 857-866. doi: 10.1111 / tpj.13903).

[0052] 2. Detection of TaMYB46L-5A gene-edited mutant plants

[0053] The genomic DNA of the TaMYB46L-5A gene-edited mutant material was extracted, and the sequence of the TaMYB46L-5A gene was amplified using a forward primer represented by a nucleotide sequence such as SEQ ID NO: 5 (CCTCCATTCCTGCCCTCACTCGAC) and a reverse primer represented by a nucleotide sequence such as SEQ ID NO: 6 (GGCGCACGCCTGCATGGTG). The sequence was sequenced. Analysis showed that compared with the wild-type control genomic sequence, the TaMYB46L-5A mutant material had a mutation in the gene sequence encoding TaMYB46L-5A on wheat chromosome 5A, resulting in a protein translation frameshift ( Figure 1 ).

[0054] Example 2

[0055] Acquisition of TaMYB46L-5A overexpression materials and detection of gene expression

[0056] 1) Construction of the recombinant vector pUbi::TaMYB46L-5A-FLAG

[0057] The TaMYB46L-5A gene shown in SEQ ID NO: 2 was ligated into the Sad site of the pUbi-FLAG vector using the infusion method to obtain the recombinant vector pUbi::TaMYB46L-5A-FLAG, which was then sequenced and verified.

[0058] The primer pair used for sequencing was pUbi-Seq-f: GCCCTGCCTTCATACGCT (SEQ ID NO: 7) and FLAG-Seq-r: CGTCGTCTTTATAATCGATG (SEQ ID NO: 8).

[0059] Sequencing showed that there was no mutation in the TaMYB46L-5A gene in the recombinant vector.

[0060] 2) Obtaining recombinant bacteria

[0061] The recombinant vector pUbi::TaMYB46L-5A-FLAG was transformed into Agrobacterium tumefaciens C58C1 to generate the recombinant strain pUbi::TaMYB46L-5A-FLAG / C58C1. The primer pair used to detect the recombinant strain was pUbi-Seq-f and FLAG-Seq-r.

[0062] The specific conversion method is as follows:

[0063] Take competent Agrobacterium C58C1 stored at -70°C and place it in room temperature or ice water bath for a while until it partially thaws. When it is in the ice-water mixing state, insert it into the ice bath; add 1 μg plasmid DNA per 100 μL competent culture, stir the bottom of the tube by hand to mix, and then stand on ice for 5 minutes, liquid nitrogen for 5 minutes, 37°C water bath for 5 minutes, and ice bath for 5 minutes.

[0064] After an ice bath, add 800 μL of TY liquid medium and culture at 28°C with shaking for 3 h; collect the bacteria by centrifugation at 5000 rpm for 1 min, retain about 100 μL of supernatant, gently pipette to resuspend the bacteria, spread the plate on a TY plate containing the corresponding antibiotics (final concentration of 50 μg / mL rifampicin and 50 μg / mL kanamycin sulfate), and place it upside down in a 28°C incubator for culture for 3 days.

[0065] 3) Obtaining pUbi::TaMYB46L-5A-FLAG transgenic wheat

[0066] The recombinant bacteria pUbi::TaMYB46L-5A-FLAG / C58C1 were used to infect the wild-type hexaploid wheat Kenong 199 with good growth status. Genetic transformation was carried out by embryo stripping method. After callus differentiation, the transgenic wheat lines carrying TaMYB46L-5A were obtained by screening.

[0067] The specific methods for wheat genetic transformation are as follows:

[0068] Remove the seeds with embryos as small as 2 mm and store at 4°C. Wash the seeds with 75% alcohol for 1 minute, shake them in 15% NaClO solution for about 10 minutes, wash them with sterile water four times, and then peel the embryos. Place the peeled embryos in WLS culture medium and centrifuge them at 7500 rpm at 4°C for 10 minutes. Remove the supernatant and add WLS to clean them.

