CN116411018B

CN116411018B - Application of TaVRT-A2 protein and related biological materials thereof in regulation and control of protein content of plant seeds

Info

Publication number: CN116411018B
Application number: CN202310549579.6A
Authority: CN
Inventors: 倪中福; 刘静; 孙其信; 刘杰; 陈朝燕
Original assignee: China Agricultural University
Current assignee: China Agricultural University
Priority date: 2023-05-16
Filing date: 2023-05-16
Publication date: 2024-03-19
Anticipated expiration: 2043-05-16
Also published as: CN116411018A

Abstract

The invention discloses TaVRT-A2 protein and application of related biological materials thereof in regulating and controlling the protein content of plant seeds. The invention relates to an application of a wheat-derived protein in regulating and controlling or improving the protein content of plant seeds, wherein the protein is named as TaVRT-A2, and the amino acid sequence of the protein is sequence 1 in a sequence table. According to the invention, the gene TaVRT-A2 is cloned from wheat and is subjected to over-expression in a wheat field to obtain a plant transformed with the TaVRT-A2 gene, and the detection shows that the over-expression of the TaVRT-A2 gene can obviously improve the total protein content in wheat seeds by 1.0% -4.6%, so that the TaVRT-A2 protein has the effect of regulating and controlling the protein content of plant seeds, and can be applied to plant breeding or quality improvement.

Description

Application of TaVRT-A2 protein and related biological materials thereof in regulation and control of protein content of plant seeds

Technical Field

The invention relates to an application of TaVRT-A2 protein and related biological materials thereof in regulating and controlling the protein content of plant seeds.

Background

Common wheat (Triticum aestivum l.) is a gramineous plant that is widely cultivated worldwide. Wheat is one of the main foods of human beings and mainly consists of two major components of starch and protein. The flour can be ground into bread, steamed bread, biscuit, noodle, etc., and can provide various proteins, minerals, vitamins, etc.

In recent years, modern agriculture is increasingly developed, the living standard of people is obviously improved, and the demand for high-quality wheat is increasing. Starch and protein together determine the nutritional quality, appearance quality, retort food quality, processing quality, etc. of wheat. High yield and high quality have been the targets of breeding research, but so far, genes with improved yields and quality have been reported. Therefore, research on the genetic basis of starch and protein and the influence of the genetic basis on the quality of wheat is important to develop genes with improved yield and quality for wheat breeding.

TaVRT-A2 has important regulation and control effects on the growth and development of plants. The prior art shows that TaVRT-A2 has a report in regulating and controlling the glume length of wheat, and can increase the glume length and thousand grain weight at the same time.

Disclosure of Invention

The invention aims to solve the technical problem of regulating or improving the protein content in plant seeds.

In order to solve the above technical problems, the present invention provides, first of all, any one of the following uses of a protein and/or a substance regulating the expression of a gene encoding the protein and/or a substance regulating the content of the protein and/or a substance regulating the activity of the protein:

p1, in regulating and controlling the protein content of plant seeds,

p2, in improving the protein content of plant seeds,

p3, in the preparation of products for increasing the protein content of plant seeds,

p4, application in plant breeding,

p5, application in plant quality improvement.

The protein may be referred to as a protein of A1), A2) or A3) as follows:

a1 Amino acid sequence is protein of sequence 1 in a sequence table;

a2 Protein which is obtained by substituting and/or deleting and/or adding amino acid residues on the amino acid sequence shown in the sequence 1 in the sequence table, is derived from A1) and has the same function or has more than 80 percent of identity with the protein shown in A1) and has the same function;

a3 Fusion proteins obtained by ligating protein tags at the N-terminus or/and the C-terminus of A1) or A2).

The protein can be synthesized artificially or obtained by synthesizing the coding gene and then biologically expressing.

Among the above proteins, the protein tag (protein-tag) refers to a polypeptide or protein that is fusion expressed together with a target protein by using a DNA in vitro recombination technique, so as to facilitate the expression, detection, tracing and/or purification of the target protein. The protein tag may be a Flag tag, his tag, MBP tag, HA tag, myc tag, GST tag, and/or SUMO tag, etc.

