CN118725065B - Application of alfalfa MsTC protein and its encoding gene in regulating plant vitamin E content - Google Patents

Abstract

The present invention discloses the application of alfalfa MsTC protein and its encoding gene in regulating the vitamin E content of plants. The present invention constructs the MsTC gene in alfalfa onto a pBI121 vector to form a pBI121-MsTC recombinant vector, and then transforms the pBI121-MsTC recombinant vector into alfalfa "Zhongmu No. 1" by a method of alfalfa leaf disk Agrobacterium transformation to obtain transgenic alfalfa overexpressing MsTC. Through transgenic experiments, it was found that overexpressing the MsTC gene can significantly increase the vitamin E content of alfalfa, which is of great significance for improving the quality of alfalfa and promoting the stable and healthy development of animal husbandry.

Description

Application of alfalfa MsTC protein and encoding gene thereof in regulating and controlling vitamin E content of plants

Technical Field

The invention relates to the field of plant genetic engineering, in particular to application of alfalfa MsTC protein and a coding gene thereof in regulating and controlling the vitamin E content of plants.

Background

Alfalfa (Medicago sativa L.) is a perennial leguminous plant, and has the characteristics of 'pasture' sing the praises of due to good palatability, high nutritive value, rich trace elements, rich protein content and the like. As the demand for high quality livestock products increases, the demand for high quality pasture is also increasing.

Vitamin E, also known as tocopherol, cannot be synthesized by the animal itself and must be taken from the diet. The vitamin E is properly supplemented, so that the immunity of animals can be improved, the meat quality can be improved, and the breeding performance of the animals can be improved. The natural vitamin E (tocopherol) content of the alfalfa can not meet the requirements of livestock and poultry, and the activity of the artificially synthesized vitamin E is far lower than that of the natural vitamin E synthesized by plants, so that the vitamin E content of the alfalfa needs to be improved, the problem of insufficient vitamin E content in the alfalfa is solved from the source, and the improvement of the quality of the alfalfa is promoted.

Disclosure of Invention

The invention aims to solve the technical problem of how to increase the vitamin E content of plants.

In order to solve the technical problems, the invention firstly provides a novel application of MsTC protein.

The present invention provides the use of MsTC proteins in any one of the following 1) -3):

1) Regulating and controlling the vitamin E content of plants;

2) Cultivating a transgenic plant with increased vitamin E content;

3) Plant breeding;

the MsTC protein is any one of a 1) or a 2) or a 3) or a 4):

a1 Amino acid sequence is a protein shown as SEQ ID No. 2;

a2 A fusion protein obtained by ligating a tag to the N-terminal or/and the C-terminal of the protein shown in SEQ ID No. 2;

a3 A protein with the same function is obtained by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence shown in SEQ ID No. 2;

a4 A protein which has 90% identity with the amino acid sequence shown in SEQ ID No.2 and has the same function.

The protein of a 2), wherein the tag refers to a polypeptide or protein which is fused and expressed together with the target protein by using a DNA in vitro recombination technology, so as to facilitate the expression, detection, tracing and/or purification of the target protein. The tag may be a Flag tag, his tag, MBP tag, HA tag, myc tag, GST tag, and/or SUMO tag, etc.

The protein according to a 3) above, wherein the substitution and/or deletion and/or addition of one or several amino acid residues is a substitution and/or deletion and/or addition of not more than 10 amino acid residues or a substitution and/or deletion and/or addition of not more than 9 amino acid residues or a substitution and/or deletion and/or addition of not more than 8 amino acid residues or a substitution and/or deletion and/or addition of not more than 7 amino acid residues or a substitution and/or deletion and/or addition of not more than 6 amino acid residues or a substitution and/or deletion and/or addition of not more than 5 amino acid residues or a substitution and/or deletion and/or addition of not more than 4 amino acid residues or a substitution and/or deletion and/or addition of not more than 3 amino acid residues or a substitution and/or deletion and/or addition of not more than 2 amino acid residues or a substitution and/or deletion and/or addition of not more than 1 amino acid residue.