[0069] Infection: The recombinant bacteria were inoculated into 4 mL of TY medium and shaken overnight at 28°C. The bacteria were collected and resuspended in WLS. The resuspended bacteria solution was added to the embryos, allowed to stand for 5 minutes, and the suspension was removed. The embryos were placed on sterile filter paper. After blotting, the suspension was placed with the scutellum facing down on AS medium and grown in the dark at 23°C for 48 hours. After tip cutting, the embryos were placed on recovery medium with the cut end facing down for 5 days. The embryos were then transferred to a first sieve plate. After 14 days, the calli were cut in half and transferred to a second sieve plate. After 20 days, the calli were observed for differentiation. After bud formation, the calli were transferred to differentiation medium. After 14 days, the plants were transferred to 1 / 2 MS medium and allowed to root. Rooting resulted in the TaMYB46L-5A overexpressing transgenic lines TaMYB46L-5A-OE1 and TaMYB46L-5A-OE2.

[0070] 4) Detection of target gene expression

[0071] The relative expression levels of the gene in the TaMYB46L-5A overexpressing transgenic plants and the control were detected. The nucleotide sequences of the primer pairs for detecting the relative expression levels of TaMYB46L-5A are as follows:

[0072] RT-VIGS-TaMYB46L-5A-f:GTAATATCTGGTCCGTATCAG(SEQ ID NO:3)

[0073] RT-VIGS-TaMYB46L-5A-r: GGATCGGTGATAACCGGTAT (SEQ ID NO: 4). The reaction system for detection is:

[0074] 2×ChamQ Universal SYBR qPCR MasterMix (Novozymes, Q711) 10.0 μL;

[0075] Forward primer (10 μM) 0.4 μL;

[0076] Reverse primer (10 μM) 0.4 μL;

[0077] Template cDNA 1 μL;

[0078] ddH2O 8.2μL.

[0079] The reaction program was: 95°C for 5 min; 95°C for 10 s, 60°C for 30 s, for 40 cycles.

[0080] Use the instrument's default melting curve acquisition program: *Fluorescence signal acquisition.

[0081] The results showed that compared with the control, the expression level of TaMYB46L-5A gene in the overexpressing transgenic plants was significantly increased ( Figure 2 ), which indicated that the overexpressing transgenic lines TaMYB46L-5A-OE1 and TaMYB46L-5A-OE2 were transgenic-positive lines.

[0082] Example 3

[0083] Determination of protein content in wheat grains

[0084] The protein content of the grains of TaMYB46L-5A-OE1, TaMYB46L-5A-OE2 and wild-type control plants after harvest was measured using a Swedish DA7200 near-infrared analyzer. The results showed that the protein content of the grains of TaMYB46L-5A overexpressing plants was significantly lower than that of the control ( Figure 3 ).

[0085] Example 4

[0086] Detection of glutenin and alcohol-soluble protein content in wheat grains

[0087] Glutenin and alcohol-soluble proteins were extracted from wheat grains, and the grain glutenin and alcohol-soluble proteins of the TaMYB46L-5A gene-edited mutant and wild-type control plants were detected by modified SDS-PAGE and A-PAGE, respectively, and quantified and analyzed using ImageJ software.

[0088] 1. The method for extracting wheat grain glutenin is as follows:

[0089] (1) Weigh 45 mg of wheat flour and place it in a 2.0 mL centrifuge tube filled with steel balls. Grind the flour using a German QIAGEN high-throughput tissue grinder.

[0090] (2) Add 1 mL of 70% anhydrous ethanol to the centrifuge tube, vertically shake and extract at room temperature for 1 hour, centrifuge at 12,000 rpm for 10 minutes, and discard the supernatant.

[0091] (3) Add 1 mL of 50% isopropanol and incubate in a 65°C water bath for 30 min, inverting the tube once during the incubation period. Centrifuge at 12,000 rpm for 10 min, discard the supernatant, and repeat this process.

[0092] (4) Add 1 mL of 50% isopropanol (containing 8% 1 M tri-HCl, pH = 8, and 1% DTT) to the precipitate and extract at 65°C for 1 h, inverting the tube twice.

[0093] (5) Add 4-VP (containing 8% 1M tris-HCl, pH=8.0) with a final concentration of 1.4% equal to that in step (4), and extract at 65°C for 30 minutes.

[0094] (6) Centrifuge at 12000 rpm for 10 min and transfer the supernatant to a new 1.5 mL centrifuge tube.

[0095] 2. The extraction method of wheat grain alcohol-soluble protein is as follows:

[0096] (1) Weigh 45 mg of flour into a 2.0 mL centrifuge tube containing steel balls and grind into powder using a German QIAGEN high-throughput tissue grinder.