In the above proteins, the identity refers to the identity of amino acid sequences. The identity of amino acid sequences can be determined using homology search sites on the internet, such as BLAST web pages of the NCBI homepage website. For example, in advanced BLAST2.1, the identity of a pair of amino acid sequences can be searched for by using blastp as a program, setting the Expect value to 10, setting all filters to OFF, using BLOSUM62 as Matrix, setting Gap existence cost, per residue gap cost and Lambda ratio to 11,1 and 0.85 (default values), respectively, and calculating, and then obtaining the value (%) of the identity.

In the above protein, the 80% or more identity may be at least 81%, 82%, 85%, 86%, 88%, 90%, 91%, 92%, 95%, 96%, 98%, 99% or 100% identity.

In the above application, the protein may be derived from wheat.

In the above application, the plant may be any of the following:

d1 A monocotyledonous plant,

d2 A plant of the order Gramineae,

d3 A) a plant of the Gramineae family,

d4 A) a plant of the genus Triticum,

d5 Wheat.

The above-mentioned substances may be the following biological materials.

The modulation may be up-regulation or enhancement or improvement.

Any of the following applications of the biological material related to the proteins described above also fall within the scope of the invention:

q1, the application of the biological material in regulating and controlling the protein content of plant seeds,

q2, the application of the biological material in improving the protein content of plant seeds,

q3, the application of the biological material in preparing a product for improving the protein content of plant seeds,

q4, the application of the biological material in plant breeding,

q5, the use of said biological material in plant improvement.

In the above application, the biomaterial may be any of the following:

b1 A nucleic acid molecule encoding a protein as described above;

b2 An expression cassette comprising the nucleic acid molecule of B1);

b3 A recombinant vector comprising the nucleic acid molecule of B1) or a recombinant vector comprising the expression cassette of B2);

b4 A recombinant microorganism comprising the nucleic acid molecule of B1), or a recombinant microorganism comprising the expression cassette of B2), or a recombinant microorganism comprising the recombinant vector of B3);

b5 A transgenic plant cell line comprising the nucleic acid molecule of B1) or a transgenic plant cell line comprising the expression cassette of B2);

b6 A transgenic plant tissue comprising the nucleic acid molecule of B1) or a transgenic plant tissue comprising the expression cassette of B2);

b7 A transgenic plant organ comprising the nucleic acid molecule of B1) or a transgenic plant organ comprising the expression cassette of B2);

b8 A nucleic acid molecule that promotes or enhances gene expression of a protein as described above;

b9 An expression cassette, a recombinant vector, a recombinant microorganism or a transgenic plant cell line containing the nucleic acid molecule of B8).

In the above application, the nucleic acid molecule of B1) may be a gene encoding the protein as shown below:

b1 A coding sequence of the coding chain is a cDNA molecule or a DNA molecule of a nucleotide of a sequence 2 in a sequence table;

b2 Nucleotide is cDNA molecule or DNA molecule of sequence 2 in sequence table,

b3 A cDNA molecule or a DNA molecule which hybridizes with the cDNA or DNA molecule defined in b 2) and which codes for a protein having the same function.

In the above application, the plant may be any of the following:

d1 A monocotyledonous plant,

d2 A plant of the order Gramineae,

d3 A) a plant of the Gramineae family,

d4 A) a plant of the genus Triticum,

d5 Wheat.

In the above biological material, the expression cassette containing a nucleic acid molecule of B2) refers to a DNA capable of expressing the protein of the above application in a host cell, and the DNA may include not only a promoter for promoting transcription of a gene encoding the protein but also a terminator for terminating transcription of the gene encoding the protein. Further, the expression cassette may also include an enhancer sequence. Promoters useful in the present invention include, but are not limited to: constitutive promoters, tissue, organ and development specific promoters, and inducible promoters.