The protein according to a 4) above, wherein the identity is the identity of an amino acid sequence. 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, by using blastp as a program, expect values are set to 10, all filters are set to OFF, BLOSUM62 is used as Matrix, gap existence cost, per residue gap cost and Lambda ratio are set to 11,1 and 0.85 (default values), respectively, and the identity of 1 pair of amino acid sequences is searched for and calculated, and then the value (%) of the identity can be obtained. Such identity includes amino acid sequences 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 homology to the amino acid sequence shown in SEQ ID No.2 of the present invention.

The protein described in the above a 1), a 2), a 3) or a 4) can be synthesized artificially or can be obtained by synthesizing the coding gene and then biologically expressing.

In order to solve the technical problems, the invention also provides a new application of the biological material related to MsTC protein.

The present invention provides the use of a MsTC protein-related biomaterial in any one of the following 1) -3):

1) Regulating and controlling the vitamin E content of plants;

2) Cultivating a transgenic plant with increased vitamin E content;

3) Plant breeding;

the biological material is a nucleic acid molecule encoding MsTC protein or an expression cassette, a recombinant vector or a recombinant microorganism containing the nucleic acid molecule.

In the above application, the nucleic acid molecule is a gene as shown in the following B1) or B2):

b1 A DNA molecule shown in SEQ ID No. 1;

b2 A DNA molecule which has 75% or more identity with the nucleotide sequence defined in B1) and which encodes the above MsTC protein.

Wherein the nucleic acid molecule may be DNA such as cDNA, genomic DNA or recombinant DNA, or RNA such as mRNA or hnRNA.

The nucleotide sequence encoding MsTC proteins of the present invention can be easily mutated by one of ordinary skill in the art using known methods, such as directed evolution and point mutation. Those artificially modified nucleotides having 75% or more identity to the nucleotide sequence encoding MsTC protein are derived from the nucleotide sequence of the present invention and are equivalent to the sequence of the present invention as long as they encode MsTC protein and have the same function.

The term "identity" as used herein refers to sequence similarity to a native nucleic acid sequence. "identity" includes a nucleotide sequence having 75% or more, or 85% or more, or 90% or more, or 95% or more identity with the nucleotide sequence of the protein consisting of the amino acid sequence shown in SEQ ID No.2 of the present invention. Identity can be assessed visually or by computer software. Using computer software, the identity between two or more sequences can be expressed in percent (%), which can be used to evaluate the identity between related sequences.

The 75% or more identity may be 80%, 85%, 90% or 95% or more identity.

In the above applications, the expression cassette refers to a DNA capable of expressing MsTC protein in a host cell, which may include not only a promoter for initiating MsTC transcription, but also a terminator for terminating MsTC transcription. 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. Suitable transcription terminators include, but are not limited to, the Agrobacterium nopaline synthase terminator (NOS terminator), the cauliflower mosaic virus CaMV 35S terminator, the tml terminator, the pea rbcS E9 terminator and the nopaline and octopine synthase terminators.

In the above applications, the vector may be a plasmid, cosmid, phage or viral vector. The recombinant vector can be a vector constructed by utilizing the existing plant expression vector and containing MsTC gene expression cassette. 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, pBin438, pCAMBIA1302, pCAMBIA2301, pCAMBIA1301, pCAMBIA1300, pBI121, pCAMBIA1391-Xa or pCAMBIA1391-Xb, etc. 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. The sources of the translational control signals and initiation codons are broad, and can be either natural or synthetic. The translation initiation region may be derived from a transcription initiation region or a structural gene. To facilitate identification and selection of transgenic plant cells or plants, the plant expression vectors used may be processed, for example by adding genes encoding enzymes or luminescent compounds which produce a color change (GUS gene, luciferase gene, etc.), antibiotic marker genes (such as nptII gene conferring resistance to kanamycin and related antibiotics, bar gene conferring resistance to the herbicide phosphinothricin, hph gene conferring resistance to antibiotic hygromycin, dhfr gene conferring resistance to methotrexate, EPSPS gene conferring resistance to glyphosate) or chemical marker genes, etc. (such as herbicide resistance genes), mannose-6-phosphate isomerase gene providing mannose metabolization ability, etc. From the safety of transgenic plants, transformed plants can be screened directly in stress without adding any selectable marker gene.