[0097] (2) Add 1 mL of 70% anhydrous ethanol to the centrifuge tube and extract by shaking at room temperature for 1 h.

[0098] (3) Centrifuge at 12000 rpm for 10 min and transfer the supernatant to a new 1.5 mL centrifuge tube.

[0099] (4) The supernatant was freeze-dried and the precipitate was dissolved with 5% methyl green acetate solution and used for A-PAGE electrophoresis analysis.

[0100] 3. The formulas of A-PAGE gel preparation solution and 12% SDS-PAGE gel preparation solution are shown in Tables 1 and 2.

[0101] Table 1 12% A-PAGE gel solution

[0102] Reagents 12％ urea 6g 50% acetic acid 2mL 10% APS 0.4mL 1MTris-HCl (pH8.5) 0.25mL 40% acrylamide (29:1) 6mL TEMED 200 μL <![CDATA[ddH2O]]> Dilute to 20 mL

[0103] Table 2 12% SDS-PAGE gel preparation solution

[0104] Reagents 12% separation gel 5% stacking gel 30% Acr-Bis (29:1) 8.0mL 1.67mL <![CDATA[ddH2O]]> 6.7mL 5.8mL 1.5MTris-HCl (pH8.8) 5.0mL / 0.5MTris-HCl (pH 6.8) / 2.5mL 10% SDS 200 μL 100 μL 10% APS 200 μL 100 μL TEMED 20 μL 10 μL Total volume 20mL 10mL

[0105] Test results such as Figure 4 ( Figure 4 (A is the glutenin detection result, B is the alcohol-soluble protein detection result). The glutenin and alcohol-soluble protein contents in the TaMYB46L-5A gene-edited mutant were significantly higher than those in the control, indicating that knocking out the TaMYB46L-5A gene can increase the gluten protein content in wheat grains.

[0106] Although the above embodiment provides a detailed description of the present invention, it is only a part of the embodiments of the present invention, not all of the embodiments. Other embodiments can be obtained based on this embodiment without creativity, and these embodiments all fall within the scope of protection of the present invention.

Claims

1. Use of a TaMYB46L-5A protein or a gene encoding the TaMYB46L-5A protein in at least one of regulating the protein content of plant seeds, creating transgenic plants with superior protein content, and breeding plant varieties with superior protein content.

2. The application according to claim 1, characterized in that The TaMYB46L-5A protein or the gene negatively regulates the protein content of plant seeds.

3. Use of TaMYB46L-5A protein or a derivative containing a gene encoding TaMYB46L-5A protein in reducing the protein content of plant seeds; The derivative containing the gene encoding TaMYB46L-5A protein includes at least one of the following: a recombinant expression vector containing the gene, a recombinant bacterium containing the gene, and a gene expression cassette containing the gene.

4. The use according to any one of claims 1 to 3, characterized in that: The plant seed protein includes glutenin and / or prolamin.

5. The application according to claim 4, characterized in that: The plants include plants of the grass family.

6. A method for regulating the protein content of plant seeds, characterized in that: Overexpressing the gene encoding TaMYB46L-5A protein in plants can reduce the protein content in plant seeds; Alternatively, the biological function of the TaMYB46L-5A protein or the expression level of the gene encoding the TaMYB46L-5A protein can be inhibited to increase the protein content of plant seeds.

7. The method according to claim 6, characterized in that The method for inhibiting the biological function of the TaMYB46L-5A protein or inhibiting the expression level of the gene encoding the TaMYB46L-5A protein comprises gene editing the gene encoding the TaMYB46L-5A protein.

8. A method for breeding plant varieties with superior seed protein content, characterized in that: detecting the expression level of the TaMYB46L-5A protein or a gene encoding the TaMYB46L-5A protein in the genome of the plant; Plants with inferior expression levels of the TaMYB46L-5A protein or the gene encoding the TaMYB46L-5A protein are selected as breeding materials for breeding.

9. The method according to claim 8, characterized in that The reagent for detecting the expression level of the gene encoding the TaMYB46L-5A protein includes a forward primer having a nucleotide sequence as shown in SEQ ID NO: 3 and a reverse primer having a nucleotide sequence as shown in SEQ ID NO:

4.

10. The method according to claim 8 or 9, characterized in that: The plants include plants of the grass family.

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