Recombinant expression vectors containing the protein-encoding gene expression cassettes can be constructed using existing plant expression vectors. The plant expression vector comprises a binary agrobacterium vector, a vector which can be used for plant microprojectile bombardment and the like. Such as pAHC25, pWMB123, pBin438, pCAMBIA1302, pCAMBIA2301, pCAMBIA1301, pCAMBIA1300, pBI121, pCAMBIA1391-Xa or pCAMBIA1391-Xb (CAMBIA Co.). The plant expression vector may also comprise the 3' -untranslated region of a foreign gene, i.e., comprising a polyadenylation signal and any other DNA segments involved in mRNA processing or gene expression. The polyadenylation signal may direct the addition of polyadenylation to the 3 'end of the mRNA precursor and may function similarly to the 3' transcribed untranslated regions of Agrobacterium tumefaciens induction (Ti) plasmid genes (e.g., nopaline synthase gene Nos) and plant genes (e.g., soybean storage protein genes). When the gene of the present invention is used to construct a plant expression vector, enhancers, including translational or transcriptional enhancers, may be used, and these enhancers may be ATG initiation codon or adjacent region initiation codon, etc., but must be identical to the reading frame of the coding sequence to ensure proper translation of the entire sequence.

In the above biological material, the recombinant microorganism may specifically be yeast, bacteria, algae and fungi.

In order to solve the above technical problems, the present invention also provides a method for increasing the protein content of plant seeds, which may include increasing or enhancing the expression level of the gene encoding the protein described above in a plant of interest or/and regulating the content of the protein or/and the activity of the protein described above, thereby increasing the protein content of plant seeds.

In the above method, the enhancement or improvement of the activity of the above-described protein in the target plant or/and the expression level of the above-described gene encoding the protein is achieved by introducing the above-described gene encoding the protein into the target plant.

In the method, the encoding gene of the protein can be modified as follows and then introduced into a target plant so as to achieve better expression effect:

1) Ligating to promoters expressed by various plants to facilitate expression thereof in plants; the promoter may include constitutive, inducible, chronologically regulated, developmentally regulated, chemically regulated, tissue-preferred, and tissue-specific promoters; the choice of promoter will vary with the time and space of expression requirements and will also depend on the target species; for example, a tissue or organ specific expression promoter, depending on the desired time period of development of the receptor; although many promoters derived from dicots have been demonstrated to be functional in monocots and vice versa, it is desirable to select dicot promoters for expression in dicots and monocot promoters for expression in monocots;

2) The expression efficiency of the gene of the invention can be improved by connecting with a proper transcription terminator; e.g., tml derived from CaMV, E9 derived from rbcS; any available terminator known to function in plants may be ligated to the gene of the present invention;

3) Enhancer sequences such as intron sequences (e.g., derived from Adhl and bronzel) and viral leader sequences (e.g., derived from TMV, MCMV and AMV) are introduced.

In the above method, the stress-sensitive plant may be a transgenic plant, or a plant obtained by conventional breeding techniques such as crossing.

In the above methods, the transgenic plants are understood to include not only first to second generation transgenic plants but also their progeny. For transgenic plants, the gene may be propagated in that species, and may be transferred into other varieties of the same species, including particularly commercial varieties, using conventional breeding techniques. The transgenic plants include seeds, calli, whole plants and cells.

In the above method, the plant and/or plant of interest may be any of the following:

d1 A monocotyledonous plant,

d2 A plant of the order Gramineae,

d3 A) a plant of the Gramineae family,

d4 A) a plant of the genus Triticum,

d5 Wheat.

The proteins described above and/or the biological materials described above are also within the scope of the present invention.

The invention discovers a new function of TaVRT-A2 by analyzing the total protein content of near isogenic lines with the difference of gene TaVRT-A2 sequences by taking Jinshi No. 8 as a background, and the gene expression quantity is positively correlated with the protein content of seeds.

The invention has the beneficial effects that the invention discovers the application of the wheat glume-growing gene TaVRT-A2 and the encoding protein thereof in improving the protein content of wheat for the first time

The invention improves the protein content of seeds by over-expressing TaVRT-A2 genes in plants, and the protein content in the seeds can be improved by 1.0% -4.6%.

Thus, the TaVRT-A2 gene and the protein encoded by the same can be used for plant breeding and quality improvement.