In a specific embodiment of the invention, the recombinant vector is a pBI121-MsTC recombinant vector, and the pBI121-MsTC recombinant vector is obtained by replacing a DNA fragment between XbaI and BamHI cleavage sites of the pBI121 vector with a CDS sequence of MsTC gene shown in SEQ ID No.1 and keeping other sequences of the pBI121 vector unchanged.

In the above application, the microorganism may be a yeast, a bacterium, an alga or a fungus, such as Agrobacterium. The recombinant microorganism refers to a recombinant microorganism with changed functions obtained by manipulating and modifying genes of a target microorganism. Such as a recombinant microorganism obtained by introducing the recombinant vector into a microorganism of interest. The recombinant microorganism is understood to mean not only the particular recombinant microorganism but also the progeny of such a cell, and such progeny may not necessarily correspond exactly to the original parent cell, but are included within the scope of the recombinant microorganism, due to natural, accidental, or deliberate mutation and/or alteration.

In a specific embodiment of the present invention, the recombinant microorganism is Agrobacterium EHA105 containing the recombinant vector of pBI121-MsTC described above.

In the above applications, the vitamin E comprises gamma-tocopherol content and/or alpha-tocopherol.

In the application, the regulation of the vitamin E content in plants is to improve the vitamin E content in plants. Further, the improvement of the vitamin E content of the plant is to improve the vitamin E content in the plant leaves.

In a specific embodiment of the invention, when the amount of MsTC gene expression in a plant is increased, the gamma-tocopherol content, the alpha-tocopherol content and the total tocopherol content in the leaves of the plant are increased.

In the above applications, the object of plant breeding is to cultivate plant varieties with a high vitamin E content.

In order to solve the technical problems, the invention finally provides a method for cultivating transgenic plants with increased vitamin E content.

The method for cultivating the transgenic plant with the increased vitamin E content comprises the following steps of increasing the content and/or activity of MsTC protein in a receptor plant to obtain the transgenic plant, wherein the vitamin E content of the transgenic plant is higher than that of the receptor plant.

In the above method, the method for increasing the content and/or activity of MsTC protein in the recipient plant is to overexpress MsTC protein in the recipient plant.

Further, the over-expression method is to introduce MsTC protein encoding gene into the acceptor plant.

Furthermore, the coding gene sequence of the protein is shown as SEQ ID No. 1.

In the above method, the transgenic plant has a higher vitamin E content than the recipient plant and is represented by any one of the following X1) -X3):

X1) the gamma-tocopherol content in the leaves of the transgenic plant is higher than that of the recipient plant;

x2) the alpha-tocopherol content in the leaves of the transgenic plant is higher than that of the recipient plant;

x3) the total tocopherol content in the leaves of the transgenic plant is higher than that of the recipient plant.

In any of the above applications or methods, the transgenic plant is understood to include not only the first generation transgenic plant obtained by transforming the MsTC gene with the recipient plant, but also its 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 any of the above applications or methods, the plant is a dicotyledonous plant or a monocotyledonous plant.

Further, the dicotyledonous plant is a leguminous plant;

Still further, the leguminous plant is a alfalfa plant;

Still further, the alfalfa plant is alfalfa;

in a specific embodiment of the present invention, the alfalfa is alfalfa ("alfalfa 1").

Compared with the prior art, the invention has the outstanding effects that MsTC genes capable of improving the vitamin E content in the alfalfa are discovered, and the transgenic alfalfa with the obviously improved vitamin E content and over-expressed MsTC is obtained by an agrobacterium transformation method.

The invention constructs MsTC gene in alfalfa to pBI121 vector to form pBI121-MsTC recombinant vector, then converts pBI121-MsTC recombinant vector into alfalfa 'alfalfa No. 1' by means of alfalfa She Pannong bacillus conversion to obtain transgenic alfalfa over-expressed MsTC. Through a transgenic test, the over-expression MsTC gene can obviously improve the vitamin E content of the alfalfa, which has important significance for improving the quality of the alfalfa and promoting the stable and healthy development of animal husbandry.

Drawings

FIG. 1 shows the phenotype and expression of pBI121-MsTC transgenic alfalfa. A, the structure of pBI121-MsTC vector is schematically shown. B, cutting phenotype map of wild alfalfa with 2 months of age and pBI121-MsTC transgenic alfalfa. WT stands for wild-type alfalfa. And C, detecting the expression condition of MsTC in the pBI121-MsTC transgenic alfalfa by RT-qPCR. NbActin is used as reference gene of alfalfa.

FIG. 2 shows vitamin E content in pBI121-MsTC transgenic alfalfa. A, cutting 2 months old wild alfalfa and gamma-tocopherol content in pBI121-MsTC transgenic alfalfa. B, content of alpha-tocopherol in wild alfalfa of 2 months of age and pBI121-MsTC transgenic alfalfa. And C, cutting the total tocopherol content of 2 month old wild alfalfa and pBI121-MsTC transgenic alfalfa.

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 alfalfa "in alfalfa No. 1" and pBI121 vectors in the examples below are both described in literature "Shi K,Liu J,Liang H,Dong H,Zhang J,Wei Y,Zhou L,Wang S,Zhu J,Cao M,Jones CS,Ma D,Wang Z.An alfalfa MYB-like transcriptional factor MsMYBH positively regulates alfalfa seedling drought resistance and undergoes MsWAV3-mediated degradation.J Integr Plant Biol.2024Apr;66(4):683-699.".

The CDS sequence of MsTC gene in the following example is shown as SEQ ID No.1 in the sequence table, and the amino acid sequence of MsTC protein coded by the CDS sequence is shown as SEQ ID No.2 in the sequence table.

Example 1 preparation of pBI121-MsTC transgenic alfalfa

1. Construction of pBI121-MsTC vector

1. Obtaining alfalfa cDNA

Alfalfa "alfalfa No. 1" leaf was sampled, RNA was extracted using Trizol method, and the RNA was reverse transcribed into cDNA.

2. And (3) using the cDNA obtained in the step (1) as a template, and carrying out PCR amplification by using a primer pair MsTC-F/MsTC-R through high-fidelity DNA polymerase to obtain a MsTC fragment. The primer sequences were as follows:

MsTC-F:gagaacacgggggactctagaATGGAAACCAAGCTATTGAATCCTCCTT;

MsTC-R:ggactgaccacccggggatccCTACAAGCCAGGTGGTTTAAACAGAG。

the PCR amplification system was as follows, 2X KOD one 10. Mu. L, msTC-F (10. Mu. Mol/L) 0.5. Mu. L, msTC-R (10. Mu. Mol/L) 0.5. Mu.L, alfalfa cDNA 1. Mu.L, and ddH ₂ O to a final volume of 20. Mu.L.

The PCR reaction was performed by 94℃pre-denaturation for 2min, 98℃denaturation for 10s,55℃annealing for 5s,68℃extension for 20min,35 cycles, 68℃extension for 5min, and 4℃for 5min.

3. And (3) performing agarose gel electrophoresis detection on the MsTC fragment obtained in the step (2), cutting and purifying and recovering the correct target band, and connecting the fragment with the pBI121 vector which is digested by XbaI and BamHI by using a Northey recombinase (Vazyme ClonExpress ll One StepCloning Kit, cat# C112) through a seamless cloning method to obtain a connecting product.

The ligation system was 1. Mu.L for MsTC fragments, 2. Mu.L for cleaved pBI121, 5x CE II Buffer 4. Mu.L, exnase II. Mu.L, and ddH ₂ O to a final volume of 20. Mu.L.

The ligation conditions were as follows, 37℃for 30min.