Drawings

FIG. 1 shows the wheat glume-growing gene TaVRT-A2 near isogenic line of the present invention, and the near infrared spectrometer is used in determining the protein content and NIL content in wheat seed ^HapI Refers to NIL-TaVRT-A2 ^JY8 ，NIL ^HapII Refers to NIL-TaVRT-A2 ³⁹⁶² 。

FIG. 2 is a schematic diagram showing the construction of a cloning vector for the plant gene TaVRT-A2 in example 2 of the present invention.

FIG. 3 is a schematic diagram showing the structure of plant expression vector pWMB110 in example 3 of the present invention.

FIG. 4 shows the results of the analysis of the expression level of TaVRT-A2 in the wheat transgenic material of example 4 of the present invention. "×" represents P < 0.01.

FIG. 5 is a graph showing the detection and analysis of protein content in wheat seeds of transgenic material measured by near infrared spectrometer in example 4 of the present invention. "×" represents P < 0.01.

Detailed Description

The following detailed description of the invention is provided in connection with the accompanying drawings that are presented to illustrate the invention and not to limit the scope thereof. The examples provided below are intended as guidelines for further modifications by one of ordinary skill in the art and are not to be construed as limiting the invention in any way.

The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to techniques or conditions described in the literature in the field or according to the product specifications. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.

The sources of the reagents and materials of the invention are as follows:

coli Trans1-T1 phase resistance: full gold company, cat number: CD501-03;

t vector:blunt Simple Cloning Kit, whole gold Corp., cat#: CB111-01;

pWMB110 vector: the laboratory is stored, and related literature: liu J, chen Z, wang Z, zhang Z, xie X, wang Z, chai L, song L, cheng X, feng M, wang X, liu Y, hu Z, xing J, su Z, peng H, xin M, yao Y, guo W, sun Q, liu J, ni Z.Ectopic expression of VRT-A2 underlies the origin of Triticum polonicum and Triticum petropavlovskyi with long outer glumes and grains.mol plant.2021Sep6; 14 (9):1472-1488.

Polish wheat 3962, jin hard number 8: the laboratory is kept and the public is available from the university of agricultural China for repeated use of the invention. Related literature: ectopic expression of VRT-A2 underlies the origin of Triticum polonicum and T. Petropavlovskyi with long outer glume and grain. Molecular Plant,2021,14:1472-1488.

Wheat material "Fielder" and china spring: the laboratory is kept and the public is available from the university of agricultural China for repeated use of the invention. Related literature: cloning of wheat 'Fielder' omega-prolamin gene and promoter and sequence analysis [ J ]. University of agricultural university, china university journal, 2020,25 (07): 1-9.

Example 1 cloning of wheat glume-growing Gene TaVRT-A2

Comparison of the polish wheat 3962 with the near isogenic line constructed in jin hard 8 in earlier work showed: constructing near isogenic line NIL (TaVRT-A2) by taking Polish wheat 3962 as donor and Jinshi No. 8 (JY 8) as acceptor ³⁹⁶² ) And NIL (TaVRT-A2) ^JY8 ) In the above, the results of comparing the protein content of the pair of near isogenic lines are shown in FIG. 1, NIL-TaVRT-A2 ³⁹⁶² Seed total protein content (NIL in FIG. 1) ^HapII Representative) are all significantly higher than NIL-TaVRT-A2 ^JY8 (NIL in FIG. 1) ^HapI Representative). Thus, the TaVRT-A2 gene in wheat was cloned and over-expressed and transformed, and phenotypically analyzed.

Extracting genomic DNA of wheat material Fielder, respectively utilizing forward primer 5'-ATGGCGCGGGAGAGGCGGGC-3' (sequence 3 in a sequence table) and reverse primer 5'-TTACTTCCAAGGTAACGCTAG-3' (sequence 4 in the sequence table), using genomic DNA of wheat as a template to perform PCR amplification, cloning and sequencing to obtain wheat TaVRT-A2 gene, wherein CDS sequence is shown as sequence 2 in the sequence table; the amino acid sequence of TaVRT-A2 protein is shown as sequence 1 in the sequence table.