4. And (3) transforming DH5 alpha escherichia coli by using the connection product obtained in the step (3), plating bacterial liquid on a culture plate screened by kanamycin, culturing the bacterial liquid upside down at 37 ℃ for overnight, selecting single colony for colony PCR identification of positive clone, selecting the single colony of the positive clone, culturing the single colony in LB culture medium containing kanamycin resistance for 12-16 hours at 37 ℃, extracting and sequencing plasmids, and marking the plasmid with correct sequencing as pBI121-MsTC recombinant plasmid.

The recombinant plasmid pBI121-MsTC is obtained by replacing the DNA fragment between the XbaI and BamHI cleavage sites of the pBI121 vector with the CDS sequence of MsTC gene shown in SEQ ID No.1, and keeping other sequences of the pBI121 vector unchanged. The structure of the pBI121-MsTC recombinant plasmid is schematically shown in FIG. 1A.

2. Cultivation of pBI121-MsTC transgenic alfalfa

1. 50Ng of pBI121-MsTC recombinant plasmid was mixed with 100. Mu.L of Agrobacterium EHA105 competent, frozen in liquid nitrogen by freeze thawing, recovered and spread on a solid medium containing kanamycin and rifampicin resistance, and the Agrobacterium EHA105 harboring the pBI121-MsTC recombinant plasmid was obtained by screening.

2. Agrobacteria EHA105 with pBI121-MsTC recombinant plasmid is infected on the leaf disc of aseptic alfalfa (alfalfa No. 1), callus is obtained through differentiation culture, and seedling is obtained through rooting culture and transferred into soil. After the growth of the plantlets which are continuously cultivated is stable, taking a leaf sample, extracting genome DNA by using a CTAB method, detecting whether the pBI121-MsTC vector is inserted into plants or not by using a primer pair 35S-F and MsTC-R (35S-F: ACTGACGTAAGGGATGACGCAC and MsTC-R: GGACTGACCACCCGGGGATCCCTACAAGCCAGGTGGTTTAAACAGAG), and obtaining transgenic plantlets with the size of about 1500bp by PCR amplification, namely positive plantlets.

3. Positive seedlings and leaves of wild alfalfa were taken, RNA was extracted using Trizol method, and cDNA was obtained by reverse transcription. And (3) performing real-time fluorescence quantitative PCR amplification by using the primer pairs q-MsTC-F and q-MsTC-R and MsActin-F and MsActin-R and using the extracted cDNA as a template, and detecting the expression quantity of MsTC. The primer sequences were as follows:

q-MsTC-F:ACTACATTGCGTGCTCCAACATC;

q-MsTC-R:TTGCTGCCGTCATATCTTCTTTCC;

q-MsActin-F:CAAAAGATGGCAGATGCTGAGGAT;

q-MsActin-R:CATGACACCAGTATGACGAGGTCG。

As shown in FIG. 1C, the expression level of MsTC in the transgenic alfalfa OE-MsTC-7, OE-MsTC-27 and OE-MsTC-28 of pBI121-MsTC is obviously higher than that of wild alfalfa. The pBI121-MsTC transgenic alfalfa OE-MsTC-7, OE-MsTC-27, OE-MsTC-28 were selected for the following biological functional assays.

Example 2, detection of vitamin E content in transgenic alfalfa pBI121-MsTC

1. Alfalfa No. 1 and pBI121-MsTC transgenic alfalfa strains OE-MsTC-7, OE-MsTC-27 and OE-MsTC-28 in wild alfalfa are cut.

2. After 2 months of growth, 50mg of fresh leaves were ground in liquid nitrogen until they were as uniform as possible, 1mL of a mixed solution of methanol and chloroform (2:1) containing 0.01% butylated hydroxytoluene was added, and the mixture was left at room temperature for 20 minutes.

3. Subsequently, 300. Mu.L of chloroform and 600. Mu.L of water were added, and after mixing uniformly, 14000g was centrifuged for 10min.

4. The upper aqueous phase was discarded and the lower organic phase was transferred to a new centrifuge tube.

5. The organic phase was dried under vacuum and 400. Mu.L of a mixture of dichloromethane and methanol (1:5) was added for vitamin E determination.

6. The vitamin E content was determined by HPLC. The tocopherol was measured using a silica gel column (4.6*250mm length,5. Mu. M PARTICLE size) with n-heptane to isopropanol (99:1) as the mobile phase at a flow rate of 1 mL/min. The content of each component was calculated relative to the standard by varying the vitamin E component from the peak time, and 3 biological replicates were set for each experiment.

The results are shown in FIG. 2, which shows that the gamma-tocopherol, alpha-tocopherol and total tocopherol content in pBI121-MsTC transgenic alfalfa were significantly increased relative to the wild-type. Wherein the average gamma-tocopherol content in the leaves of wild-type and pbi-MsTC transgenic alfalfa lines OE-MsTC-7, OE-MsTC-27, OE-MsTC-28 is 0.14mg/100g, 0.34mg/100g and 0.36mg/100g, respectively, the average alpha-tocopherol content in the leaves of wild-type and pbi-MsTC transgenic alfalfa lines OE-MsTC-7, OE-MsTC-27, OE-MsTC-28 is 4.92mg/100g, 8.74mg/100g, 6.25mg/100g and 7.84mg/100g, respectively, and the average total tocopherol content in the leaves of wild-type and pbi-MsTC transgenic alfalfa lines OE-MsTC-7, OE-MsTC-27, OE-MsTC-28 is 5.06mg/100g, 9.08mg/100g, 6.59mg/100g and 8.20mg/100g, respectively.

The experimental results show that the over-expression MsTC in wild alfalfa can obviously improve the content of each component of vitamin E in the alfalfa.

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

Claims (7)

1. Use of MsTC protein in any of the following 1) or 2): 1) Increase the content of plant vitamin E; 2) Cultivate transgenic plants with increased vitamin E content; The MsTC protein is any one of a1) or a2): a1) the amino acid sequence is the protein shown in SEQ ID No. 2; a2) a fusion protein obtained by connecting a tag to the N-terminus or/and the C-terminus of the protein shown in SEQ ID No. 2; The plant is alfalfa; The vitamin E is α-tocopherol. 2. Use of biological materials related to MsTC protein in any of the following 1) or 2): 1) Increase the content of plant vitamin E; 2) Cultivate transgenic plants with increased vitamin E content; The MsTC protein is any one of a1) or a2): a1) the amino acid sequence is the protein shown in SEQ ID No. 2; a2) a fusion protein obtained by connecting a tag to the N-terminus or/and the C-terminus of the protein shown in SEQ ID No. 2; The biological material is a nucleic acid molecule encoding the MsTC protein or an expression cassette, a recombinant vector or a recombinant microorganism containing the nucleic acid molecule; The plant is alfalfa; The vitamin E is α-tocopherol. 3. The use according to claim 2, characterized in that: the nucleic acid molecule is the gene shown in B1) or B2) below: B1) DNA molecule shown in SEQ ID No.1; B2) A DNA molecule having 75% or more identity with the nucleotide sequence defined in B1) and encoding the MsTC protein. 4. A method for cultivating a transgenic plant with increased vitamin E content, comprising the following steps: increasing the content and/or activity of MsTC protein in a recipient plant to obtain a transgenic plant; the vitamin E content of the transgenic plant is higher than that of the recipient plant; The MsTC protein is any one of a1) or a2): a1) the amino acid sequence is the protein shown in SEQ ID No. 2; a2) a fusion protein obtained by connecting a tag to the N-terminus or/and the C-terminus of the protein shown in SEQ ID No. 2; The plant is alfalfa; The vitamin E is α-tocopherol. 5 . The method according to claim 4 , characterized in that: the method for increasing the content and/or activity of MsTC protein in the recipient plant is to overexpress the MsTC protein in the recipient plant. 6. The method according to claim 5, characterized in that: the overexpression method is to introduce the gene encoding the MsTC protein into the recipient plant. 7. The method according to any one of claims 4 to 6, characterized in that the coding gene sequence of the MsTC protein is as shown in SEQ ID No. 1 in the sequence listing.