Sequence 1:

MARERRAIRRIESAAARQVTFSKRRRGLFKKAEELAVLCDADVALVVFSSTGKLSQFASSSMNEIIDKYSTHSKNLGKSDQQPAIDLNLEHCKYDSLNEQLAEASLRLRHMRGEELDGLSVGELQQMEKNLETGLQRVLCTKDRQFMQQISDLQHKGTQLAEENMRLKNQMHEVPTASTVAVAEAENVVPEDAHSSDSVMTAVHSGSSQDNDDGSDISLKLALPWK。

sequence 2:

5’-ATGGCGCGGGAGAGGCGGGCGATACGGCGGATAGAGAGCGCGGCGGCGCGGCAGGTGACCTTCTCCAAGCGGAGGCGCGGGCTGTTCAAGAAGGCCGAGGAGCTCGCCGTGCTCTGCGACGCCGACGTCGCGCTCGTCGTCTTCTCCTCCACCGGCAAGCTCTCCCAGTTCGCCAGCTCCAGCATGAACGAGATCATTGACAAGTATAGTACTCATTCAAAGAACCTGGGGAAATCTGATCAGCAGCCGGCTATTGATTTAAATTTAGAGCACTGCAAGTATGACAGTTTGAATGAACAACTCGCAGAAGCAAGTCTTCGACTTAGACACATGAGAGGTGAGGAACTTGACGGACTGAGTGTCGGTGAGTTGCAGCAGATGGAAAAGAATCTCGAAACAGGATTGCAGAGGGTGCTTTGTACAAAGGACCGGCAATTCATGCAACAAATTAGTGACCTCCAACACAAGGGAACACAGCTGGCAGAGGAAAATATGCGCTTGAAAAACCAAATGCATGAGGTGCCAACTGCTAGCACCGTGGCCGTTGCCGAAGCCGAAAATGTTGTCCCTGAAGATGCTCATTCATCTGACTCTGTGATGACGGCAGTACATTCGGGAAGCTCACAGGACAATGATGACGGTTCTGATATATCCCTGAAACTAGCGTTACCTTGGAAGTAA-3’。

the PCR reaction procedure was: pre-denaturation at 94℃for 3 min; 98℃10s,58℃15s,68℃40s,35 cycles; extending at 68deg.C for 5 min.

Example 2 construction of wheat long glume Gene TaVRT-A2 expression vector

The PCR product obtained in example 1 was directly cloned into a T vector as shown in FIG. 2 according to the TA cloning methodOn a Blunt Simple. The method comprises the following steps: the PCR product (1. Mu.L), T vector (1. Mu.L) and sterile water (3. Mu.L) were ligated at 37℃for 20min, then the ligation product was transformed into E.coli Trans1-T1 to give recombinant E.coli, which was subjected to shaking amplification, and positive monoclonal colonies were screened and sequenced.

Amplifying the recombinant escherichia coli containing the wheat glume gene TaVRT-A2 by using a forward primer 5'-AGGTCGACTCTAGAGGATCCATGGCGCGGGAGAGGCGGGC-3' (sequence 5 in a sequence table) and a reverse primer 5'-TCGAGGGTACCCGGGGATCCTTACTTCCAAGGTAACGCTAG-3' (sequence 6 in the sequence table) as a template to obtain a PCR product added with a homologous recombination arm; the vector pWMB110 (carrier schematic diagram is shown in figure 3) connected with Ubiquitin promoter (Ubiquitin) of corn is digested by utilizing restriction enzyme BamHI, and the digested product of the vector and PCR product are connected (connection condition: 50 ℃ for 20 min) to obtain recombinant plasmid, sequencing the recombinant plasmid to obtain positive recombinant plasmid pWMB110-TaVRT-A2 containing TaVRT-A2 gene, wherein the recombinant vector plasmid pWMB110-TaVRT-A2 contains CDS sequence of TaVRT-A2 shown in sequence 2 in sequence table, and can express TaVRT-A2 protein shown in sequence 1 in sequence table.

Example 3 acquisition of transgenic plants and phenotypic observations

1. Obtaining transgenic plants

1.1 recombinant plasmid transformation of Agrobacterium

The recombinant plasmid pWMB110-TaVRT-A2 is transformed into agrobacterium EHA105, and the plant transformation method is performed by adopting an agrobacterium-mediated method. The screening marker in the plants is Bar.

1. Mu.g of competent cells of Agrobacterium EHA10 transformed with the recombinant vector plasmid pWMB110-TaVRT-A2 prepared in example 2 were cultured for two days at 28℃on LB medium (containing 50mg/L kanamycin and 25mg/L rifampicin), and positive clones were selected and used as primers F:5'-TAgCCCTgCCTTCATACgCT-3' and primer R:5'-AAgACCggCAACAggATTCA-3' PCR was performed (product size 871 bp). The positive bacterial liquid obtained by PCR identification is named as recombinant agrobacterium EHA105/pWMB110-TaVRT-A2 and is preserved at-80 ℃.

Recombinant agrobacterium EHA105/pWMB110-TaVRT-A2 contains the CDS nucleotide sequence of the TaVRT-A2 gene of the sequence 2 in the sequence list.

1.2 obtaining transgenic plants

Referring to the wheat transformation method of Hayta et al (related literature: hayta, S., smedeley, M.A., clarke, M., forner, M., and Harwood, W.A. (2021) An efficient Agrobacterium-mediated transformation protocol for hexaploid and tetraploid WHEAT. Curr. Protoc.1: e 58.), the recombinant Agrobacterium EHA105/pWMB110-TaVRT-A2 obtained in step 1.1 was transformed into wheat Fielder to obtain TaVRT-A2 over-expressed transgenic wheat, and the transgenesis was performed using the detection primers (F: 5'-ATTTGGAGAGGACACGCTGA-3' and R: 5'-GAAACCCACGTCATGCCAG-3') of the Bar geneIdentification of plants to obtain TaVRT-A2 transgenic T ₀ And 3 strains of the generation positive over-expressed wheat. Will T ₀ The T is obtained by planting and harvesting the generation positive transgene over-expression wheat ₁ Transgenic overexpressing wheat seeds are planted to obtain T ₁ Substitution of individual plants, T ₁ The generation single plant is also subjected to PCR identification by using a detection primer of Bar gene to obtain positive T ₁ Single plant generation, T harvesting ₁ The single plant seeds with positive generation are continuously planted and detected to obtain 3T ₂ The generation positive transgenic wheat homozygous lines OE-TaVRT-A2- #1, OE-TaVRT-A2- #3 and OE-TaVRT-A2- #6.

For TaVRT-A2 transgene T ₂ The expression level of TaVRT-A2 gene in 3 lines of the over-expressed wheat is detected, and the experiment is set to be three times. Each replicate was set up as follows: extracting total RNA of leaves of 3 transgenic over-expression lines and Wild Type (WT) wheat field, reversely transcribing into cDNA, adopting Real-time RT-PCR method, taking heat-actin as internal standard gene, detecting relative expression quantity of TaVRT-A2 gene. The primer sequences were as follows:

qTaVRT-A2-F：5’-GTTGTCGTTTTATGTCTCCG-3’；

qTaVRT-A2-R：5’-CCTGTCTCAGCGAATTCACT-3’；

qwheat-actin-F：5’-gACCgTATgAgCAAggAgAT-3’；

qwheat-actin-R：5’-CAATCgCTggACCTgACTC-3’。

as shown in FIG. 4, the expression level of the TaVRT-A2 gene in transgenic wheat OE-TaVRT-A2- #1, OE-TaVRT-A2- #3 and OE-TaVRT-A2- #6 is obviously higher than that of wild wheat Fielder (P < 0.01), which indicates that the construction of the TaVRT-A2 over-expression wheat strain is successful.

2. Phenotypic observation

The total protein content in mature seeds of 3 TaVRT-A2 transgenic overexpressing lines and wild-type (WT) wheat field was determined using a near infrared spectrometer (Botong DA 7250).

As shown in FIG. 5, the total protein content of transgenic wheat OE-TaVRT-A2- #1, OE-TaVRT-A2- #3 and OE-TaVRT-A2- #6 seeds is obviously (P < 0.01) higher than that of wild-type plant (WT) wheat field, and the improvement is 1.0% -4.6%.

Therefore, the protein content in the wheat seeds can be obviously improved due to the overexpression of the wheat glume-growing gene TaVRT-A2, the wheat glume-growing gene and the related protein TaVRT-A2 thereof can regulate and control the protein content of the wheat seeds, and the wheat glume-growing gene and the related protein TaVRT-A2 can be applied to wheat breeding and variety improvement.

The present invention is described in detail above. It will be apparent to those skilled in the art that the present invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with respect to specific embodiments, it will be appreciated that the invention may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains.

Claims

1. Use of the protein TaVRT-A2 for any of the following applications:

p1, in the application of improving the protein content of plant seeds,

p2, in the preparation of products for increasing the protein content of plant seeds,

the protein TaVRT-A2 is a protein of the following A1) or A2):

a1 Amino acid sequence is protein shown as sequence 1 in a sequence table;

a2 Fusion protein obtained by connecting protein tags at the N terminal or/and the C terminal of the protein shown in the sequence 1 in the sequence table;

the plant is wheat.

2. The use according to claim 1, characterized in that: the protein TaVRT-A2 is derived from wheat.

3. Use of any one of the following nucleic acid molecules encoding the protein TaVRT-A2:

q1, the use of said nucleic acid molecules for increasing the protein content of plant seeds,

q2, the application of the nucleic acid molecule in the product for improving the protein content of plant seeds,

the protein TaVRT-A2 is a protein of the following A1) or A2):

a1 Amino acid sequence is protein shown as sequence 1 in a sequence table;

the plant is wheat.

4. Use of an expression cassette comprising a nucleic acid molecule encoding the protein TaVRT-A2 for any of the following:

q1, the application of the expression cassette in improving the protein content of plant seeds,

q2, the application of the expression cassette in preparing a product for improving the protein content of plant seeds,

the protein TaVRT-A2 is a protein of the following A1) or A2):

a1 Amino acid sequence is protein shown as sequence 1 in a sequence table;

the plant is wheat.

5. Use of any one of the following recombinant vectors comprising a nucleic acid molecule encoding the protein TaVRT-A2:

q1, the application of the recombinant vector in improving the protein content of plant seeds,

q2, the application of the recombinant vector in preparing a product for improving the protein content of plant seeds,

the protein TaVRT-A2 is a protein of the following A1) or A2):

a1 Amino acid sequence is protein shown as sequence 1 in a sequence table;

a2 Fusion protein obtained by connecting protein tags at the N terminal or/and the C terminal of the protein shown in the sequence 1 in the sequence table; the plant is wheat.

6. Use of any one of the following recombinant microorganisms comprising a nucleic acid molecule encoding the protein TaVRT-A2:

q1, the application of the recombinant microorganism in improving the protein content of plant seeds,

q2, the application of the recombinant microorganism in preparing products for improving the protein content of plant seeds,

the protein TaVRT-A2 is a protein of the following A1) or A2):

a1 Amino acid sequence is protein shown as sequence 1 in a sequence table;

7. Use according to any one of claims 3-6, characterized in that: the nucleic acid molecule is a DNA molecule shown in a sequence 2 in a sequence table.

8. A method for increasing the protein content of a plant seed, comprising increasing or increasing the expression level of a gene encoding the protein TaVRT-A2 in a plant of interest, thereby increasing the protein content of the plant seed;

the protein TaVRT-A2 is a protein of the following A1) or A2):

a1 Amino acid sequence is protein shown as sequence 1 in a sequence table;

the plant is wheat.

9. The method according to claim 8, wherein: the enhancement or increase of the expression level of the gene encoding the protein TaVRT-A2 in the target plant is achieved by introducing the gene encoding the protein TaVRT-A2 into the target plant.